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Many multidrug-resistant (MDR) cell lines overexpress the epidermal growth factor receptor (EGFR) as well as P-glycoprotein (P-gp). However, the role of the increased EGFR in P-gp-mediated drug resistance remains unclear. Since recent studies suggest that activation of phospholipase C (PLC) could increase the phosphorylation of P-gp, and activation of the EGFR would also activate PLC, we investigated whether the effect of epidermal growth factor (EGF) on the phosphorylation of P-gp was mediated through PLC. Treatment of the human MDR breast cancer cell line, MCF-7/AdrR, with EGF increased the phosphorylation of P-gp by 20-50%. The increased phosphorylation of P-gp was accompanied by stimulation of PLC activity, as measured by the production of inositol, 1,4,5-trisphosphate and diacylglycerol, products of phosphatidylinositol-4,5-bisphosphate hydrolysis. Treatment of MDR cells with EGF also had detectable effects on P-gp function. For example, following incubation of MCF-7/AdrR cells with ECF, we observed a consistent decrease in total vinblastine (VBL) accumulation. Kinetic analysis revealed this change to be due to an increase in membrane efflux. The latter was measured by the initial uptake velocity, which was inhibited by EGF. VBL uptake measured at 0-320 sec was inhibited by 20-40%, which was associated with a similar increase in VBL efflux. EGF had no effect on drug accumulation, uptake, or efflux in sensitive MCF-7 cells. These data indicate that EGF can modulate the phosphorylation and function of P-gp, and suggest that this effect may be initiated by the activation of PLC.

Drugs that interfere with the action of P-glycoprotein (P-gp), the membrane efflux pump responsible for multidrug resistance (MDR), should be valuable in the treatment of patients with drug-resistant cancer. We have used one class of drug, the phenothiazines, to study the structural features required for optimum interference with the function of P-gp. The structure-activity relationships revealed three important components including the hydrophobicity of the tricyclic ring, the length of the alkyl bridge and the charge on the terminal amino group. Trans-flupenthixol is a lead compound that conforms to these structural requirements and demonstrates significant activity as a sensitizer of MDR cell lines to drugs affected by the MDR phenotype. Based on these data, we have proposed a model for the binding of modulators to P-gp and have speculated on the structure of the drug-binding domain. We have developed pre-clinical models of MDR that may help predict clinical activity of chemo-modulators. L1210/VMDRC.06 is a murine lymphocytic leukemia line transformed by a retroviral expression vector containing a full-length cDNA for the human mdr1 gene. K562/VBL1-3 are clones of human myeloid blast cells that were transformed with the same vector. Resistance in these lines is not complicated by changes in the cellular content of glutathione or alterations in topoisomerase II. The transformed L1210 line grows in mice as a slowly proliferating non-metastatic peritoneal implant. Both MDR lines are restored to sensitivity by cyclosporin A or trans-flupenthixol, and the K562 clones are induced to differentiate by hemin. These lines should provide simple, sensitive screens for new drugs for use against cancers expressing P-gp. We have proposed a model to explain how the pumping activity of P-gp is activated in response to toxic drugs. In this schema, basal activity of P-gp is modulated through phosphorylation/dephosphorylation reactions mediated by protein kinase C (PKC) and calcium sensitive phosphatases. In response to the activation of phospholipase C by toxic drugs and the local production of 1,2-diacylglycerol, PKC is translocated to the cell membrane where it phosphorylates P-gp. Following the extrusion of drug from the cell membrane, phospholipase C activity returns to baseline, diacylglycerol is metabolized, PKC returns to the cytosol and serine/threonine phosphatases dephosphorylate P-gp returning it to the basal state.

We have characterized the antiproliferative effects of the phenothiazines, a group of antipsychotic drugs possessing a wide range of pharmacological actions. The phenothiazines inhibited both the proliferation and clonogenicity of L1210 leukemic lymphocytes. This effect was dependent on both time of exposure and concentration of drug. Clonogenicity of cells in the logarithmic phase of growth was inhibited by greater than 99% at a concentration of drug that had no effect on cells in the plateau phase of growth. Human and murine cell lines, grown either in suspension or in monolayers, were equally susceptible. Calmodulin (CaM), purified from L1210 cells by preparative polyacrylamide gel electrophoresis, had sensitivity to inhibition by phenothiazines similar to that reported for CaM prepared from brain. The order of potency was trifluoperazine greater than or equal to fluphenazine greater than chlorpromazine greater than chlorpromazine-sulfoxide. As a class, these drugs were less potent antagonists of CaM than was the bee venom polypeptide, melittin. The antiproliferative effects of phenothiazines were similar to the anticalmodulin effects. Thus, the same order of potencies was seen for both effects; the shapes of the dose-response curves were similarly steep and the effects of excess calcium on the inhibition of both were identical. These studies add pharmacological support for CaM being a potential intracellular target for the antiproliferative effect of the phenothiazines.