ABSTRACT: To determine the ability of UCN-01 to abrogate the cell cycle arrest induced by camptothecin (CPT) in tumor cells that lack p53 function, and therefore enhance the cytotoxicity of CPT in these cells in relation to normal cells with wild-type p53.
The responses of MDA-MB-231 and GI 101A breast cancer cells were compared to those of normal bovine endothelial cells. Cytotoxicity was assessed by the MTT assay, and the resulting data were modeled using median-effect analysis. Inhibition of DNA synthesis was determined by loss of [(3)H]thymidine incorporation, and cell cycle status was determined by flow cytometric analysis of propidium-iodide-stained nuclei.
UCN-01, a specific inhibitor of protein kinase C (PKC) presently in clinical trials, abrogated CPT-induced activation of S and G(2) checkpoints in human MDA-MB-231 and GI 101A breast carcinoma cells, both of which are mutants for the p53 gene. This abrogation occurred with the use of sublethal doses (100 nM) of UCN-01 and correlated with the enhancement of CPT-induced cytotoxicity. Median-effect analysis showed that synergistic cytotoxic interactions existed between CPT and UCN-01 against these tumor cells. In normal cells, however, abrogation of the S phase arrest caused accumulation in G(0)/G(1) phase, perhaps by the presence of wild-type p53 activity, with no change in CPT-induced cytotoxicity.
We have shown previously that the cytotoxicity of CPT is correlated with cell cycle response in normal and tumor cells. Low doses of CPT arrest cells in the G(2)/M phase and inhibit DNA synthesis, but higher doses cause arrest of cells in S phase. Thus modulation of events at the S and G(2) checkpoints may provide an opportunity to enhance CPT-induced cytotoxicity in tumor cells. The results of this study indicate that UCN-01 enhances the progression of tumor cells through S phase thus greatly increasing CPT-induced cytotoxicity. Normal cells, however, are able to arrest in G(0)/G(1) and thus avoid the increased toxicity induced by CPT. Our findings suggest potential usefulness of combining UCN-01 in topoisomerase I inhibitor-based drug therapy for the treatment of breast cancer with a dysfunctional p53 gene.
Cancer Chemotherapy and Pharmacology 02/2000; 45(3):252-8. · 2.83 Impact Factor
ABSTRACT: To assess parameters that might determine resistance to the topoisomerase I inhibitor, camptothecin (CPT), the sensitivities of three established human breast cancer cell lines (ER-) and of normal bovine endothelial cells to CPT in the free form and incorporated into liposomes (LCPT), were contrasted with topoisomerase I (topo I) content and activity, and with cell cycle response to CPT treatment.
Drug sensitivities were determined using the tetrazolium dye assay and by 3H-thymidine incorporation. Topo I levels were determined by Western blot analysis, and catalytic activity was determined with a plasmid relaxation assay, using nuclear protein from each cell line. CPT stabilization of cleavable complexes in nuclear extracts was determined using a labeled oligonucleotide with a specific topo I cleavage site. Cell cycle response to CPT was determined by flow cytometric analysis of propidium iodide-stained nuclei.
CPT was extremely potent against MDA-MB-157 cells with an IC50 value of 7 nM compared with IC50 values of 150 nM for GI 101A and 250 nM for MDA-MB-231 cells. In contrast, CPT inhibited the incorporation of 3H-thymidine at very low doses in GI 101A and MDA-MB-231 cells with IC50 values of 9 nM and 5 nM, respectively; while MDA-MB-157 cells did not stop incorporating 3H-thymidine until very high doses (500 nM) of CPT were used. When incorporated into multilamellar liposomes (LCPT), CPT retained its potency, with IC50 values similar to that of the free drug. No correlation was found between CPT-induced cytotoxicity and any of the topo I parameters determined. Cell cycle analysis, however, showed an accumulation of cells in G2/M phase after 24 h treatment with low doses (5 nM) of CPT in only GI 101A and MDA-MB-231 cells with no arrest in normal endothelial or MDA-MB-157 cells. At higher doses (50 nM), however, a dramatic accumulation of cells in the S phase was observed in MDA-MB-157, MDA-MB-231 and GI 101A cells. In contrast, a G2/M phase block was seen with the normal bovine endothelial cells using the higher doses of CPT.
The results suggest that cell cycle regulation plays an important role in determining the effect of CPT on malignant and normal cells. The possible mechanisms explaining the sensitivities of the two cellular compartments to the action of CPT are discussed.
Cancer Chemotherapy and Pharmacology 02/1997; 40(6):475-83. · 2.83 Impact Factor
ABSTRACT: We have reported earlier that camptothecin (CPT) incorporated into multilamelar liposomes of appropriate lipid composition displayed effective anti-tumor activity with minimal host toxicity in a nude mouse model xenographed with the human breast carcinoma Clouser nut 1. To investigate this observation further, we have determined the differential effects of CPT on the Clouser tumor cells as well as normal vascular (BVEC) endothelial cells in culture. We report here that Clouser cells are approximately 200-fold more sensitive to CPT (IC50 = 4.0 nM) than the normal endothelial cells (IC50 approximately 1 microM) as assayed by MTT; however, CPT demonstrates a potent anti-proliferative activity on both cell lines at low drug concentrations as measured by [3H]thymidine uptake. At higher concentrations (> 25.0 nM), however, the Clouser cells maintained a higher percentage of cells capable of incorporating [3H]thymidine. No significant differences in the levels of topoisomerase 1 protein and in vitro enzymatic activity were seen; although, the Clouser cells showed a 2-fold greater incidence of cleavable complex formation by CPT in vivo. Based on the data presented here, we propose that the selective cytotoxic activity of CPT towards tumor cells may be a function of the tumor cells' reduced ability to prevent cleavable complex formation. We also propose that the antitumor effect of CPT may be enhanced in vivo by its anti-proliferative effect on vascular endothelial cells which are normally solicited to promote tumor growth.
Anti-Cancer Drugs 11/1996; 7(8):851-7. · 2.41 Impact Factor