[CANCER RESEARCH 46, 6290-6294, December 1986] Isolation and Characterization of a Chinese Hamster Ovary Cell Line Resistant to Bifunctional Nitrogen Mustards1 Craig N. Robson, Janice Alexander, Adrian L. Harris, and Ian D. Hickson2 Departmem of Clinical Oncology, University of Newcastle upon Tyne, The Royal Victoria Infirmary, Newcastle upon Tyne, NEI 4LP, United Kingdom ABSTRACT mustards, is collaterally sensitive to nitrosoureas (10), despite the fact that both these classes of agent generate DNA inter- A drug-resistant derivative of a Chinese hamster ovary cell line has strand cross-links. been generated by chronic exposure to progressively higher concentra Although chlorambucil is widely used in the curative therapy tions of chlorambucil. The cells exhibit greater than 20-fold resistance of Hodgkin's disease (11), in modified cyclophosphamide-meth- to the cytotoxic effects of chlorambucil and comparable levels of cross- resistance to mechlorethamine and melphalan. These drugs all belong to otrexate-5-fluorouracil protocols for breast cancer (12), in ovar a class of bifunctional alkylating agents which generate DNA cross-links ian cancer (13), and in small cell lung cancer (14), cell lines by reaction at the N-7 position of guanine. However, no resistance is isolated on the basis of chlorambucil resistance have rarely been observed to several other drugs which possess a similar mechanism of reported (15). action, to en-platinum (Mamminedichloride or to bischloroethylnitrosou- Here, we describe the isolation of a CHO3 cell line which rea and mitomycin C, which cross-link DNA via the O6 position of exhibits elevated levels of resistance to the cytotoxic effects of guaninc. Lack of resistance to vincristine, colchicine, or Adriamycin chlorambucil and appears to exhibit a novel profile of drug coupled with the failure of the calcium channel blocker verapamil to resistance. The cells are cross-resistant only to a strictly limited reverse the phenotype, indicates that the mechanism of resistance is distinct from that characterized by the multidrug-resistant phenotype. number of bifunctional alkylating agents. Acquired resistance Support for this view comes from the finding that no significant alteration has been accompanied by marked overexpression of a cytosolic in melphalan uptake could be demonstrated. protein with a molecular weight of approximately 25,000. Wild- The phenotype is very stable and has been maintained during 12 type levels of sensitivity to Adriamycin, vincristine, and colchi months of continuous culture without further selection. A slightly elevated cine have been maintained, suggesting that an alternative mech basal level of glutathione is present in the resistant cells, but resistance anism to the membrane glycoprotein-mediated multidrug-re is not overcome by depletion of intracellular glutathione with buthionine sistant phenotype is responsible for the observed drug resistance sulfoximine. A cytosolic protein with a molecular weight of approximately of these cells. 25,000 is constitutively overexpressed in the resistant cells. Although these cells have an abnormal karyotype, no conclusive evidence for any cytogenetic indicator of gene amplification could be detected. MATERIALS AND METHODS INTRODUCTION Cell Culture and Media. Cells were maintained as described previ ously (16). One of the major drawbacks to effective cancer chemotherapy Isolation of a Chlorambucil-resistant Cell Line. The chlorambucil- is the emergence of tumor cell subpopulations with intrinsic or resistant cell line was selected by growing cells for 3 months in the acquired resistance to anticancer drugs. A common clinical presence of progressively higher concentrations of chlorambucil. Cells were exposed to chlorambucil for 3-4 days before subculturing and observation is that tumor cells which acquire resistance to a growing in drug-free medium for 24 h. Following this, the drug treat particular anticancer agent also exhibit resistance to a range of ment was repeated but at a higher dose. The final concentration of other drugs which may be structurally or functionally unrelated. chlorambucil used was 50 Mg/ml. This culture was plated for single In vitro studies implicate alterations in intracellular drug accumulation with the "so-called" multidrug resistance pheno colonies which were overlaid with medium containing 0.4% Noble agar. After 2 days, when the cells were growing up into the agar, they were type. An increasing level of cellular resistance to agents such as transferred onto the surface of an agar plate (0.5% agar in medium). the Vinca alkaloids, colchicine, or the anthracyclines is accom Single colonies of cells that grew were dispersed into liquid medium panied by increased expression of a family of high molecular and expanded into large scale cultures. weight membrane glycoproteins (1-6). This appears to be a Survival Curves. Survival determinations were performed as de common mechanism for acquired drug resistance in several scribed previously (16). Treatments with cytotoxic agents were as outlined below. different human and rodent cell lines. Radiation. Exposure to UV or X-rays was as described previously Although this mechanism has been reported to be responsible (16). for induced cellular resistance to some alkylating agents, such Drug Treatments. The DNA-damaging drugs were generally pur as melphalan and mitomycin C (4), other potential mechanisms chased from Sigma. BCNU and busulfan were from Dr. Narayanan, exist for mediating alkylating agent resistance, including in Natinal Cancer Institute, Bethesda, MD, and Professor Fox, Christie creased efficiency of DNA repair, or enhanced intracellular Hospital, Manchester, United Kingdom, respectively. The activated drug detoxification. For example, cell lines selected on the basis cyclophosphamide and ifosphamide were from Dr. Chapman, Boehrin- of resistance to certain DNA-damaging agents, such as the ger, Ingelheim, Federal Republic of Germany. chloronitrosoureas, appear not to be cross-resistant to other Drug storage conditions were as described previously (16), with the following additions. CdCl2 was dissolved in H2O prior to use and stored drugs unless they generate adducts recognized by a common at 4°C.Mechlorethamine, thiotepa, melphalan, BSO, verapamil, and DNA repair pathway (7-9). Conversely, the Walker carcinoma colchicine were prepared freshly in H2O. 4-Hydroxyperoxycyclophos- cell line, which exhibits resistance to bifunctional nitrogen phamide and 4-hydroxyperoxyifosphamide were freshly prepared in serum-free medium. Etoposide was dissolved in dimethyl sulfoxide and Received 3/31/86; revised 7/22/86; accepted 8/27/86. stored at 4°C.Busulfan was freshly prepared in dimethyl sulfoxide. 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. 3The abbreviations used are: CHO, Chinese hamster ovary; BCNU, bischlo- ' Supported by the North of England Cancer Research Campaign. roethylnitrosourea; c/j-Pt, e»platinum diammine dichloride; BSO, buthionine 2To whom requests for reprints should be addressed. sulfoximine; !>>-.dose required to reduce cell survival to 37% of control values. 6290 Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1986 American Association for Cancer Research. NITROGEN MUSTARD-RESISTANT CHO CELLS Normally, cells were exposed to a drug for 24 h before being washed with phosphate-buffered saline and returned to fresh growth medium. The exceptions were mechlorethamine and BCNU, with 0.5-h expo sures, and cis-Pt, for which a 2-h exposure was used. The cells were then incubated at 37°Cuntil colonies visible by eye developed (up to 12 days). These were fixed in methanohacetic acid (3:1), stained with crystal violet (400 ¿ig/ml),and counted. Colonies containing more than 50 cells were considered survivors. Verapamil was included in selected experiments at a concentration of 100 ^M. This represented the maximum tolerated dose before vera- pamil toxicity was observed. Cells were simultaneously exposed to verapamil and either chlorambucil or melphalan for 24 h. For BSO treatments, cells were incubated for 8 h in medium con taining 25 MMBSO. Following this, mechlorethamine was added and a« incubation was continued for a further 0.5 h, after which the cells were returned to drug-free medium. Each point on a survival curve represents the average of at least 3 O independent experiments. In every case, wild-type CHO-K1 cells were treated in parallel. The D37represents the average dose required to kill a cell. The D37values were estimated directly from the survival curves. Two-Dimensional Gel Electrophoresis. Two-dimensional gel electro- phoresis was carried out essentially as described by O'Farrell (17), with the exception that isoelectric focusing gels were loaded at the anode. Polyacrylamide slab gels (9%) were used for the second dimension. Following electrophoresis, the gels were soaked in 10% acetic acid for 15 min, dried onto Watman No. 3MM paper, and exposed to Kodak DEF-59 film. Gel samples were prepared by labeling approximately 5 x IO5cells 20 40 60 80 with 10 nC\ [35S]methionine for 2 h. The cells were then harvested, chlorambucil (pg/ml) washed with phosphate-buffered saline, and resuspended in sample Fig. I. Survival of wild-type CHO-KI (O) and CHO-Ch 1 (•)cells after buffer (0.8 g Nonidet P-40, 0.5 ml 2-mercaptoethanol, 2.0 ml pH 3.5- chlorambucil. Points,