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[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 (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 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 -meth- to the cytotoxic effects of chlorambucil and comparable levels of cross- resistance to mechlorethamine and . These drugs all belong to otrexate-5- 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 , which cross-link DNA via the O6 position of exhibits elevated levels of resistance to the cytotoxic effects of guaninc. Lack of resistance to , 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 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 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 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, , 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. 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. -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, mean of 3 independent experiments. 10 ampholines, 5.5 g urea, and H2O to 10 ml). Approximately 20 ng of soluble protein were loaded onto each gel. Melphalan Uptake. Accumulation of ['4C]melphalan was measured essentially as described by Bates et al. (18). Glutathione Levels. Determination of intracellular glutathione levels was kindly performed by Dr. A. Hall using the method of Griffith (19).

RESULTS AND DISCUSSION Following the selection procedure described in "Materials and Methods," a clonal population of cells, designated CHO- Chl, was recovered and tested for resistance to a range of DNA- damaging agents and anticancer drugs. Fig. 1 shows the survival response to chlorambucil of CHO- Chl cells and wild-type CHO-K1 cells. A comparison of D37 values indicates that the CHO-Ch 1 cell line is greater than 20- fold resistant to the cytotoxic effects of chlorambucil. CHO- Ch 1 cells were also tested for resistance to mechlorethamine (Fig. 2) and to melphalan (Fig. 3) and were found to be approximately 34-fold and 14-fold resistant, respectively. In addition, mild cross-resistance (2-fold) to 4-OOH hydroxype- roxyifosphamide was apparent, but no significant resistance to radiation, mitomycin C, hydroxyperoxycyclophosphamide, BCNU, vincristine, colchicine, ethylmethane sulfonate, CdCl2, thiotepa, m-Pt, busulfan, Adriamycin, or etoposide was ob 08 12 16 served (data not shown). mechlorethamine (\igjml) This pattern of resistance differs from that of another chlor- Fig. 2. Survival of wild-type CHO-KI (O) and CHO-Ch! (•)cells after ambucil-resistant cell line, the Walker carcinoma, which is mechlorethamine. Points, mean of 3 independent experiments. cross-resistant to a much wider range of bifunctional alkylating agents, including m-Pt and mitomycin C (10). Although a Cross-resistance to melphalan has been described in the mul- number of other cell lines previously isolated do show limited tidrug-resistant phenotype resistant phenotype (25). However, phenotypic similarities to CHO-Ch 1 cells (7, 15, 20-24), none CHO-Ch 1 cells show no resistance to a number of agents exhibits an identical profile of cross-resistance (Table 1). generally associated with this phenotype, such as Adriamycin, CHO-Ch 1 cells were grown for 12 months in continuous colchicine, and vincristine. Also, the calcium channel blocker culture without reexposure to chlorambucil and then retested verapamil is usually able to reverse the phenotype of multidrug- for drug resistance. No changes in drug resistance levels were resistant cells (26), but in CHO-Ch 1 cells verapamil produced observed (data not shown). only the same low level of potentiation of melphalan and 6291 Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1986 American Association for Cancer Research. NITROGEN MUSTARD-RESISTANT CHO CELLS

100 A 4-fold dose enhancement of mechlorethamine toxicity was achieved in our wild-type cells following depletion of intracel- lular glutathione levels by treatment with BSO (Table 2). The concentration of BSO used was the highest which could be tolerated by the CHO cells before the toxicity of the BSO contributed significantly to cell survival. This level of BSO depleted glutathione levels in both lines by approximately 80% (data not shown). Although CHO-Chl cells were sensitized around 1.5-fold more than wild-type cells, they were nonethe less approximately 25-fold more resistant than CHO-K1 cells to the combined cytotoxic effects of mechlorethamine and BSO. The differential effect of BSO-mediated glutathione depletion is probably a consequence of a slightly (approximately 1.8-fold) elevated basal glutathione level in CHO-Chl cells (Table 4). Because this CHO cell line shows wild-type resistance to X- rays, Adriamycin, and BCNU, BSO does not reverse the resist ant phenotype, and basal glutathione levels are similar to those of wild-type, the mechanism of resistance is unlikely to be solely a consequence of an alteration in intracellular glutathione lev els. Metallothioneins are ubiquitous low molecular weight pro teins with an unusually high cysteine content (30 mol%) (29). 4 8 12 They can bind cadmium, zinc, and copper; and are induced by melphalan (jjg/ml) these metals, and resistance to cadmium can be mediated by Fig. 3. Survival of wild-type CHO-K1 (O) and CHO-Chl (•)cells after gene amplification of metallothioneins (30). Resistance to melphalan. Points, mean of 3 independent experiments. chlorambucil and melphalan has also been associated with zinc chlorambucil cytotoxicity as in CHO-K1 cells (Table 2). pretreatment (31, 32). Since these CHO cells are not resistant to cadmium, metallothionein induction is unlikely to cause such To eliminate the possibility that resistance was a consequence of decreased drug uptake, the level of accumulation of radiola- a high degree of drug resistance. All of the drugs to which this cell line is resistant produce beled melphalan was measured in resistant and wild-type cells. DNA intrastrand cross-links, and in some cases interstrand Both during the period of linear increase in drug uptake (up to cross-links, via N-7-guanine (33). If this cell line was more approximately 10 min) and at a point after uptake had reached proficient at repairing N-7-guanine monoadducts or cross-links a plateau (30 min) there was no significant difference between than the wild-type CHO cell line, it may be expected that the the 2 lines in the level of drug accumulated (Table 3). cells would also be resistant to busulfan, c/s-Pt, and thiotepa Elevated glutathione levels have been reported in a mel- and almost as resistant to oxazaphosphorines as to aniline phalan-resistant LI210 cell line (27) and this was associated mustards. However, these cells are not cross-resistant to busul with increased melphalan breakdown. In a human ovarian fan, m-Pt, or thiotepa. The range of lesions produced by these cancer cell line, elevated glutathione levels were shown, but compounds does, however, differ markedly from the mustards these cells were cross-resistant to radiation and Adriamycin, (34-39). All of these N-7 binding compounds may have differ and their sensitivity was restored to wild-type by BSO (21). In ent repair pathways or different lethal lesions. The critical the Walker chlorambucil-resistant line, glutathione levels are lesions are unlikely to be monoadducts, since the CHO cell line marginally lower than in the wild-type and the resistant cells was not cross-resistant to monofunctional alkylating agents. show collateral sensitivity to BCNU (28). Lack of cross-resistance to mitomycin C and BCNU could

Table 1 Cross-resistance patterns of 12 cell lines to alkylating agents Two-fold or greater resistance of the cell line studied compared with the parental line is arbitrarily called resistant (R). A 50% reduction or more of the drug concentration required for an equivalent degree of toxicity in the studied cell line compared with the parental line is arbitrarily called collateral sensitivity (CS). Responses within these limits are considered equivalent to wild-type sensitivity (S). Although arbitrary, the 2-fold differences were chosen because the degree of resistance or sensitivity is likely to be clinically relevant. selective CelllineCHO agentChlorambucil4-OOH CP°SRRRRRRRR4-OOHIPcis-PtR agentsMMSSX-ray5Adriamycin5X-ray"Adriamycin1*MMSS (thispaper)L1210(20)LI210(21)LI210(7)2080 SR

CP4-OOH CSSRSRRRR CPMelphalanMelphalanMechlorethamineMechlorethamineBCNUChlorambucil4-OOH

Me(22)Raji

1(23)Raji 1(23)Raji 1(23)Walker (IS)P388 (20)Human CPc«-PtMelphalanChlorambucilRCSRRSMelphalanRCSRRRRSRRRRRMechlorethamineRCSRRRRRSR4-OOHRRBCNUSCSsssRRCSRRThiotepaSCSRRRROther (23)MelanomaSSC25 (24)Initial ' 4-OOH CP. 4-hydroxyperoxycyclophosphamide: 4-OOH IP. 4-hydroxyperoxyifosphamide; MMS, methylmethanesulfonate. 6292 Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1986 American Association for Cancer Research. NITROGEN MUSTARD-RESISTANT CHO CELLS

Table 2 Effects ofBSO and verapamil on drug resistance IEF D37values are given for parental CHO-K1 cells and CHO-Chl cells following exposure to a combination of mechlorethamine and BSO (25 ;i\i ) or to verapamil 6 (100 ¿IM)andeither melphalan or chlorambucil. SDS (ixg/ml)Mechlorethamine CHO-KI Gig/ml)2.05

Mechlorethamine + BSO 0.014 0.33 Chlorambucil 3.3 80 Chlorambucil + verapamil 1.8 30 Melphalan 0.65 8.65 Melphalan + verapamil0.059 0.33CHO-Chl 3.10

Table 3 Comparison of intracellular melphalan accumulation in wild-type and CHO-Chl cells Extracellular melphalan level melphalan cells)CHO-KI0.19(nmol/106 concentra time tion(UM)15.4 (min)3 ±0.06° ±0.04 15.4 30 0.34 ±0.06 0.41 ±0.08 46.2 3 0.35 ±0.07 0.33 ±0.08 46.2Exposure 30Intracellular0.79 ±0.11CHO-Chl0.150.74 ±0.12 " Mean ±SE of 3 separate experiments. Cells (107/ml) in suspension were exposed to [MC]melphalan for the times indicated. The reaction was terminated by the addition of ice-cold phosphate-buffered saline-1% bovine serum albumin, the cells were washed 3 times with the same buffer, and the pellet was solubili/al in 1% sodium dodecyl sulfate before scintillation counting.

Table 4 Intracellular glutathione levels Intracellular glutathi one level (nmol/mg Cell line protein) CHO-KI 18.4 ±2.2° CHO-Chl 33.0 ±5.6 " Mean ±SE of at least 3 independent assays. Fig. 4. Two-dimensional gel electrophoresis of [3!S]methionine-labeled total soluble proteins from CHO-KI cells (bottom) and CHO-Chl cells (top). Proteins be expected because of the cross-linking via O'-guanine in the were separated by isoelectric focusing (//./) (pi values are shown across the top of the gel) followed by sodium dodecyl sulfate (SflSVpolyacrylariide gel electro case of these two drugs (40, 41) and because resistance to phoresis. Curved arrow, position of the M, 25,000 polypeptide overproduced by BCNU may be mediated by the specific repair enzyme O6- CHO-Chl cells. methylguanine methyltransferase (42). To investigate whether increasing cellular resistance was Karyotype analysis on CHO-Chl cells (kindly performed by accompanied by major alterations in the level of synthesis of C. Ockey) failed to reveal conclusive evidence for cytogenic specific proteins, 2-dimensional gel electrophoresis of [35S]- indicators of gene amplification, such as double minutes or methionine-labeled polypeptides was undertaken. The results homogeneous staining regions. However, it did reveal that (Fig. 4) show that CHO-Chl cell synthesize greatly elevated CHO-Chl cells have an abnormal karyotype with a modal levels of a polypeptide with a molecular weight of approxi chromosome number of 29 (compared to 22 for parental cells). mately 25,000 with an isoelectric point around 7.5. This level While this additional genetic information appears to have re of enhanced protein synthesis was not dependent upon contin sulted largely from chromosome rearrangements, there does ued exposure of CHO-Chl cells to chlorambucil. This single not appear to be evidence of significant heterogeneity among major difference in the protein gel patterns for the 2 lines is the population (data not shown). also seen on Coomassie blue-stained gels (data not shown). Since glutathione transferases are inducible and up to a 26- The 25,000 polypeptide does not appear to be a nuclear fold increase in mRNA for a transferase has been reported (45), protein as it was absent from preparations of nuclei from CHO- it may be unnecessary for gene amplification to occur in order Ch 1 cells isolated by a number of different methods (data not to produce the level of resistance seen here. It is possible that a shown). Based on its cellular location and isoelectric point, the genetic rearrangement has constitutively activated an inducible possibility that this protein is a glutathione transferase is cur protein, in a way similar to that of c-myc activation in lympho- rently under investigation. mas. Alternatively, the extra genetic information may simply There are many different glutathione transferase isoenzymes code for extra copies of a glutathione transferase gene. which participate in a wide variety of intracellular reactions The pattern described in this CHO cell line is. compatible involving detoxification of drugs and carcinogens. If one spe with many clinical observations of alkylating agent resistance. cific transferase had increased in activity in CHO-Chl cells Chlorambucil is commonly used as a single agent in ovarian then the pattern of cross-resistance to aklylating agents and cancer treatment, and cw-Pt is an effective second line agent other drugs such as Adriamycin would depend on its substrate (46). In multiple myeloma, patients relapsing or resistant to specificity. This has not yet been defined with regard to cyto- melphalan have responded to cyclophosphamide (47, 48). In toxic drugs for any glutathione transferase. There has been a addition, the cell line is stable, as is clinical resistance. report of a chlorambucil-resistant line in which glutathione Considering the heterogeneity already described in different transferase activity was elevated (43). Also, an MCF-7 cell line cell lines (Table 1) and the variety of mechanisms discussed resistant to Adriamycin was shown to have a manifold increase above, it is clear that there are many "mullid rug-resistant in glutathione transferase but was not nearly as cross-resistant phenotypes" to alkylating agents. As these mechanisms are to alkylating agents (44). elucidated, it should be possible to biochemically profile a 6293 Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1986 American Association for Cancer Research. NITROGEN MUSTARD-RESISTANT CHO CELLS relapsed tumor to select appropriate alkylating agent therapy phorines by inhibitors of aldehyde dehydrogenase activity in cultured oxaza- (e.g., by measuring metallothionein, specific glutathione Irans- phosphorine-resistant LI210 and cross-linking agent-resistant P388 cell lines. Cancer Res., 45: 1549-1555, 1985. ferase isoenzymes, glutathione levels). This should also allow 22. Louie, K. G., Behrens, B. C., Kinsella, T. J., Hamilton, T. C., Grotzinger, specific pharmacological antagonists to be developed. K. R., McKay, W. M., Winker, M. A., and Ozols, R. F. Radiation survival parameters of antineoplastic drug-sensitive and -resistant human ovarian cancer cell lines and their modification by buthionine sulfoximine. Cancer Res., 45:2110-2115, 1985. ACKNOWLEDGMENTS 23. Frei, E., Ill, Cucchi, C. A., Rosowsky, A., Tanlravahi, R., Bernai, S., Ervin, T. J., Ruprecht, R. M., and Haseltine, W. A. 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6294 Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1986 American Association for Cancer Research. Isolation and Characterization of a Chinese Hamster Ovary Cell Line Resistant to Bifunctional Nitrogen Mustards

Craig N. Robson, Janice Alexander, Adrian L. Harris, et al.

Cancer Res 1986;46:6290-6294.

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