Cytotoxic and mutagenic response of mismatch repair-defective human cancer cells exposed to a food-associated heterocyclic amine
Warren E. Glaab1 and
Thomas R. Skopek
Department of Genetic and Cellular Toxicology, Merck Research Laboratories, WP45-320, West Point, PA 19486, USA
 |
Abstract
|
---|
The cytotoxic and mutagenic effects of 2-amino-1-methyl-6-phenylimidazo-[4,5-b]-pyridine (PhIP), a food-associated heterocyclic amine, were measured in three human cancer cell lines possessing different mismatch repair (MMR) defects and in matched cell lines corrected for the MMR deficiencies by specific chromosome transfer. Cells deficient in MMR were more resistant to PhIP-induced cytotoxicity and displayed ~3-fold more induced mutations at the hypoxanthine-guanine phosphoribosyl transferase locus. These results suggest that defects in MMR carried by patients with hereditary nonpolyposis colorectal cancer syndrome may result in enhanced sensitivity to certain dietary and environmental carcinogens such as PhIP.
Abbreviations: HAs, heterocyclic amines; HNPCC, hereditary nonpolyposis colorectal cancer; HPRT, hypoxanthine-guanine phosphoribosyl transferase; MMR, mismatch repair; PhIP, 2-amino-1-methyl-6-phenylimidazo-[4,5-b]-pyridine; 6-TG, 6-thioguanine.
 |
Introduction
|
---|
Genomic instability in human cells resulting from the loss of DNA mismatch repair (MMR) and its role in carcinogenesis have been the focus of much attention in recent years (reviewed in refs 13). The association of MMR deficiency and carcinogenesis was first recognized in the study of hereditary nonpolyposis colorectal cancer (HNPCC) (46) and later implicated in the etiology of a variety of hereditary and sporadic human cancers (13). Currently, there are five genes known to be involved in MMR in humans (hMSH2, hMSH3, hMSH6, hMLH1 and hPMS2), although additional homologs are currently under investigation (13). The roles of MMR-gene products in the repair process and their effect on cellular phenotype have been studied using human cancer cell lines with specific MMR gene defects (710). These cell lines exhibit hallmarks of MMR deficiencies including (i) microsatellite instability; (ii) elevated spontaneous mutation rates at endogenous loci; and (iii) lack of enzymatic MMR repair activity when assayed in vitro (7). Complementation of the MMR-gene defects in several of these cell lines by chromosome transfer has been shown to reverse the MMR-deficient phenotype (810).
Cells defective in MMR are more resistant than normal cells to cytotoxicity induced by DNA-damaging agents such as alkylating and chemotherapeutic agents, and are more susceptible to their mutagenic effects (715). Both resistance and hypermutability are reversed in cell culture by restoration of the defective MMR gene by chromosome transfer (811). It is believed that the greater sensitivity to killing in MMR-competent cells results from futile cycling of the repair pathway at certain damaged sites, while in cells deficient in MMR these potentially lethal pathways are avoided (1,14). Hypermutability in MMR-defective cells is likely to be the result of failure to repair promutagenic DNA mismatches induced by mis-incorporation opposite damaged bases. While the resistant/hypermutable phenotype of MMR-deficient cells has only been demonstrated with model alkylating and chemotherapeutic agents known to produce mismatched basepairs, it is logical that other types of chemicals, including dietary, environmental and endogenous compounds, may elicit a similar response. Thus, enhanced survival and mutagenesis of MMR-deficient cells in the body may also play an important role in the etiology of chemically induced tumors in vivo.
Heterocyclic amines (HAs) are common pyrolysis products produced from heating amino acids and proteins (reviewed in refs 16,17). The representative HA studied here, 2-amino-1-methyl-6-phenylimidazo-[4,5-b]-pyridine (PhIP), is found in cooked meat, beer, wine and cigarette smoke (reviewed in refs 16,17), and has been observed in urine of normal human subjects after eating charbroiled beef (18). It is a potent mutagen and has been shown to induce tumors in a number of tissues in mice and rats (16,17,19). PhIP has also been implicated in human carcinogenesis, especially in the colon (16,17,20). Considering that MMR was first implicated in colon cancer (46), we have investigated the cytotoxic and mutagenic response of PhIP in MMR-defective cells, and in cells in which the MMR defect has been complemented by chromosome transfer. Here we demonstrate that MMR-defective cells are more resistant to the cytotoxic effects of PhIP, while displaying a greater increase in induced mutations, than MMR-proficient cells. These data suggest that dietary compounds such as PhIP may enhance the risk of cancer in patients with HNPCC.
 |
Materials and methods
|
---|
Cell lines
The human cancer cell lines and cell lines derived by chromosome transfer were obtained from and characterized in the laboratories of T.A.Kunkel and J.C.Barrett (NIEHS, RTP, NC). The genotype and phenotype of the cell lines studied here are described in Table I
. The gene defect and complementation by chromosome transfer were defined previously (summarized in refs 79). The DLD-1 chromosome 2 transfer line, clone 1, is designated DLD-1+ch2 (9), the HCT116 chromosome 3 transfer line, clone 6, is designated HCT116+ch3 (8) and the HEC59 chromosome 2 transfer line, clone 4, is designated HEC59+ch2 (9). All chromosome transfer lines complement the defective MMR gene present in the parental cell lines. Cell lines were grown in D-MEM/F-12 medium (1:1) + 10% dialyzed fetal bovine serum (FBS) (HyClone, Logan, UT), and chromosome transfer cell lines maintained with G418 [400 µg(active)/ml; Gibco BRL, Grand Island, NY].
PhIP treatment
PhIP was obtained from Toronto Research Chemicals (Toronto, Canada) and resuspended at 5 mg/ml in dimethyl sulfoxide (DMSO) (Sigma, St Louis, MO) immediately prior to use. Chemical exposures were performed in normal medium (D-MEM/F-12 (1:1) + 10% FBS) in the presence of mouse liver S9 induced with phenobarbital and benzoflavone (Moltox, Boone, NC). Approximately 500 µg S9 protein was added per ml of medium (15 µl S9/ml). Metabolic enzyme cofactors NADP (Boehringer Mannheim, Indianapolis, IN) and DL-isocitric acid (Sigma) were added for a final concentration of 1 and 5.8 mM, respectively. Cells were exposed to PhIP for 4 h at 37°C, after which PhIP-containing medium was removed and replaced with fresh medium.
Cytotoxicity
The cytotoxic response to PhIP exposure was determined for each cell line by colony-forming ability. Approximately 500 cells were plated in 10 cm dishes in triplicate for each dose. After 1215 h following plating, cells were treated with various doses of PhIP as described above, then allowed to grow for 1214 days and colonies visualized by staining with 0.5% crystal violet (Sigma; in 50% methanol). Colonies with 50 or more cells were counted and survival expressed as percentage of untreated control plates. Each cytotoxicity determination was repeated two additional times, and the average of all three independent experiments reported.
Induced mutant frequency
The induced mutagenic response at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus was determined as described previously (11). Cell lines were cleansed of preexisting HPRT mutants by culturing in HAT medium (100 µM hypoxanthine, 0.4 µM aminopterin and 16 µM thymidine; Sigma), then plated in 15 cm dishes at 1.5x106 cells per dish. Twenty-four hours after plating, cells were treated with PhIP (three dishes per dose), while three additional dishes were exposed to only S9-containing medium. A parallel cytotoxicity experiment was performed with the same PhIP-containing medium to determine survival. The cells were maintained in logarithmic growth during a 10 day expression period, after which the frequency of 6-thioguanine (6-TG)-resistant cells (40 µM) was measured. The determined HPRT mutant frequencies presented are an average of two separate experiments, each performed with three independent cultures for each PhIP treatment.
 |
Results and discussion
|
---|
We first investigated the cytotoxic effects of increasing doses of PhIP in MMR-defective cell lines and MMR-proficient chromosome transfer cell lines. The survival of the chromosome transfer cell lines following PhIP treatment was significantly lower than that observed in the corresponding parental lines (Figure 1
). That is, cells with functional MMR were more sensitive to the cytotoxic effects of PhIP than cells deficient in MMR. Survival was significantly different between parental cell lines and chromosome transfer cell lines at all doses >5 µM (P < 0.05).

View larger version (12K):
[in this window]
[in a new window]
|
Fig. 1. PhIP cytotoxicity in MMR-defective cell lines and MMR-proficient chromosome transfer lines. Graphs represent the average percent survival (colony forming ability) from three independent determinations. (A) HCT116 cell lines; (B) DLD-1 cell lines; (C) HEC59 cell lines.
|
|
Next, the mutagenic response induced by exposure to PhIP at the HPRT locus was measured for two doses of compound and is presented in Table II
. The mutagenic response in HEC59 was not investigated since HPRT mutants cannot be selected in this line (21). For the MMR-defective parent cell lines, HCT116 and DLD-1, there was a significant increase in mutant frequency compared with untreated controls following a 4 h exposure to PhIP in the presence of mouse liver S9 for both doses investigated (Table II
). PhIP also increased the number of mutants in the MMR-proficient chromosome transfer lines, but the magnitude of the induced response (observed mutant frequency with PhIP minus control mutant frequency) was ~3-fold lower than that observed in the matching MMR-defective cell lines. For example, in the MMR-defective cell line HCT116, 10 µM PhIP induced 160 mutants per 106 cells, while the same dose induced only 47 mutants per 106 cells in the MMR-proficient cell line HCT116+ch3 (Table II
).
Since chromosome transfer introduces many genes in addition to the one required to complement the defect in MMR, it could be argued that the difference in the PhIP-induced response between parental and chromosome transfer lines may be unrelated to MMR. While this remains a formal possibility, the fact that defects in three MMR genes (hMSH6, hMLH1, hMSH2) were complemented by the transfer of two different chromosomes (ch2 and ch3) in three different cell lines and, in all cases, produced the same PhIP-related response, provides strong evidence that MMR is causative. It is unlikely that differences in metabolic activation/detoxification are involved due to the fact that (i) no PhIP-related toxicity or mutagenicity was seen in any cell line in the absence of S9, illustrating that all of the cell lines lack the ability to activate PhIP (data not shown); and (ii) a similar pattern of hypermutability and resistance to toxic effects has been seen with these lines using a diverse set of direct-acting alkylating and chemotherapeutic agents (811).
Metabolic activation of PhIP by N-oxidation is carried out by the cytochrome P450 1 family of enzymes, resulting in the formation of N-hydroxy-PhIP (22). N-hydroxy-PhIP can be further metabolized to form N-acetylated or sulfated conjugates (23). The current model for PhIP-induced colorectal carcinogenesis involves primary metabolism by cytochrome P450 1A2 in the liver, followed by transport of N-hydroxy-PhIP to the colon and secondary conjugation by N-acetyltransferases (16,17,23). These reactive secondary metabolites bind DNA primarily at the C8 position of guanine to form N-(deoxyguanosin-8-yl)-PhIP (dG-C8-PhIP) (24), which is considered to be the critical DNA modification involved in PhIP mutagenesis. Other bulky aromatic amine adducts at the C8 position of guanine are bound by the mismatch recognition complex, hMutS
(hMSH2/hMSH6) (25), implicating MMR in the processing of such damage.
The specificity of mutations induced by PhIP at endogenous loci such as the human HPRT locus is characterized by a high percentage of G:C
T:A transversions (26,27). This finding suggests that dG-C8-PhIP may directly miscode with adenine. The resulting mismatch intermediate, dG-C8-PhIP:A, may be recognized by MMR. Additionally, G frameshifts in homopolymeric runs of guanine bases have been seen following PhIP treatment, including a G frameshift hotspot in the sequence GGGA (26,27). The same G frameshift in a GGGA sequence has been seen in the APC tumor suppressor gene in five of eight colon tumors from PhIP-treated rats (28), suggesting that this mutagenic event may play a role in the observed carcinogenesis. It should be noted that a characteristic feature of MMR deficiencies is frameshift mutations in homopolymeric runs (13) and that this type of mutation has been observed in genes associated with neoplastic transformation in MMR-deficient tumors (29).
It is interesting to note that the mutant frequency induced by the potent mutagen PhIP in MMR-corrected cells is actually less than the spontaneous frequency in the corresponding MMR-deficient cells (Table II
). This suggests that the mutagenic potential of other agents in wild-type cells may be dwarfed when compared with the tremendous mutational burden imposed by the MMR-deficient state itself. However, the mutagenic activity of these compounds may be significantly elevated in an MMR-deficient background, as demonstrated here with PhIP. It may be that many compounds with weak mutagenic potential can only achieve significant mutation rates in an MMR-deficient background.
The strong association of the MMR-deficient phenotype with the induction of colon tumors is prima facie evidence that an enhanced level of mutagenesis is critical for tumor formation. Therefore, it is logical that any condition that significantly augments the mutation rate in MMR-deficient cells will also increase the probability of tumor formation. Given the significant mutagenic response to PhIP in MMR-deficient cells seen in this study and the high spontaneous frequency of tumor development in HNPCC individuals, it is likely that PhIP exposure and MMR deficiency will provide a synergistic effect on the carcinogenic response.
Mutation induced by PhIP in a MMR-deficient background was measured here at just one locus. The model for colorectal tumorigenesis proposed by Fearon and Vogelstein (30) suggests that there are at least five genes which must mutate before the transformed phenotype is attained. These mutations are believed to occur after the induction of the MMR-deficient phenotype. Therefore, if environmental conditions are imposed that increase the probability of mutation in MMR-deficient cells, then the probability of tumor formation will be elevated by the same factor raised to the fifth power, assuming equal mutation rates at each locus. For example, if the mutation frequency is increased by 2-fold at each of the five loci necessary for tumor formation (as observed in this study at 10 µM PhIP; Table II
), then a 25- or 32-fold increase in the probability of tumor formation would be expected. This has obvious health implications for individuals with HNPCC. These individuals are not only at risk of tumor formation due to high spontaneous mutation rates resulting from their MMR deficiencies, but they may also be subject to enhanced carcinogenic insult from dietary and environmental mutagens/carcinogens such as PhIP. The possibility that a simple change in diet may result in a substantial decrease in tumor probability in these individuals warrants investigation. Although extrapolation to humans is difficult due to the wide range of expected exposure to PhIP in the diet (16,17,20), even a modest decrease in mutant frequency may have an important impact on human health.
 |
Acknowledgments
|
---|
We acknowledge Dr Phaik Leong-Morgenthaler for help with optimizing exposure conditions involving PhIP, Dr Kenneth R.Tindall for help with material transfer agreement for chromosome transfer lines and Kristy Kort for technical assistance. We thank Drs Sheila Galloway, John Deluca, James Monroe and Diane Umbenhauer for critical evaluation of this manuscript.
 |
Notes
|
---|
1 To whom correspondence should be addressed Email: warren_glaab{at}merck.com 
 |
References
|
---|
-
Modrich,P. (1997) Strand-specific mismatch repair in mammalian cells. J. Biol. Chem., 272, 2472724730.[Free Full Text]
-
Marra,G. and Boland,C.R. (1996) DNA repair and colorectal cancer. Gastroenterol. Clin. N. Am., 25, 755772.[ISI][Medline]
-
Eshleman,J.R. and Markowitz,S.D. (1996) Mismatch repair defects in human carcinogenesis. Hum. Mol. Genet., 5, 14891494.[Abstract]
-
Papadopoulos,N., Nicolaides,N.C., Wei,Y.F. et al. (1994) Mutation of a mutL homolog in hereditary colon cancer. Science, 263, 16251629.[ISI][Medline]
-
Nicolaides,N.C., Papadopoulos,N., Liu,B. et al. (1994) Mutations of two PMS homologues in hereditary nonpolyposis colon cancer. Nature, 371, 7580.[ISI][Medline]
-
Leach,F.S., Nicolaides,N.C., Papadopoulos,N. et al. (1993) Mutations of a mutS homolog in hereditary nonpolyposis colorectal cancer. Cell, 75, 12151225.[ISI][Medline]
-
Boyer,J.C., Umar,A., Risinger,J.I., Lipford,J.R., Kane,M., Yin,S., Barrett,J.C., Kolodner,R.D. and Kunkel,T.A. (1995) Microsatellite instability, mismatch repair deficiency, and genetic defects in human cancer cell lines. Cancer Res., 55, 60636070.[Abstract]
-
Koi,M., Umar,A., Chauhan,D.P., Cherian,S.P., Carethers,J.M., Kunkel,T.A. and Boland,C.R. (1994) Human chromosome 3 corrects mismatch repair deficiency and microsatellite instability and reduces N-methyl-N'-nitro-N-nitrosoguanidine tolerance in colon tumor cells with homozygous hMLH1 mutation. Cancer Res., 54, 43084312.[Abstract]
-
Umar,A., Koi,M., Risinger,J.I., Glaab,W.E., Tindall,K.R., Kolodner,R.D., Boland,C.R., Barrett,J.C. and Kunkel,T.A. (1997) Correction of hypermutability, N-methyl-N'-nitro-N-nitrosoguanidine resistance, and defective DNA mismatch repair by introducing chromosome 2 into human tumor cells with mutations in MSH2 and MSH6. Cancer Res., 57, 39493955.[Abstract]
-
Umar,A., Risinger,J.I., Glaab,W.E., Tindall,K.R., Barrett,J.C. and Kunkel,T.A. (1998) Functional overlap in mismatch repair by human MSH3 and MSH6. Genetics, 148, 16371646.[Abstract/Free Full Text]
-
Glaab,W.E., Risinger,J.I., Umar,A., Barrett,J.C., Kunkel,T.A. and Tindall,K.R. (1998) Cellular resistance and hypermutability in mismatch repair-deficient human cancer cell lines following treatment with methyl methanesulfonate. Mutat. Res., 398, 197207.[ISI][Medline]
-
Karran,P. and Bignami,M. (1994) DNA damage tolerance, mismatch repair and genome instability. Bioessays, 16, 833839.[ISI][Medline]
-
Branch,P., Hampson,R. and Karran,P. (1995) DNA mismatch binding defects, DNA damage tolerance, and mutator phenotypes in human colorectal carcinoma cell lines. Cancer Res., 55, 23042309.[Abstract]
-
Drummond,J.T., Anthony,A. Brown,R. and Modrich,P. (1996) Cisplatin and adriamycin resistance are associated with MutL
and mismatch repair deficiency in an ovarian tumor cell line. J. Biol. Chem., 271, 1964519648.[Abstract/Free Full Text]
-
Andrew,S.E., McKinnon,M., Cheng,B.S., Francis,A., Penney,J., Reitmair,A.H., Mak,T.W. and Jirik,F.R. (1998) Tissues of MSH2-deficient mice demonstrate hypermutability on exposure to a DNA methylating agent. Proc. Natl Acad. Sci. USA, 95, 11261130.[Abstract/Free Full Text]
-
Felton,J.S., Malfatti,M.A., Knize,M.H., Salmon,C.P., Hopmans,E.C. and Wu,R.W. (1997) Health risk of heterocyclic amines. Mutat. Res., 376, 3741.[ISI][Medline]
-
Gooderham,N.J., Murray,S., Lynch,A.M., Yadollahi-Farsani,M., Zhao,K., Rich,K., Boobis,A.R. and Davies,D.S. (1997) Assessing human risk to heterocyclic amines. Mutat. Res., 376, 5360.[ISI][Medline]
-
Lynch,A.M., Knize,M.G., Boobis,A.R., Gooderham,N.J., Davies,D.S. and Murray,S. (1993) Intra- and interindividual variability in systemic exposure in humans to 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine, carcinogens present in cooked beef. Cancer Res., 52, 62166223.[Abstract]
-
Ito,N., Hasegawa,R., Imaida,K., Tamano,S., Hagiwara,A., Hirose,M. and Shirai,T. (1997) Carcinogenicity of 2-amino-1-methyl-6-phenyl-imidazo-[4,5-b]-pyridine (PhIP) in the rat. Mutat. Res., 376, 107114.[ISI][Medline]
-
Potter,J.D., Slatery,M.L., Bostick,R.M. and Gapstur,S.M. (1993) Colon cancer: a review of the epidemiology. Epidemiol. Rev., 15, 499545.[ISI][Medline]
-
Glaab,W.E. and Tindall,K.R. (1997) Mutation rate at the hprt locus in human cancer cell lines with specific mismatch-repair gene defects. Carcinogenesis, 18, 18.[Abstract]
-
Buonarati,M.H. and Felton,J.S. (1990) Activation of 2-amino-1-methyl-6-phenylimidazo[4,5-f]pyridine (PhIP) to mutagenic metabolites. Carcinogenesis, 11, 11331138.[Abstract]
-
Buonarati,M.H., Turteltaub,K.W., Shen,N.H. and Felton,J.S. (1990) Role of sulfation and acetylation in the activation of 2-amino-1-methyl-6-phenylimidazo[4,5-f]pyridine (PhIP) to intermediates which bind DNA. Mutat. Res., 245, 185190.[ISI][Medline]
-
Lin,D., Kaderlik,K.R., Turesky,R.J., Miller,D.W., Lay,J.O. and Kadlubar,F.F. (1992) Identification of N-(deoxyguanosin-8-yl)-2-amino-1-methyl-6-phenylimidazo-[4,5-f]-pyridine as the major adduct formed by the food-borne carcinogen, 2-amino-1-methyl-6-phenylimidazo[4,5-f]pyridine, with DNA. Chem. Res. Toxicol., 5, 691697.[ISI][Medline]
-
Li,G.M., Wang,H. and Romano,L.J. (1996) Human MutS
specifically binds to DNA containing aminofluorene and acetylaminofluorene adducts. J. Biol. Chem., 271, 2408424088.[Abstract/Free Full Text]
-
Leong-Morgenthaler,P.M. and Holzhauser,D. (1995) Analysis of mutations induced by 2-amino-1-methyl-6-phenylimidazo-[4,5-b]-pyridine (PhIP) in human lymphoblastoid cells. Carcinogenesis, 16, 713718.[Abstract]
-
Yadollahi-Farsani,M., Gooderham,N.J., Davies,D.S. and Boobis,A.R. (1996) Mutational spectra of the dietary carcinogen 2-amino-1-methyl-6-phenylimidazo-[4,5-b]-pyridine (PhIP) at the Chinese hamster hprt locus. Carcinogenesis, 17, 617624.[Abstract]
-
Kakiuchi,H., Watanabe,M., Ushijima,T., Toyota,M., Imai,K., Weisburger,J.H., Sugimura,T. and Nagao,M. (1995) Specific GGGA to GGA mutation of the APC gene in rat colon tumors induced by 2-amino-1-methyl-6-phenylimindazo-[4,5-b]-pyridine. Proc. Natl Acad. Sci. USA, 92, 910914.[Abstract]
-
Yamamoto,H., Sawai,H. and Perucho,M. (1997) Frameshift somatic mutations in gastrointestinal cancer of the microsatellite mutator phenotype. Cancer Res., 57, 44204426.[Abstract]
-
Fearon,E.R. and Vogelstein,B. (1990) A genetic model for colorectal tumorigenesis. Cell, 61, 759767.[ISI][Medline]
Received September 1, 1998;
revised October 14, 1998;
accepted October 29, 1998.