[CANCER RESEARCH 49, 1197-1201, March 1, 1989) Effect of Retinoic Acid on DNA Cleavage and Cytotoxicity of Topoisomerase II- reactive Drugs in a Human Head and Neck Squamous Carcinoma Cell Line1 Hoon-Kyo Kim, Leonard A. Zwelling, Peter G. Sacks, Waun Ki Hong, Diana Chan, Lynn Silberman, and Bonnie S. Glisson2 Departments of Medical Oncology [H. K. K., L. A. Z., W, K. H., D. C., L. S., B. S. GJ and Tumor Biology fP. G. S.J, The University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030 ABSTRACT Evidence from several in vitro systems indicates that cellular responses to DNA topoisomerase H-reactive compounds (i.e., the epipodophyllo- toxins and intercalating agents) may be affected by the relative rate of proliferation. Using a human head and neck squamous carcinoma cell line 183A, we have investigated the effect of /3-all-frans-retinoic acid (RA), a substance with known antiproliferative effects, on the DNA cleavage and cytotoxic activities of etoposide and 4'-(acridinyla- mino)methanesulfon-m-anisidide which interact with topoisomerase II. The effect of RA treatment on the activity of X-radiation and bleomycin, both of which produce free radical mediated effects, was also examined. RA treatment (10 to 20 ¿IM for 72 h) does not significantly influence DNA cleavage induced by X-radiation or bleomycin but decreases DNA cleavage and cytotoxicity mediated by etoposide and 4'-(acridinyla- mino)methanesulfon-m-anisidide. Further, this effect can be demon strated at a dose of RA that is minimally growth inhibitory. The inhibitory effect of RA appears to be localized to the nucleus given that similar effects on drug-mediated DNA cleavage can be demonstrated in nuclei isolated from RA-treated cells. However, both drug-stimulated DNA cleavage activity and topoisomerase II catalytic activity are approxi mately equal in crude nuclear extracts of untreated and RA-treated cells. These data suggest that the resistance to topoisomerase II-reactive drugs induced by RA treatment of 183A cells is not mediated through a direct effect on the enzyme, but, instead, is related to other changes in the nuclear milieu occurring in the initial stages of quiescence such as altered chromatin conformation. INTRODUCTION Recent evidence substantiates the role of the nuclear enzyme DNA topoisomerase II as a critical intracellular target of a variety of antineoplastic agents, many of which are quite useful clinically. This includes the nonintercalative epipodophyllotox- ins, etoposide and teniposide, and several different classes of intercalating agents, e.g., aminoacridines, anthracyclines, an- thracenediones, and others (Ref. l for review). There are several studies which indicate that topoisomerase II activity, as well as sensitivity to topoisomerase H-reactive compounds, is de creased in quiescent versus cycling cell populations. Enzyme activity is increased in mitogenized lymphocytes (2) and in regenerating mouse liver tissue after partial hepatectomy (3). More recently it has been shown that enzyme content and sensitivity to etoposide-mediated strand-breaking activity are increased in peripheral blood lymphocytes stimulated with phy- tohemagglutinin and interleukin 2 (4). However, other data suggest that cultured cell lines are not uniform in their regula- Received 12/9/87; revised 3/29/88, 8/16/88, 10/17/88; accepted 11/30/88. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1This work was supported by a Clinical Oncology Career Development Award (B. S. G.) and Grant CH-324A (L. A. Z.) awarded by the American Cancer Society, USPHS Grants CA 40090 (L. A. Z.) and RR 5511-23 (L. A. Z.), and by a gift to the M. D. Anderson Annual Fund for the Chemotherapy Research Program by Henry C. Beck, Jr., of Dallas, TX (L. A. Z.). 2 To whom requests for reprints should be addressed, at Department of Medical Oncology, Box 80, University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Blvd., Houston, TX 77030. tion of topoisomerase II content nor in their response to topo isomerase H-reactive agents in quiescence (5). Retinoids are a group of metabolites and synthetic analogues of vitamin A (retinol) which have been shown to have both antiproliferative and differentiative effects on many human squamous cell lines in culture (6-8). In the present paper we have used a HHNSCC3 cell line 183A to investigate the effect of RA treatment on cellular responses to drugs that interact with topoisomerase II, namely, etoposide and m-AMSA. For comparison we examined the effect of RA treatment on the activity of bleomycin and X-radiation, both of which produce free radical-mediated effects and are used quite commonly to treat head and neck cancer. We also determined topoisomerase II activity and drug-stimulated DNA cleavage activity in nuclear extracts of RA-treated cells. Our data indicate that RA treat ment of 183A cells induces resistance to etoposide- and m- AMSA-induced DNA cleavage and cytotoxicity that does not appear to be mediated through a direct effect on the enzyme, and they suggest that other events occurring in the early stages of decreased proliferation can influence topoisomerase H-me- diated DNA cleavage. MATERIALS AND METHODS Chemicals. Cell culture medium, fetal calf serum, trypsin, and Hanks' balanced salt solution were purchased from Grand Island Biological Co. (Grand Island, NY). [14C]Thymidine (58 mCi/mmol), [methyPH]- thymidine (20 Ci/mmol), and 3H2O (1 mCi/g) were from New England Nuclear (Boston, MA); bleomycin and m-AMSA were obtained from the Drug Synthesis and Chemistry' Branch, Division of Cancer Treat ment, National Cancer Institute; etoposide was provided by Bristol Myers (Syracuse, NY); [3H]etoposide (200 mCi/mmol) was purchased from Moravek Biochemicals (Brea, CA); [14C]m-AMSA (19.6 mCi/ mmol) was from SRI International (Menlo Park, CA); RA was provided by Dr. Reuben Lotan (Department of Tumor Biology, University of Texas M. D. Anderson Cancer Center, Houston, TX); tetrapropylam- monium hydroxide was obtained from RSA Corp. (Ardsdale, NY); closed circular SV40 DNA was from Lofstrand Labs. (Gaithersburg, MD); and other chemicals were obtained from Sigma Chemical Co. (St. Louis, MO). Cell Lines and Culture Techniques. HHNSCC lines 183A and 1483 were grown in monolayer in Dulbecco's modified Eagle's medium with 10% fetal calf serum. Details concerning the epithelial origin and characteristics of these cell lines have been published (9). Doubling time is approximately 48 h for 183A and 55 h for 1483. Murine leukemia LI210 cells were grown in suspension culture in RPMI 1630 medium with 10% fetal calf serum. Cells were grown at 37°Cin the presence of 5% CO2 with 2 mM glutamine, 50 ug/m\ of penicillin, 50 Mg/ml of streptomycin, and 100 Mg/ml of neomycin added to the culture medium. Drug Treatment. Etoposide, m-AMSA, and RA were dissolved in DMSO at 10 mM, stored at -20°C, and diluted with medium as 3The abbreviations used are: HHNSCC, human head and neck squamous cell carcinoma; RA, /3-all-Ã-ranÃ--retinoic acid; m-AMSA, 4'-(acridinyla- mino)methanesulfon-sn-anisidide; DMSO, dimethyl sulfoxide; PBS, phosphate- buffered saline; kDNA, kinetoplast DNA; SDS, sodium dodecyl sulfate. 1197 Research. on November 30, 2014. © 1989 American Association for Cancer cancerres.aacrjournals.org Downloaded from