Inhibitory effects of tumor necrosis factor-alpha and interferon-gamma on deoxyribonucleic acid and collagen synthesis by rat osteosarcoma cells (ROS 17/2.8).

Tumor necrosis factor-alpha (TNF alpha) and interferon-gamma (IFN gamma) have potent effects on bone resorption and collagen synthesis in cultured rat long bones. Since the effects of TNF alpha and IFN gamma may result from interaction with multiple cell types, we studied the effects of these cytokines on the synthesis of DNA and collagen in one cell type with osteoblast phenotype, cloned rat osteosarcoma cells (ROS 17/2.8). Recombinant human TNF alpha did not affect DNA synthesis after 48 h with concentrations of 10(-11)-10(-8) M and inhibited DNA synthesis slightly at 10(-6) M. Recombinant rat IFN gamma (5-500 U/ml) caused a dose-dependent inhibition of DNA synthesis. Coincubation with TNF alpha and IFN gamma inhibited DNA synthesis more than maximal doses of either cytokine alone. This enhanced inhibitory effect was due to the induction of a response to TNF alpha by IFN gamma, since preexposure of cells to IFN gamma for 24 h, followed by incubation with TNF alpha alone for an additional 48 h, also resulted in increased inhibition of DNA synthesis. Preexposure to TNF alpha for 24 h, followed by IFN gamma alone, did not increase the inhibition of DNA synthesis. Incubation with either IFN gamma (5-500 U/ml) or TNF alpha (10(-10)-10(-6) M) inhibited the incorporation of [3H]proline into collagen. Coincubation with intermediate concentrations of both cytokines resulted in an inhibitory effect greater than that produced by maximal concentrations of either alone. The results indicate that 1) IFN gamma and TNF alpha have direct actions on osteoblast-like cells in vitro; 2) IFN gamma modulates the DNA response to TNF alpha; and 3) the greater responses to combined cytokines than to high doses of either alone suggest that these cytokines act, at least in part, through different pathways.

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

The clinical utility of adriamycin in the treatment of patients with metastatic breast cancer is often-limited 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.

Characterization of a cis-diamminedichloroplatinum(II)-resistant human ovarian cancer cell line and its use in evaluation of platinum analogues.

Human ovarian cancer cell lines with stable cisplatin resistance have been developed by chronic exposure of the parent cisplatin-sensitive A2780 line to increasing concentrations of cisplatin. 2780CP8 (CP8 refers to this cell line's growth in medium containing 8 microM cisplatin) has several clonal cytogenetic abnormalities but lacks homogeneously staining regions or double-minute chromosomes. It has a significantly greater monolayer growth rate, cloning efficiency in agarose, and total glutathione content compared to the A2780 line, but similar activities of several glutathione-dependent enzymes. The 2780CP8 subline is 7.3-fold resistant to cisplatin compared to the A2780 line, as well as cross-resistant to irradiation and melphalan. It is not cross-resistant to Adriamycin, but this develops with increased cisplatin resistance (14-fold) obtained by further cisplatin exposure of 2780CP8. Of the cisplatin analogues tested which are of current clinical interest, carboplatin, iproplatin, and tetraplatin, only the latter is more cytotoxic than cisplatin in the A2780 and 2780CP8 lines. The 2780CP8 subline is also cross-resistant to these analogues in the relative order carboplatin greater than iproplatin greater than tetraplatin (most to least cross-resistant). Treatment of a highly cisplatin resistant cell line (2780CP70) with either melphalan or cisplatin was associated with a significant increase in [3H]thymidine incorporation into DNA in the presence of 10 mM hydroxyurea compared with the parent sensitive cell line which showed essentially no capacity to repair DNA damage by these drugs. A2780 and its cisplatin-resistant cell lines may thus be useful in studying drug resistance mechanisms, in screening new drugs for activity (especially against drug resistant tumors), and in formulating induction and salvage therapies for ovarian cancer.

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.

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

Combination 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.

Activity of tricyclic nucleoside 5'-phosphate in model systems of human ovarian cancer.

Tricyclic nucleoside 5'-phosphate (TCN-P) was evaluated in two models of human ovarian cancer. TCN-P reduced both colony number and volume in clonogenic assays employing human ovarian cancer cell lines. TCN-P cytotoxicity depended on the concentration, exposure duration and cell line studied, but not on cell line plating efficiency or growth rate in soft agarose. Comparison of experimental IC50 concentrations for 1 hour or continuous TCN-P exposure with reported clinically relevant concentrations suggests that therapeutic TCN-P levels are more likely to be achieved by continuous infusions. Cell lines and sublines with resistance to several standard chemotherapeutic agents acquired both in vivo and in vitro were at most 2.6-fold cross-resistant to TCN-P with 1 hour drug exposure. Cross-resistance was not evident with continuous TCN-P exposure. Intermittent bolus TCN-P (100 mg/kg/d X 5) was ineffective in an in vivo xenograft model of human ovarian cancer. These data suggest that TCN-P is most likely to be clinically effective against ovarian cancer, and may be non-cross-resistant with several standard agents, if administered by continuous infusion. Preclinical evaluation of new agents, such as TCN-P, in these experimental models may provide information useful in subsequent clinical trials.

Radiation survival parameters of antineoplastic drug-sensitive and -resistant human ovarian cancer cell lines and their modification by buthionine sulfoximine.

The optimum integration of chemotherapy and irradiation is of potential clinical significance in the treatment of ovarian cancer. A series of human ovarian cancer cell lines have been developed in which dose-response relationships to standard anticancer drugs have been determined, and the patterns of cross-resistance between these drugs and irradiation have been established. By stepwise incubation with drugs, sublines of A2780, a drug-sensitive cell line, have been made 100-fold, 10-fold, and 10-fold more resistant to Adriamycin (2780AD), melphalan (2780ME), and cisplatin (2780CP). Two additional cell lines, NIH:OVCAR-3nu(Ag+) and NIH:OVCAR-4(Ag+), were established from drug-refractory patients. 2780ME, 2780CP, OVCAR-3nu(Ag+), and OVCAR-4(Ag+) are all cross-resistant to irradiation, with DOS of 146, 187, 143, and 203, respectively. However, 2780AD remains sensitive to radiation, with a DO of 111, which is similar to that of A2780 (101). Glutathione (GSH) levels are elevated in 2780ME, 2780CP, OVCAR-3nu(Ag+), and OVCAR-4(Ag+) to 4.58, 6.13, 12.10, and 15.14 nmol/10(6) cells as compared to A2780, with 1.89 nmol/10(6) cells. However, the GSH level in 2780AD is only minimally higher than that in A2780 (2.94 nmol/10(6) cells). Buthionine sulfoximine, a specific inhibitor of GSH synthesis, significantly increases the radiation sensitivity of 2780ME (changing the DO from 143 to 95) and 2780CP to a lesser extent, suggesting that intracellular GSH levels may play an important role in the radiation response of certain neoplastic cells. These results suggest that the sequential use of irradiation following chemotherapy with melphalan and cisplatin may be less effective than a combined modality approach, which integrates radiation and chemotherapy prior to the development of drug resistance and cross-resistance to irradiation.

Characterization of a xenograft model of human ovarian carcinoma which produces ascites and intraabdominal carcinomatosis in mice.

We have used in vivo and in vitro procedures to select a subpopulation of cells from the human ovarian carcinoma cell line, NIH:OVCAR-3, with the capacity to grow i.p. in female nude athymic mice. After i.p. injection of these cells, animals develop metastatic spread similar to that of clinical ovarian cancer. Disease progression is characterized by the development of massive ascites, extensive invasive i.p. tumors, and pulmonary metastases. The malignant ascites cells are transplantable, manifest cytoplasmic androgen and estrogen receptors, and express the ovarian cancer associated antigen CA125 (116,000 units/ml of ascites supernatant). The cells also have the same chromosome markers which were present in the original cell line, NIH:OVCAR-3. Survival following i.p. passage of ascites is dependent on tumor cell inoculum ranging from a median survival of 39 days with 40 million cells to 84 days for 11.5 million transplanted cells. The characteristics of this unique in vivo model make it well suited for the evaluation of new drugs and novel experimental therapies in ovarian cancer. In addition, this in vivo model, together with ovarian cancer cell lines, may prove particularly useful for the study of pharmacological ways to specifically increase the cytotoxicity of anticancer agents in tumor cells while not increasing toxicity in normal tissues. The presence of hormone receptors should facilitate the experimental evaluation of hormonal therapy in ovarian cancer.

Characterization of a human ovarian carcinoma cell line (NIH:OVCAR-3) with androgen and estrogen receptors.

A cell line, NIH:OVCAR-3, has been established from the malignant ascites of a patient with progressive adenocarcinoma of the ovary after combination chemotherapy with cyclophosphamide, Adriamycin, and cisplatin. OVCAR-3 grows as a cobblestone-like monolayer with foci of multilayering, is tumorigenic in athymic mice, clones in agarose, and has an abnormal karyotype which includes a homogeneous staining region and a double minute chromosome. The cultured cells and xenografts contain cytoplasmic androgen- and estrogen-binding macromolecules with the specificity of the respective steroid hormone receptors. These components have sedimentation coefficients of 7 to 9S in low-salt sucrose-density gradients, have dissociation constants of 250 and 9.6 pM, and are present at concentrations of 30 and 28 fmol/mg cytosol protein characteristic of androgen and estrogen receptors, respectively. OVCAR-3 is resistant in vitro to clinically relevant concentrations of Adriamycin (5 X 10(-8) M), melphalan (5 X 10(-6) M), and cisplatin (5 X 10(-7) M) with survival compared to untreated controls of 43, 45, and 77%, respectively. Furthermore, there are multiple histological similarities between the patient's original tumor, the cell line, and the transplantable tumor. These data indicate that OVCAR-3 may be of use for investigations as to the significance of androgens and estrogens and the mechanisms of cytotoxic drug resistance in ovarian cancer.