Characterization of Hypersensitive Cell Lines1

Home , Treosulfan

[CANCERRESEARCH57. 454-460. February 1. 19971 High-Dose Selection with Mafosfamide Results in Sensitivity to DNA Cross-Linking Agents: Characterization of Hypersensitive Cell Lines1

Gerhard FritZ,2 Jan Georg Hengstler, and Bernd Kaina Division ofApplied Toxicology fG. F., B. K.), Institute of Toxicology Ii. G. HI, University ofMainz. Obere Zahlbacher So. 67, D-55131 Mainz, Germany

ABSTRACT Interestingly, hypersensitivity of cells to X-rays or UV light is often accompanied by cross-sensitivity to DNA cross-linking agents, such as One ofthe most frequently used alkytating drugs in the therapy ofa broad mitomycin C. This was shown for the rodent mutants irsl, irs3, irslSF, spectnim of tumors is cyclophosphamide. To elucidate the mechanisms by VC-8, UV2O, UV41, and VH-4 (4, 15â€”17).Some of these cell lines which tumor celLs acquire resistance to this agent, Chinese hamster ovary cells (CHO-Ki) were treated with a high dose of the cyclophosphamide proved to be sensitive also to the DNA cross-linking cytostatic drugs analogue mafosfamide, and survivors were analyzed as to their cell kiffing cisplatin and melphalan (16, 18, 19), which indicates that overlapping r@ponse, diromosomal aberrations, and DNA repair capacity. None of the mechanisms for the repair of DNA damage induced by UV light, X-rays, surviving clones tested were mafosfamide reststant. Surprisingly, seine of the or DNA cross-linking agents do exist. Resistance to melphalan and isolated cell lines exhibited a mafosfamide-hypersensitive phenotype Two of cisplatin is supposed to be influenced by ERCC1, which interferes with these cell variants (designated as CHO-K1-4 and CHO-K1-12) were analyzed early steps in nucleotide excision repair (19â€”21).Because expression of In more detail and proved to be cross-sensitive to other DNA cross-linking the DNA repair protein ERCC1 was found to be enhanced in cisplatin antineoplastic drugs such as N-hydroxyethyl-N-chloroethylnitrosourea, treo resistant tumors from patients with ovarian carcinoma as well as in sulfais, melphalan, ciSplatin, and mitomycin C. The hypersensitivity to the nitrogen mustard-resistant cells from patients suffering from chronic cytotoxiceffect ofmafosfamide was accompanied by a 2-3-fold Increase m the lymphocytic leukemia (22â€”24),the conclusion was drawn that overex frequency ofdiromosomal aberrations. The intracellular levels of glutathione and glutathione S-transferase activity ofthe hyperseitsitive vailants as well as pression of ERCC1 exerts a protective effect. However, transfection of growth rate were comparable to wild-type cells. Both the vanant and the ERCC1 cDNA expression vector into wild-type Chinese hamster ovary parental cells did not exhibit an Increase in the amount of p53 upon UV AA8 cells did not confer resistance to melphalan and cisplatin, but in irradiation. Furthermore, sensitive celia displayed similar UV-induced ma contrast, increased the sensitivity of the transfectants to these drugs (19). Scheduled DNA synthesis and showed identical amounts OIERCC1 mRNA as Obviously, in this cell type, ERCCI overexpression alone is not sufficient wild-type eeIls, indicating that the hyperseosifive phenotype is not due to a to mediate resistance to DNA cross-linking agents. Another cellular defect in nucleotide excision repair. The induction of DNA single-strand factor, which confers resistance to cytostatic drugs, is O@-methylguanine breaks upon mafosfamide treatment was very similar In wild-type and mu DNA methyltransferase,which removes adducts from the (P-position of tents, and the removal of mafOsfamide-induCed DNA cross-links was not reduced in hypersensitive cells. However, the hypersensitive cell vatiants guanine, thereby protecting cells from the mutagenic, recombinogenic, exhibited a less severe drag-induced block to DNA replication. From the data clastogenic, and cytotoxic effect of O@-alkylguanine (25). The protective obtained, we conclude that hypersensitivity to cross-linking agents upon effect of O'@-methylguanine-DNA methyltransferase covers a narrow mafosfamide selection is due to changes in cell cyde progression of drug range of alkylating cytostatic drugs; it is limited to nitrosoureas, such as treated ceft& l-(4-amino-2-methyl-5-pyrimidinyl)methyl-3-(2-chloroethyl)-3-nitro sourea, l,3-bis(2-chloroethyl)-l-nitrosourea, l-(2-cffloroethyl)-3-cyclo hexyl-l-nitrosourea, streptozotocin, and hydrazine derivatives like pro INTRODUCTION carbazin (26â€”28).Another mechanism contributing to cellular resistance to cytostatic drugs is gene amplification. Thus, methotrexate resistance is Preexisting and acquired drug resistance is a serious problem in cancer chemotherapy. A suitable tool for elucidating the mechanisms caused by amplification of the dihydrofolate reductase gene (29, 30), and resistance to agents like vincristine results from amplification or overex involved in cytostatic drug resistance are mutant cell lines, which pression of the multidrug resistance gene (mdr-1; Refs. 31â€”33).Further distinguish from the corresponding wild-type cells by an altered more, treatment of cells with high doses of alkylating agents, such as susceptibility to the cytotoxic effects of the agents used. Such cell MNNG and N-methyl-N-nitrosourea yields drug-resistant survivors (34â€” variants are also highly useful in analyzing the mechanism of action of genotoxic agents in general. Thus, hamster mutant cell lines char 36), which was shown to be due to defects in the mismatch repair system acterized by their hypersensitivity to UV light and X-rays (1â€”4)have (37â€”39). been used for complementation studies, resulting in the cloning of One of the best established and most widely used DNA cross ERCC3andXRCC(X-raycross-complementing)genes(5â€”8).Their linking agents in the therapy of a broad spectrum of tumors is analysis provided growing insights into the molecular mechanisms of cyclophosphamide. After its oxidative activation, acrolein and phos DNA repair as well as the underlying cause of human repair-deficient phoramide mustard are formed, which cause DNA strand breaks and disorders, such as ataxia telangiectasia (9, 10), Fanconi's anemia (11, DNA interstrand cross-links, respectively (40). It is reasonable to 12), and xeroderma pigmentosum (7, 13, 14). suppose that the appearance of cyclophospharnide-resistant tumors is due to changes in cytochrome P-450-catalyzed drug activation or GST-mediated drug detoxification, which both can affect the biolog Received 8/12/96; accepted 12/4/96. The costs of publication of this article were defrayed in part by the payment of page ically available concentration of the active drug. However, as deduced charges. This article must therefore be hereby marked advertisement in accordance with from in vitro studies, neither GSH, GSH reductase, nor GSH-trans 18 U.S.C. Section 1734 solely to indicate this fact. ferase seem to interfere with resistance of tumor cells to mafosfamide, I This work was supported by Grant SFB 519, B4, from the Deutsche Forschungsge meinschaft and Grant 8036â€”386261/170 from Stiftung Rheinland-Pfalz. which is an activated analogue of cyclophosphamide (41, 42). Fur 2 To whom requests for reprints should be addressed, at Institut fur Toxikologie, thermore, upon treatment with cyclophosphamide, no reduction in the Abteilung fÃ¼rAngewandte Toxikologie, UniversitÃ¤tMainz, Obere Zahlbacher Str. 67, D-5513lMains,Germany.Phone:06131/17-3627;Fax:06131/17-3421. level of activated metabolites was found in humans (43), indicating 3 The abbreviations used are: ERCC, excision repair cross-complementing; MNNG. that the level of metabolic drug activation does not decrease upon N-methyl-N'-nitro-N-nitrosoguanidine; GSH, glutathione; MMS, methyl methanesulfon ate; HeCNU, N-hydroxyethyl-N-chloroethylnitrosourea; GST, glutathione S-transferase; repeated cyclophosphamide administration. There is a report showing UDS,unscheduledDNAsynthesis. that defective drug transport may affect the level of resistance to 454

nitrogen mustards (44). However, taking into account the fact that the was calculated as k = â€”(lgR)/l0h,where R is the fraction of DNA remaining primary cytotoxic target of cyclophosphamide is the DNA, the repair on the filter after an elution period of 10 h. For determination of DNA of DNA cross-links may play an important role in drug resistance. For cross-links, cells were irradiated (800 cGy) before they were subjected to the most part, the mechanisms leading to cyclophosphamide resist alkaline elution (46). ance of tumor cells on the basis of alterations of DNA repair processes Determination of GSH and GST Activity. For determination of intracel lular concentrations of GSH, the protein-free fraction of 200 @gof total cellular are largely unknown. protein was photometrically analyzed for total content of GST as described (47, To investigate mechanisms of cross-linking drug resistance, we ana 48). GST activity was photometrically analyzed with 50 @gofprotein from total lyzed in a first step the fate ofa cell population exposed to a cross-linking extractsusing l-chloro-2,4-dinitrobenzeneassubstratefor GST (49). tumor-therapeutic drug. Notably, this study aimed at elucidating whether Western Blot Analysis. Cells were scraped off the plates in ice-cold PBS cells, which have survived a treatment with a single high dose of mafos and pelleted by centrifugation. Cell pellets were resuspended in lysis buffer famide, gained a drug-resistant phenotype. Here we show that survivors [0.5 NP4O, 10 mM Iris (pH 7.4), 10 mtvt NaCI, and 3 mM MgCl2] and incubated were not more mafosfamide resistant, but surprisingly, some of them for 5 mm on ice. Afterward, crude membrane and nuclear fractions were showed a hypersensitive phenotype. These cell variants, representing a pelleted by centrifugation (10 mm at 1000 X g for 4Â°C).Afterwashing, the new class of hypersensitive cell type, have been analyzed as to their pellets were resuspended in 20 masIris (pH 7.4), 1 mt@iEDTA,10 mMNaCl, cross-sensitivity to other cytostatic drugs and expression of potential and 1 mM phenylmethanesulfonyl fluoride. After sonification and centrifuga tion, protein concentration of the supematant was determined by Bradford defense functions as well as DNA repair activity. (50), and 20 i.@gof protein were separated on 10% SDS-gels. Afterwards, proteins were wet-blotted to nitrocellulose, and p53 was detected by incubation MATERIALS AND METHODS with a monoclonal antibody (PAb122; Boebringer Mannheim). For detection of p53 protein, the ECL detection system (Amersham Corp.) was used. Materials. Cisplatin, MNNG, MMS and mitomycin C were purchased Northern Blot Analysis. Total RNA was preparedfrom logarithmically from Sigma Chemical Co. HeCNU was a gift of Dr. Eisenbrand (Kaiserslau growing cells according to the method of Chomczynski and Sacchi (51). After tern, Germany). Mafosfamide and melphalan were generously provided by Dr. separation on 1.0% agarose gels, RNA was transferred overnight on Hybond Pohi (ASIA Medica, Frankfurt, Germany); treosulfan was obtained from N@ membrane (transfer buffer, 50 mM NaOH). Hybridization was performed Medac (Hamburg, Germany). Stock solutions were prepared in water and overnight at 60Â°Cinhybridization solution (7% SDS, 1% BSA, and 1 mM storedat â€”80Â°C. EDTA in 0.5 Mphosphatebuffer,pH 7.2) containing32P-labeledERCCIor Cell Calture and SelectionProcedure.Chinesehamsterovarycells glyceraldehyde-3-phosphate dehydrogenase cDNA as probe. Filters were (CHO-Kl) wereroutinelygrownin DMEM:Fl0 medium(1:1) containing5% washed 3 times for 30 mm at 60Â°Cwithwashing solution containing decreas heat-inactivated FCS (Life Technologies, Inc.) and 30 p.g/mlgentamycin (Life ing concentrations of salt (2X SSC, 1X SSC, and 0.5X SSC) and 0.5% SDS, Technologies, Inc.). High dose selection with mafosfamide was performed 1 mM EDTA. The autoradiograms were densitometrically analyzed, and 24 h after seeding of 5 X iO@cells per 10-cm dish by addition of 30 @.tMofthe ERCC1mRNA expressionwas calculatedby relatingERCC1mRNA to the drug to the medium without subsequent medium exchange. Selection with amount of glyceraldehyde-3-phosphate dehydrogenase mRNA. MNNG (25 SM), treosulfan (20 @LM),and HeCNU (60 @tM)was performed in UDS. UDS was measured as described (52). To reduce the background of the same way. Colonies appearing 2 weeks later were picked, grown, and replicative DNA synthesis, cells were cultured for 24 h in medium with 0.5% analyzed for drug resistance. This was done by seeding the cells in multiwell FCS.ThirtymmbeforeUV irradiation(60i/m2), cells werepretreatedwith10 dishes (10â€•/well)andtreating them 8â€”12hlater with three different concen mM hydroxyurea. After irradiation, cells were further incubated in the presence trations ofthe agent that had been used for selection (mafosfamide, 10â€”30,.@M; of 10mMhydroxyurea and [3H]thymidine(10 @.&Ci/m1)for4 h. Thereafter, cells HeCNU, 20â€”60,xM;treosulfan, 10â€”20,.LM;and MNNG, 15â€”30SM).Five were transferred to GF/A filters (Whatman, Inc.) using a Skatron cell harvester. days later, cells were fixed with methanol and stained with crystal violet. Filter-bound radioactivity was determined by scintillation counting. Variations in the cytotoxicity of the respective drug was inferred from differ Determination of DNA ReplicatiOn. To investigate the effect of drug ences in the density of the cell layers at a given concentration of the agent. treatment on DNA replication, cells were pulse labeled after treatment with the Survival Assay. To quantitate the level of resistance, â€”500 cells were agent by addition of [3H]thymidine(final concentration, 1 @Ci/ml)andwere seeded per 5-cm dish and treated 6â€”8h later. For UV irradiation, medium was incubated for 30 mm at 37Â°C.Subsequently,cells were washed twice with removed and added again after irradiation. Drug treatments (mafosfamide, ice-cold PBS, trypsinized, and harvested on GF/A-filters (Whatman, Inc.) MNNG, MMS, cisplatin, HeCNU, mitomycin C, melphalan, treosulfan, and using a Skatron cell harvester. Filter-bound radioactivity was determined by H202) wereperformedbyaddingtheagentfroma stocksolutiondirectlyto the scintillation counting. Drug-induced block to DNA replication was calculated medium without a subsequent medium change. Colonies were fixed with by relating [3H]thymidine incorporation of treated cells to those of the un methanol 7 days after seeding and stained with crystal violet. Survival fre treated control. [3H]Thymidineincorporation of control cells was set to 100%. quency of treated cells is expressed in relation to the untreated control, which was included in each experiment. Chromosomal Aberrations. To determine aberration frequency, logarith RESULTS mically growing cells were treated with the indicated concentrations of mafos famide, HeCNU, and MNNG, respectively. The medium was not exchanged High-Dose Selection with Mafosfamide Yields Mafosfamide after drug treatment. After a recovery period of 18h, colcemid (50 ng/ml) was sensitive Clones. We attempted to study the features of survivors of added for a time period of 2 h. Thereafter, cells were harvested by trypsiniza a cell population exposed to a high dose of cyclophosphamide, which tion. Chromosomes were stained with 5% Giemsa (in PBS). At least 50 is often used in tumor chemotherapy. Because cyclophosphamide metaphases were evaluated per treatment level. For quantitation, cells with needs metabolic activation by cytochrome P-450 to evoke its DNA multiple aberrations (more than five aberrations per metaphase) were consid binding properties and this enzyme is not expressed in CHO cells, we cml to exhibit five aberrations. used the cyclophosphamide analogue mafosfamide for treatment, Determination of DNA Single-Strand Breaks and DNA Cross-Links which in aqueous solution, spontaneously decomposes to the active Logarithmically growing cells were treated with 50 @.LMmafosfamideand form 4-hydroxy-cyclophosphamide. Exponentially growing CHO-Kl harvested by trypsinization 3â€”24hlater. Cells (1.5 X 106)were analyzed for cells were treated with a single high dose of mafosfamide (30 p@M), DNA single-strandbreaks and DNA cross-links using the alkaline elution technique as described (45). Cross-link assay was performed under deprotein which reduced colony formation by â€”[email protected] the surviving cob ized conditions. Alkaline elution was allowed to proceed for 10 h. Thereafter, flies, 19 clones were isolated and tested as to their resistance to pH of the samples was neutralized,fluorochromesolution was added, and mafosfamide. None of the clones analyzed exhibited an elevated level DNA concentrations in the eluted fractions, as well as the DNA that remained of resistance, but unexpectedly, three of them (designated as CHO on the filters, were quantified using a fluorescence detector. Elution rate (k) Kl-4, CHO-Kl-l 1, and CHO-Kl-l2) showed a clearly enhanced 455

functional alkylating agent MNNG resulted only in cell variants that were not altered or resistant to MNNG (Table 1). Cross-Sensitivity to Other DNA-damaging Agents. To analyze whether the isolated mafosfamide-hypersensitive cell lines are cross sensitive to other DNA cross-linking and noncross-linking agents, do nogenic survival of CHO-Kl-4 and CHO-Kl-12 was assayed after cx posure to various drugs. As compared with the parental control line I CHO-Ki, both variantcell lines exhibited a reduced survival after treat ment with the methylating agent MMS, and only the strain CHO-K1-4 was more sensitive to MNNG (Fig. 2). Both cell lines were cross sensitive to all other DNA cross-linking therapeutic agents tested, such as I HeCNU, melphalan, treosulfan, cisplatin, and mitomycin C (Fig. 2). CHO-Kl-4 cells were slighily more sensitive to H202 than wild-type cells, whereas CHO-K1-l2 cells exhibited an enhanced sensitivity to UV light (Fig. 2). In summary, the data reveal that both isolated cell clones displayed a general hypersensitivity to DNA cross-linking agents. 12 Chromosomal Aberrations. If the hypersensitivity of the isolated Matosfamide (pM) variants to the cytotoxic effects of DNA cross-linking agents is geneti cally determined, it should be accompanied by increased chromosomal Fig. I. Sensitivity of cell lines obtained after selection to mafosfamide. Cells that had survived the selection procedure with 30 p.ti mafosfamide were cloned and tested for their instability. To test this, cytogenetic analyses were performed. As shown resistance to mafosfamide. For testing cell survival, cells were seeded (500 cells/dish) and in Fig. 3, CHO-K1-4 and CHO-Kl-l2 cells were clearly more sensitive treated 6â€”8h later with the indicated concentrations of mafosfamide. One week later, cells were fixed and stained, and the colonies were counted. Survival in untreated control to the clastogenic activity of mafosfamide than the parental cell line. was set to 100%. CHO-Kl, wild-type cells; CHO-Kl-4, CHO-Kl-l0, CHO-Kl-l 1, Thus, after treatment with 5 p.M of mafosfamide, 2.9, 2.8, and 0.9 CHO-Kl-12,andCHO-Kl-l3,cellclonesthatwereisolatedaftermafosfamideselection. aberrations per cell were observed in CHO-K1-4, CHO-Kl-l2, and Data shown are the means of at least three independent experiments. wild-type cells, respectively. This clastogenic hypersensitivity was also observed if aberrations were expressed in terms of percentage of aberrant metaphases (data not shown). Also, both mafosfamide-hypersensitive cell sensitivity to mafosfamide. The survival of these clones together with lines exhibited an increased sensitivity to the clastogenic effect of wild-type and two other nonsensitive derivatives obtained from the HCCNU. In contrast, only CHO-K1-4 showed an enhanced frequency of selection procedure is shown in Fig. 1. Based on the D10 level, the chromosomal aberrations after MNNG treatment (Table 2). Obviously, mutant cells showed an about 2â€”3-foldenhanced sensitivity to mafos the patternof chromosomal sensitivity parallels the cytotoxic response of famide as compared with the parental cell line CHO-Ki. One of these the mutants. sensitive clones (CHO-Kl-ll) proved to be unstable; it lost its hy Cellular Growth, DNA Repair Synthesis, and p53. As shown in persensitive phenotype upon propagation, whereas two other clones Fig. 4A, mafosfamide-sensitive cell lines exhibited identical growth (CHO-Kl-4 and CHO-K1-l2) retained hypersensitivity over the en rates and saturation densities as wild-type cells. A slightly enhanced tire period of investigation (>6 months). serum dependency was observed for CHO-Kl-l2, whereas CHO To estimate the frequency of generation of mafosfamide-sensitive K1-4 behaved very similarly to wild-type cells (Fig. 4B). One rca variants, phenotypical analysis of survivors of mafosfamide selection sonable explanation for mafosfamide hypersensitivity would be a was extended by analyzing in total 35 surviving clones. About 17% of defect in the nucleotide excision repair system, which was shown to the clones propagated after the mafosfamide selection gained sensi be involved in cellular resistance to cross-linking alkylating drugs (19, tivity to mafosfamide (Table 1). The generation of sensitive cell 20). To measure the extent of overal nucleotide excision repair, variants upon high-dose drug selection appears to be agent specific. unscheduled DNA synthesis was determined after UV irradiation, Thus, cells that survived high-dose treatment with HeCNU did not which is very effective in inducing UDS. As shown in Fig. 5, A and distinguish from parental cells in respect to their sensitivity to the B, mutant cells were clearly able to perform UV-induced UDS. They selecting agent, whereas selection with treosulfan also yielded drug also did not show changes in the expression of ERCC1 mRNA as sensitive survivors (Table 1). In contrast, selection with the mono compared with wild-type cells. An involvement of p53 in the hyper

survivorsCells Table1 Effectofhigh-dosedrugselectionondrugsensitivityof wasnot (5 X l0@)were seeded per 10-cmdish, and cells were treated 24 h later with either malosfamide, HeCNU, treosulfan, or MNNG at the concentrations indicated. Medium selectionprocedureexchanged after drug treatment. After an incubation period of 12â€”14days,colonies were isolated and rechecked for resistance to the same agent that was used for the Methods.â€•Treatmentas describedinâ€œMaterialsand FrequencyaMafosfamideâ€• Dose (pM) Survivors/106 cells Clones tested Clones resistant/sensitive 17.1HeCNU 30 24 35 0/6 <5.6Treosulfanc 60 82 19 0/0 8.7MNNG 20 74 23 0/2 8.3a 25 50 24 2/0

Percentage of resistant or sensitive clones as related to the number of clones tested. bChemicalstructure: S-CH2-CH2-S03 (Cl-CH2-CH2)2Nâ€”Pâ€”NH

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MAFOSFAMIDE AND SENSITIVITY TO DNA CROSS-LINKING AGENTS

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[email protected] â€¢0 24 1@2 0 10 51 30 40 0 lÃ a@ 51 40 50 50 aâ€” TmosiiI@@(jiM) UV(J/m2) H,02(%x 10@) Fig. 2. Sensitivity to mafosfamide is accompanied by cross-sensitivity to a variety of DNA-damaging agents. Six to 8 h after seeding (500 cells/dish), cells were treated with different concentrations of MNNG, MMS, HeCNU, melphalan, treosulfan, cisplatin, UV, mitomycin C, and H202. One week later, cells were fixed and stained, and the colonies were counted. Survival in untreated control was set to 100%.CHO-Kl, wild-type cells; CHO-Kl-4 and CHO-Kl-12, mafosfamide-hypersensitivecell clones isolated by 30 ,.ui mafosfamide selection. Each point represents the mean of at least two independent experiments.

sensitive phenotype also appears unlikely because both wild-type and incorporation than wild-type cells. Similar results were obtained for mutant cells did not exhibit p53 accumulation upon UV irradiation or HeCNU (data not shown). The initial inhibition of [3H}thymidine MMS treatment (Fig. 5C). This observation indicates that p53 is mutated incorporation was most pronounced 2 h after MMS treatment, where in CHO-Kl cells. It should be noted that in these experiments, p53 [3H]thymidine incorporation was reduced by 60â€”70%in wild-type induction occurred in NiH 3T3 cells, which were included for control. and only 30â€”40%in variant cells (Fig. 7C). Similar to wild-type cells, Determination of GSH and GST Activity. Because GSH and mutant cells had recovered from the transient block to DNA replica GST are important factors in detoxification of various drugs, the tion 6 h after treatment (Fig. 7C). hypersensitive mutants were analyzed for both parameters. These measurements showed that wild-type and mutant cells have similar contents of GSH (0.5â€”0.6nmol/mg protein) and GST activities (0.21â€” 5.0 0.23 p.Mol/mg of protein per minute). Thus, differences in GSH- and GST-based detoxification mechanisms are unlikely to be the cause for the hypersensitive phenotype described here. 4.0 Induction of DNA Strand Breaks and Repair of DNA Cross I I Links. To analyze if differences in the level of mafosfamide-induced II II DNA strand breaks exist between wild-type and mutant cell lines, 3.0. AI @ cells were treated with 50 ,.LMmafosfamide, and DNA strand breaks I II were quantified up to 24 h after treatment using the alkaline elution -I I 0 technique. As shown in Fig. 6, the extent of DNA strand breaks 2.0. II II induced by mafosfamide treatment was very similar in wild-type and II hypersensitive mutants. To measure the removal of mafosfamide 1.0. induced DNA cross-links, mafosfamide-treated cells were irradiated ,;f A [email protected]@i with a high dose of X-rays (800 cGy) prior to alkaline elution. Using â€¢CHO4Ci-4 this method (46), cell line-specific variations in the removal of DNA OCHO-K1-12 cross-links can be detected by an altered increase in elution rate ib i's 20 ([email protected]).The elution rates at 3 and 24 h after mafosfamide treatment were measured, and [email protected]. As shown in Table 3, elution Mafosfamide (@tM) rates determined under these conditions were very similar in wild-type Fig. 3. The hypersensitivity of the mutants to the cytotoxic effects of mafosfamide is accompanied by an increase in the frequency of chromosomal aberrations. CHO-Kl and hypersensitive cells, indicating that the hypersensitive cells are wild-type cells and mutants (CHO-Kl-4 and CHO-K1-12) were treated with different not blocked in the repair of DNA cross-links. concentrations of mafosfamide. After an incubation period of 18 h, cells were treated with DNA Replication. Because the transient mutagen-induced block to colcemid, and metaphases were prepared 2 h later. Fifty metaphases were analyzed per treatment. Data shown are the mean values from two independent experiments. DNA replication appears to be related to a mutagen-hypersensitive phenotype (53, 54), we compared the isolated cell lines in their ability Table2 AberrationfrequencyofCHOwild-typeandhypersensitivecellvariantsafter to proceed in DNA replication after drug exposure. Exponentially @.z@w)Aberrations/cellCelltreatment with HeCNU (20 paw)and MNNG (10 growing wild-type and mutant cells were treated with increasing doses of mafosfamide or MMS. Subsequently, incorporation of [3H]thymi @LM)CHO-KIline HeCNU (20 @M) MNNG (10 dine applied as a pulse was determined. As shown in Fig. 7, A and B, 0.8 0.9 after treatment with both mafosfamide and MMS, CHO-Kl-4 and CHO-Kl-4 3.2 2.9 CHO-Kl-12 cells displayed a less severe inhibition of [3H]thymidine CHO-K1-12 2.1 1.0 457

A B

Fig. 4. Growth rate and serum dependence are similar in wild-type and mutant cells. A, cellular growth was determined by counting the number of â€˜To cells up to 7 days after seeding (4 X lO@cells/well) in medium containing 5% FCS. B, cells were seeded in low density (500 cells/dish) and grown in 10 the presence of different concentrations of FCS (0.5 up to 10%). One week later, colonies were I fixed with methanol and counted. The colony forming ability is related to the number of colonies I 0 Oi@m I grown in the presence of 10% FCS, which was set S OlOK14 to 100%. a 0404C1-12

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A induced genetic changes, which enabled the cells to survive. Whether these changes were due to loss or gain of function cannot be answered @H04C1-12 â€”@ yet, but obviously, in some clones they resulted in a permanently en hanced sensitivity to mafosfamide. The generation of sensitive clones CHO-K1-4 appears to be in contrast to the survival of the cell after the selection procedure and thus might be a late effect of the changes induced by this CHO4(1 drug. Interestingly, the phenomenon of generation of sensitive cell vat-i- ants upon drug treatment is not restricted to mafosfamide but was also 0 2000 4000 os@ 8000 10000 12000 observed after selection with treosulfan. Thus, it is tempting to speculate 3H-ThymidlneIncorporation (otcpm/10 cells) that induction of drug sensitivity increases the therapeutic efficiency of cycbophosphamide and probably other cross-linking antineoplastic agents. B C Two phenotypically stable variant clones resulting from mafos NIH3T3 KI K1-4 K1-12

EACCI 1?Lii GAP0H@ 1â€” Fig. 5. Mutant cells are not defective in nucleotide excision repair and do not distinguish from wild-type cells in p53 expression. A, logarithmically growing cells were 40 cultured in the presence of 0.5% FCS for 24 h. Thirty mm before UV irradiation (60 JIm2), cells were preincubated with hydroxyurea (10 mM).After UV treatment. cells were further incubated in the presence of 10 mti hydroxyurea and [3HJthymidine (10 pCi/mI) for 4 h. 20 After this incubation period, incorporation of [3H]thymidinewas determined as described in â€œMaterialsandMethods.â€•Datashown are mean values from two independent exper iments, each performed in triplicate. B, total RNA was prepared from logarithmically growing cells. Northern blot analysis was performed as described in â€œMaterialsand 0 10 20 Methodsâ€• C. logarithmically growing N1113T3, CHO-Kl (Kl), CHO-Kl-4 (Kl-4), and Time (h) CHO-Kl-12 (Kl-l2) cells were UV irradiated (30 JIm2) or treated with MMS (1 ntsi). Afteranincubationperiodof8 h,cellswereharvested,and20 @gofnuclearcellextracts Fig. 6. Comparison of the level of mafosfamide-induced DNA strand breaks in were analyzed for p53 level by Western blot analysis. hypersensitive mutants and wild-type cells. Logarithmically growing CHO wild-type cells and hypersensitive cell variants CHO-Kl-4 and CHO-Kl-l2 were treated with 50 [email protected] mafosfamide. After incubation periods of 4, 8, and 24 h, cells were harvested for determination of DNA strand breaks using the alkaline elution method. Data shown are DISCUSSION derived from two independent experiments.

In a first step in studying the involvement of DNA repair mechanisms Table3 Mafosfamidehypersensitivecellvariantsareofmafosfamide-induced notblockedintheremoval in cycbophosphamide resistance, we have treated CHO cells with a single cross-linksLogrithmically DNA high dose of the cycbophosphamide analogue mafosfamide and analyzed growing cells were treated with 50 p.t@oofmafosfamide. After incuba the survivors as to their level of resistance to this drug. Contrary to the lionasdescribed periods of 3 and 24 h, cells were harvested for determination of DNA cross-links leastthree in â€œMaterialsandMethods.â€•Datarepresent mean values and SDs from at expectation, mafosfamide selection did not result in resistant but rather independent experiments.Elution drug-sensitive cell clones. The generation of sensitive cell variants after 103/h)CHO-KI rate (X high-dose selection is unusual and, to our knowledge, has not been described before. The rise of sensitive cell variants upon mafosfamide CHO-Kl-l2Untreated CHO-Kl-4 treatment occurred with a relatively high frequency (17% of survivors) 5.0X-ray 11.6 Â±2.2 12.5 Â±3.1 12.6 Â± 6.8X-ray 86.5 Â±16.0 78.3 Â±13.2 84.3 Â± and was not observed after selection with either HCCNU or MNNG. The 5.6X-ray+ Maf' (50 ,LM,3h) 67.8 Â±12.3 64.6 Â±7.6 66.0 Â± unexpected generation of mafosfamide-sensitive cell lines after selection 9.8a + Maf (50 ELM,24h) 107.0 Â±6.7 119.2 Â±11.2 117.0 Â± with this drug might be due to spontaneous or, more likely, mafosfamide Maf, mafosfamide. 458

A B C 125@ 120

100

1' OGHO4CI .oIoâ€¢rn-4 acHO.K1.12 30 i:l go MMS (mM) This (Pt)

Fig. 7. Hypersensitive mutants exhibit DNA damage-resistant DNA synthesis. A and B, logarithmically growing cells were treated with 30, 60, and 90 p@Mmafosfamide(A) or 0.5, 1.0, and 1.5 mMMMS (B). After an incubation period of 2 h for MMS and 3 h for mafosfamide, respectively, the medium was replaced by fresh medium containing [3H]thymidine (1 @.tCi/ml).Anadditional 30 mm later, cells were rinsed twice with ice-cold PBS and trypsinized; DNA was harvested for scintillation counting as described in â€œMaterialsandMethods.â€• [3H]Thymidineincorporation in untreated control was set to 100%. Data shown are derived from at least two independent experiments. C, logarithmically growing CHO-KI wild-type cells and mutants (CHO-Kl-4 and CHO-Kl-l2) were treated with 1 mr@tofMMS. After an incubation period of 2, 4, and 6 h, cells were pulse-labeled with [3H]thymidineas described in A. [3H]Thymidine incorporation in untreated control was set to 100%. Mean values and standard deviations are derived from three independent experiments.

famide selection were analyzed in more detail. These clones exhibited to DNA replication as measured 2 h after treatment. Similar to the cross-sensitivity to all cross-linking cytostatic drugs tested but dif wild-type cells, they completely recovered from the block to DNA fered in their response to MNNG, UVC, and H202. The cytotoxic replication 6 h after exposure. We should note that in this respect, the effect of mafosfamide, HeCNU, and MNNG paralleled the clasto hypersensitive cells described here do not resemble the phenotype of genic activity of these agents on the cell lines, indicating that cyto Fanconi's anemia cells, which show a more severe block to DNA toxic hypersensitivity and increased sensitivity to the induction of replication after treatment with cross-linking agents (54, 56). The chromosomal aberrations are related. From the analysis of cross observation of an altered response to DNA replication arrest upon sensitivities to a variety of DNA-damaging agents, we conclude that DNA damage might be a reasonable explanation for the hypersensi different genetic changes may be involved in the mafosfamide-hyper tive phenotype, because a less severe block to DNA replication may sensitive phenotype of the isolated mutants. Our observation that the decrease the probability for prereplicative repair of DNA damage, hypersensitivity of both mutants did not pertain solely to DNA cross thereby contributing to enhanced clastogenic and cytotoxic effects. linking agents is in accordance with other data showing that hyper In summary, a single high-dose exposure ofCHO cells to mafosfamide sensitivity to mitomycin C was accompanied by cross-sensitivity to yielded survivors that were more sensitive to this agent. The isolated either UV light, X-rays, and/or MMS (4, 15â€”17,19,55). Obviously, mafosfamide-hypersensitive clones, which are stably hypersensitive to all the repair of lesions induced by different classes of DNA-damaging DNA cross-linking therapeuticalagents tested, displayed increased chro agents requires both common and agent-specific pathways. mosomal sensitivity and exhibited drug-resistant DNA synthesis. Al The most simple explanation for cellular hypersensitivity to mafos though it is not yet clear whether tumor cells respond in the same way as famide would be that the level of the induced DNA damage is CHO cells, the finding may be taken as an indication that acquired enhanced. This appears unlikely, however, because: (a) GSH content resistance of tumor cells to cyclophosphamide might be a less frequent and GST activity are similar in wild-type and hypersensitive cells; and event than induction of sensitivity. The generation of cell variants semi (b) the level of mafosfamide-induced DNA strand breaks, which can tive to a broad range of cytostatic drugs could be one of the factors be taken as an indication for the level of induced DNA damage, was contributing to the high effectivity of cyclophosphamide in cancer ther not enhanced in the mutant cell lines. Also, defects in nucleotide apy. The isolated cell variants might become helpful tools in the identi excision repair are very likely not responsible for the hypersensitive fication of cellular factors that cause sensitivity of tumor cells to DNA phenotype, because mutant cells are not blocked in DNA repair cross-linking antineoplastic agents. synthesis upon UV irradiation and do not distinguish from wild-type cells in respect to ERCC1 mRNA expression. As determined in filter elution experiments, the hypersensitive cell lines did not show a reduced ACKNOWLEDGEMENTS removal ofmafosfamide-induced DNA cross-links, as compared with the We thank Susanne Gebhard and Uta Eichhom for excellent technical parental cell line, and thus appear also not to be blocked in cross-link assistance. We are grateful to Dr. J. Pohl, Asia Medica (Frankfurt), for the repair. This fmding agrees with the observation that the hypersensitivity generous gift of mafosfamide. of the mutants was not limited to DNA cross-linking agents but also pertained to MMS, MNNG, or H202. It should be noted that wild-type and mutant cells were very similar in growth rate and serum dependence REFERENCES and did not distinguish in p53, also making these parametersunlikely to 1. Jones, N. J., Stewart, S. A., and Thompson, L. H. Biochemical and genetic analysis be involved in the hypersensitivity of the mutants. of the Chinese hamster mutants irsl and irs2 and their comparison to cultured ataxia telangiectasia cells. Mutagenesis, 5: 15â€”23,1990. The main difference between variant and wild-type cells is that the 2. Thompson,L.H.,Brookman,K.W.,Dillehay,L.E.,Carran,A.V.,Carrano,A.V., hypersensitive cell variants exhibited a less severe drug-induced block Mazrimas, J. A., Mooney, C. L., and Minkler, J. L. A CHO strain having hypersen 459

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Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 1997 American Association for Cancer Research. High-Dose Selection with Mafosfamide Results in Sensitivity to DNA Cross-Linking Agents: Characterization of Hypersensitive Cell Lines

Gerhard Fritz, Jan Georg Hengstler and Bernd Kaina

Cancer Res 1997;57:454-460.

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