High susceptibility of Scid mice to colon carcinogenesis induced by azoxymethane indicates a possible caretaker role for DNA-dependent protein kinase

Masako Ochiai1, Tsuneyuki Ubagai1, Toshihiko Kawamori2, Hiroshi Imai1,3, Takashi Sugimura1 and Hitoshi Nakagama1,4

1 Biochemistry Division and
2 Cancer Prevention Division, National Cancer Center Research Institute, 5-1-1, Tsukiji, Chuo-ku, Tokyo 104-0045 and
3 Department of Pathology, Mie University School of Medicine, Mie 514-8507, Japan


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Severe combined immunodeficiency (Scid) mice have defects in V(D)J recombination and DNA double-strand breaks repair caused by an inherited genetic defect in the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs). Scid mice are highly susceptible to development of T-cell lymphomas, and because of the nature of its association with DNA repair and recombination, DNA-PKcs is considered to belong to the caretaker class of tumor suppressor genes. In the present study, the susceptibility of Scid mice to colon carcinogenesis due to administration of azoxymethane (AOM) was investigated. Significantly higher susceptibility in terms of induction of both aberrant crypt foci (ACFs), putative pre-cancerous lesions of the colon and colon cancers was observed as compared with the isogenic strain, C.B-17 mice. The incidences of colon tumors, either adenomas or adenocarcinomas, in Scid and C.B-17 mice after administration of AOM (10 mg/kg body weight/week) for 6 weeks were 87% (26 of 30) and 50% (15 of 30), respectively, by experimental week 22 (P < 0.01). The multiplicity of colon tumors in Scid mice was also significantly higher than in C.B-17 mice, being 2.2 ± 1.5 and 0.9 ± 1.2, respectively (P < 0.001). The present study clearly demonstrated high susceptibility of Scid mice to colon carcinogenesis, which might be attributable to disruption of the caretaker role of DNA-PK in colonic epithelial cells.

Abbreviations: AC, aberrant crypt; ACF, aberrant crypt focus; AOM, azoxymethane; DNA-PKcs, catalytic subunit of DNA-dependent protein kinase; DSB, double-strand break; MN, minisatellite; MMR, mismatch repair; O6-meG, O6-methylguanine; MGMT, O6-meG DNA methyltransferase; PCR, polymerase chain reaction; Scid, severe combined immunodeficiency; SSCP, single strand conformation polymorphism.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The genetic defect in the severe combined immunodeficiency (Scid) mouse has been recently characterized as an intragenic mutation at the 3'-end of the gene encoding a catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) (1). The biological function of DNA-PKcs has been clarified as being involved in the genomic organization of the T-cell receptor and immunoglobulin genes and also in the non-homologous end-joining repair pathway for DNA double-strand breaks (DSBs) (for a review, see ref. 2). The molecular basis of the hypersensitivity of Scid mice to various DNA damaging agents, including ionizing radiation, is therefore considered to be a defect in the DSB repair system.

Recently, a subset of molecules has been characterized as being responsible for DSB repairs in mammalian cells, including ATM (the gene product of ataxia-telangiectasia mutated) (3), Rad52 epistasis gene family members (4), Ku70 (5), Ku80 (6), Brca1 (7), Brca2 (8) and poly(ADP-ribose)polymerase (9). Targeted disruption of these genes confers hypersensitivity to various DNA damaging agents in mice and/or cultured cells (8,1016). However, the precise molecular mechanisms underlying the repair processes with these gene products and their functional interactions still remain largely unresolved.

The biological role of DNA-PKcs in maintaining genomic stability at minisatellite (MN) loci has attracted attention. Minisatellites are arrays of 5–100 nucleotide repeats and are dispersed throughout the entire genome of vertebrates, and are frequently mutated in various types of tumors of both humans and experimental animals (17,18). When two fibroblast cell lines derived from Scid mice, SC3VA2 and SC1K, were subjected to multi-locus DNA fingerprint analysis using mouse MN Pc-1 as a probe, they were found to harbor genomic instability at MN loci (19). Therefore, DNA-PKcs may also have a role in maintaining genomic integrity.

Various types of genomic instability, reflected in microsatellite instability, gene amplification, chromosomal aberration and loss of heterozygosity are considered to play important roles in multi-stage carcinogenesis (20). The fact that Scid fibroblasts feature such genomic instability allows the intriguing hypothesis that Scid mice could be highly susceptible to carcinogenesis induced by genotoxic agents in different organs. In fact, T-cell lymphomas have been reported to develop spontaneously in Scid mice at a frequency of 15–30% (21), and the incidence becomes much higher on exposure to ionizing radiation (22). Li et al. (11) have also proposed Ku70, which is a component of the DNA-PK complex, to be a candidate tumor suppressor gene for murine T-cell lymphoma. Difilippantonio et al. (16) have recently reported that disruption of Ku80, another component of the DNA-PK complex, resulted in a marked increase in chromosomal aberrations and promotes the occurrence of pro-B-cell lymphomas, especially with the concomitant loss of p53. Therefore, DNA-PKcs, Ku70 and Ku80, which compose the DNA-PK complex, are considered to belong to the caretaker class of tumor suppressor genes for T- and B-cell lymphomas (23).

In the present study, we investigated whether Scid mice are susceptible to colon carcinogenesis induced by azoxymethane (AOM), a widely utilized synthetic alkylating agent and a potent colon carcinogen. Induction of aberrant crypt foci (ACFs), putative pre-cancerous lesions of the colon and colon tumors (24) was thus analyzed after administration of the carcinogen, and compared with those of the isogenic mice strain, C.B-17. After administration of AOM, Scid mice developed a significantly higher number of ACF and colon tumors than C.B-17 mice, although the majority of tumors developing in both strains were diagnosed as adenocarcinomas. Possible involvement of genomic instability caused by an abrogation of the caretaker role of DNA-PKcs was considered with regards to the preferential occurrence of colon tumors in Scid mice.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Animals
Five-week-old female Scid and C.B-17 mice were purchased from CLEA Japan (Tokyo). Scid mice have long been maintained in CLEA Japan as heterozygotes for the DNA-PKcs locus by mating with their isogenic strain, C.B-17 mice. Therefore, Scid mice should have the same genetic background as C.B-17 mice, except for the DNA-PKcs locus (1). The animals were housed six per cage with woodchip bedding in an air-conditioned animal room, and fed AIN-93G pellet diet (Dyets, Bethlehem, PA) ad libitum and allowed free access to water.

Detection of ACF
Short-term exposure to AOM was employed to analyze the induction of ACFs. Groups of 20 six-week-old Scid and C.B-17 female mice were administered 10 mg AOM (Sigma, St Louis, MO) per kg body weight by intraperitoneal (i.p.) injection twice with a 1 week interval. Six age-matched Scid and six C.B-17 mice received i.p. injections of saline without AOM as controls, and this short-term study was terminated at experimental week 4. The mice were killed, the colons resected and flushed out with neutralized 10% formalin, and cut along the longitudinal median axis. Then they were immersed in neutralized 10% formalin overnight at 4°C and then stained with 0.2% methylene blue, as described previously (24). The number of ACFs developing in each mouse was counted under a light microscope at low magnification. The total number of aberrant crypts (ACs) composing ACFs in each mouse was also assessed.

Detection of colon tumors
For the carcinogenicity experiment, long-term exposure to AOM was employed. Groups of 30 six-week-old Scid and C.B-17 mice received i.p. injections of 10 mg AOM/kg body weight once a week for 6 weeks. Ten mice each of the respective strains received i.p. injections of saline as controls. Mice were killed when they manifested anal bleeding, and this long-term study was terminated at experimental week 22. When colon tumors were evident to the naked eye, they were resected, fixed in neutralized 10% formalin overnight at 4°C and embedded in paraffin blocks according to standard procedures. Some of the tumors were bisected and one half of each was frozen and stocked at –80°C until use for DNA extraction, the other half being embedded in paraffin as detailed above. When a tumor was not detected macroscopically, the presence of microscopic tumors was carefully ascertained under low magnification after staining with 0.2% methylene blue, as described above. Paraffin sections were prepared at a thickness of 3.5 µm, then stained with hematoxylin and eosin, and histopathological analysis was performed.

Immunohistochemical staining of ß-catenin
Immunohistochemical staining with anti-ß-catenin antibody was carried out using the Vectastain ABC system (Vector Laboratories, Burlingame, CA). Briefly, tissue sections were prepared at 3.5 µm thickness as described above and mounted on slide glasses, deparaffinized with xylene and rehydrated with graded ethanol. Antigen activation was carried out by boiling for 10 min in 10 mM citrate buffer (pH 6.0). Primary mouse monoclonal antibody for ß-catenin (Transduction Laboratories, Lexington, KY) was used at a dilution of 1:400, with biotinylated goat anti-mouse IgG (Vector Laboratories) as the secondary antibody at a dilution of 1:200.

Detection of ß-catenin mutation
Ten of the colon adenocarcinomas in Scid mice and seven in C.B-17 mice were subjected to polymerase chain reaction–single strand conformation polymorphism (PCR–SSCP) analysis to detect mutations in the ß-catenin gene. Primer pairs were designed from the phosphorylation target site by GSK-3ß in exon 3 as described elsewhere (25). PCR reactions were carried out for 35 cycles in 1x reaction buffer (10 mM Tris–HCl, 50 mM KCl, 1.5 mM MgCl2, pH 8.3) at 94°C for 30 s, 60°C for 30 s and 72°C for 45 s. PCR products were subjected to SSCP analysis on the GenePhor DNA Separation System (Amersham Pharmacia Biotech, Little Chalfont, UK). After PCR–SSCP analysis, shifted bands were extracted from the gel and subjected to PCR-based direct sequencing using an ABI PRISM 310 Genetic Analyzer (PE Applied Biosystems, Foster City, CA).

Statistical analysis
Survival rates were calculated by the Kaplan–Meier method, and the difference between Scid and C.B-17 mice was analyzed by a log-rank test. Statistical analyses of tumor incidence and other parameters were performed using the {chi}2 test or Mann–Whitney U-tests (SPSS for the Macintosh; SPSS Japan, Tokyo, Japan). Statistical significance was set at values of P < 0.05.


    Results
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 Abstract
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 Materials and methods
 Results
 Discussion
 References
 
Induction of ACF after short-term exposure to AOM
As demonstrated in Figure 1Go, Scid mice developed significantly larger numbers of ACFs and total aberrant crypts (ACs) in their colons than their C.B-17 counterparts after the administration of AOM. No ACFs were detected in the untreated control groups of either strain of mice (data not shown).



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Fig. 1. Aberrant crypt foci (ACFs) induced by azoxymethane (AOM) in Scid and C.B-17 mice. Numbers of ACFs and ACs induced in each mice by AOM were plotted. Horizontal bar indicates the average number of ACFs and ACs in each group of mice. None of the Scid or C.B-17 mice without AOM-treatment developed ACFs (data not shown).

 
Survival of Scid and C.B-17 mice after long-term exposure to AOM
Survival rates of AOM-treated and untreated mice of both strains are shown in Figure 2Go. For AOM-treated groups, 19 of 30 (63%) Scid mice manifested anal bleeding and were judged moribund, and were killed by experimental day 141. In the case of C.B-17, only six mice demonstrated anal bleeding and were killed by this time point. The rest of the mice were killed and analyzed between experimental days 142 and 149. The difference in survival rates between Scid and C.B-17 mice was statistically significant (P < 0.01). All Scid and C.B-17 mice in the untreated groups stayed healthy and no signs of tumor development were detectable on external observation during the entire experimental period of 22 weeks.



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Fig. 2. (A) Survival curves of Scid and C.B-17 mice. Thirty mice were subjected to this analysis. The solid line indicates the survival curve of Scid mice and the hatched line that of C.B-17 mice. All 10 untreated Scid and C.B-17 mice stayed healthy during the entire experimental period (data not shown). (B) Distribution of tumors in colon of Scid and C.B-17 mice.

 
Induction of colon tumors by AOM
The first colon tumor was detected by anal bleeding on experimental day 86 in a Scid mouse. C.B-17 mice developed colon tumors much later and the first tumor was recognized by anal bleeding at day 102 (Figure 2Go). By the end of the experiment, 26 of 30 (87%) Scid mice had developed colon tumors, mainly adenocarcinomas, and the tumor incidence was significantly higher than the 50% observed for C.B-17 mice (Table IGo). The multiplicity of tumors was also significantly different, being 2.2 ± 1.5 in Scid and 0.9 ± 1.2 in C.B-17 mice (Table IGo). The histological features of the tumors were similar between Scid and C.B-17 mice, and 91 and 89% of tumors developed in Scid and C.B-17 mice, respectively, were diagnosed as adenocarcinomas with the remainder being adenomas (Table IIGo). In addition, most of the lesions in both strains developed in the distal half of the colon, as summarized in Figure 2Go, consistent with the preferential occurrence of ACFs in this section (data not shown).


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Table I. Incidences and multiplicities of colon tumors in Scid and C.B-17 mice induced by AOM
 

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Table II. Histological analysis of colon tumors, and immunohistochemical analysis for ß-catenin accumulation
 
Accumulation of ß-catenin in tumors
Since the Wnt–ß-catenin signaling pathway plays a key role in the development of colon tumors in both humans (26) and experimental animals (25,27), immunohistochemical analysis was carried out to determine whether differences might exist regarding the incidence and/or the mode of accumulation of the ß-catenin protein in tumor cells (Figure 3Go). Eighty percent (37 of 46) of the tumors in Scid mice and 93% (14 of 15) in C.B-17 mice demonstrated accumulation of ß-catenin in both the cytoplasm and the nucleus. The difference was not significant (Table IIGo). Mutations in the ß-catenin gene were also found in 70% (7 of 10) of colon adenocarcinomas in Scid mice and 57% (4 of 7) in C.B-17 mice, again without any significant difference (Table IIIGo). All of the mutations were G->A or C->T transitions.



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Fig. 3. Histological features of colon adenocarcinoma induced by azoxymethane in (A) Scid and (C) C.B-17 (hematoxylin and eosin staining). Expression of ß-catenin protein in tumor derived from (B) Scid and (D) C.B-17. ß-Catenin was densely stained in both nucleus and cytoplasm in tumors (left part of the section).

 

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Table III. Mutations of the ß-catenin gene in colon tumors induced by azoxymethane
 
Other types of tumors
Thymic lymphomas were observed in Scid mice, but the incidence in the AOM-treated group did not differ from that in the untreated control group, with values of 7 (2 of 30) and 10% (1 of 10), respectively. One adenoma developed in the small intestine of a Scid mouse and one squamous cell carcinoma of the skin in a C.B-17 mouse after AOM treatment.


    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
As is clearly demonstrated in our study, Scid mice show a higher susceptibility to the induction of ACFs and colon tumors after administration of AOM than the isogenic mouse strain, C.B-17. Salim et al. (28) also reported the potential advantages of Scid mice in colon carcinogenesis studies based on their finding that large ACFs (>=4 crypts), considered to be important as precursors of colon cancers, can be induced by a food-borne carcinogen, 2-amino-3-methylimidazo[4,5-f] quinoline (IQ), in a dose-dependent manner. The question to be addressed is therefore what kind of molecular mechanisms could be responsible for this elevated susceptibility to colon carcinogenesis? Considering that the induction of ACF by AOM significantly differed between Scid and C.B-17 mice, and correlating well with the development of colon tumors, causative genetic events which confer the differential susceptibility may reside at the earlier stage; namely, even before the formation of ACF.

Azoxymethane is one of the most widely used alkylating agents and formation of O6-methylguanine (O6-meG) is considered to be a crucial event leading to the induction of G->A point mutations and consequently to cancers (29). O6-meG DNA methyltransferase (MGMT) is the principle enzyme involved in the repair of O6-meG adducts by removing the methyl group from guanine bases (30). In addition, mismatch repair (MMR) genes have been demonstrated to affect the killing and tumorigenic effects of alkylating agents (31). Although DNA-PKcs has not been clearly shown to be involved in the repair process of DNA damage induced by alkylating agents, our present data suggest their possible participation. One possible mechanism is that MMR recognizes the O6-meG:T mispairs that occur due to erroneous incorporation of a thymine nucleotide opposite O6-meG during DNA replication, excises this mispair and thereby makes single-stranded regions. These could lead to double-stranded gaps when replicated during the next S phase (32). When DNA-PKcs is mutated, this gap could be repaired less efficiently or improperly by the impairment of the non-homologous end-joining repair pathway, which may confer a higher susceptibility to induction of cell death and malignant transformation of cells. Another possibility is that DNA-PKcs could support the biological functions of either MGMT or MMR through functional interactions with these molecules, and their abrogation could result in the increase of mutation rate induced by AOM. The effects of DNA-PKcs deficiency on spontaneous in vivo mutation rates in a long-term carcinogenesis experiment, however, remain to be resolved.

The other intriguing scenario is an involvement of genomic instability in Scid cells. Scid fibroblasts have been demonstrated to harbor genomic instability at MN loci and this is overcome by complementation with DNA-PKcs (19). Although the molecular mechanism underlying the induction of MN mutations in somatic cells are still largely unknown, recombination-based gene conversion at the tandem repeat could be one possible mechanism (33) and DNA-PKcs could be one molecule active in this process. The presence of genomic instability in cells is known to play an important role in the multistage carcinogenesis of various organs in both humans (20) and experimental animals (34), and genes involved in the maintenance of genomic stability can be considered as a caretaker-class of tumor suppressor genes. In the case of lymphoid cells, DNA-PKcs is considered to play a caretaker role in carcinogenesis (23). Recent studies of two other components of the DNA-PK complex, Ku 70 and Ku 80, also pointed to novel biological roles as caretaker-type tumor suppressors for the development of T- or B-cell lymphomas (11,16). In colonic epithelial cells, when initiating and crucial genetic alteration(s) are introduced into colonic epithelial cells by metabolites of AOM, the presence of genomic instability could render the initiated cells more susceptible to generation of subsequent genetic alterations, as is the case with mismatch repair deficiency (31,35). Abrogation of DNA-PKcs function in colonic epithelial cells could thereby accelerate tumor promotion and progression in colon carcinogenesis. The possible involvement of genomic instability in colon carcinogenesis in Scid mice could be strengthened, if the presence of MN instability could be demonstrated in Scid-derived tumors, and less frequently in C.B-17-derived tumors. In order to do this precisely, we need to establish several cell lines from AOM-induced colon cancers and mutation rates in MN sequences should be calculated by Southern blot fingerprint analysis. This, however, is something that remains to be performed at a future date.

Kurimasa et al. (36) recently demonstrated that genetic disruption of DNA-PKcs results in the induction of hyperproliferative pre-neoplastic epithelial lesions and cryptic hyperplasia with mild to moderate dysplasia in the colon, which also strongly supports a tumor-suppressor role in colonic epithelial cells. Although no cryptic hyperplasia or ACFs were observed in Scid mice without AOM treatment in our present study, the number of ACFs induced by AOM was significantly higher in Scid than in C.B-17 mice. Possible explanations for the lack of hyperproliferative pre-neoplastic epithelial lesions and cryptic hyperplasia in Scid mice without AOM treatment in our study could be the shorter observation period for ACF induction in our study, the small number of mice examined, differences in genetic background of mice used for the analysis or the presence of leakiness of DNA-PK activity in Scid mice. No DNA-PK activity is apparent in DNA-PKcs knockout mice generated by Kurimasa et al. (36).

The last factor which should be taken into consideration is that since Scid mice have a substantial defect in normal T-/B-cell function, consequent defective immunosurveillance could allow tumor cells to grow more efficiently and much faster. However, the previous study by Dudley et al. (37) using double mutant mice generated by mating Scid and ApcMin mice did not show any significant increase in the numbers of intestinal tumors or the onset of tumors compared with the ApcMin single mutant mice, which harbor a dominant germ-line mutation in the Apc gene at codon 850 and develop a large number of intestinal tumors spontaneously. This result suggests to us that a defect in immunosurveillance in Scid mice does not play an essential role in the development of colon tumors, at least after the induction of genetic changes in the Wnt–Apc–ß-catenin signaling pathway. Although we are not able to completely negate the possible involvement of immunosurveillance in the very early stages of colon carcinogenesis, even before the induction of ACFs, this scenario is less likely.

In conclusion, the present study demonstrated Scid mice to be more highly susceptible to colon carcinogenesis than the isogenic C.B-17 strain, in addition to the preferential occurrence of T- and B-cell lymphomas. A possible role of DNA-PKcs as a caretaker-type tumor suppressor gene in colon carcinogenesis is considered to be highly likely.


    Notes
 
4 To whom correspondence should be addressed

Email: hnakagam{at}gan2.ncc.go.jp Back


    Acknowledgments
 
We thank our colleagues, Yuko Iizuka and Yoshiko Hirao, for expert care of the mice and Dr Minako Nagao for helpful discussions and critical reading of the manuscript. This work was supported in parts by Grants-in-Aid for Cancer Research and for the Second Term of the Comprehensive 10-Year Strategy for Cancer Control from the Ministry of Health, Labour and Welfare of Japan, and by a Research Grant from the Sankyo Foundation for Life Science.


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received February 19, 2001; revised June 18, 2001; accepted June 19, 2001.





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