CALIFORNIA STATE UNIVERSITY, NORTHRIDGE
IN VIVO
- CONTROL OF ACTIN POLYMERIZATION
;.
IN SARCOMA-180 ASCITES TUMOR CELLS
A thesis submitted in partial satisfaction of the
requirements for the degree of Master of Science in
Biology
by
John Gordon Monroe
Augus-t·· , 1979
The Thesis of John Gordon Monroe is approved:
Dr : Steven B. _ Oppenheim~rr
- Dr. ~dward G. Pollock, Commi~tee Chairman
California State University, Northridge
i
TABLE OF CONTENTS
ACKNOWLEDGEMENT .
ABSTRACT
iii
iv
INTRODUCTION
1
MATERIALS AND METHODS .
7
Sarccma-180 cell line
7
Actin isolation and purification .
3
Protein determination
9
Sodium dodecyl sulfate polyacrylamide
gel electrophoresis
9
Gel scans of electrophoretic gels
9
KCl concentration
RESULTS . . . ; . . . . .
10
10
Actin isolation and purification
10
Intracellular actin quantification
15
Intracellular pH of sarcoma-180 cells
17
DISCUSSION
19
BIBLIOGRAPHY
26
PLATES
30
ACKNOWLEDGEr~ENT
I would like to thank Dr. Edward G. Pollock whose assistance
and patience made this work possible.
Also, the helpful and
interesting discussions with Dr. Steven B. Oppenheimer were very
inspiring.
Additionally, I thank Dr. Nancy H. Bishop for reading
and suggestions in the writing of this thesis.
iii
ABSTRACT
IN VIVO CONTROL OF ACTIN POLYMERIZATION
IN SARCOMA-180 ASCITES TUMOR CELLS
by
John Gordon Monroe
Master of Science in Biology
June, 1979
Actin was isolated and purified from ·sarcoma-180 ascites
tumor cells by a method employing DEAE-cellulose anion exchange
chromatography, polymerization-depolymerization and exclusion
chromatography.
vitro system.
The actin was demonstrated to polymerize in an in
SDS-polyacrylamide gel electrophoresis was perfm·med
upon crude extracts of the sarcoma-180 cells and the total amount of
actir. was determined on a per cell basis.
Thirteen percent of the
total, and 12.6% of the soluble, protein of the sarcoma-180 cell was
determined to be actin.
This corresponds to an intracellular actin
concentration of 6 mg/ml.
These results are discussed in terms of
current theories of in vivo control of tumor cell actin polymerization
and microfilament assembly.
The intracellular contents of the sarcoma-180 tumor cells were
iv
demonstrated to be very acidic by measuring the change in pH of a cell
suspension bef ore and after gentle lysis of the cells.
This result,
coupled with the experimental results on other tumor cells as reported in the literature, forms the basis for an alternative mechanism
for
the~
vivo control of actin polymerization involving intracell-
ular pH.
v
INTRODUCTION
Actin is a protein most commonly considered in relationship to
muscle cells.
It is the primary component of the I-band in the con-
tractile elements of striated muscle cells.
However, beginning with
the work of Hatano and Oosawa (1966), it is now known that actin is a
major component of non-muscle cells which neither contract nor contain
myofibri .ls.
Actin has been demonstrated in virtually every non-muscle
cell type thus far investigated .
Such diverse cell types as vertebrate
brain (Kuczmarski and Rosenbaum, 1979) and algae (Palevitz, 1976) have
been shown to contain actin.
While the actin in muscle cells exists in primarily the polymerized F-actin form, the actin in non-muscle cells is known to consist
of two molecular forms; the first, a monomeric or G-actin species and
the other the F- actin species found in muscle cells (Weber et
1975).
~·,
The non-muscle F-act i ns have been shown to resemble muscle
F-actin in their ability to bind myosin and to activate myosin
..1..2
Mg' -ATPase (Korn , 1978).
Indeed, the non -muscle actins greatly
resemble muscle F-actin in respect to their migration on SDS-polyacryiamide gels and their polymerization characteristics (Korn, 1978).
In
normal non-muscle cells a significant percentage of the intracellular
actin exists in the F-actin state (F i ne and Taylor, 1976).
In this
st ate the pol ymerized actin is found to be a primary component of the
microfil ament structures cha r acterist i c of these cells (Goldman et
1
~· ,
2
1975).
Two lines of evidence support the contention that microfilaments are composed of actin, namely that they bind to heavy meromyosin
(Ishikawa et
~·,
1969; Spooner et
~·,
1971; Goldman et
~·,
1973)
and secondly, that they bind to anti-actin antibody (Lazarides and
\_
Weber, 1974).
Microfilaments have been implicated as mediators in many events
occuring within the cell or on its surface.
An example is the media-
tion of the mobility and distribution of lectin and immunoglobulin
receptor sites by microfilaments (Paste et al., 1975).
Agents which
disrupt microfilaments have been shown to inhibit capping on the
surfaces of lymphocytes (Ryan et
~-,
1975).
This inhibition of
capping is believed to support the premise that the microfilaments
serve as contractile elements to distribute protein molecules within
the
~embrane
(Paste et
~·,
1975).
The cytoplasmic streaming which
occurs in many plant cells has been shown to be controlled by microfilaments (Nagai and Rebhun, 1966).
Loss of microfilament structures
has been shown to affect the external topography of the plasma
membrane such that cells whose microfilaments have been disrupted
exibit
11
ruffles 11 on their membrane as opposed to the microvilli of
normal cells (Wang and Goldberg, 1976).
This change has been shown
to affect the agglutinability of these cells by lectins (Oppenheimer
et
~·,
1977).
~The fact that microfilaments appear to play so many diverse and
important roles in normal cells makes it especially interesting that
tumor cells contain very few microfilaments (Pollack et
~·,
1975;
3
Weber et
~·,
1975; Wang and Goldberg, 1976; Pollock, 1978).
~1uch
work has been done comparing microfilament structures in normal and
transformed cells; in all cases, the normal cells contain complex
arrays of microfilaments while the transformed cells contain few, if
any, microfilaments.
Cells which have been transformed and then have
reverted contain microfilaments in numbers and distribution comparable
to a .normal cell (Pollack et
~·, 1975;
Weber et
~·, 1975).
This
would indicate either that the lack of microfilaments is a direct
result of the transformation process or that the lack of microfilaments
may be a prelude to transformation.
The loss of microfilaments in
tumor cells has been implicated as the causative factor for many of the
characteristics of tumor cells.
For example, one of the more obvious
characteristics of malignant cells is their lack of contact inhibition
during growth.
Microfilaments have been postulated as stabilizing
agents for plasma membrane sites of cell-cell contact functioning to
mediate contact inhibition of
Taylor, 1976).
gr01~th
(Dermer et
~·,
1974; Fine and
Moreover, the loss of microfilaments, previously
mentioned, appears to result in a redistribution of membrane proteins.
As it has been shown that membrane proteins and glycoproteins are
important in cell-cell adhesion (Rutishauser et
~·,
1976),
the lack
of microfilaments in tumor cells may be important in metastasis.
That
the metastatic properties of malignant cells is dependent on cell-cell
adhesiveness has already been established (Nicolson et
~·,
1976).
This evidence suggests that microfilaments are intimately
associated with the aberrant behavior of malignant cells.
Therefore,
a study into the intracellular control mechanisms mediating their
4
disappearance in tumor cells may be an important step toward understanding carcinogenesis.
Much of the work done to date concerning
the loss of microfilaments upon transformation has been descriptive
in nature.
An understanding of the mechanisms involved must ultimate-
ly lie in elucidation of the molecular aspects of microfilament formation and degradation, specifically actin and the intracellular control
of its polymerization.
Several possibilities exist to explain the decrease in the
degree of actin polymerization (microfilaments) in tumor cells.
Because the polymerization of actin is a concentration-dependant event
(Gordon et
~·,
1977), the most obvious explanation is that the total
amount of actin in the cell decreases as the cell is transformed.
This implies that there is a critical concentration of actin above
which polymerization occurs spontaneously (Gordon et
~·,
1977).
This
hypothesis proposes that the concentration of actin in the tumor cell
falls below the critical concentration for polymerization.
Wang and Goldberg (1976) supports this idea.
Work by
They maintain that the
synthesis of actin in the tumor cells is slowed while degradation
proceeds at the normal rate.
This results in a lowered intracellular
concentration of actin relative to the untransformed cell.
Another hypothesis to explain the decreased numbers of microfilaments centers upon the state of the actin molecule.
While there
may be normal amounts of actin in the tumor cell, the actin present
may be intrinsically defective and incapable of polymerization.
This
defect could occur at the level of the genes coding for actin.
It is
possible that in the transformation process the actin genes are
altered in such a way that they produce an actin molecule incapable
of undergoing polymerization under physiological conditions.
Another
possible mechanism for producing an unpolymerizable actin molecule
may be related to a covalent modification of actin in the tumor cell
(Weber et .!]_., 1975).
One popular explanation for the altered control of tumor cell
actin polymerization is the formation of actin binding proteins.
There
may exist in the tumor cell gene products which can specifically bind
to actin monomers and prevent their polymerization (Gordon et
1976).
~·,
For example, the src gene product produced in cells trans-
formed by Rous sarcoma virus is capable of disrupting microfilament
assembly (Wang and Goldberg, 1976).
Considerable evidence exists
implicating actin binding proteins in the control of polymerization
in normal cells (Tilney, 1976; Carlsson et .!]_., 1977).
This suggests
that the actin binding proteins exist as control mechanisms in normal
cells and that the defect in tumor cells may be related to the actin
binding protein, not to the actin molecule
per~·
In addition to proteins there are other molecules and ions
which may potentially control actin polymerization and microfilament
stability. These include ATP, KCl, Mg+ 2 and Ca+ 2. Some evidence
indicates that ATP is required for actin polymerization (Nachmias,
1976).
The addition of one G-actin molecule onto the F-actin fila-
mentis accompanied by the hydrolysis of one ATP molecule to ADP +Pi.
The suggestion of a Ca+ 2 effect on actin polymerization comes from the
finding that a threshold concentration of 7.0 x 10-? M Ca+ 2 is required
by the cells.
When the Ca+ 2 concentration falls below this level,
6
microfilament stability decreases (Condeelis et
~·,
1976).
One other parameter that may be involved in intracellular control of tumor cell actin polymerization and depolymerization is that
of intracellular pH.
Many biologically important reactions are very
sensitive to small fluctuations in pH. For example, an elevation of
pH in the presence of Ca+ 2 has been shown to trigger the acrosome
reaction in sea urchin sperm (Collins and Epel, 1977).
The charge on
the R-groups of the amino acids composing a particular preteih molecule can be altered as the environment becomes more acidic or basic,
thereby affecting its specific association with other molecules.
In
the polymerization of actin a change to an unfavorable intracellular
pH upon transformation could conceivably create a situation where the
G-actin molecules could no longer interact and polymerize.
Consider-
able evidence has accumulated indicating that the pH of cancerous
tissues is more acidic than that of the corresponding normal tissue
(Kahler and Robertson, 1943; Meyer et
1953; Eden et
~·,
1955).
~·,
1948; Naeslund and Swenson,
Meyer and coworkers (1948) indicate that
this depression in pH is as much as 0.5 pH units.
While these values
do not represent the best possible measurements of intracellular pH,
they are indications of altered acid-base balance in tumor tissues
and may have some relevance to the intracellular pH (Gershon, 1978).
The actual intracellular pH of Walker-256 carcinoma cells as measured
by Schloerb et
~·
(1965) was found to be more acidic than the
corresponding normal cells.
It has been proposed that there is a
causal relationship between the onset of carcinogenesis and the
demonstrated lowering of the intracellular pH (Flaks et
~·,
1973;
Kitagawa and Kuroiwa, 1976).
The possibility that actin polymerization may be affected by
the depressed pH of tumor cells has no precedent in the literature.
However, most if not all actin isolation procedures which rely upon
actin polymerization occur in buffers of pH 7.2 or above.
Tilney
(1976) found that actin polymerized more rapidly as the pH approached
pH 8·0.
Therefore, it is conceivable that the optimum pH for poly-
merization may be in the region of pH 7·0 - 8·0 and that lower pH
ranges may affect the polymerization characteristics of the actin
molecule.
The cell system used in this investigation was the sarcoma-180
ascites tumor cells of Swiss-Webster white mice.
Previous fine struc-
tural work in this laboratory indicates sarcoma-180 cells have very
few short-segmented microfilaments distributed in some, but not all,
of the surface microvilli (figure 1).
It is clear that the role of
microfilaments in the transmembrane system (TMS) of information transfer between the cell and its environment must be evaluated against the
backgr·ot.md of the contrast of complex networks of mi crofi laments
characteristic of normal cells versus non-ubiquitous , short-segmented
microfilament arrays in tumor cells (Pollock, 1978).
In this context ,
the aim of this work is to delineate the possible mechanisms of in
vivo control of actin polymerization in sarcoma-180 tumor cells.
MATERIALS AND METHODS
Sarcoma- 180 cell line.
The sarcoma - 180 cell line was obtained
3
orignally from Dr. Melvin Cohen of the Salk Institute and from Mr.
Samuel Parley of the National Institutes of Health (1963 SKI line).
The cell line is maintained in our laboratory by intraperitoneal passage in male, Swiss-Webster white mice.
Cells were collected from the
peritoneal cavities of mice 9 days after inoculation, washed 3 times
in cold Hanks• balenced salts solution and centrifuged between each
wash for 2 minutes at 1000 RPM in a clinical centrifuge.
Packed cells
were then suspended in an equal volume of buffer, and one ml- of tnis
suspension was used as the inoculum for each new mouse.
Actin isolation and purification.
scribed above.
The cells ':Jere harvested as de-
Following the initial collection and wash in Hanks'
balenced salts solution the cells were washed twice in 10 mM imidazole
chloride, pH 7·5.
The cells were collected following each wash by
centrifugation for 5 minutes at 5,000 RPM in a Sorvall GSA rotor.
The
isolation and purification of the sarcoma-180 cell actin was according
to the method of Gordon et
~·
(1976) with the exception that the cells
were disrupted by sonication for 2 minutes in 30 second bursts with
a Sonifier (Branson Instruments Inc., Stamford, Connecticut) set at
position 8.
The progress of the sonication was checked every 30
seconds by phase microscopy.
This isolation and purification
procedure utilized initial purification by DEAE-cellulose chromatography followed by polymerization and depolymerization of the actin.
The final stage of the purification utilized gel filtration on a
Sephadex G-150 column.
9
Protein determination.
All protein determinations were made by the
method of Lowry et al. (1951) or by absorbance at 290 nm.
Sodium dodecyl sulfate
polyacrylamide~
electrophoresis.
In all
cases the discontinuous (DISC) gel electrophoresis system was used
with a 3% stacking gel at pH 6·8 and an 11% resolving gel at pH 8·8.
The samples were prepared as follows:
50A glycerol, 15A 2-mercapto-
ethanol, 150A 10% sodium dodecyl sulfate, 25A 0.1% bromphenol blue
and 65A of a buffer containing 0.5
sulfate, pH 6.8.
~
tris-HCl and 0.4% sodium dodecyl
700A of a properly diluted sample solution was added
to the above mixture and placed in a boiling water bath for 3-10
minutes to denature proteolytic enzymes.
For all experiments, the
samples were prepared as described within several hours of collection.
The samples were placed on the gels (usually 0.1 ml) and run in the
following buffer system:
0.02 M Trizma (Sigma Chemical Company, St.
Louis, Missouri), 0.20 M glycine and 0.1% sodium dodecyl sulfate,
pH 8·3.
A current of 1 rnA/gel was applied until the samples entered
the resolving gel at which time the current was maintained at 3 rnA/gel.
The finished gels were removed from the gel tubes and stained overnight
in the following staining solution:
blue in methanol:acetic acid:water (5:1:5).
0.05% coomassie brillant
Destaining was for
48-72 hours in methanol :acetic acid:water (5:7:100).
Gel scans of electrophoretic
~·
Gel scans were performed on a
Densicord model 5099 Photovolt densitometer with a 570 nm filter.
areas of the peaks were measured with a planimeter .
The
10
KCl concentration.
The KCl concentration of the DEAE-cellulose eluted
fractions was done on a conductivity meter (Leeds and Northrup, Co.,
Philadelphia, PA ) using a Whetstone bridge conductivity cell.
RESULTS
Actin isolation and purification.
Data for the number of mice used
and the quantity of sarcoma-180 cells collected for a typical isolation run are presented in Table 1.
The cells were removed from the
mice as described in the materials and methods an& : washed two times
in 250 ml of cold 10 mfi imidizole
chloride~
pH 7! 5.
Following the
final wash, the cells were suspended in 300 ml of cold buffer G,
pH 7·6.
At this stage, the cells were .ruptured by sonication.
The
sonication was monitored by phase microscopy so that the majority of
the cells were broken open but disruption of internal organelles
was minimized.
Figure 2a,b shows the results of a typical sonication.
Figure 2a is the cells suspended in buffer G prior to sonication.
Figure 2b is the same material following a total of 2 minutes of
sonication.
Virtually all cells are disrupted.
However, the lipid-
luscent lysosomes are still intact indicating that sonication under
these conditions does not disrupt all of the intracellular organelles.
At this
11
stage, an aliquot was saved for later analysis and labeled,
Cell homogenate-total cell protein. 11
The cell sonicate was then
centrifuged for 2 hours at 75,000 x g at 4°C.
Following centrifuga-
tion, the pellets were discarded and the supernatents, which contained
considerable lipid material, were pooled.
An aliquot was again
11
Table 1.
Data from a typical actin isolation indicating the
quantity of materials used.
Number of mice
Number of days following inoculation . . . . . . . . . . . . . . . . . . .
61
9
Volume of packed cells
120 ml*
Wet weight of cells
200 g
Final amount of purified actin
*Packed for 5 min. @ 5,000 RPM in a clinical centrifuge.
t Following Sephadex G-150 purification.
.J..
89.8 mg '
12
saved at this stage and labeled,
11
Cell homogenate-soluble protein.
11
These pooled supernatents were applied to a DEAE-cellulose, anion
exchange column prepared, equilibrated and eluted as described by
Gordon et
~·
(1976).
All of the homogenate was applied to the column
within 8 hours following ultracentrifugation.
were collected.
Fifty 40 ml fractions
Within 8 hours of collection, each fraction was
warmed to room temperature, adjusted to pH 7·6 and
by the addition of 0.4 ml of 0.2
~
MgC1 2.
to 2 mM MgC1 2
The samples were allowed
to remain at room temperature for 2-3 hours.
After this time, each
sample was tested for polymerized actin by observing flow birefringence through crossed polarizers.
phenomenon.
Figure 3a,b illustrates this
Figure 3a is a sample not containing polymerized actin,
while Figure 2b contains polymerized actin and exibits marked flow
birefringence.
In addition, each sample eluted from the column was
assayed for total protein and KCl concentration.
An elution profile
for the DEAE-cellulose column is presented in Figure 4.
As can be
seen, the actin eluted from the column in four fractions at approximately 0.2
~
KCl.
This point of elution was very consistant from
experiment to experiment and agrees with the results obtained by
Gordon et
~-
(1976) using the same method.
The four fractions
exibiting flow birefringence and, hence, containing polymerized actin,
were pooled and a small aliquot saved and labeled,
actin.
11
11
0EAE-cellulose
The remaining polymerized actin was pelleted by centrifu-
gation for 4.5 hours at 75,000 x g at room temperature.
The pelleted
actin was homogenized in cold buffer G and depolymerized by dialysis
against daily changes of buffer G for 3-4 days at 4°C.
Following
13
dialysis, an aliquot was saved and labeled,
actin."
11
polymerized-depolymerized
The rest of this partially purified actin was layered onto a
Sephadex G-150 gel filtration column.
The column was eluted with 500
ml of buffer G at 0-4°C and a pressure of 30 em of water.
Five ml
fractions were collected and each was assayed for protein by their
absorbance at 290 nm.
Figure 5.
A typical elution pattern is reproduced in
The large peak represents the fractions containing the
purified actin.
During the isolation and purification steps, five samples were
saved and later assayed for total protein by the method of Lowry et
(1951) and analyzed by 50S-polyacrylamide gel electrophoresis.
electrophoretic gels are pictured in Figure 6.
~·
The
In addition, photo-
metric gel scans were made for each gel and the results of these are
presented in Figures 7-11.
The amount of actin relative to the total
amount of protein in the sample was determined
us~ng
these gel scans.
The percentage of the protein in the sample that was actin was derived
by dividing the area of the actin peak by the total area of all the
peaks and multiplying by 100%.
These results are presented in the
first two columns of Table 2.
The third column gives the amount of
actin present in the sample as calculated from the percent actin in
the sample and the total amount of protein in the same sample.
Additionally, these figures indicate that, 89.8 mg of 93.8% pure actin
may be isolated from the cells collected from 61 mice.
The last two columns in Table 2 list data regarding the
recovery of the actin throughout the purification steps.
percent recovery illustrates the amount of actin
l~ecovered
The relative
at a
Table 2.
Actin isolation and purification data from a typical isolation experiment.
Fraction
Actin *
Protein
(mg)
%
Cell homogenate, total protein
% Recoveryt
(relative)
% Recovery§
(absolute)
(mg)
12,075
13.0 1570
Cell homogenate, soluble protein
5,951
12.6 746.9
47.6
100
DEAE-cellulose
396.4
28.3
112.8
15.1
15.1
Polymerized-depolymerized
176.2
56.8
94.9
84.2
12.7
95 . 7
93.8
89.8
94.6
12.0
Sephadex G-150
*Represents the protein determined by gel electrophoresis to be migrating at a molecular weight
corresponding to actin.
-rRecovery relative to the first preceding purification step.
§Recovery relative to the amount of actin present in the cell homogenate, total protein.
f-'
~
15
particular stage relative to the stage preceding it.
This value
allows one to assess the relative merits of a particular stage in the
purification regimen in terms of the concurrent increase in purity of
the actin.
The fifth column, absolute percentage recovery, illustrates
the amount of actin recovered at each step relative to the orignal
amount present in the cell homogenate-soluble protein.
Intracellular actin quantification.
Whereas one explanation account-
ing for the lack of polymerized actin in tumor cells is an insufficient quantity of actin in the cell, it was necessary to quantify
the intracellular actin of the sarcoma-180 cells.
This was done in
terms of the amount of actin present relative to the total protein,
soluble (intracellular) protein and the actual intracellular concentration of actin in the sarcoma-180 cells.
The percent of total and soluble protein contributed
by actin is presented in Table 2.
As can be seen, actin comprises
13.0% of the total cellular protein and 12.6% of the soluble cellular
protein.
The amount of actin relative to the total protein of the
sarcoma-180
Table 3.
tumor cell compared to normal cells is presented in
In all cases, with the exception of Acanthamoeba castellanii,
the percent actin within sarcoma-180 cells is markedly higher than
that of norma 1 ce 11 s.
Once determining that the amount of actin in the intracellular
contents was 12.6%, another actin isolation was performed on a known
number of cells in order to determine the amount of actin per cell in
the sarcoma-180 cells.
The data for this experiment are presented in
Table 3.
Comparison of the percentage actin in sarcoma-180 cells with that of other, normal cell types
from the literature.
Cell type
Sarcoma-180
Rat liver
NIH-3T3
%Actin
Reference
13.2*
1.5
5-10
Gordon et
~·
, 1977
Weber et al., 1974; Fine and
TaylOr-, 1976
Chick brain
7.8
Gordon et
Chick embryo fibroblast
8.5
Anderson, 1978; Bray and Thomas,
1975
Acanthamoeba castellanii
15.0
Gordon et
~·
~·
, 1977
, 1976
*Mean value from 6 determinations derived from the data from 3 separate experiments.
1-'
0'>
17
Table 4.
The mean value for the amount of actin per sarcoma-180 cell
is 3.4 x 10- 8 mg/cell. Inasmuch as the average diameter of these cells
is 22 ~m, the average volume is 5.6 x 10- 9 cc. Using this value as
the intracellular free volume (the actual volume is probably much
smaller) the amount of actin per cell corresponds to an intracellular
actin concentration of 6.2 mg/ml.
This value indicates that 6.2 mg/ml
of actin is available within the cell for polymerization.
Intracellular pH of sarcoma-180 cells.
Intracellular pH has been
suggested as a possible regulatory mechanism in actin polymerization,
and evidence that some tumor cells have significantly lower intracellular pH than normal cells has been cited (see p. 6).
Therefore,
the following experiment was performed in order to obtain data concerning the intracellular pH of sarcoma-180 cells. A 52 ml suspension
containing 1.4 x 108 cells/ml in saline (0.85% NaCl) was sonicated
until the cells had just broken open.
The initial pH of the sus-
pension prior to sonication was determined to be pH 7·3.
pH immediately following sonication was pH 6·75.
The final
This indicates that
lysis of the cells released enough H+ ions to lower the pH of 52 ml
of solution 0.55 pH units, resulting in a final pH of less than 7·0.
This finding supports the contention that the intracellular pH may
indeed be acidic.
More definitive experiments are now in progress
in an effort to determine the precise value of the intracellular pH
of these ce 11 s .
18
Table 4.
Data from the experiment designed to determine the
intracellular actin concentration within sarcoma-180 cells.
Number of cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.6 x 108
Amount of soluble protein ..................... 98.4 mg
Percentage actin in soluble protein ........... 12.6 %*
Amount of actin/cell ..........................
3.4 x 18- 8 mg
Vol ume of sarcoma- 180 ce 11 ................... .
5.6
Concentration of actin within
sarcoma-180 cell ...........................
6.2 mg/ml
X
10 -9 cct
*Mean value from 6 determinations derived from the data of 3
separate runs.
t Assumes the cells are spherical.
19
DISCUSSION
Microfilaments, as major components of the transmembrane
system, are important mediators in many cellular events.
Tumor
cells, as opposed to normal cells, contai.n very few microfilaments
(vJeber et .!}_., 1975; Pollack et .!}_., 1975; Wang and Goldberg, 1976;
Pollock, 1978).
This non-ubiquitous distribution of microfilaments
is believed to be related to aberrant behavior characteristic of
tumor cells such as loss of contact inhibition of growth, increased
lectin agglutinability and a microvillus, pleomorphic surface
(Dermer et .!}_., 1974; Fine and Taylor, 1976; Oppenheimer et .!}_.,
1977; Pollock, 1978).
Additionally, because microfilaments are
important units of the transmembrane system (TMS), their loss in
tumor cells could significantly affect the interaction of the cell
with its external environment (Pollock, 1978).
The facts that micro-
filaments are composed of polymerized actin and that tumor cells
contain actin in an unpolymerized form (Weber et .!}_., 1974; Pollack
et al., 1975; Fine and Taylor, 1976), suggest a malfunctioning of
a control mechanism in the tumor cell which prevents actin polymerization.
The most obvious possible explanation for the lack of polymerized actin in tumor cells would be related to the amount of actin
present in the cell such that the amount to actin in the tumor cell
might be but a fraction of that in a normal cell.
This difference
20
would account for a concurrent drop in the number of microfilaments
in these cells.
To the contrary, the results of this investigation
indicate that, on a percentage basis, the sarcoma-180 tumor cells
contain as much or more actin than normal, microfilament-containing
cells.
Because actin polymerization is an equilibrium reaction that
is dependant upon the concentration of G-actin (Gordon et
~·,
1977),
the intracellular concentration of G-actin for the sarcoma-180 cells
was calculated and found to be 6.2 mg/ml.
Gordon et
~·
(1977)
determined that the critical concentration of actin necessary for
spontaneous polymerization in non-muscle cells was 0.05 mg/ml.
Therefore, the intracellular concentration of actin in the sarcoma-180
tumor cell is well over 100 times the minimum concentration necessary
for polymerization.
However, these values were derived from electrophoretic gel
scans of cell homogenates and it is possible that the actin electrophoretic band contains other proteins in significant amounts which
co-migrate with the actin by virtue of identical molecular weights.
Given this possibility, the determination of the amount of actin present could be significantly above the actual value.
Nonetheless,
using the data from the actin purification procedure, 12% of the actin
(or possibly proteins with the same molecular weight) in the whole
cell homogenate was recovered as polymerizable (93.8% pure) actin at
the end of the purification procedure .
Assuming this amount of actin
alone comprises the intracellular pool of actin, the intracellular
concentration of actin in the sarcoma-180 cell would still be 10-fold
greater than the va 1ue derived by Gordon et a1. ( 1977) for the
21
critical concentration for non-muscle cell actin polymerization.
It
does appear that the amount of actin in the sarcoma-180 tumor cell is
sufficiently high so as to preclude actin concentration as a negative
factor in polymerization.
Another possible explanation for the lack of actin polymerization in the sarcoma-180 cell concerns the state of the actin
molecule.
Even if significant amounts of actin were present in the
tumor cell, the actin molecule itself might be incapable of polymerization.
Results reported here do not support this idea however.
Both the DEAE-cellulose and the Sephadex G-150 purified actin samples
were shown to polymerize in 2 mM Mg +2 and ATP at pH 7·6 and room
temperature.
There is no reason to suspect that the sarcoma-180
derived actin molecule is capable
incapable
of~
of~
vitro polymerization but is
vivo polymerization.
Also, the popular contention that actin polymerization in
tumor cells is modulated by protein inhibitors does not appear to
apply to the sarcoma-180 tumor cell system.
Evidence for this comes
from the work of Carraway and Moore (1978).
They demonstrated that
cytoplasmic extracts of sarcoma-180 cells exhibit gelation when brought
to room temperature.
The first step in this gelation is known to be
actin polymerization followed by a complex reaction involving other
contractile proteins (Kane, 1976).
Since this polymerization occurs
in a crude extract, any intracellular protein inhibitors, if they
existed, would still be present and, therefore, would be available
to prevent polymerization.
Indeed, the fact that polymerization
occurred indicates that protein inhibitors capable of inhibiting
22
polymerization probably do not exist in the sarcoma-180 cell.
Various non-protein molecules normally found in cells have been
demonstrated to affect the polymerization of actin and/or microfilament stability. The most important of these are ATP, Ca+ 2 , and
Mg+ 2.
The work of Hatano et ~· (1967) demonstrated that actin poly-
merization in Physarum is accompanied by the hydrolysis of one ATP
to ADP + Pi for each G-actin molecule added to the polymerizing actin
chain.
This suggests that insufficient levels of ATP in the tumor cell
could account for the lack of actin polymerization, hence producing
a decrease of microfilaments in tumor cells.
This result could be a
function of a total decrease in ATP concentration within the cell or
it could be related to an altered compartmentalization of ATP in the
tumor cell.
valid.
Once again, this explanation does not appear to be
As a rule, tumor cells are very actively metabolizing cells.
For this reason alone one would assume large quantities of ATP are
being produced.
This argument does not rule out the possibility
that ATP may be very highly compartmentalized so that little, if any,
ATP is free for actin polymerization .
This possibility cannot be
excluded by the data presented here, but it seems unlikely in view of
the multiple needs for ATP throughout the cell.
Additionally, the
work of Nachmias (1976) indicates further that ATP is not a good
candidate as a control molecule for actin polymerization.
He showed
that it is not ATP alone that is important but merely the presence
of a nucleotide to bind to G-actin monomers.
Ca+ 2 concentration has been implicated also as affecting
actin polymerization as well as the stability of actin in the
23
polymerized state (Condeelis et~., 1976). Low concentrations of
Ca+ 2 appear to lower the stability of microfilaments. However,
other work indicates that aggregation of polymerized actin into filament bundles in inhibited by Ca+ 2 (Spudich and Cooke, 1975). Also,
it has been demonstrated that polymerization of actin does occur in
+2
~vitro systems in the absence or in the presence of Ca
(Hatano
et
1967; Spudich and Watt, 1971; Yang and Perdue, 1972; Spudich
+2
and Cooke, 1975; Gordon et ~·, 1976). Therefore, the role of Ca
~.,
in the polymerization of actin and microfilament assembly is unclear.
It seems clear however, that actin is capable of polymerization
+2 .
whet her or no t Ca 1s present.
While Mg+ 2 has been shown to affect the polymerization of
actin, its effect does not seem to be important ~nder physiological
conditions. Mg+ 2 removes the temperature dependence for actin polymerization (Kane, 1976). Addition of 2 mM Mg+ 2 allows the actin to
spontaneously polymerize at lower temperatures.
However, this is not
an important limiting factor at the 37°C temperature of the mice nor
at the high actin concentrations characteristic of sarcoma-180 cells.
Each of the aforementioned proposals to account for a decrease
in polymerized actin in tumor cells appears inconsistent with
experimental data reported here for
the sarcoma-180 tumor cell system.
Consequently, an alternative proposal for the control of actin polymerization in tumor cells based upon pH is proposed.
intracellular pH of tumor cells (Schloerb et
~·,
The lowered
1965; Gershon, 1978)
results in a chemical environment unfavorable for actin polymerization
and, therefore, also for microfilament assembly.
The possibility of
24
a pH regulatory mechanism in this system is strongly supported by
the fact that small changes in pH can drastically affect the function
of proteins such as hemoglobin and many enzymes.
Results of this work indicate that the intracellular environment of the sarcoma-180 tumor cell is also acidic as measured on
gently lysed cells, although the technique used to measure this does
not allow for a precise quantitative value.
The major argument
against this technique is that intracellular organelles are also
disrupted in lysed cells which possibly influence the final pH value.
In this work, the cells were broken with as little disruption of the
internal organelles as is possible.
many lysosomes and mitochondria.
The sarcoma-180 cells contain
Review of Figure 2a,b indicates that
a major type of lysosome, the lipidluscent lysosome, remained intact
following sonication.
These lysosomes are not membrane bound organ-
elles and are, therefore, very delicate (Schmidt and Pollock, 1977;
Pollock, 1978).
The fact that they were not disrupted attests to the
gentleness of the sonication.
Additionally, if the mitochondria
were disrupted they would have served to increase the apparent
intracellular pH rather than further lowering it, since the intramitochondrial pH is alkaline (Addanki et
~·,
1967a,b).
Whether or not this change in pH affects the polymerization
capabilities of actin in tumor cells has not been previously investigated:
However, the fact that all actin polymerization systems
reported in the literature occur above pH 7· 0 indicates a need to
further explore the possibility of a pH requirement in this event.
Additiona ·lly, the acrosome reaction , which has been shown to involve
25
actin polymerization (Tilney and Flicker, 1978) in sea urchin sperm
has been shown to be triggered by an elevation in pH in the presence
of Ca+ 2 (Collins and Epel, 1977).
Accurate measurements of the intracellular pH of the sarcoma-180
cell is in progress.
If the intracellular pH is a major factor in
the inhibition of actin polymerization, given the importance of microfilaments in carcinogenic behavior, attempts to modify the intracellular pH of tumor cells may have important clinical potential in terms
of the prophylactic control of cancer.
26
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- -- -- -
-
-
-
30
Plate I
Figure 1.
Sarcoma-180 ascites tumor cell indicating the paucity
of microfilament structures (arrows).
of Dr. E.G. Pollock.
Magnification X37,000.
Courtesy
-
------.-
••
32
Plate II
Figure 2.
a) Sarcoma-180 cells prior to sonication.
b) The same sarcoma-180 cells following sonication
for approximately 2 minutes in 30 second bursts.
Note the presence of intact lipidluscent lysosomes
(arrows).
34
Plate III
Figure 3.
a) DEAE- cellulose fraction 11ith 2 mt!_ MgC1 2 , adjusted to
pH 7·6 and brought to room temperature. The picture
was taken through crossed polarizers and indicates no
flow birefringence hence, does not contain polymerized
actin.
b) Another DEAE-cellulose fraction under the identical
conditions described above.
Note the marked flow
birefringence patterns indicating this fraction
contains polymerized actin.
36
Plate IV
Figure 4.
Typical elution pattern from the DEAE-cellulose anion
exchange column.
The crude homogenate was applied to a previously
equilibrated column and initially eluted with 50 ml of buffer G,
pH 7·6 followed by 200 ml of 0.1
~
KCl in buffer D, pH 7·6.
At this
point, the column was eluted with 2 liters of a 0.1 M to 0.5 M
KCl gradient in buffer D, pH 7·6.
40 ml fractions were collected
and assayed for total protein by the Lowry method (Lowry et
~-,
and for KCl concentration based upon solution conductivity.
The
1951)
shaded region depicts those fractions which upon addition of 2 mM
MgC1 2 exhibited flow birefringence, and hence contained polymerized
actin.
...
0
0
KCI Concentration
(Molar)
0
0
0
0
0
0
c-.
C")
~
.,0
...
0
0
0
0
~
{JWj6w)
UO!JDJjUii»lUO) U!8j0Jd
38
Plate V
Figure 5.
Typical elution from the Sephadex G-150 column.
The column
was eluted with buffer G, pH 7·6 and 5 ml fractions were collected.
Each fraction was monitored for protein content by the absorbance
at 290 nm.
In this case, fractions 21-30 were pooled and shown to
contain the purified actin.
07 -
0.6 '""
0.5
~
0.4
~
·0.3
1-
f
E
c
0
oN
~
0.2 •
0.1
~
I
10
)
20
Fraction
I
30
40
Plate VI
Figure 6.
Electrophoretic gels of the samples taken at various
stages in the isolation and purification procedure.
bands were stained in coomassie brillant blue.
1) cell homogenate-total cell protein
2) cell homogenate-soluble protein
3) DEAE-cellulose purified acti n
4) polymerized-depolymerized actin
5) Sephadex G-150 purified actin
The protein
; "'::·
1
2
3
4
5
42
Plate VII
Figure 7.
Densitometer scan of the sarcoma-180 sample; cell
homogenate-total cell protein.
The sample was resolved on 11% sodium
dodecyl sulfate polyacrylamide gels by electrophoresis.
The resulting
protein bands were stained with coomassie brillant blue and scanned
at 570 nm .
The actin peak is the prominent peak at 0.625.
•
CIO
c)
c)
wu OLS sqy
N
c)
44
Plate VIII
Figure 8.
Densitometer scan of the sarcoma-180 sample; cell
homogenate-soluble protein.
The sample was resolved on 11% sodium
dodecyl sulfate polyacrylamide gels by electrophoresis.
The
resulting protein bands were stained with coomassie brillant blue
and
scanned at 570 nm.
0.625.
The actin peak is the prominent peak at
Ql
>
·.;:
0
""i
~
N
0
wu OLS
sqy
46
Plate IX
Figure 9.
Densitometer scan of the sarcoma-180 sample; DEAE-cellulose
purified actin.
The sample was resolved on 11% sodium dodecyl
sulfate polyacrylamide gels by electrophoresis.
The resulting
protein bands were stained with coomassie brillant blue and
scanned at 570 nm.
0.625.
The actin peak is the prominent peak at
Ql
>
·.;
..2
Ql
a.:
CIO
0
wu OLS sqy
Plate X
Figure 10.
Densitometer scan of the sarcoma-180 sample; polymerized-
depolymerized actin.
The sample was resolved on 11% sodium dodecyl
sulfate polyacrylamide gels by electrophoresis.
The resulting
protein bands were stained with coomassie brillant blue and
scanned at 570 nm.
The actin peak is the prominent peak at 0.625.
.~
..a
0
"'0
~
Cll
·~
...!!
Cll
1111:
wu OLS
sqv
50
Plate XI
Figure 11.
Densitometer scan of the sarcoma-180 sample; Sephadex
G-150 purified actin.
The sample was resolved on 11% sodium dodecyl
sulfate polyacrylamide gels by electrophoresis.
The resulting
protein bands were stained with coomassie brillant blue and
scanned at 570 nm.
The actin peak is the prominent peak at 0.625.
co
0
wu OLS sqy