Differential phosphorylation of sites in the linker region of P-glycoprotein by protein kinase C isozymes alpha, betaI, betaII, gamma, delta, epsilon, eta, and zeta.

To determine whether individual protein kinase C (PKC) isozymes differentially phosphorylate sites in the linker region of human P-glycoprotein (P-gp), we used a synthetic peptide substrate, PG-2, exactly corresponding to amino acid residues spanning the region 656-689 of the multidrug resistance gene (MDRI). All tested PKC isozymes phosphorylated PG-2. The maximum phosphate incorporation by calcium-dependent PKC isozymes alpha, betaI, betaII, and gamma was 3, 2, 2, and 3 mol phosphate/mol PG-2, respectively. The maximum phosphate incorporation by calcium-independent isozymes delta, epsilon, eta, and zeta was 1.5, 0.5, 1.5, and 1.5 mol phosphate/mol PG-2, respectively. Two-dimensional tryptic phosphopeptide mapping indicated differential phosphorylation of the PKC consensus sites Ser-661, Ser-667, and Ser-671 by individual isozymes, which may be functionally significant. These data suggest that differential phosphorylation by PKC isoenzymes of PKC sites within the P-gp linker region may play a role in modulating P-gp activity.

Effects of sphingosine stereoisomers on P-glycoprotein phosphorylation and vinblastine accumulation in multidrug-resistant MCF-7 cells.

To investigate the role of protein kinase C (PKC) in the regulation of multidrug resistance and P-glycoprotein (P-gp) phosphorylation, the natural isomer of sphingosine (SPH), D-erythro sphingosine (De SPH), and its three unnatural stereoisomers were synthesized. The SPH isomers showed similar potencies as inhibitors of in vitro PKC activity and phorbol binding, with IC50 values of approximately 50 microM in both assays. Treatment of multidrug-resistant MCF-7ADR cells with SPH stereoisomers increased vinblastine (VLB) accumulation up to 6-fold at 50 microM but did not alter VLB accumulation in drug-sensitive MCF-7 wild-type (WT) cells or accumulation of 5-fluorouracil in either cell line. Phorbol dibutyrate treatment of MCF-7ADR cells increased phosphorylation of P-gp, and this increase was inhibited by prior treatment with SPH stereoisomers. Treatment of MCF-7ADR cells with SPH stereoisomers decreased basal phosphorylation of the P-gp, suggesting inhibition of PKC-mediated phosphorylation of P-gp. Most drugs that are known to reverse multidrug resistance, including several PKC inhibitors, have been shown to directly interact with P-gp and inhibit drug binding. SPH stereoisomers did not inhibit specific binding of [3H] VLB to MCF-7ADR cell membranes or [3H]azidopine photoaffinity labeling of P-gp or alter P-gp ATPase activity. These results suggest that SPH isomers are not substrates of P-gp and suggest that modulation of VLB accumulation by SPH stereoisomers is associated with inhibition of PKC-mediated phosphorylation of P-gp.

Chlorpyrifos oxon interacts with the mammalian multidrug resistance protein, P-glycoprotein.

Multidrug resistance (MDR) to chemically unrelated therapeutic anticancer agents in mammalian cells is mediated by the overexpression of an ATP-dependent 150- to 180-kD membrane glycoprotein P-glycoprotein (P-gp). Although the complete physiological role of P-gp is unknown, it is proposed to function in cellular detoxification of xenobiotics. In this study, we investigated whether the organophosphorus insecticide chlorpyrifos (O,O-diethyl O-3,5,6-trichloro-2-pyridinyl phosphorothioate) or its metabolites interact with P-gp. Immunohistochemical analysis of tissues from male Fischer 344 rats administered chlorpyrifos (7.6 mg/kg gavage) showed increased P-gp expression in the kidney, adrenal, liver, jejunum, and stomach (tissues associated with elimination of xenobiotics), compared to control tissues. The most prominent increase was detected in the large bile ducts of the liver and the proximal tubule region of the kidney. P-gp expression was increased throughout the adrenal medulla and cortex, while a moderate increase was detected in the epithelial layers of the stomach and jejunum. To examine further the interaction between chlorpyrifos and P-gp, we evaluated whether chlorpyrifos or its active metabolite, chlorpyrifos oxon, could inhibit [3H]azidopine labeling of P-gp in MDR1 baculovirus-infected insect Sf9 cells. A concentration-dependent inhibition of [3H]azidopine labeling of P-gp was detected with chlorpyrifos oxon, while significant inhibition was not detected with chlorpyrifos. To correlate the binding of chlorpyrifos oxon to P-gp with a biochemical effect, we examined its ability to stimulate P-gp-mediated ATPase activity in these Sf9 cells. Chlorpyrifos oxon stimulated P-gp ATPase activity 1.75 times that of the positive control (10 microM verapamil). Taken together, these results suggest that chlorpyrifos oxon interacts with P-gp, and support the hypothesis that P-gp may play a role in the cellular detoxification of insecticides in mammalian tissues. To our knowledge this is the first report of an organophosphorus insecticide interacting with and increasing the expression of P-gp.

P-glycoprotein, multidrug resistance and protein kinase C.

The multidrug resistant (MDR) phenotype is a well-studied subject that has been recognized as a determinant underlying specific types of drug resistance in human cancer. Although it is clear that the P-glycoprotein plays a major role in MDR, it is not clear whether post-translational modifications such as phosphorylation have any major impact on its modulation. The laboratory of Dr. Bruce Chabner was one of the first to describe increased expression and activity of protein kinase C (PKC) associated with the MDR phenotype. Since that time, a similar correlation has been observed in many other MDR cell lines. Most of these studies have been performed with doxorubicin-selected cells that have acquired MDR and have shown increased PKC activity, mainly for PKC-alpha isoenzyme. Intrinsic MDR in human renal cell carcinoma lines has been shown to correlate directly with PKC activity, but further studies with intrinsic MDR cell lines are needed before any conclusions can be drawn. More recent evidence suggests that there is a complex biochemical process by which PKC isoenzymes differentially phosphorylate specific serine residues in the linker region of P-glycoprotein which may lead to alterations in P-glycoprotein ATPase and drug-binding functions. To further complicate matters, PKC plays an important role in anti-apoptotic pathways, which can confound the dissection and elucidation of drug-resistance mechanisms. However, these areas are still under active investigation and not fully answered. Further studies are needed to specifically answer the question of whether PKC directly modulates basal and/or drug-stimulated P-glycoprotein function. This manuscript reviews the majority of the literature on PKC and MDR, as well as offers caveats for interpretation of these studies to answer the above questions.

Partial inhibition of multidrug resistance by safingol is independent of modulation of P-glycoprotein substrate activities and correlated with inhibition of protein kinase C.

Safingol is a lysosphingolipid protein kinase C (PKC) inhibitor that competitively interacts at the regulatory phorbol binding domain of PKC. We investigated the effects of safingol on antineoplastic drug sensitivity and PKC activity of MCF-7 tumor cell lines. Safingol treatment of 32P-labeled MCF-7 WT and MCF-7 DOXR cells inhibited phosphorylation of the myristoylated alanine-rich protein kinase C substrate in both cell lines, suggesting inhibition of cellular PKC. However, only in MCF-7 DOXR cells did safingol treatment increase accumulation of [3H]vinblastine and enhance toxicity of Vinca alkaloids and anthracyclines. Drug accumulation changes in MCF-7 DOXR cells treated with safingol were accompanied by inhibition of basal and phorbol 12,13-dibutyrate-stimulated phosphorylation of P-glycoprotein (P-gp). Expression of P-gp and levels of mdr1 message in MCF-7 DOXR cells were not altered by safingol treatment alone or in combination with vinblastine. Treatment of MCF-7 DOXR cell membranes with safingol did not inhibit [3H]vinblastine binding or [3H]azidopine photoaffinity labeling of P-gp. Furthermore, safingol did not stimulate P-gp ATPase activity in membranes prepared from MCF-7 DOXR cells. We conclude that enhanced drug accumulation and sensitivity in MCF-7 DOXR cells treated with safingol are correlated with inhibition of PKC rather than competitive interference with P-gp drug binding through direct interaction with P-glycoprotein.

Characterization of prenylcysteines that interact with P-glycoprotein and inhibit drug transport in tumor cells.

Prenylcysteine methyl esters that represent the C-terminal structures of prenylated proteins demonstrate specific substrate-like interactions with P-glycoprotein (Zhang, L., Sachs, C. W., Fine, R. L., and Casey, P. J. (1994) J. Biol. Chem. 269, 15973-15976). The simplicity of these compounds provides a unique system for probing the structural specificity of P-glycoprotein substrates. We have further assessed the structural elements of prenylcysteines involved in the interaction with P-glycoprotein. Carboxyl group methylation, a modification in many prenylated proteins, plays an essential role of blocking the negative charge at the free carboxylate. Substitution of the methyl ester with a methyl amide or simple amide does not change the ability of the molecule to stimulate P-glycoprotein ATPase activity, but substitution with a glycine is not tolerated unless the carboxyl group of glycine is methylated. The presence of a nitrogen atom, which is found in many P-glycoprotein substrates and modifiers, is also essential for prenylcysteines to interact with P-glycoprotein. The structure at the nitrogen atom can, however, influence the type of interaction. Acetylation of the free amino group of prenylcysteine/results in a significant loss in the ability of prenylcysteines to stimulate P-glycoprotein ATPase activity. Instead, certain acetylated prenylcysteines behave as inhibitors of this activity. In studies using MDR1-transfected human breast cancer cells, the acetylated prenylcysteine analogs inhibit P-glycoprotein-mediated drug transport and enhance the steady-state accumulation of [3H]vinblastine, [3H]colchicine, and [3H]taxol. These inhibitors do not, however, affect drug accumulation in parental cells. These studies provide a novel approach for designing P-glycoprotein inhibitors that could prove effective in reversing the phenotype of multidrug resistance in tumor cells.

Interaction of prenylcysteine methyl esters with the multidrug resistance transporter.

The multidrug resistance transporter is an integral membrane protein, termed P-glycoprotein, which can function as an ATP-dependent drug efflux pump to reduce intracellular drug accumulation in treated cells. The physiologic function of this protein in normal cells, however, is not completely understood. We report here that prenylcysteine methyl esters, which represent the C-terminal structures of prenylated proteins, both stimulate the transporter's intrinsic ATPase activity and compete for drug binding. The structural elements of prenylcysteine methyl esters involved in their interaction with P-glycoprotein include the isoprenoid moiety, the carboxyl methyl group, and the free amino group. These findings indicate that these molecules are potential physiologic ligands of the transporter. Furthermore, as the structures of the active prenylcysteines are distinct from the known substrates of P-glycoprotein, this information may facilitate design of novel inhibitors of the transporter.

Selective regulation of expression of protein kinase C (PKC) isoenzymes in multidrug-resistant MCF-7 cells. Functional significance of enhanced expression of PKC alpha.

The multidrug resistance (MDR) phenotype induces cross-resistance to many chemotherapeutic agents in cancer cells. Protein kinase C (PKC) has been implicated in the regulation of the MDR phenotype. In order to determine the role of specific PKC isoenzymes in regulating the MDR phenotype, the expression and activity of PKC isoenzymes in the human breast cancer cell line, MCF-7-WT, and an MDR subline, MCF-7-MDR, were examined. The MDR phenotype was associated with a 10-fold increase in calcium-dependent PKC activity as well as a 10-fold decrease in calcium-independent activity was due to a selective increase in the activity was due to a selective increase in the expression of PKC alpha as determined by Western blot analysis and hydroxylapatite chromatography. This increase in expression of PKC alpha was regulated at the message level as demonstrated by Northern blot analysis. The decrease in calcium-independent activity was caused by a decrease in the expression of PCK delta and epsilon. The significance of the increase in PKC alpha expression was then demonstrated by a commensurate 11-fold increase in the basal and stimulated phosphorylation of the myristolated alanine-rich C kinase substrate. Phosphorylation of P-glycoprotein, the cellular mediator of the MDR phenotype, was increased > 20-fold in the unstimulated MCF-7-MDR cell line and its phosphorylation was further increased 2-fold in response to phorbol 12-myristate 13-acetate. These changes paralleled the increases in P-glycoprotein pump function and the MDR phenotype underscoring the role for PKC alpha in regulating P-glycoprotein phosphorylation and function.

Neutrophil elastase activity and superoxide production are diminished in neutrophils of alcoholics.

Several lines of evidence indicate that neutrophil elastase and oxidants secreted by phagocytic inflammatory cells play key roles in the development of centrilobular emphysema. A recent report suggests that ethanol consumption may have a protective role against this disease in smokers. The aim of this study was to examine the effect of ethanol consumption on neutrophil elastase activity and superoxide production of peripheral blood neutrophils. These activities were measured in neutrophils from 52 male intoxicated patients and compared to activities in neutrophils from 20 male volunteers. Neutrophils from intoxicated patients contained 31% less elastase activity than that found in controls, 0.99 +/- 0.27 versus 1.44 +/- 0.23 micrograms/10(6) neutrophils (p less than 0.0001) and produced 25 to 27% less superoxide than controls in response to phorbol 12-myristate-13-acetate, 0.90 +/- 0.17 versus 1.2 +/- 0.21 nmol/min/10(6) PMN (p less than 0.0001) or N-formylmethionylleucylphenylalanine, 0.64 +/- 0.19 versus 0.88 +/- 0.24 (p less than 0.001). In follow-up studies of 10 patients admitted for acute alcoholism, elastase activity and superoxide production remained low for 2 to 4 days. After 6 to 10 days, elastase activity and superoxide production were significantly greater than they were at Day 0 and approached normal levels. Neutrophils isolated from blood samples of healthy abstaining donors, which had been exposed to ethanol or to plasma from inebriated patients for 16 to 20 h, showed no loss of elastase activity or superoxide production.(ABSTRACT TRUNCATED AT 250 WORDS)