Search PubMed⌕ Search

Biomedical subjects

T C Hamilton

Publications and source records attributed to T C Hamilton.

At least 55 records · Page 3Linked to original sources
PubMed1996

Cross-resistance, cisplatin accumulation, and platinum-DNA adduct formation and removal in cisplatin-sensitive and -resistant human hepatoma cell lines.

The BEL7404 human hepatoma cell line was selected in vitro for primary cisplatin resistance. A panel of four cisplatin-resistant sublines were generated which exhibited resistance to cisplatin (up to 34-fold) but were not cross-resistant to adriamycin, taxol, etoposide, or mitomycin C. Further characterization of this panel of cell lines revealed that increased cisplatin resistance was associated with decreased cisplatin accumulation in the selected sublines (up to 14-fold) relative to the parental BEL7404 cell line. A significant reduction in platinum-DNA adduct formation (9-fold) and ribosomal RNA gene-specific interstrand crosslink formation (12-fold) were also observed in the 7404-CP20 cell line. No differences in the rate of platinum efflux from the BEL7404 and 7404-CP20 cell lines were detected following a 4-h loading period, and total platinum-DNA adduct and gene-specific interstrand crosslink removal rates were similar in both cell lines. There were approximately 3-fold more total platinum-DNA adducts present in the BEL7404 cells relative to the 7404-CP20 cells at equitoxic concentrations of cisplatin, suggesting that DNA damage tolerance also contributes to the cisplatin resistance phenotype. Overall, these results indicate that decreased cisplatin accumulation is the major cisplatin resistance mechanism present in the in vitro-selected cell lines. This model system of acquired cisplatin resistance may be valuable in determining the molecular basis for decreased cisplatin uptake and be useful for the study of potential resistance modulators.

Carcinoma, Hepatocellular↗
PubMed1996

Identification of zinc finger mRNAs using domain-specific differential display.

An oligonucleotide primer specific for a conserved amino acid region of the Cys2/His2 zinc finger proteins was used in conjunction with mRNA differential display to amplify related mRNAs from a human ovarian cancer cell line. Six of the 12 cDNAs analyzed from the differential display polyacrylamide gel exhibited zinc finger homology at the nucleotide and predicted amino acid sequence level. None of these cDNA fragments, however, shared complete homology with genes encoding any known zinc finger proteins. All 6 cDNA fragments with zinc finger homology had a poly-A tail and 3 of these fragments contained a putative polyadenylation signal. Northern blot analysis was performed using radiolabeled probes prepared from the 12 cDNA fragments. Two of the 6 cDNA fragments with zinc finger homology hybridized to 3.6- and 6.0-kb mRNAs. In addition, 2 of the fragments which did not contain significant homology to zinc finger or any other known sequences hybridized to 4.1- and 5.8-kb mRNAs. These results suggest that domain-specific differential display may be a useful approach for the identification of novel gene family members as well as for the analysis of changes in gene expression of family members between related cell lines or tissue samples.

Amino Acid Sequence↗
PubMed1996

Identification of two candidate tumor suppressor genes on chromosome 17p13.3.

A second tumor suppressor locus on 17p that is distinct from TP53 has been identified in brain, breast, lung, and ovarian tumors. Using allelic loss mapping and positional cloning methods, we have recently identified two novel genes, which we refer to as OVCA1 and OVCA2, that map to 17p13.3. The two genes are ubiquitously expressed and encode proteins of 443 and 227 amino acids, respectively, with no known functional motifs. Sequence comparison of OVCA1 and OVCA2 revealed extensive sequence identity and similarity to hypothetical proteins from Saccharomyces cerevisiae, Caenorhabditis elegans, and Rattus species. Northern blot analysis reveals that OVCA1 and OVCA2 mRNA were expressed in normal surface epithelial cells of the ovary, but the level of this transcript is significantly reduced or is undetectable in 92% (11/12) of the ovarian tumors and tumor cell lines analyzed. The location, high degree of amino acid conservation, and reduced expression in ovarian tumors and tumor cell lines suggest that decreased expression of these two genes contributes to ovarian tumorigenesis and should be considered candidate tumor suppressor genes.

Alleles↗
PubMed1996

Neuroprotective effect of recombinant neutrophil inhibitory factor in transient focal cerebral ischaemia in the rat.

The neuroprotective effects of recombinant neutrophil inhibitory factor (NIF) have been assessed with temporary (2 h) middle cerebral artery (MCA) occlusion in the rat using the intraluminal suture technique. Administration of NIF (1.5 mg/kg, i.v.) immediately after the onset of reperfusion and at 4 and 6 h post-MCA occlusion significantly reduced both hemispheric infarct volume (P < 0.001) and brain swelling (P < 0.001), and improved neurological outcome (P < 0.02) when assessed at 24 h. These results demonstrated the marked neuroprotective efficacy of NIF in a rat model of transient focal cerebral ischaemia.

Animals↗
PubMed1996

Motor dysfunction in a photothrombotic focal ischaemia model.

The study of behavioural deficits resulting from cerebral infarction in animal models of stroke has in the past taken second place to histological assessment. This is particularly true of the photothrombotic lesion model. Most tests currently used to measure motor deficits use a scoring system to quantify parameters such as beam walking. The present study set out to characterise a simple and objective assessment for motor impairment in the photothrombotic cortical lesion model. Rats were assessed on a number of motor function tests, i.e. gross locomotor activity, rotarod, and grip strength. After the establishment of stable baselines, cortical photothrombotic lesions were induced, after which the animals were re-tested for a further 18 days. The presence of cortical photothrombotic lesions significantly imparied the rats' performance on the rotarod and grip-strength tests. The deficit observed with the grip-strength task appeared 24 h postsurgery, but was much reduced by day 18 postsurgery. The rotarod test revealed an effect that took longer to establish, but which was more persistent. Gross locomotor activity was not affected. These data suggest that bilateral photothrombotic lesions of the prefrontal cortex produce deficits that can be detected by rotarod and grip-strength tasks.

Animals↗
PubMed1996

Development and characterization of a clinically useful animal model of epithelial ovarian cancer in the Fischer 344 rat.

OBJECTIVE: Our purpose was to develop and characterize a spontaneously arising, nonimmunogenic experimental animal model of epithelial ovarian cancer. STUDY DESIGN: NuTu-19 is a cell line derived from a poorly differentiated adenocarcinoma formed in a female athymic mouse after subcutaneous injection of spontaneously transformed Fischer 344 rat ovarian surface epithelial cells. This cell line was injected intraperitoneally into naive, immunocompetent Fischer 344 rats to determine tumor growth and animal survival. Immunogenicity of this cell line was determined by repetitive vaccination of naive rats with either mitomycin C-treated or irradiated (5000 cGy) NuTu-19 cells, followed by intraperitoneal rechallenge with viable tumor cells. Kaplan-Meier survival analysis was used to analyze survival data. Major histocompatibility complex class I and class II and intercellular adhesion molecule-1 cell surface antigens were determined by fluorescence-activated cell sorting analysis. RESULTS: NuTu-19 cells injected intraperitoneally grew progressively as numerous serosal nodules (peritoneum, omentum, diaphragm, liver, bowel), exhibited local tissue invasion and formed malignant ascites in a manner typical for human ovarian epithelial carcinomas. Animal survival was dosage dependent where as few as 10(4) cells were fatal when introduced intraperitoneally; mean animal survival was noted to be approximately 49 days when 10(5) cells were injected intraperitoneally. Repetitive immunizations of animals with large doses (10(7)) of inactivated NuTu-19 cells did not confer immunity to the animals, which all died on subsequent challenge with viable parental tumor cells. NuTu-19 cells expressed high levels of major histocompatibility complex class I and intercellular adhesion molecule-1 cell surface antigens and very low levels of major histocompatibility complex class II antigens. CONCLUSION: This is the first report of a reliable, spontaneously arising, nonimmunogenic epithelial ovarian cancer animal model. Because this model exists in an immunocompetent animal, it will be useful for studying the biologic and immunologic features of ovarian cancer.

Adenocarcinoma↗
PubMed1996

Phase I trial of buthionine sulfoximine in combination with melphalan in patients with cancer.

PURPOSE AND METHODS: Resistance to alkylating agents and platinum compounds is associated with elevated levels of glutathione (GSH). Depletion of GSH by buthionine sulfoximine (BSO) restores the sensitivity of resistant tumors to melphalan in vitro and in vivo. In a phase I trial, each patient received two cycles as follows: BSO alone intravenously (i.v.) every 12 hours for six doses, and 1 week later the same BSO as cycle one with melphalan (L-PAM) 15 mg/m2 i.v. 1 hour after the fifth dose. BSO doses were escalated from 1.5 to 17 g/m2 in 41 patients. RESULTS: The only toxicity attributable to BSO was grade I or II nausea/vomiting in 50% of patients. Dose-related neutropenia required an L-PAM dose reduction to 10 mg/m2 at BSO 7.5 g/m2. We measured GSH in peripheral mononuclear cells (PMN), and in tumor biopsies when available, at intervals following BSO dosing. In PMNs, GSH content decreased over 36 to 72 hours to reach a nadir on day 3; at the highest dose, recovery was delayed beyond day 7. The mean PMN GSH nadirs were approximately 10% of control at BSO doses > or = 7.5 g/m2; at 13 and 17 g/m2, all but two patients had nadir values in this range. GSH was depleted in sequential tumor biopsies to a variable extent, but with a similar time course. At BSO doses > or = 13 g/m2, tumor GSH was < or = 20% of starting values on day 3 in five of seven patients; recovery had not occurred by day 5. We measured plasma concentrations of R- and S-BSO by high-performance liquid chromatography (HPLC) in 22 patients throughout the dosing period. Total-body clearance (CLt) and volume of distribution at steady-state (Vss) for both isomers were dose-independent. The CLt of S-BSO was significantly less than that of R-BSO at all doses, but no significant differences in Vss were observed between the racemates. Harmonic mean half-lives were 1.39 hours and 1.89 hours for R-BSO and S-BSO, respectively. CONCLUSION: A biochemically appropriate dose of BSO for use on this schedule is 13 g/m2, which will be used in phase II trials to be conducted in ovarian cancer and melanoma.

Adult↗
PubMed1996

Evaluation of carboplatin pharmacokinetics in the absence and presence of paclitaxel.

In a clinical trial of paclitaxel (Taxol) and carboplatin in combination, the severity of thrombocytopenia was less than would be expected with an equivalent dose of carboplatin alone. To determine whether a pharmacokinetic interaction was responsible for this observation, the effect of pretreatment with Taxol on the pharmacokinetics of carboplatin was examined in 11 patients. Each patient was randomized to one of two treatment groups that determined the order of drug treatments. The treatments were carboplatin as a 30-min infusion alone or immediately following 175 mg/m2 Taxol administered as a 3-h i.v. infusion. The treatments were separated by 1 week. The carboplatin dose was chosen to produce a target area under the concentration-time curve (AUC) of 3.75 mg-min/ml according to a previously published formula (A. H. Calvert et al., J. Clin Oncol., 7: 1748-1756, 1989). The mean administered dose of carboplatin was 338 mg. Serial blood samples were collected over 24 h and analyzed for total and free platinum, and, in some patients, Taxol. The pharmacokinetics of carboplatin (i.e., total clearance and volume of distribution at steady state), was not significantly affected by pretreatment with Taxol. Total clearances of carboplatin were 67.2 +/- 28.8 ml/min and 64.6 +/- 27.9 ml/min in the absence and presence of Taxol, respectively (P = 0.56). The AUC of free carboplatin (3.45 mg-min/ml) obtained in the absence of Taxol was not significantly different from that measured in the presence of Taxol (3.27 mg-min/ml). The AUC of carboplatin in both the absence and presence of Taxol agreed with the projected target AUC of 3.75 mg-min/ml. In conclusion, the application of an individualized dosing strategy is valid for the calculation of the carboplatin dose in this combination. The pharmacokinetics of carboplatin is not altered by pretreatment with Taxol at a standard dose, and a pharmacokinetic interaction is not responsible for the altered toxicity of the combination.

Adult↗
PubMed1995

Time-dependent pharmacodynamic models in cancer chemotherapy: population pharmacodynamic model for glutathione depletion following modulation by buthionine sulfoximine (BSO) in a Phase I trial of melphalan and BSO.

The development of time-dependent pharmacodynamic models in cancer chemotherapy has been extremely limited. A population approach was used to develop such a model to describe the effect of buthionine sulfoximine (BSO), via its active S-isomer (S-BSO), on glutathione (GSH) depletion in peripheral mononuclear cells. The Phase I trial utilized escalating doses of BSO, from 5 to 17 gm/m2, as a multiple infusion regimen. The population model consisted of a linear 2-compartment pharmacokinetic model coupled to an indirect response model. The indirect response model consisted of a GSH compartment with input and output rate processes that are modulated as a function of S-BSO and GSH concentrations. The model predicted the observed gradual depletion of GSH, a nadir at approximately 30 h after the last dose of BSO, and a return to baseline GSH levels. On the basis of an IC50 estimate of about 1.6 microM for inhibition of gamma-glutamylcysteine synthetase, the target enzyme of BSO, the population model predicted near identical GSH concentration time profiles over the dose range studied. Time-dependent pharmacodynamic models are seen as a powerful means to design dosing regimens and to provide a mathematical platform for mechanistic based models.

Adult↗
PubMed1995

Evidence for altered regulation of gamma-glutamylcysteine synthetase gene expression among cisplatin-sensitive and cisplatin-resistant human ovarian cancer cell lines.

We have shown previously that tumor cell resistance to cisplatin is associated with elevated intracellular levels of glutathione, which is accomplished at least in part by increased expression of the heavy subunit of gamma-glutamylcysteine synthetase (gamma-GCS). To investigate the mechanism by which gamma-GCS expression is elevated, we have examined four related human ovarian cancer cell lines with increasing cisplatin resistance. Relative amounts of steady-state gamma-GCS mRNA in CP70, C30, and C200 were 4.8, 6.0, and 10.6, respectively, compared to the parental A2780 cell line, and a proportional increase in the transcriptional rate but not RNA stability was demonstrated. In contrast, no increase in mRNA for the gamma-GCS light subunit was found. To determine the mechanism of upregulation of this mRNA, we have cloned the promoter of the gene that encodes the heavy subunit of gamma-GCS. This region contains AP-1, NF-kappa B, XRE, AP-2, EpRE, CAAT, and TATA box elements upstream of the transcription initiation site and two MREs between this site and the start codon for the protein. Using gel mobility shift assays, we have found nuclear extract binding activity to the AP-1 response element to be closely associated with the level of gamma-GCS gene expression. A supershift assay showed that the AP-1 DNA-binding complexes are predominantly formed by dimers of JUN family members. Consistent with this finding, the expression of c-JUN was found to be elevated in the resistant cells. In contrast to AP-1 binding, AP-2 and NF-kappa B binding were inversely related to resistance. Furthermore, we have examined a partial revertant of the C200-resistant cells, which shows lower glutathione levels, and found decreased gamma-GCS expression associated with decreased AP-1 binding activity.

Antineoplastic Agents↗
PubMed1995

Characterization of chromosome 9 in human ovarian neoplasia identifies frequent genetic imbalance on 9q and rare alterations involving 9p, including CDKN2.

We have examined 41 forms of ovarian cancer for genetic alterations on chromosome 9 using a combination of five RFLP DNA probes and 15 simple tandem repeat polymorphisms. Genetic imbalance (i.e., loss of heterozygosity, microsatellite instability, amplification) for 1 or more informative markers on chromosome 9 was observed in 66% (27 of 41) of our tumor panel. Genetic imbalance was observed on 9q in 59% (24 of 41) of tumors informative for at least one locus. In contrast, only 13% (5 of 40) of informative tumors demonstrated a genetic alteration involving 9p. Furthermore, allelic loss on 9q was more common in late stage tumors (63%, 17 of 27) and poorly differentiated tumors (75%, 15 of 20) as compared to benign and early stage tumors (30%, 3 of 10). Evaluation of 15 tumors showing limited regions of genetic imbalance has identified 2 candidate tumor suppressor regions on 9q and 1 on 9p. Interestingly, the regions defined to 9p21-p24, 9q31, and 9q32-q34 all overlap with several known disease loci. In this aspect, the potential role of the CDKN2 gene at 9p21-p22 in ovarian carcinogenesis was assessed in an extended panel of ovarian tumors, 11 human ovarian carcinoma cell lines, and 1 cervical tumor cell line. With the use of comparative multiplex PCR, homozygous deletions were detected in 16 of 115 (14%) fresh tumors and 3 of 12 cancer cell lines. For those tumors demonstrating allelic loss for markers on 9p no somatic mutations were observed in the retained allele of CDKN2, as determined by single-strand conformation polymorphism analysis, but a mutation was observed in an additional cell line. Furthermore, CDKN2 mRNA levels were similar in the 9 cancer cell lines that retain CDKN2, as compared to normal human ovarian surface epithelial cell lines. Overall, our results suggest the potential involvement of a gene or genes on chromosome 9q and de-emphasize a significant role for the CDKN2 gene on 9p in the initiation and progression of ovarian cancer.

Adenocarcinoma, Mucinous↗
PubMed1995

Determinants of cisplatin sensitivity in normal versus spontaneously transformed rat ovarian surface epithelial cells.

The chemosensitivity of spontaneously transformed rat ovarian surface epithelial (ROSE) cell lines was compared to that of the parental cells from which they were derived. Cisplatin cytotoxicity was determined in three nontransformed (early passage) and three transformed (late passage) ROSE cell lines. Transformed cells were uniformly more sensitive to cisplatin than parental cells (1.5- to 2.6-fold) and grew more rapidly (doubling time range 15-22 hr) than parental cells (28-37 hr). Increased doubling time correlated with decreased cisplatin sensitivity (rho = 0.771). Cisplatin accumulation did not correlate with cisplatin sensitivity. Glutathione (GSH) levels were higher in two of three early passage cell lines, but the correlation between GSH and decreased cisplatin sensitivity in the overall panel of cell lines was modest (rho = 0.549). No statistically significant differences in DNA-platinum binding were observed between early and late passage cell lines. However, initial levels of DNA-bound platinum correlated with cisplatin sensitivity (rho = 0.812) in the six-cell-line panel. GSH levels were inversely correlated with cisplatin accumulation (rho = -0.829) and DNA platination (rho = -0.786). Increased cisplatin sensitivity in spontaneously transformed ROSE cell lines showed a weak, inverse relationship with GSH levels, but more strongly correlated with their increased growth kinetics and to DNA platination.

Animals↗
PubMed1995

Expression of two mucin antigens in cultured human ovarian surface epithelium: influence of a family history of ovarian cancer.

OBJECTIVE: The genetic changes in hereditary ovarian cancer syndromes suggest that the phenotype of ovarian surface epithelium in women with familial ovarian cancer might be altered. To test this hypothesis, we compared two tumor markers, CA 125 and 2G3, in cultures of overtly normal epithelium from patients with and without familial ovarian cancer. STUDY DESIGN: Surface epithelia from 18 patients with no family history of ovarian cancer, five with family histories that were insufficient to be classified as familial, and seven with strong family histories were examined by immunofluorescence, immunocytochemistry, and radioimmunoassay. The influence of cell density, morphologic features, propagation in culture, and immortalization with SV40 on the expression of the markers was investigated. RESULTS: CA 125 occurred in cells with epithelial rather than atypical morphologic features. In cultures with no family history and minor family history, CA 125 was present in up to 45% cells in passage 1 but in only < 5% cells in 14 of 16 cultures by passages 3 to 4. In contrast, nine of 10 cultures with family history retained > 5% CA 125-positive cells in passages 3 to 4. This prolonged presence of CA 125 correlated with a persisting epithelial phenotype, whereas most cells with no family history and minor family history became atypical by passage 3. Immortalization eliminated CA 125 in all three types of cells. 2G3 bound to few cells in low passage, independent of family history and morphologic features. The proportion of 2G3-expressing cells increased significantly with immortalization in all cultures, independent of family history. Ovarian carcinoma lines expressed both markers. CONCLUSION: In cultures of ovarian surface epithelium 2G3 expression increases with immortalization, whereas CA 125 is lost with immortalization but correlates with epithelial cell morphologic features and with family history. The results suggest that there may be phenotypic changes in overtly normal ovarian surface epithelium of women with family histories of ovarian cancer.

Antigens, Neoplasm↗
PubMed1995

Failure of isradipine to reduce infarct size in mouse, gerbil, and rat models of cerebral ischemia.

BACKGROUND AND PURPOSE: The dihydropyridine L-type calcium channel blocker isradipine has been reported to exhibit neuroprotective properties in some, but not all, studies performed in the rat middle cerebral artery occlusion (MCAO) model. In the present study, we examined isradipine in several other models of focal and global ischemia: rat rose bengal, mouse MCAO, and gerbil bilateral carotid artery occlusion (BCAO). For comparison, a novel calcium channel blocker, SB201823A, that we have previously shown to be neuroprotective in rat and gerbil models was also examined in the mouse. METHODS: In the gerbil BCAO model, isradipine was administered at 2.5 mg/kg i.p. as a single dose 60 minutes after ischemia (n = 10). Corresponding controls received vehicle (n = 10), and sham-operated animals received no treatment (n = 6). Locomotor activity and histological assessments were made at 4 days after ischemia. In the rat photothrombotic occlusion model, isradipine was administered at 2.5 mg/kg i.p. as a single dose 60 minutes after ischemia (n = 10), and corresponding controls (n = 10) received vehicle. Histological assessment was made at 7 days after ischemia. In the mouse MCAO model, isradipine was also administered at 2.5 mg/kg i.p. as a single dose 60 minutes after ischemia. Histological assessments were made at 1 (n = 13), 2 (n = 9), and 4 (n = 9) days after ischemia. Vehicle numbers were n = 10, n = 6, and n = 8, respectively. Isradipine and SB201823A were also examined using a combined preischemia and postischemia regimen. Isradipine was administered at 2.5 mg/kg i.p. before occlusion, 1.25 mg/kg i.p. 1 hour after occlusion, 1.25 mg/kg i.p. 2 hours after occlusion, and 2.5 mg/kg twice a day for 3 days after occlusion (n = 16). Corresponding controls received vehicle at the same time points (n = 14). SB201823A was administered 30 minutes before occlusion, 30 minutes after occlusion, and twice daily for 3 days (n = 12). Corresponding controls received vehicle (n = 9). Histological assessment was performed at 4 days after ischemia. RESULTS: When given after ischemia, isradipine failed to affect lesion volume in both the rat and mouse models. In the gerbil, locomotor hyperactivity and hippocampal cell loss were unaffected. Given before and after ischemia in the mouse, isradipine was also ineffective, whereas SB201823A produced a significant reduction in lesion volume. CONCLUSIONS: The L-type calcium channel blocker isradipine was devoid of neuroprotective activity in focal and global models of cerebral ischemia in three species of normotensive animals. These results were compared with data for the novel calcium channel blocker SB201823A, which exhibited a significant effect after pre- and postocclusion administration in the mouse model of permanent focal ischemia.

Animals↗
PubMed1995

Retroviral-like sequences specifically expressed in the rat ovary detect genetic differences between normal and transformed rat ovarian surface epithelial cells.

We have identified a repetitive DNA element in the rat genome that we demonstrate to be suitable to detect molecular genetic differences between normal and malignantly transformed rat ovarian surface epithelial cells by genome scanning. With fluorescence in situ hybridization, we show that these elements are widely distributed in the rat genome, and that a member of this family is present within a homogeneously staining chromosomal region of tumorigenic rat ovarian surface epithelial cells. The homogeneously staining chromosomal region infers the presence of an amplified DNA sequence in the tumor cells, and we provide molecular evidence for an amplicon in this DNA by genome scanning using a probe related to these elements. Sequence analysis revealed that these elements have the structural features of retroviral DNA. We show that they are transcriptionally active in the rat ovary, but not in a wide range of other normal rat tissues. In situ hybridization to ovarian tissue sections revealed that the elements are expressed in granulosa and theca interna cells and in the surface epithelial cells adjacent to preovulatory follicles. Based on this property, we refer to these retroviral-like elements as ovary-specific transcribed sequences. Partial sequence analysis of multiple members of this family and the range in transcript sizes strongly suggest that multiple members of this retrovirus-like family are transcriptionally active. We speculate that binding by one or a combination of ovary-specific transcription factors to regulatory sequences within the genomic ovary-specific transcribed sequences units contributes to the tissue specificity of their expression.

Animals↗
PubMed1995

Modulation of glutathione and related enzymes in reversal of resistance to anticancer drugs.

Eukaryotic cells have evolved several mechanisms to protect cellular constituents, especially DNA, from highly reactive molecules entering from without. The greater affinity of electrophiles for thiol groups than for hydroxyl or amine groups provides a teleologic rationale that the availability of high concentrations of thiol could be protective of these other important entities. The major intracellular nonprotein thiol is the tripeptide glutathione.

Animals↗
PubMed1994

Relationship between platinum-DNA adduct formation and removal and cisplatin cytotoxicity in cisplatin-sensitive and -resistant human ovarian cancer cells.

We examined several aspects of platinum-DNA adduct formation and repair in cisplatin-sensitive and -resistant human ovarian cancer cell lines. The formation of cisplatin-interstrand crosslinks (ICLs) was measured in five DNA sequences by renaturing agarose gel electrophoresis. There were considerable differences (up to 4-fold) in ICL levels in these DNA sequences following a 4-h incubation with cisplatin; however, the pattern of ICL formation did not depend on whether the region was transcriptionally active or gene encoding. Incubation of purified DNA with cisplatin yielded an ICL pattern with considerably less variability between the regions examined. Cisplatin ICL and total DNA platination levels were significantly higher (up to 20- and 40-fold, respectively) in cisplatin-resistant cell lines as compared to the parental, cisplatin-sensitive cell line at equivalent levels of cisplatin cytotoxicity. Under cisplatin exposure conditions which yielded similar initial levels of sequence-specific ICLs, the cisplatin-resistant cells removed up to 2.5 times more ICLs by 12-h posttreatment than the parental cell line. Increased removal of the individual platinum-deoxyribonucleosides of platinum-DNA adducts was also observed in the highly resistant C200 cell line as determined by high performance liquid chromatography separation and quantitation by atomic absorption spectrometry. These results indicate that DNA repair contributes significantly to cisplatin resistance and that increased DNA-damage tolerance may also be a component of the resistance phenotype in this model system.

Cisplatin↗