Raymond P. Perez

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.

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.

Resistance to alkylating agents and cisplatin: Insights from ovarian carcinoma model systems

The curative potential of chemotherapy for ovarian cancer is frequently not realized due to platinum and alkylating agent resistance. Mechanisms which may contribute to the resistant phenotype include alterations in drug transport, increased levels of sulfhydryl molecules (and/or related enzymes), and enhanced DNA repair. We have developed several ovarian cancer cell lines resistant to platinum compounds and alkylating agents. Increased levels of glutathione and enhanced DNA repair are major determinants of chemoresistance in these cells. Modulation of these processes with buthionine sulfoximine (BSO), aphidicolin, arc-C, etc. partially reverses in vitro resistance. Similar clinical treatment strategies are under investigation.

Antierythrocyte autoantibody formation after therapy with interleukin-2 and gamma-interferon

The cardiovascular, renal, pulmonary, and dermatologic toxicities of interleukin‐2 (IL‐2) and gamma‐interferon (IFN) are well described. However, autoimmune toxicities have only recently been noticed. The authors report the development of warm autoantibodies against erythrocytes in a patient receiving IL‐2 (3.75 × 106 cetus units/m2 intravenous bolus three times per week) and gamma‐IFN (0.1 mg/m2 subcutaneously three times per week) for metastatic renal cell carcinoma. Other potential causes of autoantibody formation, such as drugs, infection, and collagen vascular disease, were excluded. Both gamma‐IFN and IL‐2 have the potential to trigger or exacerbate autoimmunity due to either aberrant expression of restricted antigens or inhibition of normal cellular immune suppressor mechanisms.

Increased DT-diaphorase expression and cross-resistance to mitomycin C in a series of cisplatin-resistant human ovarian cancer cell lines

In a series of ovarian carcinoma cell lines selected in vitro for resistance to cisplatin by continuous exposure to increasing drug concentrations, the level of resistance is proportional to the expression of gamma-glutamylcysteine synthetase (gamma-GCS). To determine if other detoxicating genes are coordinately expressed, we measured the activity of DT-diaphorase and cytochrome P450 reductase. The specific activity of DT-diaphorase, but not that of cytochrome P450 reductase, increased with increasing resistance to cisplatin. Steady-state mRNA levels for DT-diaphorase correlated with enzyme activity and hence with cisplatin resistance. Since the activity of DT-diaphorase has been associated with sensitivity to quinones, we studied the cytotoxicity of mitomycin C under oxic conditions. Unexpectedly, resistance to mitomycin C increased proportionally with that to cisplatin (r = 0.997). Pretreatment with buthionine sulfoximine, which inhibits glutathione (GSH) synthesis, failed to sensitize either the sensitive or the resistant lines to mitomycin C. Thus, the basis for collateral resistance to mitomycin C in the cisplatin-resistant lines under oxic conditions is unrelated to overproduction of GSH. Under hypoxia, the toxicity of mitomycin C to the most sensitive (A2780) cell line was unchanged. However, the most resistant (C200) line was 2-fold more resistant to mitomycin C under hypoxic conditions. The coordinate overexpression of DT-diaphorase and gamma-GCS in the resistant cell lines is thus associated with hypoxic cell resistance, and supports the involvement of shared mechanisms of gene regulation in the observed resistant phenotype.

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

SummaryIn 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.

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.

Phase II study of amonafide in advanced and recurrent sarcoma patients.

SummaryThe activity of amonafide, a benzisoquinoline-1,3-dione, was assessed in 15 patients with advanced or recurrent sarcoma (11 previously treated). Eligible patients had ECOG performance status 0–2, and acceptable renal, hepatic and bone marrow function. Amonafide 300 mg/m2 was given intravenously over one hour daily on five consecutive days, every 3 weeks. Leukopenia and granulocytopenia were the most common and severe toxicities (grade 3 or 4 toxicity in 20% and 27% of patients, respectively). Local irritation and nausea/vomiting, the most common nonhematologic toxicities, were generally mild. No objective responses were seen, though 2 patients had brief stabilization of disease. Amonafide at this dose and schedule has no activity against advanced, recurrent sarcoma.