Genetic, biochemical, and cross-resistance studies with mutants of Chinese hamster ovary cells resistant to the anticancer drugs, VM-26 and VP16-213.

The effects of the anticancer drugs 4'-demethylepipodophyllotoxin thenylidene /3-o-glucoside and 4'-demethylepipodophyllotoxin ethylidene 0-o-glucoside on the growth and viability of Chinese hamster ovary cells have been examined. Stable mu tants which are from 4to 12-fold more resistant to these drugs (VpmR mutants) are obtained in a single selection step in Chinese hamster ovary cells at a frequency of between 0.2 x 1(T6 and 0.5 x 10~6. However, treatment of cells with the mutagen ethyl methanesulfonate (300 /¿g/ml for 20 hr) increased the frequency of resistant mutants by about 20to 30-fold in different experi ments. The two VpmR mutants which have been studied in detail exhibited significantly increased cross-resistance to a number of anticancer drugs and other compounds, including actinomycin D, Adriamycin, bruceantin, chromomycin Aa, colchicine, daunomycin, ellipticine, ethidium bromide, 5-fluorouracil, ftorafur, mithramycin, puromycin, and vinblastine. However, the level of re sistance of these mutants towards several podophyllotoxin de rivatives (namely, podophyllotoxin, 4'-demethylepipodophyllotoxin, and podophyllotoxin ß-o-glucoside)and a number of other anticancer drugs, namely, 9-0-D-arabinofuranosyladenine, chlorambucil, hexamethylmelamine, hydroxyurea, maytansine, methotrexate, methylglyoxal (bis)guanylhydrazone, mitomycin C, and taxol, was found to be unaltered. Interestingly, the sensitivity of the above mutant cell lines toward a number of other anticancer drugs, namely, bleomycin, c/s-diamminedichloroplatinum(ll), and 1-ß-o-arabinofuranosylcytosine, was found to be enhanced in comparison to the parental cells. The mutant cell lines showed reduced uptake of [3H]daunomycin in comparison to the sensitive cells, and this result, together with the cross-resistance of the mutants to unrelated drugs, indicates that the genetic lesion in these mutants is most probably affecting the membrane perme ability of various drugs. In cell hybrids formed between the resistant and the sensitive cells, the drug-resistant phenotype of both of the above mutants behaved in a codominant manner. The results of these studies are discussed in relation to the mechanism of action of 4'-demethylepipodophyllotoxin thenyli dene 0-D-glucoside and 4'-demethylepipodophyllotoxin ethyli dene /3-D-glucoside. INTRODUCTION VM-262 and VP16-213 are 2 structurally related semisynthetic derivatives of podophyllotoxin which have proven effective 1Work supported by a research grant from the Medical Research Council and the National Cancer Institute of Canada. 2The abbreviations used are: VM-26, 4'-demethylepipodophyllotoxin thenyli dene /i-D-glucoside; VP16-213, 4'-demethylepipodophyilotoxin ethylidine fi-D-glucoside; CHO, Chinese hamster ovary; Vpm" mutants, mutants of CHO cells which show stable, high levels of resistance toward VM-26 and VP16-213; WT, wild-type; c/s-platin, c/s-diamminedichloroplatinum(ll); ara-C, 1-fJ-o-arabinofuranosylcytosine; methyl-GAG. methylglyoxal (bis)guanylhydrazone. Received July 23. 1982; accepted December 10,1982. against a number of different animal and human cancers (5, 21, 36,37). In view of their favorable therapeutic responses in human clinical trials, these compounds, either alone or in combination with other drugs, are currently being used in the treatment of a number of human cancers, including Hodgkin's and non-Hodgkin's lymphomas, leukemias, brain tumors, bladder carcinomas, and small-cell bronchogenic carcinomas (3, 5, 18, 25, 27-29, 31). Although VM-26 and VP16-213 are derivatives of podo phyllotoxin (see Chart 1 for chemical structures), their mecha nism of action appears to be quite different from that of the parental compounds podophyllotoxin and 4'-demethylepipodophyllotoxin. While the latter compounds inhibit microtubule as sembly and arrest cells during mitosis, VM-26 and VP16-213 have no effect on microtubule assembly, and cells treated with these drugs are blocked in a premitotic phase (late S or G2) of the cell cycle (20, 22, 23, 26, 36, 38). Earlier studies on the effects of VM-26 and VP16-213 in mammalian cells indicate that treatment with these compounds causes a reduction in nucleoside uptake and preferential inhibition of thymidine incorporation into DNA (8, 9, 23). In human hematopoietic cell lines exposed to VP16-213, a high incidence of chromosomal aberration has been reported by Huang et al. (19). More recently, Loike and Horowitz (24) have observed that, in HeLa cells treated with VP16-213 or VM-26, cellular DNA is converted to a lowermolecular-weight form, suggesting that these compounds prob ably induce single-strand lesions in the DNA. Although all of the above observations indicate that the primary effect of these compounds is on DNA metabolism, their precise mode of action remains unclear at present. To understand the mechanisms of action of various cytotoxic chemotherapeutic drugs, we have been using a genetic approach in which cellular mutants resistant to various agents are selected and subsequently characterized genetically and biochemically. Using this approach, recently mutants of CHO cells exhibiting greatly increased resistance towards podophyllotoxin and which were affected in a microtubule-associated protein were de scribed (10, 14). The various podophyllotoxin-resistant mutants which were examined exhibited proportionally increased resist ance towards various podophyllotoxin analogues which pos sessed microtubule-inhibitory activity. However, these mutants did not show any cross-resistance toward VM-26 or VP16-213, thus providing further evidence that the mechanism of action of these compounds is different from that of the parental podophyllotoxin-like compounds (12, 14). In the present paper, we describe the selection and partial characterization of mutants of CHO cells which show stable, high levels of resistance toward VM-26 and VP16-213 (Vprn" mutants). The results of crossresistance studies with 2 of the Vpm" mutants, which behave codominantly in cell hybrids, toward several podophyllotoxin derivatives and a wide variety of other antineoplastic drugs are also presented in this paper. 1568 CANCER RESEARCH VOL. 43 on April 13, 2017. © 1983 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from PodophylloîO VP-16-213 4'-D.m.lh,l «pipodophylloio.m