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 in- cluding 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.

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