The optimal measure of linkage disequilibrium reduces error in association mapping of affection status.

We have developed a simple yet powerful approach for disease gene association mapping by linkage disequilibrium (LD). This method is unique because it applies a model with evolutionary theory that incorporates a parameter for the location of the causal polymorphism. The method exploits LD maps, which assign a location in LD units (LDU) for each marker. This approach is based on single marker tests within a composite likelihood framework, which avoids the heavy Bonferroni correction through multiple testing. As a proof of principle, we tested an 890 kb region flanking the CYP2D6 gene associated with poor drug-metabolizing activity in order to refine the localization of a causal mutation. Previous LD mapping studies using single markers and haplotypes have identified a 390 kb significant region associated with the poor drug-metabolizing phenotype on chromosome 22. None of the 27 Single nucleotide polymorphisms was within the gene. Using a metric LDU map, the commonest functional polymorphism within the gene was located at 14.9 kb from its true location, surrounded within a 95% confidence interval of 172 kb. The kb map had a relative efficiency of 33% compared with the LDU map. Our findings indicate that the support interval and location error are smaller than any published results. Despite the low resolution and the strong LD in the region, our results provide evidence of the substantial utility of LDU maps for disease gene association mapping. These tests are robust to large numbers of markers and are applicable to haplotypes, diplotypes, whole-genome association or candidate region studies.

Linkage disequilibrium mapping identifies a 390 kb region associated with CYP2D6 poor drug metabolising activity.

The cytochrome p450 enzyme, CYP2D6, metabolises approximately 20% of marketed drugs. CYP2D6 multiple variants are associated with altered enzyme activities. Genotyping 1018 Caucasians for CYP2D6 polymorphisms (G1846A, delT1707, delA2549 and A2935C), known to result in the recessive CYP2D6 poor drug metaboliser (PM) phenotype, identified 41 individuals with predicted PM phenotype. These 41 individuals were classified as 'cases'. Single nucleotide polymorphisms (SNPs) mapping within an 880 kb region flanking CYP2D6, were identified to evaluate potential association between genetic variation and the CYP2D6 PM phenotype. The 41 PM cases and 977 controls were genotyped and analysed for 27 SNPs. Associations were observed across a 390 kb region between 14 SNPs and the PM phenotype (P values from 6.20 x 10(-4) to 4.54 x 10(-35)). Haplotype analysis revealed more significant levels of association (P = 3.54 x 10(-56)). Strong (D' > 0.7) linkage disequilibrium (LD) between SNPs was observed across the same 390 kb region associated with the CYP2D6 phenotype. The observed phenotype:genotype association reached genome-wide levels of significance, and supports the strategy for potential application of LD mapping and whole genome association scans to pharmacogenetic studies.

Single-nucleotide polymorphism alleles in the insulin receptor gene are associated with typical migraine.

We have identified a migraine locus on chromosome 19p13.3/2 using linkage and association analysis. We isolated 48 single-nucleotide polymorphisms within the locus, of which we genotyped 24 in a Caucasian population comprising 827 unrelated cases and 765 controls. Five single-nucleotide polymorphisms within the insulin receptor gene showed significant association with migraine. This association was independently replicated in a case-control population collected separately. We used experiments with insulin receptor RNA and protein to investigate functionality for the migraine-associated single-nucleotide polymorphisms. We suggest possible functions for the insulin receptor in migraine pathogenesis.

DNA sequencing of the MHC class II region and the chromosome 6 sequencing effort at the Sanger Centre.

The human Major Histocompatibility Complex (MHC) is located on the short arm of chromosome 6 (6p21.3) and spans about 4 Mb. According to different gene families the MHC is subdivided into a class I, class II and class III region and many of its gene products are associated with the immune system and the susceptibility to various diseases. To date, we have sequenced about 40% (400 kb) of the class II region between HLA-DP and HLA-DQ and a coordinated effort to sequence the entire MHC is well underway. Analysis of the sequence revealed several novel genes and provides new insights into the molecular organisation and evolution of the MHC. All our data are publicly available via the MHC database (MHCDB) which allows rapid access, retrieval and display in the context of other MHC associated data. MHCDB is online available at (http:(/)/www.hgmp.mrc.ac.uk/) and, together with all our sequences also via anonymous ftp (ftp.icnet.uk/icrf-public).

Evolutionary dynamics of non-coding sequences within the class II region of the human MHC.

About 40% (350 kb) of the human MHC class II region has been sequenced and a coordinated effort to sequence the entire MHC is underway. In addition to the coding information (22 genes/pseudogenes), the non-coding sequences reveal novel information on the organisation and evolution of the MHC as demonstrated here by the example of a 200 kb contig that has been analysed for local and global features. In conjunction with cross-species comparisons, our results present new evidence on the structure of isochores, the evolutionary dynamics of repeat-mediated recombination and its effect on certain MHC encoded genes, and a higher than average degree of natural polymorphism that has implications for sequencing the human genome. We also report the finding of a class I-related pseudogene (HLA-ZI) in the middle of the class II region, which provides the first direct evidence for DNA exchange between these two related regions in man.

Multiple mechanisms of resistance in a series of human testicular teratoma cell lines selected for increasing resistance to etoposide.

Mechanisms of resistance to VP-16 were monitored in a series of sublines of the human testicular teratoma cell line (SuSa) derived following exposure either to fractionated X-irradiation (DXR-10) or to VP-16 using pulsed 24-hr exposures (VP10) or continuous exposure conditions (VPC2, VPC3 and VPC4). Orders of resistance expressed (ranging from 3- to 33-fold based on IC50 values derived from colony forming assays) were comparable with those likely to be encountered clinically. All of these resistant sublines showed some cross-resistance to VCR, and the 3 drug-selected sublines tested also proved cross-resistant to ADR. Resistance was not associated with modified 3H-VP-16 accumulation. However, decreased VP-16-induced SSBs were detectable in all the resistant sublines and a strong positive correlation was noted between the extent of SSB formation and VP-16 resistance by linear regression analysis. Topo II alpha protein content, as judged by Western blotting, was significantly decreased only in the sublines derived by continuous exposure to VP-16, but this was not progressive with increasing levels of resistance expressed. RNase protection assays also showed no significant differences in Topo II alpha expression in the low-level resistant DXR-10 and VP10 sublines, contrasting with the 2-fold decreases identified in the VPC2, VPC3 and VPC4 sublines. Significantly, however, mRNA levels of two alternately spliced Topo II beta mRNAs were markedly decreased (2- to 9-fold) in all the drug-selected resistant sublines. No mutations in consensus ATP-binding sequences or in the DNA-binding region of Topo II alpha were detected by single strand conformational polymorphism analysis. Significant Pgp overexpression was only identified in the most highly resistant sublines VPC3 and VPC4, which both showed 4-fold cross-resistance to VCR. Decreased 3H-VCR accumulation and partial reversal of resistance by VPM (6.6 microM) addition was also identified, consistent with a functional Pgp being overexpressed in these sublines. Modifications of Topo II expression therefore appear to precede Pgp overexpression in this series of sequentially derived VP-16 resistant sublines and to represent the predominant mechanism underlying low level (< 10-fold) resistance.

Characterisation of the unusual expression of cross resistance to cisplatin in a series of etoposide-selected resistant sublines of the SuSa testicular teratoma cell line.

Three etoposide-selected resistant sublines of the SuSa testicular teratoma cell line expressing 9-, 21- and 33-fold levels of resistance, proved increasingly cross resistant to cisplatin with levels approximating to 3-, 4- and 6-fold in sublines VPC2, VPC3 and VPC4, respectively. Cisplatin resistance was not associated with any significant modifications in levels of total glutathione or associated enzyme activities. Decreased platinum (Pt) accumulation was detected, although this did not correlate either with total platination levels judged immunochemically or with peak induction of interstrand crosslinks (ISC) determined by alkaline elution. Following exposure to cisplatin in the least resistant subline, VPC2, total platination levels were markedly decreased (3-fold) relative to those of the parental cells, whilst peak ISC levels were markedly increased (4-fold). In the most highly resistant subline, VPC4, peak levels of ISCs were even higher (9-fold), although total platination levels remained comparable with those in parental cells. Both VPC2 and VPC4 cells appeared highly proficient in removing ISCs, unlike the parental cells. However, whilst VPC2 cells appeared to share deficient removal of the intrastrand platinated lesions with parental cells, VPC4 cells proved proficient in removing specific adducts in the sequence pApG. This unusual expression of cross resistance to cisplatin in a series of etoposide-selected resistant sublines derived from an inherently repair deficient parental cell line, SuSa, therefore appears to be associated with enhanced removal of the specific intrastrand crosslinks in the sequence pApG and/or of DNA-DNA ISCs. Similar mechanisms have been implicated in two other cisplatin resistant SuSa sublines selected following in vitro exposure to the drug itself or to fractionated X-irradiation.

Differential cytotoxic effects of docetaxel in a range of mammalian tumor cell lines and certain drug resistant sublines in vitro.

The in vitro cytotoxic effects of docetaxel (Taxotere; RP56976, NSC688503) proved both time and concentration dependent. Amongst thirteen human cell lines from various tumor types, exposure to increasing concentrations of docetaxel over 24 hrs resulted in a plateau-shaped dose response curve, suggesting that increased cell kill becomes more dependent on increased exposure duration than on concentration. IC50 concentrations (reducing survival by 50%) ranged from 0.13-3.3 ng/ml, with three neuroblastoma lines proving most sensitive and three breast and two colon carcinoma lines showing least sensitivity. There was significant cross-resistance to docetaxel in the classic multidrug resistant (MDR) Chinese hamster ovarian (CHO) CHRC5 line and the human lymphoblastoid CCRF-CEMVLB1000 line, as well as in two vincristine (VCR)-selected MDR MCF-7 sublines. All four of these MDR sublines overexpress P-glycoprotein (Pgp), as did a 6-fold docetaxel-selected resistant CHO subline. As an apparent corollary, in two human teratoma lines selected for etoposide resistance and showing some cross-resistance to VCR and in two CHO sublines expressing low levels of VCR resistance, yet all proving Pgp positive, no docetaxel cross-resistance was identified. Verapamil modulated docetaxel resistance only in sublines expressing resistance to the drug and overexpressing Pgp. Four other human tumor sublines selected for resistance to 5-fluorouracil, cisplatin or teniposide, showed a lack of cross-resistance to docetaxel. Furthermore, cross-resistance to docetaxel was not apparant in four epipodophyllotoxin-selected resistant sublines with alterations in topoisomerase II, indicating its effectiveness against tumor cells expressing the topoisomerase II-related MDR phenotype. Our observation that docetaxel cross-resistance was not automatically expressed by classic MDR tumour cells appears of interest and of potential clinical relevance.

Differential effectiveness of a range of novel drug-resistance modulators, relative to verapamil, in influencing vinblastine or teniposide cytotoxicity in human lymphoblastoid CCRF-CEM sublines expressing classic or atypical multidrug resistance.

A series of five potential modulators of resistance were tested for their relative ability, as compared with verapamil, to sensitize CEM lymphoblastoid leukemia drug-resistant tumor sublines expressing either the classic or the atypical multidrug-resistance (MDR) phenotype to vinblastine or teniposide. Maximal non-cytotoxic concentrations of each modulator were tested and sensitization induces (SIs) were derived by comparing the drug concentration required to inhibit growth by 50% in their presence or absence. Like verapamil (10 microM) itself, three of the other modulators tested, namely, S9788 (4 microM), flunarizine (20 microM) and quinidine (30 microM), resulted in 2- to 3-fold sensitization of vinblastine against the parental CEM cells, and comparable effects were noted in the CEM/VM-1 cells, which were not cross-resistant to vinblastine. In contrast, cyclosporin A (0.5 microM) and B859-35 (2 microM) did not enhance vinblastine growth inhibition in these lines. However, the greatest sensitization with all the modulators was noted in the classic MDR VBL1000 cells, with SIs ranging from 40- to 350-fold, except for cyclosporin A, which proved ineffective at the concentration tested (SI, 2.6). The greatest extent of differential sensitization of these VBL1000 tumor cells occurred with quinidine or B859-35, which proved significantly more effective than verapamil alone. Combinations of modulators resulted in additive effects, with B859-35 plus cyclosporin A proving superior to B859-35 plus verapamil. In contrast, none of these compounds proved effective as a sensitizer to teniposide. The growth-inhibitory effects of this drug were not modified significantly in either the 92-fold teniposide-resistant VM-1 cells or in the parental cells. Addition of verapamil itself also failed to modulate teniposide growth inhibition in the VBL1000 cells, which express significant cross-resistance to this drug (36-fold). However, SI values of 3- to 5-fold were obtained using quinidine or B859-35. These results serve (a) to emphasise the need to monitor the effects of modulators not only on drug-resistant cells but also on their drug-sensitive counterparts so as to ensure differential sensitization such that normal sensitive tissues are not likely to be adversely influenced and (b) to highlight the observation that the extent of modulation differs depending not only on the antitumor drug used but also on the mechanism of drug resistance expressed. This in vitro model system appears to provide a useful screening system for resistance modulators and certainly could be used in attempts to identify alternative agents that may influence teniposide sensitivity in these drug-resistant sublines.

Evaluation of S9788 as a potential modulator of drug resistance against human tumour sublines expressing differing resistance mechanisms in vitro.

Significant activity has been identified using S9788, a triazineaminopiperidine derivative, as a new modulator of multi-drug resistance against a series of drug-resistant human tumour-cell lines in vitro. Maximal non-cytotoxic concentrations (i.e., those resulting in < or = 10% cytotoxicity) of S9788 or verapamil were tested in combination with vinblastine, Adriamycin or vincristine and cytotoxicity was evaluated using a clonogenic assay, or the metabolic dye reduction MTT assay, or by monitoring growth inhibition. Under these conditions, the extent of resistance modulation by verapamil and by S9788 was comparable in the various tumour cell lines tested, although a definite concentration-dependent modulation was noted with both compounds. The highest dose-modification factors were noted in the highly vinblastine-resistant classic multi-drug-resistant subline CEM/VLB100, although resistance reversal was only partial. Resistance modulation by both verapamil and S9788 was noted in 4 drug-selected resistant sublines and 4 "intrinsically" resistant human tumour cell lines, which all exhibited significant P-glycoprotein expression. In contrast, in 2 drug-resistant human tumour sublines (GLC4/ADR and CEM/VM-1) characterized by altered topoisomerase-II activity and proving to be P-glycoprotein-negative, no resistance modulation relative to parental cells was observed. These data are consistent with the proposal that resistance modulation is mediated by interaction between S9788 and P-glycoprotein and support its clinical evaluation in patients with P-glycoprotein-positive tumours.

Expression of P-glycoprotein-mediated drug resistance in CHO cells surviving a single X-ray dose of 30 Gy.

We reported previously that Chinese hamster ovary (CHO) cells surviving exposure to repeated doses of 9 Gy of X-irradiation in vitro expressed a multiple drug resistance phenotype characterized by cross-resistance to epipodophyllotoxins and to Vinca alkaloids, and by P-glycoprotein (Pgp) overexpression (Hill et al. 1990). We have now shown that exposure of these CHO cells to a single 30-Gy X-ray dose similarly resulted in the survivors expressing resistance to vincristine and to etoposide and overexpressing Pgp. In agreement with data obtained on cells which received repeated X-ray exposures, this Pgp overexpression occurred in the absence of any significant elevation of Pgp mRNA. However, the reduced ability to accumulate rhodamine 123 identified in these sublines, and the ability of verapamil to reverse this accumulation defect, implies that the Pgp which was overexpressed was functional. These findings indicate that a series of X-ray exposures is not necessary for expression of this distinctive multiple drug resistance phenotype, suggesting that this results not from a general 'stress-type' response but rather more specifically from the radiation exposure itself, with both single-dose and repeated X-irradiation selecting for similar genetic mutants.

Characterization of the P-glycoprotein over-expressing drug resistance phenotype exhibited by Chinese hamster ovary cells following their in-vitro exposure to fractionated X-irradiation.

Exposure of the Chinese hamster ovarian AuxB1 cell line in vitro to fractionated X-irradiation generated sublines designated DXR-10, which proved resistant to multiple drugs and overexpressed P-glycoprotein (Pgp), as judged by Western blotting using the C219 monoclonal antibody. Further characterization of these irradiated DXR-10 sublines has provided evidence for: (i) the expression of cross-resistance to gramacidin D, taxol, puromycin and Navelbine, but not to daunomycin or mitoxantrone; (ii) overexpression of the class I Pgp, as judged by Western blotting using the C494 monoclonal antibody; (iii) decreased accumulation of 3H-vincristine, which could be enhanced by verapamil addition; (iv) unaltered accumulation and subcellular distribution of adriamycin; (v) significantly increased rhodamine 123 accumulation in the presence of verapamil; (vi) plasma-membrane ultrastructural modifications resulting in a significantly increased surface area; (vii) numerous clonal karyotypic alterations, with abnormalities involving the long arm of chromosome 1 being consistently identified; (viii) a lack of overexpression of sorcin; (ix) increased total glutathione levels and overexpression of glutathione S-transferase pi. The fact that only certain of these features are considered characteristic of the 'classic' multidrug-resistant CHRC5 cell line supports our earlier proposal that exposure to fractionated X-irradiation results in the expression of a unique drug-resistance phenotype.

Dominant expression of multiple drug resistance after in vitro X-irradiation exposure in intraspecific Chinese hamster ovary hybrid cells.

BACKGROUND: Exposure of Chinese hamster ovary (CHO) cells to fractionated x irradiation in vitro has led to expression of a distinctive multiple-drug-resistant phenotype. This phenotype is characterized by overexpression of P-glycoprotein without an increase in P-glycoprotein messenger RNA or gene amplification; increased glutathione S-transferase (GST) activity; and resistance to vincristine, colchicine, and etoposide but not to doxorubicin. PURPOSE: To investigate whether this phenotype is dominant or recessive, we established intraspecific hybrids by fusion of x-ray-treated, drug-resistant CHO cells (DXR-10I or DXR-10II) with drug-sensitive CHO cells (E29). METHODS: Drug resistance levels were determined in the wild-type CHO cell line AuxB1, the drug-sensitive E29 line, the x-ray-pretreated lines, and the hybrid lines by colony-forming assay of cells grown in increasing concentrations of colchicine, vincristine, or doxorubicin. The hybrids were characterized by analysis of DNA content, P-glycoprotein expression by Western blotting, GST activity by use of 1-chloro-2,4-dinitrobenzene as substrate, and sensitivity to reversal of resistance to vincristine by exposure to verapamil. RESULTS: These hybrids proved resistant to colchicine (two-fold) and vincristine (five- to seven-fold) but not to doxorubicin. After the hybrids were exposed to verapamil, vincristine cytotoxicity was increased 10- to 12-fold. The hybrid lines exhibited levels of P-glycoprotein comparable to those of the unfused x-ray-treated parent cell line, suggesting that P-glycoprotein overexpression is a dominant trait in these hybrid lines. Interpretation of the role of increased GST activity in these hybrids was inconclusive because of the very high levels of GST in the drug-sensitive cell-fusion partner. CONCLUSIONS: The multiple-drug-resistant phenotype following x-ray treatment of CHO cells in vitro was dominantly expressed. Overall, these data are consistent with the hypothesis that this phenotype is a consequence of the dominant genetic alteration resulting from exposure to x irradiation. IMPLICATIONS: This work adds weight to our hypothesis that there is a biological basis for the expression of clinical drug resistance in certain patients whose tumors have been previously irradiated.

Over-expression of P-glycoprotein and glutathione S-transferase pi in MCF-7 cells selected for vincristine resistance in vitro.

This study has provided evidence that exposure of the wild-type MCF-7 human breast carcinoma cell line to the mutagen ethyl methane sulphonate (EMS), followed by selection in vincristine (VCR), resulted in a stably-resistant subline, designated VCREMS, which expressed an approximately 14-fold level of resistance to VCR. This VCREMS subline showed cross-resistance (3-fold) to adriamycin (ADR) and to etoposide (3-fold), but not to cisplatin. The addition of a non-toxic concentration of verapamil (6.6 microM) significantly enhanced VCR cytotoxicity only in the resistant subline. This resistance was associated with over-expression of P-glycoprotein (Pgp), but without a concomitant increase in Pgp mRNA or gene amplification. In addition, activities of total glutathione S-transferases (GST) and glutathione peroxidase were elevated in this resistant subline, with over-expression of the GST-pi isozyme and its associated mRNA being identified, without gene amplification. This VCR-selected resistant MCF-7 cell line therefore provides another example of a breast carcinoma subline in which there is co-ordinate over-expression of both Pgp and GST-pi, without attributing a causal relationship to either event, and extends the range of anti-tumour drugs known to elicit modifications in glutathione metabolism.

Mechanisms associated with the expression of cisplatin resistance in a human ovarian tumor cell line following exposure to fractionated X-irradiation in vitro.

Interactions between cisplatin (CDDP) and irradiation are of potential significance for the combined modality treatment of cancer. Previous data have indicated that following in vitro exposure to X-irradiation certain tumour cells expressed resistance to CDDP. To identify parameters associated with this CDDP resistance, the human ovarian carcinoma cell line SK-OV-3/P was pre-exposed to fractionated X-irradiation (total dose: 50 Gy) in vitro. The resultant subline (SK-OV-3/DKR-10) proved 2-fold resistant to CDDP, but not to acute X-irradiation. Consistent with unaltered dihydrofolate reductase and thymidylate synthase activities, SK-OV-3/DXR-10 cells were neither cross-resistant to methotrexate nor to 5-fluorouracil. Verapamil (6.6 microM) significantly (P less than 0.05) enhanced CDDP-induced cytotoxicity in the resistant DXR-10 subline, but not in the parental cells. Total glutathione levels were significantly (P less than 0.01) lower in the resistant subline and BSO pretreatment failed to influence cytotoxicity, whilst related enzyme activities were not consistently modified in the SK-OV-3/DXR-10 cells. Resistance in these cells was associated with significantly decreased cisplatin uptake (P less than 0.002). Immediately following drug exposure the total platination level of the DNA, quantitated immunochemically, was higher (P less than 0.05) in the resistant subline indicative of increased tolerance to DNA damage. After an 18 h post-treatment incubation the parental cell line appeared proficient in the removal of the intrastrand adduct Pt-AG, but deficient in removing the major adduct Pt-GG and the difunctional Pt-(GMP)2 lesion, whilst the DXR-10 resistant subline appeared proficient in removal of all four Pt-DNA adducts. DNA polymerases alpha and beta activities, however, were comparable in both cell lines. These data implicate both enhanced repair and increased tolerance of DNA damage as mechanisms of resistance to CDDP resulting from in vitro exposure of a human ovarian carcinoma cell line to fractionated X-irradiation.

Characterization of a cisplatin-resistant human ovarian carcinoma cell line expressing cross-resistance to 5-fluorouracil but collateral sensitivity to methotrexate.

In vitro exposure of the TR170 ovarian carcinoma cell line to six intermittent 24-h treatments with a 90% inhibitory concentration of cisplatin (CDDP) (0.15 micrograms/ml; 0.5 microM) resulted in a 2-fold stably resistant subline designated TR170/CP+ (B.T. Hill et al., Int. J. Cancer, 39: 219-225, 1987). Resistance to CDDP in these CP+ cells has now been associated with reduced uptake of 195mCDDP (2-fold; P less than 0.01) and decreased removal of specific Pt-DNA adducts, quantitated immunochemically, indicative of an apparent increased tolerance of CDDP-induced DNA damage. Specifically these resistant cells appeared deficient in removal of the major cis-Pt-(NH3)2d(pGpG) adduct and the difunctional cis-Pt(NH3)2d(GMP)2 lesion, showed less efficiency in removing cis-Pt(NH3)2d(pApG) adducts, but proved as proficient as the parental cell line in removing DNA-DNA interstrand cross-links. Activities of DNA polymerase-alpha and -beta were comparable in both lines, and no significant alterations in glutathione metabolism were identified. Response to acute X-irradiation was not modified in these TR170/CP+ cells, but they showed marked (10-fold) cross-resistance to 5-fluorouracil and, unusually, proved collaterally sensitive (12-fold) to methotrexate. Resistance to 5-fluorouracil was associated with significantly increased thymidylate synthase activity (P less than 0.01), but this was not reflected in altered gene expression, while increased sensitivity to methotrexate was accompanied by increased drug uptake but by unaltered activity and expression of dihydrofolate reductase. These results indicate that exposure to CDDP can result in numerous alterations, both intracellularly and at the cellular membrane, reflected in significant changes in the tumor cells' responses to the cytotoxic effects of a range of antitumor drugs. The clinical relevance of these observations remains to be established.

Expression of collateral sensitivity to cisplatin, methotrexate, and fluorouracil in a human ovarian carcinoma cell line following exposure to fractionated x-irradiation in vitro.

Examples of collateral sensitivity, even in experimental tumor systems, remain few. Preliminary data from this laboratory indicated that certain tumor cells expressed increased sensitivity to cisplatin after exposure in vitro to x-irradiation. To further clarify whether the type of fractionated radiation procedure used clinically can induce hypersensitivities to certain antitumor drugs we have pre-exposed the human ovarian carcinoma cell line JA-T/P derived from a tumor from an untreated patient to fractionated x-irradiation (total dose 50 Gy) in vitro. The resultant subline JA-T/DXR-10 expressed collateral sensitivity to cisplatin (CDDP), methotrexate (MTX) and fluorouracil (5-FU), but not to acute x-irradiation. Hypersensitivity to CDDP was associated with decreased activity of DNA polymerase beta (3.5-fold, P less than .01), but unaltered glutathione metabolism. Pre-incubation with cyclosporin A or with 3-aminobenzamide significantly enhanced (twofold, P less than .01) CDDP-induced cytotoxicity in JA-T/P cells, but not in the DXR-10 subline. Consistent with MTX hypersensitivity dihydrofolate reductase activity was significantly decreased (2.9-fold, P less than .01). Despite collateral sensitivity to 5-FU, however, thymidylate synthase activity was increased (twofold, P less than .05) suggesting alternative mechanisms for 5-FU-induced cytotoxicity in these JA-T/DXR-10 cells. These data demonstrate that DNA repair and associated reduced folate metabolism can be modified not only by drugs but also by fractionated x-irradiation.

Anomalous relationship between cisplatin sensitivity and the formation and removal of platinum-DNA adducts in two human ovarian carcinoma cell lines in vitro.

Two human ovarian tumor cell lines (SK-OV-3 and TR175), established from patients previously treated with alkylating agents, but not with cisplatin, expressed greater than 23-fold differences in cisplatin sensitivities in vitro. Cisplatin resistance in SK-OV-3 cells appeared to be associated with increased levels of glutathione and activities of glutathione reductase and glutathione peroxidase, with reduced catalase activity. No significant modification of drug uptake was noted and there was only marginally lower (16%) total platination of DNA, measured immunochemically, in these cells compared with the more sensitive TR175 cell line. SK-OV-3 cells, however, showed a significantly lower overall ability to remove drug-induced DNA damage, with an apparent inability to remove either the major DNA-DNA intrastrand cross-links in the sequence pGpG or the adducts cis-Pt(NH3)2d(GMP)2, although by alkaline elution repair of DNA-DNA interstrand cross-links was demonstrated. Significantly more of these interstrand cross-links were induced in these resistant cells. These data provide evidence for the involvement of altered glutathione metabolism and increased tolerance of certain types of drug-induced DNA damage as factors associated with the resistance phenotype of SK-OV-3 cells. Paradoxically, however, although the highly cisplatin-sensitive TR175 cells had lower glutathione levels this was not reflected in significantly higher total platination of DNA, and these cells appeared to be proficient in removing all the major platinum-DNA adducts quantitated in this study. Mechanisms responsible for this relative sensitivity to cisplatin remain to be identified.