Mutation Research, DNA Repair, 254 (1991) 161-165 © 1991 Elsevier Science Publishers B.V. 0921-8777/91/$03.50 ADONIS 092187779100057K

161

MUTDNA 06420

Isolation and characterization of nitrogen mustard-sensitive mutants of Chinese hamster ovary cells Raymond E. Meyn, David Murray, Susanna C. vanAnkeren, Gary S. Bernard, David N. Mellard and Marvette L. Hobbs Department of Experimental Radiotherapy, Universityof Texas M.D. Anderson Cancer Center, 1515 Holcombe Blvd, Houston, TX 77030 (U.S.A.) (Received 21 November 1989) (Revision received 16 July 1990) (Accepted 18 July 1990)

Keywords: Nitrogen mustard-sensitive mutants; Chinese hamster ovary cells; DNA-damaging agents; Cis-diamminedichloroplatinum

Summary Three nitrogen mustard-sensitive lines of Chinese hamster ovary cells were isolated from mutagenized cultures using the procedure of Thompson et al. (1980). The lines, designated NM1, NM2 and NM3, were 2.1-, 17- and 6.8-fold more sensitive to nitrogen mustard, respectively, than their parent, wild-type, line as determined by the dose required to kill 90% of the cells, IC90. Patterns of cross-sensitivity to other DNA-damaging agents including ultraviolet light, cis-diamminedichloroplatinum, and other alkylating agents were determined for each line. Analysis of these results suggests that the phenotypes of the mutant lines are different from those lines reported previously.

Although our current understanding of the mechanisms by which DNA-damaging agents exert their mutagenic and cytotoxic effects in mammalian cells is still far from complete, progress in this area has been greatly aided by the development of mutant lines of defined rodent origin that are defective in various steps of the DNA-repair pathway. Such lines have usually been isolated on the basis of their sensitivity to a DNA-damaging agent and the repair defect subsequently shown by

Correspondence: Dr. R.E. Meyn, Department of Experimental Radiotherapy, University of Texas M.D. Anderson Cancer Center, 1515 Holcombe Blvd., Houston, TX 77030 (U,S.A.).

biochemical analysis. Mutant lines sensitive to a variety of agents, including ultraviolet light (UV), mitomycin-C (MMC), ethyl methanesulfonate (EMS) and X-rays have now been isolated (Thompson et al., 1980; Busch et al., 1980; Wood and Burki, 1982; Robson et al., 1985; Stamato et al., 1983; Jeggo and Kemp, 1983; Jones et al., 1987; Fuller and Painter, 1988). One of the most intriguing observations reported by Thompson et al. (1980) concerning their original panel of UV-sensitive mutant lines was the cross-sensitivity of some of these lines to MMC. These UV-sensitive lines were subsequently shown to be cross-sensitive to a variety of chemical mutagens, including .additional cross-

162

linking agents such as cis-diamminedichloroplatinum (CDDP), nitrogen mustard (HN2), and L-phenylalanine mustard (L-PAM) (Meyn et al., 1982; Meyn and Murray, 1984; Hoy et al., 1985). These results suggest that the excision-repair pathways in mammalian cells responsible for removing UV-induced photoproducts and chemically induced bulky adducts have at least some steps in common. The question that arises, however, is whether mutant fines that have the same defective genes would be isolated from mutagenized cultures if bifunctional alkylating agents are used as the selecting agents or whether new mutant lines would emerge that have defects in repair steps separate from those for photoproducts. This question was addressed in the present study by isolating mutant lines of Chinese hamster ovary (CHO) cells selected on the basis of sensitivity to HN2 and determining their patterns of cross-sensitivity. Materials and methods

Cells and culture conditions CHO cells were maintained in exponential monolayer culture in McCoy's 5A medium (Hsu's modification; Gibco, Grand Island, NY) supplemented with 15% fetal bovine serum. The mutant lines isolated as part of this study were compared to 4 lines generously provided by Dr. Larry Thompson, Lawrence Livermore Laboratories, Livermore, CA: two UV-sensitive lines, UV-20 and UV-41; an EMS-sensitive line, EM9; and line AA8, the wild-type cells from which his mutants were derived (Thompson et al., 1980). The AA8 line was also used as the parent line for selecting the HN2-sensitive mutants in the studies to be described. All incubations were carried out at 37 ° C in a humidified atmosphere of 95% air/5% CO 2. Mutant isolation Wild-type AA8 ceils were mutagenized in monolayer culture with either ICR-191 or UV light (5.0 j/m2). ICR-191 was dissolved in medium with serum at a concentration of 1.75 ~ g / m l and was left in contact with the cells for 18 h. The cells were then rinsed and incubated in fresh medium for 5-7 days to allow mutant expression. The procedure used for mutant selection was identical

to that originally published by Thompson et al. (1980). Briefly, 300-400 mutagenized cells were plated into 100-mm culture dishes (a total of 50100 dishes). Colonies were allowed to develop for 4-5 days, at which time the selecting agent, 20 nM HN2, was added to the culture medium, and incubation continued. Three days later, each dish was stained with 0.4% erythrosin B and scanned under a dissecting microscope. Mutant colonies appeared as small heterogeneous groups of cells containing some nonviable cells stained pink by the erythrosin B. Such colonies were isolated with cloning rings and further growth was allowed in fresh medium. 20 subclones were selected from each original clone and subsequently tested for sensitivity to HN2. Drug treatments and survival analysis The degree of cross-sensitivity of the mutants to a variety of DNA-damaging agents was tested by survival curve analysis. Known numbers of freshly trypsinized cells were plated at various dilutions in 60-mm culture dishes 4 h prior to treatment to allow attachment. The attached cells were then treated with various concentrations of the drugs of interest for 30 min or 1 h. UV- and T-irradiations were performed as previously described (Meyn et al., 1974; vanAnkeren et al., 1988). Following treatment the dishes were returned to the incubator for 8-10 days to allow colony development. Results

3 different HN2-sensitive lines were isolated from 3 separate screening experiments. Each experiment was conducted in an identical manner except for the mutagen used (Table 1). The fact that only one line was isolated from each screen TABLE 1 ISOLATION

OF HN2-SENSITIVE

CLONES

Expt. No.

Mutagen

Number of clones screened

Number of clones retested

Clones isolated

1 2 3

UV UV ICR191

- 25000 - 25000 - 25000

4 4 9

NM1 NM2 NM3

163 1.0

0.1

.01

.001 .OOl

.()1

011

I

L

1.0

10

DRUG CONCENTRATION (BM) Fig. 1. HN2-survival curves for wild-type AA8 (o) cells and HN2-sensitive mutant lines NM2 (o) and NM3 ([]). UV-sensitive, UV-41 (zx), and EMS-sensitive, EM9 (A), mutant strains isolated by Thompson et al. (1980) are shown for comparison.

does not accurately reflect the mutation frequency since clones that did not grow well were discarded and some clones were lost to contamination. The HN2 sensitivity of 2 of the lines is presented in Fig. 1. The HN2-sensitive lines are compared to their wild-type parent, AA8, and 2 mutant lines isolated by Thompson et al. (1980) based on

sensitivity to UV (UV-41) or EMS (EM9). The response of the NM1 line, while not shown for the sake of clarity, was similar to that of the EM9 line. While all of the lines tested were sensitive to HN2, they differed greatly in their relative degree of sensitivity. The survival data (Fig. 1) are displayed on a log-log plot because of the very large differences (nearly 100-fold) in sensitivity among the various lines. It was evident that the HN2 sensitivities of some of the mutant lines were indistinguishable from one another. In order to ascertain whether the newly isolated mutants had unique phenotypes, the patterns of cross-sensitivities of the 3 HN2-sensitive mutants to a variety of DNAdamaging agents were compared to the patterns for the pre-existing mutant lines. A wide variety of DNA-damaging agents were chosen for this study, including UV, ~-irradiation, bi- and mono-functional alkylating agents. Complete dose-response curves were generated for each agent tested and the IC90 values (i.e., the dose required to kill 90% of the cells) were calculated. A summary of the IC9o values determined from these families of survival curves is presented in Table 2. Examination of the results in Table 2 reveals that the three HN2-sensitive, two UV-sensitive and the EMS-sensitive mutants all had different phenotypes. For example, while lines NM2 and UV-20 showed similar sensitivities to HN2, they had quite different sensitivities to UV and CDDP.

TABLE 2 CROSS-SENSITIVITIES OF MUTANT LINES TO VARIOUS CYTOTOXIC AGENTS Agent

UV ( J / m 2) HN2(#M) HN1 (#g/ml) CDDP(vg/ml) MNU (mM) MMS (mM) MMC (#g/ml) ~,-Rays (Gy)

Cell line AA8

UV-20

UV-41

EM9

NM1

6.5 a 3.4 14 8.4 0.34 1.25 2.5 5.6

0.92 (7) b 0.2 (17) 4.7 (3) 0.16 (53) NT c NT NT NT

0.92 (7) 0.04(85) 2.5 (5.6) 0.16 (53) NT NT NT NT

4.5 (1.4) 2.07 (1.5) 0.5 (28) 6.6 (1.3) 0.20 (1.7) 0.14 (9) NT 3.4 (1.6)

6.25 1.6 11 8.4 NT NT NT NT

NM2 (1.04) (2.1) (1.3) (1.0)

4.5 0.2 1.9 2.7 0.086 0.45 1.6 4.0

NM3 (1.4) (17) (7.4) (3) (4) (2.8) (4) (1.4)

2.5 0.5 3 1.6 0.033 0.14 0.25 3.3

(2.6) (6.8) (4.7) (5.3) (10) (9) (10) (1.7)

a These numbers represent the ICgo values, i.e. the dose required to kill 90% of the cells. The survival curves from which the IC90 values were determined were performed at least in duplicate and in most cases triplicate. b The numbers in parentheses represent the dose-reduction factors, i.e. ICgo (wild-type)/IC~ (mutant). c Not tested.

164

It is also of interest to note that the NM3 line was less sensitive to HN2 than was the NM2 line but more sensitive than that line to several other agents, including UV, CDDP, MNU, MMS and ,/-rays. Discussion

To our knowledge, the mutant lines described here, designated NM1, NM2, and NM3, are the first to be specifically isolated for sensitivity to HN2. HN2 is a particularly interesting compound, representing one of the structurally simplest bifunctional alkylating agents, and was used in studies that first demonstrated the importance of DNA-repair mechanisms for cell sensitivity to interstrand cross-links (Kohn et al., 1965). The 3 new fines have quite different sensitivities to HN2 and when compared to other mutant lines illustrate the broad range of sensitivities to this agent (Fig. 1 and Table 2). This pattern is in contrast to the response to UV where many lines having different genotypes have similar sensitivities (Thompson et al., 1980). The sensitivity comparisons summarized in Table 2 suggest that the 3 new HN2-sensitive mutants have unique phenotypes that are different both from each other and from 3 mutant lines isolated in another laboratory (Thompson et al., 1980). Several other mutant lines of Chinese hamster origin have been reported in the literature and, while they were not available for direct comparison, their reported sensitivities to various agents allow a comparison with the patterns of crosssensitivity for the fines reported here. Jeggo and Kemp (1983) isolated several X-ray-sensitive C H O lines that display some cross-sensitivity to monofunctional alkylating agents. Two of our lines, NM2 and NM3, were moderately sensitive to Xrays (Table 2 and vanAnkeren et al., 1988) but they were not as radiosensitive as Jeggo and Kemp's lines. Robson and Hickson (1987) isolated CHO-K1 lines sensitive to MMS. While they did not test the sensitivity of their lines to HN2, none of their MMS-sensitive lines had a similar phenotype to NM3 based on the observation that none of their lines was as sensitive to UV as was NM3 (Fig. 2). Their MMS-2 line may be similar to our NM2 line, however.

X-Ray-sensitive mutants of Chinese hamster cells have also been isolated by Jones et al. (1987) and Fuller and Painter (1988). Again, to the extent that comparisons can be made, our lines are not similar to any of their lines based on their intermediate sensitivity to 7-rays (vanAnkeren et al., 1988) or their hypersensitivity to alkylating agents (Table 2). It should be pointed out that while these comparisons give a useful impression of the uniqueness of any newly isolated mutant line, the ultimate test for a unique genotype is complementation analysis. We are currently conducting such an analysis in order to place our existing (and any future) HN2-sensitive lines into complementation groups. In addition to providing patterns of cross-sensitivity for comparative purposes, analysis of a mutant line's response to a panel of cytotoxic agents with different mechanisms of action may also provide clues to the line's particular biochemical defect. In the case of HN2, such defects might be expected to fall into 3 broad categories. First, the defect may be at the membrane level, affecting uptake or efflux of the drug. Such a defect would not explain the sensitivity of NM2 cells to y-rays nor of NM3 cells to UV (Table 2). Second, decreased cellular levels of protective activities such as glutathione (GSH) and GSH-Stransferases may make the cells sensitive to alkylating agents (e.g. Green et al., 1984 and Wang and Tew, 1985). While these mechanisms cannot be totally ruled out, such mechanisms would not explain the UV sensitivity of the NM3 line. Third, defects in the DNA-repair pathways responsible for removing lesions from the DNA might make cells sensitive to DNA-damaging agents. Indeed, many of the mutant fines isolated in other laboratories, discussed above, have been shown to have defects in DNA-repair processes (Thompson et al., 1987; Kemp et al., 1984). The cross-sensitivity of some of the UV-sensitive lines isolated by Thompson et al. (1980) to CDDP, MMC or diepoxybutane appears to correlate with the respective line's reduced ability to remove cross-links from D N A (Meyn et al., 1982; Hoy et al., 1985). The abilities of the NM2 and NM3 mutant lines to repair various D N A lesions is currently under investigation. In summary, three HN2-sensitive CHO lines

165

have been isolated and characterized in terms of their patterns of cross-sensitivity to a variety of DNA-damaging agents. Two of these lines display sufficient degrees of sensitivity to HN2 to be of continued interest. While the exact nature of their biochemical defects must await more detailed analysis, their cross-sensitivity to either UV or ionizing radiation is consistent with possible defects in DNA-repair processes. The examination of such mutants should ultimately aid in the elucidation of those biochemical pathways important in modulating the cytotoxicity of bifunctional alkylating agents.

Acknowledgments This investigation was supported by grants CA23270 and CA-26312 awarded by the National Cancer Institute, DHHS. S.C. vanAnkeren was supported by the Katharine M. Unsworth Chafftable Annuity Lead Trust.

References Busch, D.B., J.E. Cleaver and D.A. Glaser (1980) Large-scale isolation of UV-sensitive clones of CHO cells, Somat. Cell Genet., 6, 407-418. Fuller, L.F., and R.B. Painter (1988) A Chinese hamster ovary cell line hypersensitive to ionizing radiation and deficient in repair replication, Mutation Res., 193, 109-121. Green, J..A., D.T. Vistica, R.C. Young, T.C. Hamilton, A.M. Rogan and R.F. Ozols (1984) Potentiation of melphalan cytotoxicity in human ovarian cancer cell lines by giutathione depletion, Cancer Res., 44, 5427-5431. Hoy, C.A., L.H. Thompson, C.L. Mooney and E.P. Salazar (1985) Defective DNA cross-link removal in Chinese hamster cell mutants hypersensitive to bifunctional alkylating agents, Cancer Res., 45, 1737-1743. Jeggo, P.A., and L.M. Kemp (1983) X-Ray-sensitive mutants of Chinese hamster ovary cell line, isolation and crosssensitivity to other DNA damaging agents, Mutation Res., 112, 313-327. Jones, N.J., R. Cox and J. Thacker (1987) Isolation and cross-

sensitivity of X-ray-sensitive mutants of V79-4 hamster cells, Mutation Res., 183, 279-286. Kemp, L.M., S.G. Sedgwick and P.A. Jeggo (1984) X-Ray-sensitive mutants of CHO cells defective in double-strand break rejoining, Mutation Res., 132, 189-196. Kohn, K.W., N.H. Steigbigel and C.L. Spears (1965) Crosslinking and repair of DNA in sensitive and resistant strains of E. coli treated with nitrogen mustard, Biochemistry, 53, 1154-1161. Meyn, R.E., and D. Murray (1984) Cell cycle effects of alkylating agents, Pharmacol. Ther., 24, 147-163. Meyn, R.E., S.F. Jenkins and L.H. Thompson (1982) Defective removal of DNA cross-links in a repair-deficient mutant of Chinese hamster cells, Cancer Res., 42, 3106-3110. Meyn, R.E., D.L. Vizard, R.R. Hewitt and R.M. Humphrey (1974) Fate of pyrimidine dimers in the DNA of ultraviolet-irradiated Chinese hamster cells, Photochem. Photobiol., 20, 221-226. Robson, C.N., and I.D. Hickson (1987) Isolation of alkylating agent-sensitive Chinese hamster ovary cell lines, Carcinogenesis, 8, 601-605. Robson, C.N., A.L. Harris and I.D. Hickson (1985) Isolation and characterization of Chinese hamster ovary cell lines sensitive to mitomycin C and bleomycin, Cancer Res., 45, 5304-5309. Starnato, T.D., R. Weinstein, A. Giaccia and L. Mackenzie (1983) Isolation of cell cycle-dependent gamma ray-sensitive Chinese hamster ovary cells, Somat. Cell Genet., 9, 165-173. Thompson, L.H., J.S. Rubin, J.E. Cleaver, G.F. Whitmore and K. Brookman (1980) A screening method for isolating DNA repair-deficient mutants of CHO ceils, Somat. Cell Genet., 6, 391-405. Thompson, L.H., E.P. Salazar, K.W. Brookman, C.C. Collins, S.A. Stewart, D.B. Busch and C.A. Weber (1987) Recent progress with the DNA repair mutants of Chinese hamster ovary cells, in: The Molecular Biology of DNA Repair, J. Cell Sci., Suppl., 6, 97-110. vanAnkeren, S.C., D. Murray, P.M. Stafford and R.E. Meyn (1988) Cell survival and recovery processes in Chinese hamster AA8 cells and two radiosensitive clones, Radiat. Res., 115, 223-237. Wang, A.L., and K.D. T¢W (1985) Increased glutathione-Stransferase activity in a ~gll line with acquired resistance to nitrogen mustards, C a n e r Treatment Reports, 69, 677-682. Wood, R.D., and H.J. !~urki (1982) Repair capability and the cellular age response for killing and mutation induction after UV, Mutation Res., 95, 505-514.