Proficient Human Colorectal Cancer Cells

Differential cytotoxicity of anticahcer agents in hMutSα-deficient and -proficient human colorectal cancer cells

lichiro UCHIDA and Xiaoling ZHONG1

1Department of Molecular Biology and 21st Department of Surgery , Toho University School of Medicine, Tokyo 143-8540, Japan

Abstract: Mismatch repair (MMR)-deficient cells exhibit drug resistance to several anticancer agents including N-methyl-AP-nitro-N-nitrosoguanidine (MNNG), cisplatin, and adriamycin. Since these agents are potent mutagens, it is possible to select resistant clones of tumor cells during chemotherapy. Prior to determining whether drug cytotoxicity was altered by MMR-deficiency, mutation in the (A)8 repeat region of the hMSH3 gene of the MMR-deficient human colorectal cancer cell line HCT116 and the MMR-proficient human chromosome 3-transferred HCT116 (HCT116+ch3) was comfirmed. A screening method was then determined using MNNG cytotoxicity in both cell lines and 20 additional anticancer agents were examined. Clonogenic cytotoxic assay revealed in 8 anticancer agents (streptozotocin, 5-fluorouracil, tegafur, bleomycin, mitomycin C, vinblastine, vincristine, and nidoran) maintaining the desired level of cytotoxicity required a higher concentration in HCT116 than in HCT116+ch3. Cytosine0-13- arabinofuranoside, chlorambucil, and epirubicin were more cytotoxic to HCT116. Dacarbazine, nitrogen mustard, 31-azido-3'-deoxythymidine, aclarubicin, neocarzinostatin, actinomycin D, and peplomycin possessed similar cytotoxicity. These results suggest that drugs with higher or uncompromised sensitivity can circumvent drug resistance due to MMR-deficiency in tumor cells.

Key words: mismatch repair deficiency, drug resistant, human colorectal cancer cells, circum- vention

hMSH23'4),hMSH35), hMSH6 (GTBP or p160)", Introduction hA4LH18'9),hPMS2, and hPMS11°) have been defined by cytogenetic, biochemical, and molecu- The mismatch repair (MMR) system plays an lar biological studies. hMSH2, dimerized with ei- important role in an increase in DNA replication ther hMSH67) or hMSH311), recognizes mis- and transcription fidelity1). MMR is a highly con- matched bases and subsequently recruits the served cellular process found in organisms from hMLH1 and hPMS2 complex, hMutLa12),prolifera- Escherichia co/ito humans. It results in the rec- tive cell nuclear antigen13), and other MMR-related ognition, excision, and re-synthesis of a newly synthesized DNA strand containing mismatched proteins. The hMSH2 and hMSH6 complex, hMutSa, recognizes one base mismatch and one bases2). Six human MMR genes such as unpaired base. The hMSH2 and hMSH3 complex, hMutSp, binds to two or more unpaired correspponding author: lichiro Uchida, M. D. Department of bases11). A defective MMR system is associated Molecular Biology, Toho University School of Medicine, 5-21-- with hereditary nonpolyposis colorectal cancer 16, Ohmori-Nishi, Ohta-ku, Tokyo 143-8540, Japan. Tel: +81-3-3762-4151 (FINpcc )3,4,6-8,14,15)and certain sporadic colon Fax: +81-3-3764--6923 cancers1'16-18). MMR deficient cells have a higher E-mail: [email protected],ac.jp frequency of both spontaneous mutation and alterations in microsatellite repeat sequences (the

301 lichiro Uchida et al.

microsatellite instability (MSI) phenotype1i19-22) effective on hMutSa-deficient cells; and those with and are resistant to some antitumor agents such no difference in effectiveness. The potential util- as N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)23, ity of drugs for treatment of MMR-deficient tumors 6-thioguanine24,25), cisplatin26'27), adriamycin27), is then discussed. and etoposide28). Materials and Methods One pair of the human colorectal cancer cell lines, HCT11629)and normal human chromosome Materials 3-transferred HCT116 (HCT116+ch3)23), provides MNNG, streptozotocin, dacarbazine, an excellent experimental system to understand chlorambucil, 3'-azido-3'-deoxythymidine (AZT), the biological roles of MMR gene products be- 5-fluorouracil (5-FU), tegafur, cytosine /3-D- cause of their similar genetic and cytological arabinofuranoside (Ara-C) , aclarubicin, mitomy- composition. HCT116 has the homozygously cin C, actinomycin D, vincristine, vinblastine, mutated hMLH1 gene located in 3p21-238) and bleomycin, and 3-(4,5-dimethyl-thiazole-2-yI)-2-5- exhibits microsatellite instability, lack of MMR diphenyl tetrazolium bromide (MIT) were pur- and transcription-coupled excision repair (TCR) chased from Sigma Chemical Co. (St. Louis, MO, activities23'3°)and resistance to some of the anti- USA). Neocarzinostatin was kindly donated by cancer drugs mentioned above. Conversely, Dr. Shigehiro Matsumoto of the Kayaku Co. Ltd., HCT116+ch3 restores these 'MMR-deficient Tokyo. Nitrogen mustard, nidoran, peplomycin, phenotypes23,24,26,30-32)because of adequate epirubicin, ifomide, and ranimustine were gener- expression of the wild-type hMLH1 gene on the ous gifts of Yoshitomi Pharmaceutical Industries, transferred chromosome18). Recently, another Ltd., Osaka, Sankyo Co., Ltd., Tokyo, Nippon mutation in a microsatellite sequence, (A)8,in exon Kayaku Co., Ltd., Tokyo, Kyowa Hakko Kogyo Co., 7 of the hMSH3 gene located in 5q11-135 of Ltd., Tokyo, Shionogi & Co., Ltd., Osaka, and HCT116 has been identified33). Thus, hMutSa, Tokyo Tanabe Co., Ltd., Tokyo, respectively. but not hMutS/3, is assumed to be active in HCT116, while the MMR pathway is blocked at the Cells and cell culture hMutLa step. HCT116 was maintained in minimum essen- Drug resistance due to MMR deficiency is a tial medium (Earle's salt) supplemented with 10% serious problem in cancer chemotherapy. Since fetal bovine serum. HCT116+ch3 was cultured anticancer agents are potent mutagens, treatment in the same medium with 400 ,ug geneticin (Gibco- with these agents may lead to the regeneration or BRL)/ml. Cells were grown in a 5% CO2 in air in a proliferation of drug-resistant MMR-deficient tumor humidified atmosphere. cells. A mere two-fold difference in cisplatin sensitivity has been demonstrated to be sufficient Mutation analysis to regenerate a resistant tumor in a mouse DNA was isolated from cultured cells with a mode134). Knowledge concerning the sensitivity DNAzol Reagent (GIBCO BRL Life Technologies, of anticancer agents in MMR-deficient cells is Inc., Grand Island, NY). A set of primers (sense limited. Thus, it is important to determine the sen- 5'-GAGATAATGACTGATACTTCTACC-3'; sitivity of cancer chemotherapeutic agents fre- antisense 5'-CATTTGTTCCTCACCTGCAAAG-3') quently used in treating MMR-deficient malignant for polymerase chain reaction (PCR) and sequenc- tumor cells. In this study, MNNG cytotoxicity in a ing was specific to a part of the hMSH3 gene pair of cell lines, HCT116 and HCT116+ch3, (Gene Bank accession J104810). Reaction mix- was used to determine the best of 3 assay ture of PCR contained 0.1 ,ug template DNA, prim- methods. The sensitivity of 20 anticancer agents ers, 0.2 mM each dNTP mix, and 2.5 U Taq DNA was then screened by clonogenic cytotoxic assay polymerase, and amplification buffer consisting of using the pair of cell lines. The agents were clas- 10 mM Tris-CI, 50 mM KCI, and 1.5 mM MgCl2, pH sified into three groups: those less effective on 8.8, in a total volume of 100 pl. Thermal condi- hMutSa-deficient than -proficient cells, those more tion was 30 cycles of denaturing at 96°C, anneal-

302 cytotoxity of anticancer agents in hMutSa-deficient cells

ing at 58°C, extension at 72°C for 1 min, and final For clonogenic assay, 200 single cells sus- extension for 8 min in a PCR Thermal Cycler pended in 2 ml medium in a well of a 6-well plate (model PJ2000, Perkin-Elmer Co., Foster City, were cultured in a CO2 incubator. After 24 hrs, CA). The PCR product was used for single cells were exposed to various concentrations of strand conformational polymorphism (SSCP) as an agent for 1 hr, 24 hrs, or continuously (168 described35) with small modifications. Aliquot of hrs). Culture was continued another 7 days. the PCR product was mixed with denaturing solu- Cells were fixed and stained with a mixture con- tion containing 95% formamide and the mixture sisting of 0.05% crystal violet, 4.2% neutralized was heated at 95°C for 3 min, immidiately placed formalin, and 10% ethanol. Colonies consisting on an ice-bath for 5 min, and electrophoresed with of more than 50 cells were counted. The number a 12% polyacrylamide gel (20 x 40 x 0.1 cm) at of colonies in the culture without agents was de- 20°C for 20 hrs with 3 W constant power. The fined as 100% and 50% inhibitory concentration abnormal bands were detected by comparing (IC50)was estimated in triplicate. Colony efficien- those of normal DNA. DNA sequencing was per- cies of HCT116 and HCT116+ch3 were 0.7-0.75 formed using a Taq DyeDeoxy Terminator Cycle and 0.6-0.65, respectively. Sequence kit (Perkin-Elmer) as recommended by M17 assay was carried out as described36) the supplier. The reaction mixture was analyzed with small modifications. Cells were plated into a on an automated DNA sequencer (Perkin-Elmer, well of a 96-well plate at a cell density of 104/well Applied Biosystems Division, model 373S). in a total culture volume of 100 ,u1. After 24 hrs, cells were treated with various concentrations of Preparation of single cell suspension MNNG in quadruplicate for 1 hr and washed with Exponentially growing cells in a 25 cm2 flask medium. After 3 day cultivation, 10 ,u1of M I I so- were harvested by trypsinization, washed with lution was added and incubated for 3 hrs. After medium supplemented with 10% fetal bovine se- removal of the supernatant by suction, 50 ,u1of rum by centrifugation, and resuspended with 10 fresh medium and 100 1.11of 0.04 N HCI in isopro- ml of medium in a 15 ml tube. To make a single panol were added and thoroughly mixed with a cell suspension, the tube was left to stand for 15- microplate mixer. The absorbance at 545 nm 20 min at room temperature and 6 ml of top super- was measured by a microplate reader (model 550, natant was removed to another tube. The tube Bio-Rad Laboratories, Hercules, CA. USA). was centrifuged and 100 dal of medium was Dye-staining was performed as described added. After the cell suspension was up and with small modifications37). Cells (104) in 200 pl down several times with a 200 p1 tip, fresh medium of culture medium were added to each well of a was added for further dilutions. This step was 96-well microplate. After incubation for 24 his at repeated until cells became a single cell 37°C, cells were treated with MNNG for 1 hr in suspension. Confirmation of a single cell suspen- quadruplicate and culture was continued for sion and determination of the cell number were another 3 days. After cultivation, cells were done with a hemocytometer. washed, fixed, and stained with crystal violet. The absorbance at 610 nm was measured by a Cytotoxic assays microplate reader. MNNG was dissolved with dimethylsulfoxide at a concentration of 20 mM and further dilutions Statistical analysis were made with sterile phosphate-buffered saline The values in triplicate or quadruplicate (PBS) consisting of 136.9 mM NaCI, 2.7 mM KCI, samples are expressed as means and 8.1 mM Na2PO4, and 1.5 mM KH2PO4, pH SD. Paired Student's t-test 1-tail p values were 7.3. Other cytotoxic agents were dissolved in calculated from 3 to 5 independent experiments. Milli-Q grade water and filtered with a 0.22 pm p value less than 0.05 was determined as signifi- pore-sized membrane. Further dilutions were cant difference. made with sterile PBS.

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Results HCT116+ch3 has the same mutation as that of HCT116 and the hMutSf3 heterodimer consisting Mutation in the (A)8 repeat region of the hMSH3 of hMSH2 and hMSH3 in both cell lines is gene dysfunction. Therefore, the MMR pathway medi- We studied the mutation in the (A)8 repeat ated by hMutSa in HCT116+ch3, but not HCT116 , region of the hMSH3 gene of the HCT116+ch3 is assumed to be proficient. cells with PCR-SSCP analysis. As expectedly , the band pattern of DNA of HCT116+ch3 was iden- Comparison of cytotoxic assays tical to the parent cell line, HCT116 (Fig. 1). To determine an appropriate assay method Sequence analysis showed homogygous one A for the drug sensitivity in the hMutSa-deficient deletion in both cell lines (data not shown). These HCT116 cell line and the hMutSa-proficient results indicate that the hMSH3 gene of HCT116+ch3 cell line, the clonogenic, MTT, and dye-staining cytotoxic assays were evaluated , using MNNG,which is known to be approximately 5-fold resistant to MMR-deficient cell lines23). Though HCT116 was shown to be resistant to MNNG by all the assay methods, the clonogenic cytotoxic assay was the most sensitive and reli- able (Fig. 2). Thus, the clonogenic assay was selected to determine the cytotoxicity of the other antitumor agents.

Cytotoxicity of anticancer agents The cytotoxic effects of 5 alkylating agents on HCT116 and HCT116+ch3 were studied to determine the resistance of hMutSa-deficient cells. HCT116 was resistant to streptozotocin, a potent DNA-methylation agent, at all concentrations tested. The survival curves of both cell lines treated with various concentrations of streptozotocin are shown in Fig. 3A. Four independent experiments with streptozotocin revealed an approximately 2.7-fold resistance in hMutSa-deficient cells (p = 0.002), when comparing the IC50values for both cell lines (Table 1). Although more HCT116 cells than HCT116+ch3 cells survived when treated with nidoran and dacarbazine (Fig. 3B and C), as well as nitrogen mustard, no signifi- cant differences in IC50values of these agents were observed (Table 1). In contrast to the DNA methylating agents, such as MNNG and streptozotocin, HCT116 was more sensitive Fig. 1. Mutation of the (A)8 repeat region of the hMSH3 to chlorambucil than HCT116+ch3 (Fig. 3D). gene in HCT116+ch3. Genomic DNAs isolated Similar results were obtained in 3 independent ex- from normal peripheral lymphocytes (lane 1), HCT116 (lane 2), and HCT116+ch3 (lane 3) were periments: an approximately 1.3-fold resistance subjected to nonradioactive PCR-SSCP analysis. was noted in HCT116+ch3 cells (Table 1). After electrophoresis, a gel was visualized by stain- Ifomide and ranimustine displayed no cytotoxicity ing with ethidium bromide. at any concentration examined.

304 cytotoxity of anticancer agents in hMutSa-deficient cells

Fig. 2. Comparison of sensitivity of clonogenic (A), M I 1 (B), and dye-staining (C) assays in determining MNNGcytotox- icity to the human colorectal cancer cell lines, HCT116 and HCT116+ch3. Single cell suspensions of HCT116 (open symbols) and HCT116+ch3 (closed symbols) were inoculated into wells of a 6 well-culture plate, and the cytotoxicity of various concentrations of MNNG indicated in figure was determined as described under Materials and Methods. Bars show SD.

Fig. 3. Survival curves of HCT116 and HCT116+ch3 treated with alkylating agents. Percent survival of HCT116 (open symbols) and HCT116+ch3 (closed symbols) treated with various indicated concentrations of streptozotocin (A) for 1 hr, nidoran (B) for 24 hrs, dacarbazine (C) for 7 days, and chlorambucil (D) for 1 hr, determined by the clonogenic assay in triplicate. Bars show SD.

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The IC50 values were determined from the survival curves and are expressed as means ± SD of the number of independent experiments in triplicate. Index was calculated by following formula: mean of the 1050 value for HCT116/mean of the IC50 value for the HCT116+ch3. p values were caluculated using paired t-test with 1-tail p. The concentration of neocarzinostatin is expressed as pg per ml.

Since 6-thioguanine, a purine derivative, is Bleomycin and mitomycin C were less cytotoxic to known to be less effective in HCT11625),the drug HCT116 than HCT116+ch3 (Fig. 5A and B). sensitivity of 5 nuclear base-related analogs was These antibiotics were approximately half as then examined. HCT116 was 2.5- and 1.7-fold effective with HCT116, as determined by IC50 more resistant to 5-FU and tegafur, respectively values (Table 1). Like chlorambucil and Ara-C, (Fig. 4A and B, Table 1). Unexpectedly, Ara-C HCT116 had a 1.2-fold sensitivity to epirubicin (Fig. was more cytotoxic to HCT116 than HCT 116 + ch3 5D, Table 1). Actinomycin D, neocarzinostatin, (Fig. 40, Table 1). Aclarubicin and AZT had and peplomycin were almost equally cytotoxic to similar cytotoxicity to HCT116 and HCT116+ch3 both cell lines (Fig. 5C, Table 1). Vinca alkaloids were more cytotoxic to HCT116+ch3 than HCT116 (Fig. 4C, Table 1). Many antitumor antibiotics cause DNA dam- (Fig. 6, Table 1). age, such as double or single strand cleavage, cross-linking, and/or DNA adduct formation.

306 cytotoxity of anticancer agents in hMutSa-deficient cells

Fig. 4. Survival curves of HCT116 and HCT116+ch3 treated with nucleoside analogs. Percent survival of HCT116 (open symbols) and HCT116+ch3 (closed symbols) treated with various indicated concentrations of 5-FU (A) for 1 hr, tegafur (B) for 7 days, aclarubicin (C) for 1 hr, and Ara-C (D) for 1 hr, determined by clonogenic assay in triplicate. Bars show SD.

and vinblastine in HCT116 cells, as determined by Discussion IC50 values, were approximately 2- to 3-fold higher The present study aimed to determine the than those in HCT116+ ch3 cells. The cytotoxic sensitivity of cancer chemotherapeutic agents in effects of nidoran, tegafur, mitomycin C, and vinc- MMR-deficient cells. Twenty-one cytotoxic ristine on HCT 1 16 were less than those with agents, including MNNG, were screened using a HCT116 + ch3. Fink et al. have demonstrated that clonogenic cytotoxic assay system with a pair of an approximately 2-fold difference in cisplatin human colon cancer cell lines: HCT1 16, an hMLH1 cytotoxicity between MMR-deficient and -proficient deficient cell line, and HCT1 16+ch3, which is pro- cells in vitro is sufficient to enable regrowth of ficient in MMR due to microcell fusion of a wild type MMR-deficient tumor cells in mice treated with copy of the hMLH1 gene23). The cytotoxic agents cisplatin34). Furthermore, proliferation of MMR- were either more, less or equally cytotoxic to MMR- deficient cells cocultured with proficient cells32) deficient cells. The efficacies of 5-FU, bleomycin, and an increased mutation rate in MMR-deficient

307 lichiro Uchida et al.

Fig. 5. Survival curves of HCT116 and HCT116+ch3 treated with antitumor antibiotics. Percent survival of HCT116 (open symbols) and HCT116+ch3 (closed symbols) treated with various indicated concentrations of bleomycin (A), mitomycin C (B), actinomycin D (C), and epirubicin (D), for 1 hr, determined by the clonogenic assay in triplicate. Bars show SD.

Fig. 6. Survival curves of HCT116 and HCT116+ch3 treated with vinca alkaloids. Percent survival of HCT116 (open symbols) and HCT116+ch3 (closed symbols) treated with various indicated concentrations of vinblastine (A) and vincristine (B), for 1 hr, determined by clonogenic assay in triplicate. Bars show SD.

308 cytotoxity of anticancer agents in hMutSa-deficient cells

cells38) have been demonstrated when the cells cause both depolymerization of mitotic interpolar were drugs to which they possessed only limited microtubules and cell metaphase block46'47) resistance such as platinum compounds, adria- and do not appear to result in direct DNA mycin, and etoposide. Thus, it is likely that the 8 damage. Therefore, the mechanism responsible additional chemotherapeutic agents with lower for MMR-deficient cell resistance to vinka alkaloids cytotoxicity to HCT 1 16 cells in this study may may differ from the DNA damaging agents men- produce similar results to other drugs with com- tioned above. Possibly, resistance is triggered promised cytotoxicity such as MNNG, platinum by events downstream of the MMR signaling path- compounds, adriamycin, 6-thioguanine, and way, events which to lead to arrest the cell cycle. etoposide, namely, proliferation of MMR-deficient Unexpectedly, Ara C, chlorambucil, and tumor cells and an increase in the mutation rate in epirubicin were significantly more cytotoxic to tumor cells. MMR-deficient cells. To our knowledge, antican- The mechanism of MMR-deficient cell resis- cer drugs belonging in this category have not been tance to alkylating agents and platinum com- reported previously. These DNA damaging pounds has been extensively studied. hMutSa, agents inhibit DNA replication. Ara-C inhibits the a heterodimer consisting of hMSH2 and hMSH6, action of DNA polymerase a48). Chlorambucil binds to methylated DNA and platinum adducts, inhibits DNA replication by alkylation and induces and MMR-proficient cells cease to proliferate be- G2 phase arrest49.5°). Epirubicin acts as a DNA cause they are unable to pass the G2 check- intercalator and its maximal cell kill occurs during point23,24,39-41). In MMR-deficient cells, because the S phase of the cell cycle". Because no com- of the lack of cell cycling signalling, most cells mon target molecule or pathway is known, further pass through the checkpoints, leading to resis- detailed investigation is required. During the tance for certain drugs. Streptozotocin, an anti- preparation of this manuscript, the modulating biotic derivative of MNNG42), which was least activity of aphidicolin, a DNA polymerase inhibi- cytotoxic to HCT 1 16 among the drugs tested here, tor, was observed in MMR-deficient cells52). may act in a similar manner to MNNG. Major DNA These agents appear to have utility in treating drug damage induced by antitumor antibiotics such as resistant tumor due to MMR deficiency either alone bleomycin, mitomycin C, neocarzinostatin, and or in combination with less cytotoxic drugs such actinomycin D includes cross-linking and/or cleav- as cisplatin, 5-FU, and tegafur, which have been age of single or double strand DNA. Besides widely utilized in solid tumor chemotherapy. cleavage of DNA strands, mitomycin C also Acknowledgments generates DNA adducts43), which may bind to hMutSa. Though there is no report describing We would like to thank Drs. Hiroyuki bleomycin-related DNA adducts, it is possible to Shimatake, Kazunori Tsujita, and Toshifumi Yoshio generate bleomycin-specific adducts or cleavage for their continuing supports, Dr. Hiromichi Hemmi sites in DNA, which may be recognized by for his useful suggestions and discussion through- hMutSa. Neocarzinostatin is reported to gener- out this study and for his critical review of the ate DNA adducts"), though no difference in manuscript, and Dr. Minoru Koi of the National In- cytotoxicity was observed. Perhaps cleavage stitute of Environmental Health Sciences, NIH, USA action to DNA by the neocarzinostatin-chro- for his useful suggestions and generous gift of mophore is more rapid than other antibiotics45). HCT 1 16 and HCT1 16+ch3. This work was sup- Actinomycin D, a DNA intercalator, which gener- ported in part by a Project Research Grant of Toho ates no DNA adduct showed no difference in University School of Medicine. cytotoxicity. The cytotoxicity of 5-FU and its metabolic analog, tegafur, were lower in HCT 1 16 References than in HCT 1 1 6+ch3, indicating that 5-FU incor- 1) Loeb, L. A.: Cancer cells exhibit a mutator pheno- porated into the thymidine residue of a DNA strand type. Adv. Cancer Res., 72, 25-56, 1998 . may be recognized by hMutSa. 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