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.

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.

Spontaneous transformation of rat ovarian surface epithelial cells results in well to poorly differentiated tumors with a parallel range of cytogenetic complexity.

We previously used rat ovarian surface epithelial cells subjected to repetitious growth in vitro to provide experimental evidence in support of a role for incessant ovulation in the etiology of ovarian cancer. We have now initiated a series of 30 independent rat ovarian surface epithelial cell lines. This report describes findings in eight of the cell lines that, based on tumor formation in athymic mice, have undergone malignant transformation. Each of the tumors exhibited chromosomal alterations. Two well to moderately differential tumors had only one or two cytogenetic changes, and they had in common the presence of numerical gains. Each of five poorly differentiated tumors had complex karyotypes with three to eight clonal aberrations, prominent among them being unbalanced rearrangements and numerical losses. Several poorly differentiated tumors also had marker chromosomes, double minutes, or homogeneously staining regions. These findings demonstrate that the malignant tumors produced by spontaneously transformed rat ovarian surface epithelial cell lines range in degree of differentiation, which is paralleled by the cytogenetic complexity. Thus, this model system may fill an important void in future efforts to define the genetic basis of common epithelial tumors of the ovary and many features characteristic of these neoplasms.

Role of platinum-DNA adduct formation and removal in cisplatin resistance in human ovarian cancer cell lines.

A series of cisplatin-resistant cell lines were used to examine the formation and removal of platinum-DNA adducts from the overall genome and the formation and removal of cisplatin-interstrand cross-links from specific genomic regions. Cisplatin accumulation and DNA platination levels, which correlated linearly, were similar in three of the resistant cell lines despite differences in their primary cisplatin resistance. Increased platinum removal from total genomic DNA was found to be associated with increased resistance. Interstrand cross-link levels were found to be 2- to 4-fold lower in the 28S ribosomal RNA gene and a non-coding genomic region of the resistant cell lines as compared with the parental A2780 cell line. In addition, 1.2- to 2.7-fold more cross-links were formed in the non-coding region than in the ribosomal RNA gene in all of the cell lines. Interstrand cross-links were removed more rapidly from both regions of the highly cisplatin-resistant C80 and C200 cells and from the ribosomal RNA gene only in the cell lines of lower resistance. The results support a role for DNA repair and alterations in interstrand cross-link formation in cisplatin resistance and provide evidence for heterogeneous interstrand cross-link formation in the genome.

Transformation of rat ovarian epithelial and Rat-1 fibroblast cell lines by RAST24 does not influence cisplatin sensitivity.

Recent reports suggest that expression of an activated c-Ha-ras oncogene is associated with cisplatin resistance in NIH-3T3 fibroblasts. To investigate the generality of these observations, cisplatin cytotoxicity was determined in a series of clonal Rat-1 fibroblast and rat ovarian surface epithelial (ROSE) cell lines carrying a zinc-inducible metallothionein-RAST24 fusion gene, MTRAST24. Cisplatin sensitivity in RAS-transformed fibroblast sublines did not differ from parental controls. Induction of mutant RAST24 expression by zinc sulfate did not affect the cisplatin sensitivity of individual cell lines. Expression of mutant p21Ha-RAS varied more than 40-fold in these fibroblast sublines. Similarly, there was no difference in cisplatin sensitivity between parental ROSE controls, neomycin phosphotransferase transfected controls, or MTRAST24 transfectants. Finally, the cisplatin sensitivity of RAS-transformed ROSE cells was similar to that of spontaneously transformed ROSE cells. Overall, these observations suggest that there is little relationship between mutant ras expression and cisplatin sensitivity in rat epithelial and fibroblast cell lines.

A comparison of clonogenic, microtetrazolium and sulforhodamine B assays for determination of cisplatin cytotoxicity in human ovarian carcinoma cell lines.

An assay based upon quantitative staining of cellular protein by sulforhodamine B (SRB) has recently been adopted by the NCI for large-scale screening of new drugs. However, there are few data available regarding whether the SRB assay is comparable to other established methods. Cisplatin cytotoxicity was determined in 16 human ovarian carcinoma cell lines by both SRB and clonogenic assays, and by microtetrazolium (MTT) assay in seven cell lines. Cell lines were derived from untreated patients (some of which were selected for cisplatin resistance in vitro) and from patients clinically refractory to cisplatin-based chemotherapy. There was excellent linear correlation between SRB staining and cell number in all cell lines (r = 0.972-0.999). IC50 values obtained by the SRB and clonogenic assay (r = 0.824, P = 0.000022) were highly correlated, although values obtained in the SRB assay were uniformly higher. IC50 values obtained by SRB assay also correlated well with results obtained by MTT assay (r = 0.906, P = 0.0010). Overall, the SRB assay permitted rapid and reliable assessment of cisplatin sensitivity in these cell lines and compared favourably with clonogenic and MTT assays.

Potentiation of cisplatin cytotoxicity in human ovarian carcinoma cell lines by trifluoperazine, a calmodulin inhibitor.

Chemotherapy for ovarian cancer is frequently limited by cisplatin (CDDP) resistance. Enhanced DNA repair is one of several mechanisms which may cooperate to produce resistance in human ovarian carcinoma cell lines. Published reports suggest that calmodulin inhibitors, such as trifluoperazine (TFP), may inhibit one or more steps in DNA repair. The effects of TFP alone or in combination with CDDP were determined by clonogenic assay of six human ovarian carcinoma cell lines, derived from untreated patients (some of which were selected for cisplatin resistance in vitro) and from patients clinically refractory to cisplatin-based chemotherapy. TFP produced dose-dependent cytotoxicity in all cell lines. In addition, TFP (10 microM) produced approximately two-fold enhancement of CDDP cytotoxicity in three of the six cell lines (A2780, 2780-CP8, and 2780-C30). TFP and CDDP had additive or synergistic cytotoxicity in four of the six cell lines by median effects analysis, while clear antagonism was apparent in the remaining cell lines. These results suggest that TFP may enhance CDDP cytotoxicity in some, but not all, human ovarian carcinoma cell lines. The potential utility of trifluoperazine in ovarian cancer, either alone or in combination with cisplatin, remains to be defined in xenograft models and in clinical trials.

Spontaneous transformation of rat ovarian surface epithelial cells: association with cytogenetic changes and implications of repeated ovulation in the etiology of ovarian cancer.

BACKGROUND: Ovarian surface epithelial cells undergo several rounds of division to repair the wound created by follicular rupture at the time of ovulation. This cyclical requirement for cell division, when not interrupted by the long anovulatory rest periods that occur during pregnancy and lactation, may contribute to the development of ovarian cancer. PURPOSE AND METHODS: To test this hypothesis, we isolated rat ovarian surface epithelial cells from 10 adult female Fisher rats, initiated two mixed-population and seven clonal cell lines, and repeatedly subcultured these cells in vitro for more than 20 passages. We then tested them for the acquisition of the following four features associated with transformation: 1) the loss of contact inhibition, 2) the capacity for substrate-independent growth, 3) the ability to form tumors when injected subcutaneously and/or intraperitoneally into athymic mice, and 4) cytogenetic abnormalities. RESULTS: Loss of contact inhibition was observed in all nine late-passage cell lines. Six of the nine late-passage, but none of the early-passage, cell lines tested exhibited a capacity for substrate-independent growth that was augmented in a dose-dependent manner by epidermal growth factor. Two late-passage cell lines (clone 2 and mixed-population 2) generated tumors in athymic BALB/c mice within 3 weeks following subcutaneous injection of 5 x 10(6) cells, whereas similar numbers of early-passage cells from the same cell lines failed to generate palpable tumors. Late-passage clone 7 cells were tumorigenic when 5 x 10(7) cells were injected intraperitoneally. Two of the cell lines analyzed exhibited alterations involving losses of part or all of one member of the chromosome 5 pair. Clone 2 possessed an interstitial deletion, del(5)(q21.3q24), consistent with the loss of an uncloned putative tumor suppressor gene at 5q22q23 previously reported to reside near the loci for the interferon alpha, interferon beta, and c-jun genes. Early-passage clone 7 cells exhibited chromosome 5 monosomy, while late-passage cells contained one normal chromosome 5 and a derivative (5q12q). Southern analysis of the three cell lines revealed no consistent loss of loci for the interferon and c-jun genes, although early-passage clone 7 cells had one half the gene copy number for the interferon beta and c-jun genes and both early- and late-passage clone 7 cells lacked DNA sequences hybridizing with the probe for interferon alpha. CONCLUSION: This pattern of passage-dependent spontaneous transformation of rat ovarian surface epithelial cells in vitro supports the hypothesis that repetitious ovulation contributes to the etiology of human ovarian cancer.

In vitro interactions between platinum analogues in human ovarian-carcinoma cell lines.

In vitro and clinical data suggest that cisplatin and carboplatin resistance may be overcome in some cases by dose escalation, although clinical toxicities limit this approach. Administration of platinum analogues in combination is an alternative dose-intensification strategy that has been little studied. The cytotoxicities of cisplatin (CDDP), carboplatin (CBDCA), and tetraplatin (TP, ormaplatin) alone and in combination were assayed by inhibition of the clonogenic survival of human ovarian-carcinoma cell lines (a) from an untreated patient (A2780), (b) selected for CDDP resistance in vitro (2780-CP70), and (c) from patients presenting with clinically refractory disease (OVCAR3, OVCAR10). The sensitivity patterns of these cell lines to platinum analogues were consistent with the existence of at least two platinum-resistance phenotypes - one being moderately resistant to CDDP and CBDCA but highly resistant to TP and the other being highly resistant to CDDP and CBDCA but only partially cross-resistant with TP. Effects of drug combinations were determined by median-effect analysis. Interactions between platinum analogues were variable in different cell lines. Synergistic cytotoxicity was apparent for the CDDP-CBDCA combination in the A2780 and OVCAR-3 cell lines and for the CDDP-TP combination in 2780-CP70 and OVCAR-3. Strong antagonistic effects were seen for CBDCA-TP in 2780-CP70. Platinum analogues showed additive effects in the remaining cell lines. These data suggest that there may be distinct sensitivity phenotypes for platinum-analogue combinations. The demonstration of in vitro synergy between platinum analogues supports their combined clinical use.

Comparative cytotoxicity of CI-973, cisplatin, carboplatin and tetraplatin in human ovarian carcinoma cell lines.

The clinical efficacy of cisplatin-based chemotherapy for ovarian cancer is frequently compromised by drug resistance or dose-limiting renal and neurologic toxicities. CI-973 (NK-121), a 2-methyl-1,4-butanediamine analogue of carboplatin, has shown little nephro- and neuro-toxicity in pre-clinical model systems and in phase-I trials. Its in vitro spectrum of activity against ovarian cancer cell lines has not been previously characterized. The in vitro activities of CI-973, cisplatin, carboplatin and tetraplatin were compared in several platinum-sensitive and -resistant human ovarian carcinoma cell lines. Cytotoxicity was assessed by inhibition of clonogenic survival in soft agar with continuous drug exposure. On a molar basis, cisplatin and tetraplatin were the most potent analogues, while carboplatin was consistently less potent. Cisplatin, carboplatin and CI-973 elicited a very similar response pattern by Spearman rank correlation, distinct from that seen with tetraplatin. The magnitude of resistance to CI-973 was comparable to cisplatin in 5 cell lines but was substantially lower in the highly cisplatin-resistant 2780-CP70 and OVCAR-10 cell lines. These results suggest that CI-973 and tetraplatin may have potential utility in some cases of cisplatin-resistant ovarian cancer. In addition, our data are consistent with the existence of at least 2 platinum-resistance phenotypes--one with moderate levels of resistance to cisplatin, carboplatin and CI-973 but highly resistant to tetraplatin, the other highly resistant to cisplatin and carboplatin but only partially cross-resistant with tetraplatin and CI-973. The recognition of different resistance phenotypes may facilitate the study of cellular resistance mechanisms to cisplatin and newer platinum analogues.

Metallothionein gene expression and resistance to cisplatin in human ovarian cancer.

Intracellular thiols have been proposed as mediators of resistance to alkylating agents and cisplatin. As metallothionein is the predominant protein thiol, we examined its relationship to cisplatin resistance in human ovarian cancer cell lines. A human ovarian carcinoma cell line, A2780, derived from an untreated patient, was treated with cisplatin in several ways and the induced resistance to cisplatin ranged from 13- to 68-fold. The degree of resistance was dependent upon the method of selection. The drug-resistant cell lines also developed low levels of cross-resistance to cadmium. Additional cell lines established from untreated patients or ovarian cancer patients refractory to cisplatin- and/or carboplatin-containing combination chemotherapy were studied. The most cisplatin-resistant cell lines, OVCAR-8 and -10, were from patients previously treated with intensive chemotherapy. OVCAR-8 was relatively cross-resistant to cadmium while OVCAR-10 appeared relatively sensitive. Cell lines were examined for expression of metallothionein mRNA to evaluate the relationship between cisplatin resistance, cadmium cross-resistance and metallothionein expression. Only two of the cell lines with in vitro-induced resistance to cisplatin, 2780E80 and 2780CP70B3, had detectable metallothionein mRNA. The other cell lines selected in vitro for cisplatin resistance, as well as the parental A2780 ovarian cancer cell line, showed no expression at our level of detection. There was variable expression of metallothionein among the OVCAR cell lines. Cell lines from untreated patients, OVCAR-5 and -7, did express metallothionein, while the most cisplatin-resistant cell lines, OVCAR-8 and -10, did not. We also examined cisplatin induction of metallothionein mRNA in the cell lines. Only 2780CP70B3 among the cell lines with in vitro-induced cisplatin resistance showed increased expression after short-term exposure to cisplatin. OVCAR-4 also had a slight increase in expression after exposure to cisplatin. Mouse C127 cells transfected with a bovine papilloma virus-metallothionein gene construct were compared for cisplatin sensitivity to the same cell type transfected with bovine papilloma virus alone. In this model system, metallothionein expression did not influence cisplatin cytotoxicity. On the basis of these studies, we conclude that there is no causal relationship between metallothionein expression and cisplatin resistance.

Elevated expression of pp60c-src alters a selective morphogenetic property of epithelial cells in vitro without a mitogenic effect.

Madin-Darby canine kidney (MDCK) cells are highly differentiated and have retained the morphogenetic properties necessary to form polarized, multicellular epithelial structures (cysts) in vitro that resemble epithelial tissues in vivo. We introduced the c-src gene into MDCK cells to elevate the level of the plasma membrane-associated cellular tyrosine kinase, pp60c-src, to levels two- to ninefold higher than that expressed in parent MDCK cells. Our results revealed a highly discriminatory biological action of pp60c-src on the morphogenetic properties of MDCK cells. Elevated expression of pp60c-src conferred on MDCK cells the ability to undergo dramatic changes of cell shape that includes the formation of long cell processes (100 to 200 microns), never observed in control MDCK cells. The morphogenesis of multicellular epithelial cysts was altered by elevated levels of pp60c-src and led to predictable distortions of their three-dimensional architecture. However, these cells established morphologically normal cell polarity, formed adhesive epithelial cell-cell contacts indistinguishable from those of control MDCK cells, and exhibited neither focus-forming ability or anchorage-independent growth potential. Finally, we showed that MDCK cells expressing elevated levels of pp60c-src exhibit increased phosphorylation of a more limited number of phosphotyrosine-containing proteins than MDCK cells expressing pp60v-src. We suggest that a natural function of pp60c-src is to regulate the morphogenetic properties which determine the shape of differentiated cells and multicellular structures.