Karen R. Grotzinger

Augmentation of adriamycin, melphalan, and cisplatin cytotoxicity in drug-resistant and -sensitive human ovarian carcinoma cell lines by buthionine sulfoximine mediated glutathione depletion

Abstract The development of acquired resistance to antineoplastic drugs and the associated broad cross-resistance to other agents frequently limits the effectiveness of chemotherapy. Ling and coworkers have demonstrated that Chinese hamster ovary (CHO) cells develop the phenotype of pleiotropic drug resistance which is manifest by a decrease in drug accumulation in these cells and hence a decrease in cytotoxicity (1). The role of drug accumulation and membrane glycoproteins in the expression of primary resistance and cross-resistance in human tumors is an area of active investigation (2–4). We have developed a series of human ovarian cancer cell lines with acquired resistance to melphalan, cisplatin, or adriamycin (5). These cell lines exhibit sensitivity/resistance profiles characteristic of pleiotropic drug resistance. In addition, the melphalan and cisplatin resistant variants are also cross-resistant to irradiation (6). Both the primary resistance to melphalan and the cross-resistance to irradiation in these cell lines can be reversed by lowering glutathione (GSH) levels in the cells with buthionine sulfoximine (BSO) (6,7). In the present study, the role of GSH in the expression of sensitivity to agents other than melphalan was examined by BSO-mediated depletion of GSH. In addition, the patterns of both primary resistance and cross-resistance were compared following GSH depletion in these cell lines.

Adriamycin accumulation and metabolism in adriamycin-sensitive and -resistant human ovarian cancer cell lines

Adriamycin accumulation and metabolism were studied in three distinct groups of human ovarian cancer cell lines: those derived from previously untreated patients, those from clinically refractory (relapsed) patients, and those with induced resistance to adriamycin in vitro. The 2-hr [14C] adriamycin accumulation in cell lines from previously untreated patients (A2780 and A1847 [Eva et al., Nature, Lond. 295, 116 (1982)] and OVCAR-5 [National Institutes of Health human OVarian CAR-cinoma cell line no. 5]) was 11-14 ng/10(6) cells. 2780AD and 1847AD (variants with in vitro induced resistance to adriamycin) accumulated one-third as much adriamycin after 2 hr (4 ng/10(6) cells). However, three cell lines derived from clinically refractory patients accumulated the same amount of adriamycin as cell lines from untreated patients (8-13 ng/10(6) cells). A high-performance liquid chromatography (HPLC) assay for adriamycin and its analogs confirmed these results and demonstrated only parent drug (no metabolites) in any of the cell lines tested. These results demonstrate that the primary mechanism of adriamycin resistance in some ovarian cancer cells from clinically refractory patients is not enhanced metabolism of drug or a transport defect leading to a decreased net accumulation such as has been described for cells with in vitro induced resistance to adriamycin.

Modulation of induced resistance to adriamycin in two human breast cancer cell lines with tamoxifen or perhexiline maleate

SummaryThe clinical utility of adriamycin in the treatment of patients with metastatic breast cancer is oftenlimited by the development of drug resistance. It has been recognized that in addition to the development of primary resistance against adriamycin, malignant cells can simultaneously develop cross-resistance to other agents. An adriamycin-resistant human breast cancer cell line (MCF 7Ad) was developed by exposing the parent line (MCF 7) to gradually increasing concentrations of adriamycin while the cells were being grown in monolayer. Using these lines in a clonogenic assay, the relative drug sensitivities to adriamycin, vinblastine, melphalan, 5-fluorouracil and methotrexate were studied. MCF 7Ad was 12.5-fold more resistant to adriamycin than MCF 7 and 500-fold cross-resistant to vinblastine. There was no cross-resistance to melphalan, 5-fluorouracil or methotrexate. The resistance of MCF 7Ad was decreased by simultaneous exposure to tamoxifen (by a factor of 3.33) or perhexiline maleate (by a factor of 7.50). This decreased resistance was evidenced by a shift to the left of the sensitivity curves. However, there was no consistent change in the sensitivity curves of MCF 7. At the selected concentration of tamoxifen and perhexiline maleate, the cloning efficiency of MCF 7 and MCF 7Ad was 80%–90% of control values in medium without tamoxifen, perhexiline maleate or cytotoxic drugs. The resistance of MCF 7Ad to adriamycin was associated with a lower accumulation of [14C]adriamycin than exhibited by the sensitive MCF 7 line. There was no consistent change in [14C]adriamycin accumulation in MCF 7 or MCF 7Ad when tamoxifen was added, but when perhexiline maleate was added the [14C] accumulation increased. These results suggest that the tamoxifen-induced change in MCF 7Ad adriamycin resistance was not due to an increase in the amount of cell-associated adriamycin, but rather to some other mechanism that increased the cytotoxicity of the adriamycin.

Preclinical evaluation of aclacinomycin A for the intraperitoneal treatment of human ovarian carcinoma

SummaryCombination chemotherapy regimens have produced a pathological complete response rate of only 1%–25% in patients with advanced ovarian cancer. Patients with small-volume residual disease after treatment are refractory to further systemic therapy, and most eventually die of their disease. Intraperitoneal (i.p.) chemotherapy, particularly with adriamycin or cisplatin has shown promise in these patients. However, the dose-limiting painful peritonitis associated with i.p. adriamycin makes this regimen potentially too toxic for many patients. Aclacinomycin A, another anthracycline antibiotic, has been found to have activity against a wide variety of murine tumors and human xenografts. It has also demonstrated clinical efficacy in phase I and II trials against refractory ovarian cancer and has less pronounced vesicant properties than adriamycin, making it an ideal candidate for i.p. use in ovarian cancer patients. In vitro clonogenic assays utilizing a battery of adriamycin-sensitive and-resistant human ovarian carcinoma cell lines have shown that aclacinomycin a is more cytotoxic than adriamycin in all cell lines tested. In addition, aclacinomycin A was found to prolong survival in a nude mouse xenograft of i.p. human ovarian cancer. These results have provided the experimental rationale for an ongoing clinical trial of i.p. aclacinomycin in refractory ovarian cancer patients at the Medicine Branch, NCI.

ALTERATIONS IN 3H‐THYMIDINE INCORPORATION INTO DNA INDUCED BY METHYL CCNU [1‐(2‐CHLOROETHYL)‐3‐(4‐METHYL CYCLOHEXYL)‐1‐NITROSOUREA] IN NORMAL AND TUMOROUS TISSUES IN VIVO

Methyl CCNU produces a suppression of tritiated thymidine (3H‐TdR) incorporation into DNA in vivo in normal bone marrow and gastrointestinal tissues which is different in magnitude and duration from that seen in L1210 ascites tumor in the same animals. This suppression and recovery pattern is not seen in animals bearing L1210 ascites tumor resistant to MeCCNU. Where a different pattern of recovery is seen between normal host target tissues and tumor, the pattern can be exploited to increase the cure rate of animals bearing advanced L1210 ascites tumor with properly spaced second doses of MeCCNU. Additional information on the potential toxicity of second doses of MeCCNU can be predicted from knowledge of the time of recovery of DNA synthesis in the normal host target tissues.

Alterations in [3h]thymidine incorporation into dna and [3h]uridine incorporation into rna induced by 5-azacytidine in vivo.

Abstract Administration in vivo of 5-azacytidine (5-aza-CR) caused suppression of [3H]thymidine ([3H]TdR) incorporation into DNA of bone marrow and gastrointestinal mucosa of mice and a more prolonged suppression of L1210 ascites tumor. Single doses of 5-aza-CR caused a modest and short-lived suppression of incorporation of [3H]uridine ([3H]UR) into nuclear RNA of L1210 ascites tumor cells. No suppression of [3H]UR incorporation into RNA of bone marrow or gastrointestinal mucosa was observed. L1210 tumor cells resistant to the other active cytidine analogue, cytosine arabinoside, demonstrated less disruption of [3H]TdR incorporation after exposure to 5-aza-CR, suggesting some cross resistance in the effects of these two drugs on DNA synthesis. Survival studies carried out in mice bearing both the sensitive and resistant L1210 tumor cell lines confirmed cross resistance of the anti-tumor effects of the two cytidine analogues. Second doses of 5-aza-CR, with the timing og administration based upon the differing patterns of recovery of [3H]TdR incorporation between normal tissues and tumor cells, led to a prolongation of survival in mice bearing the sensitive L1210 ascites tumor.