Studies on Citrobacter rodentium-enhanced
Colonic Tumorigenesis in Mice
by
Zhifeng Zhang
B.S. in Biochemistry
Peking University, 1988
Submitted to the Division of Toxicology
in Partial Fulfillment of the Requirements for the Degree of
DOCTOR OF PHILOSOPHY
in Toxicology
at the
MASSACHUSETTS INSTITUTE OF TECHNOLOGY
September 1996
01996 Zhifeng Zhang. All Rights Reserved
The author hereby grantspermission to MIT to reproduce and to distributepublicly
paperand electronic copies of this thesis document in whole or in part.
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Signature of Author................††.
Division of Toxicology. September. 1996
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C ertified by.................. ..... ......................................................................................
Dr. David B. Schauer. Thesis Advisor
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Dr. Peter C. Dedon. Chairman.
7, ;>C•;ikhifi•ittee on Graduate Students. Division of Toxicology
A ccepted by...................V.........................................
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SEP 1 21996
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This doctoral thesis has been examined by a committee of the Division of
Toxicology as follows:
Professor James Fox
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Chairman
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Professor David Schauer
Thesis Supervisor
Professor Gerald Wogan
Professor Tyler acks
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STUDIES ON CITROBACTER RODENTIUM-ENHANCED
CGLONIC TUMORIGENESIS IN MICE
by
Zhifeng Zhang
Submitted to the Division of Toxicology in September, 1996 in partial fulfillment of the
requirements for the degree of Doctor of Philosophy in Toxicology
ABSTRACT
Citrobacterrodentium, a murine pathogen that causes transmissible colonic hyperplasia.
has been suggested to promote colonic tumorigenesis in DMH-treated mice. To further
investigate the cocarcinogenesis event, CB6FI mice were infected with C. rodentium and
treated with azoxymethane (AOM). A two-fold increase in colonic tumor burden was
observed in infected mice, compared to that of uninfected controls. To study the
mechanism of tumor promoting effect of C. rodentium, CB6FI Min mice were infected
with this pathogen. A significant increase in colonic tumor incidence was observed
compared to uninfected CB6FI Min mice. These observations confirm that C. rodentium
infection enhances colonic tumorigenesis. Furthermore, to analyze the genetic basis of
colonic tumorigenesis. some of these tumors were subjected to K-ras 12th codon mutation
detection and genome-wide loss of heterozygosity (LOH) screening. Approximately 20%
of large adenomas from AOM-treated animals. including AOM-treated Min mice, were
found to carry K-ras mutations. These mutations were mainly G to A transitions, which is
consistent with the theory that AOM causes mutation by inducing 0 6 -methylguanine. In
contrast. no K-ras mutations were found in tumors harvested earlier from AOM-treated
mice or in spontaneous tumors from Min mice, regardless of the status of the C.
rodentium infection. These findings suggest that K-ras mutations were associated with
AOM treatment, but they are not required in the early stage of tumorigenesis. LOH
screening revealed that all tumors from AOM-treated animals exhibited a very low
frequency of random allelic loss, whereas nearly 100% tumors from Min or infected Min
mice lost the APC+ allele. Two other LOH hot-spots were identified in non-AOM-treated
Min mice. C. rodentium infection did not change the K-ras mutational frequency or the
LOH patterns. These data support an epigenetic basis of tumor enhancement by C.
rodentiunm infection, and suggest that elevated cell proliferation increases cancer risk.
Thesis Advisor: David B. Schauer
Professor in Division of Toxicology and Division of Comparative Medicine
Massachusetts Institute of Technology
ACKNOWLEDGMENT
First of all, I am grateful to my mentors, Dr. David Schauer, and Dr. Gerald
Wogan. I give special thanks to David for always providing me with practical advice on
designing experiments, and for insightful discussions of experimental data. I thank Dr.
Wogan for teaching me the scientific attitude and being my mental support.
I would like to specially thank my committee members. Dr. James Fox, and Dr.
Tyler Jacks for their helpful and invaluable comments and advice.
I would like to thank the current and the past members of Wogan lab. especially
colleagues in the basement of building 26. for their friendship and support. I have spent
many' years in the company of Rony Gal and John Zhuang, and I am grateful to their help
and companion.
I would like to thank the current and past members of Schauer Lab. Sharda. Brian.
Elaine. Lori. Steve. Sam. Vince. Isabel. and of course, all the UROP students. for their
friendship and support. I am indebted to Brian Zabel. Elaine Chin. and Laura Trudel. who
suffered themselves in helping me revise the introduction part of the thesis.
For the passing two years I received continuous supports from people in Division
of Comparative Medicine. I would like to thank all members in DCM for their
unconditional help and friendly smiles.
Finally. I thank my wife Elaina and my parents, for all of their love and support.
and my son Arno. who has made my thesis writing more enjoyable. I dedicate this work
to them. for the love they gave and sacrifice they made to make all these possible.
TABLE OF CONTENTS
Title Page ................................................................
...............
..........................
Com mittee Page..............................................................................................
2
A bstract ........................................................... ................................................. 3
Acknow ledgm ent..............................................
.............................................. 4
Table of C ontents ................................................................................................
5
List of Sym bols and Abbreviations......................................................................... 8
List of Tables..................................................................................................
L ist of F igures...
9
.............................................................................................. 10
Chapter 1. INTRODUCTION
COLORECTAL CANCER IN HUMAN................................................... I1
A genetic basis for cancer
Colorectal cancer: a multi-step pathway
A genetic model of colorectal tumorigenesis
Environmental factors in colorectal tumorigenesis
CHEMICAL INDUCTION OF COLONIC TUMORS.............................
29
Colon specific carcinogens in rodents: DMH and its metabolites
Mutagenic effects of DMH family of carcinogens
Histopathologic changes after carcinogen administration
Genetics affecting susceptibility to colonic carcinogenesis
Epithelial cell turnover and colonic carcinogenesis
CITROBA CTER RODENTIUM AND TMCH...................................
37
The Bacteria
The Disease
Cocarcinogenesis of TMCH
Bacteria and cancer
MIN MICE: A MODEL FOR COLORECTAL CANCER RESEARCH..... 46
INTRODUCTION TO THE SPECIFIC AIMS..................................
49
Chapter 2. CITROBACTER INFECTION ENHANCES COLONIC
TUM ORIGENESIS IN M ICE.............................................................................. 54
ABSTRACT .............................................................................................
55
INTRODUCTION...........................................................................
56
MATERIALS AND METHODS...................................
............ 57
Animals
Tumor induction
DNA isolation
K-ras 12th codon mutation detection
LOH analysis by PCR-SSLP
Statistical analysis
RE SU L TS.................................................................................................. 6 1
Enhancement of colon tumor development by infection
Detection of K-ras 12th codon mutations
LOH analysis
DISCUSSION ........................................................
................................. 63
Chapter 3. CITROBACTER INFECTION AFFECTS COLONIC TUMOR
IN CIDENCE IN M IN M ICE.................................................................................
71
ABSTRA CT ...................................................................................
72
INTRODUCTION...........................................................................
73
MATERIALS AND METHODS...................................
............ 76
Mice
C. rodentium infection and AOM treatment
Genotyping for Min mice
Necropsy and tumor harvest
Small intestine neoplastic index (SINI) evaluation
DNA extraction
K-ras 12th codon mutation detection
LOH screening by PCR-SSLP
RESULTS........................................................
80
Tumor incidence
SINI and Tumor histology
Tumor distributions
K-ras 12th codon mutation detection
LOH screening
DISCUSSION ........................................................
................................. 83
Chapter 4. ON THE ROUTE TO UNDERSTANDING THE TUMOR
ENHANCING EFFECTS OF C. RODENTIUM.............................
..........
93
IN TROD U CTION ................................................................................... 94
MATERIALS AND METHODS...................................
............ 95
Animal experiments
Histological Evaluation
BrdU Immunocytochemistry
Labeling index measurement
iNOS and F4/80 immunohistochemical staining
Statistical analysis
RESU LTS........................................................................................
98
Genetic difference in symptoms after infection
C. rodentium increase LI in all infected mice
Min mutation and AOM-treatment do not change LI
Macrophages infiltrate after infection but no iNOS observed
D ISC U SSIO N .........................................................................................
100
Chapter 5. SUM MAR Y ........................................................................................ 106
References...............................................
............................... 118
LIST OF SYMBOLS AND ABBREVIATIONS
ACF (aberrant crypt foci)
AEEC (attaching and effacing E. coli)
AOM (azoxymethane)
APC (adenomatous polyposis coli gene)
B6 (C57BL/6)
C. r. (Citrobacter rodentium)
DCC (deleted in colorectal cancer)
DMH (1,2-dimethylhydrazine)
ENU (ethylnitrosourea)
FAP (familial adenomatous polyposis)
GALT (gut-associated lymphoid tissue)
GI tract (gastrointestinal tract)
GS (Gardner syndrome)
H. h. (Helicobacterhepaticus)
HNPCC (hereditary non-polyposis colorectal cancer)
iNOS (inducible form of nitric oxide synthase)
LB (Luria-Bertani broth)
IBD (inflammatory bowel diseases)
LFS (Li- Fraumeni syndrome)
LOH (loss of heterozygosity)
MAM (methylaxoxymethanol)
MAMA (methylazoxymethanolacetate)
MCC (mutated in colorectal cancer)
Min (multiple intestinal neoplasia)
MNM- (methylnitrosourea)
NO (nitric oxide)
PAGE (polyacrylamide gel electrophoresis)
PBS (phosphate-buffered saline)
PCR (polymerase chain reaction)
Rb (retinoblastoma gene)
SINI (small intestine neoplastic index)
SSLP (simple sequence length polymorphism)
TMCH (transmissible murine colonic hyperplasia)
LIST OF TABLES
Table 2-1
Experimental study design
Table 2-2
Citrobaclerrodentium infection increases tumor burden
but not tumor size
Table 2-3
87
Low incidence of K-ras 12'1-codon mutations correlates
with AOM treatment
Table 3-3
68
C. rodentium infection increase tumor incidence
in female CB6FI Mine mice
Table 3-2
67
K-ras 12'h codon mutations and loss of heterozygosity
in colonic tumors
Table 3-1
66
88
AOM affects LOH pattern in tumors from female
CB6FI Min mice
89
Table 3-4
List of 43 SSR markers screened for LOH
90
Table 4-1
Experimental Overview
102
Table4-2
Labeling indexes in experimental animals
103
LIST OF FIGURES
Fig. 1-1
A genetic model for colorectal tumorigenesis
Fig. 1-2
Proposed hypothetical scheme for the role of NO
27
and other reactive species in the multistage
carcinogenesis process, triggered by chronic infection
and inflammation.
28
Fig. 1-3
Metabolic pathway for DMH family of carcinogens
36
Fig. 1-4
A transmission electron micrograph of attaching and
effacing lesion caused by C. rodenltium
Fig. 2-1
C rodentium infection significantly increases
tumor numbers per mouse
Fig. 2-2
45
69
Histopathologic changes in mice infected with
C. rodentium and treated with azomethane
70
Fig. 3-1
Different tumor distribution in AOM-treated Min mice
91
Fig. 3-2
LOH in SSR marker Dl8mitl20 (APC)
92
Fig. 4-1
Cytokinetical changes after C. rodentium infection
in a C3H/HeJ mouse
Fig. 4-2
104
Macrophages infiltrate after C. rodenlium infection
in an AC3FI mouse
105
Chapter One
INTRODUCTION
COLORECTAL CANCER IN HUMAN
A genetic basis for cancer
Cancer is a result of uncontrolled growth. A normal
living organism maintains and perpetuates itself through highly organized growth and
differentiation. A tumor cell, although originally derived from a normal cell, has escaped
from the forces that govern normal cell growth. It grows and divides at the expense of the
organism. until the organism collapses. The question what makes a normal cell progress
to a neoplastic one continues to baffle many cancer researchers.
Historical observations favor a genetic basis for cancer. Early in this century.
studies have shown that some cancers might be clustered in families (reviewed in (Hansen
and Cavenee. 1987)). These observations suggested an inherited basis for cancer.
Perhaps the first persuasive evidence was the observations by Rous in 1911 (Rous, 1911).
that a cell-free filtrate from a chicken sarcoma could induce sarcomas in other chickens.
Sixty years later the oncogenicity of the Rous sarcoma virus has been attributed to v-src. a
viral transduced cellular gene (Martin, 1970; Stehlin el al., 1976). Direct. non-viral
evidence fobr the genetic basis of cancer came from reports that DNA from human bladder
carcinomas caused the transformation of the NIH3T3 line of mouse embryo cells
(Krontiris and Cooper, 1981; Shih et al., 1981). It turned out that the transforming gene
was a codon 12 mutant of Ha-ras gene, an activated proto-oncogene (Der et al., 1982;
Parada et al., 1982; Santos et al., 1982). Today, some 50 different cellular oncogenes
have been identified. Most of them can be classified into growth factors or receptor
homologs, cytoplasmic oncoproteins or nuclear oncoproteins, which promote growth and
cell cycle deregulation with resultant tumor development (Weinberg, 1989). Nevertheless,
oncogene is only part of the story. It can not completely explain the inheritance property
of some cancers.
In 1971. Knudson proposed a tumor suppressor gene concept (Knudson, 1971)
based on studies of childhood retinoblastomas. He postulated that patients with early
presenting tumors inherited one defective copy of the retinoblastoma susceptibility
gene. This hypothesis was later proven by identifying the Rb gene, which was inactivated
in the inherited form of retinoblastoma (Cavenee et al., 1983; Friend el al., 1986; Lee el
al.. 1987). Since then other tumor suppressor genes, namely p53. APC (and MCC, DCC).
?7l].
p l6 etc. have also been identified. When these genes were involved in tumor
development. very often specific LOH (loss of heterozygosity) was observed, indicating
allelic losses. These allelic losses occur by a variety of mechanisms including
chromosomal nondisjunction, mitotic recombination. or gene conversion (Weinberg, 1989:
Weinberg. 1991). Regardless of the mechanism. the allelic losses serve to unmask the
prior mutation. resulting in the outgrowth of cells which have completely lost the tumor
suppressing function. Allelic losses involving particular chromosomal regions have been
observed in many different human tumor types (Weinberg, 1991). However. even to date.
only a small subset of the observed specific LOH can be explained by allelic loss of known
tumor suppressor genes.
A number of distinctions now are recognized between oncogenes and tumor
suppressor genes. The mutations that activate proto-oncogenes to oncogenes may reside
in the coding sequence of a gene, producing proteins with altered function, or in some
cases are found in the regulatory portion of a gene. leading to the overproduction of a
normal protein. In both cases a gain of function results. often leading to a continuous
signal or an abnormal signal for cell proliferation or growth. These types of mutations are
dominant, and there is no further selection pressure on this gene to accumulate other
mutations in the same locus. In contrast, the tumor suppressor gene products act in some
way to stop cell proliferation or growth, even when abnormal signals for cell division are
presented to the cell cycle regulatory machinery. For example, in cells after irradiation,
the level of p53 protein increased (Maltzman and Czyzyk, 1984) and cells remained in GI
phase (Kuerbitz et al., 1992). suggesting that p53 protein detected DNA damage and
triggered cell cycle arrest. In any case, the tumor suppressors become negative regulators
of cell proliferation or progression through the cell cycle. Thus the loss of one allele via
mutation is expected to have little impact on function of the second. wild-type allele.
These mutations are loss of function mutations and act in a recessive manner to the wildtype allele. In this case both alleles of a tumor suppressor gene must be mutated to
inactivate the tumor-suppressing function. As previously stated. the most common way
for this to happen is via a mutation in one allele. then followed by a reduction to
homozvgosity (LOH) and lost of the wild-type second allele (Weinberg. 1994).
With the rise of molecular genetics, it became widely accepted that carcinogenesis
results from mutagenesis. Therefore the genetic basis of cancer was quickly substantiated.
Nevertheless, an alternative hypothesis for cancer does exist. It argues that neoplasia may
be the result of epigenetic mechanisms rather than from DNA mutations, and these
mutations are possibly the by-products of cancers, which may arise from lack of
prevention or repair (Prehn, 1994; Rubin, 1994). It is proposed that epigenetic
mechanisms produce neoplastic phenotypes by producing persistent abnormalities in the
patterns of gene expression. This hypothesis is mainly based on results from cultured cell
transformations (Rubin, 1992; Rubin and Xu, 1989) or chemical carcinogenesis studies
(Faber and Rubin. 1991). However, the experimental observations can not support the
epigenetic hypothesis nor damage the genetic hypothesis, because the experimental models
themselves are poorly understood. Cultured cells are somewhat different from cells in
vivo, and properties and mechanisms for many chemical carcinogens are still unclear.
Most of the questions doubting the genetic hypothesis can be explained directly or
indirectly without shaking the foundation of the genetic hypothesis of cancer, especially
with the improved understanding of angiogenesis and apoptosis in tumorigenesis.
Furthermore, the epigenetic hypothesis cannot perfectly explain the inherited property of
some cancers and the specific mutations or LOH in many tumor types. Thus. it seems
reasonable to say that epigenetic factors play important roles in tumorigenesis through
modifying the phenotypic expression of genetic materials.
In summary, it is generally accepted that tumorigenesis is a multistage process
involving accumulation of genetic changes producing altered functions of two classes of
genes. proto-oncogenes and tumor suppressor genes (Vogelstein and Kinzler, 1993;
Weinberg. 1994). The formation and the tumorigenecity of these genetic alterations are
modified by environmental, sometimes epigenetic factors such as diet, cell proliferation.
chronic inflammation and bacterial infection. A relatively well characterized model for the
multi-step properties of cancer, colorectal cancer, will be discussed in detail in the
following section.
Colorectal cancer: a multi-step pathway
Colorectal Cancer is currently the
second most common cause of cancer death in the US (ACS, 1995). Unlike many other
types of human cancers, colorectal cancer can be relatively easily studied through
colonoscopy and colectomy. In terms of epidemiological significance, colorectal tumors
are limited to hyperplastic polyps, adenomas, and adenocarcinomas. Other types of benign
and malignant tumors of the large intestine comprise less than 5% of the tumor population
(Fenoglio-Preiser el al., 1990). Colonic hyperplastic polyps consist of a localized zone of
proliferation of intestinal mucosa associated with exaggerated cellular differentiation and
maturation. Colonic adenomas are benign glandular neoplasms originating from intestinal
mucosal epithelium. characterized by incomplete cell differentiation and by unrestricted
cell division. Adenocarcinomas of the large intestine are malignant tumors arising from
the mucosal glandular epithelium. Abundant evidence from clinical and histopathological
studies demonstrates that the majority of colorectal cancers arise from preexisting
adenomas over a long period of time. In fact, very often a continuous histological
spectrum with increasing degrees of atypia and progressive invasion can be seen in an
adenoma containing a carcinoma (Fenoglio and Pascal. 1982; Morson, 1968). This is
often referred to as an adenoma-to-carcinoma sequence (Morson. 1974: Muto el al..
1975). On the other hand. there appears to be little evidence to support an evolutionary
relationship between hyperplastic and adenomatous polyps, thus hyperplastic polyps
should not be included in the polyps-cancer sequence (Fenoglio-Preiser et al.. 1990; Lane
et al.. 1971). However. mixed hyperplastic adenomatous polyps have been observed
(Fenoglio and Pascal, 1982), and hyperplastic areas in adenomas have been reported
(Goldman et al., 1970). leading some investigators to hypothesize that some adenomas
may arise from preexisting hyperplastic polyps.
Studies on colorectal cancer support the hypothesis that cancer progress through a
multi-step process. A small benign adenomatous polyp may arise preferentially from.
instead of normal mucosa, the hyperproliferative mucosa or abnormal tissue architecture.
such as an aberrant crypt focus (ACF) (Lipkin, 1988; Tudek et al., 1989). These abnormal
crypts can be identified by increased size, thicker epithelial lining, and increased pericryptal
zone after staining the colon with methylene blue (Tudek et al., 1989). Subsequent
progression of a small adenoma to a large adenoma with increased malignant potential
may occur in some cases. Finally, a fraction of larger adenomas may progress to invasive
and metastatic cancer (Vogelstein and Kinzler, 1993). If 1000 epithelial polyps could be
randomly selected. 900 of them would be hyperplastic polyps, while 100 would be
adenomas. Of those adenomas, 90 would be small and fully benign, 10 would be larger
adenomas with malignant potential. Of the 10 large adenomas. only one would harbor a
carcinoma (Fenoglio-Preiser et al., 1990). This type of pyramid structure, which can be
observed in both sporadic and inherited colonic polyps. supports the hypothesis of a multistep pathway for colorectal cancer in humans.
A genetic model of colorectal tumorigenesis
Specific molecular events
underlying the initiation of colorectal tumorigenesis are poorly understood. However.
studies from human and animal models suggest that colorectal adenomas and carcinomas
arise after somatic mutational events. In fact, even very small benign tumors from either
sporadic or familial origin are clonal (Fearon et al., 1987). Similar results were found in
early dysplastic lesions and small adenomas in mice treated with AOM (Ponder and
Wilkinson. 1986). Thus, observations from both human and murine models are consistent
with the hypothesis that a somatic mutational event causes one or a small number of cells
from within a single intestinal crypt to initiate the neoplastic process by clonal expansion
(Nowell. 1976). Recently, extensive genetic alterations including both tumor suppressor
genes and oncogenes. e.g.. abnormalities in APC, MCC, DCC,p53, ras, MSH2, MLH1.
have been reported. Accumulation of these genetic alterations, instead of the sequence of
these mutations, is thought to be most important in the development of colorectal tumor
(Hamilton, 1992).
Familial adenomatous polyposis (FAP) is an autosomal dominant syndrome
estimated to affect nearly 1 in 10,000 individuals in the U.S. (Bussey, 1975). Affected
individuals develop hundreds to thousands of adenomatous polyps in their colon and
rectum by age 40. A small fraction of these adenomas may progress to cancer. A related
variant to FAP. Gardner syndrome (GS). produces a similar autosomal dominant
phenotype. The gene responsible for FAP and GS was mapped to chromosome 5q21
(Bodmer et al.. 1987) and recently was identified as the APC gene (adenomatous
polyposis coli gene) (Kinzler et al., 1991; Nishisho et al.. 1991). To date. all inherited
mutations identified in the APC gene appear to result in inactivation of the gene. including
gross deletions in some cases, and more often localized mutations that cause null
mutations. or frameshifts. or missense mutations (Miyoshi et al.. 1992). Recently a few
mouse mutant models that carry a mutated murine APC gene homolog were found to
exhibit a dominantly inherited predisposition to the multiple intestinal neoplasia (Min)
phenotype (Fodde et al.. 1994; Oshima el al., 1995; Su et al., 1992). Thus, present data
provide compelling evidence that germ-line inactivation of the APC gene predisposes to
colorectal neoplasm in human and mice, and argue that the APC gene is a tumor
suppressor gene. Moreover, studies demonstrated that inactivation of both APC alleles is
necessary for adenoma development (Ichii et al., 1992; Levy et al., 1994), which is
consistent with the recessive nature of a tumor suppressor gene.
The importance of APC gene in colorectal cancer research is not limited to FAP or
hereditary colorectal cancers. As much as 35-60% of tumors from patients with no
familial predisposition to colorectal cancer display allelic losses involving chromosome
5q21 (Ashton-Rickardt et al., 1989; Solomon et al., 1987; Vogelstein et al., 1988).
Chromosome 5q allelic losses are the most frequently detected genetic alteration in small,
early adenomas from patients without polyposis (Powell et al., 1992), whereas LOH of 5q
can only rarely be noted in adenomas from patients with polyposis (Vogelstein et al.,
1988). This suggests that in the majority of colorectal tumors, one or both alleles of the
APC gene may be inactivated by somatic mutation, and this inactivation may occur at an
early stage of tumorigenesis. Considering the existence of small deletion and pointmutations that are not detectable by allelic analysis, the actual mutation frequency could be
much higher. Moreover, a second gene at chromosome 5q21. the MCC (mutated in
colorectal cancers) gene, has also been implicated in the development of some sporadic
colorectal cancers by somatic mutations that appear to inactivate the gene in about 15% of
cases (Kinzler et al., 1991 ). Pulse-field gel electrophoresis of constitutional DNA from
two FAP patients revealed cytogenetically invisible deletions that exclude the MCC from
within the region containing the FAP predisposing mutation (Joslyn et al., 1991).
However, little is known about the function of the APC or MCC protein. It is speculated
that APC protein is a multifunctional microtubule-associated protein and may somehow
regulate cellular growth of colonocytes (Bhattacharya and Boman, 1995; Munemitsu et
al.. 1995; Munemitsu et al., 1995).
Studies from in vitro and animal models have implicated that ras genes are critical
to tumor development in a number of tumor types (reviewed in (Bos, 1989)), including
colon cancer (Shirasawa et al., 1993). Mutations in ras genes can be identified in about
50% of colorectal carcinomas and adenomas larger than 1 cm in diameter (Forrester et al.,
1987; Gallinger et al., 1995; Shaw et al., 1991; Vogelstein et al., 1988). Adenomas of
this size are thought to have an increased malignant potential (Muto el al., 1975). In
smaller adenomas. ras mutations have been identified in fewer than 10% of the cases
(Vogelstein et al.. 1988). Studies of adenomas with various grades of dysplasia have also
found that ras mutations are more frequently detected in tumors with increased dysplasia
regardless of their polyposis or non-polyposis origin (Miyaki et al., 1990; Ranaldi et al..
1995). These data favor the hypothesis that K-ras mutation are involved in intermediate
to late stage in colorectal carcinogenesis. However, studies from chemically induced
colon cancer models in rats suggested that K-ras mutations appeared early in the
neoplastic lesions (Stopera and Bird, 1992; Stopera et al., 1992). Pretlow et al. observed
that mutations in codon 12 of K-ras gene alone were present in 73% of the ACF they
obtained from grossly normal colonic mucosa of colon cancer patients (Pretlow et al..
1993). If we accept ACF as the precursor lesion for colorectal cancer, these data would
suggest, more confusingly, an early appearance of the K-ras mutation in colonic
tumorigenesis. The 12th codon mutations of K-ras gene account for about 70 to 80% of
total ras mutations detected in colorectal cancer (Fearon, 1993). Currently, there exists
two possible hypotheses for the role of observed ras mutations in colorectal cancer
development. Mutations in the ras genes may be an early initiating event in the
development of a small fraction of adenomas. but these adenomas possess increased
potentials to progress to larger adenomas or adenocarcinomas than do those without ras
mutations. Alternatively, ras mutations may contribute at a later stage when small
adenomas progress to larger ones with elevated malignant potential. Further studies are
necessary to understand the roles of ras oncogene mutations in colorectal tumor
development.
Allelic losses or LOH in chromosome 17p and 18q are frequently observed in
colorectal cancers. Therefore it is reasonable to assume that other tumor suppressor
genes in these regions are involved (Vogelstein el al., 1988: Vogelstein et al., 1989).
LOH on chromosome 18 centering 18q21 can be detected in more that 70% of colorectal
carcinomas. in about 50% of advanced adenomas. but rarely in earlier stage adenomas. A
candidate tumor suppressor gene from this region. DCC. has been identified (Fearon el
al.. 1990). This large gene encodes a product of approximately 190KD with amino acid
sequence homology to a neural cell adhesion molecule. suggesting a role in cell-to-cell and
cell-to-matrix interaction. The gene product has consensus transmembrane domains and
an intracytoplasmic domain of unknown function (Fearon et al., 1990). Perhaps
inactivation of the DCC gene is responsible for some altered growth properties seen in
advanced colorectal tumor cells, including invasiveness, altered adhesion and increased
metastatic potential. Currently only a limited number of colorectal cancers have been
found to harbor DCC gene alterations (Kikuchi-Yanoshita et al., 1992). and knowledge
about DCC protein and its tumor suppressor property is very limited.
Allele losses in chromosome 17p were found in more than 75% of colorectal
carcinomas, but were rarely detected in adenomas at any stage (Baker et al., 1989; Shaw
et al., 1991). In a few cases. allele loss at 17p has been found to be associated with the
progression of individual tumors from adenoma to carcinoma (Kikuchi-Yanoshita el al..
1992: Vogclstein et al.. 1988). Evidence supports that p53 is one of the tumor
suppressor genes (if not the only one) at 17p that is associated with the development of
colorectal carcinomas. The p53 gene was found to be mutated in patients with LiFraumeni syndrome (LFS). which in an autosomal dominant fashion predispose affected
individuals with early onset of cancers in many organs (Malkin el al., 1990). This gene
encodes a 53 KD nuclear phosphoprotein. The wild-type protein appears to form a
homodimer that binds to specific DNA sequences and acts as a transcription factor (Fields
and Jang. 1990; Raycroft et al.. 1990). and seems to function by reacting to DNA damage.
causing cell-cycle arrest and leading to apoptosis (Canman et al.. 1994: Guillouf et al..
1995: Lane et al.. 1994: Lee and Bernstein, 1995: Lowe el al., 1993). Dimerization of the
p53 gene product may in part explain the dominant negative effect seen in some cases
when wvild-type and mutant p53 co-exist (Eliyahu et al.. 1988). However. in almost 90%
of colorectal carcinomas with LOH on 17p. a single missense point mutation could be
detected in the remaining p53 allele (Baker et al., 1990). Thus. a point mutation in one
p53 allele. coupled with the loss of the remaining wild-type allele. both occur frequently in
later stages of the progression to colorectal carcinomas. Finally, p53 mutations are not
rate-limiting for the development of early stage colorectal adenomas. since patients with
LFS are not at a greatly increased risk for colorectal cancer (Malkin el al., 1990). Also.
p53 deficient mice did not spontaneously develop tumors in the intestine (Donehower et
al.. 1992). Mutations in the other genes may be necessary before mutated p53 alleles can
exert a phenotypic effect in colonic epithelial cells.
In addition to adenomatous polyposis, hereditary non-polyposis colorectal cancer
(HNPCC or Lynch syndrome) also predisposes affected individuals to colorectal cancer,
and accounts for up to 10% of the colorectal cancers (Bodmer el al., 1994; Lynch and
Lynch, 1995; Lynch el al., 1995). This syndrome has few adenomas, and the
adenocarcinomas occur mainly in the right and transverse colon rather than the left-sided
predominance found in FAP or sporadic colorectal cancer patients. It is more difficult to
define than adenomatous polyposis because of its dramatic heterogeneity of phenotypes.
Some evidence has shown that HNPCC patients form adenomas at about the same rates as
the general population and it is hypothesized that HNPCC follows an accelerated adenoma
to carcinoma sequence (Lynch et al.. 1995). Advances in molecular genetics during the
past 3 years have led to the cloning of four HNPCC genes, namely MHS2 (Leach et al.,
1993). MLHI (Bronner et al., 1994), PMS1 and PMS2 (reviewed by (Lynch el al.. 1995;
Marra and Boland, 1995)). which all belong to a class of genes in charge of DNA
mismatch repair. Mutations in each of the four genes have been found in the germline
cells of HNPCC families. These mutations are mostly nonsense mutations that ultimately
results in the synthesis of a truncated protein, which in turn weakens the ability for DNA
mismatch repair. This inactivation of the mismatch repair function leads to RERphenotype. which is observed primarily as frameshift mutations resulting in altered length
of microsatellite sequences (microsatellite instability) (Chong el al., 1994: Jacoby et al..
1995; Mao et al.. 1994). The lack of a DNA repair system observed in HNPCC matches
the heterogeneity status of HNPCC, for the rate of mutations is so high that many genes
can be mutated. It is now possible to provide presymptomatic DNA testing followed by
genetic counseling for HNPCC gene carriers. This will open a new era of cancer
presymptomatic diagnosis and prevention.
The above reviewed genetic alterations in both oncogenes and tumor suppressor
genes structure a multistep genetic model for colorectal tumorigenesis. This is sometimes
referred as "Vogelgram" (Fig. 1-1), a summary of molecular genetic alterations during the
adenoma-adenocarcinoma sequence, based on studies by Vogelstein and colleagues of
colorectal mucosa. adenomas, and carcinomas. Mutation of the APC gene and MCC gene
on the chromosome 5q leads to abnormal epithelial proliferation and differentiation in FAP
patients. An increase in the prevalence of ras gene mutations. especially in codon 12 of
the K-ras gene on chromosome 12 p,is evident in intermediate adenomas (if not earlier).
Deletion of the DCC gene on chromosome 18q occurs more frequently in late adenomas.
Deletion and mutation of the p53 gene on chromosome 17p are associated with the
development from adenomas to carcinomas. All these molecular genetic alterations rarely
occur together in an individual tumor, and alterations in the other genes have also been
reported (reviewed in (Hamilton., 1992)). In addition. alterations in the DNA methylation
system are evident in the grossly normal colonic mucosa of patients with large bowel
neoplasia and may represent important early events(Goelz el al.. 1985; Silverman el al..
1989). DNA hypomethylation was reported to suppress intestinal neoplasia in Min mice
(Laird el al.. 1995). It is noted that accumulation of these genetic alterations, rather than
occurring in the implied order, is more important in colorectal tumorigenesis.
Environmental risk factors in colorectal tumorigenesis
The incidence of
colorectal cancer is much higher in the Western countries, than it is in African and Asian
countries (Bremner and Ackerman, 1970: Painter and Burkitt. 1971). As stated by
Burkitt. cancer of the colon was "among the rewards of the westernized lifestyle" (Burkitt.
1969). The differences in the incidence of colon cancer in different cultures are believed
to be related to diet rather than hereditary influences. except in cases of familial polyposis.
which will be discussed in detail later. Japanese who live in Hawaii exhibit similar
morbidity and mortality rates for colon cancer to those of Hawaiian Caucasians. which are
much higher than those of native Japanese who live in Japan (Doll et al.. 1970; Haenszel
and Correa. 1971). It is commonly believed that populations with a high crude-fiber and
loxN dietarN fat diet have lower incidences of colonic adenomas and cancer (Hill. 1974:
Wvnder and Shigematsu. 1967). Individuals on a high fiber diet are believed to have a
more rapid transit time of the bowel contents. thus reducing the opportunity for bacterial
production of carcinogens and minimizing the interaction time of carcinogens with the
intestinal mucosa. Dietary fat determines the fecal concentrations of bile acids. High fecal
concentrations of lipid metabolic intermediates and bile acids promote the proliferation of
certain anaerobic bacteria. which may influence the fecal production of carcinogens or
cocarcinogens from the bile acids (Reddy et al.. 1975). In addition. increased intake of
dietary calcium and vitamin D was shown to reduce the risk of colonic cancer (Garland et
al.. 1985).
Inflammatory bowel diseases. such as ulcerative colitis and Crohn's disease of the
colon, are also believed to increase the risk for colorectal carcinoma (Gressnstein et al..
1980: Riddell et al.. 1983). The cause of this increased incidence of colorectal cancer is
unknown but may be associated with or be the result of chronic inflammation (Levin.
1991). as chronic infection and inflammation have long been recognized as risk factors for
many human cancer types (Ohshima and Bartsch. 1994). Several hypotheses exist which
may explain the increased cancer incidence in combination, which are depicted in Fig. 12. First. active oxygen species such as superoxide anion, hydrogen peroxide and hydroxyl
radical generated in inflamed tissues can cause injury to target cells and also damage
DNA, which could result in mutations and contribute to tumor development. Second,
there is now increasing evidence to imply that NO and its derivatives produced by
activated phagocytes may also play a role in the tumorigenesis (Ohshima and Bartsch,
1994). Endogenous NO is produced by activated inducible form of NO-synthase (iNOS).
NO has a double-edged role in specialized cells and tissues (Beckman, 1991). On one
hand. it is an essential physiological signaling molecule. mediating various cell functions.
such as a neurotransmitter in the brain (Bredt et al.. 1990) and a dilatation / relaxation
regulator in the GI tract (Desai el al., 1991). On the other hand, NO is a potentially toxic
molecule with free radical properties and may induce cytotoxic and mutagenic effects
when present in excess (Beckman et al., 1990). Indeed, elevated mutation frequency was
observed in some bacteria and cell culture systems, including base deamination and DNA
breaks (Liu and Hotchkiss, 1995; Maragos el al., 1993; Nguyen el al., 1992; Wink et al..
1991). Lastly, elevated cell proliferation during inflammation, perhaps caused by
proinflammatory cytokines and/or growth factors. may also contribute to the dynamics of
tumorigenesis (Cohen and Ellwein, 1990; Deschner, 1988). The truth about the
mechanism of the increased cancer risk in IBD patients may lie in the combination of all
above hypotheses.
Normal Epithelium
I
SI 4
APC gene mutation
Proliferative Changes
And Micro-adenoma
I .4Cho
sme5 Ls
Chromosome 5a Loss
Early Adenoma
I
4
K-ras Mutation?
Intermediate
Adenoma
I•
I
Chromosome 18q Loss
.
IA
·
·
(And DUU mutation'?)
Late Adenoma
I
Ir~
-- i-,*
~a a
-
-
po.3Mutation ana
Chromosome 17p Loss
Carcinoma
•.'
.
J
U
J....
-
.
.
.
.
I.·..
Metastasis
Fig.1-1. A genetic model for colorectal tumorigenesis.
Fig. 1-2. Proposed hypothetical scheme for the role of NO and other reactive species in
the multistage carcinogenesis process, triggered by chronic infection and inflammation.
28
CHEMICAL INDUCTION OF COLON TUMORS
To better understand the pathogenesis of colorectal cancer in humans, appropriate
animal models are necessary. Unfortunately, colon tumors rarely occur spontaneously in
experimental animals. Thus, since the 1960s, chemical-induced colonic tumor models
were the only ones available. It was not until 1990 that the Min mouse was established
and characterized. The Min mice, which are described in detail later in this chapter,
possess a dominant phenotype that predisposes carriers of this phenotype to development
of numerous spontaneous polyps throughout the intestine (Moser et al.. 1990).
Colon specific carcinogens in rodents: DMH and its metabolites
One compound that has high carcinogenic activity for the colon and rectal tissue is
cycasin. which is extracted from the cycad plant (Hoffmann and Morgan. 1984). The
cycad plant. once a dominant plant form that covered the surface of the continents, is now
found exclusively in warm areas of the earth. The cycad nut is still eaten by some poor
people in various tropical areas. and certain cycad plants are eaten by cattle. resulting in a
disease known as the "wobbles". In 1964 cycasin (methylazoxymethanol-beta-Dglucoside. see Fig. 1-3 for structure), isolated from the cycad nut, was fed to rats in a
chronic toxicity test. The compound was found to induce tumors in the liver, kidney. and
more frequently. in the colon (Laqueur, 1965; Laqueur and Spatz, 1969). This was the
first time that a naturally occurring compound was found to induce colon tumors in
experimental animals. The breakdown of cycasin to its reactive form
methylazoxymethanol (MAM) requires a bacterial beta-glucosidase; thus intestinal
microflora are necessary for the formation of active mutagenic and carcinogenic
metabolites of cycasin (Matsushima et al., 1979; Mayer and Goin, 1983). Based on the
structure and metabolic information of cycasin, DMH and AOM, a DMH metabolite, were
chosen as the ideal compounds for colon tumor induction. They were shown to be
metabolized to the same proximate carcinogen, MAM, as was cycasin (Fig. 1-3), and were
later shown to induce colon tumors in rats and mice (Druckrey, 1970; Thurnherr et al.,
1973: Ward el al.. 1974). Today, DMH and AOM are the most popular compounds to
selectively induce colon tumors in experimental animals.
Mutagenic effects of DMH family of carcinogens
DMH/AOM are alkylating
agents and hence potentially mutagenic. Similar to cycasin, they do not react directly with
DNA in vitro but require bioactivation (Fiala, 1977; Kerklaan el al.. 1984). They are
among the fewer than 10% of known carcinogens that are negative in the Ames
Salmonella/microsome mutagenicity test. MAM. a downstream DMH metabolite. is
positive in the sensitive test of the Ames assay, suggesting MAM may not require
enzymatic activation (Fiala et al.. 1984; McCann and Ames, 1976). It is hypothesized that
DMH/AOM may be activated by enzymes of the intestinal microflora, since fewer tumors
(21% vs. 93%) develop in treated germ-free rats compared to conventional control rats
(Reddy et al., 1975). Metabolic studies indicate a common metabolic activation pathway
for DMH and AOM. The metabolism of DMH proceeds via AOM to
methylazoxymethanol (MAM), the deacetylated form of methylazoxymethanolacetate
(MAMA) (see Fig. 1-3 for metabolic pathway of DMH) (Fiala. 1977). In vitro, MAM is
hypothesized to give rise to a methylcarbonium ion, a highly reactive intermediate with
DNA-damaging and mutagenic properties (Matsumoto and Higa. 1966: Nagasawa et al..
1972). When [C"']-DMH was administrated to Sprague-Dawley rats, a number of
methylated purines was detected in hydrolysates of DNA, including 7-methylguanine, O6 methylguanine. 3-methyladenine, 1-methylguanine and 7-methyladenine. Although 7methylguanine was the major alkylated product, 0 6 -methylguanine was thought to be
more important in DMH induced mutagenesis (Rogers and Pegg, 1977). This was
supported by a mutational spectrum study of the lacI gene of E. coli injected i.v., then
recovered from the livers of Swiss mice exposed to DMH, AOM and MAMA. In this
system 94% of the mutations found were G:C to A:T mutations, which is consistent with
the hypothesis that O0-methylguanine leads to G to A mutation through mispairing with T
(Pegg. 1984: Toorchen and Topal. 1983). The same experiment also demonstrated that
DMH. AOM and MAMA had similar if not the same mutational spectra. Additional
evidence supporting O"-methylguanine as the key adduct for mutation is that G to A
mutations in the 12th and the 13th codon of the K-ras gene have been identified in the
DMH/AOM induced colonic neoplasia (Singh el al., 1994; Stopera el al.. 1992: Vivona et
al.. 1993). This led to the hypothesis that the DMH family of carcinogens cause colonic
neoplasia by introducing K-ras mutations to preneoplastic cells. Besides point mutations.
DMH has also been shown to cause DNA single strand breaks in vitro (Parodi el al..
1981).
Histopathologic changes after carcinogen administration The histopathology
of DMH/AOM induced colon adenocarcinomas is similar to those seen in colorectal
cancer patients (Pozharisski el al.. 1979). There is evidence that induced colon tumors
follow an adenoma to carcinoma sequence, resembling that of human colorectal tumors
(Madara et al.. 1983). The early histopathologic changes after DMH administration
include the following: 1) aberrations of nuclei of epithelial cells at the bases and the
proliferative compartments of the crypts, which peak at 24 hours after DMH
administration; 2) DNA synthesis depression, down to 20% of its normal value only 30
minutes after the administration of the drug; 3) elevated cell proliferation, characterized by
elongated heights of the mucosal crypts and expansion of the proliferative compartment of
the colonic crypts after repeated weekly injections; 4) the development of aberrant crypt
foci (ACF) after repeated weekly injections (Blakey el al., 1985; Wargovich el al.,
1983). ACF are commonly believed to be the precursor lesions of colonic neoplasm
(Mclellan and Bird. 1988) and are frequently used as a quantitative biomarker for colonic
neoplasia (Mclellan and Bird, 1988). However, counter-arguments also exist. The
distribution of ACF does not seem to match that of arising colon tumors, instead, it seems
to match well with the distribution of gut-associated lymphoid tissue (GALT) (Carter el
al.. 1994: Hardman and Cameron. 1994). About 80% ACF collected from mice or
humans were shown to harbor K-ras mutations (Pretlow et al.. 1993; Singh et al., 1994).
yet the occurrence of K-ras mutation was rare in dysplastic crypts (Jen and Powell. 1994)
and early stage adenomas (Vogelstein et al., 1988). This did not seem to consistent with
the hypothesis that ACF were tumor precursors. In several experiments ACF were found
to correlate poorly with colonic tumor occurrence (Hardman et al., 1991; Trorup et al.,
1994). Very recently, the susceptibility genes for ACF and adenomas to DMH induction
were shown to be different (Moen et al., 1996), which implies that ACF and precursors of
adenomas might be two non-related parallel events. The following three possibilities
could explain these inconsistencies: 1) only a small fraction of ACF represent true
premalignant lesions progressing to colon cancer; 2) factors associated with the GALT
promote carcinogenesis and thereby the few ACF in the region subsequently progress to
tumors at a much higher rate than elsewhere in the colon; 3) ACF and premalignant
lesions of adenomas represent two parallel and distinct events that are initiated by colon
carcinogens. and ACF seldom if ever progress to adenomas. Nevertheless, there seems to
exist little doubt that ACF are indicators of colon carcinogen exposure.
Genetics affecting susceptibility to colonic carcinogenesis
Different
susceptibilities to DMH/AOM colonic tumor induction exist among different rodent
species (Sohn et al., 1985) and mouse strains (Deschner et al., 1983; Diwan el al., 1977;
Evans et al.. 1974). Among the mouse strains, SWR/J. A/J, CFI. P/J. GR. ICR/Ha are
most susceptible, C57BL/6, C57BL/Ha. BALB/C, C3H are moderately sensitive, and
AKR/J. DBA/2 are relatively resistant (Deschner et al.. 1983; Diwan el al., 1977; Evans et
al.. 1974: Ward el al., 1974). By testing the susceptibility ofF1 and F2 hybrids of
ICR/Ha and C57B6 strains. susceptibility to colonic tumor induction by DMH was shown
to be a dominant phenotype and displayed rather precise 3:1 and 1:1 Mendelian
segregation. implying that susceptibility was controlled by a single autosomal gene (Evans
et al.. 1977). Two chromosomal loci, one linked to murine chromosome 12 (Ccs-1)
(Jacoby ei al.. 1994). one linked to chromosome 2 (Scc-1) (Moen el al., 1992). have been
suggested to possibly harbor the colon cancer susceptibility gene. These loci are not
linked to any known tumor suppressor genes. It has also been suggested that a gender
related gene affects the susceptibility (Deschner et al., 1989). Further investigation is
necessary to identify, this hypothesized colon cancer susceptibility gene.
Epithelial cell turnover and colonic carcinogenesis
The DMH and AOM
induced rodent colon cancer models are frequently used in searching for colon cancer
chemoprevention drugs. Dietary calcium and NSAIDs (non-steroidal anti-inflammatory
drugs). both shown to suppress the DMH/AOM induced colonic carcinogenesis, are
among the subjects of intensive investigation (Appleton et al., 1987; Craven and
DeRubertis. 1992; Llor et al., 1991; Reddy et al., 1992; Sitrin el al., 1991). The
suppression of colonic tumors by increased intake of dietary calcium is hypothesized to
somehow relate to reduction of mucosal crypt cell proliferation (Barsoum et al., 1992;
Guo et al.. 1990; Wargovich and Lointier, 1987). Sulindac sulfide, an aspirin-like
NSAID. suppresses colonic tumor induction (Rao et al., 1995; Singh and Reddy, 1995).
and also inhibits cellular proliferation (Piazza et al.. 1995; Shiff et al.. 1995). Elevated
epithelial cell proliferation has long been hypothesized to somehow relate to increased risk
in colonic tumorigenesis (Deschner. 1988; Deschner. 1987; Deschner et al., 1988). This
hypothesis fits well with tumor cell dynamics: more cell division. more chance for
mutations. more neoplastic foci, more tumors. As suggested in a mathematical model for
colonic tumorigenesis. if we assume tumor development to be at least a two-step process.
then by increasing the number of S-phase cells in the colon, one can significantly increase
the number of neoplastically initiated cells produced with a chemical carcinogen (Maskens.
1978). There is abundant evidence supporting this hypothesis. The differential
susceptibility to DMH-induced colonic tumorigenesis can be well predicted by the
proliferative status of colonic epithelial cells after DMH treatments (Deschner el al., 1984;
Deschner et al., 1983; Glickman et al., 1987). The cellular damaging effect of cholic acid
(Deschner et al., 1981) and the abrasive action of wheat bran (Jacobs and Schneeman.
1981; Jacobs and White, 1983) cause massive cell loss and compensatory cell replacement.
The resulting hyperproliferative state significantly increases susceptibility to carcinogen
treatment and tumor yield. Another example is the colonic hyperplasia caused by a murine
pathogen Citrobacter rodentium (formerly C. freundii biotype 4280). Under ordinary
conditions, ACF arise with an incidence of 70% 3 months after weekly DMH
administration. But in the presence of the C. rodentium-associatedhyperplasia stimulus.
these lesions are detectable in equal numbers after only one month of DMH
treatment. The mucosal hyperplasia promotes the DMH-initiated cells, thereby
significantly reducing the latent period and increasing the expression of neoplasia
(Barthold and Jonas. 1977). This cocarcinogenic effect of C. rodentium infection will be
discussed in greater detail in the following sections.
H3C
H
H
N--N
H3C-NN--CH2--- R
CHz-R
R = H 1,2-Dlmethylhydrazine
H3 C-N=N-CH--R
O
S
R OH
HC-N=N-C--O
H
Cycasin
R=H
_ _
OH
HO
OH
3
OH
I
H3C-N=N-CH-R
MAM
CH2OH
DNA
Methylation
I CH3-N=N-OH
Fig. 1-3. Metabolic pathway for DMH family of carcinogens.
CITROBACTER RODENTIUM AND TM CH
Transmissible murine colonic hyperplasia (TMCH) is a naturally occurring disease
of laboratory mice, caused by Citrobacterrodentium. Infection of mice with C.
rodentium results in an increased rate of epithelial cell proliferation in the distal large
bowel and in turn leads to mucosal hyperplasia, which is characterized by cytokinetic
changes similar to those seen in patients suffering from proliferative bowel disorders
(reviewed in Schauer. 1994). TMCH promotes the onset of chemically induced colon
cancer in infected mice (Barthold and Jonas, 1977; Barthold and Beck, 1980; Barthold el
al.. 1978), which may be analogous to proliferative bowel diseases, such as ulcerative
colitis, which are known to be related with a higher risk of colorectal cancers in humans
(Barthold, 1981). It is hoped that elucidation of the relationship of TMCH and murine
large bowel carcinogenesis will facilitate the understanding of colorectal cancer in man.
The bacteria The genus Citrobacterwas previously classified into 3 species:
C. diversus (C. koseri), C. amalonaticus. C. freundii (Schauer, 1994). They occupy a
distinct taxonomic position, intermediate between E. coli and Salmonella species in the
family of Enterobacteriaceae. Forty to fifty percent of the CitrobacterDNA content is
closely related to both E. coli and Salmonella (Brenner, 1984). It is very common to
isolate Citrobacterfrom the feces of humans and animals. and from water, soil. and food
(Sakazaki, 1984). Most isolates are considered to be typical opportunistic pathogens,
producing infection at a variety of extraintestinal sites, the most common of which is in the
urinary tract (Zwadyk, 1992). Occasionally, isolates of Citrobacterfireundiiare
associated with diarrheal disease in humans (Guarino et al., 1987). Strains producing
heat-stable enterotoxin have been reported (Guarino el al., 1987). others isolated from the
stools of humans with diarrhea and from beef samples, have been shown to produce a
Shiga-like toxin (Schmidt el al., 1993). Although the true significance of C. freundii in
human diarrheal disease is not yet clear, it would appear that some strains have the ability
to cause primary gastrointestinal disease in otherwise healthy individuals. Recently, using
DNA relatedness studies, I1 genomospecies of citrobacteria were identified (Brenner el
al.. 1993). C. amalonalicusbiogroup numberl was shown to be a separate
homogeneous species named C. farmeri. (Farmer et al.. 1985). Strains previously
classified as C. freundii were broken down into 7 species. Among them C. rodentium.
previously classified as C. freundii biotype 4280. causes TMCH (transmissible murine
colonic hyperplasia). produces a primary gastrointestinal disease in otherwise healthy mice
(Schauer el al.. 1995).
Sporadic outbreaks of rectal prolapse and diarrhea associated with moderate
mortality were occasionally observed in several mouse colonies in the late 1960s and
1970s (Barthold el al.. 1976; Brennan et al.. 1965; Brynjolfsson and Lombard. 1969;
Ediger et al.. 1974). A strain of C.freundii was isolated from affected mice by Brennan et
al.. and it was used to fulfill Koch's Postulates (Brennan et al.. 1965). This nonmotile
strain differed from the typical ones in that it did not utilize citrate as a sole carbon source.
it did not produce H 2 S in the butt of Klingler iron agar, and it produced only slight H 2 S on
lead acetate paper. An almost identical strain was isolated later by Ediger, except it did
not produce H 2S on lead acetate paper (Ediger et al., 1974). In 1976, Barthold et al.
characterized their isolates more extensively (Barthold el al., 1976). This atypical
C. freundii strain was then designated biotype 4280. In addition to the previous findings
by Brennan el al.. Barthold et al. found C. freundii biotype 4280 to decarboxylate
ornithine. In contrast. most C. freundii isolates are motile, produce H2S. readily utilize
citrate as a sole carbon source and seldom decarboxylate ornithine (Farmer el al.,
1985). C. freundii biotype 4280 fulfilled Koch's Postulates, causing TMCH when a pure
culture was orally inoculated into either conventional or germfree mice (Barthold et al.,
1976). An E. coli eaeA gene homolog was identified in this type of C. freundii and was
found to be associated with the ability to infect mice and cause TMCH (Schauer and
Falkow. 1993: Schauer and Falkow, 1993). The taxonomic status of C. freundii biotype
4280 has been recently determined. With a battery of biochemical tests and DNA
relatedness studies. three isolates of C. freundii biotype 4280 were shown to be members
of an unnamed Citrobacterspecies, designated C. rodentiumn (Schauer et al., 1995).
Different from other Citrobacterspecies, C. rodentium was found to possess the eaeA and
the eaeB genes. homologs of genes found in attaching and effacing E. coli (AEEC).
AEEC is known to cause small bowel diarrhea, or hemorrhagic colitis and hemolytic
uremic syndrome in humans (Moon et al., 1983).
The disease
TMCH is primarily a disease of young mice. Although adult mice
are also susceptible to infection, they rarely show any clinical signs of disease. Affected
young mice exhibit nonspecific clinical signs, including listlessness, ruffled pelage, hunched
posture. and retarded growth. Occasionally, mice may develop accumulation of pasty
feces. and may even develop rectal prolapse. Upon postmortem examination, the distal
colon of young and adult mice exhibit pale appearance, and profound thickening and
rigidity. Frequently the affected region includes the entire colon, sometime even the
cecum. Typically. the colon contains few formed feces, and the cecum may be empty and
contracted (Barthold et al., 1978). In young mice, the onset of clinical signs may start as
early as 5 or 6 days post-infection, and reach peak of severity and mortality approximately
10 to 15 days after infection, after which the disease quickly decreases in severity.
Likewise. detectable colon thickening is first observed at about one week following
infection, and becomes most apparent at two to three weeks, and gradually diminishes
thereafter. Mucosal hyperplasia, characterized by elongation of the colonic crypts,
exaggerated mitotic activity, extension of proliferative zone and reduction of number of
goblet cells, are detectable microscopically by the fourth day post-infection (Barthold.
1981). By the end of the first week, colonic crypts can reach some 40 cells in height,
compared to about 25 cells in crypts of healthy mice. At the peak of the hyperplasia, the
crypts are crowded throughout, often appearing pseudostratified. and can reach 80 cells in
height. Following maximal hyperplasia, the regression of the colonic lesions may last
another 2 to 4 weeks. then return to normal. By 6 to 8 weeks postinfection. the lesions
are usually completely resolved. Adult mice follow a similar onset and progression of
hyperplasia. with less severe clinical symptoms and inflammatory changes. There appears
to be no correlation between the presence of inflammation and the severity of hyperplasia,
although inflammation does correlate with morbidity and mortality (Barthold et al., 1978).
In addition to age related differences, the genetic background of mice also influences the
response to C. rodentium infection (Barthold et al., 1977). Inflammation and mortality
are greater in C3H/HeJ mice than in DBA/2J, NIH Swiss, or C57BL/6J mice. In contrast,
hyperplasia is no greater in C3H/HeJ mice than in other strains of mice used in the study
(Barthold et al.. 1977). The basis for genetic and age-related differences in susceptibility
to disease, and in the severity of hyperplasia, is not clear.
Conversely. the number of C. rodentium recovered from the colon does not reach
its maximal level at the same time as the peak period of clinical signs. In both adult and
suckling mice, the presence of C. rodentium in the large bowel is first detectable 3 to 4
days after oral inoculation. The number of organisms reaches its peak at 7 to 10 days
postinfection. at which time the population of C. rodentium may reach 1010 CFU/g of
colon tissue (Barthold, 1980). The rapid expansion of C. rodentium in the colon seems to
be at the expense of other resident flora. More than 90% of aerobic flora in the colon are
made up of C. rodentium one week postinfection (Brennan et al., 1965). The
spontaneous reduction in the number of C. rodentium is also dramatic. By 2 weeks
postinfection. at the time of maximum hyperplasia, the number of C. rodenlium present
in the colon is greatly reduced. In fact, it is rare to recover the organism from the GI tract
at all (Barthold. 1980) 3-4 weeks following infection. The longer the bacteria are present.
the greater the degree of hyperplasia.
When C. rodentium colonizes the large bowel in mice, it adheres to the mucosal
epithelial surface in a characteristic manner. Using transmission electron microscopy. the
bacteria appear to be partially embedded in the apical plasma membrane of intestinal
epithelial cells (Fig. 1-4). This intimate adherence implies that the plasma membrane is
forming a pedestal or cup for the bacteria to rest in. There is dissolution of the brush
border at the site of bacterial attachment, and destruction of the terminal web. which is a
characteristic of mucosal adherence by AEEC. In fact, C. rodenlium does have two
important genes homologues to those in AEEC, eaeA and eaeB, which are responsible for
the intimate attachment (Foubister et al., 1994; Schauer et al., 1995). The formation of
attaching and effacing lesions by C. rodentium involves a complex interaction of many
gene products. The mechanism has yet to be elucidated.
Cocarcinogenesis of TMCH
The transient hyperplastic state induced by
C. rodentium infection has a profound effect on the cytokinetics of the mucosal epithelial
cells in the large bowel. These changes also serve to increase the susceptibility of the
colonic epithelial cells to the carcinogenic effect of DMH. ACF (aberrant crypt foci),
believed to be the earliest discernible neoplastic lesion induced by DMH,. develop with a
70% incidence in mice treated weekly with DMH. In mice infected with C. rodentium
early in the course of DMH administration, the latent period for carcinogenesis is reduced.
In these animals a 70% incidence of ACF is reached after only one month of weekly DMH
administration (Barthold and Jonas, 1977). If C. rodentium is introduced into untreated
mice or mice carrying established DMH-induced lesions, it does not change the ACF
incidence. In addition, the hypothesis assuming a direct relationship between DMH and C.
rodentium has been ruled out. Mice infected with C. rodentium one or two weeks after a
single DMH treatment still readily develop ACF (Barthold and Beck. 1980). Since DMH
is metabolized and excreted rather rapidly, the temporal relationship between DMH
administration and C. rodentium infection support a model of cocarcinogenicity. C.
rodentium induced hyperproliferation may serve as a promoting step to DMH initiated
cells, thereby dynamically increasing the rate for neoplasia. The characteristics of TMCH.
increased labeling index, and expansion of the proliferative zone, have also been observed
in patients with ulcerative colitis (Bleiberg et al., 1970; Isbell and Levin, 1988).
Individuals with this disease are known to suffer an increased risk and an early onset of
colorectal cancer (Gressnstein et al.. 1980; Riddell et al., 1983). It may be that TMCH is
an appropriate model for some aspects of IBD. Nevertheless. TMCH and its
cocarcinogenesis with DMH present a good model for investigating the relationship
between cell proliferation and cancer.
Bacteria and cancer
The relationship between bacterial infection and
cancer remains poorly understood. In the late 19' h century, it was widely believed that
cancer was caused by infectious microorganisms. Many microorganisms were isolated and
thought by their discoverers to be causative. Most often, however, these organisms
turned out to be common saphrophytic bacteria. Disappointingly, vaccines made from
them generally had no demonstrable therapeutic effect. Pathologists had already
demonstrated histologically that cancerous tumors were composed of altered tissue cell,
possessing the power of growth without apparent external stimulus. It was later
discovered that cancers can be induced by certain chemicals and irradiation. Experimental
and epidemiological evidence suggesting an infectious nature of some cancer, was limited.
and the little that was known was usually ignored or misinterpreted.
Recently. Helicobacierpyloriinfection has been associated with gastric cancer in
humans (Correa, 1992). The hypothesis of a causal relationship of H. pylori and gastric
cancer is a new concept given that the organism was first isolated in 1982. It is now
known that the bacteria cause active, chronic gastritis and peptic ulcers, and may lead to
the development of gastric adenocarcinomas (Blaser, 1995; Parsonnet, 1993). It has also
been shown that infection of H. pylori greatly enhances proliferation in gastric mucosal
cells. which may contribute in part to the carcinogenicity of H.pylori (Lynch and Axon,
1995). These findings open a new era in studying the relationship between bacteria and
cancer. Recent findings in mice have linked a new type of Helicobacier,H. hepaticus, to
chronic active hepatitis, hepatic adenomas and adenocarcinomas (Fox et al., 1994). H.
hepaticus also resides in the lower gastrointestinal tract and may produce a tumor
promoting effect in these locations (Fox el al. in press, Werd el al. 1996). However, the
mechanism of the causal relationship between bacteria and cancer is still poorly
understood.
Currently there exists several hypotheses about how microorganisms can cause
cancer: 1) by producing bacterial carcinogens and cancer promoters; 2) by directly
introducing mutations or inserting bacterial plasmids; 3) by accelerating cell proliferation:
4) by causing inflammation which leads to the production of local nitric oxide and oxygen
radicals. and causing DNA damage (Macomber, 1990; Ohshima and Bartsch, 1994: Rosin
et al.. 1994). The fact that C. rodentium promotes DMH induced colonic carcinogenesis
through induced TMCH suggests that C. rodentium is not a carcinogen itself. but rather a
cocarcinogen. It promotes cell proliferation through induction of hyperplasia. a
hyperproliferate state in which there are more cells and cell divisions, therefore mutations
are more likely to occur. In contrast to mucosal hyperplasia, inflammation effects caused
by C. rodentium are less significant in most strains of mice, especially in adult mice
(Barthold et al., 1978). This suggests that inflammation may not be a major contributing
factor to the C. rodenlium promoted tumorigenesis. It is also possible that C. rodentium
affects colonic epithelial cells by producing secreted products. Investigations on C.
rodentium-induced colonic cancer promotion may lead to better understanding of
relationship between bacteria and cancer.
Fig. 1-4. A transmission electron micrograph of attaching and effacing lesion
caused by C. rodentium. (Bar represents 0.5 ýtm.)
MIN MICE: A MODEL FOR COLORECTAL CANCER RESEARCH
Despite the rather established DMH/AOM induced rodent colon cancer model, a
spontaneous colonic tumor animal model has not been available until recently. A
dominantly acting nonsense mutation in the murine APC gene results in an allele called
Min (for multiple intestinal neoplasia), predisposing heterozygotes to many spontaneous
adenomas throughout the small and large intestine. The Min phenotype in affected mice, a
homolog to FAP in humans. was identified in a pedigree established during a mutagenesis
project in which B6 males were treated with ENU and then mated to AKR/J females
(Moser et al.. 1990). The mutation was established in a B6 background and maintained
by mating Min/+ males with wild-type B6 females, since Min females are rarely healthy
enough to support a pregnancy. Min/Min homozygotes die early in pregnancy, as a result
of disrupted development prior to gastrulation (Moser et al.. 1995). B6 Min/+ mice have
an average life span of approximately 160 to 180 days. Bloody feces are frequently
observed in adult animals. and the resulting anemia due to chronic blood loss is likely to be
the major cause of death. Upon postmortem examinations, both male and female mice
are found to have tens to hundreds of polyps throughout the intestine, with small bowel
tumors being predominant. These polyps are primarily polypoid, papillary or sessile
adenomas. with small areas of adenocarcinoma occasionally found in older animals. The
single dominant allele, as suggested by simple Mendelian inheritance in continuous
backcrosses, was named Min, for multiple intestinal neoplasia. Later studies identified the
Min allele as a mutant copy of the murine APC gene, a nonsense point mutation in exon
15. codon 850, resulting in a truncated and inactivated APC product (Su et al..
1992). This truncated murine APC protein, or Min protein, may have dominant negative
effects to normal APC translates, as APC knockout mice (APC')have a less severe
intestinal neoplasia phenotype (A. Fazeli, personal communication). Min mice, a murine
homolog of FAP in humans, provide a good animal model for studying the role of APC
and interacting genes in the initiation and progression of intestinal tumorigenesis (Moser et
al.. 1995).
The significance of the single mutation in murine APC gene was further confirmed
by artificially introducing nonsense mutations into the exon of murine APC gene in two
other experiments. In both cases a similar Min phenotype was observed (Fodde el al.,
1994: Oshima et al.. 1995). The role of APC gene as a "gatekeeper gene'" for intestinal
neoplasia was further demonstrated by crossing a battery of mice transgenic with SV-40 T
antigen. human K-ras Val-1 2 mutation. and a dominant negative mutant of human p53
Alal43. either singly or in all combinations. onto Min/+ mice. Only mice carrying the Min
mutation developed intestinal tumors, while the presence of SV40 TAg increased the
number of tumors by several fold (Kim et al.. 1993). There also appears to be a genetic
factor affecting the Min phenotype. When backcrossing B6 Min/+ males to AKR females.
the resulting hybrids had fewer intestinal tumors and a longer lifespan. A gene locus,
termed Mom-I for hypothesized modifier of Min. was later mapped to murine
chromosome 4. This locus links to a human homolog at 1p35-36, a region of frequent
somatic LOH in a variety of human tumors including colon cancer (Dietrich et al.,
1993). The Mom-I gene accounts for approximately 50% of the genetic variance in the
backcrosses. and a secretory type II phospholipase gene (Pla2s) was proposed as a
candidate gene (MacPhee et al., 1995). A mutation in Pla2s in Min mice was
hypothesized to account for an increased polyp number compared to mice without the
mutation (MacPhee et al., 1995; Praml et al., 1995). Min and other APC knockout mice
are now frequently used in intestinal cancer studies. Scid (severe combined
immunodeficiency) mice carrying the Min alleles did not seem to differ in polyp frequency
and progression when compared with other Min mice, implying that neither genomic
instability nor lack of immunosurveillance conferred by Scid contributes to intestinal polyp
formation (Dudley el al., 1996). ENU has been demonstrated to affect tumor formation in
Min mice in an age-related manner, suggesting that the majority of intestinal tumors in
Min mice are initiated relatively early in life (Shoemaker et al., 1995). On the other hand.
feeding PhIP (2-amino- 1-methyl-6-phenylimidazo[4,5-b]pyridine, IQ (2-amino-3methylimidazo[4.5-flquinoline) and MelQ (2-amino-3. 8-dimethylimidazo[4,5-f]quinoline)
to APC-knockout mice resulted in an increase in tumor size but not in the number of
polyps. And the tumors from treated mice still showed a complete loss of the full-length
APC allele, the same as tumors from untreated Min mice, suggesting that PhIP affects
intestinal polyp development by accelerating the growth rate of neoplastic lesions (Oshima
et al.. 1996). Chemopreventive agents have also been tested in APC-knockout
mice. DHA (docosahexaenoic acid), an active ingredient in fish oil, was shown to
significantly reduce tumor number and size (Oshima et al., 1995). In all cases,
spontaneous adenomas and dysplasia lesions were shown to have lost the APC+ allele, and
most often the entire chromosome 18 where murine APC, MCC and DCC reside (Levy et
al.. 1994: Luongo el al., 1994; Oshima et al., 1995). Min and related types of APC
knockout mice provide an excellent system for studying colon cancer in human and are
gaining steadily in their importance in studying genetic basis of colon carcinogenesis.
INTRODUCTION TO THE SPECIFIC AIMS
The mouse is a powerful tool in modem biomedical research. The cocarcinogenic
murine model of colon carcinogen and C. rodentium-inducedhyperplasia provides
researchers with a manipulatable system to study the relationship between cell
proliferation and cancer. The Min mouse, a recently discovered murine FAP homolog, has
already been used extensively in cancer research of the large bowel (Moser et al., 1995).
A combination of these models may create a powerful system to correlate a germline
mutation. a chemical carcinogen and an environmental cell proliferative stimulus, thus
bringing a better understanding to colorectal tumorigenesis in humans, especially in FAP
patients.
Our current knowledge concerning the cocarcinogenesis of DMH and C.
rodentium infection is based upon ACF as the histological marker of premalignancy
(Barthold and Beck. 1980). As mentioned earlier, many recent findings argue against
using ACF as a quantitative measurement for precancerous neoplasia. Therefore, it is not
clear whether C. rodentium infection will ultimately promote carcinogen-induced colonic
tumor formation, let alone provide a quantitative understanding in this cocarcinogenesis
event. On the other hand, although it is generally believed that increased cell proliferation
is correlated with an elevated risk in cancer development, this causal relationship has not
been demonstrated directly. Arguments against this relationship also exist (Farber, 1995).
C. rodentium induced mucosal proliferation is a transient event. The bacteria start to
diminish before the hyperplasia reaches its peak, and disappear before the appearance of
tumors. Furthermore. the bacteria themselves do not induce ACF or adenomas.
Therefore. studying C. rodentium induced hyperplasia and cocarcinogenesis effects may
bring a better understanding of the relationship between cell proliferation and
tumorigenesis.
It is widely accepted that carcinogenesis is a multistage process involving the
accumulation of genetic changes which produce altered functions of two classes of genes,
proto-oncogenes and tumor suppressor genes (Weinberg. 1994). Activation of protooncogenes is thought to increase positive proliferative signals in neoplastic cells, while
inactivation of tumor suppressor genes reduces the effectiveness of their negative
influences on cell proliferation. DMH/AOM was assumed to initiate colonic
tumorigenesis through introducing G to A mutations in the
12th
codon of the K-ras gene
by forming O0-methylguanine. But the doubtful role of the K-ras mutation in the initiating
event of the earliest adenomas. and lack of direct proof of the mutagenic properties of
DMH/AOM makes this hypothesis less convincing. Furthermore, the possible tumor
suppressor(s) involved in this colonic carcinogenic system. characterized frequently by
LOH. has not been reported at all. Lack of powerful molecular biological tools to identify
chromosomal genetic changes may be the reason for this blank spot in cancer research in
animal models.
Recent advances have made it possible to use simple sequence length
polymorphism to perform genome-wide LOH screening in mice (Dietrich el al.. 1992:
Dietrich et al.. 1996). The mammalian genome contains tens of thousands of randomly
distributed simple sequence repeat (SSR. such as (CA)n). In inbred animals, which have
two identical sets of chromosomes, the lengths of the locus-specific SSRs are the same.
But in non-inbred animals or in hybrids of inbred strains, the lengths of the locus-specific
SSRs may be polymorphic. The advances in the murine genome project bring available
data from some 7.000 SSR markers for 8 strains of inbred mice. Using Fl hybrid mice of
two inbred strains. LOH analysis can now be achieved through a powerful technique
called PCR-SSLP (PCR based single strand length polymorphism). In this technique PCR
is used to amplify the SSR regions then the length polymorphism is compared. PCRSSLP is now frequently used to identify non-specific LOH in murine cancer research. For
example. SSLP analysis has been used to detect allele loss in the p53 gene locus from
colon tumors from mice treated with DMH (Okamoto et al., 1993). No LOH in p53 locus
was detected in this study. suggesting that the p53 gene is probably not involved in DMH
induced colonic carcinogenesis. unless both alleles had point mutations. In other
examples. butadiene-induced mammary and lung tumors from B6C3FI mice have been
characterized for LOH by PCR-SSLP, using 78 SSR markers (Davis et al.. 1994; Hegi el
al.. 1994). Microsatellite instability, which is predominantly represented by frameshift
mutations in the SSR region, can also be detected using PCR-SSLP, but in this case the
polymorphism of the SSR markers is not required (Kemp et al.. 1993).
The overall goal of this thesis project is to investigate the effects of mucosal
hyperplasia and chemical carcinogen on mice with or without germline APC mutations, in
an attempt to elucidate the relationship of genetic and environmental factors involved in
murine colonic tumorigenesis. To reach this goal, C. rodentium infection and AOM
treatment were used alone and in combination to treat wild-type and Min mice on a
background of CB6FI hybrids (B6 x BALB/c). AOM was chosen because it is more
potent and less toxic compared with DMH. and the CB6FI mouse was chosen because the
polymorphism between B6 and BALB/c are greater than most other available inbred
strains. Two molecular analyses was used to detect genetic changes in harvested colon
tumors: 12th-codon mutation detection of the K-ras gene, which is a good biomarker
representing genetic changes in proto-oncogenes, and genome-wide LOH screening for
possible tumor suppressor genes that are involved.
The specific aims are:
1) To study mouse colon tumorigenesis with 3 factors (Min, AOM-treatment, C.
rodentium infection) acting singly and in all combinations.
2) To characterize LOH and/or possibly microsatellite instability in tumor
samples. and correlate the results with treatment and tumor progression.
3) To perform K-ras 12 th codon mutation screening in tumor samples and
elucidate the role of K-ras mutations in murine tumorigenesis.
I expected outcomes of the proposed experiments:
1) C. rodentium infection promotes AOM initiated colonic tumor growth;
2) C. rodentium infection should not change the K-ras mutational frequency and
LOH patterns, if the tumor promoting effect of C. rodentium infection is
simply due to an increased cell proliferation; alternatively, if hyperplastic
epithelial cells become neoplastic through different genetic pathways, or if C.
rodentium can somehow be mutagenic, the mutational frequency and LOH
pattern may be changed.
3) AOM, C. rodentium infection and Min probably act in an cooperative manner.
The presence of Min predisposes the host to a critical mutation for colon
tumorigenesis-murine APC mutation; AOM is believed to be mutagenic,
thereby increasing the chances for a second mutation to occur: C. rodentiuminduced hyperplasia dynamically accelerates tumorigenesis.
4) Spontaneous colonic tumors from CB6FI M,"" are likely taking longer to
develop than B6 Min mice. This is suggested by results from B6 Min x AKR/J
backcrosses (Dietrich et al., 1993). Nevertheless, these tumors are expected to
lose the APC+ allele, which is the BALB/c allele in murine chromosome-18.
AOM induced tumors, including tumors from DMH treated Min mice. are
likely follow a different genetic pathway. or the same pathway but with a
different mutational pattern. Thus, the K-ras mutational spectrum and the
LOH pattern in AOM-induced tumors could be different from those of
spontaneous ones from Min animals.
5) We may be able to identify some non-specific LOH loci from tumors with
different treatments. which may suggest possible tumor suppressor genes that
are involved. including known or possibly new tumor suppressor genes.
Chapter Two
Citrobacter rodentium Infection Enhances Colon Tumorigenesis in Mice
Zhifeng Zhang. Gerald N. Wogan. and David B. Schauer
This chapter was submitted to Cancer Research
ABSTRACT
Citrobacterrodentium, previously designated C. freundii biotype 4280, causes
transmissible murine colonic hyperplasia. Forty-eight CB6F1 mice were used to
study the co-carcinogenesis of C. rodentium infection and azoxymethane
(AOM). Mice receiving 6 weekly incremental doses of AOM developed colon
tumors by the 16th week after the initial treatment. Mice infected with C.
rodentium at the time of the first AOM treatment developed more tumors (16.3 +
6.3, n = 20) than mice treated with AOM alone (9.3 ± 4.6, n = 18; P = 0.0003). No
K-ras 12th codon mutations were detected in 53 tumors collected 17 weeks after the
first AOM treatment. Larger tumors > 3 mm in diameter, collected at or beyond
the 19th week, exhibited a 19% (5 of 27) prevalence of K-ras 12th codon
mutations. Simple sequence length polymorphism analysis revealed loss of
heterozygosity in only one of 77 tumors, which was found to have lost part of
chromosome 18. These results indicate that epithelial cell hyperplasia caused by C.
rodentium infection enhances colon tumorigenesis induced by AOM, and that K-ras
mutations and allele loss are not necessary for tumor development in this system.
INTRODUCTION
Citrobacterrodentium infection causes transmissible murine colonic hyperplasia
(TMCH), a naturally occurring bacterial disease of laboratory mice (Barthold et al.,
1976). Infected mice exhibit transient epithelial cell hyperplasia in the distal colon, of
approximately 6 week duration, which is maximal some 2 weeks after infection. The
epithelial cytokinetics in affected mice are similar to those seen in human patients suffering
from idiopathic inflammatory bowel disease, such as Crohn's disease and ulcerative colitis
(Barthold. 1979). These individuals are known to suffer a high incidence and an early
onset of colorectal cancer. The mechanism by which C. rodentium induces epithelial cell
hyperplasia is not known. but intimate attachment of bacteria to the apical surface of
colonic enterocytes and signal transduction events between the bacteria and the colonic
epithelial cells appear to be involved (Schauer and Falkow, 1993). The hyperplastic state
that results from C. rodentium infection has been shown to promote the development of
aberrant crypt foci (ACF) following 1,2-dimethylhydrazine (DMH) treatment (Barthold
and Jonas, 1977). It has been hypothesized that ACF are early precursors of colonic
tumors (Mclellan and Bird, 1988). However. ACF have been reported to regress
(Barthold and Beck, 1980). and studies on the anatomical distribution of ACF and
adenomas (Carter et al., 1994), as well as on the prevalence of K-ras mutations in ACF
compared to colon tumors (Pretlow et al., 1993), suggest that only a subset of ACF goes
on to become malignant neoplasms.
In order to determine whether TMCH enhances the formation of colon tumors,
mice were treated with 6 weekly incremental doses of azoxymethane (AOM) alone, or in
combination with C. rodenlium infection. To begin to characterize the genetic events
associated with C. rodenlium co-carcinogenesis. the prevalence of K-ras 12th codon
mutations and of loss of heterozygosity (LOH) in the tumors was determined.
MATERIALS AND METHODS
Animals
Forty-eight 12-week-old, female CB6FI mice, specific-pathogen-
free for C. rodentium, were purchased from The Jackson Laboratory (Bar Harbor,
ME). Mice were housed in microisolator cages in a room with an environment of
controlled temperature, humidity, and lighting (12 h cycle), in an AALAC-accredited
facility. Water and rodent chow were provided ad libitum. All animal procedures were
approved by the MIT Committee on Animal Care.
Tumor induction
AOM (Ash Stevens Inc.. Detroit. MI) was diluted with PBS
to the appropriate concentration. Group P (n = 4) received 0.1 ml of PBS once a week
for 6 weeks by i.p. injection, and 0.01 ml of sterile Luria-Bertani broth (LB) p.o. at t =
0. Group C (n = 4) received 0.1 ml of PBS once a week for 6 weeks by i.p. injection, and
0.01 ml of C. rodentium grown to a density of 2 xl09 colony forming units/ml in LB
p.o. at t = 0. Animals in group A and in group AC received weekly i.p. injections of
AOM in PBS at doses of 5. 10, 10, 15, 15, and 20 mg/kg body weight from the first week
(t = 0) through the sixth week (t = 5) respectively (Table 2-1). At t = 0, group A received
0.01 ml of sterile LB, and group AC received 0.01 ml of C. rodentium. Colonization by
C. rodentium was verified by fecal culture on MacConkey lactose agar (Difco
Laboratories. Detroit, MI) as previously described (Schauer and Falkow, 1993).
Tumor harvest
All animals were subjected to a postmortem
examination. At the time of necropsy, the entire gastrointestinal tract was examined for
the presence of tumors. The colon was removed and incised longitudinally, then placed in
an ice-chilled petri dish for gross examination. The number of tumors, and the size and
the distance from the anus of each, was recorded. Representative tumors, approximately 5
per animal. were dissected free of the surrounding mucosa and frozen on dry ice for DNA
isolation. The remaining tumors and the normal colonic tissue were fixed in formalin for 3
- 4 h. routinely processed, and embedded in paraffin. Blocks were sectioned at 5 lIm and
stained with hematoxylin and eosin.
DNA isolation
DNA was isolated either from frozen tissue or from
paraffin-embedded tissue. Frozen tissue was incubated with 0.1 ml of 100 mM Tris-HCI
(pH 8.5). 5 mM EDTA. 200 mM NaC1, 2 mg/ml sodium dodecyl sulfate, and 100 jPg/ml
proteinase K at 60 0 C for 4 - 18 h. Lysates were subjected to phenol-chloroform
extraction, followed by ethanol precipitation, and DNA was resuspended in 0.1 ml of 10
mM Tris-HCl (pH 8.0), 1 mM EDTA. For paraffin-embedded tissue, tumor samples were
sectioned from wax blocks, and paraffin was removed by incubating in xylene for 10
min. The sections were rehydrated by sequential 5-min incubations in 100%, 95%. 80%.
and 70% ethanol. followed by a brief incubation in PBS. DNA was then isolated as
described for frozen tissue.
K-ras 12th codon mutation detection
PCR amplification using a
mismatched primer designed to introduce a diagnostic MspI restriction enzyme site into
amplicons containing the wildtype K-ras 12th codon nucleotide sequence was performed.
essentially as described by Jacobson and Moskovits (Jacobson and Moskovits.
1991). This was accomplished using primer MKI (5'-AACTTGTGGTGGTrGGAGCC-3')
(mismatched at the 3' nucleotide) and primer MK2 (5 '-TACCTCTATCGTAGGGTCGTA3'). Two pl of each DNA sample were added to a 5-jl reaction containing: 0.2 pM MKI,
0.2 jiM MK2, 200 jiM each dCTP. dGTP, dTTP, and dATP, 2.5 mM MgCl2. 10 mM TrisHCI (pH 9.0 at 25 0 C). 50 mM KCI, 0.1% Triton X-100, and 0.125 units of Taq
polymerase. Reactions were overlaid with mineral oil, and amplified in a model 480
thermocycler (Perkin-Elmer, Foster City, CA) using 30 cycles of the following
temperature profile: 30 s at 94°C, 30 s at 500 C. and 30 s at 72 0 C, followed by I cycle at
72oC for 7 min.
PCR products were digested with 10 units of Mspl (New England BioLabs,
Beverly. MA) at 370 C for 24 h, then separated by electrophoresis on a 6% denaturing
polyacrylamide gel. Amplicons digested to an 82-bp fragment were judged to contain the
wildtype K-ras 12th codon nucleotide sequence, while amplicons which resisted digestion
and remained 102 bp in length were judged to contain K-ras 12th codon mutations. To
avoid false-positive results due to incomplete restriction digestion, duplicate PCR
amplification of each DNA sample was performed using MK2 which had been
3 2P
end-
labeled. End-labeling was performed using 20 gIM MK2, 10 gLCi [y- 3 2 P]ATP (DuPont
NEN. Boston. MA). 70 mM Tris-HCI (pH 7.6). 10 mM MgCI2, 5 mM dithiothreitol, and
0.2 units/pl T4 polynucleotide kinase (New England BioLabs). The reaction mixture was
incubated at 370 C for 30 min, followed by a 5-min incubation at 750 C to inactivate the
enzyme. Following electrophoresis. signal was quantitated using a Phosphorimager
(Molecular Dynamics. Sunnyvale. CA). Full-length amplicons were recovered and reamplified under the same PCR conditions for 20 cycles. Five plof each product was
digested with MspI and applied to a gel for electrophoresis. Samples containing fragments
which resisted digestion under (i) non-radiolabeled. (ii) radiolabeled. and (iii) re-amplified
conditions. were subjected to nucleotide sequence determination using [y- 3 2 P]-labeled
MK2 and a DNA cycle sequencing kit (Promega. Madison, WI).
LOH analysis by PCR-SSLP
PCR was performed in a 5-pl reaction. as
described above. except that the reaction mixture contained 0.5 pCi of [oa- 3 2 P]dCTP
(DuPont NEN). and 10 pM unlabeled dCTP. The reaction mixture was heated to 940 C
for 5 min before beginning the following temperature profile: 5 cycles of 30 s at 94 0 C. 30
s at 60 0 C. and 30 s at 72 0 C; followed by 5 cycles of 30 s at 94 0C. 30 s at 580 C. and 30 s
at 720 C; followed by 15 cycles of 30 s at 94 0C, 30 s at 550 C. and 30 s at 720 C. On
occasion.
32P
end-labeled primers, labeled as described above, and 200 pM unlabeled
dCTP without [a- 3 2 P]dCTP were used. PCR products were added to 5 pl of 95%
formamide. I mM EDTA (pH 8.0), were heated to 95 0 C for 10 min.and were then applied
to pre-warmed. 4-mm-thick denaturing polyacrylamide gels (6% Sequel-Gel. National
Diagnostics. Atlanta. GA). Electrophoresis was carried out at 800 V for I h on a 15-cm-
long vertical gel, and signal was measured using a Phosphorimager (Molecular
Dynamics). Typically. a 5-bp difference could be detected under these conditions. In
some instances. resolution was increased by performing electrophoresis on a 38.5-cm-long
gel.
Statistical analysis
Statistical differences in tumor burden and tumor size
between group A mice and group AC mice were analyzed by two-tailed student's t
test. In all cases a value ofP < 0.05 was considered to be significant.
RESULTS
Enhancement of colon tumor development by infection All AOM-treated mice
exhibited moderate weight loss during the 6-week treatment period, and increased water
consumption beginning at t = 11 weeks (data not shown). At t = 13 weeks. AOM-treated
mice began to pass sticky feces. occasionally containing fresh blood. At t = 17 weeks,
25% of the animals from each group were killed for necropsy and tumor harvest; at t = 19
weeks. another 25% of the animals were killed; and at t = 26 weeks, the remaining animals
were killed. Extensive autolysis in two AOM-treated mice precluded collection of tumor
samples.
All AOM-treated mice developed colon tumors. C. rodentium infection
significantly increased the tumor burden (i.e. the number of tumors per animal) in AOMtreated mice (Table 2-2), as illustrated in Fig. 2-1. As expected. no tumors developed in
any of the mice receiving vehicle alone, whether they were infected with C. rodentium or
not (Table 2-2). At necropsy, mice treated with AOM typically exhibited splenomegaly.
peritoneal effusion, pallor. and emaciation. Figure 2-2 depicts the typical microscopic
colonic lesions in AOM-treated animals. Tumors were clustered in the distal colon, and
ranged in size from 0.5 - 5 mm in diameter. There was no significant difference in tumor
size (Table 2-2) or in histologic appearance of the tumors (data not shown) between mice
in group A and mice in group AC. At t = 26 weeks, 2 mice in group A and 1 mouse in
group AC were found to have multiple hepatic nodules consisting of small, pleomorphic
hepatocytes.
Detection of K-ras 12th codon mutations Twenty-six colon tumors isolated
from group A mice and 27 colon tumors isolated from group AC mice, all collected at t =
17 weeks. were analyzed for K-ras 12th codon mutations. No K-ras 12th codon
mutations were detected.
Fourteen colon tumors from group A mice and 13 colon tumors from group AC
mice. each tumor > 3 mm in diameter, were harvested at or following t = 19 weeks. and
were analyzed for K-ras 12th codon mutations. Five of the 27 larger tumors were found
to contain K-ras 12th codon mutations. The results are summarized in Table 2-3.
LOH analysis
All 53 tumors harvested at t = 17 weeks and evaluated for K-
ras 12th codon mutations were also analyzed for LOH using SSLP markers D4mit 145,
D7mit247. D 10mit80. Dl Imit5, Dl lmit29 (p53 ). DI 8mit64, DI 8mit79 (DCC), and
D1 8mit 120 (APC). all of which are polymorphic between the BALB/c and the C57BL/6
strain (Dietrich el al.. 1992). No LOH was detected at any of these loci.
All 27 tumors collected at or following t = 19 weeks and evaluated for K-ras 12th
codon mutations were also analyzed for LOH using SSLP markers D3mit I 1, D4mit226.
D5mitl82. D10mit80. D12mit37. Dl4mit115 (Rb), DI 5mitl7. Dl6mit106. DI7mit28.
D18mit64. Dl8mit79 (DCC). Dl8mitl20 (APC). and D19mit34. One sample from group
A exhibited 78% loss of the C57BL/6 allele at Dl8mitl20 and 84% loss of the C57BL/6
allele at DI 8mit79. The same sample also exhibited 75% loss of the BALB/c allele at
D5mit 182. No LOH was detected in any of the other samples. These results are
summarized in Table 3-3.
DISCUSSION
We have established a successful tumor induction method that achieves 100%
tumor incidence in less than 25 weeks, with 100% survival during the treatment. Using
this method. colon tumors have also been induced in outbred Swiss Webster mice. and in
C3D2F1 and B6AFI mice with similar results (ZZ, unpublished data).
Our results demonstrate that C. rodentium infection increases tumor burden in
AOM-treated mice (P = 0.0003), but does not effect tumor size. These results confirm
and extend the findings of Barthold and Jonas. who reported that TMCH increases the
incidence and the severity of ACF following treatment with DMH. if the hyperplasia
occurs early in the course of DMH treatment, but has no effect on established DMHinduced lesions (Barthold and Jonas. 1977). These observations are consistent with the
hypothesis that benign hyperplasia increases the number of cells susceptible to the
carcinogenic effects of DHM and AOM. but does not directly influence the evolution of
neoplastic cells. Quantitatively, the two-fold increase in tumor burden we observed is in
agreement with the two-fold increase in labeling index reported in the hyperplastic mucosa
of mice with TMCH (Barthold, 1979).
The absence of K-ras 12th codon mutations in small AOM-induced tumors
suggests that mutation of this oncogene is not necessary for colon tumorigenesis in this
system. although the possibility that mutations may be present in other K-ras codons has
not been eliminated. Jen el al. have suggested that human colonic polyps can develop
without K-ras mutations, and that some cells will eventually acquire K-ras mutations as
the tumors progress (Jen and Powell, 1994). In our study, larger tumors collected at or
after t = 19 weeks. did have a moderate prevalence of K-ras mutations. We did not
determine the relative number of wild-type and mutant cells within each tumor. however
we favor the hypothesis that K-ras mutations occur late in tumor
development. Consistent with this notion is the observation that one tumor contained two
independent K-ras 12th codon mutations.
LOH was detected in one tumor, indicating allele loss at the APC and the DCC
loci. Both of these genes are thought to encode tumor suppressor proteins involved in
human colorectal tumorigenesis, with APC mutations occurring as early events (Powell et
al.. 1992). While allele loss is a frequent mechanism of tumor suppressor gene
inactivation in both human and mouse colon tumors, point mutations induced by the
methylating agent AOM have also been implicated in tumor suppressor gene inactivation
(Zaidi el al.. 1993). and could account for the low frequency of LOH in this study. On the
other hand. the possibility that somatic APC mutations are less frequent in ulcerative
colitis-associated colon tumors than in sporadic colon tumors has been raised (Benhattar
and Saraga. 1995). It may be that C. rodentium infection in mice is therefore an
appropriate model for some aspects of colorectal cancer susceptibility in patients with
Crohn's disease and ulcerative colitis.
C. rodentium infection is not unique in its ability to enhance carcinogenesis by
inducing epithelial hyperplasia. Bombesin has been shown to have a similar effect on
enhancement of DMH-induced colon tumors in rats (Ide el al.. 1994). While bombesin is
known to act as a growth factor on some types of cells. the mechanism of C. rodentiumassociated epithelial cell hyperplasia remains unclear. Tyrosine phosphorylation of a 90kDa protein. inositol phosphate fluxes, and cytoskeletal rearrangements have been shown
to occur following intimate attachment of enteropathogenic Escherichiacoli to human
epithelial cell lines (Foubister et al.. 1994). C. rodentium shares several bacterial virulence
factors with this human pathogen (Schauer and Falkow, 1993; Schauer et al.. 1995) and
appears to cause similar changes following attachment to colonic enterocytes in the mouse
(Johnson and Barthold. 1979; Schauer and Falkow. 1993). It is tempting to speculate that
bacterial attachment and signal transduction might activate ras p21 and provide, at least
temporarily. a proliferative stimulus. On the other hand. C. rodentium achieves a high
density of colonization in the distal colon, attaching largely to differentiated enterocytes
(Johnson and Barthold. 1979; Schauer and Falkow, 1993). Mucosal hyperplasia is
perhaps more likely to be a nonspecific, protective response to luminal cell
damage. Further characterization of the mechanism of tumor enhancement by C.
rodentium infection should provide additional insight into the role of epithelial cell
hyperplasia in colon cancer susceptibility.
Table 2-1 Experimental study design
Group
n
Oral inoculuma
Intraperitoneal injectionsb
P
4
Sterile broth
Vehicle alone
C
4
C. rodentium
Vehicle alone
A
20
Sterile broth
Azoxymethane
AC
20
C. rodentium
Azoxvmethane
a Oral inocula were given once. at t = 0 weeks. and consisted of 0.01 ml of either
sterile Luria-Bertani broth (Sterile both). or an 18 h growth of C. rodentium, with a
bacterial concentration of 2 x 109 colony forming units/ml.
b Intraperitoneal injections were given 6 times. at t = 0. 1, 2, 3. 4. and 5 weeks. and
consisted of either 0.1 ml of PBS (vehicle alone), or azoxymethane in PBS at a dose of 5.
10. 10. 15. 15. and 20 mg/kg of body weight. respectively.
Table 2-2 Citrobacterrodentium infection increases tumor burden but not tumor size
Number of colonic tumors a
Group
17 weeksb
19 weeks
22 weeks
26 weeks
P
0
0
0
0
C
0
0
0
0
A
7.2 + 5.1
8.9 ± 4.4
8.4 ± 4.5
9.3 ± 4.6
2.0 + 1.1 mm
AC
13.3
15.1 + 5.8
16.7 ± 6.4
16.3 + 6.3
1.8 ± 1.0 mm
±
7.2
Tumor sizec
a Cumulative number of tumors per mouse in each group. expressed as the mean
±
standard deviation. AC group has significantly more tumors/mouse than A group at all
time points after t = 17: P = 0.0181 at 19 weeks, P = 0.0018 at 22 weeks. and P = 0.0003
at 26 weeks.
b Number of weeks after the initial azoxvmethane treatment.
c Diameter of all tumors. expressed as the mean ± standard deviation.
Table 2-3 K-ras 12th codon mutations and loss of helerozygositv in colonic tumors
19 weeksa
17 weeks
K-rasb
LOHC
K-rasd
LOH e
A
0/26
0/26
3/14
1/14
AC
0/27
0/27
2/13
0/13
Group
a Cumulative data from tumors harvested at or following 19 weeks after the initial
azoxvrnethane treatment.
b K-ras 12th codon mutations expressed as the number of tumors with
mutations/tumors tested.
c Loss of heterozygosity detected by PCR-SSLP. expressed as the number of
tumors with LOH/tumors tested.
d All 3 group A tumors with K-ras mutations contained GGT to GAT transition
mutations: one group AC tumor had a GGT to AGT transition, and the other had both
GAT and GAA mutations
e One tumor had 78% loss of the C57BL/6 allele at D18mitl20. 84% loss of the
C57BL/6 allele at D18mit79. and 75% loss of the BALB/c allele at D5mit182.
a20
15
0
E
10
0
0
Fig. 2-1. C. rodentium infection significantly increases tumor numbers per mouse.
White bar: Average number of colonic tumors per mouse induced by AOM treatment
alone. Black bar: Average number of colonic tumors per mouse induced by AOM/Cr.
P<0.05 after 17 weeks in student t-test. Tumor data are calculated in accumulative basis.
See Table 2-2 for more details.
-C-:--;:-Vcc--..,
._-
__~._
__
~-t·r
_n
· ·-, ·-
Fig. 2-1. Histopathologic changes in mice infected with C. rodentium and treated with
azoxymethane. ACF apparently arising from individual crypts (A); more advanced ACF
composed of several coalescing crypts (B); colon tumor (C); and detail of colon tumor
showing irregular acinar arrangement of epithelial cells.
Chapter Three
Citrobacter rodentium Infection Affects Colonic Tumor Incidence
In Min Mice
ABSTRACT
Citrobacterrodentium (previously designated C.freundii biotype 4280) infects
laboratory mice and causes transmissible murine colonic hyperplasia. We have
previously demonstrated that C. rodentium infection significantly increases the
number of tumors per AOM-treated mouse, and observed a low frequency of K-ras
12th codon mutations and LOH. We investigated the relationship between C.
rodentium infection and colonic tumor incidence of CB6FIMin' (BALB/c X C57B6/J
Mn-)
hybrid Min mice. Infected Min mice showed significantly (p<0.02) higher
colonic tumor incidence (47%) than uninfected controls (5%). Similar increase was
also found in AOM-treated CB6F1 Min mice (60% vs. 100%). In addition, AOMtreatment accelerated colonic tumorigenesis in Min mice and these tumors had a
different distribution pattern in the colon. When analyzing tumors collected for Kras 12th codon mutation and LOH, all spontaneous colonic adenocarcinomas
showed APC chromosome deletions but no K-ras mutations, whereas AOMinduced colonic adenocarcinomas show <10% APC' chromosome deletions and 2030% K-ras mutations (All G to A transitions). C. rodentium infection did not alter
LOH or K-ras mutation patterns. Spontaneous and C. rodentium promoted tumors
exhibit a higher rate of LOH when compared to AOM-induced tumors in Min mice,
and two LOH hot spots D3mitll and D10mit180 were identified. These data
support the hypothesis that C. rodentium-induced mucosal proliferation enhances
tumorigenesis in mice.
INTRODUCTION
Citrobacterrodentium causes transmissible murine colonic hyperplasia (TMCH) in
laboratory mice (Schauer, 1994; Schauer et al., 1995). The bacteria colonize the distal
colon, and produce an attaching and effacing lesions that are indistinguishable from those
seen in attaching and effacing E.coli infections in the human intestine (Schauer and
Falkow. 1993). Although the clearance of the bacteria occurs 7 to 10 days post-infection,
mucosal hyperplasia reaches its peak approximately 2 weeks after infection, then gradually
resolves in another 2 to 4 weeks. The bacterial-induced mucosal cytokinetic changes
including increased labeling index, expansion of the proliferative zone, and elongation of
the crypt height, which are similar to those seen in proliferative bowel disorders in
humans. such as Crohn's disease and ulcerative colitis (Barthold, 1979; Barthold, 1981).
Patients with these diseases are known to suffer a high incidence and an early onset of
colorectal cancer (Gressnstein et al., 1980). TMCH has been shown to promote
development of aberrant crypt foci (ACF) induced by 1,2 dimethylhydrazine (DMH)
(Barthold and Jonas, 1977; Barthold and Beck, 1980). C. rodentium induced TMCH
provides a manageable system to help elucidate the relationship between cell proliferation
and cancer.
Germline mutations of the APC (adenomatous polyposis coli) gene have been
observed in patients with familial adenomatous polyposis (FAP) (Miyoshi et al., 1992). In
sporadic colorectal cancer, APC gene mutations are frequently observed and seem to
occur early during colorectal tumorigenesis (Powell et al., 1992). Although the function
of the APC protein is still under investigation, APC is hypothesized to be the 'gatekeeper'
tumor suppressor gene for colorectal cancer (Fearon and Vogelstein, 1990).
Min (multiple intestinal neoplasia) is a mutant allele of the murine APC gene.
consisting of a nonsense mutation at codon 850 (Su et al., 1992). While mice
homozygous for Min mutation die before gastrulation (Moser et al.. 1995), B6 APCMin mice have a lifespan of 160 to 180 days and are predisposed to numerous adenomas
distributed throughout the small intestine and colon (Moser et al., 1990). Min mice
provide a good spontaneous inbred animal model for human colorectal cancer studies.
Recent progress in murine genome research has made it possible to use polymorphism of
simple sequence repeats (SSR) between different inbred mouse strains to perform
genome-wide loss of heterozygosity (LOH) screening (Dietrich et al., 1996). Using this
powerful tool. studies on F1 hybrid AKRxB6 APC Mn revealed that entire APCchromosome has been deleted in all spontaneous occurring adenomas in small intestine
and colon (Luongo et al.. 1994) (Levy et al.. 1994). These studies strongly suggested
that complete inactivation of the APC gene is a necessary step in colonic tumorigenesis.
We have previously demonstrated that C. rodentium infection enhances colonic
tumorigenesis in AOM-treated mice (ZZ, submitted). The infected mice exhibit
significantly greater numbers of tumors per mouse. while average tumor size. tumor
nmice. we
histology,. and mutational pattern remain unaffected. Using CB6FI APCVM
investigated the possibility that C. rodentium infection promotes colonic tumorigenesis in
Min mice. and analyzed and compared tumors from different treatments for K-ras
mutations and genome-wide LOH screening. B6 Min animals from Jackson Laboratory
are know to carry Helicobacterhepaticus (Shames et al., 1995), a bacterium colonizing
the liver and the intestine of mice and causing chronic hepatitis in susceptible mice (Fox et
al.. 1994). Recently, H. hepaticus infection has also been shown to cause chronic.
proliferative typhlitis and colitis in immunodeficient mice (Ward et al., 1996), and to be
associated with intestinal hyperplasia and inflammation in monoassociated Swiss Webster
mice (Fox. 1996). To better characterize the effects of the C. rodentium, we bred H.
hepaticus-free B6-Min mice through embryo-transfer. This is the first report of an
experiment in which H. hejaticus-free Min mice were used.
MATERIALS AND METHODS
Mice Mice were bred and maintained in a H. hepaticus-free environment. The
C57BL/6J (B6) APCMn" males were originally purchased from The Jackson Laboratory
' males were obtained
(Bar Harbor, ME). The H. hepaticus-free B6 APC in•
by embryo-
transfer. performed by the Division of Comparative Medicine, MIT. The BALB/c females
were purchased from Taconic Laboratory (Germantown, NY), and are H. hepaticus-free
(Shames et al., 1995). Mice were housed (4-5 per cage) in an environment of controlled
temperature. humidity and lighting. with water and chow provided ad libilum.
C. rodentium infection and AOM treatment
Sixty H. hepaticus-free
female CB6F1 mice at 4-5 weeks of age, bred from B6 APC "- males x BALB/c females.
were randomly separated into two groups of 30 mice each. One group of mice was orally
inoculated with C. rodentium (ca. 2x1 07 CFU). and the control group were given sterile
LB broth. One half of these mice were expected to be Min mice.
Fifty-two H. hepaticus-free male CB6FI mice. bred from B6 APCMI"
-
males x
BALB/c females. were genotyped for Min allele and then separated into the following 4
groups. Group one mice, consisted of 5 Min mice and 5 wild-type ones. were subjected to
4 weekly AOM (Ash Stevens Inc., Detroit. MI, freshly diluted in PBS to proper
concentration) i.p. injections at 10 mg/kg beginning at 4-5 weeks of age. with 10 Pl of
sterile LB given at the time of the first injection. Group two mice consisted of the same
number of mice and received the same treatments as mice in group one. except that they
were also infected with C. rodentium as described previously (ZZ submitted), at the time
of the first AOM injection. Group three mice, consisted of 5 Min mice and 5 wild-type
ones. were given 4 weekly PBS i.p. injection and sterile LB p.o. at the time of the first
injection. They served as vehicle controls. Group four mice consisted of 12 Min mice and
10 wild-type mice. and did not receive any treatment.
Genotyping for Min mice
Mice were all identified by ear-punch, and the
excised ear tissue was saved for Min-genotyping using an allele-specific PCR assay. DNA
was isolated by incubating tissue in 100 mM Tris buffer, pH8.5, 5mM EDTA, 200 mM
NaCl. 0.2% SDS. and 100 jig/ml proteinase K (added immediately prior to use) at 60 0C
overnight. Samples were then centrifuged for 5 min at top speed, followed by ethanol
precipitation. The isolated DNA was then used in 25-p1 standard PCR reactions to
identify the Min allele. The primers sequences are: MAPC-9. 5'-GCC ATC CCT TCA
CGT TAG: MAPC-15. 5'-TTC CAC TTT GGC ATA AGG C: MAPC-MT. 5'-TGA
GAA AGA CAG AAG TTA. The PCR cycle program consisted of 30 cycles of 30" at
94°C. 30" at 550C. and 1'at 720C. with an extension time of 7' at 72 0 C following the last
cycle. The PCR products of approximately 600 bp in length served as an internal PCR
control. while the appearance of a product of approximately 300 bp indicated the presence
of a Min allele.
Necropsy and tumor harvest
All mice that either died or were killed
during and at the end of the experiment were subjected to a complete postmortem
examination. At the time of necropsy, the entire gastrointestinal tract was examined for
tumors. The colon was removed, measured, incised longitudinally, and then placed in a
Petri-dish for gross examination for tumors. The size, location (distance from anus) and
number of tumors were recorded. Tumors were partially dissected free of surrounding
normal tissue and quick-frozen on dry ice for DNA extraction. One piece of normal colon
tissue per mice was also taken as a control. The remaining tissue was fixed in formalin for
3-4 hours prior to processing and paraffin embedding. Embedded tissue blocks were
sectioned at 5 microns and were stained with hematoxlin and eosin for histopathologic
evaluation done by pathologist Dr. X. Li in Division of Comparative medicine, MIT.
Small intestine neoplastic index (SINI) evaluation
To measure the
frequency of neoplasia in the small intestine, we introduce a grading system, namely SINI.
At necropsy. the polyps in the small intestine were enumerated. SINI was scored as the
following: A SINI score 0 represented no polyps, 1 represented approximately 5 polyps,
2 represented 10 polyps. 3 represented > 15 polyps.
DNA extraction
One hundred microliters of buffer, containing 100 mM Tris.
pH8.5. 5mM EDTA. 200 mM NaC1. 0.2% SDS. and 100 Cpg/ml proteinase K (added
immediately prior to use). were added to frozen tumor and normal tissues and digested at
60"C overnight. The lysate was then subjected to phenol-chloroform extraction and
ethanol precipitation.
K-ras 12th codon mutation detection
A PCR-RFLP-based assay was used
to identif ' 12th codon mutations in the K-ras gene. The primer sequences are the
followings:
MK3: 20-mer. 5'-AAC TTG TGG TGG TTG GAC CT (GGT...) and MK2: 22-mer. 5'TTA CCT CTA TCG TAG GGT CGT A. The third 3' nucleotide of the primer MK3 was
designed to be mismatched, and thereby introduced an BstNI site (underlined) into the
PCR products from a wild-type K-ras allele. After a standard I0-tl PCR amplification
(30 cycles of 94"C-55oC-720C, 30 seconds for each step), the products were digested with
10 units of BstNI (New England Biolabs Inc., MA) at 370C for 24 hours (BstNI was used
in excess to ensure a complete digestion), then separated on 6% polyacrylamide gels. The
undigested products of 102 bp in length were recovered from gels and re-amplified for 20
cycles. After re-confirming K-ras mutation by digesting part of the re-amplified products,
the remaining PCR products were used for sequence determination using [y32 P]-labeled
MK2 primer using Promega cycle sequencing kit (Promega, Madison, WI).
LOH screening by PCR-SSLP
Data containing SSR length polymorphism
and primer sequences were obtained from searching genomedatabase d-genome.wi.mit.edu. PCR-SSLP were performed as described (ZZ,. submitted).
A total of 43 SSR markers (Table 3-4 for details) was screened for LOH.
RESULTS
Tumor incidence
Mice that were not treated with AOM were killed by CO 2
asphyxiation 6 months after the infection (or about 7 months of age). AOM-treated mice
and their control animals were sacrificed 3 months after the first AOM treatment or when
they became moribund. With C. rodentium infection, the combined tumorigenic effects
were dramatic. One mouse died as early as 6 weeks after the initial AOM treatment (70
days of age). with multiple colon tumors clustered in the distal colon. The tumor
incidence is summarized in Table 3-1. While none of the wild-type mice had any colon
tumors. C. rodentium-infected CB6FI-Min mice had a significantly greater (p<0.02 in
Chi-square test and Fisher's exact test) colonic tumor incidence (7/15, with total of 9
tumors). compared to uninfected controls (1/19).
AOM/C. rodentium-treated CB6F1
Min mice also had a greater (5/5) tumor incidence than AOM-treated Min mice (3/5). In
addition. the average number of tumors in AOM/C. rodentium-treatedCB6F1-Min mice
was 11.4. while that in AOM-treated tumor-bearing mice was 5.3.
SINI and Tumor histology The SINI are also summarized in Table 3-1. AOMtreatment and C. rodentium infection did not seem to affect the SINI scores. The male
CB6FI-Min mice had a higher (1.3 + 0.8) SINI than that of females (1.1 + 0.5). but the
difference was not significant. When evaluated microscopically, colonic tumors from Min
mice in different treatment group appeared to be quite similar, all were primary papillary
adenomas to adenocarcinomas . Tissue sections from AOM-treated Min animals
displayed multiple aberrant crypt foci (ACF) in the colon. The density of these ACF is
much greater than those seen in AOM-treated wild-type mice or untreated Min mice.
Tumor distributions
At necropsy, the mouse colons measured 9 to 13 cm
in length. Tumor locations (distances from anus) were all normalized to a 10 cm length.
The tumor distribution of different treatment groups was plotted in Fig. 3-1. For purposes
of comparison, normalized colonic tumor locations from parental B6 Min mice and AOMtreated CB6FI wild-type mice (H. hepaticus positive) were also plotted in the same figure
(ZZ. previous observations). Interestingly, tumors from non-AOM-treated CB6FI Min
mice have a quite different location pattern compared to tumors from AOM-treated mice.
Although the number of spontaneous colonic tumors from uninfected Min mice was too
small to be compared to those from infected Min mice. C. rodentium infection did not
seem to change the tumor distribution in AOM-treated Min mice.
K-ras 12th codon mutation detection
Colon tumor samples were analyzed
for K-ras 12th codon mutation. and the results are summarized in Table 3-2. For
purposes of comparison. ten colonic tumors from B6 Min and 10 small intestine tumors
from untreated CB6F1 Min were also analyzed for mutations. Only colonic tumors from
AOM-treated animals exhibit K-ras 12th codon mutations. Three out of 11 tumors from
AOM-treated CB6FI -Min mice and 2 out of 10 tumors from AOM-treated C. rodentiuminfected CB6FI -Min mice were positive for the K-ras mutation. All mutations were found
to be G to A transitions (one GGT to AGT, 4 GGT to GAT). Again, C. rodentium
infection did not change the K-ras mutational frequency.
LOH screening
Selected colonic tumors were also screened for LOH by
PCR-SSCP. A total of 43 SSR markers was screened, covering all 19 autosomes (see
Table 3-4). The results were summarized in Table 3-3. Almost all colonic tumors from
non-AOM-treated mice displayed APC+ chromosome- 18 (BALB/C allele) loss. regardless
of whether the mice were infected or not. Other LOH hot-spots were D3mitl I and
D1 Omit 180. both occurring in approximately 30% of the tumors. In contrast. colonic
tumors from AOM-treated animals showed less than 10% of APC+ allele loss, and LOH in
other markers was infrequent and random. It's noteworthy that once again C rodentiuminfection did not change the LOH pattern created by Min and AOM treatment.
DISCUSSION
This investigation is the first to used H. hepalicus-free hybrid Min mice to study
the effects of C. rodentium infection and AOM-treatment. The results demonstrated two
distinct ways to accelerated tumorigenesis, one is epigenetic, namely C. rodentium
induced TMCH. the other is mutagenic, namely AOM treatment. In tumors, AOM leaves
its own characteristic 'fingerprint' mutations and tumor distribution pattern, while the
epigenetic acceleration of C. rodentium infection is somewhat 'invisible' in terms of tumor
characteristics. Our results strongly supported the hypothesis that C. rodentium-induced
hyperproliferation rather than a direct mutagenic effect is the force promoting colonic
tumorigenesis in mice. C. rodentium reaches a high density of colonization in the distal
colon. attaching largely to differentiated enterocytes. The induced mucosal hyperplasia is
perhaps more likely to be a non-specific protective response to luminal cell damage.
similar to that seen in compensatory hyperplasia after surgery. which has also been shown
to stimulate colonic carcinogenesis (Williamson and Rainey, 1984). Although C.
rodentium infection does cause inflammatory effect. characterized by significant increase
of macrophages in basal matrix and in-between the crypts (ZZ, unpublished data), the
combined effects on tumorigenesis are mainly epigenetic. This may be an analogue to the
observations by Oshima el al. (Oshima el al., 1996), in which 2-amino-l-methyl-6phenylimidazo[4.5-b]pyridine (PhlP) was given to APC knockout mice. PhIP increased
the average tumor size. but it did not affect the number of tumors and the allelic loss
property. The likely explanation could be the difference in the onset of the tumorigenesis.
due to the difference in the APC mutations and the mouse strains. The true relevance of
these finding for human colorectal cancer remains to be determined.
On the other hand. AOM seems to induce primary neoplasia through its mutagenic
effects. Compared to AOM-treated wild-type CB6FI mice, AOM-treated CB6FI Min
mice developed colonic tumors much earlier. In combination with C. rodentium infection.
Min mouse died of multiple colonic tumors as early as 6 weeks after the initial-AOM
treatment. In contrast. the first mouse death occurred 15 weeks after the initial treatment
and C. rodentium infection. under a higher dose regime (ZZ, submitted). This result,
combined with low-frequency of LOH and APC-allele loss observed in tumors, suggests
that a) the combined effects of Min phenotype and AOM-treatment is synergistic: b) to
explain this synergistic effect. it is tempting to assume that AOM create point-mutations in
the APC-allele: c) because APC chromosome 18 loss was unlikely the effect of AOM
treatment (ZZ submitted). the observed low rate of APC-allele loss in AOM-treated Min
mice maybe from spontaneous mutations. The early emerging of these tumors may
indicate that AOM can also accelerate tumorigenesis by targeting genes other than APC.
or through other effects. Repeated AOM treatment may induce an elevated cellular
proliferation. similar to those reported from mice treated repeatedly with DMH
(Wargovich et al.. 1983).
K--ras mutations have been found in ACF and adenomas induced by DMH/AOMtreated rats (Stopera et al.. 1992) and mice (Singh et al., 1994). The G to A transition
pattern fits with the model that AOM induces G to A mutation through forming 06methylguanine (Pegg. 1984; Toorchen and Topal, 1983), which mispairs with T.
Considering that K-ras mutations can occur at loci other than the 12th codon. the actual
frequency of K--ras mutations in our experiment could be somewhat higher. Our data
showed that G to A mutations in the 12th codon of K-ras mutations are presented in
about 25% of colonic tumors from AOM-treated Min mice, but no 12th codon K-ras
mutation was observed in spontaneous occurring colonic or SI tumors from Min mice.
These data suggest the following: 1) G to A mutations in the 12th codon of K-ras gene
are characteristics of AOM-induced colon tumors; and 2) K-ras mutations, at least the
12th codon mutations. are not a necessary step in early stage of colonic tumorigenesis in
mice. It also possible that K-ras mutations are associated with advanced stage of
tumorigenesis. as what have been suggested by observations from AOM-treated wild-type
mice (ZZ. submitted) and in human studies (Miyaki et al., 1990; Ranaldi et al.. 1995).
Our observations on spontaneous arising colonic tumors from Min mice and those
from C. rodentium infected Min mice were consistent with recent observations that the
APC+ allele was lost from all spontaneous tumors in Min mice (Levy et al., 1994: Luongo
et al.. 1994). We also observed significantly higher LOH in tumors from non-AOMtreated Min mice than those from AOM-treated Min mice. Two likely explanations for
this could be the followings: 1)AOM-induced mutations inactivated necessary tumor
suppressor genes. abrogating the need for allele loss: and 2) LOH events are a relatively
slow process, ant the rapidity with which AOM-induced tumors develop precludes allele
loss from occurring at the frequency seen without AOM-treatment. These results are
different from observations by Sheomaker et al. (Shoemaker et al., 1995). which
suggested an earl)y appearance of the allelic loss event. This may be explained by the
different type of Min mice used in our experiment. BALB/c mice have two normal alleles
of Pla2s gene. a candidate gene for the Mom-i locus. It may be that the existence of a
copy of this gene delays the appearance ofAPC allelic loss. We also identified two LOH
sites as hot-spots in colonic tumors from non-AOM-treated Min mice. One of the two
sites. DI Omit 180, is located close to murine homologs of genes such as Mdm2. and
Interferon-y. Further investigations are needed to elucidate the significance of these nonspecific LOH loci.
Table 3-1. C. rodentiuni infection increase colon tumor incidence in CB6FI Min mice"
No Treatmentb
C. rodentiumb
AOM'
AOM +C. r.c
Tumor Incidenced
1/19
7/15
3/5
5/5
Total # of Tumors
1
9
16
57
Average SINI
1.1 (0.5)e
1.1
0.5
0.6
a Only
Min animals are displayed.
bFemale
CB6FI Min mice were used. Mice were killed at 7 months of age. Tumor
incidence in C. rodenlium infected group is significantly higher than that in non-treatment
group. P<0.02 in Chi-square test.
c Male CB6FI Min mice were used. Mice were killed at 4 months of age.
d Incidence
is shown as (tumor-bearing mice) / (total Min mice in a group). Data
not shown in this table: Five Male CB6F1 Min mice killed at 4 months of age had no
colonic tumors. and 2 out of 12 Male CB6FI Min mice killed at 7-month of age developed
3 colon tumors.
'The number in the bracket is the average SINI of 5 male Min mice used as vehicle
controls and killed at 4 months of age. Data not shown in this table: 12 male CB6FI Min
mice had an average SINI of 1.3 when killed at 7-month of age.
Table 3-2. Low incidence of K-ras 12th-codon mutations correlates with AOM treatment
Mice / Treatment
K-ras Mutation'
Mutated Sequence
CB6FM'"- / None
0/4
CB6FI
M 'n"
/ C. rodentium
0/9
CB6FI
M 'n
/ AOM
3/11
GAT (2); AGT
CB6F1 M,'-/ AOM/ C. r.
2/10
GAT (2)
CB6FI-- /AOMb
3/13
GAT (3)
CB6F1 - / AOM'
2/14
GAT. AGT
C57B6M 'n- / None'
0/11
CB6FI Mn / None /SI
a Mutations
h
d
0/10
are expressed as (# of mutants) / (tumors screened)
Data are from ZZ submitted. These adenocarcinomas are >3 mm in diameter.
Colon tumors from spontaneous B6 Min mice.
d
Small intestine tumors
Table 3-3. AOM affects LOH pattern in tumors from female CB6FI Min mice'
None
# of tumors analyzed
C. rodenlium
AOM
9
11
10
10%
4
APC- Chr- 18 lossb
100%
89%
9%
Other LOH
hot spotsc
DlOmitl80
D3mitl 1
D3mitl 1
DlOmitl8O
<10%
random
a Total
AOMIC. r.
<10%
random
of 43 SSR markers (Table 3-4) were screened for LOH.
APC- chromosome 18 loss was characterized by the loss of BALB/c allele in all
h
three SSR marks (D18mit64, Dl8mit120. D18mit79) in chromosome 18.
cTumors
fiom Non-AOM-treated mice had a higher prevalence of overall LOH
than those from AOM-treated mice. LOH occurs at D Omit 180 and D3mit I in 31% of
colon tumors from non-AOM-treated mice.
Table 3-4. List of 43 SSR markers screened for LOH
Chromosome
Chr-1
Chr-2
Chr-3
Chr-4
Chr-5
Chr-6
Chr-7
Chr-8
Chr-9
Chr- 10
Chr-I 1
Chr-12
Chr-13
Chr-14
Chr-15
Chr-16
Chr- 17
Chr-18
SSR Markersa
D mit3, Dlmit 187
D2mit249
D3mit 164, D3mit 1, D3mit 19
D4mit 145, D4mit226
D5mit 182. D5mit95
D6mit 166. D6mit 14
D7mit247. D7mit 105
D8mit4
D9mit91. D9mit 104, D9mit24
D10Omit80. D1 Omit 180
D11 mit78. DI Imit29(p53), DI lmit203
D12mit37. D12mit34, Dl2nds2
DI 3mit57. Dl2mitl51
D14mit133. D14mit115 (Rbl)
D15mit53. Dl5mitl7
D16mit154. D 6mit 106
D 7mit28. D 7mit66. D17mit129
D18mit64. Dl8mitl20(APC).
D1 8mit79(DCC)
D19mit59. D19mitl9. D19mit34
Chr-19
a Markers are displayed in an order away from
centromere. Genes displayed in parentheses are within
2 cM distance.
Fig.3-1. Different tumor distribution in AOM-treated Min mice a
Colon Tumor distribution
14
12
10
0
E
4
2
0
10
20
30
40
50
60
70
80
90
Tumor position (% of colon length)
a Tumor
position is determined by each tumor's normalized distance from anus. Position
10% contains number of tumors within the first 10% of length of colon, and so on.
Diamond line depicts colon tumor position from 10 B6 Min mice from our previous data:
square line, from 10 tumor-bearing non-AOM-treated CB6F1 Min mice; triangle line,
from 5 female CB6F1 Min mice treated with AOM; cross line, from 5 C. rodentiuminfected AOM-treated female CB6F1 Min mice; star line, from 5 AOM-treated female
CB6FI wild-type mice (ZZ, previous observations). Tumors from AOM-treated mice
show quite different distribution pattern comparing to non-treated Min mice.
B6 N1 T1 N2 T2 N3 T3 N4 T4 N5 T5 N6 T6 N7 T7 N8 TS N9 T9 N1oTjo B/c
Fig. 3-2. LOH in SSR marker Dl8mitl20 (APC).
(B6, C57BL/6; N,, normal tissue samples; Tn, colon tumor samples; B/c, BALB/c. Ti to
T4 are four spontaneous colon tumors from CB6FI Min mice. All BALB/c alleles (APC+)
were lost in these samples. T5 to T10 are 6 tumors from AOM-treated CB6F1 Min mice.
Only one tumor, T7 , showed allelic loss.)
Chapter 4
On Route To Understanding The Tumor Enhancing Effects
Of C. rodentium
INTRODUCTION
In the previous chapters C. rodentium has been shown to enhance tumorigenesis in
AOM-induced colonic carcinogenesis and spontaneous colonic tumors in Min mice, but
the exact mechanism remains undefined.
In both reports, the effect of C. rodentium infection is mainly epigenetic. The
infection did not seem to affect K-ras mutations or LOH patterns, nor did it affect the
distribution or size of colon tumors. Only the number of tumors increased. These results
are consistent with the hypothesis that cell proliferation induced by C. rodentium infection
increases tumor number and incidence.
The cytokinetics of colonic mucosal hyperplasia during C. rodenlium infection had
been studied in NIH Swiss mice (Barthold, 1979). It has been suggested that the
abnormal cyvtokinetic changes during the hyperplasia are similar to those seen in IBD
patients. It would be interesting to compare the cytokinetics of Min mice to the wild-type
controls. Also. a systematic study of cytokinetic changes during C. rodentium-induced
hyperplasia in different genetic backgrounds has not been conducted.
Chronic inflammation has been correlated with increased cancer risk in IBD patient
(Levin. 1991). It is hypothesized that NO and other oxidative species formed by the
inflammatory response cause DNA damage and lead to mutations (Ohshima and Bartsch.
1994). C. rodentiunm infection has been reported to cause acute inflammatory response
particularly in some strains of mice, such as C3H/HeJ (Barthold et al.. 1977). It would be
interesting to study the significance of NO production in mucosal epithelial cells during C.
rodentium infection.
MATERIAL AND METHODS
Animal experiments
Five different types of mice were used in the experiments.
They are: C3H/HeJ (Taconic Laboratory, Germantawn, NY), C57BL/6 (Taconic
Laboratory). AC3FI (A/J x C3H/HeJ), CB6FI (BALB/C x B6 APCMin.
BCF2 (backcross F2. BALB/C x CB6F1 APCMin',
2 are
,
V2 are Min),
Min). All mice were H.
hepaticus-free except the AC3F 1 mice. Mice were housed in filtered cages in an
environment of controlled temperature. humidity and lighting. with water and certified
chow provided ad libitum.
At 4 to 6 weeks of age. mice in the experiments were genotyped for Min and then
grouped. and thereafter infected with DBS434 (a chloramphenicol-resistant strain of C.
rodentiumr) and/or treated once with AOM at 10 mg/kg. as detailed in earlier chapters.
The experimental procedures are summarized in Table 4-1. At different intervals
postinfection. mice were killed with CO2 and subjected to necropsies. One hour prior to
killing. mice received a single intraperitoneal injection of BrdU (from Sigma. freshly
dissolved in PBS at 5mg/ml) at a final dose of 50 mg/kg body weight.
Histological Evaluation
At the time of necropsy, distal colons were removed
and incised longitudinally, rinsed with PBS, fixed in Carnoy's fixative overnight, and then
embeded in parafin wax. Five-micron-thick cross-sections were made from these parafin
blocks and used for immunohistochemical stains or hematoxlin and erosin stains for
histological evaluation. A piece of stomach tissue was also embeded with the distal colon
tissue as a control for BrdU staining.
BrdU lmmunocytochemistry
Immunohistochemical detection of the BrdU
incorporation was performed on 5-gm sections of embeded, Carnoy's-fixed tissue, and
visualized using the following avidin-biotin monoclonal antibody immunohistochemical
technique. After deparaffinization in xylene to ethanol, sections were passed through a
graded series of ethanol to water, then incubated in 4 N hydrochloric acid at room
temperature for 30 minutes. to denature the DNA to single strands (BrdU antibody only
recognizes single strand DNA). After extensive washes with water, the sections were
incubated with I drop of Peroxoblock (Zymed Co.. South San Francisco. CA) for 45
seconds. to block endogenous peroxidase activity. The slides were then rinsed with water
and incubated with 5% normal rabbit serum for 10 minutes to block nonspecific binding of
the primary antibody. Then. without washing off the serum. 1:50 diluted (with Trisbuffered saline) monoclonal mouse-anti-BrdU antibody (Zymed Co.. South San Francisco.
CA) was added to the slides and incubated for 1 hour. Control slides were incubated with
Tris-buffered saline instead. The sections were washed in Tris-buffered saline and
incubated with 1:100 diluted peroxidase-conjugated rabbit-anti-mouse immunoglobulin G
antibody (Dako Co.. Carpinteria. CA) for 30 minutes. After thoroughly washing with
Tris-buffered saline. stained slides were visualized by the diaminobenzidine reaction and
lightly counter-stained with hemotoxylin. The nucleus of cells containing BrdU. an
indication that cell are at the S-phase of the cell cycle, were stained to dark brown
color. The average crypt height for each animal was also recorded.
Labeling index measurement
Under microscopic inspection, pictures of
longitudinally sectioned colonic crypts were taken for labeling index (LI) counting. A
minimum of 3 photos with viewable full length of the crypts were taken. A mean number
of 10 well-orientated crypts per photo were chosen for LI counting. LI was measured by
counting the number of BrdU-positive cells and expressing the result as a percentage ratio
of total number of cells in chosen crypts.
iNOS and F4/80 immunohistochemical staining Five gtm thick tissue slides
were also used in iNOS and F4/80 staining. Deparaffinized and rehydrated slides were
fixed in formalin for 1 minute, washed extensively, then incubated with peroxoblock for 45
seconds. Slides were then incubated with CAS block (Zymed) for 10 minutes. to block
the non-specific binding. then 1:200 PBS-diluted polyclonal rabbit anti-mouse iNOS
antiserum (or 1:200 PBS-diluted rat anti-mouse F4/80 antibody) were added and
incubated together for 2 hours at room temperature. Thereafter goat anti-rabbit IgG
conjugated to biotin (Dako) or rabbit anti-rat IgG conjugate in case of F4/80 (Dako) was
incubated with the sections for 10 minutes. followed by incubation with alkaline
phosphatase-conjugated strepavidin (Dako) for 10 minutes. Color was developed by'
dianminobenzidine reaction and counter-stained with Mayer's hematoxylin. In all cases. a
slide of spleen from tumor-bearing SJL mice was stained as a positive control (Gal et al..
1996).
Statistical analysis
The total labeling index was compared between the study
groups and different intervals. Significance was analyzed using the two-tailed Student's ttest for unpaired data. In every case, P values of <0.05 were considered statistically
significant.
RESULTS
Genetic difference in symptoms after infection
All mice inoculated with
overnight culture of C rodentium strain DBS434 were infected. Except for C3H/HeJ
mice. no significant clinical symptoms were found in these mice. C3H mice showed signs
of listlessness and ruffled pelage starting 6 days postinfection. and became moribund at the
ninth day after infection. At necropsy. colon of all infected mice displayed typical signs of
TMCH: pale. thick and rigid. often empty. with colons of C3H/HeJ and AC3FI mice the
most severe.
C. rodentium increase LI in all infected mice
LI in the colon of all infected
mice were found to be elevated. Results are summarized in Table 4-2. BrdU-labeled cells
in control mice were limited to the lower 1/3 of the height of the crypt. LI was between 7
and 8% in control mice. regardless of strains. but was significantly elevated by I- to 2-fold
in infected mice. The increase in LI was most dramatic in C3H/HeJ and AC3FI mice.
The average crypt height was also increased in all infected mice. Similar to LI. crypt
height increases were the greatest in C3H/HeJ and AC3FI mice (data not shown). A
demonstration of the cytokinetic change is presented in Fig. 4-1.
Min mutation and AOM-treatment do not change LI
In infected
BCF2 Min mice. LI and crypt height were also found to be elevated, and the level was
comparable to that of BCF2 wild-type mice. In BCF2 wild-type mice three days after
AOM injection, regardless of whether or not they were infected, LI and crypt height
decreased (Table 4-2). but the decrease was not significant. Min BCF2 exhibited similar
properties.
Macrophages infiltrate after infection but no INOS observed
Staining for
macrophage surface antigen F4/80 revealed a significant increase in macrophage numbers
starting 6 days after infection. These macrophages seem to cluster in the basal matrix and
in-between the crypts. Again, in C3H/HeJ and AC3FI mice, the macrophage influx was
greatest. while in other mouse types, similar increases were also observed but somewhat
less dramatic. These changes are demonstrated in Fig. 4-2. Surprisingly. no positive
iNOS co-stained in the same region in any mouse, regardless of strains, although the
positive control worked well.
DISCUSSION
In this experiment. we demonstrated that C. rodenlium infection induced murine
colonic mucosal hyperplasia and quantitatively observed the elevated proliferation by
BrdU incorporation. This is the first time a quantitative measurement of cell proliferation
has been done on C rodentium-infected mice of different genetic backgrounds. The
results. an increase of I- to 2- fold in LI and a dramatic increase in crypt height. were
consistent with the findings by Barthold et al. using [methyl-'H]thymidine
autoradiography in NIH Swiss mice (Barthold. 1979). Mice with different genetic
backgrounds showed differences in the magnitude of increases in cell proliferation. with
C3H./HeJ and AC3FI mice exhibiting the greatest magnitude. Although no previous C.
rodentium infection data are available for A/J mice. it is tempting to assume a dominant
susceptibility for C. rodentium induced hyperplasia. Further studies on effects of genetic
background to induced TMCH and mechanisms of mucosal hyperplasia are necessary to
evaluate this hypothesis.
Our results also demonstrated that the presence of Min allele did not change cell
proliferation promoted by the C rodentium infection. The APC gene product is thought
to somehow control cell growth. Clearly the loss of one allele in Min mice did not affect
this cell growth control. which is also in agreement with the recessive nature of this tumor
suppressor gene. Spontaneous tumors in Min mice exhibit an 100% loss of the APC+
allele (ZZ. Chapter 3) (Levy et al., 1994: Luongo et al., 1994). It would be interesting to
study the cell proliferation of these self-arising tumors under the effect of C. rodentiumn
infection. which may provide a valuable insight in the function of the APC protein.
The observed moderate decrease in LI shortly after AOM administration is also
consistent with the previous observations (Deschner el al., 1983; Glickman el al., 1987).
The purpose of this part of the experiment was to examine the effect of Min allele on cell
growth in AOM treatment. Again, no significant effect on LI was detected.
F4/80 is a macrophage-specific surface glycoprotein of unknown function (Haidl
and Jefferies. 1996). Using a molecular marker, the observation in this report is the first
time that macrophage infiltration has been identified after the C. rodentium infection. The
inducible NO-svnthase (iNOS) is the sole biological nitric oxide producer in the
macrophages. My results with iNOS immunohistochemical staining suggested that the
infiltrated macrophages in response to C. rodentium infection did not produce detectable
amount of nitric oxide. The reason for this observation is unclear. Although C3H/HeJ
mice are LPS non-responder (Watson el al.. 1978). other strains of mice are all LPS
normal responders. Immunohistochemical staining of iNOS in murine colon tissue is still
a novel field and deserves more scientific attention.
Table 4-1. Experiment Overview: Number of mice used in the experiment'
# of Mice
Wild-Type Mice
Min Mice
Used
C3H/HeJ
AC3FI
B6
CB6F1
BCF2
CB6FI
BCF2
Vehicle
3
3
4
5
5
3
5
Cr. 6 days
3
3
5
-
-
-
-
Cr. 9 days
4
3
5
5
5
4
5
AOM
-
-
-
-
5
-
5
AOM/Cr
-
-
-
-
5
-
5
1 Both
sexes of mice are used. except AC3FI mice which are all females.
2 Mice
were killed 3 days after i.p. injection of AOM at 10 mg/kg.
·
Six days postinfection of C.rodentium (Cr.). mice were given AOM i.p. at 10
mg/kg. Three days later mice were sacrificed.
Table 4-2. BrdU labeling indexes in experimental animals
Labeling
Wild-Type Mice
Min Mice
Index(%)
C3H/HeJ
AC3FI
B6
CB6FI
BCF2
CB6FI
BCF2
Vehicle
8.6+1.7
8.4+2.8
7.8+0.8
7.7+0.9
8.2+1.1
7.3+1.5
8.0+0.5
Cr.6 days
14.8+4.5
13.3+3.2
10.7+2.2
Cr. 9 days
19.5+6.4
21.0+6.8
11.8+0.6
13.3+1.3
13.4+0.8
13.6+1.6
12.3+1.7
AOM
-
-
-
-
7.4+2.1
-
7.1+1.3
-
10.9+2.3
-
10.7+1.9
AOM/Cr.
Note: All results expressed as mean + SD. A mean of 25 colonic crypts was analyzed per
mouse. Cr.. . rodentiunm.
Fig. 4-1 Cytokinetical changes after C. rodentium infection in a C3H/HeJ mouse
BrdU immunohistochemical staining of a distal colon from A) an uninfected C3H/HeJ
mouse. B) an infected C3H/HeJ mouse. Notice the increase in LI and crypt height.
104
Fig. 4-2 Macrophages infiltrate after C. rodentium infection in an AC3F1 mouse.
Macrophages were stained brown by using F4/80 antibody. A) Colon of an uninfected
female AC3F1 mouse. B) Colon ofa C. rodentium infected female AC3F1 mouse.
105
Chapter Five
Summary
Citrobacterrodentium, an naturally-occuring murine pathogen, colonizes the distal
colon of mice and causes transmissible murine colonic hyperplasia. This transient
epithelial hyperplasia is likely to be a nonspecific, protective response to luminal cell
damage. The resulted cytokinetic changes, including increase in mucosal labeling index,
elongation of proliferative zone and crypt height, are similar to those seen in humans
suffering from proliferative bowel disorders. If infected mice are dosed with DMH
concurrently, the hyperplastic state serves to promote the formation of ACF, which are
believed to be early colonic neoplastic lesions. C. rodentium-inducedmucosal hyperplasia
may be a good model for cancer susceptibility in patients suffering from ulcerative colitis.
a disease with a high risk of developing colorectal cancer. It is hoped that studying the
relationship of C. rodentium and murine colon tumorigenesis will provide valuable insight
to how bacteria affect cancer development in humans. I chose to carry out this
investigation one step further by studying the tumor promotion effect of C. rodentium. and
characterizing the molecular basis of induced tumors.
I have demonstrated that C. rodentium infection eventually increases the tumor
burden by nearly two fold in AOM-treated mice. In Min mice. compared to uninfected
animals. I observed a significant increase in colonic tumor incidence as a consequence of
C. rodentium infection. While studying the cell proliferation in this hyperplastic state, I
showed that the hyperplasia resulted in a significant two fold elevation in BrdU labeling
index. and this elevation is affected by genetic backgrounds of mice. but not the presence
of a Min allele. Although an infiltration of macrophage was seen, no inducible-form of
nitric oxide synthase has been found to co-stain with the macrophages. suggesting possibly
that NO is not involved in C. rodentium promoted colonic tumorigenesis. The 2 folds
107
elevation in BrdU uptake and tumor burden provides a basis for quantitative modeling cell
proliferation and carcinogenesis.
I performed 12h' codon K-ras mutation detection in harvested colonic tumors. My
results indicated that approximately 20% of tumors from AOM-treated mice possessed Kras mutations, whereas no K-ras mutation was found in spontaneous colonic tumors or
small bowel tumors from Min mice. These mutations are mainly G to A transitions,
which are consistent with the theory that AOM causes G to A transitions through inducing
O-methylguanine. In addition. tumors that harbor K-ras mutation were larger in size,
suggesting that K-ras 12th codon mutations appear relatively late in tumorigenesis. One
tumor had two independent K-ras mutations. Taking all these findings together, K-ras
mutation is very likely not required in the early events of colonic tumorigenesis, but may
play a role in the later stages. Because AOM was administrated in a repeated weekly
regime. these K-ras mutations may arise from the neoplastic crypts instead of normal cells.
and provide a growth advantage accelerating further tumor development. Certainly. K-ras
proto-oncogene can be activated by mutations at codons other than the
.
12 th or
simply by
over-expression. These possibilities need to be investigated to reach a clearer
understanding on the roles of K-ras in colonic tumor development. Genome-wide LOH
screening was also performed on these colon tumor samples. Tumors from AOM-treated
mice. including those from Min mice, showed low frequency of random LOH. In contrast.
in tumors from untreated or C. rodentium infected Min mice, nearly 100% of the tumors
exhibited APC+ allelic loss. Two other specific LOH hot spots were also identified.
These loci are not linked to known tumor suppressor genes or to LOH hot spots in human
colorectal cancers. Further studies are necessary to evaluated the significance of these
108
loci. It is possible that AOM may induce point mutations and thereby inactivate the
critical tumor suppressor gene(s). so that LOH is not necessary in tumorigenesis. Above
all. C. rodentium infection. although it did enhance tumorigenesis in both AOM induction
and Min mouse model, did not seem to alter the K-ras mutational frequency or LOH
patterns in harvested tumors. These data support the hypothesis of an epigenetic basis of
tumor promotion by C. rodentium. and suggest that an elevation in cell proliferation is
correlated with an increase in cancer risks.
Importantly. the established Citrobaciertumor promotion model opens many
opportunities for further investigation. Some of the possibilities are listed in the following:
1) Colorectal tumorigenesis proceeds through an accumulation of specific genetic
alterations. Studies of the mechanism by which these genetic changes affect malignant
transformation have focused on the deregulation of cell proliferation. However. colorectal
epithelial homeostasis is dependent not only on the rate of cell production but also on
apoptosis. a genetically programmed process of autonomous cell death. Apoptosis has
been suggested to play a role in colonic tumorigenesis (Bedi et al., 1995; Ijiri. 1989: Shiff
et al.. 1995; Tatebe et al.. 1996). It maybe that C. rodentium infection modifies the
apoptosis status in mucosal epithelial cells, thereby given mutant cells a higher chance to
grow and survive. Inhibition of apoptosis can potentially contribute to the elevated cell
numbers in C. rodentium induced hyperplasia. AOM treatment and the loss ofAPC+
allele in Min mice may also have their effects on apoptosis. Very little work has been
done on apoptosis in colonic tumorigenesis. It would be very informative to study
apoptosis in the hyperplastic state and in tumors either induced by AOM or self-arising
from Min mice.
109
2) data from my experiments support the epigenetic basis of tumor promotion by
C. rodentium. This hypothesis can be directly tested by studying mutational frequencies in
infected 'Blue' mice, an engineered transgenic murine system that enables in vivo
mutagenesis studies. These mice have a plasmid containing a reporter LacZ gene in their
genome. thus enable color mutant selection in bacteria. In addition, AOM has been
implied to cause G to A transitions. but this theory has never been directly demonstrated.
These 'Blue' mice can potentially be used for in vivo AOM mutagenesis studies, to answer
the question whether or not the mutagenicity of AOM is affected by Citrobacter infection.
3) I used H. hepaticus-free Min mice in my experiments. H. hepaticus has been
shown to colonize the intestines. Previously. I have noticed a reduction of tumor number
and elongated latent period for tumorigenesis in H. hepaticus-free B6 Min mice.
comparing to those of infected ones. To investigate the possibility that these bacteria may
affect tumorigenesis, I infected 10 male CB6F
Mn-
mice with H. hepaticus, and killed
these mice 6 months postinfection. At necropsy, no increase in colon tumor incidence was
found. The SINI scores in these mice were not significantly different from those of
uninfected mice. This preliminary result suggested that H. hepaticus infection might not
affect the tumor development in the intestine of a Min mouse as I suspected. However. it
maybe that the effects of H hepaticus take long to appear. A carefully designed
experiment with longer duration (or with B6 Min mice) and larger animal numbers are
necessary to reach a more conclusive result.
4) in tumors from AOM-treated animals, including Min mice, LOH was seldom
found. This is in contrast to nearly 100% APC+ allele loss found in spontaneous tumors.
My explanation is that AOM causes point mutations in the critical tumor suppressor
110
gene(s). therefore no LOH is necessary. APC gene seems very likely to be the critical
gene guarding the route to colonic neoplasm. It is therefore reasonable to believe that
AOM causes mutations in the APC gene thereby inactivate the suppressor function. There
are several approaches to investigate this possibility. One of the practical ones is called
IVSP method. for in-vitro synthesized protein method (Levy el al., 1994). This method is
used to detect null mutations in selected DNA sequence. The idea behind this technique is
to amplifqy suspected mutational hot-spot region with a pair of oligo primers containing a
transcription promoter. Then using a labeled protein expression system followed by
electrophoresis. one can identiA, the existence of any truncated protein band, which may
arise from a nonsense mutation. I tried this method on some tumor samples from AOMtreated mice using Promega TNT system. but failed to find any protein band other than the
full-length product one. Although the IVSP system can only detect null mutatiOns. it still
worth some in-depth efforts of investigation. This experiment needs to be repeated with
appropriate positive controls.
5) SJL mice were derived from the Swiss Webster mouse stain. These mice
spontaneously develop B cell lymphomas by 12 months of age and inflammatory muscle
disease (myositis) by 6 months of age. The spontaneous myositis, which resembles human
idiopathic myositis. is characterized by various abnormalities in skeletal muscle, including
infiltration with inflammatory cells consisting primarily of macrophages. SJL mice exhibit
an elevated NO production as early as 7 weeks of age compared to BALB/C mice (Tamir
et al.. 1995). As a mouse model with an exaggerated in vivo NO production. SJL mice
may provide a good system to study the effects of nitric oxide on C rodentium induced
mucosal hyperplasia. The first question is whether these mice overproduce NO in the
colon after infection. This can be answered by immunohistochemical staining for iNOS
and measuring urinary nitrate. If NO is overproduced as an reaction to Citrobacter
infection, then the function of NO can be investigated by comparing characteristics to
those of Swiss Webster mice, or to mice received NMA (N--methyl-L-arginine acetate),
an iNOS inhibitor. SJL mice are also potentially useful in evaluating roles of NO in Min
mice or AOM-induced carcinogenesis model.
6) my results and previous data demonstrated that all spontaneous tumors in Min
mice have lost their APC+ allele. These tumors then provide an in vivo APC knockout
system. which can be used to study the function of the APC proteins. It has been
suggested that APC protein may control cell growth. It would be meaningful to see how
these tumor cells react to proliferation stimulus such as C. rodentium induced mucosal
hyperplasia. There is some evidence suggested that neoplasia in Min mice started very
early in life. Therefore the observed tumor promotion by C. rodentium could be the effect
of the proliferation stimulus over the APC - cells. Immunohistochemical analysis for BrdU
and apoptosis will be useful in this experiment, to evaluate the cellular homiostatsis.
7) (C.rodentium infection and AOM treatment in Min mouse form a nice one-hit
system for cancer modeling. Min mouse has already inherited one faulty APC gene. As a
potential mutagen. AOM may provide the second hit which in turn inactive the APC+
allele. The proliferation stimulus from C. rodentium infection can accelerate tumor
development. Mutational frequency of AOM can be estimated from the 'Blue" mice. while
BrdU and apoptosis analysis may provide quantitative measurement for cell turnover.
Different strains. doses and timing may be used as variables. This highly manageable
system for in viiro cancer modeling offers features that few other systems can match.
112
8) to continue studying the role of K-ras in colonic tumorigenesis. it is important
to also screen other codons for mutations. K-ras mutations at the 12th codon cover
approximately 70 to 80% of total ras mutations found in tumors from DMH/AOM-treated
animals. It is possible that mutations may arise at other hot-spots of ras mutations, codon
13 and 61. for example. but unlikely in H-ras. Screening for mutations at these codons
can be done by applying similar designed mismatched primer PCR-RFLP methods to
available tumor DNA. Also, tumors that harbored K-ras mutations seemed to be larger in
size. More tumors with K-ras mutations should be examined to better correlate the K-ras
mutations with the degree of tumor progression. In addition, ras proto-oncogene can be
activated by over-expression. Thus it is also important to establish an
immunohistochemical method to detect ras proteins in colon tissue samples and tumors.
There are commercial monoclonal antibodies (e.g.. Oncogene Science Inc.) available
against ras and rasASP-l" (GGT to GAT mutation). To my knowledge. inadequate efforts
have been made to apply these approaches to a murine colonic tumor model.
9) to overcome the poor breeding performance of the B6 Min mice. I started to
transfer the Min allele to H. hepaticus-free BALB/c background by continuously
backcrossing the CB6FI Min mice. So far, mice are at their 5th backcross. These mice
exhibit less severe Min phenotype. perhaps due to the lack of Mom-1 (modifier of Min
gene) mutation in the BALB/c mice. Recent findings suggested that non-pancreatic
secretory type II phospholipase (Pla2s or sPLA2 or snpPLA2) maybe a good candidate for
the Mom-I gene (MacPhee et al., 1995; Praml el al., 1995). B6 and 129/sv mice carry
homozygous frameshift mutations in their Pla2s genes. whereas BALB/c. C3H/He and
DBA mice have normal genotype. Pla2s is upregulated to orders of magnitude by
cytokines during infection or inflammation, but its physiological role is largely unknown
(Vadas el al., 1993). An anti-inflammatory drug has been shown to also inhibit Pla2s
activity (Burke et al.. 1995). Besides it potential usefulness in H. hepaticus studies, H.
hepaticus-free BALB/c Min mice provide a good animal model to evaluate the role of
Pla2s in intestinal tumorigenesis.
10) Min mice and AOM-induced murine colon carcinogenesis models are
potentially useful in testing chemopreventive drugs for colorectal cancer. For example. no
one has demonstrated the effects of NSAIDs in Min mice. Similar studies have previously
been done mostly on rat carcinogenesis models. Mouse alternatives are less expensive
with a smaller, better-characterized genome. Also. the availability of various knockout
mice offers even greater advantages. The H. hepaticus-free Min mice in our lab will
provide more convincing results in these experiments.
11) the significance of the two observed LOH hot-spots in Min mice needs to be
further investigated. A higher number of spontaneous tumors and more SSR markers
around the hot-spot area are necessary to carry on the investigations. CB6FI Min mice
have one copy of the normal Pla2s gene which may be inactivated during tumorigenesis.
Since Mom-I locus is on chromosome 4, it is worthwhile to select a few markers to
evaluate the possibility that LOH may occur at regions closely linked to Mom-I locus.
Besides its implications for colorectal cancer in humans, my thesis work
established a good example for studies the relationship of bacteria and cancer. Similar
approaches can readily be adopted and used in Helicobacterresearch. The research
opportunities opened by this thesis work are not limited to the list above. Further
investigation will lead to better understanding to many questions in cancer research.
115
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