Clonal Analysis of Cancer Cells that Survived Anticancer Drug Treatment

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1 Osaka City Medical Journal Vol. 43, No , 1997 Clonal Analysis of Cancer Cells that Survived Anticancer Drug Treatment TETSUJI TANAKA, LING CHANG, NAOHIKO UMESAKI and SACHIO OGITA Department of Obstetrics and Gynecology, Osaka City University Medical School, Asahi-machi, Abeno-ku, Osaka, 545, Japan Key Words: Drug resistance, Recurrent cancer, Chemotherapy, Cervical cancer Summary We hypothesized that postchemotherapeutic recurrent cancer cells consist of sensitive cells and resistant cells, To examine this hypothesis, the clonality of the postchemotherapeutic surviving cancer cells was characterized, The postchemotherapeutic surviving cancer cells was established using a human cervical carcinoma cell line, ME180, in which cells were treated with either SN38, VP16, or THP for more than 8 weeks, The surviving cells were subcloned by limi ting dilutions yielding the following cloning efficiencies: 18% for SN38, 26% for VP16, and 57 % for THP, Characterization of the established subclones proved that the postchemotherapeutic recurrent cancer cells consisted of both sensitive cancer cells and resistant cancer cells, This result supports the hypothesis and hence points toward improvement of chemotherapeutic effect through an understanding of the dual types of surviving cells, Introduction Postchemotherapeutic recurrent cancers are not always resistant to the pnor ffi=.. &. ffi$a Corresponding Author: Tetsuji Tanaka MD, PhD, Department of Obstetrics and Gynecology, Osaka City University Medical School, Asahi-machi, Abeno-ku, Osaka 545, Japan TEL: FAX:

2 TETSUJI TANAKA, et al. chemotherapy. In most cases of postchemotherapeutic recurrent cancers, the patients have the same levels of drug-responsiveness as they had during the initial chemotherapy. These clinical facts suggest that most postchemotherapeu tic recurrent cancers are sensitive recurrent cancers, and that the induction of sensitive recurrent cancer cells is based on a different mechanism from that of drug-resistant recurrent cancer cells. Few analyses have been done on such differences in inducibility of recurrent cancer cells. Moreover, there is no report of an in vitro assay to predict the incidence of cancer recurrence after chemotherapy or on factors to control cancer recurrence. In this study we established an in vitro experimental model to analyze cellular and molecular mechanisms in postchemotherapeutic recurrence of cancer. Materials and Methods Anticancer Drug Sensitivity Test Cell proliferation was assayed by a non-ri colorimetric cell proliferation assay kit, XTT (Boehringer-Mannheim, Mannheim, Germany). Briefly, the cultured log phase cells were detached by 0.25% trypsin/l mm EDTA (GIBCO-BRL Japan, Kyoto), and cultured at 100.ul/well (5000 cells/well) in 96-well culture plates overnight. On the following day stepwisely diluted anticancer drugs were added to the cultures, and after a 72-hour culture, viable cells were assayed with the kit. Anticancer drug sensitivity was evaluated by comparing 50% growth-inhibitory concentration (IC50) of the anticancer drug for surviving cancer cells to those for the parent cancer cells. Establishment of Recurrent Cancer Cells ME180, (1) a human cervical squamous cell carcinoma cell line, was obtained from JCRB (Japan Cell Resources Bank, Tokyo, Japan). All cells were cultured with OPTI-MEM (GIBCO-BRL) with 5% Fetal Bovine Serum (MBL, Nagoya, Japan), 100 D/ml Penicillin (GIBCO-BRL), and 100.ug/ml Streptomycin (GIBCO-BRL). In the study, three anticancer drugs were used to establish recurrent cancer cells. SN38 (courtesy of Yakult Co. Ltd., Tokyo, Japan) is a topoisomerase I inhibitor, that is a major active in vivo metabolite of camptothecin-ll. (2) VP16 (Etoposide) (courtesy of Japan Bristol-Meyers-Squib Co. Ltd., Tokyo, Japan) is a topoisomerase II inhibitor. (3) THP (4'-o-tetrahydropyranyladriamycin) (courtesy of Meiji-Seika Co. Ltd., Tokyo, Japan) is a derivative of anthracycline. (4-6) In order to establish recurrent cancer cells, stepwisely dilu ted anticancer drugs were added to the culture cells. Then each anticancer drug in which surviving cancer cells had grown for 2 weeks was maintained at its highest concentration to suppress cell growth for more than 8 weeks. The drug concentrations used to establish the recurrent cancer cells were

3 Clonal Analysis of Recurrent Cancer ng/ml for SN38, 80 ng/ml for VP16, and 1.2 ng/ml for THP. Surviving cells were subcloned by limiting dilutions with drug-free culture media. Drug-sensitivities of all established subclones were examined by the assay described above, and the su bclones showing more than threefold the lc50 of the parent cells were diagnosed as resistant subclones. Results When ME180 cells were cultured with one of the anticancer drugs, almost all the cultured cells died, and the few surviving cells started to grow 4 weeks later. Limiting dilution analyses were performed after 8-week cultures with the anticancer drugs, and different cloning efficiencies were found between the three drugs. Three types of surviving cells were observed: 1. cells that survived but did not proliferate, 2. cells that were drug-sensitive and proliferated (sensitive recurrent cancer cells), 3. cells that were drug-resistant and proliferated (drug-resistant recurrent cancer cells). Cell Concentration (CellslWell) VP16 r;f:l -QJ QJ = 0I:l QJ Z 10 1 rthp57% Figure 1. Limiting dilution analysis of surviving cancer cells treated with anticancer drugs

4 TETSUJI TANAKA, et al. Table 1. Differential induction of two kinds of drug-resistant cells in survived cells after chemotherapy. Anticancer Drug Cloning No. of Subclones No. of No. of Efficiencies Examined Resistant Cell Sensitive Cell SN38 VP16 THP 18% % % r...,.q e =z , Resistant Clone 0 Sensitive Clone 0 Sensitive Clone Resistant Clone...1!Jt... Sensitive Clone Parent Cell Line Figure ,---,---, VP 16 (x 1/3 f.lg1mi) Drug sensitivity tests of the subclones established from the surviving cancer cells treated with VPl6. Thick solid lines with closed figures show VPl6-resistant subclones, and thick dotted lines with open figures show VPl6-sensitive subclones. The thin solid line with closed diamonds is a VPl6-sensitivity curve of the parent MEl80 Cells. Cloning efficiency was evaluated by limiting dilution analysis for both the sensitive recurrent cells and the resistant recurrent cells. (7) Surviving cells treated with one of the three anticancer drugs were subcloned by limiting dilution. Their cloning efficiencies yielded 18% for SN38. 26% for VPI6, and 57% for THP (Figure 1). Not all s'ubclones showed resistance to the anticancer drug. Actually. many sensitive cancer cells were found among the established recurrent subclones (Table 1). The resul ts of drug-sensi tivi ty tests for established -262-

5 Clonal Analysis of Recurrent Cancer subclones are shown in Figure 2. Discussion The present study provides clear evidence of recurrent cancer cells that are sensitive to an anticancer drug and have nontheless survived in culture media containing the anticancer drug for more than 8 weeks. Induction of the sensitive recurrent cancer cells is a highly probable cause of clinical unresponsiveness to chemotherapy. However, the induction mechanism of the sensitive recurrent cells has not been clarified. MEl8a cells grow in a monolayer fashion with a doubling time of 2-3 days. The sensitive recurrent cells from MEl8a survived in drug-containing media and did not lose their sensitivity to the drug over more than 8 weeks. From these facts we speculate that exposure of MEl8a cells to anticancer drugs may have induced some cells into a long resting stage of the cell cycle and kept these cells unresponsive to the drug. Most recent analytical studies concerning how to overcome recurrent cancer after chemotherapy are analyses of the molecular mechanism of drug-resistant mutant cancer cells. In fact, however, in recurrent cancer patients mutated drugresistant cells are not so frequent as sensitive cancer cells. In other words, the majority of clinical recurrent cancer cells can be sensitive recurrent cells. Our present study found two distinct types of postchemotherapeutic recurrent cancer cells, and we determined that limiting dilu tion analysis can be a useful assay for evaluating the inducibility of postchemotherapeutic recurrent cells. Since a limiting dilution analysis takes a long time to complete, an easier method to predict inducibility of recurrent cancer is needed. Molecular markers to control cancer recurrence may be developed in the future. Clues toward predicting recurrence rate may lie in the characterization of the sensitive recurrent cells exemplified in this study. This study also revealed a distinct difference between anticancer drugs in cloning efficiencies of recurrent subclones. These results indicate that differential therapeutic effects of anticancer drugs may be due largely to the differential inducibility of the drug-sensitive recurrent cancer cells in addition to the effect of the native drugsensitive characteristics of the cancer cells. Therefore, when choosing an anticancer drug for therapy, the inducibility of recurrent cells, which is also a latent characteristic of the cancer cells, needs to be considered. This study has shown experimental evidence that postchemotherapeutic recurrent cancer cells consist of two types of cancer cells: sensitive recurrent cancer cells and resistant recurrent cancer cells. The inducibilities of these recurrent cells may depend on concentration or administration route. But understanding that there are two -263-

6 TETSUJI TANAKA, et al. kinds of recurrent cells must be very important for establishing the most effective chemotherapeutic protocol, since both kinds of recurrent cells can develop in recurrent cancer patients. In fact, some recurrent cancer patients with resistant cells are treated repetitively with the same chemotherapy as the previous treatment. Other recurrent cancer patients with sensitive cells are sometimes treated with another chemotherapy than the previous sensitive. therapy. Wrong decisions can be made by clinicians who do not consider the ratio of the two kinds of recurrent cancer cells in individual patients. Based on the results of the present study, a chemotherapeutic analysis of recurrent cancer raised the following hypothesis. The sensitive recurrent cancer cells may have survived due to insufficient dosage of an anticancer drug during the primary chemotherapy, and therefore patients may have experienced a long cell cycle that was unresponsive to the anticancer drug. For patients with sensitive recurrent cancer cells, a bolus administration of a large amount of the drug will have a low effect on the cancer because the sensitive recurrent cancer cells can have a long unresponsive cell stage. In order to treat those patients who have sensitive recurrent cancer cells, frequent administrations of drugs or a long and continuous exposure to a low concentration of them may be necessary. In contrast, for patients with primarily resistant recurrent cancer cells, the same chemotherapy regimen described above may have no clinical effect on the cancer. To have a similar anticancer effect for such patients, the same anticancer drug used in the prior therapy must be administered at extremely high doses. This, however, is likely to cause lethal side effects. If the same anticancer drug as was used before is to be administered to patients with resistant recurrent cells, one of the following will be necessary: administration of a bolus massive dose of drug over a short time, or combining the drug with another drug to enhance the sensitivity of the recurrent cells to the prior chemotherapeutic drug. The decision concerning what drug should be selected for the combined chemotherapy to enhance drug-sensitivity in the resistant cell, can be done based on the method described in the drug-sensitivity tests in this study. To conclude, for clinical chemotherapy to recurrent cancer patients, an understanding of the two types of recurrent cells and analysis of the clonality of recurrent cells in patients by examining the clinical responses to chemotherapy are the two most viable considerations for determining the best chemotherapeutic protocol for each recurrent cancer patient. Acknowledgment We would like to thank Yakult Co. Ltd., Bristol-Meyers-Squib Co. Ltd., and Meiji-Seika Co. Ltd. for their kind donations of anticancer drugs. This study would -264-

7 Clonal Analysis of Recurrent Cancer not have been possible without their support. References 1. Lancillotti, F., Giandomenico, V., Affabris, E., Fiorucci, G., Romeo, G., and Rossi, G.B.: Interferon alpha-2b and retinoic acid combined treatment affects proliferation and gene expression of human cervical carcinoma cells. Cancer Res. 55: (1995) 2. Taniguchi, K., Kohno, K., Kawanami, K., Wada, M., Kanematsu, T., and Kuwano, M.: Drug-induced down-regulation of topoisomerase I in human epidermoid cancer cells resistant to Saintopin and Camptothecins. Cancer Res 56: (1996) 3. Schneider, E., Horton, J.K., Yang, C-H., Nakagawa, M., and Cowan, K.H.: Multidrug resistance-associated protein gene overexpression and reduced drug sensitivity of topoisomerase II in a human breast carcinoma MCF7 cell line selected for etoposide resistance. Cancer Res 54: (994) 4. Umezawa, H., Takahashi, Y., Kinoshita, M., Naganawa, H., Masuda, T., Ishizuka, M., Tatsuta, K., Takeuchi, T.: TetrahydropyranyI derivatives of daunomycin and adriamycin. J Antibiotics 32: (979) 5. Tsuruo, T., Iida, H., Tsukagoshi, S., and Sakurai, Y.: 4'-0-Tetrahydropyranyladriamycin as a potential new antitumor agent. Cancer Res 42: (982) 6. Hisamatsu, T., Suzuki, K., Sakakibara, S., Takeuchi, T., and Umezawa, H.: Antitumor spectrum of a new anthracycline, (2 n R) -4'-0-Tetrahydropyranyladriamycin, and effect on the cellular immune response in mice. Jpn. J. Cancer Res. 76: (1985) 7. Lefkovits 1: Limiting dilution analysis. in Immunological Methods. Academic Press (979) Received August 29,