Colorectal cancer is one of the most predominant cancers in the Western World. By
the age of 70, 1 in 2 people will have a colon tumour. The genetic analysis of both
spontaneous and hereditary forms of this disease have greatly added to our
understanding of colorectal cancer, with mutations in a range of genes now strongly
linked to neoplasia. In recent years, several different transgenic models of colorectal
cancer have been generated. Key amongst these are strains mutant for the Ape
(.Adenamatous Polyposis Coli) gene and strains mutant for different members of the
mismatch repair (MMR) gene family, the majority of which show predisposition to
intestinal neoplasia. In this thesis, these transgenic models are used in an attempt to
systematically characterise the nature of the genetic control over a series of end
points. These include the apoptotic response of enterocytes to cytotoxic agents; the
effect of genotype upon clonogenic survival and mutation and ultimately the effect of
genoytype upon the development of intestinal neoplasia.Previously analyses of enterocyte apoptosis had established roles for p53 and Msh2
following DNA damage of methylation type. This prompted an analysis in mice
deficient for two other members of the MMR family, Mlhl and Pms2. Mlhl and Pms2
deficient mice were seen to have a significantly reduced apoptotic response to
temozolomide, confirming again an association between the MMR family and
apoptosis. However, both Mlhl'" and Pms2~/~ mice were found to possess a normal
apoptotic response to high levels of the alkylating agent NMNU, even though they are
deficient for functional MMR. This unexpected finding dissociates normal mismatch
repair from MMR dependent apoptosis and raises fundamental questions about the
nature of the death signal following damage of methylation type.Perturbations to the normal apoptotic response would be predicted to impact upon
longer term survival as determined through the microcolony assay. Therefore
clonogenic survival was examined using this approach in Msh2 and p53 null mice.
Despite being necessary for apoptosis for all cytotoxic agents studied, loss ofp53 only
led to an increased in survival following cisplatin treatment and not following NMNU
or Nitrogen Mustard treatment.The above data was obtained from morphologically normal tissue. Therefore this
analysis was extended to the apoptotic response within intestinal lesions. This
displayed that there was both lesion type dependent differences and genotype
dependent differences in the apoptotic response. As there were high basal levels of
apoptosis in the smallest lesions whilst virtually no apoptosis in adenomas, this
highlighted that loss of this apoptotic programme may be crucial to tumour
progression.The battery of in vivo analyses used throughout this thesis were applied to a new
candidate tumour suppressor, Mbd4. Mbd4 deficient mice have no overt phenotype,
but fail to mediate normal apoptosis following a wide variety of DNA damage.
Following cisplatin treatment, Mbd4 treatment confers increased clonogenic survival
Surprisingly, Mbd4 mice are not characterised by an in increase in either spontaneous
or induced mutation rate, but when crossed to ApcMm mice they accelerate tumour
development. These studies demonstrate that Mbd4 is a central mediator of the
response to DNA damage and that it functions as an intestinal tumour suppressor in
the mouse.Finally the ability of aspirin to suppress intestinal neoplasia in murine models of
colorectal cancer was examined. Numerous epidemological and animals studies have
shown that Non-Steroidal Anti-Inflamatory Drugs (NSAIDS) are associated with
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lower risks of colorectal cancer. However studies using aspirin in the ApcMm/+ mouse
have yielded contrasting results. Here it is shown that aspirin does not reduce
tumourigenesis when ApcMl + mice are put on diet containing aspirin post weaning.
However when parents were put on aspirin, a significant suppression of
tumourigenesis was observed in the min offspring. In fact there was incomplete
penetrance of the ApcMm/+ phenotype (40%). To test whether in utero administration
of aspirin could also suppress murine models of HNPCC, Msh2 deficient and
(ApcMm/+, Msh2~/~) deficient mice were examined. In both cases a significant
attenuation of tumourigenesis was observed. Taken together this raises the exciting
prospect of prophylactic treatment of FAP and HNPCC patients and highlight the
power of using transgenic models to investigate intestinal neoplasia.The interaction between Msh2 and p53 in tumourigenesis was also investigated. Both
homozygosity and hemizygosity for p53 were found to dramatically accelerate
tumourigenesis on a mismatch (Msh2) deficient background. Significantly, the levels
of micro-satellite instability (MSI) were highest in tumours which were additionally
heterozygous for p53. EMSA, Western and immunohistochemisty analysis of these
tumours indicated retention of p53 function in at least a proportion of these tumours.
Similar data were obtained from primary cultures, with again increased microsatellite
instability and retained p53 functionality in cultures derived from p53 heterozygotes.
Taken together, this data shows that hemizygosity for p53 increases microsatellite
instability and that, at least in a percentage of tumours, complete loss of p53 is not a
required event. These findings have particular relevance to our understanding of cross
talk between p53 and MMR deficiency in human colorectal disease
