Hindawi Publishing Corporation
`Journal of Biomedicine and Biotechnology
`Volume 2011, Article ID 473964, 14 pages
`doi:10.1155/2011/473964
`
`Review Article
`Morphological and Molecular Alterations in
`1,2 Dimethylhydrazine and Azoxymethane Induced Colon
`Carcinogenesis in Rats
`
`Martina Perˇse and Anton Cerar
`
`Institute of Pathology, Medical Experimental Centre, Medical Faculty, University of Ljubljana, Korytkova 2, 1105 Ljubljana, Slovenia
`
`Correspondence should be addressed to Martina Perˇse, martina.perse@mf.uni-lj.si
`
`Received 14 September 2010; Revised 30 October 2010; Accepted 29 November 2010
`
`Academic Editor: Andrea Vecchione
`
`Copyright © 2011 M. Perˇse and A. Cerar. This is an open access article distributed under the Creative Commons Attribution
`License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly
`cited.
`
`The dimethyhydrazine (DMH) or azoxymethane (AOM) model is a well-established, well-appreciated, and widely used model of
`experimental colon carcinogenesis. It has many morphological as well as molecular similarities to human sporadic colorectal cancer
`(CC), which are summarized and discussed in this paper. In addition, the paper combines present knowledge of morphological and
`molecular features in the multistep development of CC recognized in the DMH/AOM rat model. This understanding is necessary in
`order to accurately identify and interpret alterations that occur in the colonic mucosa when evaluating natural or pharmacological
`compounds in DMH/AOM rat colon carcinogenesis. The DMH/AOM model provides a wide range of options for investigating
`various initiating and environmental factors, the role of specific dietary and genetic factors, and therapeutic options in CC. The
`limitations of this model and suggested areas in which more research is required are also discussed.
`
`1. Introduction
`
`Colorectal cancer (CC) is one of the leading causes of cancer-
`related morbidity and mortality in humans in western
`developed countries [1]. In recent years, increasing attention
`has been paid to environmental and food components, with
`the hope of identifying its preventive or carcinogenic effects
`[2, 3]. Much effort has been dedicated to a search for natural
`or pharmacological preventive agents, which would block or
`attenuate CC process [4, 5].
`There are several experimental models of CC, providing
`a wide range of options for investigating various initiating
`and environmental factors, the role of specific dietary and
`genetic factors, and therapeutic options in CC. These models,
`which can be broadly divided into induced and transgenic
`animal models, vary in their suitability for investigating
`each of these factors. Homologous recombination of random
`chemically induced mutagenesis has been used to generate
`a series of APC (+/−) mice that exhibit mutation in
`codons 474, 716, 850, or 1638 of the APC tumour sup-
`pressor gene and encode a truncated gene product. Similar
`
`recombination approaches have also generated a mouse
`model with a mutation in the DNA mismatch repair genes.
`Mlh1 (+/−), Mlh1 (−/−), Msh6 (+/−), and Msh6 (−/−)
`mice exhibit accelerated small
`intestinal carcinogenesis.
`These animal models have been generated to study familial
`adenomatous polyposis (FAP) and hereditary nonpolyposis
`colorectal cancer (HNPCC) syndromes and are described
`elsewhere [6]. Among chemically induced animal models,
`1,2 dimethylhydrazine (DMH) and azoxymethane (AOM)
`animal models are most frequently used [4, 7–9]. Induction
`of colon cancer by other chemical carcinogens, such as
`nitrosamines or heterocyclic amines is less frequently used
`[10–12], because DMH and AOM (DMH/AOM) are less
`expensive, more potent and more convenient to use [7, 9].
`The DMH/AOM model of colon carcinogenesis is a valid,
`well-appreciated, and widely used model of experimental
`colon carcinogenesis. It shares many similarities to human
`sporadic CC, including similarities in the response to some
`promotional and preventive agents [4]. Today, it is a widely
`used model for the evaluation of environmental, dietary, and
`chemopreventive agents in the colon cancer process. It is also
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`Journal of Biomedicine and Biotechnology
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`used to study morphologic and molecular mechanisms of the
`multistage development of colon cancer in order to elucidate
`new targets for chemoprevention [12, 13].
`A number of excellent reviews on animal models of
`colon carcinogenesis [6, 12], including chemically induced
`carcinogenesis [7, 13, 14], have recently been published.
`Various aspects of the applicability of the DMH/AOM animal
`model are demonstrated in these papers. However, in order
`accurately to identify and interpret alterations that occur
`in the colonic mucosa when assessing natural or pharma-
`cological compounds in an animal model, understanding
`both the morphologic and molecular development of the
`CC process in this model is required. This paper, therefore,
`summarizes the present knowledge of morphological and
`molecular features in multistep development of CC in the
`DMH/AOM rat model and its similarities to human sporadic
`CC. In addition, features and limitations of this model and
`suggested areas in which more research is required are also
`discussed.
`
`2. Induction of DMH/AOM Colon
`Carcinogenesis
`
`DMH and its metabolite AOM are widely used agents for the
`induction of colorectal carcinogenesis in rodents. DMH is
`metabolically activated in the liver by a series of reactions
`through intermediates AOM and methylazoxymethanol
`(MAM) to the ultimate carcinogenic metabolite, highly reac-
`tive methyldiazonium ion [15]. MAM can be excreted into
`the bile and transported to the colon (the development of
`small intestinal tumours distal to the entrance of the bile duct
`into the intestine is ascribed to this path) or enter directly
`into epithelial cells of the colon from blood circulation [15].
`Some studies have also demonstrated that rat colon epithelial
`cells are capable of metabolizing DMH into the carcinogenic
`metabolite without previous metabolism by other tissues or
`colon bacteria [16, 17]. The ultimate carcinogenic metabolite
`of DMH is responsible for methylation of the DNA bases of
`various organs, including epithelial cells in the proliferative
`compartment of the crypts, which result in a great loss of
`colonic cells by apoptosis, an increase in proliferation, and
`an apparent increase in mutations of colonic epithelial cells
`[18].
`However, DMH/AOM are highly specific carcinogens
`that induce colorectal tumours in a dose-dependent manner
`[19]. Various rat and mouse strains differ in susceptibil-
`ity to these carcinogens [12, 20]. The susceptibility for
`DMH/AOM-induced colorectal carcinogenesis is also sex
`[21] and age dependent [22, 23]. Most commonly, 6 weeks
`old male F344, Wistar and Sprague-Dawely rats are used
`[7, 8]. Colon carcinogenesis is usually induced by two s/c
`applications of DMH (150 mg/kg) or AOM (15 mg/kg) given
`one week apart. Using these protocols, animals are scored
`for intermediate biomarkers of colon carcinogenesis, termed
`aberrant crypt foci (ACF), 8–12 weeks after the application
`(short-term study) or for the number of colonic tumours 40
`weeks later (long-term study). Chemopreventive treatment
`can begin before exposure to the carcinogen and during
`
`the initiation phase, during the promotion or progression
`phase, or through both phases. These protocols are used
`to assess the promotional or protective effects of the tested
`factor and when followed closely provide data that are quite
`reproducible (protocol explained in detail by Femia and
`Caderni [7] and Bird [9]).
`Nevertheless, tumour outcome depends on the total
`amount of carcinogen administered and the latency period.
`In long-term studies, therefore, DMH is frequently admin-
`istered weekly for 15 weeks in a relatively low concentration
`(20 mg/kg). Repeated injections of carcinogen are evident at
`a later stage of cancer development and not in the stage of
`preneoplastic lesions, that is, ACF [24] and result in higher
`tumour incidence and multiplicity than following one or two
`injections of a colon carcinogen [19, 24].
`
`3. Histopathological Nomenclature of
`Colorectal Epithelial Lesions
`
`DMH/AOM colon carcinogenesis is a multistep process with
`morphological and histological features similar to those seen
`in human sporadic colon carcinogenesis [25, 26]. It is widely
`accepted today that the adenoma to carcinoma sequence is
`characterized by recognizable histological changes that start
`with dysplastic aberrant crypts or intraepithelial neoplasia
`(IEN) [27, 28]. These lesions then have the potential to
`progress to advanced adenomas, which have a significant
`potential to transform into adenocarcinomas [26].
`
`3.1. Intraepithelial Lesions. The first lesions in the multistep
`development of CC cannot be seen grossly. They can be
`identified in histological sections of colon mucosa after
`careful histological examination as hyperplastic or dysplastic
`epithelial lesions [29] or on the surface of whole mount colon
`under low-magnification stereomicroscope as ACF [24, 25]
`or mucin depleted foci (MDF) [28, 30]. It is important to
`bear in mind that visualization and identification of ACF
`or MDF on whole mount colon does not yield specific
`information on the histological features of these lesions. The
`structural and cytological features of ACF or MDF can be
`recognized or confirmed only after histological examination.
`Nevertheless, in order better to understand the histological
`background of ACF and MDA (which are described in the
`next section) as well as molecular alterations recognized and
`described at different stages of colon carcinogenesis, we will
`briefly summarize the histological criteria and classification
`of colorectal epithelial lesions in rodents, which share many
`similarities with human pathology [26, 29].
`
`Intraepithelial Lesions. Hyperplastic
`3.1.1. Hyperplastic
`epithelial lesions are composed of a mixture of goblet and
`absorptive cells, with enlarged or sometimes crowded nuclei
`without stratification. Mitotic figures are limited to the
`lower two thirds of the crypts and are never observed on the
`surface of crypts. Nuclei are basally located, ovoid or round,
`with occasional visible nucleoli and usually uniformly
`dark. The luminal opening of crypts is slightly elevated
`from the surrounding normal mucosa and the crypts are
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`elongated and occasionally branching, with partial mucin
`depletion [29]. It is worth mentioning that hyperplastic
`epithelium has never been observed in colonic tumours
`of the DMH/AOM rat model. It has only been found in
`intraepithelial lesions composed of a various number of
`crypts. The role of hyperplastic aberrant crypts in the
`process of colon carcinogenesis in DMH/AOM models is not
`clear and is a matter of debate and further investigation.
`
`3.1.2. Intraepithelial Neoplasia/Dysplasia. Inraepithelial neo-
`plasia is a histological term for dysplastic lesions in the
`epithelial layer of colon mucosa that cannot be identified
`macroscopically but only after careful histological exam-
`ination. The presence of dysplasia is regarded as early
`histopathological changes in the precursor lesions of colon
`cancer. The word dysplasia is used to describe structural
`and cytological alterations in the epithelium that predispose
`an organ to cancer development. IEN is synonymous with
`the terms atypical hyperplasia, microadenoma, carcinoma
`in situ, and dysplasia. Depending on the cytological and
`architectural features, IEN is classified as low grade or high
`grade. The differential criteria involve hypercelularity with
`enlarged, hyperchromatic nuclei, varying degrees of nuclear
`stratification, loss of polarity, high nuclear/cytoplasmic ratio,
`nuclear crowding, increased mitotic index, and decreased
`mucine excretion [29].
`
`3.2. Tumours. Pathological changes that can be seen macro-
`scopically on the surface of colon mucosa as pedunculated
`or broad-based, slightly elevated, flat, or depressed (sessile
`or nonpolypoid) masses/lesions are called tumours. The
`incidence of colon tumours is the most reliable endpoint
`for evaluation of the chemopreventive effects of substances.
`Further histological examination is required to determine the
`malignant or benign character of tumours.
`
`3.2.1. Adenomas. Tumours confined to the mucosa are
`benign neoplasms that are called adenomas. On the basis
`of the histologic type, adenomas can be tubular (when
`more than 75% of the adenoma is composed of branching
`tubules), villous (more than 75% of adenoma is composed of
`villous structures), or tubulovillous (25%–75% of adenoma
`composed of both tubular and villous structures). Depend-
`ing on the degree of dysplasia on the most severely dysplastic
`area of each tumour, adenomas are graded as low or high
`[29].
`
`3.2.2. Adenocarcinomas. Tumours that penetrate through
`the muscularis mucosa are malignant lesions, histologically
`denoted adenocarcinomas (well, moderately, or poorly dif-
`ferentiated). Based on the histological type, they are further
`classified into tubular, tubulovillous, or villous adenocarci-
`nomas (as with adenomas), mucinous adenocarcinomas (if
`more than 50% of the lesion is composed of extracellular
`mucin and signet-ring cell can be present), signet-ring
`cell adenocarcinomas (if more than 50% of tumour cells
`with prominent intracytoplasmic mucin are present), and
`
`undifferentiated carcinoma (if no glandular structure is
`present) [29].
`
`4. Biomarkers of Colon Carcinogenesis
`
`In the past, assessment of chemopreventive substances was
`based on the incidence of tumours. Since the development
`of tumours is a relatively lengthy process, taking around
`6–8 months to develop in the DMH/AOM rat model,
`preneoplastic lesions can be used as biomarkers for assessing
`the risk of developing colon cancer or for identifying
`modulators of colon carcinogenesis in short-term studies
`[9]. The use of preneoplastic lesions as biomarkers was not
`possible until 1987, when Bird [31] developed a simple,
`rapid and cheap methodological approach to detecting ACF
`[25, 31]. In the last decade, additional biomarkers of colon
`carcinogenesis have been identified, such as dark ACF [32],
`flat ACF [33], dysplastic ACF [34], MDF [30], and β-catenin
`accumulated crypts (BCAC) [35]. Their characteristics and
`application in short-term studies are briefly described.
`
`4.1. ACF. ACF are the first lesions in the development of
`CC that can be identified microscopically on the surface
`of the whole mount colon mucosa after methylene blue
`staining. They have been identified in carcinogen treated
`rodents [25, 31] and in humans at high risk of developing CC
`(personal or familial history) [36, 37]. A number of studies
`in rodents and humans, including molecular analysis, have
`shown that ACF are lesions that are a valuable intermediate
`biomarker in the development of colon carcinogenesis [38].
`ACF have to date been used as an endpoint in identifying
`and assessing the preventive or promotional role of natural
`and pharmacological compounds, as well as dietary and
`environmental factors, in the process of colon carcinogenesis
`[4, 5].
`An increasing number of studies have demonstrated
`that ACF in both animals and humans are a heterogeneous
`group of lesions containing multiple genetic, epigenetic,
`and phenotypic alterations [37–40]. Histologically, ACF
`exhibit variable features, ranging from mild atypia to
`severe dysplasia. Most ACF show a hyperplastic character,
`while only a small subgroup of ACF has been found to
`contain intraepithelial neoplasia (such as severe dysplasia,
`microadenoma, or carcinoma in situ). It has been shown that
`ACF with hyperplasia possess different genetic and epigenetic
`alterations than ACF with dysplasia, and some studies have
`suggested that ACF possessing hyperplastic feature may not
`be directly related to tumorigenesis [27, 28]. However, there
`are reports suggesting that some ACF possessing hyperplasic
`features may progress to ACF with dysplasia [40, 41].
`Nevertheless, ACF are useful biomarkers for the screen-
`ing of compounds for their chemopreventive activities [5,
`42]. When using ACF as biomarkers, it is important to
`take into consideration that ACF are a heterogeneous group
`of lesions. The total number of ACF may be considered
`to be a valid biomarker only at a very early stage of
`carcinogenesis, while, in subsequent weeks, ACF with higher
`crypt multiplicities (more than 4 crypts) are considered
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`Journal of Biomedicine and Biotechnology
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`a more specific biomarker than the total number of ACF. In
`more advanced stages of colon carcinogenesis, ACF may not
`be a reliable intermediate biomarker of colon carcinogenesis
`(explained in detail by Bird and Good [24] and Raju [42]).
`It is also important to mention that ACF are not equally
`distributed among the proximal, middle, or distal colon. The
`majority of ACF develop in the middle and distal colon [43–
`45], which needs to be taken into account when using ACF as
`biomarkers (comprehensively discussed by Bird [24, 25] and
`Raju [42].
`
`4.2. Subgroup of ACF with Dysplastic Features (Dark, Flat,
`and Dysplastic ACF). Since dysplasia is widely accepted as an
`indication of an increased risk of progression to cancer, it has
`been suggested that dysplastic crypts may be more directly
`associated with tumorigenesis than ACF [27]. Dysplastic
`ACF have recently been identified by various investigators
`using different approaches. Ochiai et al. [34] developed
`a differential staining method to identify dysplastic ACF,
`while identification of flat ACF [33] and dark ACF [32]
`were based on the surface morphology of ACF. However,
`all these lesions have been observed as subgroups of ACF
`with thicker epithelial lining, compressed luminal openings
`and mildly enlarged crypts, which were not elevated from
`surrounding epithelium. Histologically, all these subgroups
`of ACF have possessed dysplastic features with absent or
`scarce mucin production and have shown cytoplasmic and
`nuclear accumulation of β-catenin [32–34]. Based on a
`description of surface morphology (except the description
`of staining intensity) and histological characteristics, it is
`likely that flat, dark, and dysplastic ACF may represent the
`same group of ACF with dysplastic features. If each of these
`lesions represents a different subgroup of dysplastic ACF,
`their use as biomarkers would probably be questionable or
`confusing. Further investigations and determination of their
`relations are certainly needed before they can be used as
`biomarkers.
`
`4.3. MDF. MDF are identified on the mucosal surface of
`unsectioned colon after staining with high-iron diamine
`alcian blue (HID-AB), which visualizes crypts with mucous
`production [30]. Identification of MDF is based on a scarce
`or absent production of mucous, which is a common feature
`of severe dysplasia. In contrast to ACF, which are histologi-
`cally heterogenous, MDF are composed of dysplasic crypts,
`which display frequent genetic and epigenetic alterations
`observed also in colon cancer [46–49]. It has been shown
`that MDF appear 7 weeks after carcinogen administration
`and their number and multiplicity increases with time.
`MDF have been demonstrated as a potential biomarker
`for evaluation of the chemopreventive effects of natural or
`pharmacological compounds in colon carcinogenesis [50].
`Since few studies have evaluated MDF as a biomarker, further
`investigations are needed to evaluate their role in colon
`carcinogenesis.
`
`4.4. BCAC. In contrast to ACF and MDF, which can be iden-
`tified on the surface of the whole mount colon, identification
`
`Table 1: Nomenclature of colorectal lesions according to their
`morphological appearance on whole mount colon (low magnifica-
`tion) or according to their histological characteristics identified in
`embedded and stained colon sections under a high-magnification
`microscope.
`
`Morphological
`description
`Tumour
`(i) polypoid
`
`(ii) nonpolypoid
`
`ACF (methylene
`blue)
`(i) “dysplastic”
`
`(ii) dark
`
`(iii) flat
`
`Histological description
`
`Adenomas:
`(i) low-grade dysplasia
`(ii) high-grade dysplasia
`Adenocarcinomas:
`(i) well, moderate, and poorly
`differentiated adenocarcinomas
`(ii) mucinous adenocarcinomas
`(iii) signet-ring cell adenocarcinomas
`(iv) solid or undifferentiated carcinomas
`
`Intraepithelial lesions:
`
`(i) hyperplastic
`(ii) dysplastic/intraepithelial
`neoplasia/microadenoma/carcinoma
`
`in situ
`
`MDF (HID-AB)
`
`(a) BCAC (immunohistochemical
`staining)
`ACF, aberrant crypt foci; BCAC, β-catenin accumulated crypts; MDF, mucin
`depleted foci; HID-AB, high-iron diamine alcian blue.
`
`of BCAC is based on an immunohistochemical method
`in sectioned colon [35]. BCAC are intraepithelial lesions
`that accumulate β-catenin protein in the cytoplasm and
`nucleus and harbor frequent β-catenin (Ctnnb1) mutations.
`Histologically, BCAC shows dysplasia with reduced or absent
`mucin production [35, 51].
`Based on the assumption that mutations in the β-catenin
`gene or accumulation of β-catenin are the necessary first
`step in rat colon carcinogenesis, crypts with increased β-
`catenin expression have been proposed as a more relevant
`biomarker of colon cancer than ACF [27, 28, 35, 51].
`However, histological identification and quantification of
`BCAC is relatively costly, tedious, and time consuming,
`which limits the use of BCAC as a biomarker.
`However, we do not know at present whether BCAC,
`MDF, dark, flat, and dysplastic ACF are related lesions.
`The development of
`these dysplastic lesions is clearly
`related to the early development of tumours. Histologically,
`all
`these lesions show dysplasia with scarce or absent
`production of mucin and accumulate β-catenin in the
`cytoplasm and/or nucleus. Accordingly, it is possible that
`all these lesions are only dysplastic subgroups of ACF,
`which may predict tumour outcome better than ACF itself.
`However, first investigations suggest that this assumption
`may not be correct, because these lesions may not overlap
`completely [52, 53]. Further investigations are, therefore,
`needed to elucidate their features, evaluate relations and
`discrepancy among them and to identify the reason for
`discrepancy.
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`
`Hyperplastic
`crypts
`
`?
`
`Normal
`mucosa
`
`Dysplastic crypts
`
`Adenoma
`
`Carcinoma
`
`Biomarkers
`
`Hyperplastic ACF ?
`
`Dysplastic ACF, flat ACF,
`dark ACF, MDF, BCAC
`
`Tumours
`
`Mutations
`
`↑expression
`
`↓expression
`
`K-ras
`
`Cyclin D1,
`PPAR-δ,
`COX-2
`
`K-ras, β-catenin, Apc
`
`K-ras, β-catenin, Apc, α-catenin
`
`β-catenin, cyclin D1, c-myc, c-fos,
`c-jun, Akt, PPAR-δ, mmp-7
`LMW PTP, FAS, MAPK
`
`β-catenin, cyclin D1, c-myc, c-fos,
`c-jun, Akt, PPAR-δ, mmp-7, LMW
`PTP, FAS, MAPK , iNOS, COX-2
`TGFα, TGFβ,
`
`TGFα, TGFβ
`
`TGFα, TGFβ , E-cadherin
`
`E-cadherin, Smad3,
`
`Figure 1: Phenotypic, genetic, and epigenetic alterations involved in multistep development of colon carcinogenesis in the DMH/AOM rat
`model.
`
`5. Gene Mutations in DMH/AOM Colon
`Carcinogenesis
`
`involving
`Colon carcinogenesis is a multistage process,
`multiple genetic and epigenetic changes that provide tumour
`cells with a selective advantage to expand their clones
`[54]. The stepwise development of CRC from dysplastic
`crypts, adenomas to carcinomas provides opportunities for
`the investigation and identification of molecular alterations
`at various stages of tumour development [24, 25, 55].
`Genes that are mutated at different stages of colorectal
`carcinogenesis in human sporadic CC have been found to be
`also mutated in DMH/AOM-induced colon carcinogenesis
`and are described and discussed below.
`
`5.1. Apc/β-Catenin. Mutations in the tumour suppressor
`gene, APC, are responsible for an inherited predisposition
`to colon cancer, FAP. APC mutations are also believed to
`be the earliest events in the formation of sporadic colon
`adenomas [54]. They have been identified in up to 80%
`of sporadic colon tumours in humans [56]. The most
`common APC mutation in human colon adenomas is APC
`loss of heterozygosity (LOH), which causes truncation of
`the protein and its inactivation. It is believed that the
`main function of APC is the regulation of free β-catenin
`in concert with glycogen synthase kinase 3β (GSK-3β) and
`other proteins [54, 57]. It has been found that half of human
`colon tumours with intact APC protein have a mutation in
`the β-catenin gene [26].
`Apc mutations have also been identified in colorectal
`epithelial lesions in the DMH/AOM rat model, albeit to a
`
`lesser degree and in a different region from that observed
`in humans [58]. In DMH/AOM-treated rats, up to 33% of
`colon tumours harbour Apc mutations. These Apc mutations
`have frequently been found located upstream from the region
`corresponding to the human APC mutation cluster region
`(nt 3,186–3,810; nt 3078 and 3835 in exon 15). They have
`mostly been missense or truncated point mutations (G to
`A and C to T transition) [58–60]. A deletion of the region
`containing the Apc gene has recently been found in one out
`of ten tumours, suggesting that LOH may also be involved in
`inactivation of Apc in this model [61].
`In the DMH/AOM rat model, β-catenin mutations are a
`more frequent event than Apc mutations, occurring in up to
`77% of tumours (Table 2). They are mainly point mutations
`(G to A transitions) localized in the GSK-3β phosphory-
`lation consensus motif, which result in the inhibition of
`GSK3 β-dependent phosphorylation of β-catenin. However,
`Apc mutations or mutations in β-catenin have only been
`observed in the DMH/AOM rat model and in human CC in
`neoplastic/dysplastic lesions, that is, microscopic dysplastic
`epithelial lesions, adenomas, and adenocarcinomas (shown
`in Table 2) but not in hyperplastic lesions [35, 48, 58, 59, 62,
`63].
`
`5.2. K-Ras. The K-RAS gene encodes membrane bound
`protein with intrinsic GTPase activity, which is involved in
`the regulation of a number of important normal cellular
`functions, including proliferation, differentiation, and apop-
`tosis. Single point mutations at specific sites within ras genes
`activate their oncogenic potential [64]. K-RAS mutations
`have been observed with various frequencies (∼40%–50%)
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`in human colorectal neoplastic lesions, as well as ACF with
`hyperplastic epithelium. It has been reported that K-RAS is
`mutated in fewer than 10% of small adenomas (less than
`1 cm in size), in about 50% of large adenomas (larger than
`1 cm) and in approximately 50% of carcinomas [56].
`In DMH/AOM rat colon carcinogenesis, mutations in
`the K-ras gene are point mutations (G to A transitions),
`observed mostly in codon 12, rarely in codon 13 or 59. The
`frequency of K-ras mutations in rat colon carcinogenesis has
`been found to be variable, particularly in studies without
`a description of the histological features or size of the
`analyzed tumours [65–68]. Nevertheless, some studies have
`demonstrated that K-ras mutations are less frequent in small
`adenomas (on average ∼16%), while more frequent in large
`adenocarcinomas (∼53%) [62, 69]. On the basis of those
`studies, it appears that K-ras mutations in the DMH/AOM
`rat model are as frequent as in human colon carcinogenesis.
`As in humans, a K-ras mutation has been observed more
`frequently in ACF with hyperplastic epithelium than in ACF
`with dysplastic features [40, 62, 70, 71]. The mutation of
`K-ras may thus be involved in the formation of the ACF
`lesion and promotion of lesion growth but is probably not
`essential. The fact that K-RAS mutation is frequently and
`APC mutation very rarely observed in ACF in humans has
`triggered debate about the role of K-RAS mutation in colon
`carcinogenesis (discussed by Pretlow [37]).
`
`5.3. p53. Although aberration of the tumour suppressor
`P53 is a very common genetic lesion identified in human
`carcinomas, no mutations in the p53 gene in DMH/AOM rat
`colon carcinogenesis have been found by the single-strand
`conformation polymorphism method, direct sequencing or
`immunohistochemical analysis [59, 66, 72, 73]. Mutations
`in the p53 gene have been investigated in regions (exon 5–
`7) corresponding to the most commonly mutated regions
`in human colon cancer (exon 5–8) [59]. It is interesting
`that positive reactivity for p53 in ACF (27/65; 42%) and
`adenocarcinomas (6/8; 25%) by immunohistochemically
`was one of the first reported alterations in ACF [74].
`
`5.4. Genomic Instability. Two types of genomic instability
`have been found in human colon cancer, that is, microsatel-
`lite instability (MSI) and chromosomal instability (CIN).
`MSI leads to an increase in the rate of subtle DNA changes
`whereas CIN enhances the rate at which gross chromosomal
`changes occur during cell division, such as chromosome
`breaks, duplication, rearrangements, and deletions such as
`LOH on varies sites in the genome [75].
`In the DMH/AOM model, one or two chromosomal
`aberrations (amplification or deletion from 66 to 2135 kb
`in length) were found in 4/10 of tumours using a-CGH
`analysis. In one case, even a LOH of the Apc gene on
`chromosome 18 was found [61]. On the other hand, an
`investigation based on RAPD analysis with 21 random
`primers (GC rich) detected small alterations in the genome
`in 100% of analyzed tumours (16/16) and even in 70%
`of ACF (7/10) [67]. When tumours were analyzed on MSI
`using 10 different microsatellite DNA markers, it was found
`
`that 29% of colonic tumours (13/45) showed MSI in at
`least one locus and 15% of tumours (7/45) showed MSI at
`multiple loci (MSI-high tumours) [76]. Using a PCR-based
`approach with 6 DNA markers, MSI was reported in 21%
`of tumours (4/20 adenomas and 2/8 carcinomas), mostly as
`single-microsatellite change (insertion or deletion), and only
`one tumour showed instability at three loci [67].
`These results suggest that genomic instability is an
`important molecular event in DMH induced colon carcino-
`genesis. It seems that, unlike in human colon cancer, chro-
`mosomal alterations in this model are relatively infrequent.
`Much more frequent events in the DMH/AOM model are
`small alterations in the genome that are involved in the
`very early stages of colon carcinogenesis. Single nucleotide
`changes, which have been found to be responsible for Apc,
`β-catenin and K-ras mutations, are apparently widespread
`and very frequent alterations in the genome of this model.
`This carcinogen generates the methylated DNA adducts O6-
`methylguanine, which, if not repaired, may mispair with
`thymidine during DNA replication, resulting in a G:C to A:T
`transition.
`
`6. Altered Expression in DMH/AOM Colon
`Carcinogenesis
`
`Not only mutations but also many of the cellular and
`molecular defects found in human colon carcinogenesis have
`also been observed in DMH/AOM rat colon carcinogenesis.
`These alterations have been found to be involved in various
`pathways, such as the Wnt pathway, K-ras pathway, tumour
`growth factor β (TGFβ) signaling pathway, and inflamma-
`tory related process, which play important role in colon
`cancer.
`
`6.1. Wnt Pathway. The Wnt pathway has been implicated as
`a crucial step in the initiation and development of colonic
`tumorigenesis. In the absence of the extracellular Wnt signal,
`free β-catenin is bound to the APC-axin-conductin-GSK3β
`complex. Phosphorylation of β-catenin by this complex
`marks it for ubiquitination and subsequent proteolytic
`degradation by the proteasome. When APC or β-catenin is
`mutated, β-catenin cannot be degraded but accumulates in
`the cytoplasm and translocates into the nucleus, where it
`binds to T-cell factor (TCF) and activates the Wnt target
`genes [80–82].
`In normal colon epithelial cells, β-catenin is highly
`expressed in the cell membrane and not detected in the
`cytoplasm or nucleus of cells [63]. In all dysplastic colorectal
`epithelial
`lesions (dysplastic ACF, dark ACF, flat ACF,
`BCAC, MDF, adenomas, and adenocarcinomas), but not
`in hyperplastic lesions, β-catenin expression is found to be
`increased in the cytosol and nucleus [14, 77, 83]. Aberrant
`expression of β-catenin has been associated with mutations
`in Apc or β-catenin, but has also been present in lesions
`without either Apc or β-catenin mutations [68], suggesting
`that aberrant expression of β-catenin may be the result
`of altered expression of one of the many proteins with
`which β-catenin interacts, such as axin, conductin, or E-
`cadherin [82]. In fact, inactivation of E-cadherin, which
`
`

`

`Journal of Biomedicine and Biotechnology
`
`7
`
`Table 2: Frequencies of gene mutations found in biomarkers and different stages of colon carcinogenesis in the DMH/AOM rat model.
`
`Tumours (n)
`14.3% (27)
`—
`
`Adenomas (n)
`(17)
`—
`
`Carcinomas (n)
`(10)
`—
`
`Carcinogen
`AOM
`DMH
`
`8.8% (57)
`30.4% (23)
`
`—
`33.3% (12),
`
`—
`27.3% (11)
`
`—
`33% (6)
`
`75% (8)
`77% (26)
`
`Gene
`Apc
`
`β-catenin
`
`K-ras
`
`ACF/MDF (n)
`0% ACF (66)
`—
`
`—
`0% ACF (24)
`25% MDF (24)
`—
`15.4% ACF (13)
`
`20% ACF (15)
`—
`—
`7% ACF (27)
`25% MDF (28)
`—
`32% ACF (19)
`7.4% ACF (27)
`—
`53.8% ACF (13)
`
`P53
`
`Histologic diagnosis (n)
`—
`22.2% dysplasia (18)
`15.4% AC (13)
`—
`—
`