CANCER
VOLUME
25
RESEARCH
NOVEMBER
1965
NUMBER
10
Mitotic and Functional Homeostasis:
A SpeculativeReview
w. S.BULLOUGH
Birkbeck College, University of London, London, England
CONTENTS
I. Introduction
II. Theories of gene expression
A. Genetic control mechanisms
B. Embryonic differentiation
C. Cell division and cell function
III. Mitotic honteostasis in adult tissues
A. Control by chalones
1684
1684
1684
1685
1686
1687
1687
B. The role of the adrenals
1687
1. The diurnal mitotic cycle
2. Adrenalin and the glucocorticoid
3. Conclusions
C. The wound reaction
1687
1688
1689
1689
hormones
D. Regeneration
1690
1. Local and general mitotic reactions
2. Hormones and regeneration
3. Chalones and regeneration
4. The experimental evidence
5. Mitotic inhibition and stimulus
6. The effects of metabolic damage
7. The effects of the adrenal hormones
8. Other evidence on regeneration
9. Conclusions
E. The mitotic cycle
1690
1691
1692
1692
1693
1693
1694
1695
1695
1695
1 . The phases
of the cycle
1695
2. The initiation of the prosphase
3. The phase of DNA synthesis
4. Antephase and mitosis
5. RNA and protein synthesis
6. The action of the chalone complex
F. Conclusions
1696
1696
1696
1697
1697
1698
Iv. Mitotic and functional homeostasis
A. The basic mechanism
1.
2.
3.
4.
1699
1699
Mitosis and cell function
Life expectancy of cells
The nature of the dichophase
Conclusions
B. The versatility
1. Methods
1699
1700
1701
1702
of the mechanism
of alteration
1703
of tissue mass
1683
@
1703
This
One
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@
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IIll@II@
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2C17-TSN-2XGG
Vol. 25, November
Cancer Research
1684
1703
1704
1707
1708
1709
1710
1710
1711
1712
1712
1713
1714
1715
1715
1716
1716
1716
2. The mitogenic hormones
3. The poietins
4. Mitotic control in lymphoid tissue
5. The growth of hair
6. Conclusions
C. The breakdown of the mechanism
1. The mechanism itself
2. Carcinogenesis
3. The chalone complex and the latent period
4. Promoting agents
5. Retarding agents
6. Conclusions
V. General summary and conclusions
A. The role of inducing substances
B. The role of the chalones
C. The role of the hormones
D. The problem of gene damage
II. THEORIES
I. INTRODUCTION
One of the most important
cerns both the manner
tissues and the manner
entiation
is maintained
In recent
steps
problems
con
have taken
an understanding
a number
of this
problem,
of new
and
al
though much of what is now being written is admittedly
hypothetical,
it nevertheless
seems worthwhile
at this
moment
to attempt
to review
these
new
ideas
and
to
place them in a logical context.
Perhaps
the most significant
of recent biologic discov
eries in this field have been those that have provided new
knowledge and understanding
of the genetic manipula
tion of protein synthesis (see, for instance, Ref. 262), of
the changes that occur in the expression of the genome
when
an embryonic
cell differentiates
(see, for instance,
Refs. 165, 166), and of the homeostatic mechanisms by
which both specialized function and mitotic activity are
controlled in the differentiated
cells of adult mammals
(see, for instance,
Refs. 73, 75).
These
3 fields of research
are evidently closely interrelated, and the conclusions that
have emerged probably apply generally to all metazoan
animals.
However,
has been restricted
sues of adult
expression
controlled
in the
present
to the situation
mammals
review
consideration
that exists in the tis
and to the manner
in which
the
of the genome in a differentiated
cell may be
by mitotic and functional homeostatic mecha
nisrns.
It appears
these
self-evident.
mechanisms
the problem
must
of cancer,
that
have
and indeed that
understanding
of the changes
genesis is likely to be achieved
previous
knowledge
sues maintain
their
any conclusions
important
in
little or no real
occurring
during carcino
without some considerable
of the manner
organization.
regarding
implications
in which normal
It seems
reasonable
Received for publication February 4, 1965.
The most important biologic discoveries of recent years
have concerned the structure of the DNA' molecule, and
the most important hypotheses have concerned the possi
ble manner in which genetic information may be coded in
the base sequences of this molecule.
This work is outside
the scope of the present review except in so far as it forms
the background for our understanding,
first, of the man
ner in which instructions may emanate from the nucleus
t.o induce the synthesis of specific types of proteins, and
second, of the manner in which, from time to time, these
instructions
may be changed
to take account
of the
changed
conditions,
whether
inside or outside
the cell.
Regarding these 2 points, most of our information has been
derived from studies of microorganisms ; recent reviews are
those of Monod el al. (262, 355, 356), whose conclusions
have been discussed in a more general chemical context by
Dean and Hinsheiwood (131). There are, however, strong
indications that similar mechanisms must also exist in the
differentiated cells of adult mammals, in which they may
act as an essential part of homeostatic mechanisms.
As regards microorganisms,
current hypotheses may be
summarized
as follows. Two main types of genes are
recognized : the structural genes and the regulator genes.
A structural gene, acting through the intermediary of an
RNA messenger, is stimulated to initiate the synthesis of
a particular protein.
The stimulus to commence activity
originates
in a particular
region
of the
gene
called
the
“operator,―and each operator may be in control of 1 or
of several related structural genes. Those genes, whether
1 or many, that are under the control of a single operator
are together
known as an “operon.― When an operon
tis
to
conclude that the main, and perhaps only, reason why
cancer has remained such a mystery for so long is our lack
of any adequate understanding
of these normal mecha
nisnis.
OF GENE EXPRESSION
A. GENETICCONTROL
l\1ECHANISMS
to form
in which, in such tissues, differ
and mitotic activity
is controlled.
years biologists
towards
of all biologic
in which cells differentiate
1965
contains several structural genes, these are usually adjacent
to each other so as to form a cluster on the chromosome
(134).
Each opei ator, and therefore each operon, is under the
‘The
following
abbreviations
are
used
:
DNA,
deoxyribo
nucleic acid; RNA, ribonucleic acid; ACTH, adrenocorticotropin;
ATPase, adenosine triphosphatase; ATP, adenosine triphosphate.
BULL0uGH—Mitotic
and Functional
negative control of a substance called a “repressor,―
which
may be a protein.
Each repressor, which acts only on a
specific operator, is produced in response to the activity
of a regulator gene, probably through the normal iries
senger RNA mechanism.
It is because the repressor has
the ability to react with particular substances, which in
the case of microorganisms
are metabolites of low mo
lecular weight, that the whole system is so remarkably
versatile in its response to the needs of the moment . A
metabolite
that reacts with a repressor in this way is
called an “effector―;
in the reaction the repres.sor is either
inactivated,
so that the relevant messenger RNA and
proteins begin to be synthesized, or it is activated, so that
the synthesis of messenger RNA and protein is inhibited.
Thus in microorganisms
the expression of the genoine is
determined by the type of metabolite available, and it is
important to note that the reaction to a new situation is
almost instantaneous.
A reaction time of less than 15 sec
has been established (262).
It is interesting to note that an effector of this type may
not necessarily exert its influence on the repressor by
binding at the same active site as that which binds the
operator.
A recent hypothesis suggests the possibility
that a repressor may be an allosteric protein, which
possesses
2 separate
active
sites
(355).
In this situation
the activation or inactivation of the repressor is considered
to depend on the molecular distortion caused when the
effector becomes bound to its own site. Because of this
an effector need not bear any obvious chemical relation
ship to the synthetic pathway it influences.
Although these conclusions and suggestions relate almost
entirely to the reactions of unicellular organisms to sub
strates, they are also obviously relevant. to any consider
ation of cellular activity in metazoans.
In spite of the
fact that in the case of the metazoans the evidence is only
slight, Monod and Jacob (356) have concluded that it may
still be sufficient to “justifythe conclusion that the rate of
structural gene expression is controlled, in higher organisms
as well as in bacteria and bacterial viruses, by closely
similar mechanisms,
involving regulator
genes, apore
pressors, operators and operons.― Some of the still in
adequate mammalian evidence relates to the control of the
agouti
hair pattern
(43, 118) and to certain
liver enzymes
Homeostasis
process of differentiation
on the expression
1685
and also the effect it. must have
and control
of the genome.
The nucleus of a fertilized egg contains a gene comple
ment. that is caj)able of directing the construction of each
and every part of the fully differentiated
adult; in other
words, the genome is totipotent.
It. is also evident, at
least
iii some
animals
such
as molluscs
(408)
and
am
l)hibialls (164, 165), that certain cytoplasmic constituents
that. may originate in the cortex (126) of the newly ferti
lized egg are already so organized and distributed
as to
predetermine
the positions of such major adult organs as
brain, intestine, or muscles, and that as the egg undergoes
repeated cleavage this basic patterti becomes even more
firmly stabilized by the developing cell walls (133, 167).
It is also evident that as development proceeds the cyto
l)lasmic cOmI)onents themselves undergo increasing spe
cialization and diversification.
Thus the cells in each region of the developing body are
characterized by a particular tyj)e of cytoplasm, and it has
beeii
suggested
that
it is the
contents
of this
which,
by
reacting with the genonie, determine the type of different i
at.ion that will occur.
It is interesting that in the higher
plants
differentiation
situations
is also evidently
determined
in which the cells find themselves
by the
(460).
One of the methods by which cytoj)laslmc
may be induced,
so that. it in turn may
specializat
determine
ion
the
pattern of differentiation within a group of cells, is through
the influence exerted by son@e neighboring
preformed
tissue.
Thus
it is commonly
said
that
one
tissue
will
induce the appearance of another (see Refs. 231, 430, 431).
Such a tissue interaction may be envisaged as involving
either a stimulus or an inhibition : in the sense of a stimulus
it may be considered that the differentiating cells, following
intrinsic
genotypic
and amplify
these
instruct ions
instructions
‘
‘select among, translate
in response
to extrinsic
influences― (213) ; in the sense of an inhibition it is possible
to regard the reaction as the outcome of the suppression of
intrinsic genotyj)ic instructions
when the “products of
differentiating
tissues act. as inhibitors of like activities in
surrounding tissues― (50). Some indication of the com
plexity of the induction process is given by Saxén(430),
who stresses that the inducing factors may act as triggers
only and that the main part of the differentiation
mecha
(315, 367, 392). The existence of typical messenger R.NA
in the mammal has also been demonstrated
in association
with the ribosomes in rat liver cytoplasm (300, 362).
However, apart from the existence of this evidence, it
This situation is immediately reminiscent of the effect or
repressor-operator
type of mechanism described in micro
organisms.
The simplest suggestion would be that. in
seems
embryonic
most improbable
from what
is already
known
of the
remarkable uniformity of the biochemical organization in
all living matter that the mechanisms of gene expression
and gene control will prove to be fundamentally
different
in the cells of the various groups of organisms, even when
these are as different as mammals and bacteria.
B.
EMBRYONIC DIFFERENTIATION
In considering metazoan animals it is obvious that,
apart from their multicellular
nature, their outstanding
peculiarity lies in the manner in which their component
cells become differentiated and in the way in which these
differentiated
cells are grouped together to form tissues.
It is extremely important to determine the nature of the
IlisIfl
evidently
lies
inside
the
reacting
cells all the specialized
cells.
genot.ypic
instructions
remain suppressed until, during differentiation,
one spe
cific repressor is inhibited by a particular effector.
If this
suggestion is valid, then, at least in the higher animals, it
must also be accepted that this proces@s involves the
permanent suppression of all or most of the other spe
cialized potentialities of the genome (53, 166).
It is interesting to note that in the higher plants it is
possible to stimulate a single fully differentiated
cell to
such a degree that it will give rise to a new plant (460), and
this imist indicate that within such a differentiated cell the
genome is still potentially totipotent.
However, there is
at the moment no evidence at all that differentiated
mammalian
cells, with which
this review
is primarily
con
certied, can act in this way. In such cells differentiation
commonly appears to be irreversible.
From what has been said it is clear that differentiation
is a irocess that, in mammals, takes place almost entirely
in the embryo.
Once organogenesis is complete all the
cells of the body,
except.
for instance
the germ
cells and
the stem cells of the hemopoietic systems (see p. 1706),
are specialized to such an extent that each must continue
ever afterwards to breed true to type.
It is with this type
of
differentiated
concerned,
that
cell
that
the
present
review
and in such a cell it is the genetic
remain
after differentiation
iml)ortance.
It
now
appears
that
that
maliaii tissues the only remaining
functional
genes are those
that
are of outstanding
functional
dictate
Unfortunately,
in the
literature
is mainly
potentialities
in most
adult
or potentially
it is common
in the manner
of the
are regarded
to which
they
belong
for
to find
that. only those cells that fuiiction
tissue
of the
pathways,
typical
of
their
genome
has
become
severely
Except
and
CELL DIVISION AND CELL FUNCTION
for a few highly
specialized
mitosis
or in protein
synthesis
for function
may
take
the
forni of a diffusible chemical n@essenger (377), which may
act. as part of a negative feedback mechanism
to suppress
mit.osis within
the cells concerned
(259, 348).
In the
language of the microbiologists
such a substance
might be
considered
to be an effector.
as.sociat.ed
with chromosomes
Stedman
and St.edman
are capable
of suppressing
still remains
whether
they
RNA
unanswered
synthesis.
(see Refs.
The
5, 24, 504),
but it is already clear that the histones are not as tissue
specific as was originally thought and as would be necessary
if they were to form an integral part of a tissue-specific
control mechanism (272).
In the 2nd line of research, Bullough and Laurence
(86, 87) have extracted from mouse epidermis a substance
that inhibits epidermal mitosis and has the necessary
characteristics to act as part of a negative feedback mecha
nism,
and
they
have
also
obtained
evidence
indicating
that similar tissue-specific mitotic inhibitors are associated
with the hypodermis and the hair bulbs (82). In addition,
tissue-specific mitotic inhibitors have been extracted by
Saetren
cells such as neurones
but also exist free in the cell.
(458) questioned
may be concerned
in the tissue-specific
control of gene
expression,
and Huang and Bonner (253) have shown that
they
typically
limited.
For an excellent discussion of the
meaning of the term “differentiation―see Weiss (513).
C.
which the commencement of 1 type of specialized synthetic
activity may be accompanied by the cessation of another.
On theoretical grounds it has been thought for some time
that. an integral part of the control mechanism that de
termines whether a cell indulges in protein synthesis for
question
as differ
entiated.
Cells that belong to the tissue but have never
functioned in the typical manner are described as undiffer
entiated, while cells that have ceased to be able to function
in the ty@)ica1 manner are described as dedifferentiated.
In fact., almost all the somatic cells of an adult mammal,
whatever may be their appearance or function, must be
regarded as fully differentiated in the sense that the power
exl)res.sion
1965
Recently 2 lines of research have converged on this
problem.
In the 1st, there has been an investigation
of
the possible functions of the histones, which not only are
main
the synthesis
enzymes needed for the general metabolic
mitotic activity, and for tissue function.
of
Vol. 25, November
Cancer Research
1686
(425) from liver amid kidney
and by Rytomaa
and
and for a number of aging cells such as circulating erythro
cytes and granulocyt.es, the tissue cells in an adult mammal
are commonly capable of at least 3 basic programs of
protein synthesis, of which 1 is essential while the others
Kiviniemi
(423) from mature granulocytes.
All these
substances have similar physical properties, and Bullough
(73) has proposed that they should be called chalones, a
appear
are discussed
to be optional.
The
essential
program
ensures
a
name that
refers to their inhibitory
in detail
below
actions.
; here
These
it is necessary
actions
only
continuous supply of the enzymes needed in such vital
metabolic l)ath\vays as that of the tricarboxylic acid cycle
(125, 169, 363). In this connection it is interesting to
note that Davidson et a]. (129) have shown that, when
stress that they result in the suppression
of synthesis
mitosis within the tissues in which they originate.
whereby
a choice
actinomycin
programs,
it must
D is added
to connective
to block
the synthesis
of messenger
enzymes
as succinic
dehydrogenase
tissue
RNA,
cells in vitro
such
continue
generalized
their
action
unaffected.
This suggests that essential enzymes may
not be subject to any immediate genetic control, which
may indicate either that they have an unusually long
functional life or that their synthesis is dictated at the
ribosome level and needs no repeated genetic stimulus for
its continuance.
By contrast,
synthesis
synthesis
are
the other
highly
of those
2 possible
specialized.
enzymes
on which
programs
These
mitosis
are,
of protein
1st,
depends,
the
and
2nd, the synthesis of those enzymes that direct the specific
activities typical of the tissue.
It appears that, although
cells are capable of undertaking these 2 types of syntheses,
none are capable of undertaking them both simultaneously;
the operation of one synthetic program always seems to
exclude the operation of the other (see p. 1699). This is
again reminiscent of t.he situation in microorganisms
in
However,
if the chalones
do form
is made
part
between
be emphasized
that
to
for
of the mechanism
possible
synthetic
in some tissues
the
choice is not simply between 2 alternatives.
In some
tissues, such as striped muscles and nerves, there is evi
dently no choice at all, and at all times the cells continue
synthesis
for
function.
In
other
tissues,
such
as epi
dermis (44, 339, 347), the cells are at least potentially
capable of more than 2 types of synthesis.
The many
varieties of epidermis can be classified into 3 major groups:
surface epidermis, which produces the outer sheet of “soft―
keratin; hair bulbs, which produce “hard―
keratin; and
sebaceous glands, which produce fatty sebum.
In all
normal situations the cells either undergo mitosis or pro
duce their typical proteins, but in abnormal situations the
various
types
(357).
Thus, after the destruction
of these
epidermal
formation
after
the
of cells
types
are
may
found
contribute
of a new and entirely
destruction
cholanthrene
of the
to be interchangeable
of the skin surface any
cells
towards
the
typical surface epidermis;
sebaceous
glands
by
methyl
new glands may be produced from the cells of
BULL0uGH—Mitotic and Functional
the outer follicle sheath (357, 358) ; and in certain circum
stances the surface epidermis may even give rise to new
hair follicles, each of which will possess a sebaceous gland
(40, 42, 52, 350). Thus it appears that at least some of
the epidermal cells retain a certain residual versatility that
can be exploited in an emergency.
Other examples of this type of reaction are found in
liver cells (308) and in melanoblasts,
which
do not synthe
size melanin until they receive an appropriate
stimulus
(43). Indeed, as Willis (526) has emphasized, any study
of pathologic
histology
soon
indicates
what
the
various
cell types “canbe or do in all manner of abnormal environ
ments . . . and shows that great transformations
of cellular
structure . . . are possible in most tissues.― It. has been
suggested by Weiss (512, 513) that when a differentiated
cell changes its activities in this way the phenomenon
should be called modulation.
In the case of epidermis it is
clear that such modulation occurs only as the result. of
some drastic change in the cellular envitonment., which
must in turn cause a significant intracellular change.
III.
MITOTIC
HOMEOSTASIS
A.
CONTROL
IN ADULT
TISSUES
BY CHALONES
It is evident that in any “typicaltissue― the alternative
programs of protein synthesis for niitosis and for tissue
function differ in at least 1 important respect.
Any cell
that begins to prepare for mitosis is thereby committed to
complete the process, but any cell that is synthesizing the
proteins
needed
for normal
tissue
function
is commonly
able to revert to a relatively nonfunctional
state from
which it can then proceed to mitosis.
Such reversion is
seen in most tissues after wounding, during regeneration,
in tissue culture, and in neoplasia, and it follows that the
state of functional activity typical of most differentiated
tissues may be regarded as unstable.
Indeed, the impli
cation is that not only the functional state of a tissue but
the whole structure of an adult mammal may need at all
times to be actively maintained against the possibility of
collapse into mitotic anarchy.
By contrast, the process of
recurrent
cell growth and mitosis is evidently entirely
stable, and both in vitro and in neoplasia, cell populations
that are behaving in this way appear to be potentially
immortal.
Thus each of the tissues of an adult mammal seems to
be in a state of equilibrium and all except the most. highly
modified types of cells, to be poised between an unstable
functional state and a stable routine of recurrent mitosis.
It is obvious that an understanding
of the nature of the
mechanisms that determine and maintain the actual point
of balance
within each tissue is of the greatest
possible
theoretical
amid practical
importance.
One
common
theory has been that tissue mitotic activity
may be con
trolled by a tissue mitotic inhibitor
greater
the tissue
mass
within
in such a way that the
the body
space,
the lower
will be the rate of cell production (see, for instance, Refs.
259,348,377,378).
The earliest indications that this concept may indeed be
broadly correct came from observations on organ regener
ation
and in particular
from studies
of the regeneration
of
Homeostasis
1687
which contains much contradictory
evidence, has been Fe
viewed by, for instance, Swanmi (467, 468) and Bullough
(73), amid it is also discussed below (see p. 1691). The 1st
significant success in the extraction of tissue-specific mi
totic inhibitors with the necessary characteristics
appears
to have been that of Saetren (425, 426), who studied
niitotic control iii kidney and liver; the 2nd was that of
Bullough and Laurence (86, 87), who studied epidermis
and suggested
that such inhibitors
should be called
chalones; and the 3rd was that of Rytomaa arid Kiviniemi
(423), who have extracted a granulocytic chalone.
The epidermal chalone is water soluble, and it can
easily be extracted when the epidermis is macerated.
It
is not species specific, amid it is capable of depressing
mitotic activity in mouse epidermis both in vivo and in
vitro (87). The epidermal chalone will also depress mitotic
activity
in cornea, sebaceous glands, and eso@)hageal
lining el)ithehum, but not in other tissues tested.
Con
versely,
aqueous
extracts
of a variety
of other
tissues
do
not. lossess the power to depress epidernial mitotic ac
tivit.y.
More recently it has been shown (78) that
the epidermal chalone is nondialyzable
and that. it. is
destroyed by boiling and precipitated
by alcohol.
It is
unstable in water solution but stable after lyophilizatiomi.
By fractional precipitation most. of the chalone present in
the original solution has been recovered in the small ire
cipitate obtained when the alcohol concentration
is raised
from 70% to 80%.
An in vitro analysis of the mode of actiomi of the epi
dermal chalone has demonstrated
1 particularly important
point, namely that this chalone is unable to exert its full
power as a mitotic inhibitor in the absence of adrenalin
(75, 87), and conversely it can be shown that. adrenalin
has
no effect
on the
epidermal
mitotic
rate
unless
the
epidermal
chalone is present.
This raises the whole
question of the relation of the adrenal glands to mitotic
activity, which it is convenient to consider before returning
to the question of the chalones.
B.
THE ROLE OF THE ADRENALS
1. The diurnal initotic cycle.—This discovery of the role
of the adrenal glands in the control of epidermal mitotic
activity has provided an explanation for the old mystery
of the diurnal mitotic rhythm, which is so prominent a
feature in the epidermis
(64, 73).
It is now evident that
when an animal rests or sleeps the rate of secretion
of
adrenalin falls, the power of the chalone is reduced, and
the epidermal mitotic rate rises; conversely, when an
animal wakes and beconies active the rate of adrenalin
secretion rises, the power of the chalone is increased, and
the epidermal mitotic rate falls (83). As would be ex
pected, adrenalectomy
destroys this diurnal rhythm and
causes the epidernial mitotic rate to rise to a constantly
high level (83).
Diurnal variations in the rate of adrenalin secretion
have
been
described
in man
(275),
rats
(161),
and
mice
(284), and in all cases it is evident that the adrenalin
concentration
is greatly reduced during rest and sleep.
The same is true of noradrenalin, but it is now clear that
kidney and liverin adultmice and rats. This literature,this substance
cannot
act as an effective
substitute
for
Cancer Research
1688
adrenalin in preventing epidernial mitotic activity.2
After
adrenalectomy
the marked rise in the epidermal mitotic
rate is associated with a reduction to about 0. 1 of the
normal adrenalin concentration,
but there is little if any
that
Vol. 25, November
no such diurnal
tissues,
including
rhythms
epidermis
appear
1965
to exist in fetal
(509).
Although more evidence is needed, it nevertheless begins
to appear that a diurnal rhythm in the rate of DNA
change in the concentration
of noradrenalin
(162).
The
synthesis may be found in any tissue that, having only a
question whether the hormones
of the adrenal cortex may
moderate mitotic rate, shows a diurnal rhythm in the
also play some part in controlling the mitotic rate is dis
nunibers of cells engaged in mitosis, and that in tissues
such as duodenal mucosa, in which the mitotic rate is high,
cussed in the next section.
Although the diurnal mit.otic cycle has been analyzed imi there may be little if any sign of either rhythm.
greatest detail in epidermis,
it is evident that similar cycles
occur iii many other tissues.
When the mitotic behavior
of a variety
of tissues
and
organs
is considered,
it is found
Finally,
diurnal
cycle
iii agreement
mitotic
with
the
cycle is inversely
in the rate
of adrenalin
suggestion
related
secretion,
that
the
to the diurnal
it is well known
that the range extends from those like kidney and liver,
which rarely show any niitotic activity, through those like
ej)idermiS, which show moderate mitotic activity, to those
like active hair roots, which show very high mnitotic
activity.
Reviewing this range, and setting aside any
hormone-dependent
tissues (see p. 1703), it is immediately
obvious that a diurnal niit.otic rhythm is commomily, if
not universally, present except. when the mitotic rate is
that any stressful situation results in both increased ac
tivity of the adrenal glands (16, 159, 161, 162, 221, 313)
and decreased mitotic activity of the tissues.
Such de
creased mitotic activity has been described in epidermis
(66, 77, 83), cornea (361), liver (307), and erythrocyte
either
500) during excitement
or pain ; and in all the tissues of
young rats after general disturbance
imiduced by repeated
changes of boxes, diet, or companions
(459).
In juvenile
rats stress by starvation
has also been shown to depress
very
low or very high.
In adult miianinials diurnal mitotic cycles, in which the
highest rat.e of cell division develops during periods of rest
or sleep,
have
been
described
in the epidermis
of mice
and
rats (70, 73) and in that of men (433), in cornea (6, 197,
501), iii lens epitheliuni (428), in oral epitheliumu (6, 490),
in esophagus (6, 64, 137), in stomach mucosa,3 in intestinal
niucosa (6, 64, 197), in rectal mucosa,3 in pancreatic
exocrine epit.helium (136, 331), in epididymis3 (64), and
possibly iii erythrocyte production (223).
In addition, in tissues that are mitotically inert in normal
adult mammals, typical diurnal mitotic cycles may de
velop during regeneration after partial hepat.ectomy (21,
266, 287, 421); in mammary gland during pregnancy (319);
and iii liver (60, 136), kidney (6, 136), adrenal cortex
(138), outer orbital gland (136), and epiphyseal cartilage
(447, 448) during the juvenile growth 1)eliod.
In tissues like active hair roots, which show an extremely
high niitotic rate, no diurnal rhythm is present. ; in tissues
like those of the adult liver and kidney, which show an
extremely low mitotic rate, no diurnal rhythm is de
tect.able, but this may be due to the inadequacy of the
usual
technics.
Of considerable theoretical importance are the recent
demonstrations
that the diurnal mitot.ic rhythm is not
confined
to the visible
stages
affects the preceding
of mitosis
but
that
phase of DNA synthesis.
has described
rhythmic
changes
undergoing
DNA synthesis
and
in the numbers
also in the rate
it also
Alov (6)
of cells
of RNA
synthesis in cornea, tongue, liver, and lymphocytes,
and
he has indicated that both these rhythms coincide with the
mitotic rhythm ; he failed to find any similar phenomenon
in intestinal
mucosa,
which is a highly active tissue in
which the mitotic rhythm is poorly defined.
The existence
of a diurnal cycle in DNA synthesis has also been found
in ej)idermis and tongue by Oehlert and Block (375), in
cornea by Tchoumak
(480), and in epidermis, tongue,
esophagus,
and forestomach,
but not in duodenal
mucosa,
production
(51) during stress by starvation
; in epidermis
during prolonged
exercise (65, 83) ; in cornea during pro
longed swimming
S.
Bullough
and
E.
3 M.
J.
Coleman,
personal
B.
Laurence,
communication.
unpublished
(68) and cornea (174,
DNA synthesis in epithelium of tongue, forestomach, and
hindstomach,
amid in liver, pancreas, and adrenal gland
(286).
2. A drenalin and the glucocorlicoid horrnones.—Turning
next
to
the
adrenal
hormones
themselves,
it
shown, both in vivo and in vitro, that adrenalin
potent
epidermal
niitotic
inhibitor
114), that the glucocorticoid
exerting
an anti-mitotic
known
hormones
action
if they
has
been
is the most
(68, 71, 83, 87,
are also capable of
are
given
in suffi
ciently large doses (68, 482), and that the mineralocorticoid
hormones are without any apparent effect (68).
Besides epidermis, adrenalin has been shown to depress
mitotic activity in cornea (48, 174), in lens epithelium,4
and
in intestinal
mucosa
its high mitotic
(158).
In this
rate the depression
small and short
lived,
and in active
last
caused
hair bulbs,
tissue
with
is relatively
which
have
perhaps the highest mitotic rate shown by any adult
tissue, adrenalin has no effect at all even when it is injected
in near-lethal doses.2 This last observation is of critical
importance
in that it indicates clearly that, by itself,
adrenalin is not a mitotic inhibitor.
The fact that its
inhibitory action on mitosis is obviously tissue specific
may be taken as indicating that before adrenalin can act
as an inhibitor it must either combine or otherwise co
operate with some substance present in the tissue.
On
the
basis
of the
evidence
obtained
suggested that this substance
from
epidermis,
is the tissue-specific
it is
chalone.
Regarding the glucocorticoid hormones, it is generally
believed that their secretion rate is directly related to the
secretion rate of ACTH from the anterior pituitary gland,
and it is also known that stressful conditions
lead both to
a higher rate of ACTH secretion
(281, 387, 427) and to an
increased
blood
flow through
the adrenal
gland
(230).
The rate of ACTH secretion is increased by the most
by Pilgrimetal.(388). Itisinteresting,
however,to note diverse
2 W.
(6) ; in epiderniis
types
of
stress—chemical,
results.
4 M.
Towlson,
personal
communication.
environmental,
and
BuLL0uGH—Mitotic
and Functional
psychologic—and the implication is that the various types
act on the anterior pituitary gland through a common
nervous pathway,
which l)OsSiblY includes the hypo
thalamus (see Refs. 229 and 427).
In such circumstances
it is not surprising to discover
that the secretion rate of the glucocorticoid
hormones
shows a pronounced diurnal rhythm (160, 209, 216, 222,
424, 497, 498) ; in addition it has been found that the
reactivity of the adrenal cortex to ACTH is higher when
an aninial is awake (182). The details of the diurnal
changes
are complex
and more
difficult
to interpret.
than
those of the adrenalin rhythm.
However, the evidence is
such as to support the possibility that the glucocorticoid
hormones
may play some role in mitotic control.
If they
act similarly to adrenalin
then 2 things should follow : 1st,
they should inhibit mitosis selectively in the tissues that
normally show a diurnal mitotic rhythm; and 2nd, when
the stimulus of ACTH is removed by hypophysectomy
there should be a rise in the mitot.ic rate of these same
tissues.
The published evidence is unfortunately
not extensive,
but on the 1st point it is already known that cortisol, or
hydrocortisone,
does inhibit mitosis in epidermis (68, 115,
258, 482), gastric mucosa (406), and regenerating
liver
(60) ; all these tissues show a diurnal mitotic rhythm.
Conversely, it is known that cortisol has little or no effect
on mitosis in duodenal crypts, intestinal lymph nodes
(406, 413), and active
hair bulbs (352) ; these tissues
show
little if any diurnal mitotic rhythm.
Thus it is already
obvious that, as with adrenalin, the action of the gluco
corticoids is not mitosis specific but tissue specific. Again
the implication may be that a tissue-specific cofactor, such
as a chalone, may be needed before the anti-mitotic action
can develop, but the information available is still insuffi
cient
to indicate
whether
the
chalone-adrenalin
inhibitory
complex also normally contains a glucocorticoid element.
In passing it is interesting to note that interference with
the adrenal mechanism in embryos, whether by cortisol in
jection or by adrenalectomy,
may cause serious growth
abnormalities
(see Refs. 245, 345, 346).
Regarding the effects of hypophysectomy,
this operation
is so drastic that it is not surprising to find conflicting
evidence.
However, although the disordered metabolism
that follows removal of the pituitary has been found to
depress mitosis in regenerating liver (60), there is strong
evidence that this operation can result in a dramatic
increase
(145,
in the
147, 481),
mitotic
rate
of epidermis,
and stomach
mucosa
sebaceous
glands
(30), all 3 of which
normally show a diurnal mitotic cycle. Conversely, hy
pophysectomy
has been found to cause no change in the
mitotic activity of the duodenal crypts (405), which
normally show only slight signs of a diurnal mitotic cycle.
It seems probable that in an operation of this magnitude
the results obtained may be greatly affected by the technic
used.
It is unfortunate that no study of the effect of hypophys
ectomy on the diurnal niitotic cycle appears to have been
made with mammals.
However, with amphibians,
in
which hypophysectomy
is an easier operation, Scheving
and Chiakulas (434) have found that in the corneal epi
thelium of salamander tadpoles, not only is the over-all
mitotic
Homeostasis
rate increased,
1689
but the diurnal
niitot.ic rhythm
is
eliminated.
3. Conc1u@sions.—The evidence concerning diurnal mi
totic rhythms, the anti-mit.ot.ic effects of stress, amid the
actions of adrenalin arid other adrenal hormones has been
listed at some length, partly because so much of it has not
previously been extracted from the large Russian literature
and partly because it illustrates so clearly that. close simi
larities may exist between the mitot.ic control niechanisms
of different tissues.
In particular it shows that the type
of mitotic control mechanism described for the epidermis
by Bullough amid Laurence (83, 87) is unlikely to be
unique.
Since it is now evident that in ei)idermis the
hour-by-hour
variations in the mitotic rate are inversely
related to the hour-by-hour variations iii the concentration
of the chalone-adrenalin
inhibitor within the cells, it may
be presumed that similar chalones, which show similar
relations with adrenalin, may exist. in other tissues that
show a diurnal niitotic rhythm, arid perhaps even in all the
tissues of the body.
It follows that, after allowance is made for the effects
of the varying adrenalin concentrations,
the degree of
mnitotic activity typical of any tissue may be explicable
either in terms of the chalone concentration
within the
cells or of the degree of stability of the chalone-adrenalin
complex.
Thus, a tissue with a naturally
low mit.otic
rate may have either a high chalone cell content or a
chalone with a high affinity for adrenalin; a tissue with a
naturally
high
mit.otic
rate
dition.
In both these
changes in the adrenalin
to exert
much
may
show
the
opposite
con
extreme situations
the diurnal
concentration
would be unlikely
effect.
A possibility exists that. the glucocorticoid
hormones
may also be important
factors in the mitotic control
mechanism.
C.
In considering
attention
THE WOUND REACTION
the
is confined
reaction
to the
of tissues
changes
to wounding,
iii the mitotic
rate.
Other aspects of wound healing, including the iniportamit
question of cell migration, have been reviewed by, for
instance, Giliman and Penn (189), Johnson and McMinn
(273), Slome (453), and Abercrombie (2).
The account given above of the recognition and ex
traction
of the epidermal
of a study
of the
chalone
mitotic
was itself
reactions
of the
the outconie
cells
closely
ad
jacent to an epidermal wound (81, 84). It is well known
that such cells may develop a mitotic rate niore than 10
times the normal maximum rate seen during sleep, and
for a long
time
it has
been
commonly
held
that
this
is the
consequence of the diffusion outwards from the wound of
a mitosis-stimulating
“woundhormone.― This old theory
was
critically
examined
by
Bullough
and
Laurence
(81),
who showed that the mitotic reaction in the neighborhood
of an epidermal wound is in fact due not to the presence
of a mitotic
stimulant
but
to the
temporary
absence
of a
mitotic inhibitor.
By means of differential
wounding
they also showed that the epidermiial mitotic inhibitor is
tissue specific and that at least 2 other tissue-specific
mitotic inhibitors also exist in mouse skin—i each in the
hypodermis and the hair roots (82).
A.s regards
el)idermlS,
it now appears
that
the mitotic
inhibitor is the epidermal chalone, amid it follows that a
hypodermal chalone amid a hair root chalone (see p. 1686)
may also exist. If the high mitotic activity associated
with local damage is due to a reduction in the chalomie
concentration,
then as the mitot.ic rate rises the mitoses
should show an increasing independence of the inhibiting
action of adrenalin, and when the mitot.ic activity reaches
its niaximum they should be unaffected by adrenalin and
should show no diurnal rhythm.
It has been found that
this is in fact the case, although it is possible that in
epidermis the niitotic activity never achieves coniplet.e
independence.
The situation may be similar to that in
the duodenal mucosa, which may respond to au adrenalin
injection by a small and short-lived mitotic depression
(158) and may show slight signs of a diurnal mitotic
rhythm (64). Epidermal cells adjacent to a wound may
therefore always retain a small quantity of chalone with
which adrenalin can react.
It may be noted that the mit.otic activity adjacent to
an epidermal wound niay riot reniaimi unifornily high from
hour to hour as has commonly been believed, but that it
may proceed in a series of waves that have nothing in
coniniori with a diurnal rhythm (235). This phenomenon
has been clearly demonstrated
by Harding and Srinivasan
(226), who have found waves of DNA synthesis passing
outwards from the edge of a corneal wound at an aj)proxi
mate rate of l7Whr.
Hell and Cruickshank (235) suggest
that this phenomenon is the outcome of an induced tempo
rary synchrony in the cell cycle, amid since it may be sug
gested that a normal function of the chalone-adrenalin
coniplex is to inhibit the duplication of DNA, the sudden
withdrawal of the chalone would be expected to have this
effect.
The failure of adrenalin to liniit the high mitotic rate
associated
importance
damage
since
with wounds is a point of considerable
practical
since it is obviously
essential that any tissue
should
be repaired
it is evident
that,
as quickly
in addition
as possible,
to any normal
and
stress,
the presence of a wound may itself be a stressful factor.
Iii this connection it is intemesting to find that from about
1 to 8 hr after the infliction
of a skin wound
a high concen
tration of monoaniirie oxidase develops around the cut
(402, 457), arid it is possible that, by destroying adrenalin
at art unusually rapid rate, this may help to stimulate the
preparations
for mitosis during the period before the
chalone concentration
is significantly reduced.
It niust be eniphasized that the pattern of the mitotic
reaction seen after mechanical injury is essentially similar
to
Vol. 25, November
Cancer Research
1690
that
seen
after
damage
by chemicals,
radiation,
or a
variety of parasites (see Refs. 73, 277). After any kind of
injury the affected cells, if they are not too heavily
damaged, will themselves react by mitosis ; if they are too
heavily damaged, of if they are killed or removed alto
gether, then it is the closely adjacent cells that react.
In
either case the reaction could be due to the failure of the
damaged cells to synthesize adequate amounts of their
characteristic chalone or to an unusually rapid diffusion of
the chalone away from the damaged area [it is known that
the water relations of the cells are severely disturbed (see
Ref. 492)1, or to these 2 factors combined.
Even in
normal epidermis there is clearly a steady loss of chalone
1965
into the dermis and the blood, amid it follows that it must
be constantly synthesized within the cells (81).
It is important
to note that the mitotic respomise to
daniage in the epidermis does not begin until after a delay
of
about
24—36 hr.
However,
a much
earlier
reaction,
beginning in about 3 hr, leads to a great increase in the
RNA content of the damaged cells (492, 494, 495, 523—25),
and in about 12—24hr there is a considerable increase in
the DNA content (226, 235). The increase in the RNA
content may be related to an increased rate of protein
synthesis as the cells begin to alter their pattern of activity,
perhaps especially in preparation
for niitosis, while the
increase in the DNA content is undoubtedly
due to
chromosonie duplication in preparation for mitosis.
It may also be noted that considerable changes occur
in the concentration
and distribution of various enzymes
as shown by histochemical
methods (see Refs. 401—4),
but the significance of these changes is not yet understood.
Although the details of the wound reactions are best
known in the case of the epidermis, it is important to
emphasize that both the premitot.ic and the niitot.ic re
actions follow an essentially similar pattern in all tissues
that are capable of mitotic activity and that have been
studied from this point of view. This has been confirmed,
for instance,
in hair
follicles
(17,
82),
tongue
(45),
esoph
agus (342), hypodermis
(82), gall bladder and urinary
bladder (340, 341), gastric mucosa (255), liver (100, 101,
330),
these
uterus
tissues
(508), amid mammary
gland (368).
the mitotic reaction to local damage
In all
is itself
strictly local, and as in epidermis (45, 81), it comiimonly
takes the form of a gradient with the highest mitotic
activity close to the wound edge. However, it is obvious
that in different tissues the time takemi for the full de
velopment of this reaction may vary widely; in epidermis,
for instance, the response is relatively rapid, while in
hypodermis it is relatively slow (82). It is possible that
such differences may reflect the differing times required
for different cell types to change fiom one program of
synthesis to another, and they may also be at least partly
dependent on the times taken for the local chalone concen
trations to fall to a low enough level.
However,
the
most
important
single
point
emerging
from a survey of the mitotic reactions shown by a variety
of tissues to damage of any kind is that they are all
essentially similar.
It may therefore be concluded that
the results obtained from an analysis of the reactions of
any one tissue, such as wounded epidermis, may apply
equally to most, if not all, other tissues that are capable
of a mitotic reaction.
In particular, since in the case of
damaged epidermis the evidence now available favors the
theory that the high mitotic activity is due to a reduced
concentration of the epidermal chalone, it may follow that
essentially similar chalone mechanisms may also be present
in a wide variety of other tissues.
D.
REGENERATION
1 . Local and general milotic reactions.—It is evident that
the effect of a small wound can only be to damage or
destroy a minute proportion of the total cell mass of the
tissue, and that the consequent local reduction in the
chalone concentration
is unlikely to have any significant
general effect on the chalone concentration
in the tissue
BuLL0uGH—Mitotic and Functional
as a whole. This may be why all attempts to show that
the presence of 1 wound can affect the process of healing
of a 2nd woumid made at a distance have always failed
(1, 42).
Since
diffuses
chalone
highest
it is known that the epidermal chalone normally
into the adjacent tissues, it appears that each
may show a pattern of distribution in which the
concentration
is in the tissue of origin, a lower
concentration
is in the intercellular
fluid, and the lowest
concentration
is at the point or points where the chalone
is either degraded or eliminated.
This iniphes a steady
synthesis within each tissue, a steady rate of diffusion
from that tissue, and a relatively short life for each chalone
molecule.
Thus
in any
normal
tissue
there
may
exist
a
state of balance in which the rates of chalone production,
diffusion, arid loss are adjusted so that the chalone concen
tration within the cells is adequate to maintain the mitotic
rate typical of that tissue.
It is obvious that such a system could be disturbed
either locally, owing to a reduced chalone concentration
within a small area, or generally throughout
the whole
tissue, owing either to a generally reduced rate of chalone
production or to a generally increased rate of chalone loss.
A local disturbance
would, of course, be the result expected
of a wound, while a general disturbance would be expected
to follow extensive tissue damage or destruction
or re
moval.
Thus in the case of the liver a small wound leads
only to a local mitotic reaction, as mentioned above, but
when about 10 % of the liver is removed a general niit.otic
response is seen throughout
the whole organ, and when
from
10 % to about
70 % is removed
the mitotic
that develops is in direct proportion
tissue excised (see Refs. 60, 192).
results
have
been obtained
reaction
to the amount. of
Essentially
similar
by measuring
the rate of DNA
synthesis; not until some 9—12% of the liver is removed
does a general response develop, and beyond this threshold
the number
of cells that
synthesize
DNA
is directly
pro
portional to the amount of liver removed (62, 325).
The reactions of the liver remnant after partial hepa
tectomy have been closely studied, especially by histo
chemical technics (see Ref. 60). These details are largely
but in stressing
the
essential similarity between the general regeneration
action and the local wound reaction it is important
irrelevant
to the present
re
to
note that the nature
argument,
and sequence
are closely similar in both
the rate of RNA synthesis
after partial hepatectomy
wounded epidermis (494) ;
is seen from about 12 hr
extensive
literature
of the cellular reactions
cases. In particular, a rise in
becomes obvious within 6 hr
(232, 436) and within 3 hr in
an increased synthesis of DNA
both in regenerating liver (for
see Ref. 60) and in wounded
epidermis
(226, 235) ; mitotic activity rises to a high level in 24—30hr
in regenerating liver (59, 109, 110, 228) and in 24—36hr in
wounded epidermis (81) ; and there are many other simi
larities.
It must be noted that the figures given here for
regenerating
liver refer to the parenchymal
parenchymal
cells have a reaction
1 day (212, 228).
Similarly
cells ; the non
that is slower by about
in wounded
skin the reaction
of the hypodermal cells is slower than that of the epidermal
cells (82).
Descriptions
of the cellular changes during kidney
regeneration are less detailed, but these changes are also
Homeostasis
1691
evidently similar (108, 143, 144, 150, 163, 276) ; indeed in
all cases of wounding or of regeneration, the essential cell
reaction is a temporary change from synthesis for tissue
function to synthesis for mitosis.
It, is now clear that neither in the liver (see Ref. 60) nor
in the kidney (204, 449, 514) can this change be due to an
increased functional load or to an increased blood supply.
Also, since the degree of the mitotic response is in direct
relation to the amount of tissue removed, it is clear that
it is related to an absence of tissue arid riot., for instance, to
the amount of tissue damaged.
In both organs it is gener
ally believed that some humoral agent is the controlling
factor, though opinion is sharply divided as to whether
this agent is a mitotic stimulant (see especially Ref. 380)
or a mitotic inhibitor (see especially Ref. 73) produced by
the tissue or organ involved.
Indeed it is often con
sidered that. both mitotic stimulants arid mitotic inhibitors
may be involved (see Refs. 60, 366, 510), arid it has also
been suggested that regenerative
growth may be under
the control of hormones produced by some endocrine
gland arid transported
to the reacting tissue in the blood
(see Ref. 1).
The most important single piece of evidence in favor of
the humoral nature of the controlling agent conies from
experiments with parabiosis in rats.
fri such experiments
it is found that partial hepatect.omy
in one partner is
followed by a regenerative response in the livers of both
partners, amidit is therefore most probable that the message
passing from the operated to the unoperated rat niust. be
carried in the blood (61, 116, 256, 515). The few reported
failures to obtain such responses may be explicable in
terms of technic (60). It niay be added that essentially
similar reactions have recently been obtained with liver
autogralts
in rats, in terms of both mitosis (503) arid
DNA synthesis (310).
2. Hormones
and
regeneralion.—The
suggestion
that
regenerative growth by mitosis may be under the control
of “orthodox―horniones has been considered,
for in
stance, by Abercronnbie (1) arid Weinibren (510). In par'
ticular, attempts have been made to demonstrate
sonic
influence on liver regeneration
by the hormones of the
anterior pituitary gland (106, 173, 243). In brief, it has
been found that, although hypophysectomy
may result in
a reduced liver size (391), the effects of the pituitary
hormones on the regenerative process are not clearly de
fined. Weinbren (510) has concluded that any effects
seen
are
more
likely
to “reflect. the
changes
in general
met abolisnri in hypophysectomized
animals rather than
any interference with the basic restorative process,― and
similarly,
in relation to normal rej)lacement
growth,
Bullough (73) has concluded that “mostof the pituitary
hormones, as well as such other hormones as thyroxin and
insulin, are important to mitotic activity only in so far as
they ensure the balanced working of the general metabolic
complex.―
There is, of course, a special group of tissues and organs
that are under the direct control of particular niitogenic
hormones (see p. 1703), but with these exceptions, and
also with the exception of certain adrenal horniories, there
is at present no indication that any of the orthodox
hormones plays a direct part in the regenerative response.
The growing belief is that those humoral agents that are
Cancer Research
1692
evidently involved in the control of the regenerative re
sponse will prove to be organ or tissue specific, to be
synthesized in the organs or tissues on which they act,
arid therefore to be internal secretions that cannot be
classed with the orthodox hormones.
It is the thesis of
this review that these agents may be chalones that act
as tissue-specific mitotic inhibitors in the manmier of a
negative feedback mechanism.
3. Chalones and regeneration.—If this theory is correct
then it should be possible to explain mitotic homeostasis
in organs such as kidney or liver in the following manner.
When the mitotic activity of the component tissues is
low, this is because of the high chalone concentration
existing within the cells. This high concentration
is
maintained
by a rate of chalone production that is in
balance with the rate of chalone loss, perhaps mainly into
the blood. This rate of loss must be at least partly de
pendent on the steepness of the diffusion gradient from
the tissue to the blood, and therefore the lower the chalone
concentration
in the blood
the greater
will be the rate
of
loss. When a large part of an organ system such as the
liver is removed, the blood chalone level must also fall,
and this must result in an increased rate of chalone loss
from the remaining
part of the organ.
that in this situation the survivimig
cantly increase their rate of chalone
lone concentration
within these cells
level at which mitotic activity is
In such circumstances,
the observed
degree of tissue destruction
On the assumption
cells cannot signifi
production, the cha
may be reduced to a
no longer inhibited.
relation between the
and the degree of the mitotic
response, whether local or general, is readily understand
able.
Since it is also suggested that each tissue is controlled
by its own specific chalone, it is obviously possible that the
concentrations of the various chalones within the surviving
part of an organ system may be reduced at different rates,
or that
different
degrees
of reduction
may
different
tissues
before
the
response
In either
case
the differing
mitotic
reaction
be needed
times
in
develops.
observed
for
instance in the tissues of the liver (3) would also be readily
understandable.
In this connection, it may be recalled
that in regenerating liver, mitotic activity develops first
in cells around the branches of the portal vein as though
this
is the area from
which
the chalones
are first drained
(227).
The end of the regenerative
response will, of course,
come when the organ system is restored to its normal
size in relation to the total body space, and when, in
consequence, the normal chalone balance is re-established.
Vol. 25, November
who induced
mitotic
activity
1965
in miormal liver by diluting
the blood, amid by Glinos (190, 191), who performed
the
converse
experiment
of inhibiting
mitotic
activity
in
regenerating
liver by increasing
the concentration
of the
blood, which he did by limiting the water intake.
How
ever, it must be noted, first, that in carefully controlled
experiments
involving massive changes in blood con
centration Bucher (60) has found “noconsistent activa
tion or suppression of regeneration,― and second, that
increasing the concentration
of the blood by limiting the
water intake may result in a state of stress similar to that
produced by limiting the food intake (83). The belief
that stress may inhibit mitotic activity in regenerating
liver is supported
by Jaffe's
of a diurnal mitotic rhythm.
Contradictory
of the literature
particular,
regeneration
mitotic
of the existence
interpretations
of results are a feature
on liver and kidney regeneration.
In
while many
authors
have
concluded
is initiated
by a reduced concentration
inhibitor,
controlled
(266) report
by
an equal
the
number
production
of
consider
a mitotic
that
of a
that
it is
stimulant.
The literature has been extensively reviewed on many
occasions (see especially Refs. 60, 73). Here it may be
briefly noted that the serum of normal rats is reported
to be capable
of limiting
mitotic
activity
in regenerating
liver (261, 283, 461, 462, 466) ; that the serum of rats with
regenerating livers may stimulate mitosis in the livers of
normal
rats
(177, 249, 254, 299, 301, 455, 536) ; that
the
serum of hepatectomized
rats may produce a greater than
usual mitotic rate in livers of other hepatectomized
rats
(4, 445, 455, 461, 462); and that the dilution of serum, for
instance with saline, is said to be able to induce mitosis
in normal liver (60, 195, 365).
However,
in none
of these
experiments
has
it been
critically established
whether the results obtained are
explicable in terms of a mitotic inhibitor or of a mitotic
stimulant.
It may be suggested that part at least of the
confusion may be due to the fact that the concentration
of any humoral agent in the blood is likely to be relatively
low and that, consequently,
it might be more promising
to investigate
the actions of tissue macerates
or extracts.
There is a considerable
literature derived from this type
of experiment,
but again the results
recorded
are con
tradictory, some for instance indicating that normal liver
macerates or extracts contain a mitotic inhibitor, some
that they are inactive, and some that they contain a
mitotic stimulant.
Considering
first
containing
a mitotic
those extracts
that are described
as
inhibitor,
the earliest positive results
4. Theexperimentalevidence.—A
considerableproportion appear to be those of Saetren (425, 426), who showed that
of the recorded
liver
experimental
and the kidney
results relating
can be interpreted
to both the
to fit a theory
of
this type, which is closely similar to that put forward by
Glinos (192, 193). Most of the experimental
work has
when a kidney macerate
is introduced
into the body cavity
after unilateral
nephrectomy
it is capable
of inhibiting
of liquid
in the
regenerative mitotic activity in the remaining kidney and,
similarly, that when a liver macerate is used it inhibits
mitotic activity in a regenerating liver fragment.
Each
body or injections
of macerates
or aqueous extracts
of the
of these
organs
in attempts
to change
involved
either
alterations
concerned
tion of the humoral
in the volume
the
concentra
agents.
Considering
first the question of the concentratiomi
of a
humoral agent in the blood, some of the most important
pioneer work was performed
by Glinos amid Gey (195),
actions
is described
as organ
specific,
and macer
ates of organs other than kidney and liver did not produce
any similar effects. Saetren's work is almost unique in
that
he paid particular
attention
to dosage,
and he noted
that the most powerful inhibitions were obtained when
the amount of macerate introduced
was equivalent
to
BULL0uGH—Mitotic and Functional Homeostasis
the amount
active
of organ removed.
agents
were heat
He also noted that
labile
and nondialyzable
the
; and in
this respect they resemble the epidermal chalone (78).
Support for these conclusions has been provided by
Stich and Florian (462) and Molimard (353). It has also
been noted by Saetren (425) that damaged liver contains
no
appreciable
Stich
erating
liver.
have
and
amount
and Florian
of the
(462) that
However,
been recorded
inhibitory
directly
(496),
and
contradictory
by Blomqvist
Tumanischvili
agent
by
the same is true of regen
who
results
(46), Paschkis
consider
that
(380),
maccrates
or extracts of liver, whether normal or regenerating,
contain
some mitosis-stimulating
agent.
For a full
survey
of this
literature
(510), and Bucher
Finally,
mention
Malmgren
obtained
see Paschkis
(380),
Weinbren
(60).
must
be made
of the
suggestion
by
and Mills (329) that a mitotic stimulus may be
only from a liver maccrate
that
has been heated.
They believe that liver tissue may contain both a mitotic
inhibitor and a mitotic stimulant
and that in normal
circumstances their actions cancel each other out. When,
however, the inhibitor
is destroyed,
the stimulant
is
unmasked and is able to act.
The only conclusion that can be drawn from all this
literature is that, in the particular contexts of the various
experiments,
each and all of the results obtained may be
correct and that therefore the conditions in which the
experiments were carried out, and in which the observa
tions were made, may be of critical importance in evalu
ating the conclusions that have emerged.
In particular
a survey of the literature shows that in both the serum
experiments arid the organ extract experiments little at
tention has usually been paid to dosage, which commonly
appears to have been very low ; or to the degree of stability
or instability of the extracts; or to the age of the animals,
which were commonly young rats that had not yet reached
their
may
full size so that a varying degree of mitotic
naturally
have been present in the kidney
activity
arid the
liver; or to the state of the reacting tissue, in which the
mitotic rate may vary from hour to hour according to a
diurnal rhythm (266) and from day to day during the
process of regeneration ; or to the proportion of the organ
system removed, which determines the magnitude of the
mitotic
response
and
for optimal
experimental
purposes
should be only 50 % of the possible maximum ; or to the
time lag allowed either after the operation or after the
injection before the mitotic response was measured ; or
in some cases to the adequacy of the criteria used in
measuring the response; or to the manner in which the
experimental
manipulations
may have affected the ani
mals, especially
in terms of the degree of stress imposed.
These and other difficulties
that stand in the way of the
proper
evaluation
of the available
evidence
have been
stressed by Weinbren
and MacDonald et al.
experiments
involving
more critical technics
various contradictions.
5. Mitotic inhibition
(510), Bullough (73), Bucher (60),
(326), and it is obvious that further
a more systematic
approach and
are now needed to resolve all the
and slimulus.—One
possible
cx
planation
for the conflicting
results that have been ob
tamed emerges from the suggestion
that both an inhibition
1693
and a stimulus may normally be produced after a maccrate
injection and that which of these is recorded depends
On the time
observation.
lag allowed between
the
Thus
Weinbren
(510),
injection
amid the
using
rats
with
regenerating
livers, found a mitotic depression after 1
day and a mitotic stimulus after 2 days, while Wilson arid
Leduc (527), using young and adult normal mice, found a
mitotic depression during the 2nd arid 3rd days arid a
mitotic stimulus during the 5th—8th days.
The differ
ences in the timing of these responses may reflect differ
ences between normal arid regenerating
livers, between
the dosages used, and perhaps also between the species.
If it is confirmed that the reaction to the injection of a
tissue homogenate is a rapid mitotic depression followed
by a later mitotic stimulus, then 3 obvious possibilities
exist. First, the homogenate
may contain both an in
hibitor arid a stimulant (329), arid these 2 elements may
act successively; 2nd, if the homogenate contains a mitosis
inhibiting chalone, the initial depression it induces may
be followed by a later compensating
stimulus; and 3rd,
if the tissue homogenate is injurious to the cells, or if it
becomes injurious when it autolyzes in the body cavity,
the initial mitotic depression may be due to tissue damage,
which, through reduced chalone production,
ultimately
leads to an increased mitotic rate.
Regarding the 1st possibility, little or nothing can be
added to the evidence already given.
However, regarding
the 2nd possibility, it is known in the case of epidermis
that the chalone-adrenalin
complex, when fully active,
imposes a complete inhibition at a point just prior to the
onset of mitosis, although it evidently has no similar
effect at any point in the long phase of DNA duplication
(83, 88). After such an inhibition has been imposed for
several hr abnormally large numbers of cells accumulate,
all fully l)repared to enter niitosis together as soon as the
effectiveness
of the chalone-adrenalin
inhibition
is ye
duced.
Thus, if the mitotic counts are made a few hr
after the experiment
begins, a mitotic depression may
be recorded ; whereas if the counts are made later, when
the inhibition
is losing its power, an apparent,
but
unreal, mitotic stimulus may be found.
It must also be
added that this double effect may result from the action
of endogenous adrenalin if for any reason the experi
mental treatment results in serious stress, but in this case
the mitotic reactions should be equally obvious in all the
tissues that normally show a diurnal mitotic cycle.
The 3rd possibility—that
the injected maccrate
is
injurious, or that it becomes so—raises again the old
theory that the stimulus to regenerative
growth by
mitosis, which develops after tissue damage of any kind,
is due to the production of “necrohormones―(see Refs.
290, 483).
It seems reasonable
to regard
such substances,
if indeed they can be proved to exist, as essentially the
same as the hypothetical “woundhormones.― However,
since Bullough and Laurence
(81, 82) have provided
evidence that “woundhormones― do not exist at all, some
doubt as to the existence of “necrohormones―also arises.
6. The effects of metabolic damage.—It does, however,
remain possible that tissue extracts may contain sub
stances that might be derived, for instance, from the
lysosomes (see Ref. 370), that would not normally be
Cancer Research
1694
present free in the body, that are injurious to the cells
in a specific (483) or nonspecific (527) manner, and that
may not form any significant part of the basic mechanism
of healing and regeneration.
Such substances may act
in the generally toxic manner of chloroform (438), trypan
blue (386), or carbon
t.et.rachloride
(117, 309), all of which
cause damage to the liver cells, as well as to certain other
cells, amid consequently
result in a raised mitotic rate.
When such damaging agents are applied to mice clear
signs of cell damage in the liver precede the mitotic re
action (528), and similarly injections of liver homogenates
have been described as causing “somedegree of necrosis―
before
the development
of the mitotic
response
(15, 290).
This
(527),
by a
liver,
l)Oirit was also considered by Wilson and Leduc
who recorded an initial mitotic depression followed
mitotic stimulus after injections of maccrates of
kidney, and even boiled egg yolk and concluded
that
although
“noinjury
was
evident
in most
of our
experiments, it would be difficult to rule this possibility
out completely.― With any cytotoxic agent the question
of dose is obviously important..
If it is too high the
damage may be too great, and indeed Wilson and Leduc
(527) have imidicated that the higher the dose of liver
maccrate the lower the ultimate mitotic stimulus.
Ac
cording to Bullough
(73), the correct dose for the
stimulation
of mitotic activity can “bedefined as that
which is sufficient to cause some cellular damage but not
sufficient to damage the cells so seriously that they can
rio longer divide.―
Iii considering the wide variety of chemical and physical
factors the application
of which leads through tissue
damage to increased mitot.ic activity, Bullough (73) has
emphasized that the “commonfactor in all these seem
ingly diverse situations is evidemitly a particular level of
disturbance
of, or damage to, some aspect of cell me
tabolism― arid that in all probability “suchdamage results
not in the production of a stimulating ‘woundhormone'
but in the failure to produce some factor which had pre
viously prevented the cells from indulging in their basic
functions of growth arid mitosis.― An essentially similar
conclusion has been expressed by Tsarievm (492, 494, 495),
who has shown that when mouse skin is damaged by
pressure in such a mariner that no cells are killed, and
therefore
no mitosis-stimulating
‘
‘necrohormones― are
produced, there is a sharp rise in epidermal mitotic ac
tivity after about 24 hr.
This agrees with the observa
tion of Bullough
and Laurence
(81) that massage
is all
that is needed to disturb the metabolism
of the cells and
to raise the mitotic rate in ear epidermis.
Other situations in which cell damage may lead to an
increased mitotic rate are those in which ligatures have
been applied
or tension
has been induced.
Thus
it is well
known that a raised mitotic rate occurs in the kidney
after the ligature of either the ureter (31, 239, 240) or
the renal artery (144). If it is assumed that such treat
ment results in a reduced rate of chalone production in
the damaged kidney, then the rise in the mitotic rate
seen both in that kidney and in its opposite partner is
easily explicable.
Some confirmation
for this point of
view comes from Saetren (425), who found that a kidney
5 R.
Tsanev,
personal
communication.
Vol. 25, November
1965
damaged for 10 days by ureter obstruction
no longer
contained any of the organ-specific mitotic inhibitor that
he was able to extract from normal kidneys.
In experi
merits by Herlant (239, 240), the mitotic reaction of the
damaged kidney lasted for about a week, when atrophy
began, while the mitotic reaction of the opposite kidney
continued for more than 4 weeks, showing the same
level and duration as that seen after unilateral nephrec
tomy.
Similar results have been obtained in the bile duct
system (102, 324, 511) and in the gall bladder (265)
during distension caused by ligature, in the endometrium
and myometrium
during distension of the uterine lumen
(412), and also most probably in the epidermis during
tension (41). The mitotic reaction of the epidermis of
limbs imiwhich the blood flow has been stopped by liga
ture has been studied by Tsanev (493). This treatment
so damages the cells that after the ligatures are removed
high mitotic
activity
repeated
applications
develops
over the whole area;
of ligatures
this high mitotic
by
ac
tivity can be maintained for long periods.
Tsanev (493)
has also shown that after blood stasis a similar reaction
develops in the ducts of the mammary glands.
7. The effectsof the adrenal horinones.—Inspite of all the
contradictory
conclusions that have been reached re
garding mitotic control during regeneration,
none of the
available evidence directly contradicts
the suggestion
that such control may be exercised in terms of a chalone
type of negative feedback mechanism.
If this suggestion
is correct, and if the chalones of the liver and kidney are
similar to that of the epidermis, then the adrenal hormones
should also be involved.
The actions of these hormones
may not be apparent in normal liver and kidney because
the mitotic activity is negligible, but as the mitotic rate
rises during regeneration they may become more obvious.
In the 1st place the regenerating
tissues may begin to
show a diurnal mitotic rhythm, and in the case of the liver
such
a rhythm
has indeed
been described
(23, 105, 266).
Second, a mitotic depression should develop in regenerat
ing tissues during stress and after adrenalin injections,
but unfortunately
such situations do not seem to have
been
studied.
Third,
adrenalectomy
should
lead
to
increased mitotic activity, and this has been described in
normal liver (249), in regenerating liver (236, 237), and
in compensatory
renal hypertrophy
(19). However, it
must be added that this operation may sometimes lead
to such a degree of metabolic disturbance,
especially
regarding the sodium balance, that mitotic inhibition may
sometimes be seen (204).
Finally,
the glucocorticoid
hormones
may
be expected
to exert some influence, and it is now well known that these
hormones do strongly inhibit mitotic activity in normal
liver (249) and in regenerating liver (see Ref. 60). Hem
ingway (238) has concluded that mitotic control in the
liver is partly
partly through
exercised
through
plasma
proteins
and
the corticosteroids
that are bound to them,
and this is in obvious
ment.
inhibit
agreement
with the present
argu
It is also known
that glucocorticoid
hormones
DNA synthesis
in regenerating
liver (see Ref. 60),
and Guzek (219) has suggested that the anti-mitotic
actions of these substances are in fact the secondary conse
BuLL0uGH—Mitotic
quence of their “director indirect interference in DNA
synthesis.― The question of the points of action of
chalones,
adrenalin,
and glucocorticoid
hormones
is
discussed below on p. 1710.
8. Other evidence on regeneration.—In conclusion 2 other
points of interest may be mentioned.
First., it is evident
that the mitotic reaction to partial hepatectomy, including
the DNA synthesis reaction, develops much more rapidly
in young animals than it does in old animals (see Ref. 60).
The livers of these young animals still normally show
many
@
mitoses
and
may
therefore
contain
1695
and Functional Homeostasis
a relatively
low chalone concentration.
A similarly low chalone con
centration
may also exist in livers damaged,
for in
stance, by cirrhosis, and it is therefore interesting to note
that the mitotic reaction to partial hepatectomy develops
most rapidly in the livers that have suffered the greatest
amount of such damage (400). Again this is in accord
with Saetren's
observation
(425) that damaged
liver
contains less than the normal chalonie concentration.
The 2nd point of interest is that after unilateral mie
phrectomy
in a pregnant rat, the regenerative
rnitot.ic
response is seen only in the remaining maternal kidney;
there is no reaction in the fetal kidneys (203). It thus
appears that maternal chalones are unable to pass the
placenta, arid this would appear to be a necessary pre
caution
that prevents
the maternal
mitotic
control
mechanisms from interfering with fetal growth.
There
are also no diurnal rhythms in DNA synthesis in fetal
tissues, including epidermis (509).
9. Conclusions.—In view of the conflicting evidence and
the conflicting opinions it is particularly difficult to reach
any general conclusions regarding the mechanisms under
lying regenerative growth.
It is clear that much of the
confusion arises from inadequately
planned experiments
and especially from a general failure to study the mitotic
reactions from hour to hour during a sufficiently long
period of time. There is also the possibility that tissue cx
tracts may contain not only the typical chalone but also
certain toxic substances which, by damaging cells, niay in
duce complex pathologic changes in the mitotic rate.
However, in spite of these difficulties, the following
points may be emphasized.
First, it is evident that with
increasing tissue damage the local mitotic reactions seen
alongside wounds grade imperceptibly into the general mi
totic reactions of regeneration ; there is such a close and
detailed similarity between these local and general reac
tions that it is reasonable to conclude that they must be
essentially similar.
Since the mitotic reaction alongside a
wound is most readily explicable in terms of the chalone
theory, and since the evidence concerning the mitotic re
action in regeneration is not inconsistent with this theory,
it is also reasonable to believe that regenerative growth
may be basically explicable in terms of a negative feedback
chalone mechanism.
The 2nd point, which lends support
to this conclusion, is the evident involvement of adrenalin
and the glucocorticoid
hormones in the control of re
generative growth.
series of phases of activity, amid it is of considerable impor
tance that these should be adequately defined arid that. the
point or points of action of the chalone-adrenalin
complex
should be determined.
There is a large literature devoted
to attempts to disentangle amiddefine these various phases
(for early evidence see Ref. 252; for later evidence see
Refs. 334—37) arid also to determine the times spent by
different types of cells both in the whole niitotic cycle arid
in each of the phases within the cycle (see Refs. 37, 132,
306). Technics for determining the duration of the miii
totic cycle arid of its various parts are described by, for
imistance, Fry et al. (178), Smith amid Dendy (454), Puck
and Steffan (396), Quastler (399), and Hof and Ying (247).
The 4 phases commonly recognized u.s constituting
the
mitotic cycle are: mitosis itself (also called “M―)
; the
“1stgap―(or “G1―);
the phase of DNA synthesis (or “8―);
and the “2ndgap― (or “G2―),
from which the cell passes
into mitosis once more. However, with increased knowl
edge this simple analysis of the mitotic cycle is no longer
adequate, amid on present evidence it is possible to recog
nize at least 6 phases, which were recently defined by
Bullough (74). Still more recently, it has become possible
to suggest slight modifications to these definitions to mdi
cate more precisely
what
may be happening
in each of the
phases, and to relate them to that other important cell ac
tivity, which is specialization for tissue function.
The 6
phases, arranged in sequence, are : apopha.se (a new term
defimied below) ; dichophase (from which a cell passes either
to specialization for function or for mitosis) ; prospha.se,
which is itself divisible into 3 phases (early I)rOsl)hase,
phase of DNA synthesis, and anitephase) ; and mitosis
itself (see Chart 1).
Apophase : This is a term proposed here for the first. time
(and derived from amro, which implies “moving away
from―) to describe the period (commonly included iii G1)
between the end of mitosis and the beginning of the dicho
phase.
Although this is a phase about which little is
known, it is reasonable to suggest that the main preoccu
pations of the cell at this time may be recovery from the
previous mitosis and growth in bulk so that the cell re
covers the size typical of its kind. Although it is well
known that cell growth does not necessarily follow mitosis,
it seems probable that it may commonly do so in the cells
apophase
dichophase I¼..@
I
tissue
function
early prosphase
DNA synthesis
prosphose
antephase ______
mitosis
E. THE MITOTIC CYCLE
1 . The phases of the cycle.—It is well known
that
cell, in passing from one mitosis to the next, traverses
any
a
CHART
1.—The
phases
of the cell
cycle.
From
dichophase
cell may specialize either for tissue function or for mitosis.
the
of adult mammalian
somatic tissues.
This phase may
therefore be regarded as the final part of the mitotic cycle,
in which the cell returns to its normal state.
Dichophase : This is the period (commonly included in
G1) during which the cell is committed either to specialize
for tissue function or to specialize for mitosis (the term is
derived from Sr@o,which expresses doubt between 2 ways
ahead).
It appears that the dichopha.se may sometimes
be short but that commonly it may be protracted.
Its
significance is discussed on p. 1701.
Prosphase : This is the period during which the cell com
pletes all the necessary syntheses to enable mitosis to begin
(the term is derived from @rpoa,which expresses movement
towards).
At the moment it can be subdivided into the
early prosphase (commonly included in G1), the phase of
DNA synthesis (or S), and the antephase (or G2). The
most dramatic of these phases, and consequently the one
that
has been
Vol. 25, November
Cancer Research
1696
most
intensively
studied,
is that
of DNA
synthesis.
It is commonly forgotten that this cannot be
the actual beginning of the preparation
for mitosis but
that at least 1 earlier phase must exist.
2. The initiation of the prosphase.—The prosphase may
be defined as beginning with the synthesis of the 1st of the
sequence of enzymes that underlie the mitotic process, and
it is already known that accelerated RNA and protein
syntheses do occur before the onset of the phase of DNA
synthesis (250, 251, 257, 316, 322). It has been suggested
that 2 enzymes in particular are likely to be of critical im
port.ance at. this early stage.
The 1st is that enizynie, or
group of enzymes, which promotes histone synthesis; it is
evident, for instance during liver regeneration,
that the
histone content of the nucleus begins to increase before
the onset of DNA synthesis (217, 257). The 2nd is the
DNA polymerase, without which the phase of DNA syn
thesis could riot begin.
Mazia (337) has questioned
whether enzymes of these kinds may be synthesized di
rectly on the chromosomes or whether, like other proteins,
they are produced in the cytoplasm.
He has also ques
tioned whether the DNA polymerase alone is capable of
inducing DNA synthesis arid has shown that another
emizyme may also be needed to prime the DNA before it
can respond.
Busch et al. (99) have suggested that DNA
may be inactive when combined with historic arid that the
DNA strands are “releasedfor replication by the removal
of specific histories protecting against their replication,―
and it is possible that this removal may be the essence of
the priming process.
However, emphasis must be placed
on the concluding words of Busch et al. (99) that “itis
painfully apparent that we are just at the earliest stages of
comprehension
of the role of proteins in regulation of
synthetic activities of the cell arid in mitosis.―
3. The phase of DNA synthesis.—It is significant that
once the phase of DNA synthesis has begun it evidently
always proceeds to completion.
This is, of course, also
true of the whole process—DNA synthesis-antephase
mitosis—but it is riot yet certain whether the actual point
of no return is at the beginning of the phase of DNA syn
thesis or at sonic previous point in early prosphase.
It
follows that although active RNA and protein synthesis
are necessary to initiate DNA replicatiomi, they may not
be necessary to sustaimi it (225, 322). Indeed there is some
1965
evidence that during this phase the rate of RNA synthesis
is normally depressed ; that it is not stopped entirely may
be due to the fact that the DNA molecules do not appear
to replicate simultaneously
and that consequently at any
given time a certain number are always mi a state in which
they can initiate RNA synthesis
(372, 394, 451). In
microorganisms
it has been suggested that the process of
replication may jass in a linear manner along the entire
genome (321), and in HeLa cells there is evidence that the
average rate of DNA synthesis may reach its maximum at
approximately
the midpoint of the phase (485). It has
also been postulated that, since the start of the phase of
DNA synthesis may be dependent on the separation of the
DNA from the histones, so also the end of the phase may
involve their final recombination
(217).
4. Antephase and mitosis.—The final period of prepara
tion for mitosis is the antephase, which extends from the
end of DNA synthesis to the beginning of mitosis.
In dif
ferent tissues and in different circumstances it has a dura
tion of from about 1 to many hr, and with the chromo
somal syntheses already completed, it appears to be the
time when the final cytopla.smic syntheses are completed.
Taylor (479) has shown that a partial inhibition of protein
symithesis results in a prolonged antephase.
Unfortunately
relatively
little
is known
of these
final
syntheses, but it has been suggested that they probably
include the production of those molecules that, at the ap
propriate moment, become reversibly aggregated to form
the spindle apparatus (see Ref. 516). The possible bind
ing mechanism used in the assembly of these macromolecu
lar units has been discussed by 1\Iazia (333, 334).
Another suggestion has been that the antephase is the
time when an adequate store of energy-rich molecules is
established to support the cell throughout mitosis.
This
suggestion has been widely accepted (70, 335, 467)—and
indeed it has been shown that the spindle apparatus car
ries an ATPase
(336)—but
recently
some important
con
trary evidence has been published (see Ref. 337). The
theory originally arose from a study of mouse epidermis
(see Ref. 79) that showed that mitotic activity is depend
emit on glucose arid oxygen, but that any cell entering
mitosis becomes independent of these substances.
It al
SO became
apparent
that
the
actual
point
of
inhibition
caused by glucose or oxygen lack is towards the end of
anitephase.
Later, similar results were obtained with cleaving sea
urchin eggs (see Ref. 467), but recently these results have
been criticized by Epel (153), who has shown that these
eggs at all times possess an energy store sufficient to sup
POrt full
activity
for about
20 mm,
by which
time
the
ATP
level falls to about 50 % of the normal.
If at this moment
the cells are part way through the process of mitosis, then
they fail to complete the division.
Doubt as to the estab
lishment of an energy store has also been expressed by
Amoore (8—13),who has studied pea root tips. In these
he has shown that the effect of oxygen lack appears to be
exercized through some interference
with a “mitotic
ferrous complex.―
However, although these conclusions may be accepted,
they do miot at the moment seem to apply to such adult
mammalian tissues as epidermis, or even to embryonic tis
BULL0uGH—Mitotic
1697
and Functional Homeostasis
sues (128). In adult epidermis the blockage imposed by
oxygen lack develops immediately in any cells in late ante
phase, but it does not develop in any cells actually in
mitosis, even though this process may last for several hr
(88). Thus, in the case of epidermis, no evidence exists
to contradict the conclusion that the antephase may be a
time when the energy debts of the dividing cell are paid
in advance.
However, this is certainly a matter that needs
further investigation ; the results of the experiments of
Gelfant (184), which may appear to be relevant, have al
ready been criticized (85) and need not be discussed here.
It remains to consider the manner in which a cell passes
from antephase
into mitosis.
It is possible that this
happens simply as the natural consequence of the comple
tion of all the necessary preparations,
but it is also possi
ble, as suggested,
for instance, by i\Iazia (335) and
Taylor (479), that the transition to mitosis may be trig
gered by the synthesis of some particular enzyme.
These
suggestions are hypothetical,
and the question remains
unanswered.
The activities of a cell in mitosis are dramatic arid in
dude especially the assembly of the spindle apparatus, the
separation and movement apart of the chromosomes, and
the production of new cell walls. It is remarkable how
little general agreement exists as to the manner in which
any of these tasks is accomplished
(see especially Refs.
335—37). However, there does appear to be general agree
ment that during mitosis both RNA amid protein synthesis
are depressed and even arrested (285, 393), and it is be
lieved that this may be the consequemice of the condensa
tion of the chromosomes, the breakdown of the nuclear
membrane, the disappearance
of the nucleolus, and the
disruption of the endopla.smic reticulum.
It is evidently
because of this dismantling of the cell machinery that all
preparations for mitosis must be completed in advance and
that, once a mitosis has begun, it is so immune to outside
interference as to give the impression of being ami all-or
none reaction (see Refs. 70, 73).
5. RNA and protein synthesis.—From this survey the
suggestion emerges that the essential RNA arid protein
syntheses of the mitotic cycle may take place in a sequence
of periods of high and low activity.
Setting aside the
phase of general protein synthesis that may commonly
characterize
the apophase and that may be regarded
merely as a final phase of recovery from the previous divi
sion, the following periods can be recognized : a phase of
protein synthesis, which initiates preparations for the next
mitosis (early prosphase) ; a phase during which the chro
mosomes, being concerned with DNA synthesis, are not in
a state to induce active protein synthesis (phase of DNA
synthesis) ; a 2nd phase of protein synthesis in preparation
for mitosis (antephase) ; and finally the phase of mitosis
itself, during which protein synthesis is again depressed.
known that the phases of both DNA synthesis
It seems probable that if the early prosphase and the ante
phase of DNA synthesis and that there also appears to be
an accumulation of cells, in dichophase or early prosphase,
that enter the phase of DNA synthesis in abnormally
large numbers when food is again provided (104, 286).
To this may be added the obvious evidemice that after
wounding or during regeneration, when it is suggested that
the chalone concentration
falls, unusually large numbers
of cells enter the phase of DNA synthesis.
The implica
phase are the times when protein synthesis is most active,
then they may also be the times when metabolic inhibitors
are able to exert their maximum effects and in particular
when any natural mitotic homeostatic mechanism, acting
through an inhibition of the syntheses that precede mi
tosis, may be expected to operate most powerfully.
In agreement with these conclusions it is already well
show a high degree
of independence
and mitosis
once they have
begun
and that the 2 points in the mitotic cycle that are most
sensitive to interference are located before the onset of
DNA synthesis arid before the onset of mitosis.
Thus
Mazia (337) has comicluded that no case is known mi which
DNA synthesis fails to be completed once it has begun,
and Bullough (70) has reached a similar conclusion re
garding mitosis itself. By contrast it is well known that
cells may be arrested for days or months or years before
they enter the l)hase of DNA synthesis, amid Bullough and
Laurence (83), by strengthemiing the chalone-adrenalin
inhibitor, have shown that cells in antephase may be pre
vented from entering mitosis for periods of at least 2 days.
6. The action of the chalone complex.—This raises the
question of the l)Oinit or points in the mitotic cycle at which
the chalone-adrenalin
complex exerts its inhibitory action.
It is at once clear that any interference in apophase is most
improbable.
If apophase can be partly defined as a period
of general cell growth, and if the chalone complex sup
presses the special syntheses needed for mitosis, then there
is little reason to suspect any action at this time.
The first l)Ossible points of action of the chalone complex
may be the dichophase, the transition from dichophase to
early prosphase, arid the early prosphase.
Although no ade
quate investigation has yet been made, certain evidence is
already available.
Thus Bullough arid Rytomaa (90) have
shown that the granulocytic chalone, iroduced by mature
granulocytes,
reduces
the numbers
of bone marrow
myelo
cytes that enter the phase of DNA duplication,
while
Brown et al. (57) have shown that in a stressful situation,
which may be presumed to involve the production of cx
cessive amounts of adrenalin, there is also a reduction in
the numbers of intestinial cells entering the phase of DNA
duplication.
It has been shown above that stress and
adrenalini provide the explanation for the diurnal mitotic
cycle, arid it is therefore significant that diurnal variations
in the miumbers of cells commencing DNA synthesis have
been described in a wide variety of mammalian
tissues
(388). It is alsointeresting that these variations are found
only in tissues that also show a diurnal mitotic cycle; in
tissues in which the mitotic rate is so high that no clear
cycle is evident, there is also miodiurnal variation in the
numbers of cells entering the phase of DNA symithesis.
If these results are interpreted to mean that the chalone
adrenalin complex controls entry into the Phase of DNA
synthesis in the same way as it controls entry into mitosis,
then another parallel may be expected.
It is known that
in a stressful situation, such as starvation,
cells may be
arrested in amitephase in such a way that large numbers
accumulate and are ready to enter mitosis together as soon
as the adrenalin concentration
falls (83) ; it is now known
that
starvation
reduces
the numbers
of cells entering
the
Cancer Research
1698
tion is that these cells had previously been prevented from
taking this step by the inhibitory action of the chalone,
and it follows that all the available evidence combines to
support the suggestion that the chalone-adrenalin
complex
acts to inhibit the entry of a cell into the prosphase.
Comisidering next the phase of DNA synthesis, the cvi
dence of Pilgrim amid \Iaurer (389) suggests that there is
no diurnal rhyt.hm in the duration of this phase.
This
suggestion in turn may be taken to indicate that adrenalin,
amid therefore the chalone-adrenalin
complex, has little if
any effect on a cell once it has begun to synthesize DNA.
This conclusion is strengthened
by the observation
of
Bullough and Laurence (83) that, both in vivo and in vitro,
a high concentration of adrenalin results in the accumula
tion of abmiormally large numbers of cells in antephase.
This can oniy mean that adrenalin does not prevent the
cells that are synthesizing DNA from progressing into the
antephase.
However, while not contradicting
this con
elusion, it has been rioted by Rytomaa6 that not only does
the granulocytic
chalone reduce the number of cells en
tering the phase of DNA synthesis, but that it also slows
the passage of cells through this phase.
This is a matter
for further investigation but already it is clear that cells
in the phase of DNA duplication are not readily suscepti
ble to inhibition.
In contrast, the evidence reviewed above points over
whelmingly
to the conclusion that the antephase
is
highly susceptible to the inhibitory action of the chalone
adrenalin complex in a wide variety of tissues.
This is
shown by the existence of diurnal rhythms ; by the anti
mitotic effects of stress in vivo, and of adrenalin and cha
lone
in vivo and
in vitro ; and
by the raised
mitotic
rate
after adrenalectomy arid after chalonie reduction by wound
ing. It is also interesting, and important to emphasize,
that the main point of inhibition appears to be towards the
end of antephiLSe, perhaps only a matter of minutes before
the cells enter the earliest recognizable stage of mitosis.
Thus, in experiments in which epidermis containing no mi
toses was taken from stressed mice and placed in a saline
medium, high mitotic activity developed immediately
(83). This may lend some support to the idea that miii
tosis is initiated by some trigger synthesis (335, 479) that
is particularly sensitive to inhibition.
As regards mitosis itself, it has been found in epidermis
that the chalone complex can act to slow the process but
not to stop it and, conversely, that in the close neighbor
hood of wounds, where the chalone concentration
is cvi
dently reduced, the cells pass through mitosis more quickly
than usual (88) . However, when the situation is studied
more closely
it is found
that
the chalone
complex
Vol. 25, November
that the progress of a cell through
enced only by earlier events.
1965
mitosis is usually influ
F. CONCLUSIONS
The evidence reviewed above suggests that mitotic
homeostasis in the tissues in an adult mammal may corn
rnonly depend on the anti-mitotic actions of tissue-specific
chalones and that these actions are strengthened
by
adrenalin and perhaps also by the glucocorticoid
hor
monies. It appears that the chalone may be synthesized
at a constant rate within the cells on which it selectively
acts, while the concentration
of the adrenal hormones
varies according to the degree of stress felt by the animal.
It may be noted that, since the chalone mechanism evolved
to suit conditions in wild animals, which may commonly
be subject to considerable stress, mitotic horneostasis may
operate at less than optimum efficiency in well-kept do
mesticated animals.
The diurnal mitotic cycle appears to depend on the in
stability of a chalone-adrenalin
complex, which evidently
breaks down when the adrenalin concentration falls during
rest or sleep. The details of this cycle are now seen to be
more complex than was originally thought.
When the
adrenalin concentration
rises after a period of sleep, cells
are inhibited from entering the prosphase, but those cells
that are already in the prosphase continue their prepara
tions until they are arrested in the antephase ; the few that
manage to enter mitosis pass through it relatively slowly.
When the adrenalin concentration
falls during rest or
sleep, cells enter freely into both the prosphase and mi
tosis. The cells that enter mitosis are a mixed group,
being partly those that are then completing the prosphase
and partly those that have accumulated in antephase dur
ing the previous waking hours.
The cells that complete
prosphase during sleep pass through mitosis relatively
quickly, but those that had previously been held in ante
phase pass through mitosis even more quickly.
The result
is that the beginning of a sleep period is characterized by
a particularly high rate of mitosis; later in the sleep period
the mitotic rate falls to a lower level that is determined by
the rate at which cells are then completing the prosphase
(see Chart 2).
In considering the different types of diurnal mitotic
cycles seen in the various tissues it is evident that they
can be arranged to show every gradation between 2 cx
tremes.
These extremes are illustrated, on the one hand,
by tissues such as those of the kidney and liver, in which
has little
or no action on a cell once it has entered mitosis ; the slower
passage through mitosis is the result of the action of the
cOmj)leX in antephase.
It is interesting
to note
that
the
slower passage through mitosis that results from a lack of
oxygen or glucose is also partly or wholly due to an inhibi
tion
in antephase,
and it may
be recalled
that
colchicine
blocks mitosis in metaphase only as a result of its previous
actiomi on the cell in antephase.
Although the full signifi
canice of these observations
is unknown, it does appear
CHART 2.—Diagram
illustrating
a typical
diurnal
mitotic
cycle.
The highest mitotic activity is at the beginnin'@ of the sleep
6 T.
Rytomaa,
personal
communication.
period
(from
Coleman').
BuLL0uGH—Mitotic and Functional
@
the mitotic rate is so low that the counting technics now in
use are inadequate to determine whether or riot a diurnal
cycle exists, and on the other hand, by tissues such as that
of active hair root, in which the mitotic rate remains con
stantly at the maximum.
In an idealized form the range
of types of diurnal rhythms lying between these extremes
is illustrated in Chart 3, and it appears logical to suggest
that the mitotic rate, which determines the type of cycle
seen, may be in inverse proportion to the chalone content
of the tissue.
The evidence also suggests that when, after
tissue damage, the chalone concentration
is reduced amid
the mitotic rate rises, the type of diurnal cycle also changes
and that in extreme cases, when the mitotic rate rises to a
maximum, the diurnal cycle disappears.
It has been stressed that when the chalonie concentration
falls after tissue damage there is a collapse of functional
specialization amid a reversion to specialization for mitosis,
and the hypothesis has been advanced that the choice be
tween specialization
for function and specialization
for
mitosis is normally made in terms of the chalone conicen
tration within the cells. If this is true, then a chalone may
be regarded as acting on the genome in the manner in
which an effector is believed to act in a microorganism.
This hypothesis
differs fundamentally
from that of
Weiss (514), which explains tissue growth in terms of
tissue-specific
‘
‘templates'‘amid ‘
‘anti-templates. ‘
‘The
“templates―are regarded as catalysts in the production of
that specialized “diversity of compounds― that is typical
of the particular
cell type, while the “anti-templates,―
which diffuse throughout
the body and have a certain
superficial resemblance to the chalones, act by inhibiting
the “templates.― This hypothesis
implies that when,
within the cells of a tissue, there is active synthesis of the
specialized tissue proteins, then mitosis leading to tssue
growth naturally follows, whereas in fact it is evident that.
when a cell indulges in synthesis for tissue function, this
automatically
involves an inhibition of synthesis for nuito
sis.
Homeostasis
Iv. MITOTIC
1699
AND FUNCTIONAL
A. THE BASIC MECHANmsM
1 . Mitosis and cell function.—If the chalone-adrenalin
complex acts in the manner of an effector to determine
which region of the facultative genome shall be active at
any given moment, then when the efficiency of the cha
Ionic complex is high cells may tend to prepare for tissue
function, while when the efficiency of the chalonie complex
is low they may tend to prepare for mitosis.
Thus the
primary function of the chalone complex may be the Pro
motion arid maintenance
tioni of niitosis, by which
of tissue function,
arid the inhibi
the existence of chalones has been
recognized, may be merely the secondary outeome of this.
There may even be a hidden diurnal rhythm in the number
of cells preparing for tissue function, and if @oit would
of course be the reverse of the diurnal mitotic rhythm.
It has often been suggested that mitotic activity amid
functiomial activity are mutually exclusive.
Thus; con
sidering normal epidermis, it could be suggested that mi
totic activity is the exclusive function of the unspecialized
cells of the basal layer, and that once a cell lia.s moved into
the more superficial layers arid has commenced keratin
synthesis, mitosis becomes impossible.
However, when
this example is examined in greater detail, it is found that
the basal cells of the germinative layer already contain
bundles of fine fibrils, which are regarded as the precursors
of keratin (54, 347, 374, 442). In fact the cells are clearly
recognizable as epidermal cells, and indeed mitotic activity
may commonly occur, as in human epidermis, in the basal
layer of the stratum spinosum in which the fibrils are even
more conspicuous.
Evidence that. cells showing clear signs of their special
nature arid function may also indulge in mitotic activity
has often been given ; examples of this are seen durimig
endochondral
osteogenesis (535), eryt.hrocyte production
(373), and granulocyte production (381). However, in all
cases of this kind it is clear that
It is also difficult to perceive any relationships between cell specialization
the chalones, which are regarded as tissue-specific inhibi
tors, and such substances as “retine,―
which has been de
scribed by Szent-Gyorgyi
and his colleagues as a nonspe
cific inhibitor (233, 470—72).
HOMEOSTASIS
beyomid a certain
stage
of
mitosis no longer occurs.
Mäkeläamid
Nossal (328), studying the transformation
of lymphocyte
to plasma cell, noted an inverse relation between the anti
body-forming and the DNA-synthesizing
capacity of the
cell, and Wessels (517) has made the important point that
“thefact that mitotic figures are occasionally found in
cells in the early
stages
of specific
synthesis
may
be less
significant. . . . . than the fact that rio mitotic figures are
seen in the more fully differentiated cells.― It seems that
this comment, made especially in relation to pancreas
acinar cells, is generally true of all cell types, arid as Weiss
(513) has said, “thegeneral impairment of proliferative
capacity
iii cells undergoing
ascribed
to the fact that
niitotic prerequisites,
entiation products.―
terminal
specialization
most of their substance,
is diverted
can be
including
to the building of differ
It thus appears that mitotic activity arid specific cell
sleep period
CHART 3.—The
hour-by-hour
mitotic
tissues; for explanation see the text.
activity
of a variety
of
function are indeed mutually exclusive but that mitosis
only ceases beyond a certain point in the range of increas
ing preparation for tissue function.
It is obvious that a
graded effect of this type could be related to the effects of
a steadily increasing concentration of the chalone complex
within the cell, arid certainly the evidence does not run
Cancer Research
1700
Vol. 25, November
counter to the suggestion that mitotic homeostasis and
functional homeostasis may be achieved by one and the
same control mechanism.
The maintenance of any tissue
in a functional state automatically
involves the control of
its mitotic activity.
In this connection it is interesting to note that when cer
taini tissues
are incubated
in vitro the
cells tend
to lose
their specialization for function and to prepare for mitosis.
This collapse of organization is readily explicable if it is
postulated that a small fragment of tissue immersed in a
relatively large volume of nutrient medium suffers such a
marked reduction in the intracellular chalone concentra
tion
that
the
cells revert
to mitotic
activity.
It is also
relevant to note that when, as a result of this activity, a
large enough cell mass is built up, the cells limit their prep
arationis for mitosis arid recommence
their specialized
syntheses.
This has been described in vitro in mouse fibro
blasts,
which, when their numbers
approach
a critical
level, begin to produce collagen (210, 211), and in pan
cress acinar cells, which in similar circumstances begin to
produce zymogeni (517, 518). This latter case is especially
interesting since it is the innermost cells that first cease
mitotic activity and commence zymogeni production ; the
peripheral cells, which might be expected to possess a lower
chalone content, continue to undergo mitosis (see also
Ref.364).
One problem in such in vitro reactions is the source, if
any, of the adrenalin cofactor.
No information on this
point is available, but there is evidences that a chalone may
itself possess considerable activity which, in vivo, is merely
augmented by the adrenalin.
When
small
specialized,
most
groups
nondividing
probable
that
of already
specialized,
or partly
cells are placed in vitro, it seems
the process
by which
they
reacquire
a
mitotic ability must be the same process by which similar
cells in vivo reacquire a mitotic ability after wounding or
during regeneration.
It is this kind of evidence that
serves to stress the normal instability of the specialized
tissue functions arid also to emphasize that functional
homeostasis is merely the obverse of mitotic homeostasis.
However, it is also obvious that in many, if not all,
functional tissues there are cells that have entirely lost any
capacity for mitosis.
It seems improbable that the epi
dermal cells of the stratum granulosum, or those granulo
cytes that have been released from the bone marrow, can
ever divide again.
It is obvious that this must be true of
the nonnucleated erythrocytes,
amid it is also obvious that
this phase must be the final one in the life of a cell and
that it can end only in death.
It is evident that the pro
portion of the cell population in this final phase varies from
tissue to tissue: it is relatively high in the granulocyte sys
tem and relatively low in the epidermis.
In the nervous
system amid in striped muscle, all cells might be considered
to be in this phase.
It appears that in any typical tissue there exist at least
4 main categories of cells (see Chart 4) : the progenitor cells
(P) that are involved
in mitotic
cycles ; the immature
cells
(I) that have ceased, or are ceasing, to divide, amidare pre
paring for tissue function ; the mature cells (M) that may
or may not be functional and that, if circumstances die
tate, are capable of reversion to mitosis ; and the mature
1965
D
CHART
4.—Diagram
illustrating
the
relations
between
the
different cell types in a typical tissue. P, progenitor cells; I,
immature cells; M, mature cells; D, mature cells moving towards
death.
For explanation
see the text.
cells (D) that are perhaps always functional but cannot
revert to mitosis arid are approaching death.
It must be
emphasized that the boundaries between the 4 cell cate
gories are probably riot as sharply defined as they appear
in Chart 4 and that in fact each category may merge im
perceptibly into the next. Epidermis is an unusual tissue
in which these cell categories are stratified and in which a
5th category of cells—dead ones—are retained as the
stratum corneum.
In most other tissues the various cell
types seem to be mixed indiscriminately.
2. Life expectancy of cells.—It is also important to note
that the life expectancies of the 4 categories of cells are
quite different.
Cells of type P form a potentially irn
mortal population, as is shown by their behavior in vitro
and in neoplasia.
Cells entering I and reaching M thereby
acquire a certain limited life expectancy since M normally
leads only to D and to death, and this life expectancy is
evidently typical of the tissue to which they belong.
However, in unusual circumstances involving a fall in the
chalone concentration
or, in certain tissues, a rise in the
concentration of some specific mitogenic hormone, the cells
in I and M can return to P and thus apparently become
rejuvenated.
Cells in D can only go forward to death,
and their life expectancy may perhaps prove to be rela
tively constant in any one tissue.
in considering the balamice within a tissue between the
processes of cell production, cell function, and cell death
it is obviously inadequate
to consider only mitosis and
function.
The life expectancy
of the cells entering
I and
M arid the consequent rate of death from D are also criti
cally important factors.
It must be concluded that in any
adult tissue not only is the average rate of cell gain by mi
tosis in exact balance with the average rate of cell loss by
death, but the proportion of the cell population that is pre
paring to be or actually is functional (I, M, and D) is de
pendent on the life expectancy of the cells entering I.
Since the chalone complex appears to play an important
role in the maintenance of tissue homeosta,sis it is interest
ing to consider what influence, if any, it may have on the
life expectancy of the cells in I, 111,and D and thus on the
rate of cell death.
From many examples of tissues with
widely differing mitotic rates it is possible to suggest that,
as in duodenal mucosa, a low chalone concentration
may
lead to a high mitotic rate combined with a short life ex
pectancy in I, M, and D, while a high chalone concentra
tion, like that in the liver, may lead to a low mitotic rate
combined with a long life expectancy, especially perhaps
in M.
It thus becomes important
chalone mechanism
to consider whether the
may act not only to promote
the func
BuLL0UGH—Mitotic
tional maturity of the cells but also to prolong their life
expectancy.
In this connection it may be recalled that
when, by long-continued
stress induced by partial starva
tion, Bullough and Ebling (76) maintained the epidermal
mitotic rate of adult male mice at a quarter of its normal
level for 4 weeks, no change was recorded in either epi
dermal thickness or sebaceous gland size. It was obvious
that in these 2 tissues the rate of cell loss must have been
reduced to a quarter of its normal level. The converse
situation has been described by van Scott and Ekel (439),
who have shown that with increased epidermal mitosis in
psoriasis the life expectancy of the cells was reduced from
the normal 27 days to only 4 days, and by Skjaeggestad
(452), who, from a study of the epidermis of hairless mice,
has concluded that all hyperplasia-producing
agents also
induce an increased rate of cell loss. Similarly in the rat
liver MacDonald
(323) has described how normal cells
divide at intervals of from 191 to 453 days, while with a
raised mitotic rate in cirrhosis the cells have a life span of
only about 26 days.
The conclusions must be that when
the efficiency of the chalone complex is decreased, the
raised mitotic rate is matched by a shorter life expectancy
and therefore a higher rate of cell death, and that when the
efficiency of the complex is increased, the reduced mitotic
rate is matched by a longer life expectancy and therefore
a lower rate of cell death.
It must, however, be emphasized that only within limits
is a change in the mitotic rate exactly matched by an op
posite change in the life expectancy of the functional cells.
In epidermis Bullough and Laurence2 have noted that
when, in middle age, the number of mitoses increases to
about 3 times the normal figure the thickness of the epi
dermis remains unchanged, while van Scott and Ekel (439)
have noted that when, in psoriasis, the number of mitoses
increases to almost 30 times the normal figure the epi
dermis becomes thickened.
In this latter case it is evident
that the decrease in the cell life-span was inadequate to
offset completely the increase in the mitotic rate, and the
tissue increased in size until it became stabilized at a new
point of balance.
Thus,
@
the evidence
suggests
that the horneostatic
1701
and Functional Homeostasis
media
nism of which the chalone complex forms an important
part controls the mitotic rate, the degree of synthesis for
tissue function, and the length of life of the cells in I, M,
and D, and the question arises how one mechanism could
exert such a variety of actions.
The simplest suggestion
is that they all may be the outcome of the one basic cia
lone action, which is to promote the active synthesis of
those proteins on which the specialized functions of the cell
depend—an action that not only involves blocking any
alternative types of synthesis, but also maintains the func
tional efficiency of the cells at a higher level for a longer
period.
This hypothesis is illustrated in Chart 5. The
examples shown are taken from mouse data and include
the duodenal mucosa, in which the mitotic rate is high and
the life expectancy of the cells in M and D is about 2 days
(311) ; the ear epidermis, in which the mitotic rate is
moderate and the life expectancy of the cells in ill and D
is about 25 days (439, 443) ; and the liver, in which the
mitotic rate is low and the life expectancy of the cells in
M and D is about 3000 days (323). These are all, of
LiHweak
hchalon
@HIJ
H@ ‘
chalone
action—,——
.moderate
@!I'@M JiJ
strong chalone action
I'
LMI
CHART
5.—Diagrams
illustrating
cell types in different tissues.
the
different
P, progenitor
proportions
of
cells; I, immature
cells; M, mature cells; D, mature cells moving towards death.
Top, duodenal
mucosa with a high rate of cell production
and
loss; middle, epidermis with a moderate rate of cell production
and loss; and bottom,
liver
with
a low rate
of cell production
and
loss.
course, normal amidstable situations; but if, by tissue dam
age leading to a lowered chalone concentration,
the mitotic
rate rises in either the epidermis or the liver, then it may
be expected that temporarily these tissues will move closer
to the condition shown by the duodenal mucosa.
If this argument is taken to its theoretical limits then at
one extreme, with no chalone present at all, M and D
would disappear and all cells would remain in P; sonic such
situation may perhaps be found in cases of advanced malig
nancy.
At the other extreme, with an excess of the cha
lone complex, all cells would enter and remain in Al, and
their life span would be infinite; in effect this may be the
case in the mouse liver, in which the estimated cell life
span of 3000 days greatly exceeds the possible life-span of
the animal.
It is, of course, obviously important that the
life expectancy of the cells in Al should be directly related
to the degree of efficiency of the chalone mechanism.
If
this were not so then any tissue in which mitotic activity
declined and finally ceased would be in danger of total
diasppearance
as its cells passed steadily through M to D
and death.
3. The nature of the dichophase.—Ithas been suggested
above that the decision whether to specialize for mitosis
or for tissue function is taken by a cell in dichophase iii
terms of the intracellular
chalone concentration.
How
ever, it is evident that while in some tissues this decision
may be taken promptly, in others the cells may remain in a
state of indecision for a long time.
Tissues in which the decision is taken promptly are
those with a high mitotic rate. An example is an ac
tively growing hair root, the cells of which appear to lack
any effective chalone control and therefore to pass almost
directly from apophase into prosphase.
The speed at
which this occurs, together with the shorter duration of
Cancer Research
1702
prosj)ha.se
amid mitosis
(88),
results
in recurrent
mitotic
cycles, each of which is completed in the shortest time
needed for the essential symitheses. In an adult mam
malian tissue it does not seem possible to reduce the
duration of the mitotic cycle to less than about 12 hr (80,
Vol. 25, November
1965
chance and may vary widely between cells, but in any
particular tissue the average length of time a cell remains in
dichophase
may be precisely definable.
The variable
effects of chance on individual (Yells are shown when a
cell population
is artificially synchronized
so that the
440).
cells leave mitosis
As the effectiveness of the chalonie control increases in a
tissue the duration of the whole mitotic cycle also increases,
arid it has been shown by Bullough amid Laurence (88)
that this is at least partly due to the increased time spent
by the cells in l)rosPhase arid mitosis.
However, it is
well known that the duration of the phase of DNA syn
thesis is not likely to exceed about 10 or 12 hr (see Refs.
desymichronization occurs in the l)art of the cell cycle that
may now be recognized as the dichophase (485).
When the chalonie concentration
falls after any type of
cell damage, not only do the cells in dichophase tend to
move towards the prosphase, but the functional cells in M
103, 291, 312), the amitephase about
commencing DNA symithesis in as little as 4 hr (235);
functional
liver acinar cells, after hepatectomy,
pass
back through dichophase to commence DNA synthesis
in about 20 hr (see Ref. 60).
Thus on the present hypothesis the dichophase emerges
about. the maximum
remain
awake
time that
16 hr [which is
an animal may normally
(see Ref. 83)], and mitosis
about
6 hr [which
is about the longest time taken by an epidermal mitosis
with excess adrenalin (see Ref. 88)]; and although little
is known of the length of the apophase, it is possible that
this too may only last for a matter of hours.
Thus even
in extreme conditions the total duration of the whole
sequence,
l)rosphasc-mitosis-apophase,
seems
unlikely
to
exceedsome 2 or 3 days.
However, it is well known that in tissues with moderate
or low mitotic rates a mitotic cycle may take very much
longer than this (see Refs. 37, 306) ; examples are the cells
of the basal epidermal layer, which undergo mitosis about
once in 25 days (439, 443) and the cells of rat liver, which
may undergo mitosis only once in about 200—400 days
(323).
It therefore appears probable that in such tissues
the cells must spend a considerable
cisively in the dichophase.
time
resting
mdc
It seems probable that any cell entering the dichophase
is exposed to conflicting urges towards mitosis and to
wards functional
maturity;
this is expressed diagram
matically in Chart 6. With a low chalone concentration
the decision to enter mit.osis again would be quickly taken,
in unison.
It is found
also tend to move back into the
cells already
in dichophase
may
as perhaps
the most critical
that
progressive
prosphase.
Epidermal
react to wounding
by
of all the phases
through
which
a cell passes and also as a phase in which, if the chalone
concentration
is at sonic indeterminate
level, a cell may
remain indecisively
poised between the 2 possibilities
that are open to it.
4. Conclusions.—In any stable adult tissue the balance
between cell gain and cell loss appears to be determined,
1st, by the characteristic
chalone concentration,
and 2nd,
by the characteristic life expectancy of the maturing cells.
The primary function of a chalone may be to promote
maturity, and since it is commonly believed that chalones
are produced mainly by mature cells (73, 260, 348),
chalone production may be self-promoting.
The characteristic
life expectancy
of the maturing
cells varies, perhaps widely, among different tissues, but
within any one tissue it changes inversely with the mi
totic rate.
It is important to note that when such a change
takes place it appears to apply equally to the new cells
then being produced amid to the already mature cells.
the dichophase would be very short, and few if any cells
would enter I, M, amid D; with a high chalone concentra
tion the dichophase would be equally short, the cells
would rapidly enter ill and become functional,
and P
would be depleted and even eliminated; but with an inter
mediate chalone concentration
the dichophase might be
very long and might consequently contain a large number
of cells. The actual length of time that any particular
cell remains in dichophase appears to be a matter of
@
M
1'
.
\7@dichophase
@apophase
,mitosis
@@%%%
prosphose
CHART 6.—Diagram
phase.
illustrating
the
significance
of the
log. cell life span
dicho
Cells entering this phase may remain there for an mdc
terminate period
or tissue function.
before moving towards either the prosphase
I, immature cells; M, mature cells; D, mature
cells moving towards death.
CHART
production
7.—The
inverse
relationship
between
the
rate
of cell
and the length of life of the mature cells of the seba
ceous gland [taken from Ebling (146, 147)1.
BuLL0uGH—Mitotic
and Functional
It is also probable that the mitotic rate amidthe life expect
ancy of the mature
ship
(see Chart
cells have an inverse log-log relation
7).
The
result
is that
beyond
a certain
1703
Homeostas'is
siderable
informat.iomi already exists regarding
thyro
tropini (1, 429). It is, however, surprising how little of
the
vast
literature
that
has
been
devoted
to these
sub
point the increasing mitotic rate outpaces the increasing
ccli death rate so that the tissue increases in mass until
it reaches a new point of balance.
However, in order to
obtain a small increase in tissue mass it is necessary that
there be a great increase in the mitotic rate.
stances is concerned with the rnitot.ic response of the tar
get tissues, arid this is especially true in the case of the
androgemis, which are normally vaguely described as stimiiu
latinig the “growth―
of the male accessory sexual organs
It follows that the proportion of tissue mass to total
dent that such growth does involve a raised mnitot.ic rate
and also that the mitogenic stimulus of the androgens is
body mass tends to remain remarkably
constant.,
amid in
(see, for instance, Refs. 376, 395). It is, however, cvi
deed it seems possible that tissue mass itself might be de
steadily
termined by the combined actions of the rate of chalone
production and the life expectancy of the mature cells.
Thus it might be suggested that a tissue of large mass and
productive
by studies
terone on such nonsexual
tissues as the epidermis
low mitotic rate may be characterized by a relatively low
arid the sebaceous
(146, 360).
rate of chalone production and a long cell life ; a tissue of
small mass and low mitotic rate, by a relatively high rate
of chalone production and a long cell life ; a tissue of large
mass and high mitotic rate, by a relatively low rate of
chalone production and a short cell life; amid a tissue of
small mass and high mitotic rate, by a relatively high
rate of chalone production arid a short cell life.
It is also evident that the mitotic stimulus exerted by
the estrogens, which is felt es@)ecially in the female acces
sory sexual tissues but also in other nionsexual tissues,
must
be steadily
maintained.
However,
when the
stimulus is first applied it results not iii a constantly
elevated mitotic rate but in a series of sharply defined
waves of mitotic activity (see Refs. 63, 154, 157). The
reaction is complex, arid it also differs in its timing in dif
fererit tissues.
Thus the lining epithelia of vagina arid
uterus show their earliest reaction in about 12 hr (39, 385)
and reach maximum mitotic activity in from 24 to 36 hr,
while the lining epithelium of the Fallopian tube amid the
B. THE VERSATILITYOF THE MECHANmSM
1 . Methods of alteration of tis&ue mass.—It is evident
that the point of balance between cell gain arid cell loss,
which may also determine the tissue mass, is normally
remarkably
stable.
Only after wounding or during re
generation, when the mitotic rate is greatly increased, is
this normal balance disturbed.
However, the disturb
ance is temporary and self-cancelling, and as the chalonie
concentration
returns to normal the original I)Oinit of
balance between cell gain and cell loss amid the original
tissue mass are both regained.
Only if cell damage is
constantly
inflicted, for instance by any form of tissue
irritant,
can a new point
tissue of increased size.
However,
of balance
be established
in a
certain tissues and organs must be able read
ily to increase their mass in response to particular needs.
Thus the tissues of the accessory sexual organs become
enlarged during the breeding season in response to the
action of certain mitogenic hormones, and the mass of
the erythrocytic
tissue is increased during oxygen lack in
response to erythropoietin.
From such examples the
impression is gained that the mitogenic hormones amid the
poietins
may
constitute
a 2nd order
of mitotic
and func
tional control imposed on the basic chalone control mecha
nisms of the target tissues.
2. The mitogenic hormones.—It is well known that a
number of hormones can exert a profound effect on the
mitotic activity of certain so-called target tissues.
It
must be emphasized that the present argument is con
cerned only with those hormones that, in physiologic con
centrations, exert a direct effect on the mitotic rate.
Other
hormones, not considered here, may be important to
mitosis
but
only
in so far
as they
ensure
the
balanced
working of the general metabolic complex; in a normal
adult mammal these are always present in adequate con
centrations.
The best known examples of mitogenic
hormones are the androgens and estrogens, although con
smooth
maintained
throughout
activity.
of the steady
muscle
the
whole
This conclusion
glands
mitotic
stimulus
cells of the uterus
period
of re
has been confirmed
may
exerted
take
by testos
(89, 359)
2—3days
to
reach maximum mitotic activity (63). Similarly, in a
normal estrous cycle the vaginal amid uterine epit.helia
reach their 1st mitotic peak in late diestrus, while the other
2 tissues do riot do so until estrus (38, 63, 154, 155).
It is also obvious that, at the peak of the mitotic reaction,
those tissues that react the fastest contain the highest
proportion of cells in mitosis.
This situation is reminiscent of the different reaction
times of the tissues of the skin after wounding (81, 82),
and indeed there are obvious similarities between tissue
reactions to a reduced chalone concentration
and tissue
reactions to an increased mitogenic hornione concentra
tion.
After the 1st mitotic reaction to the estrogenic stimulus
there
is usually
a period
of at least
a day
during
which
the mitotic rate falls to a low level (63, 154, 155). Then
if the estrogen concentration remains high, this is followed
by a 2nd wave of mitotic activity, which is commonly
smaller thami the 1st wave. After this the waves are
damped down and the mitotic rate settles at a moderate
level, which however is comisiderably higher than that of
the ovariectomized
controls
(383).
A full explanation of this phemiornenionis not yet avail
able, but Epifanova (156) has argued that the high imiitial
mitotic stimulus may be reduced because of an estrogen
induced rise in the adrenalin amid glucocorticoid hornionie
content of the blood. It is well known that estrus is a
time of stress when the adrenal glands increase consider
ably in size (69) and when unusually large amounts of
adrenalin are absorbed by the uterus (530, 531). How
ever, it has been shown that, in mice stressed for long
periods by starvation,
“theuterus responded normally
to physiological amounts of estradiol― (49), arid this sug
Cancer Research
1704
gests that the stimulus of an estrogen may be more
powerful than the inhibition of the adrenal hormones.
As an alternative explanation it may be suggested that
the mitotic waves are closely similar in form to those
which occur in the epidermis alongside wounds (226, 235)
and which have been plausibly explained in terms of an
imposed mitotic synchrony.
It seems that any type of
sudden stimulus to mitotic activity may result mi an unu
sually large number of cells entering prosphase and mitosis
approximately
in unison, arid if the stimulus continues
the cells may also enter a 2nd mitotic cycle while still
preserving some of their synchrony.
The evident similarities between wound reactions and
estrogen-induced
reactions help to suggest a possible
relationship
between the chalones and the mitogenic
hormones.
Since chalones are regarded as tissue specific
arid since mitogemiic hormomies tend to be target-tissue
specific, the simplest explanation
would be to suggest
that each mitogenic hormone selectively neutralizes the
chalories of its target tissues.
In this connection Jensen
(271) has already remarked that “itis curious that so
little attention
has been given to the possibility that
estrogen may inhibit some growth-restraining
factor, for
many aspects of its action are more in keeping with an
inhibitory rather than a stimulating
role― arid that the
key property of anìy
estrogen “mightwell be an ability to
associate with sonic cellular factor and inhibit its func
tion.― The result of such an action would be a reduction
in the effective chalone concentrations
in the target tis
sues, arid if this interpretation
is correct then certain con
sequences should follow.
In the 1st place, while the mitotic rate of the vaginal
arid uterine epithelia in the ovariectomized
animal may
be too low to show any diurnal rhythm, as the effective
chalonie concentration
is reduced by the rise in concen
trationi of the est.rogenic hormone, a diurnal rhythm may
appear [compare the situation in regenerating liver (266,
421)]. That this is indeed so is indicated by Bertalanify
arid Lau (38), who have shown that diurnal mitotic cycles
arc evident in most l)hases of the estrous cycle in the
vaginal arid uterine epithelia.
In the vagina the diurnal
cycle disappears when mitotic activity is at its maximum,
as it. is also known to do in epidermis during the j)eriOd of
mnaximnum reaction to a wound, arid in both cases this
could indicate a severe reduction in the effective chalone
conicenitration.
Innthe 2nd place, if a hormone-induced mit.ot.ic reaction
is basically
similar to a wound reaction,
then in threshold
or subthrcshold situations the 2 should be additive.
In
this connection it has recently been shown by Prop7 that,
when mammary gland tissue is maintained in vitro, mitotic
activity may indeed be stimulated when a subthreshold
hormone stimulus is combined with a subthreshold wound
stimulus.
This is an important
point on which further
evidence is needed.
With a normal wound in a hormone
dependent tissue the mit.otic reaction is so great that the
hormone is unable to induce the development of airy more
mitoses (for example, see Refs. 63, 368), and indeed in a
full wound reaction there should be little or no chalone
left for the hormone to neutralize.
7 F.
J.
A.
Prop,
personal
communication.
Vol. 25, November
1965
In the 3rd place, if a mitogenic hormone normally acts
by neutralizing a chalone, then the increased mitotic rate
should result in a shorter dichophase and in a shorter ex
pectation of life in the cells in the functional phase.
Re
garding the 1st point, Epifanova
(156) has noted that
estrogenic treatment
may cause a 7-fold increase mi the
numbers of mitoses seen and a 7-fold decrease in the length
of the interphase, and Peckham and Kiekhofcr (383) have
shown that the whole mitotic cycle may be completed
in about 13 hr. Regarding the 2nd point, in an ovarec
tomized animal the rate of mitotic activity in the vaginal
lining is very low and the life expectancy of the mature
cells is very long; after treatment with estrogen the life
span of the miondividing cells is reduced to between 30 and
45 hr (383). Even more remarkable figures demonstrat
ing an inverse log-log relationship
between hormone
dependent mitotic activity amid the life expectancy of the
nondividing cells are given by Ebling (146, 147) in rela
tion to the sebaceous glands of normal, castrated, hypo
physectomized
and testosterone-treated
adult rats (see
Chart 7).
These results are particularly
important
since they
emphasize that although a target tissue, and the organ of
which it forms a part, may become larger in size when
stimulated by a mitogenic hormone, they do not become
as large as would be expected from a consideration of the
mitotic rate alone.
It is, of course, obvious that mitogenic hormones in
duce a variety of cell reactions besides the increased mi
totic rate; in particular, the new cells that are produced
begin to function in a new way (see Refs. 151, 271). For
example, under sex hormone influence, considerable cyto
plasmic changes occur in the uterine cells (180), the vagi
nal epithelium produces keratin and thus becomes corni
fled (97), the cells of the male accessory glands secrete
the seminal fluid (395), and in the thyroid the pituitary
thyrotropic
hormone also stimulates
the synthesis and
release of the thyroid hormone (198). Such reactions
involve the synthesis of unusual quantities of messenger
RNA (107, 200, 282), which leads to the active synthesis
of new proteins both fri accessory sexual tissues (502) and
in nonisexual tissues (435, 441).
It appears that when a target tissue is unstimulated
and the mitotic rate is negligible the majority of the cells,
which have an indefinitely long life-span, are mature but
nonfunctional.
They may be regarded as belonging to
type M, shown in Chart 8. The hormonal
stimulus
causes them to revert to type P and thus to begin mitosis.
Many of the newly formed cells then pass to maturity
again, but now because of a severely reduced life expect
ancy they pass from type ill, which is nonfunctional,
to
type D, which is functional.
Thus they are able to func
tion actively in the mariner typical of the tissue in the
hours or days or weeks that remain before they die. The
sequence of this reaction is shown in Chart 8.
When the hormone is withdrawn and the chalone con
centration
is again high the mitotically
active type P
cells are transformed to type Al and the functional type
D cells must go forward to death, with the result that the
tissue shrinks in mass.
3. The poietins.—\Iost of the dispersed cells of the
BuLL0uGH—Mitotic
±lt
CHART8.—The sequence of events in a target tissue when it is
stimulated by a mitogenic hormone. The mature cells (M) are
nonfunctional, and the consequence of the production of mitoti
cally active
progenitor
cells (P) is the production
of mature
functional cells (D) that are moving towards death.
body fall into 3 groups—the
granulocytes,
eryt.hrocytes,
and lymphocytes—and the manner in which these are
produced
is of particular
interest.
The gramiulocytes
arc the end product of the process of granulopoiesis,
which is generally believed to be stimulated
amid con
trolled by a substance called granulopoictin,
erythrocytes
are the product
stimulated
by erythropoietin.
while the
of erythropoiesis,
which is
The more complex prob
1cm of the lymphocytes is dealt with in the next section.
The mechanisms by which control is exercised over the
processes of cell production and cell loss in the granulo
cytic and erythrocytic
systems are evidently complex,
and they are not yet fully understood.
Regarding the
granulocytic system, Patt amid Maloney (381) have stated
that “littleis known about its beginnings and endings,
its dimensions and functions, and even less about its
controls.― In this system most of the available iniforma
tion refers to the neutrophils,
which are polymorpho
nuclear leukocytes.
These are considered to origimiate
from a population of stem cells, which give rise in the boric
marrow to a sequence of cell types—the mycloblasts,
promyelocytes,
and myclocytes—that
show increasing
degrees of maturation
but also undergo mitosis (90).
Finally, they give rise to the metamyclocytes, which show
no mitosis and mature into granulocytes. These are
stored mi the bone marrow until they are released for a
limited life in the blood and the tissues (sec Ref. 122).
The situation in the erythrocytic
similar
system is essentially
(see Ref. 292), and the cells arc usually considered
to originate from the same stem cells as those that give
rise to the granulocytes.
In the bone marrow the matura
tion of the erythrocytic progenitor cells proceeds through
a sequence of cell types—the pronormoblasts, basophilic
normoblasts, and polychromatic normoblasts—that show
increasing degrees of maturation and that all undergo
mitosis.
Finally,
into reticulocytes,
the
oldest
cells become
1705
and Functional Homeostasis
transformed
which are incapable of mitosis and
mature into erythrocytes.
These are released into the
blood immediately after they are formed, and they then
have only a limited life-span.
Described in this way, the granulocytic
amid crythro
cytic systems each constitute
a typical tissue system,
like that for instance of the epidermis, in that they con
tam cells that are mitotically active, cells that show in
creasing maturation
accompanied
by decreasing niit.otic
activity, and mature functional cells with a limited life
expectancy.
However, they differ markedly from a nor
ma! tissue in that they evidently share the same group of
mitotically
active stem cells, which must therefore be
considered to be pluripotential.
Although the emphasis in the literature has been mainly
on the l)ossible mariner of control of these 2 systems by
their respective poictins, in the i)resenit context it is mieces
sary to consider whether each may contain a typical
chalomie mechaniisni.
Since in both systems the mature
cells are dispersed it would, of course, be necessary to
suppose that any chalone produced by them would have
to lass via the blood stream to reach arid influence the
dividing arid maturing
cells in the boric marrow.
It
would also be expected, since in both systems the rate of
ccli production is very high, that the chalone concentra
tion in the blood may be low. In such circumstances
a
reduction in the already low chalone content of the blood
might have relatively slight stiniulatory
effect, but an
increase either in the chalone comicentrationi or in the ac
tivity of the adrenals might have a marked inhibitory
effect.
As regards the gramiulocyte tissue, the evidence in
favor of the existence of a negative feedback control of
the chalorie type has been assembled by Osgood (377,
378). In particular it has been shown that a decrease
in leukocyte
numbers leads to increased granulocyte
production (see Ref. 122) amid that an increase in leuko
cyte numbers inhibits granulocyte production, that fresh
blood contains some factor that inhibits granulocyte
production while old blood does not (520), amid also that
fresh serum inhibits mitosis in bone marrow in vitro (423).
From evidence of this kind and from a study of leukemia,
Osgood (377, 378) has proposed a theory of niitotic comi
trol in the granulocyte system amid in tissues in general
that is fundamentally
similar to the chalomic theory out
lined above.
However, in addition to the basic micgat.ive
feedback mechanism, Osgood lists a long series of second
ary mitotic comitrols, which includes vitamins, electrolytes,
nutrients, temperature,
CO2 temision, pH, and blood amid
lymph flow. Such factors, if they influence mitosis at
all, are most likely to do so in a nonspecific manmier arid
are umilikely to form any significant part of a tissue-specific
mechamiism.
Some attempts have been made to extract a granulo
cytic chalonc from mature gramiulocytea (122, 123, 423),
and recently Rytomaa and Kiviniemi8 have obtained a
substance
having
the required
characteristics
when
tested on the mitotic rate of bone marrow cells in vitro.
This substance is water soluble, unstable, nondialyzable,
and relatively low in molecular weight, all of which charac
teristics it shares with the epidermal chalone.
S T.
Rytomaa
and
K.
Kiviniemi,
personal
communication.
1706
Cancer Research
As regards the crythrocytic
tissue, relatively
little
attention has been paid to the possible existence of an
eryt.hrocytic chalone.
However, it is well known that
cryt.hrocyte production is depressed after the transfusion
of large numbers of red blood cells, amid it seems probable
that the erythrocyt.ic tissue will prove to be similar to
the granulocytic tissue mi possessimig a chalomie mechanism
(see Rcfs. 218, 295, 464). If this is so then the chalone
may be produced in the circulating erythrocytes
amid it
may inifluemiceonly the mitotic activity of the crythrocyte
progenitor cells.
If such a mechanism is present in both the gramiulocytic
and erythrocytic
systems, it follows that these systems
may show a reduced mitotic rate during stress, and pos
sibly even a diurnal mitotic rhythm.
Similarly, they
may show changes in the mitotic rate after adrenalectomy
or after injections of adrenalin or glucocorticoid hormones.
It is unfortunate that the literature on these various points,
while voluminous, mainly records variations in the num
bers of gramiulocytes and erythrocyt.es in the blood, and
these may commonly depend on processes other than the
mitotic rate (see, for instance, Rcfs. 27, 56, 199).
The granulocytic
and erythrocytic
tissues differ from
most other tissues so far considered in that they must be
able to adjust their rates of cell production not only to
changes in tissue mass but also to changes in functional
demand.
In the case of the gramiulocytes, it is evident
that mature circulating cells arc eliminated riot according
to their age but according to the work they have to do in
combatting
bacterial invasion, since in these cells func
tiomi leads to death (122, 422, 484). In the case of the
erythrocytcs
the mature circulating cells are eliminated,
as in a normal tissue, according to their age, but the cell
numbers must be able to imicrease if, for any reason, the
oxygen supply to the tissues becomes inadequate
(see
Refs. 263, 464). In both these tissues it is evident that
these adjustments
are made through the actions of the 2
l)Oiet.inis. The sites of synthesis of these 2 subst.amiccs
are not yet certain, but granulopoictin
may be produced
in functioning
or postfunictioniing granulocytes,6
while
crythropoietin
may be produced in the kidney or other
tissues (264, 351, 384, 411).
Vol. 25, November
1965
quire different controlling mechanisms,― and it is worth
considering what these might be.
If the stem cells arc indeed pluripotent, this may imply
that, unlike most tissue cells, they contain more than 1
set of genies that are potentially capable of directing the
maturation
of more than 1 type of cell. It appears in
fact that in these cells the final step in differentiation
has
not been taken and that when it is taken, whether in the
direction of the granulocyte or of the crythrocyte, the proc
ess must be akin to tissue induction in the embryo.
In
their final form the cells may be expected to contain only 1
functional set of genes, which will lead to only 1 type of
maturation.
From what is now known of the chalones it seems most
improbable that they could control this type of induction,
and indeed specific chalonc synthesis may itself be the
consequence of a specific type of induction.
It is the poie
tins that evidently act as inducers.
Erythropoietin
has
no significant effect on either the mitotic activity or the
rate of maturation
of the erythrocytic
progenitor cells
(7, 294) ; its primary effect is merely to promote the con
version of the stem cells into erythrocytic progenitor cells
(7, 294). In a similar way granulopoietin
appears to pro
mote the conversion
of stem cells into granulocytic
progenitor cells. The probable relations of the poietins
and chalones of the erythrocytic and granulocytic systems
arc illustrated in Chart 9.
Thus the actions of the poietins are fundamentally
dif
ferent from those of the mitogenic hormones, although
both groups of substances are similar, first, in that they
arc produced in response to a trigger mechanism operated
by circumstances
external to the animal, and second, in
that they both induce the increased mass of their par
ticular target tissues.
This theory leaves unanswered the problem of mitotic
control in the stem cells themselves, but this is a problem
that can hardly be attacked until these cells have been
recognized and localized.
Possibly they may synthesize
A second and more surprising way in which the granulo
cytic amid crythrocytic
tissues appear to differ from ordi
nary tissues is that throughout
life both may continue to
be formed from a commomi stem cell l)oPulationi, which
may j)erhaps be located in the bone marrow.
It is even
possible that the stem cells may form part of the lympho
cytic system (see Ref. 533). Although in the adult mam
mal the stem cells may prove to comprise 2 or more sepa
rate groups, each leading to only 1 mature cell type, the
Present evidence is generally held to indicate that they
are pluripotent cells (29, 293). If this is accepted then,
u.s Lajtha (292) has pointed out, there are 2 ways in
which an increased number of mature cells may be ob
taimied. First, an increased number of stem cells may
differentiate
to enter either the granulocytic or the cry
throcytic tissue systems, and second, there may be,
imicrease in the number of mitoscs through which each
maturing cell passes.
Lajtha concludes that “itis quite
l)Ossible arid indeed likely that the two mechamiisms re
E@D
Gran.D
CHART 9.—Diagram illustrating the probable method of con
trol of cell recruitment
in the erythrocytic
and granulocytic
tissues. The stem cells (8) differentiate into erythrocytic pro
genitor cells (P) under the influence of erythropoietin (E.P.), and
into granulocytic
progenitor cells (P) under the influence of
granulopoietin (G.P.). Mitotic activity is limited in the erythro
cytic progenitor cells by chalone (E.Ch.) from the mature dying
erythrocytes
(Ery.D.), and in the granulocytie progenitGr cells
by chalone (G.Ch.) from the mature dying granulocytes (Gran.D.).
BuLL0uGH—Mitotic
their own chalonc or possibly they may respond to one or
all of the chalones of their daughter
tissues.
Jun this
cominiectiomiit may be imj)Ortant that the mitotic activity
of lymphocytes,
which are evidently stem cells, can be
suppressed by granulocytes
(123, 124), possibly through
the action of the granulocytic
chalone.
4. Mitotic control in lymphoid tissue.—Froni a comi
sideration of a wide range of evidence, Yoffey (533) has
suggested that although the lymphoid tissue may appear
to be entirely separate from the granulocytic and eryth
rocytic tissues, in fact all 3 may together constitute
1
large tissue system—the lymphomyeloid
complex.
He
has concluded “thatthe different parts of the complex
are in close functional relationship, and that through the
blood stream there is in all probability a constant miter
change of cells between them.― At the moment this
remaimis only an imiteresting possibility.
The lymphoid system is obviously highly complex, but
it evidently produces omily 2 types of “mature―
cell, the
small lymphocyte amid the plasma cell [see the review by
Yoffey (534)]. The production
pathway of the small
lymphocyte is often believed to begin with the ret.icular
cell, which remains static in the lymphopoictic center amid
which, by a series of successive mitoses, gives rise to the
large lymphocyte,
the medium lymphocyte,
and finally
the small lymphocyte.
If this is the basic mitotic system
of the lymphoid tissue, and if the small lymphocyte
is
considered to be a “mature―
cell, themi the system displays
the usual pattern, whereby increasing maturity is matched
by decreasing mitotic activity.
However, it is by no means certaimi what role the small
lymphocyte plays in the body.
Its rate of j)roductiori is
high.
hi the thymus Leblond and Saimit.e-Marie (305)
consider that the division of 1 rcticular cell usually results
in the production of 128 small lymphocytes,
and iii the
mesemiteric lymph node Grundmann
(214) estimates that
1 reticular cell may give rise to 64 small lymphocytes.
This rate of production argues either a short life-span or a
rapid transformation
into some other type of cell, or both.
All that is known is that large numbers of small lynipho
cytes migrate via the lymph amid the blood to the tissues,
and it is believed that large numbers may settle in the
boric marrow (206, 207).
This system is essentially similar to that seen in the
erythrocytic
and gramiulocytic tissues, and this suggests
the possible existence of a chalone control mcchamiism.
There is no direct evidence to support this suggestion, but
considerable
indirect
evidence
has
been
reviewed
1707
and Functional Homeostasis
by
Dougherty (139), who shows that the glucocorticoid hor
mones, administered
in excess, result mi an involution of
the lymph nodes, thymus, and spleen that is characterized
by a sharply reduced mitotic rate and a lower lymphocyte
output.
The same effect is seen after any kimid of stress,
and there is also a marked diurnal rhythm in the lympho
cyte content of the blood. These stress effects are elimi
nated by adrenalectomy,
which increases the mitotic rate
and raises the number of lymphocytes in the blood while
simultaneously
reducing their life-span.
Furthermore,
during recovery after several hr of imposed stress, the blood
lymphocyte
content not only quickly returns to normal
but for a few hr exceeds it, an effect which could be the re
sult of a stress-induced accumulation of cells in aiit.cphase.
It is evidemit that all these effects are closely simiiilar to
those described for epidermis amid other tissues in which
they are dependent on the existence of a cha@mie mech
anism.
The 2nd and apparently separate niitotic system of the
lymphoid tissue is that by which the plasma (Yellis pro
duced.
Leblomid amid Saimite-Marie (305) regard this as arm
alternative
form
of maturation
to that
which
gives
rise
to the small lymphocyte, and they describe the sequence
of niitoses and of cytoplasmic maturation which they be
lieve
plasma
point
to occur
between
the
large
cell (see also Ref. 369).
of view expressed
lymphocyte
and
1mmsharp contrast
by Yoffey
the
is the
(533, 534), who regards
the plasma (Yellas being derived from the small lym@)ho
cyte (see also Refs. 205, 208). He describes the way in
which this cell grows imi size amid becomes transformed
into a pyroniiniophilic cell, which thermundergoes successive
mitoses to form a group of daughter plasma cells. Cer
tainly it appears that the small lymphocyte can be stiniu
lated to grow in size and undergo mitosis both in vivo
(205, 390) amid with @)hytoheniagglutimiini in vitro (338,
371).
It is now generally agreed that the function of the plasmiia
cell is the synthesis of amitibody. Whether the small
lymphocyte or the ret.icular cell is the l)aremit (Yell,it. is the
sudden stimulus of the invading antigen that results inn
the production of the pyronimiophilic cell amidin the burst of
mitot.ic activity that ceases only when the I)hLsma cell
matures amid fills with antibody (22). It is well known
that each invading antigen calls forth the production of
an appropriate
antibody, arid therefore it must be con
eluded that each plasma cell is narrowly committed to only
1 program of protein synthesis.
Yoffey (534) has de
scribed the plasma cell as “conditioned―amid has com
merited that “whatever theory of antibody production
may ultimately
prove
to be correct,
it. is clear that
inn the
primary response there is some conditiorunig of the lymnph
oid cells, whether genetic or otherwise.― Medawar (343),
expressing a similar l)Oimitof view, has emphasized that
“theantigen, like the inducer, is armagent that commits a
cell to a certain pathway of differentiation—to one path
way among several that it might have taken, arid that lie
within the gemictic capability
of the cell.― This point. of
view is closely similar to that expressed above when the
actions of the l)Oietimiswere likened to those of embryonic
inducers.
If it is temit.atively accepted that the small lymphocyte
may be the immuniologically competent cell therm a specu
lative theory can be developed as follows. The supply of
smnall lymphocytes is maintained by the mnitotic activity
of a population of stem cells, which are the larger lympho
cytes amidthe reticular cells. To maimitaimiitself mi balance
this population
must be controlled by sonic feedback
mcchamiism that limits the mit.otic rate amid promotes the
maturation
of the small lymphocytes.
The role l)layed
by the adrenals indicates that this may prove to be a
typical chalone mechanism, arid if so then the system
will be self-balancing simice the loss of small lymphocytes,
whether by death or differentiation,
will lead to a lowered
chalomic concentration
in the blood arid lymph.
1708
Cancer Research
On this view the small lymphocyte is regarded as a cell
in which the final step of differentiation
has riot been
taken and which therefore
still possesses a number,
perhaps a very large miumber, of gemictic possibilities.
The
anit.igcmi, acting as arminducer, limits these possibilities to
only 1—the synthesis of the appropriate
amit.ibody—and
at the same time evidently frees the cell from the mitotic
control that had previously held it inert.
If final differ
enitiation always includes an acquired ability to synthesize
a n@pecificchalomie, then a newly differentiated
cell may
always lack sufficient chalone amid therefore always under
go mitosis until, in the plasma cell, the intracellular
chalomie concentration
rises to ann effective level. Once
again, although there is no direct evidence available to
support the theory that a chalonie mechanism is present,
there is some indirect evidence derived from a study of the
actions of glucocorticoid
hormones.
White (522) has
recently reviewed evidence that these hormones reduce the
production rate of the plasma cells, but not the rate of
antibody production in army cells already formed.
A plasma cell is immunologically
active arid, being
fully differentiated,
it is unable ever again to revert to a
state of immunologic competence.
Imideed it is generally
believed that plasma cells may be short lived (121, 369,
437). The basic response of the lymphoid system to
anit.igen stimulation is in fact the creation of a new type
of tissue that was riot present in the body before, and
although the plasma cells may all belong to type D (see
Chart . 4) amid therefore die, some cells, perhaps of type
1%!,may survive for long l)criOds. These lomig-lived cells
may superficially resemble the short-lived umiconditioned
lymphocytes
(see Refs. 168, 207), amid they may be able
to take l)art in a secondary response.
Such a respomise
would then involve a double reaction : the increased ac
tivity, perhaps including mitosis as well as amitibody pro
duct.ioni, in already existing tissue cells; and the induction
of new cells into the tissue.
This would imply that an
antigen may normally act, directly or indirectly, on both
the induction and the production of the mature plasma
cells.
These suggestions tend to run counter to the clonal
theory of Burnet (95, 96), at least when this theory is
stated innits most extreme form.
Indeed, an outstanding
unanswered question of the momenit is the actual degree
of competence of the immunologically
competent cells.
As mentioned above, it. has even been suggested (see Ref.
534) that such cells may be imiduced by the poietins to
give rise to either granulocytes or erythrocytes.
The bone
marrow contains large numbers of small lymphocytes, and
there is evidemice to suggest that they may be induced to
differentiate in this way. The most sigmiificant argument
against this suggestion arises from the evident inability
of t.horacic duct lymphocytes to repopulate bone marrow
from which the cells have been eliminated by irradiation
(see Ref. 186).
Although much of this discussion of the mitotic activity
of the lymphoid tissue is speculative, one important point
is clear. As was the case with the granulocytic arid eryth
rocytic tissues, the lymphoid tissue is capable of adjusting
its mitotic rate both to the demands of tissue mass, which
may be mediated through a chalone mechanism, and to
Vol.
25, November
1965
the demands of function, which are mediated through the
inducer-like action of an antigen.
With the great variety
of possible antigens, such a system must be unusually corn
plex, amid the homeostatic mechanisms controlling mitosis
amid cell maturation
are also likely to be more complex
than in any other tissue system of the body.
5. The growth of hair.—It has been suggested above
that the relatiomiship of tissue mass to body mass is a
function of the rate of chalone production in the mature
cells. The question now arises how the mitotic rate may
be controlled in such a tissue as hair, in which the mature
cells die so rapidly that no significant mass of chalone
producing cells can be built up. The high mitotic ac
tivity of the hair bulb results in the production of a shaft
of dead cells, which are obviously incapable of chalone
production, but when the hair reaches its correct length,
or mass, the niitotic activity ceases. It is obvious that
some mechamiism must control the phases of activity and
imiactivit.y of the cells, and since it seems unreasonable
to expect that. this mechanism may be unique, it is worth
considering how a typical chalone mechanism might be
modified to produce this effect.
The complex l)attern of hair growth has often been de
scribed (see Refs. 80, 112, 357). Reduced to its simplest
terms it involves 4 successive phases of inactivity and
activity.
In the 1st phase the cells of the hair bulbs are
in a resting comiditioni (called telogen) mi which no mitosis
is ever seen ; in the 2nd phase (early anagen) the cells
show inicreasimig mitotic activity leading to the lengthening
of the follicle ; in the 3rd l)hase (anagen) the cells show con
tinuous high mitotic activity, and it is the duration of this
phase that determines the length of the hair; in the 4th
phase (catagen) the mitotic activity decreases and the
follicle shortens to enter the resting condition once more.
In any 1 area of skin all the follicles may follow this
rhythm in unison, as is the case in mice and rats (141,
142) ; alternatively,
each follicle may follow its own in
dividual rhythm, as is the case in guinea pigs and men
(1 12) . In either case it seems that the control mechanism
must be intrinsic in the individual follicle and not systemic
(141,142).
Considering first the resting follicle, it is evident that
the cells are similar to those of any ordinary inert tissue in
that they are able to react by high mitotic activity to any
form of wounding, and thus it appears that they must
normally be controlled by a high chalone concentration.
In contrast, the cells of the actively growing follicle show
an extremely high rate of mitosis, a mitotic cycle length
of only about 12 hr (80, 111), and a complete lack of re
sponse to stress or adrenalin.2
Indeed the cells react as
though little or no chalone exist within them.
One obvious possible explanation for the rhythm of hair
growth lies in the suggestion that it depends inversely on
a rhythm of chalomie production, and this agrees with the
suggestion of Chase (112) and Chase and Eaton (113)
that the onset of hair growth may be due to “amore or
less rapid loss or leaching of an inhibitor.― This theory
is expressed diagramatically
in Chart 10, and the evidence
both for and against it has recently been summarized by
Ebling and Johnson (149).
If this theory is basically correct then it should follow
BuLL0uGH—Mitotic
teloqen
anaqen
C
C.)
C
0
C.,
@
and Functional Homeostasis
teloqen
I
a)
C
0
0
C.,
trol
CHART 10.—Diagram
of the hair growth
illustrating
the possible
cycle by the fluctuating
manner
chalone
of con
concen
tration in the hair root cells.
that the adrenal hormones should be without army effect
in either the resting or the actively growing phases, but.
should exert a pronounced action during the 2 transition
periods.
Thus, for instance, when the chalone concemitra
tion falls towards that critical level at which mitot.ic ac
tivity begins, the enhancement
of the efficiency of the
chalone complex by stress or by adrenalin injectiomi should
delay the onset of the phase of hair growth, while the
weakening of the efficiency of the chalone complex by
adrenalectomy
should accelerate it. As regards stress,
Blumenthal
(47) has shown that when mice arc main
tamed on a reduced diet the initiation of new waves of hair
growth is delayed or even prevented (see also Chase, 112).
As regards the adrenal hormones, Mohn (352) amid Ebling
and Johnson (149) have shown that the initiation of hair
growth is accelerated
by either hypophysectomy
or
adrenalectomy
and that it is delayed by injections of
ACTH, cortisone, or adrenalin.
Once again it appears
that a glucocorticoid hormone may be involved as a 3rd
partner in the control niechamiism.
Thus the available evidence tends to support the theory
that hair growth may be controlled by the normal type
of mechanism, but, whereas in other tissues the rate of
chalone production may be relatively constant, in the hair
bulb it appears to be rhythmic.
This rhythm may be
inherent in the genes directing chalone synthesis or it
may be imposed or modified by factors in the follicular
environment that affect the activity of these genes. The
available evidence strongly suggests that the ultimate
control lies in a gene or group of genes that oscillate be
tween activity and inactivity.
For any particular type of
hair the durations of the various phases of the rhythm arc
evidently constant, but between different types of hair
they vary widely.
Oscillating genetic comitrol of this
type may be analogous to the oscillating genetic control
of estrous cycles (see Ref. 215).
It is, however, evident from the latest results of Ebling
and Johnson (148, 149) that the follicular environment
may exercise a moderating
influence on the basic hair
growth rhythm.
In a similar manner the follicular en
vironment may modify gene expression at the agouti locus
within the melanocytes associated with the hair root (43,
446) ; perhaps the most spectacular example of this is in
such northern species as the varying hare and the arctic
fox. In these the color of the growing hair is dependent
on the expression of the melanocyte genes, which is de
termined according to the season by the physiologic condi
tion of the animal.
6. Conclusions.—Fromthis survey it appears that the
1709
mechanism of mitotic and functional honmeostasis in certain
adult mammalian tissues is subject to a 2mid order of con
trol by mitogenic hormones amid that in a few tissues the
situation is also modified by the actions of l)Oietins or
antigens that augment the tissue mass by imiducinig the
final differentiation
of stem cells.
Regarding the mitogenic horniones, it. is clear that. be
cause of the effective inverse balance between cell gain
and cell loss a great increase in the mitotic rate is needed
before army significant chamige in tissue mass cant occur.
It seems that the effects of these hormones are dramatic
maimily because the target tissues inn their unst.inmulat.ed
comiditiomi connmomily show rio niitosis at all. Their
manmier of action supports the suggestion that they niay
mieutralize the chalonies of the target tissues, arid their
actiomi on the genonie is therefore indirect.
It is miter
esting that in insects Kroeger (288) has also concluded
“thathorniomies . . . camimiotact directly on the genetic
loci, arid that there must be an intermediate system which
relates the hormonal stimulus to the lOci.―
It seems possible that the hormone-dependent.
t.is@ues
are merely those that are characterized by armexaggerated
dependence on hormones, which also oj)crate to sonic
degree in all nornial tissues (63) . The evidence suggests
that target-tissue specificity is expressed in a quantitative
rather than a qualitative manner, and regarding the sex
horniones Bullough (63) has emphasized that. most tissues
may jossess some sensitivity, which gives them the po
temitiality for enlargement inn relation to the sexual proc
esses should the riced ever arise. Examples of accessory
sexual tissues that seem to have evolved inn this way are
the posterior region of the stickleback kidney, the thumnb
pads of the frog, the comb amid wattles of the cock, the
sexual skin of the monkey, amid the mammary gland.
In
all such cases it is evidently the tissues that become modi
fled, through changes in their nmitot.ic amid functional
homeostatic
mechanisms, to emiable them to react more
strongly with the horniones.
The hormones themselves
have evidently remained unchanged.
It must also be
emphasized that the mitogenic hormones probably exert
a wide variety of actions inside cells, amid that only one or
sonic of these are directly related to mitosis.
It is interesting that all the known mitogenic hormones,
such as androgemis, estrogens, prolactin, amid thyrotropin,
are under the control of the anterior pituitary
gland,
which is itself imifluemicedby the nervous system amid thus
by inforniation entering through the sense organs.
Al
though mmmost laboratory mammals the rate of secretion
of the mitogenic hormones may be wholly controlled by
inherent genetic mechanisms, as mi the case of the estrous
cycle (see Ref. 215), in most wild mammals the rate of
secretion of these hormones is modified by environmemital
changes that act as trigger mechammisms. This is part.icu
larly obvious in relation to seasonal breeding (@yclcs (see
Ref.72).
It is interesting to note that in laboratory animals the
mechanism
comitrolling hair growth is also evidently
genetically comitrolled, while in most wild mammals the
growth of a new coat is a sea.sonmal phenomenomi.
Here
again gene action is evidently
modified by seasonal
changes.
However, although the time of onset of hair
Cancer Research
1710
growth may be determined in this way, the emidof anagen
appears to be determined solely by gene action.
It, has been stressed that increased tissue mass cannot
easily be obtained through imicreased mitotic activity but
that in the granulocytic
and erythrocytic
systems in
creased tissue mass is sim@)ly achieved by the incorpora
tion of new cells that were not previously part of these
tissues.
This is essemitially an exploitation
of an em
bryonic mechanism whereby, under the direction of either
graniulopoictin
or erythropoietimi , relatively
unidiffer
enitiated stem cells are induced to undergo their final dif
ferenit.iatiomi. The i)Oietimis, like the antigens that act in
a similar manner on the relatively undifferentiated,
im
munologically competent lymphocytes,
are also part of a
niechaniismii whereby the animal can respond to emiviron
mental change.
Although
the manner of control of
eryt.hropoietin
production
is not yet clear (see Refs.
20, 379), granulopoietimi production seems to depend on
the rate of granulocyte destruction,
which is a function
of the work the graniulocytes have to do. Similarly in
the case of the lymphocytes,
antigen induction is de
l)emident oh unpredictable
circumstance.
It is evident that mammals possess only limited stem
cell populations,
amid iii this they differ markedly from
many amphibianis in which the regeneration,
for in
stance, of entire new limbs indicates the existence of stem
cells with great potentialities.
However, the recurrent
growth of antlers in deer has not yet been analyzed from
this l)Oinmtof view (see Refs. 201, 202).
C. THE BREAKDOWN OF THE i\IECHANISM
1. The mechanism itself.—The essential features of the
theory of mitotic amid functional homeosta.sis outlined
above are summarized
diagrammatically
fri Chart 11;
for simplicity,
regulator genes are not included.
The
process of induction, by which the tissue originated, in
volved the suppression of all genes that are relevant to
other types of tissue (the “blockedgenes―). The remain
inig genies that are, or may become, functional form the
CHART 11.—Diagram
illustrating
Vol. 25, November
1965
facultative
genome (170), and they include genes that
dictate the synthesis of general metabolic enzymes (the
“essential genes―), genes that dictate the synthesis of
those special enzymes on which the tissue organization
depends (the “tissuegenes―), and genes that dictate the
synthesis of those enzymes that underlie the mitotic cycle
(the “mitosis
genes―).In the chart the syntheses con
trolled by the tissue genies and the mitosis genes are re
garded as occurring in a series of steps, each of which is
under separate gene control.
In each case only one
synthetic pathway is indicated although, of course, there
must be many.
One group of tissue genes (the “chalone
genes―) are regarded as specifying both the structural
details amid the rate of synthesis of the tissue-specific
chalone, which then promotes the activity of the remain
inig tissue genies. If the chalone concentration
falls (as
in wounds) not only does this promotion cease but many
of the already functional cells lose some or all of their
special characteristics.
It is not clear whether in pro
moting tissue specialization
the chalonc acts alone or
whether adrenalin (and perhaps a glucocorticoid hormone)
is also involved.
It is, however, clear that the power of a chalone to
inhibit the activity of the mitosis genes is strengthened by
adrenalin.
Since it seems reasonable to believe that the
mitosis genies may be the same in the cells of all tissues, it
is difficult to understand how within any one tissue they
could develop the ability to respond only to the chalone
of that tissue.
One possibility is that a tissue-specific,
chalone-sensitive
trigger mechanism may operate at the
beginning of the l)haSe of DNA synthesis and another at
the beginning of the phase of mitosis.
Both these phases,
once begun, are self-supporting.
Finally, it should be emphasized
that although
a
chalone-adrenalimi complex appears to be a central feature
of the homeostatic mechanism, the effects of withdrawing
chalone and those of withdrawing
adrenalin are very
different.
When most of the adrenalin is withdrawn
(as
after adrenalectomy)
the epidermal mitotic rate rises 2-
the
homeostasis in a typical mammalian tissue.
theory
of
mitotic
and
functional
For explanation see the text.
BuLL0uGH—Mitotic
and Functional Homeostasis
or 3-fold (83) ; when most of the chalone is withdrawn (as
after wounding) the epidermal mitotic rate rises perhaps
10- to 50-fold (81). Also, the adrenalin effect is im
mediate, evidently because this hormone is so rapidly
gained or lost, whereas the chalone effect takes many hr,
perhaps because it is more stable and less easily lost, but
perhaps also because it may act most powerfully in early
prosphase.
Although in Chart 11 the situation shown is obviously
oversimplified and inadequate,
it is at least useful in in
dicating some of the main points at which the homeostatic
mechanism may be broken, either temporarily
or per
mamiently.
1711
normality
is usually associated with chronic niyeloid
leukemia (58). If the gene damage leads to the production
of “foreign―proteins,
then presumably
the immune
mechanism will be activated, arid as Burniet (95) has sug
gested, this may be one of the main defences against
cancer.
If, however, the mutation leads to the weakening
and ultimate cessation of genie action therm the immune
niechaniisni will presumably
not operate, arid there is
indeed considerable evidence that tumor cells are deficient
in specific proteins amid show a loss of immunologic spe
cificity (see Ref. 93).
In a carefully reasoned criticism of the theory of somatic
mutation, Rous (415) has emphasized the relative lack of
2. Carcinogenesis.—The effects of a temporary break
evidence that such mutation does commonly occur arid
down of the homeostatic mechamiism have already been
has also stressed the important. loinit that all carcinogens
considered at lemigth ; the question of a permanent break
arc riot miiutagenis arid all mutagenis are riot carcinogens.
down leads to a consideration of the relation of the chalone
This latter point is also emphasized by Trainini et al. (489),
theory to the cancer problem.
The current theories of who however conclude that it is “probable that only a
carcinogenesis
have been reviewed and criticized
by small proportion of mutagenmic agents would share the
Hiegcr (241, 242), but even more valuable for present
(@omnioni @@ite
or sites of action which one would postulate
purposes arc the recent statements
by Foulds (170—72). to be involved in the initiation of carciniogenesis.―
It is clear that one of the most important theories con
Hieger (241, 242), too, has objected that genie daniage
cerning carcinogenesis derives from the work of Bereni
must. be a comparatively
rare-event and that. if, for cancer
blum (34) amid of Rous amid Kidd (416), who distinguished
to develop, several distinct niutationis are needed, u.s
between a process of initiation, whereby a number of indeed seems to be the case, therm the chances of inntiation
dormant amidunrecognizable tumor cells are formed within
decrease “t.opractically
zero.― However, Burniet (94,
the tissue, and a process of promotion, whereby these
95), while agreeing that “at.any given genetic locus an
dormant cells are caused to multiply to produce a visible
error mi replication occurs with a frequency iii the range
tumor.
Initiation,
which is irreversible,
may perhaps
10@—10@per replication,― has pointed out that. in ann
occur spontaneously
or may be the result of any kind of animal as large as a man some 10'@cells miiay be constantly
carcinogenic action ; l)rOniOtiOn, which at least in its early
reproducing
themselves.
However, all such arguments
stages is reversible, may also be spomitaneous or may be omit a number of innportanit considerations.
First, they
hastened by a promoting agent.
The inadequacy of such
do riot take into account the effects of carcinogenic agents
a simple analysis has been stressed by Foulds (171, 172),
in increasing the mutation rate (see Ref. 296), arid it must
who has pointed out, first, that the action of a carcinogen
be emphasized that many so-called spontaneous
tumors
is quantitative.
With smaller doses fewer tumor cells may have been induced in this way by as yet undetected
are produced, and these, after promotion, develop only
carcinogens.
Second, they do not consider the POSsibilitY
into papillomas, nearly all of which regress.
With larger
that genie damage, whether spontaneous or induced, may
doses more tumor cells are iroduced,
amid these, after
occur in nondividing cells, amid indeed Curtis (127) has
promotion, give rise either to papillomas with the lower
presented
evidence that nondividing
mammalian
cells
to progress into carcinomas or occasionally to carcinomas
accumulate deleterious mutatiomis at such a rate that by
ab initio.
Second, Foulds emphasizes that, whether spomi late middle age “virtually all cells carry many genie niuta
taneously or after treatment with carcinogens, tumors may
tions.― Third, they omit consideration of the Possibility
that gene damage or failure may be at least partly de
arise at one or many sites within a tissue, appearing at
pendenit on inherited genie weaknesses, amid that mmsuch
random both in space and time, and that occasionally they
animals as man it may even be “thatthe majority of
may even involve a whole organ.
Third, Foulds con
malignancies are of this kind― (see Refs. 91—93).
eludes that a benign tumor is subject to 4 possible fates
If for any of these reasons gene function is impaired
“namelyregression, indolent persistence, growth without
then the reaction of the damaged cells would be expected
qualitative
change, and progression to malignant
neo
to vary according to the type of genie or genes involved.
plasia―,—but that the commomi tendency is for any tumor
For the present argument the simplest situation would be
to become progressively more malignant.
one involving some failure mi the genes that determine
To explain the cellular chamiges that underlie these
the production rate or the structure of the tissue chalone,
phenomena one widely held theory is that initiation in
and even a relatively slight failure would tend to give the
volves 1 or more somatic mutations, and it is evident that
cells some proliferative
advantage.
According to the
these mutations might involve damage to the genes that
Darwinian point of view advocated by Burnet (96), this
previously controlled mitotic and functional homeostasis
(55). In Haddow's (220) words, it is possible to “regard might seem sufficient to ensure the appearance of a tumor,
but in fact it is now clear that certain couniterchecks would
the problem as one of the impact, upon the genomal in
be expected to come into operation to limit amid even to
tegrity, of any of a host of reagents―, and it is now evident
First, if only single
that similar damage can be inflicted on cells in vitro (25, prevent any extra mitotic activity.
cells, or small groups of cells, are damaged, the chalone
26). It is even known that a specific chromosome ab
1712
Cancer Research
concentration
within them should remain miormal because
of diffusion inwards from the surrounding
undamaged
cells. An increased mitotic rate should not be seen until
the group has grown to such a size that its central cells
are beyond the effective range of chalone diffusion, amid
judging from the length of the chalone diffusion path ad
jacemit to a wound (81), this critical cell mass might have
a diameter of a little less than 1 mm.
Beyond this point, so long as the rise in the mitotic
rate is only moderate, a 2nmd countercheck
might begin
to oj)erate.
This would depend on the expectation that
any rise in the mitotic rate would be offset by a reduction
in the life expectancy of the mature cells. Thus the latent
period described by Bereniblum (34) and Rous amid Kidd
(416) may include the whole of the period from initiation
to the end of the period of effectiveness of this 2nd counter
check.
This question is considered further in the next
sectiomi.
So far only the expected consequences of damage to the
chalonie genes have been considered.
It. is obviously
possible that genie damage might also involve, or even
only involve, those other tissue genies that dictate the
synthesis of the typical tissue proteins.
The result of
this would be expected to vary widely according to which
genes were damaged amid in what degree.
In the 1st
place the damage might involve the genie operators through
which the chalommemechanism ultimately
acts, and this
could have similar consequences to chalone lack. Second,
the damage might upset the balance between cell gain and
cell loss either by reducing the rate at which the tissue
genies synthesize tissue protein (183, 414) or by increasing
the life expectancy of the mature cells. It seems probable
that delayed or inadequately
realized maturity may in
crease the proportion of the cell population that remains
capable of division, amid in this connection Berenblum (36)
has emphasized that promoting agents commonly appear
to operate “byproducing a delay in maturation
of the
dormant tumour cells―. Regarding the life expectancy
of the mature cells, it is known that this is enhanced by
the chalonme complex, but it is possible that this action
may operate through a mechanism that might be sepa
rately subject to damage.
In the 3rd place, the variety of possible damage to the
tissue genies could account for Foulds' (170) conclusion
that “tumoursderived from the same tissue by the same
carcinogenic
procedure
may be extremely
varied and
intergraded― so that “itis probable indeed that no two
tumours are exactly alike in every respect.― Foulds
(170) also emphasizes that tumor characters are not static
but tend to change progressively,
that such progression
occurs independently
in different characters in the same
tumor, that it occurs in tumors that are miolonger growing,
and that it may be continuous in large numbers of small
steps or discontinuous
in small numbers of large steps.
Commonly such progressive changes evidently also involve
further gradual breakdown in the mitotic and functional
homeostatic mechanism, which may lead to greater malig
nancy.
Although the evidence is still inadequate,
the
view of Klein and Klein (279, 280) that this is due to
progressive gene damage may be tentatively accepted.
One effect of such tissue gene damage might be seen at
Vol.
25, November
1965
the cell surface.
It is commonly believed that the cell
surface structure is tissue specific, with the result that the
cells of each tissue tend to hold together.
With pro
gressive malfunction
of the genes controlling
surface
structure,
a point may be reached when that structure
becomes so modified that the cells separate and metastasis
results.
One final possibility may be mentioned—namely,
that
the genie damage might involve the mitosis genies. In this
case it is probable that the cells would be unable to divide
or that they would die during attempted division, and it
is indeed one of the aims of radiotherapy
to inhibit tumor
growth in this way.
From this very brief review of the evidemice it appears
that both initiation and progression may involve sudden
amid irreversible changes in the genome ; that with increas
ing age such gene damage seems to occur at random in
most if not all tissues; that this process may be augmented
by carcinogens and supported by imiherited gene weak
niesses ; and that if, by any route, such damage leads to
malfunction
of the mitotic amid fumictional homeostatic
mechanism, so that the balance between cell gain and cell
loss is disturbed, a tumor may result.
3. The chalone complex and the latent period.—Between
the process of initiation amid the appearance of a tumor
there is the latent period, which can be shortened by the
application of a promoting agenit.. This process of promo
tion is usually neither sudden nor irreversible, and it is
importarTit to question whether the speed at which it pro
ceeds may be related to the state of the mitotic homeo
static mechanism, not only in the damaged cells but also
in the surrounding
normal tissue.
It has already been
noted that the latent period may be shortened either by
hyperplasia (220, 242) or by a delay in the maturation of
the dormant tumor cells (36), and it has also been em
phasized that in normal tissues any weakness of the
chalone complex leads both to increased mitotic activity
and to delayed maturity.
The length of the latent period is evidently determined
by chance, and in most epithelia there is the added possi
bilit.y that the damaged cells may be shed. Since, if the
ammimallives long enough, all those latent tumor cells that
are not shed may produce tumors, the difference between
an increased chance and a decreased chance should lie
mainly in an earlier or a later average time of appearance
of the tumors.
The question to be answered, therefore,
is whether with an inefficient chalone complex, as after
tissue damage or adrenalectomy,
the latent period will be
shortened, while with an efficient chalone complex, as
during stress, the latent period will be lengthened.
4. Promoting agents.—Totake first the question of
tissue damage, there is an old hypothesis that, given suffi
cient time, irritation or ulceration or scarring automati
cally leads through hyperplasia to cancer.
Although this
theory of the origin of cancer is inadequate today (242), it
does remain possible that an increased mitotic rate may
increase the chance of a genetic mistake and tend through
chalone lack to favor the multiplication of any tumor cells
that may be so formed.
There is certainly strong cvi
dence that epidermal woumiding or irritation will promote
the earlier appearance of epidermal tumors in carcinogen
BuLL0uGH—Mitotic
and Functional Homeostasis
treated skin (175, 176, 327, 397, 398, 416, 417, 450).
Similarly it has been reported that regeneration following
partial hepatectomy
promotes the earlier appearance of
tumors in carcinogen-treated
liver (194, 303, 304) and that
primary hepatic carcinoma occurs with greater frequency
in cirrhotic livers.
However, both wound repair amid re
generation
are normally short-lived episodes, amid it is
therefore not surprising that a stronger promoting action
can be obtained by repeated tissue damage (397, 398).
A 2nd way in which mitotic homeostatic mechanisms
may be damaged is by adrenalectomy,
and because of the
increased mitotic rate this may also be expected to act
in the manner of a promoting agent.
Unfortunately
most
investigations have failed to distinguish between the proc
esses of initiation and promotiomi, or have related solely to
the growth of established tumors, which may be adversely
affected by the general metabolic disturbance (32, 33, 409,
469). Recently,
however, Trainin
(488) has made a
careful analysis of the effects of adrenalectomy
amid has
shown that, although it has no effect on initiation, it
strongly enhances promotion.
This is in agreement with
Law (302), who found that adrenalectomy
increases the
incidence of spontaneous
lymphoid leukemia, amid with
Metcalf (349), who found that high leukemia AKR mice
are characterized
by low productiomi of glucocorticoid
hormone.
The 3rd type of promoting agemit includes irritant chemi
cals, such as croton oil, which are often considered to act
through cell damage and consequent hyperplasia.
How
ever, the theory that hyperplasia is the essence of promo
tion has been critically discussed by Bercnblum (35, 36),
who has suggested that promotion must depend on a
dislocation of the normal balance between cell gain and
cell loss and that a promoting agent may exert its action
more by preventing cell maturation
than by promoting
mitosis.
On present evidence it is difficult to distinguish
the relative importance
of these 2 processes, amid it is
possible that in different circumstamiccs either or both may
have the same consequence, which is the build-up of a
group of damaged cells sufficiently large for the central
‘u'eato be freed from chalone control.
After successful promotion the tumor cells are com
monly able to continue
their multiplication
without
further assistance.
It follows that the act of promotion
must either have altered the nature of the tumor cells mm
the direction of autonomy or have enabled them to realize
characteristics
they already possessed.
As they emerge
from their dormancy the tumor cells may give the impres
sion either that they are lacking sufficient chalone or that
they are no longer able to respond to whatever chalone is
present.
If the 2nd alternative is correct then the cells
should be unable to respond to induced changes in either
the chalone content or the adrenalin content of the tissue.
In fact, however, it is evident that newly formed tumors
are commonly able to respond to such changes.
Thus
Trotter (491) has shown that in the liver partial hepatec
tomy results in a greatly increased mitotic rate in small
adenomatous nodules (see also Refs. 185, 320), and further
she has pointed out that as malignancy
increases this
power of reaction is lost. In a similar manner certain
types of tumors continue to show a diurnal mitotic
1713
rhythm (317—19,505), while others that are perhaps more
malignant show no diurnal rhythm at all (135, 197).
It may therefore be concluded that both the dormant
tumor cells and the cells of benign tumors may miormally be
capable of responding to the chalonie mechanism.
Indeed,
evcmi after a tumor has appeared, it may still possess such a
relationiship between cell gain amid cell loss that it may
reach a new Poimit of balance, arid, at least for a time, it
may remain mi the state of “indolent persistence― de
scribed by Foulds (171). Commonly, however, a pro
gressive loss of functional maturity may lead to a progres
sive reduction in the chalone concemitration amid so to an
increased mitotic rate, which is dependent
both on a
progressive shortening of the cell cycle length (410) amid a
progressive increase in the proportion of cells involved in
mitosis.
In addition, those cells which do manage to ma
ture should die more quickly, amid at the theoretical cx
treme, growth would be explosive.
An approximation
to this extreme may be seen in many ascitcs tunmors, in
which the cell cycle may be reduced to as little as 11.5 hr
(132) and in which rio mature cells arc present. Un
fortunately,
relatively little precise imiformation is avail
able on the mitotic rates or on the durations of the cell
cycles of tumor cells as compared with those of their tissues
of origin (see Refs. 132, 344).
It seems probable that at some point during progres
sion the cells may lose their ability to react to army re
mainiing chalonic amid that from therm onwards they are
fully autonomous.
It has been found, for imistance, that
malignant leukemic granulocytes may continue to produce
a chalone-like substance capable of inhibiting mitosis in
normal granulocytic progemmitor cells8 (423) but that they
themselves are unable to respond to it (123, 124). With
respect to those few tumors that are already malignant
when they first appear, it may be assumed that their loss
of ability to responid to the normal homeostatic mechanism
occurred in 1 large step during either initiation or dor
mancy.
In either case promotion would be unnecessary
and growth would be rapid.
5. Retarding agents.—If a weakened mitotic arid func
tional homeostatic
mechamiism is armessential feature of
promotion then a strengthened mechanism may prolong the
period of dormancy of the tumor cells. It is apparent
that in those tissues, such as liver amid kidney, which have
a naturally low mitotic rate, amid presumably therefore a
naturally powerful mitotic control mechanism, the muta
tion rate may be high (127) but the chances of promotion
are so low that the cancer incidence is also low ; innthose
tissues, such as epidermis, which have a weaker mitotic
control mechanism, the chances of promotiomn arc much
greater; while in those tissues, such as the basal cells of
the duodenal crypts, which apparently possess a very weak
mitotic control mechanism, the high chances of promotion
may be powerfully offset by the high rate of cell loss.
Curtis (127) has noted that, while a mitotically inert tissue
spontaneous
mutations
build up to a high level, iii a
mitotically active tissue they remain rare.
The simplest way to strengthen the homeostatic mitotic
mechanisms of the tissues is by means of stress, which
leads to a higher rate of secretion of adrenalin amid gluco
corticoid hormones.
If the chalone niechamiism in the
1714
Cancer Research
damaged cells is even partly active, this should retard the
multiplication
of the latent tumor cells and delay the
growth of bemmigmi
tumors.
Direct studies of the effects of
stress are few, but it has already been shown that a longer
latent period is found in mice stressed by high tempera
tures (179, 506, 507), low temperatures
(513), or excessive
noise (354), amid recently Anderson (14) has concluded
that “apprehension [gives@ some protection against car
cinogenesis.― Stressful conditions have also been shown
to delay the growth of existing tumors, which may there
fore have been relatively benign (289, 332, 407, 419).
However, the most extensive amid important work on
the effects of stress on carcinogenesis is that of Tannen
baum and Silverstomie (475—78)and of Rusch et al. (418,
420), although none of this work was originally considered
mitermsofstress. Tanmnmenbaum
wasthefirsttoshowthat
with a wide variety of latent tumor cells, both spontaneous
arid induced, promotion is delayed amid even prevented by
a restricted diet, but determined attempts to explain this
effect in terms of the diet itself have failed.
It has been
noted, however, that such treatment results in an increase
in the size of the adrenal gland (49, 432, 478) and in an
inicrea.se in the secretion rate of both adrenalin (196) and
the glucocorticoid hormones (182).
A survey of the large literature on the effects of re
stricted diets amid carcinogemiesis has been made by White
(521). From this it is clear that riot only may promotion
be prevented but the growth of what arc l)rObably rela
tively benign tumors may be delayed (see also Ref. 248).
One important observation, made by Pearson (382), shows
that when undcrfecding
retards the growth of an im
planted tumor, the cells may show signs of reacquiring
some degree of functional maturity, and this is, of course,
exactly what would be expected with a strengthened
chalonme complex if the process of progression towards
malignancy had not gone too far. It is also clear that
when progression has gone too far a restricted diet has no
effect (521).
If this retardation
is indeed due to a strengthened
chalone complex then a similar action should be exercised
by adremialin or the glucocorticoid hormones.
The effect
of adrenalin does riot seem to have been tested, but the
effect of the glucocorticoid hormones is well known.
In
particular, the careful work of Trainin (488) has clearly
shown that, while hydrocortisone
has no effect on initia
tion, it strikingly imihibits promotion.
The cortisone in
hibition of spontaneous leukemia (267, 499, 529), of in
duced skin tumors (28, 152, 187), and of induced skin and
lung tumors (188) is probably due in each case to a similar
inhibition of the process of promotion.
Contrary cvi
dence that cortisone acts to increase the yield of induced
skin tumors (456) may be due to changed hair follicle
activity (444).
The effect of glucocorticoid hormones on the growth of
existing tumors differs in different cases, but a reduced
growth rate has been described in a lymphosarcoma
(234,
463), in lymphoid leukemia (181, 244, 274), amid in various
other tumors (465). However, such glucocorticoid-sensi
tive tumors commonly progress to a state of glucocorticoid
resistance
(18, 297, 298). This change has the stable
character of a mutation, amid it presumably denotes pro
Vol. 25, November
1965
gression towards greater malignancy.
There are several
known cortisone-resistant
types of tumor, but it is in
teresting that their cells may still bind glucocorticoid
hormones in a normal manner (98) . This may possibly
suggest that the chalonc mechanism remains and that the
cells have lost their power to react to it.
Such evidence tends to confirm the conclusion that both
latent tumor cells and relatively benign tumors may nor
mally be subject in some degree to the mitotic homeostatic
mechanism of their tissue of origin amid consequently that
anything that strengthens this mechanism may delay the
appearance and retard the early growth of a tumor.
The
only known retarding agemnts are glucocorticoid hormones,
and it is obviously important to discover whether adrenalin
and the various chalones are similarly active.
Regarding
chalones it has been noted that when excess blood, pre
sumably containing the granulocytic
chalone, is intro
duced by transfusion, chronic mycloid leukemia may be
suppressed (520) while acute leukemia is unaffected (123).
Such unresponsive malignant cells may fail either to re
spond to a normal chalone concentration
or to produce
significant amounts of chalone: the 1st alternative may
be illustrated
by malignant lcukemic granulocytes
that
continue to exert a chalone-like inhibition of mitosis in
granulocyte progcmiitor cells,6 and the 2nd by other malig
nant leukemic granulocytes,
which are no longer able to
inhibit lymphocyte mitosis (see Rcfs. 122—24).
6. Conclusions.—Thc theory that the processes of mi
tiation and progression may commonly be due to gene
damage, acquired or inherited, leads to the pessimistic
view of the cancer problem described, for instance, by
Rous (415) and Burch (93). In Burnet's words (94),
“Everything suggests that, somatic cells being what they
are, the impact of the environment must inexorably lead
to an accumulation
of mutant cells, some of which will
have malignant descendants if life persists long enough.―
However, it is already clear that the average time of
tumor appearance may be considerably delayed by retard
ing agents.
The agents so far known are those adrenal
hormones that are produced in response to stress and that
act to strengthen
the mitotic homeostatic
mechanism.
Their potential power has been strikingly illustrated in
the experiments of Tannenbaum
(473, 474), in which the
latent tumor cells have remained suppressed throughout
the entire lifetime of the animal.
These experiments,
which hold out the hope of large-scale cancer prevention,
may yet prove to be some of the most important in the
history of cancer research.
It is also evident that re
tarding agents that act by strengthening
the homeostatic
mechanism may delay the growth of visible tumors, at
least as long as these remain in the earlier stages of pro
gression.
In the converse situation, the promoting agents, which
advance the time of tumor appearance, may all act by
weakening the homeostatic mechanism with effects that
are evident in terms of mitotic rate, cell maturation,
or
cell life expectancy.
One natural factor that may act in
this way is increasing age. It has been noted in a number
of species, including man, that from middle age onwards
there is commonly a progressive degeneration
of the
adrenal cortex (130, 140, 268—70), and it has also been
BuLL0uGH—Mitotic and Functional Homeostasis
reported in both mice and men that middle age may be a
time of increased mitotic activity (67, 487). However,
the effect of middle age on adrenal function @i@not yet
elear, and the situation is further confused by the differing
reactions of individuals and of strains within 1 species
(486). Little is known of changes in the rate of adrenalin
secretion, although in men Kärki (275) has comicluded that
“theadrenal medulla maintains its activity
@pto a high
age,―while in mice Bullough arid Laurence2 have indirect
evidence indicating that the adrenalin output may be
much reduced.
In the case of the glucocorticoid
hor
mones the evidence is also inconclusive.
On the one
hand, it is generally agreed that mmman the rate of cx
cretion of the hormone derivatives declines progressively
during middle age (224, 278, 519), but on the other, it
appears clear that this does not reflect any significant.
change in the plasma hormone level (246, 519). It up
pears that a reduced adrenal secretion rate may be offset
by an impaired capacity to metabolize arid excrete the
glucocorticoid hormones and possibly also to utilize them.
Although this is a matter that needs further investigation,
it remaimis possible that in middle age adremial deficiency,
perhaps combined with a quieter amid more settled life,
may provide a more favorable environment for the multi
plicationi of latent tumor cells.
Other factors that may affect the situation innparticular
tissues are the mitogemmic hormones and the poietinms. If
the theory that one function of a mitogenic hormone is to
neutralize the chalone of its target tissue is correct therm
the resulting mitotic stimulus may be more powerfully felt
in any latent tumor cells that are producing less than the
normal amount of chalonc or reacting less adequately to
whatever chalonme is present.
The problem of hormone
dependemit tumors amid of the progressive changes that
occur in them has recently been discussed by Foulds (170,
171). While a tumor remains responsive to a hormone, it
may “growso long as an appropriate
(hormone) stimulus
is maintained, regress when it is withdrawmi arid recur, at
the same place and with the same characters, when it. is
restored (170).― This is, of course, exactly what would
be expected of armygroup of cells which arc omilymoderately
damaged amid which maintain just sufficient control to
limit their owmi growth.
The hormone would theni act
rather in the mariner of a promoting agent, increasing
mitotic activity and reducing maturation,
but when the
hormomie is withdrawn these processes would be reversed
amid the tumor might even regress com@)letcly. When,
however, progressive chamiges towards greater malignancy
occur, sonic of the cells might become so free from the
normal mitotic control that they no longer would riced the
hormone stimulus and ultimately might even lose all their
ability to react to the hormone.
By contrast, it seems improbable
that the poictinis,
which may not affect the mitotic rate, play any significant
part in the cancer i)rOblcm. Theoretically
a breakdown
in mitotic amid functional homeostasis may occur in the
progenitor
cells of the crythrocytic,
granulocytic,
arid
even the plasma cell systems, but these cells arc riot af
fected by the poietimms. Alternatively
the breakdowmi
could occur in the relatively undifferentiated
steni cells,
1715
amidin this case, too, the presence or absence of the poietins
would be unlikely to exert any significant effect.
V. GENERAL
SUMMARY
CONCLUSIONS
AND
The problem of mitotic horneostasis, with which this
review began, is now seen to constitute only a part of the
far larger 1)roblem of tissue homeostasis, which may be
analyzed most profitably in terms of the control of genie
expression.
The programs of syntheses that lead to the
creation and maimitenmance of a tissue are evidently de
termined by a sequence of genie actions that are in turn
determined
by a sequence of humoral controls.
There
are at least. 3 main levels of these controls : the basic in
duction mechanisms by which the tissues are formed, the
chalone mechammisms by which they are maintained inn a
functional state, arid in some cases the hormone mecha
nisms by which tissue function may be modified.
A.
THE
ROLE
OF INDUCrNG
SUBSTANCES
There is a general belief that tissue induction is achieved
through the initeractioni of an inducing sub.stanice with
cells that arc conipetenit to react, and that the process
involves the activation of omie region of the genonic arid
the imiactivatiomi of most of the other regions.
1mmmum
mals this inactivation may be pernianienit, arid innthe case
of most tissues it. is completed in the embryo.
However,
it is also evident that in the adult mammal, with which
this review is primarily concerned, (@crt.aingroups of stern
cells, which retain at least part of their emiibryologic
coniipetenice, are always present.
These stem cells appear to belomig to 2 related groups,
the 1st PerhaPs retaining only the dual potcnitiality of
induction
by poictinis to form either graniulocytes or
erythrocyt.cs, amid the 2nd PerhaPs retaining the multiple
potentiality of induction by antigens to form a variety of
plasma cells. It is interesting that mieit.her the granulo
cytic nor the erythrocytic
cell PoPulations are regarded
as self-mnainmtainiing in the manner of normal tissues since
their rnit.otic potentialities
are inisufficicmmtto meet their
high rates of cell death.
Thus innboth cases maintenmance
of tissue mass depends on continued induction of the stem
cells. 1mmthese unusual circunistanices it is Possible that.
the tissue chalone mechanisms may be relatively poorly
developed, but it is nevertheless clear that sonic consider
able degree of chalomic control does exist arid that it. ninny
also inifluemice the stem cell population.
The organization of the lymphocyte-pla.srmia cell system
is less well kniowmm. It. is l)rObablc that. it may contain the
most complex systeni of interrelated
tissues in the body,
and that the details of its mitotic arid functional homeo
static mechaniisms may be equally coniplex.
However,
it. would be sur@)risinmgif these mechanisms proved to in
volve anything more thani variations on the 3 niaini types
of homeostatic control.
It has been rioted that. the graniulocytic, crythrocytic,
and plasma cell systems are all tissues that must be able to
meet sudden amid urgent denianids for large niunibers of
extra cells. It. has been emphasized that such sudden
tissue augmcnitationm is riot readily achieved through inn
creased niitotic activity amid that. recruitment
by iniduc
1716
Cancer Research
tioni from the large stem cell populatiomis is a more efficient
method.
B. THE ROLE OF THE CHALONES
It is evident that the creation of a tissue does not
guarantee its continued functional efficiency and that at
all times most mammalian
tissues must be supported
against the tendency of their cells to revert to a stable
program of recurrent mitosis.
It appears that, with in
ductionm complete, the tissue cells normally retain at most
only 3 groups of active or potentially active genes: the
1st concerned with the synthesis of general metabolic
enzymes, the 2nd with tissue-specific function, and the
3rd with synthesis for mitosis.
Together these groups of
genes constitute
the facultative
genome, although it is
possible that the 1st group may sometimes be nonfunc
tional and that the synthesis of the general metabolic
enzymes may be directed at the ribosome level. The
2nd amid 3rd groups are never fully functional simul
taneously, and it now appears that the choice between
tissue function and mitosis may be decided in terms of the
intracellular
concentration
of the tissue-specific chalone.
Thus the main task of the chalone systems of the body
may be to maintain the whole functional organization of
the animal against collapse into cellular anarchy.
It is also evident that mitotic and functional homeo
stasis is not a static affair; it responds to changing cir
cumstances amid is subject to various checks and counter
balances.
The
horneostatic
mechanism
continually
maintains the tissue mass in correct relation to the body
mass and reacts appropriately
in conditions of tissue
damage, whether local or general.
Once the tissue mass
is correctly related to the body mass, the situation is
stabilized by the counterbalance
provided by the inverse
ratio between the mitotic rate and the life expectancy of
the functional cells. Because of this, the point of balance
between cell gain and cell loss is riot easily disturbed and
tissue mass tends to remain constant.
When the mitotic
rate is greatly imncrea.sed, for instance by chalone lack
through persistent cell damage, the tissue mass increases
only moderately ; when the mitotic rate is reduced towards
zero, the increased length of life of the functional cells
is such as to prevent army change in the tissue mass. It
is miot yet clear how the length of life of the functional
cells may be imifluenced in this way. However, since
nothing seems to affect the life expectancy of the non
nucleated erythrocytes,
it is possible that the time of cell
death may be controlled at the gene level, possibly through
the imntermediary of the lysosomes.
The homeostatic
mechanism is also responsive to the
effects of stress.
Adrenalin (but riot noradremialin) and
the glucocorticoid hormones strengthen the action of the
chalonies, and this, by reducing the rate of cell gain amid
cell loss, may have sonic survival value, especially perhaps
during starvation.
C. THE ROLEOF THEHORMONES
Froni time to time, in certain mammalian
tissues,
mitotic arid functional activity must be modified to meet
particular needs, amid it is evident. that this must involve
some initerferenice in the chalone mechanism.
In the
Vol. 25, November
1965
case of the hair growth rhythm such interference may be
dependent on genes that oscillate spontaneously
between
activity and inactivity, but usually it seems to depend on
the actions of diffusible substances that are synthesiaod
in other @ti@sucsand may perhaps all be classified as hor
mones.
Of these the best known are the various mito
genie hormones, such as the estrogens, which act to pro
mote both mitosis and tissue function, perhaps through
the neutralization
of the chalones of the target tissues.
When such tissues are unstimulated,
it is evident that
the mature cells are long-lived and nonfunctional ; when
they are stimulated by chalone neutralization,
the raised
mitotic rate is accompanied by a reduced life expectancy
of the mature cells, which become functional as they pass
towards death.
It is important to recall that such mitogenic hormones
as the estrogens are widespread throughout
the animal
and plant kingdoms in situations in which their functions
must be radically different from those they exert in an
adult mammal.
It seems indeed that certain types of
molecules, which possess a pronounced ability to interfere
in biochemical reactions, may have been incorporated
in
an opportunist
manner in the control mechanisms of a
wide variety of cellular activities.
In these cases it is
clearly the mechanisms that have evolved while the active
substances have changed little.
Although at the moment only relatively few hormones
are known to be active in mammals as part of this 3rd
order of- tissue control, it seems probable that a wide
variety of other active substances may also be involved.
These may perhaps include, for instance, promine and
retine (470), the nerve growth factor (314), and the skin
factor (119, 120), all of which have been omitted from
the present discussion because they form such a hetero
geneous group.
Indeed, it appears that this 3rd order of
tissue control may prove to be mediated through the ac
tivities of such a varied assortment
of substances that
there may be little conimomi pattern except in their ulti
mate disturbance of the chalone mechanism.
It may be emphasized that many adult hormone mecha
nisms are so organized as to regulate tissue functions
terms of seasonal or other environmental
changes.
in
D. THE PROBLEM OF GENE DAMAGE
If all levels of mammalian
tamed
through
the
activities
tissue organization
of
particular
are main
substances
which, directly or indirectly, control the expression of the
genome, then some at least of these substances may be
regarded as acting in the traditional manner of the effec
tors of the microorganisms.
In these organisms versa
tility in the face of a changing environment
is obtained
at the gene level by the variety of the possible alternative
synthetic programs that may be activated by a wide range
of effectors.
In the metazoans such versatility
is cvi
dently obtained by the variety of the restricted genomes
represented by the various tissues, amid in this situation
the part played by effectors is less obvious.
In this con
nection it is important to attempt to determine how re
stricted the geniome of a typical tissue may be. Of the
possible syntheses a cell may undertake,
it has already
been mentioned that those relating to the general meta
BuLL0uGH—Mitotic and Functional
bolic enzymes may perhaps sometimes be directed by
long-lived messenger RNA, but those relating to tissue
function and to mitosis, being effector-cont.rolled alt.erna
tives, must be directed at the gene level.
6. Alov,
(532) and in mneurones.In this situation cell replaceniemit
is impossible and molecular replacement is directed at. the
ribosome level ; the accumulation of molecular damage at
this level may cause amiincreasimig burden of malfunctionm.
nonfunction, and even cell death, but it cannot lead to the
rapid death of the animal itself through unregulated
growth by mitosis.
In most tissues, for a variety of obvious reasons, it is
necessary to replace cells rather than molecules, amid the
possibility of genie damage leading to cancer may be the
price that such tissues must pay for the retention of a
facultative
genome capable of directing synthesis for
mitosis.
It
is an indication
of the homeostatic
of the
mechanism
remarkable
efficiency
that this price is so rarely
paid in the tissues of wild animals; that it is more often
paid in the tissues of domestic animals
be directly related to their relatively
stressful lives.
amid of niani may
longer and less
ACKNOWLEDGMENTS
The ideas here put forward have emerged during a long study
of mitotic control in adult mouse tissues, a study that would
never have been possible but for the continuous and generous
financial support of Organmon Laboratories
Limited.
These ideas
have also been tested and often radically modified in the course of
innumerable discussions, especially with Dr. E. B. Laurence, I)r.
W. J. Tindall, Dr. C. L. Hewett, and Dr. L. Foulds in London,
with Dr. J. I). H. Homan, Mr. H. de Jager, and Dr. W. Hondius
Boldingh in Holland, and with Professor H. Teir and l)r. T.
Rytomaa in Helsinki.
Later, supported by a grant from the
Damon Runyon Memorial Fund for Cancer Research, New York,
long and fruitful discussions with Dr. Rytomaa were continued
in London.
Finally,
the stimulus
to consider
the possible
con
nection
between
these new biologic theories
and carcinogenesis
was strengthened by an invitation to attend the Fiftieth Aniniver
sary Meeting of the Netherlands Cancer Institute in Amsterdam.
For all this varied assistance I am deeply grateful.
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