The role of DNA repair in resistance of L1210 cells to isomeric 1,2-diaminocyclohexaneplatinum complexes and ultraviolet irradiation

Resistance to cisplatin in several murine leukemia L1210 cell lines is due to enhanced DNA repair. Other platinum complexes, particularly those containing 1,2-diaminocyclohexane (DACH) are of interest as they effectively kill both sensitive (L1210/0) and cisplatin-resistant (L1210/DDP) cell lines. An L1210/DACH cell line has been developed that is preferentially resistant to DACH-Pt complexes. In the current experiments, we investigated the role that DNA repair has in resistance to DACH-Pt compounds. The DACH ligand exists in 3 isomeric forms which exhibit markedly different activities in the various resistant cell lines. Generally, R,R-DACH-Pt was the most effective isomer. DNA repair was assayed by host-cell reactivation of platinated pRSVcat. DNA damage induced by all the isomeric DACH-Pt-SO4 complexes markedly reduced CAT expression in sensitive L1210/0 cells. One adduct per transcribed strand of the cat gene inhibited CAT expression demonstrating that the sensitive cells exhibited no detectable DNA repair. All the resistant cell lines reactivated the plasmid DNA whether damaged with cisplatin or any of the 3 DACH-Pt isomers. Therefore, resistance to both cisplatin and DACH-Pt appears to be mediated by enhanced DNA repair, but the level of reactivation of the transfected plasmid did not correlate with the toxicity of each analogue. These results suggest that some additional event(s) is responsible for the substrate specificity of repair of genomic DNA. These resistant cell lines also exhibited resistance to UV irradiation but this was much less than, and did not correlate with the degree of resistance to either cisplatin or DACH-Pt. However, there was a good correlation between resistance to UV irradiation and reactivation of UV-damaged plasmid DNA. This enhanced reactivation suggests that enhanced repair may be the sole reason for the resistance to UV irradiation.     

Enhanced repair of a cisplatin-damaged reporter chloramphenicol-O-acetyltransferase gene and altered activities of DNA polymerases α and β, and DNA ligase in cells of a human malignant glioma following in vivo cisplatin therapy

Current evidence suggest an important role for increased repair of drug-induced DNA damage as one of the major mechanisms involved in tumor cell resistance to cis-DDP. In this study, we examined the DNA repair capacity and the activities of three DNA repair related proteins, namely, DNA polymerases α and β, and total DNA ligase in cells of a malignant oligodendroglioma obtained from a patient before therapy and compared it with those of a specimen of the tumor acquired after the patient had failed cis-DDP therapy. DNA repair capacity was quantitated as the extent of reactivation of the chloramphenicol-O-acetyltransferase (CAT) gene in a eukaryotic expression vector that has been damaged and inactivated by prior treatment with cis-DDP and then transfected into the tumor cells. The extent of DNA-platinum adduct formation in the expression vector was determined by flameless atomic absorption spectrometry. The level of cis-DDP resistance of cells of the two tumors was determined with the capillary tumor stem cell assay. We observed a 2.8-fold increased capacity to repair Pt-DNA adducts and reactivate the CAT gene in cells of the tumor obtained after cis-DDP therapy, compared to cells of the untreated tumor. This was associated with increases of 9.4-fold and a 2.3-fold, respectively, in DNA polymerase β and total DNA ligase activities in cells of the treated tumor. At 5 μM cis-DDP, there was a 5.9-fold increase in the in vitro cis-DDP resistance of post-therapy tumor cells relative to cells of the untreated tumor. No significant difference in DNA polymerase α activity was observed between the two tumors. These data suggest that the enhanced ability to repair cis-DDP induced DNA damage, mediated, in part, by increased tumor DNA polymerase β and DNA ligase activities, plays an important role in the in vivo acquisition of cis-DDP resistance in human malignant gliomas, and that these proteins and/or their encoding genes may represent critical targets for strategies to overcome such resistance clinically.     

Transformation depending on intermolecular homologous recombination is stimulated by UV damage in transfected DNA

DNA-mediated gene transfer (DMGT) was performed in DNA repair-proficient and UV-hypersensitive, repair-deficient Chinese hamster ovary (CHO) cell lines using the UV-irradiated thymidine kinase gene from herpes simplex virus (HSV-TK). Transformation frequencies in repair-deficient CHO cell lines declined relative to repair-proficient cells with increasing UV damage in transfected DNA; approximately 3-fold higher UV fluence was required to inactivate 50% of irradiated HSV-TK plasmid molecules in repair-proficient cells. In cotransfection experiments performed with pairs of HSV-TK plasmids containing linker insertion mutations in TK coding sequences, moderate UV damage in plasmid DNA enhanced the yield of TK+ transformants resulting from homologous recombination between HSV-TK sequences up to 4-fold. These results suggest that UV damage in DNA can stimulate transformation of mammalian cells dependent on intermolecular DNA homology.     

DNA-protein cross-linking in L1210 cells sensitive and resistant to cis-diamminedichloroplatinum (II)

The role of restricted cellular accumulation of cis-diamminedichloroplatinum(II) (cis-DDP) and altered repair of DNA-Pt-protein cross-links in the mechanism of L1210 murine leukemia cell resistance was examined. An immunochemical method was used to analyze the formation and removal of DNA-Pt-protein complexes in L1210 cells sensitive and resistant to cis-DDP. The accumulation of Pt into the cells and the binding of Pt to the DNA was measured by atomic absorption spectroscopy. The results demonstrated that both decreased accumulation of the drug and the rate of DNA-Pt protein cross-link removal may be important factors in L1210 cell resistance to cis-DDP.Abbreviations AAS atomic absorption spectroscopy - cis-DDP cis-diamminedichloroplatinum(II)     

Transformation of UV-hypersensitive Chinese hamster ovary cell mutants with UV-irradiated plasmids

Transfection of UV-hypersensitive, DNA repair-deficient Chinese hamster ovary (CHO) cell lines and parental, repair-proficient CHO cells with UV-irradiated pHaprt-1 or pSV2gpt plasmids resulted in different responses by recipient cell lines to UV damage in transfected DNA. Unlike results that have been reported for human cells, UV irradiation of transfecting DNA did not stimulate the genetic transformation of CHO recipient cells. In repair-deficient CHO cells, proportionally fewer transformants were produced with increasing UV damage than in repair-proficient cells in transfections with the UV-irradiated hamster adenine phosphoribosyltransferase (APRT) gene contained in plasmid pHaprt-1. However, transfection of CHO cells with UV-irradiated pSV2gpt resulted in neither decline in transformation frequencies in repair-deficient cell lines relative to repair-proficient cells nor stimulation of genetic transformation by UV damage in the plasmid. Blot hybridization analysis of DNA samples isolated from transformed cells showed no dramatic changes in copy number or arrangement of transfected plasmid DNA with increasing UV dose. We conclude that the responses of recipient cells to UV-damaged transfecting plasmids depend both on the type of recipient cell and the characteristics of the genetic sequence used for transfection.     

Mutational hotspot variability in an ultraviolet-treated shuttle vector plasmid propagated in xeroderma pigmentosum and normal human lymphoblasts and fibroblasts

The mutagenesis shuttle vector, pZ189, was treated with ultraviolet (u.v.) radiation in vitro and passed through a DNA repair-deficient lymphoblastoid cell line derived from a patient with xeroderma pigmentosum complementation group A (XP-A) (XP12BE(EBV)) and a DNA repair-proficient lymphoblastoid cell line (GM606(EBV)). After u.v. treatment, plasmid survival was lower and mutation frequency higher with the XP-A cells mirroring the survival and mutagenesis of the host cells. The nature of the mutations in the suppressor tRNA marker gene was determined by direct sequence analysis. The G.C to A.T transition was the dominant (85%) base substitution mutation with the XP lymphoblasts and was the major (56%) base substitution mutation with the repair-proficient lymphoblasts. We found a G.C to A.T transition mutational hotspot with the XP lymphoblasts not seen in our previous experiments with fibroblasts from the same patient. Comparison of the data presented here with our results with DNA repair-deficient and DNA repair-proficient fibroblasts suggests that hotspot variability is not due to genetic polymorphism or repair capacity of the cells. Instead it appears that cellular factors can influence the probability of mutagenesis of modified DNA at particular sites.     

Phenotypic reversion of cisplatin resistance in human cells accompanies reduced host cell reactivation of damaged plasmid

Revertant cell lines were established from cisplatin (CP) resistant HeLa cells. Expression of CP damaged plasmid DNA carrying bacterial chloramphenicol acetyltransferase (CAT) gene after transfection into cells was measured. Revertant cells showed reduced host cell reactivation of damaged plasmid, as compared to resistant cells. Addition of aphidicolin, an inhibitor for DNA polymerase alpha, to resistant cells effectively blocked enhanced plasmid reactivation and acquired resistance. The results are consistent with the notion that cellular ability in repair CP-damaged DNA is a mechanism for CP resistance.     

Enhanced DNA repair as a mechanism of resistance to cis-diamminedichloroplatinum(II)

Murine leukemia L1210 cells, either sensitive or resistant to the toxic action of the cancer chemotherapeutic agent cis-diamminedichloroplatinum(II), have been studied for potential differences in the formation and repair of drug-induced DNA damage. The sensitivity for these experiments was obtained by using the radiolabeled analogue [3H]-cis-dichloro(ethylenediamine)platinum(II). The resistant cells demonstrated a 40% reduction in drug accumulation but a qualitatively similar profile of DNA-bound adducts. These adducts resembled those previously characterized in pure DNA and represented intrastrand cross-links at GG, AG, and GNG (N is any nucleotide) sequences in DNA. Repair of these cross-links occurred in a biphasic manner: rapid for the first 6 h and then much slower. The resistant cells removed up to 4 times as many adducts during the rapid phase of repair. The extent of this repair did not directly correlate with the degree of resistance in that cells with 100-fold resistance were only slightly more effective at repair than cells with 20-fold resistance. Therefore, although enhanced DNA repair is thought to contribute markedly to drug resistance, other mechanisms for tolerance of DNA damage may also occur in these cells.     

Cloning and nucleotide sequence analysis of a chloramphenicol acetyltransferase gene from Vibrio anguillarum.

The chloramphenicol resistant gene (cat) encoding chloramphenicol acetyltransferase (CAT) in a transferable R plasmid (pJA7324) isolated from the fish pathogen Vibrio anguillarum strain PT24 was cloned into the plasmid vector pUC19. The nucleotide sequence analysis of 1,348 base pair DNA identified an open reading frame encoding a protein of 216 amino acid residues with a calculated molecular mass of 25,471 daltons. The predicted amino acid sequences for this cat gene are 37-69% homologous with other CAT proteins of both Gram-negative and -positive bacteria. Colony hybridization performed with a PvuII-BamHI fragment including this cat gene as a probe, revealed that the same or similar chloramphenicol resistance genes existed among V. anguillarum isolates.     

DNA-mediated cotransfer of excision repair capacity and drug resistance into chinese hamster ovary mutant cell line UV-135.

We have investigated DNA-mediated transfer of aminopterin resistance conferred by plasmid and UV resistance conferred by genomic DNA to the Chinese hamster ovary (CHO) cell line UV-135, a UV-sensitive mutant defective in nucleotide excision repair. Plasmid pSV2gpt-CaPO4 coprecipitates induced aminopterin resistance with equal efficiency in the 6-thioguanine-resistant, aminopterin-sensitive, repair-proficient parental line AA8-4(tg-1) and in UV-135(tg-2). Genetic and molecular evidence for genomic DNA-mediated transformation of UV-135(tg-2) cells with a putative excision repair gene were obtained by demonstrating that: (i) UV resistance transformation is dependent upon and specific for genomic DNA from excision repair-competent CHO cells: (ii) UV and drug coresistant colonies are bona fide transferants as verified by hybridization and Southern blotting analysis of pSV2gpt sequences in their genomic DNAs: (iii) confirmed transferants exhibit partial to near normal UV resistances for colony formation: and (iv) UVr transferants have near normal levels of excision repair capacity. The overall frequency of drug and UV resistance cotransformation was 8 X 10(8) per cell plated. This frequency was ca. 200- to 500-fold greater than that expected from coincident but independent UVr reversion and plasmid gene transfer events. DNA transfer techniques with this CHO system will be useful for further analysis of the essential structural DNA sequences, gene cloning, and expression of functional excision repair genes.     

Theophylline reversal of alkylator-induced replicon initiation inhibition: no correlation with DDP-induced cytotoxicity

The synergistic cytotoxic activity exhibited by bifunctional alkylating agents in the presence of methylxanthines has been associated with methylxanthine-induced reversal of alkylator-induced DNA replicon initiation inhibition. This has also been seen with methylxanthines and ionizing irradiation. Methylxanthines do not appear exacerbate drug or ionizing radiation-induced damage. We report here a situation in which methylxanthine-induced reversal of DNA replicon initiation inhibition is not associated with increased cytotoxicity of the alkylator. Murine L1210 leukemia cells were assayed for cytotoxicity following treatment with either L-PAM or cis-DDP in the presence or absence of theophylline. Theophylline increased the cytotoxicity seen after L-PAM treatment but failed to increase the cis-DDP induced cytotoxicity. Analysis of pulse-labeled DNA on alkaline sucrose gradients revealed the expected decrease in DNA replicon initiation in L1210 cells treated with either L-PAM or cis-DDP. Theophylline had no effect on replicon initiation in untreated cells. Theophylline reversed the replicon initiation inhibition in cells treated with either L-PAM or cis-DDP. The reason for the apparent lack of added toxicity of the replicon initiation inhibition reversal in L1210 cells treated with theophylline and DDP is unknown.     

An XPG DNA repair defect causing mutagen hypersensitivity in mouse leukemia L1210 cells.

One of the most widely used antitumor drugs is cis-diamminedichloroplatinum(II) (cisplatin), and mechanisms of cisplatin resistance have been investigated in numerous model systems. Many studies have used mouse leukemia L1210/0 as a reference wild-type cell line, and cisplatin-resistant subclones have been derived from it. Increased DNA excision repair capacity is thought to play a key role in the acquired cisplatin resistance, and this has influenced development of drugs for clinical trials. We report here that the L1210/0 line is in fact severely deficient in nucleotide excision repair of damaged DNA in vivo and in vitro. L1210/0 cell extracts could be complemented by extracts from repair-defective human xeroderma pigmentosum (XP) or rodent excision repair cross-complementing (ERCC) mutant cells, except for XPG/ERCC5 mutants. Purified XPG protein could restore repair proficiency to L1210/0 extracts. Expression of mouse XPG mRNA was similar in all L1210 lines studied, suggesting a point mutation or small alteration of XPG in L1210/0 cells. The DNA repair capacity of a cisplatin-resistant subline, L1210/DDP10, is similar to that of type culture collection L1210 cells and to those of other normal mammalian cell lines. Nucleotide excision repair of DNA is thus clearly important in the intrinsic cellular defense against cisplatin. However, in contrast to what is generally believed, enhancement of DNA repair above the normal level in these rodent cells does not appear to be a mechanism of acquired resistance to the drug.     

Transient inhibition of DNA synthesis by 5-fluorodeoxyuridine leads to overexpression of dihydrofolate reductase with increased frequency of methotrexate resistance

Transient but incomplete suppression of DNA synthesis by a single exposure of an asynchronous population of cells to 5-fluoro-2'-deoxyuridine (FdUrd) increases the frequency of appearance of methotrexate (MTX)-resistant colonies. This increase was greater than 10-fold following a 6-h incubation of cells with 3 microM FdUrd prior to selection in MTX, an interval one-half the normal L1210 cell cycle time. During this period of exposure to FdUrd, DNA synthesis decreased to 25% of control rates and cells accumulated at the G1/S interface. The 6-h incubation with FdUrd resulted in greater than a 2.5-fold increase in the dihydrofolate reductase protein level in the treated cell population, which was accounted for, at least in part, by increased de novo synthesis of the enzyme as assessed by [35S]methionine labeling. This increase in dihydrofolate reductase was associated with a decrease in growth inhibition by MTX. A brief reversal (2 h) of FdUrd-induced DNA synthesis inhibition by the addition of thymidine eliminated the amplification of dihydrofolate reductase and the enhanced emergence of MTX-resistant clones. Beyond this, an analysis of clones that survive MTX selection indicates that the dihydrofolate reductase gene copy in cells spontaneously resistant to 50 nM MTX and those which resulted after the additional pretreatment with FdUrd for 6 h are comparable with a 2-4-fold amplification of enzyme in most clones. These studies demonstrate that FdUrd enhancement of dihydrofolate reductase expression can have a profound effect upon the incidence and expression of MTX resistance and that dihydrofolate reductase gene amplification may be another basis for antagonism between these agents.     

Repair synthesis by human cell extracts in cisplatin-damaged DNA is preferentially determined by minor adducts.

During reaction of cis-diamminedichloroplatinum(II) (cis-DDP) with DNA, a number of adducts are formed which may be discriminated by the excision-repair system. An in vitro excision-repair assay with human cell-free extracts has been used to assess the relative repair extent of monofunctional adducts, intrastrand and interstrand cross-links of cis-DDP on plasmid DNA. Preferential removal of cis-DDP 1,2-intrastrand diadducts occurred in the presence of cyanide ions. In conditions where cyanide treatment removed 85% of total platinum adducts while approximately 70% of interstrand cross-links remained in plasmid DNA, no significant variation in repair synthesis by human cell extracts was observed. Then, we constructed three types of plasmid DNA substrates containing mainly either monoadducts, 1,2-intrastrand cross-links or interstrand cross-links lesions. The three plasmid species were modified in order to obtain the same extent of total platinum DNA adducts per plasmid. No DNA repair synthesis was detected with monofunctional adducts during incubation with human whole cell extracts. However, a two-fold increase in repair synthesis was found when the proportion of interstrand cross-links in plasmid DNA was increased by 2-3 fold. These findings suggest that (i) cis-DDP 1,2-intrastrand diadducts are poorly repaired by human cell extracts in vitro, (ii) among other minor lesions potentially cyanide-resistant, cis-DDP interstrand cross-links represent a major lesion contributing to the repair synthesis signal in the in vitro assay. These results could account for the drug efficiency in vivo.     

Use of an autonomous parvovirus vector for selective transfer of a foreign gene into transformed human cells of different tissue origins and its expression therein.

In this work, we report the transduction of a chloramphenicol acetyltransferase (CAT) reporter gene into a variety of normal and transformed human cells of various tissue origins. The vector used was MVM/P38cat, a recombinant of the prototype strain of the autonomous parvovirus minute virus of mice (MVMp). The CAT gene was inserted into the capsid-encoding region of the infectious molecular clone of MVMp genome, under the control of the MVM P38 promoter. When used to transfect permissive cells, the MVM/P38cat DNA was efficiently replicated and expressed the foreign CAT gene at high levels. By cotransfecting with a helper plasmid expressing the capsid proteins, it was possible to produce mixed virus stocks containing MVM/P38cat infectious particles and variable amounts of recombinant MVM. MVM/P38cat viral particles were successfully used to transfer the CAT gene and to express it in a variety of human cells. Both viral DNA replication and P38-driven CAT expression were achieved in fibroblasts, epithelial cells, T lymphocytes, and macrophages in a transformation-dependent way, but with an efficiency depending on the cell type. In transformed B lymphocytes, however, the vector was not replicated, nor did it express the CAT gene.     

Inhibition of transient gene expression in Chinese hamster ovary cells by cyclobutane dimers and (6-4) photoproducts in transfected ultraviolet-irradiated plasmid DNA

Using a transient gene expression assay to measure host cell reactivation, the effects of cyclobutane dimer and noncyclobutane dimer uv photoproducts on expression of a reporter gene were examined in normal and repair-deficient Chinese hamster ovary (CHO) cell lines. Ultraviolet damage in plasmid pRSV beta gal DNA, containing the Escherichia coli beta-galactosidase gene, resulted in reduced reporter gene expression in both uv-hypersensitive mutant CHO cell lines UV5 and UV61 relative to wild-type, parental AA8 cells. However, the effects of uv irradiation of transfected plasmid DNA on gene activity were reduced in UV61, a mutant with normal (6-4) photoproduct repair, compared to UV5, which is deficient in (6-4) photoproduct repair; this reduction correlated with the intermediate uv-hypersensitivity of UV61. Selective removal of cyclobutane dimers by in vitro photoreactivation of uv-irradiated plasmid DNA prior to transfection substantially increased reporter gene activity in both uv-hypersensitive mutant cell lines. This increase was significantly greater in UV61 than in UV5, consistent with UV5 being deficient in repair of both (6-4) photoproducts and cyclobutane dimers. These results suggest that unrepaired (6-4) photoproducts in transfected pRSV beta gal plasmid DNA are responsible for a significant fraction of the reduction in transient gene expression observed in recipient uv-hypersensitive CHO cell mutants.