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Calmodulin (CaM) is involved in cellular processes that are vital to cell proliferation and viability. Elevated CaM content is seen in transformed cells. Anti-CaM compounds alone are cytotoxic to tumor cells and are synergistic with certain cancer chemotherapeutic agents. However, all known CaM antagonists are nonselective, complicating interpretation of these studies. To more rigorously analyze the relationship between CaM protein expression and the behavior of cancer cells, tumor-derived cell lines were engineered such that CaM concentration could be manipulated by overexpressing CaM RNA. A full-length rat CaM I cDNA was inserted into the mammalian expression vector pMTCB6+ so that either CaM mRNA (sense) or antisense RNA was expressed under the control of an inducible metallothionein promoter. Constructs were introduced into C6 glioblastoma cells by liposome-mediated transfection and colonies were selected in G418. Significantly fewer clones were recovered from transfections with antisense vectors compared to CaM sense RNA or control (empty) vector alone. This difference was attributed to the cytotoxic effects of antisense CaM RNA as opposed to differences in transfection efficiencies. CaM expression was analyzed at the RNA level by Northern blotting and CaM protein concentrations were quantitated by immunofluorescence. Clones were identified in which CaM protein could be increased or decreased following exposure to zinc ions. Changes in CaM mRNA preceded changes in CaM protein by several hours. Overexpression of CaM had no significant effects on the growth of C6 cells. However, reductions in CaM lead to decreased growth rates of C6 cells and lowered cell viability.

Modulators of P-glycoprotein (P-gp) are often themselves transported out of cells, thereby limiting their effectiveness. It may be possible to develop more effective modulators of multidrug resistance by designing drugs that irreversibly block the function of P-gp. Therefore, we studied the effect of the mustard derivatives of fluphenazine (FPN) and trans-flupenthixol (FPT) on P-gp function. Both fluphenazine-mustard (FPN-M) and trans-flupenthixol-mustard (FPT-M) possessed alkylating activity, as assayed using 4-(p-nitrobenzyl) pyridine. Multidrug-resistant MCF-7/AdrR cells were incubated with FPN or FPN-M, or FPT or FPT-M for 1 h, washed for varying number of times in phosphate-buffered saline (PBS), then resuspended in medium containing [3H]vinblastine (VBL), and assayed for steady-state accumulation of the drug. Washing had far less of an effect on the ability of FPN-M and FPT-M to increase VBL accumulation compared to their parent compounds. After eight washes in excess PBS, the cells initially exposed to FPN or FPT accumulated only 30% and 50% of the initially accumulated drug, whereas the FPN-M- or FPT-M-treated cells accumulated approximately 75% and 90% of the control, respectively. FPN-M and FPT-M also increased the uptake and decreased the efflux of VBL from MDR cells despite repeated washing. We also examined the effects of these modulators on sensitivity of MDR cells to cytotoxic agents. FPN-M and FPT-M sensitized MCF-7/AdrR cells to VBL and doxorubicin to a greater extent than their parent compounds. These studies point out the potential of "irreversible" P-gp modulators to produce prolonged chemosensitization.

Multidrug resistance (MDR) is the phenomenon in which cells become resistant to several classes of structurally and functionally diverse drugs after exposure to a single cytotoxic agent. One form of MDR is associated with the overexpression of a large plasma membrane phosphoglycoprotein, P-glycoprotein, which acts as an energy-requiring drug transport pump. Protein kinase C may participate in MDR through posttranslational modification of P-glycoprotein. The purpose of this study is to critically evaluate P-glycoprotein as a substrate for protein kinase C and to determine whether phosphorylation leads to changes in drug transport. Protein kinase C from rat brain phosphorylated immunoprecipitated P-glycoprotein in a manner dependent on the activation of the exogenous kinase. Phorbol 12-myristate 13-acetate (PMA) increased the phosphorylation of P-glycoprotein 6-fold and selectively decreased the accumulation of vinblastine in resistant MCF-7/AdrR cells. PMA selectively decreased the cellular association of vinblastine with MDR cells after brief periods of incubation, but only after critical concentrations of drug were achieved. The actions of PMA did not require new synthesis of P-glycoprotein. PMA had similar effects in MCF-7/BC-19, a cell line transfected with a cDNA for P-glycoprotein. Staurosporine inhibited the effects of PMA on the phosphorylation of P-glycoprotein and on the accumulation of vinblastine. These data demonstrated that immunoprecipitated P-glycoprotein can be a substrate for protein kinase C, and that phosphorylation of the transporter is associated with significant changes in drug transport.

We conducted a study evaluating the efficacy of simultaneous administration of 5-fluorouracil, mitomycin C, and X-irradiation in unresectable Stage III non-small cell lung cancer. Radiation was delivered in a split course to a total of 55 Gy. In the first course, radiation was administered daily in 250 cGy fractions for 12 treatments. Following a two week rest, an additional 10 treatments of 200 cGy fractions were delivered. Chemotherapy consisted of 10 mg/M2 of mitomycin C on day 1, and 1000 mg/M2 per day of 5-fluorouracil by continuous infusion on days 1 through 5 and 29 through 33. Twenty-one enrolled patients were evaluable. The overall response rate was 52% (95% confidence intervals 31-73%), with a median duration of response of 42 weeks. There were 5 complete responders (24%) and four of these patients remain disease-free with follow-up between 24-54 months. The median survival time for all patients was 59 weeks. For the complete responders the median survival time has not yet been reached, but is in excess of 42 months. Toxicity consisted of moderate stomatitis and myelosuppression. The sustained remissions obtained by 4 of 21 patients (18%) treated with concurrent mitomycin C, 5-fluorouracil and x-irradiation are encouraging and deserve further study.