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M S Center

Publications and source records attributed to M S Center.

At least 37 records · Page 2Linked to original sources
PubMed1990

Characterization of a membrane-associated protein kinase of multidrug-resistant HL60 cells which phosphorylates P-glycoprotein.

Cells containing increased levels of the membrane phosphoprotein P-glycoprotein exhibit a multidrug-resistant phenotype. In the present study we have analyzed protein kinases capable of phosphorylating P-glycoprotein in membranes of HL60 cells isolated for resistance to vincristine. Analysis of this system demonstrates that in isolated membranes the protein kinase inhibitor staurosporine greatly reduces P-glycoprotein phosphorylation. In contrast, the kinase inhibitor H-7 does not affect this reaction. Fractionation of solubilized membrane proteins from sensitive and resistant cells on DEAE-cellulose reveals a major protein kinase (PK-1) which exhibits optimal activity in the presence of Mn2+ and histone H1. This enzyme fraction does not contain detectable levels of protein kinase C or cAMP-dependent protein kinase. PK-1 phosphorylation of two endogenous proteins is, however, greatly enhanced in the presence of phosphatidylserine or phosphatidyl-inositol. In reaction mixtures containing Mg2+ or Mn2+ in the absence of phospholipid, PK-1 from resistant cells phosphorylates an endogenous protein of 180 kilodaltons (P180), which exhibits an electrophoretic mobility identical to P-glycoprotein. In parallel experiments with PK-1 from sensitive cells there is no detectable phosphorylation of a P180 protein. P180 phosphorylated by PK-1 from resistant cells is immunoprecipitated by antibody against P-glycoprotein. Additional studies demonstrate that PK-1 is capable of phosphorylating specific synthetic peptides which correspond to the sequence of P-glycoprotein. Peptide phosphorylation occurs at both serine and threonine residues. These studies thus identify a novel membrane-associated protein kinase in HL60 cells which is capable of phosphorylating P-glycoprotein. This enzyme may have an important role in regulating levels of multidrug resistance.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1990

Mechanisms of multidrug resistance in HL60 cells: detection of resistance-associated proteins with antibodies against synthetic peptides that correspond to the deduced sequence of P-glycoprotein.

HL60 cells isolated for resistance to Adriamycin are multidrug resistant and defective in the cellular accumulation of drug. These cells do not however overexpress mdr1 and do not contain detectable levels of P-glycoprotein. In the present study we have prepared antisera against synthetic peptides that correspond to various sequence domains of P-glycoprotein and have examined by Western blot analysis the reactivity of these antisera with proteins contained in membranes of HL60/Adr cells. All antisera are highly reactive with a Mr 180,000 (p180) P-glycoprotein contained in membranes of HL60 cells isolated for resistance to vincristine (HL60/Vinc). In contrast, of 13 antisera tested 12 do not react with any resistance-associated protein in the HL60/Adr isolate. One antiserum (ASP14) is however highly reactive with a Mr 190,000 protein (p190) contained in HL60/Adr membranes. This protein is not detected in drug-sensitive cells. ASP14 also reacts with proteins p195 and p50 contained in a second independent HL60/Adr isolate. Analysis of membrane subfractions shows that p190 is located primarily in the endoplasmic reticulum with only low levels contained in plasma membranes. Additional studies demonstrate that endoplasmic reticulum of HL60/Adr cells contain a major Mr 190,000 protein that is capable of binding the photoaffinity agent 8-azido[alpha-32P]ATP. p195 contained in a second HL60/Adr isolate is also labeled with 8-azido[alpha-32P]ATP. These results thus demonstrate that antiserum against a specific P-glycoprotein sequence detects a p190 (p195) resistance-associated membrane protein in two independent HL60/Adr isolates. p190 (p195) and P-glycoprotein thus contain a minor sequence homology and based on the specificity of ASP14 this occurs in a region which may be involved in nucleotide binding. Possibly this sequence is common to and essential for the functionality of proteins which contribute to resistance by reducing cellular drug levels.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1989

Mechanisms of multidrug resistance in HL60 cells. Analysis of resistance associated membrane proteins and levels of mdr gene expression.

HL60 cells isolated for resistance to Adriamycin do not contain P-glycoprotein, as determined with immunological probes. These cells, however, are multidrug resistant and defective in the cellular accumulation of drug. In view of these findings, we have examined in greater detail certain properties of the HL60/Adr cells and have compared these properties to an HL60 drug-resistant isolate (HL60/Vinc) which contains high levels of P-glycoprotein. The results of these studies demonstrated that verapamil induces a major increase in cellular drug accumulation in both HL60/Adr and HL60/Vinc isolates. An 125I-labeled photoaffinity analog of verapamil labeled P-glycoprotein contained in membranes of HL60/Vinc cells. In contrast, this agent did not label any protein selectively associated with drug resistance in membranes of the HL60/Adr isolate. The photoactive dihydropyridine calcium channel blocker [3H]azidopine and [125I]NASV, a photoaffinity analog of vinblastine, labelled P-glycoprotein in membranes from HL60/Vinc cells, whereas in experiments with the HL60/Adr isolate there was no detectable labeling of a drug resistance associated membrane protein. Additional studies have been carried out to analyze membrane proteins of HL60/Adr cells labeled with the photoaffinity agent 8-azido-alpha-[32P]ATP (AzATP32). The results demonstrate that this agent labeled a resistance associated membrane protein of 190 kilodaltons (P190). P190 is essentially absent in membranes of drug-sensitive cells. Labeling of P190 with AzATP32 in membranes of resistant cells was blocked completely when incubations were carried out in the presence of excess unlabeled ATP. Additional studies were carried out to analyze mdr gene amplification and expression in sensitive and resistant cells. Experiments carried out with human 5',mdr1 (1.1 kb) and mdr3 (1.0 kb) cDNAs demonstrate that both of these sequences were highly amplified in the HL60/Vinc isolate. Only the mrd1 gene sequence however, was overexpressed. In contrast, there was no detectable amplification or overexpression of mdr1 or mdr3 sequences in HL60/Adr cells. The results of this study thus identify a new nucleotide binding protein which is overexpressed in membranes of HL60 cells isolated for resistance to Adriamycin. P190, which exhibits properties distinct from P-glycoprotein, possibly functions in the energy-dependent drug efflux system contained in the HL60/Adr resistant isolate.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1989

Multiple mechanisms of adriamycin resistance in the human leukemia cell line CCRF-CEM.

CEM cells exhibiting a 25-fold (C25X) or 80-fold (C80X) increase in resistance to adriamycin were isolated and characterized. C25X cells were cross-resistant to daunomycin and etoposide (VP-16) but not to vincristine or colchicine. These cells were not defective in the cellular accumulation of drug and did not contain detectable levels of P-glycoprotein. Continued exposure of C25X cells to adriamycin resulted in increased levels of resistance and additional phenotypic changes. These cells (C80X) now contained high levels of P-glycoprotein and were cross-resistant to a variety of agents including vincristine and colchicine. A fluorometric assay for DNA unwinding was used to measure levels of drug-induced DNA breaks in sensitive and C25X resistant cells. Studies carried out with VP-16, 4'9-acridinyl-aminomethanesulfon-m-anisidide (m-AMSA), adriamycin, or daunomycin showed that the level of drug-induced DNA strand breakage in resistant cells was considerably less than that occurring in drug-treated sensitive cells. These studies, therefore, show that treatment of CEM cells with adriamycin resulted in a nuclear alteration that contributed to drug resistance. They also demonstrate that prolonged treatment of cells with adriamycin resulted in membrane alterations that affect cellular drug accumulation. Adriamycin resistance in CEM cells can thus occur as a result of at least two distinct mechanisms.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1988

Mechanisms of multidrug resistance in HL60 cells: evidence that a surface membrane protein distinct from P-glycoprotein contributes to reduced cellular accumulation of drug.

HL60 cells exhibiting a 140-fold increase in resistance to vincristine contain three surface membrane proteins with molecular weights of 210,000 (P210), 180,000 (P180), and 150,000 (P150) which are highly phosphorylated in vivo and in an in vitro system in the presence of Mn2+ and [gamma-32P]ATP. These phosphorylated proteins are either absent or present in very low levels in membranes of drug-sensitive cells. Growth of the vincristine-resistant isolate in the absence of drug results in a decrease in the level of resistance and a major reduction in the phosphorylation of P210 and P180. The phosphorylation of P150 is not altered in the revertant which still exhibits substantial levels of resistance. Further studies show that P210 and P180 are highly reactive with a monoclonal antibody against P-glycoprotein. These two proteins are present in only very low levels in revertant cells. The monoclonal antibody exhibits no reactivity with P150. In HL60 cells isolated for a 25-fold increase in vincristine resistance proteins reactive with P-glycoprotein monoclonal antibody are essentially absent. P150 is however highly phosphorylated in these cells. Additional experiments using lectin binding of 32P-labeled proteins demonstrates that P150 has properties distinct from P210 and P180. Analysis of drug uptake patterns in the vincristine-resistant isolates and the revertant shows that resistance is related to a reduced intracellular accumulation of drug. Reduced accumulation of vincristine is also found in HL60 cells isolated for resistance to Adriamycin. These cells are devoid of P-glycoprotein but contain phosphorylated P150. These results suggest that proteins P150, P180, and P210 may contribute to multidrug resistance in HL60 cells through a mechanism which involves reduced cellular accumulation of drug. P180 and P210 are structurally related whereas P150 is distinct from these two proteins.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1987

Adriamycin resistance in HL60 cells and accompanying modification of a surface membrane protein contained in drug-sensitive cells.

HL60 cells resistant to Adriamycin contain a 32P-labeled, Mr 150,000 surface membrane protein (p150) which is not detected in cells sensitive to drug. The levels of phosphorylation of this protein increase with increasing levels of resistance. Analysis of plasma membranes prepared from cells labeled with [14C]glucosamine shows, however, that both sensitive cells and those exhibiting an 80-fold increase in drug resistance contain essentially identical levels of a highly glycosylated Mr 150,000 protein. Identical results are obtained when cells are labeled with [14C]galactose or [14C]mannose. Limited proteolytic digestion of [14C]glucosamine-labeled p150 from sensitive and resistant cells shows that the glycopeptides formed are identical. Additional studies involving binding of proteins to insolubilized lectin indicate that 32P-labeled p150 is glycosylated. Polyacrylamide gel electrophoresis of p150 followed by silver staining shows no difference in the levels of this protein in sensitive and 80-fold drug-resistant cells. Further studies show that two-dimensional tryptic peptide maps of 125I-labeled p150 of sensitive and resistant cells are essentially the same. It has also been found that treatment of cells with 12-O-tetradecanoylphorbol-13-acetate followed by [14C]glucosamine labeling results in a selective decrease in the glycosylation of p150 of sensitive and resistant cells. TPA has an identical effect on the phosphorylation of p150 in cells resistant to drug. HL60 cells have also been examined for the presence of the Mr 170,000 to 180,000 P-glycoprotein. Using immunoblot analysis with a monoclonal antibody directed against the P-glycoprotein we did not detect the presence of this protein in membranes of drug-sensitive or -resistant HL60 cells. The results of this study suggest that Adriamycin resistance in HL60 cells may be related to a modified form of a protein contained in cells sensitive to drug. Proteins active in drug resistance in this system may be distinct from those described for other cell lines.

Cell Line↗
PubMed1987

Adriamycin resistance in HL60 cells in the absence of detectable P-glycoprotein.

Previous studies have shown that the development of multi-drug resistance in cell lines treated with chemotherapeutic agents is closely associated with the overexpression of a 170-180 kilodalton surface membrane glycoprotein (P-glycoprotein). In the present study a monoclonal antibody against the P-glycoprotein was used to determine if this protein is overexpressed in multi-drug resistant HL60 cells. Using either indirect immunofluorescent staining or immunoblot analysis P-glycoprotein could not be detected in HL60 cells isolated for resistance to adriamycin. In contrast HL60 cells isolated for resistance to vincristine contain the P-glycoprotein and the amount of this material increases with increasing levels of resistance. These studies thus demonstrate adriamycin resistance in P-glycoprotein negative HL60 cells. Furthermore adriamycin and vincristine are found to have distinct effects in inducing overexpression of P-glycoprotein in the HL60 cell line. This information could be useful in the development of therapeutic strategies for the treatment of certain forms of cancer.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1986

Dimethylsulfoxide, retinoic acid and 12-O-tetradecanoylphorbol-13-acetate induce a selective decrease in the phosphorylation of P150, a surface membrane phosphoprotein of HL60 cells resistant to adriamycin.

Studies have been carried out to analyze protein phosphorylation in membranes isolated from adriamycin resistant HL60 cells which have been grown for various time periods in the presence of dimethylsulfoxide (DMSO), retinoic acid (RA) or 12-O-tetradecanoylphorbol-13-acetate (TPA). The results show that membranes isolated from cells treated with these agents are defective in the phosphorylation of P150, a membrane phosphoprotein associated with drug resistance in HL60 cells. This response is highly selective since only a few membrane proteins show decreased phosphorylation levels under these conditions. Magnesium dependent protein kinase activity in membranes from cells treated with DMSO, RA or TPA is not altered relative to untreated membranes under conditions where there is a major decrease in P150 phosphorylation. Additional studies also show that treatment of resistant cells with TPA results in a major decrease in the in vivo phosphorylation of P150. These results thus demonstrate that agents capable of inducing differentiation in HL60 cells can selectively modulate the phosphorylation of P150. This system should be of value in clarifying mechanisms involved in the phosphorylation of this protein.

Dimethyl Sulfoxide↗
PubMed1986

Isolation and characterization of adriamycin-resistant HL-60 cells which are not defective in the initial intracellular accumulation of drug.

Two human leukemia cell lines (Molt-4 and HL-60) have been used for establishing cells which exhibit a low level resistance to Adriamycin. Analysis of drug uptake patterns shows that the Molt-4 resistant cells are defective in the initial intracellular accumulation of drug. In contrast to Molt-4 the levels of drug which accumulate in the sensitive and resistant HL-60 cells during a 60-min incubation period are essentially the same. However, when incubations are continued there is a major reduction in intracellular drug levels in the resistant cell. Further studies show that resistant cells incubated in the presence of drug for extended time periods efflux drug at a rate considerably greater than that exhibited by the sensitive parent line. Similar efflux patterns are obtained with nuclei isolated from drug-sensitive and -resistant cells. Additional studies using an in vitro phosphorylation system demonstrate distinct protein changes in membranes of Molt-4 and HL-60 resistant cells. Thus, we have found that a membrane fraction from the Molt-4 resistant line contains a Mr 170,000 protein which is not detected in a similar fraction from cells sensitive to drug. HL-60 resistant membranes contain two proteins with molecular weights of 150,000 and 120,000 which are also not found in membranes from drug-sensitive cells. The results of this study suggest that drug resistance in HL-60 cells is related to an efflux mechanism which is triggered only after cells are exposed to drug for prolonged periods.

Cell Line↗
PubMed1985

Mechanisms regulating cell resistance to adriamycin. Evidence that drug accumulation in resistant cells is modulated by phosphorylation of a plasma membrane glycoprotein.

Incubation of adriamycin resistant Chinese hamster lung cells with the calmodulin inhibitor trifluoperazine (TFP) resulted in a significant increase in the cellular accumulation of drug. When resistant cells were prelabeled with 32Pi and then treated with TFP, a major increase also occurred in the phosphorylation of a plasma membrane glycoprotein (P-180). The concentration of TFP required for inducing the superphosphorylation of this protein correlated well with the TFP concentration required for inducing an increase in drug accumulation in resistant cells. In addition to TFP, the Ca2+ channel blocker verapamil also induced drug uptake and enhanced the phosphorylation level of P-180. Additional studies showed that, when resistant cells reverted to drug sensitivity, there was a parallel loss in the TFP-induced P-180 phosphorylation. The results of this study indicate that the trifluoperazine-induced uptake of drug in resistant cells is mediated by a mechanism which involves an enhanced phosphorylation of P-180. It is suggested that, when this protein is superphosphorylated, it becomes biologically inactive, and that this results in the conversion of the resistant cell to one having a drug sensitive phenotype.

Animals↗
PubMed1985

Evidence for the involvement of two distinct membrane proteins in adriamycin resistance in Chinese hamster lung cells.

Chinese hamster lung cells resistant to Adriamycin were labeled with inorganic [32P]orthophosphate and thereafter incubated with low levels of N-ethylmaleimide. Plasma membranes and endoplasmic reticulum were isolated and the phosphorylated proteins were analyzed after polyacrylamide gel electrophoresis. The results demonstrate that both plasma membranes and endoplasmic reticulum from resistant cells contain two highly phosphorylated proteins [Mr 180,000 (p180) and Mr 220,000 (p220)] which are present in very low levels in these membrane fractions prepared from drug sensitive cells. p220 is present in much higher levels in the endoplasmic reticulum as compared to the plasma membranes whereas p180 is equally distributed in these two membrane fractions. When resistant cells revert to drug sensitivity there is a parallel loss in the phosphorylation levels of p180 and p220. Labeling of membrane proteins with 125I in the presence of chloramine-T also reveals that p180 and p220 are present in significantly greater levels in resistant membranes as compared to similar fractions prepared from drug sensitive cells. Partial digests of phosphorylated p180 and p220 produced with chymotrypsin or V8 protease reveal that each protein has a distinct phosphopeptide pattern. Both p180 and p220 are phosphorylated exclusively at serine residues. The results of this study therefore suggest that resistance to Adriamycin in Chinese hamster lung cells requires the involvement of two distinct proteins which are both bound to cell membranes.

Animals↗
PubMed1984

Involvement of plasma membrane lipid structural order in adriamycin resistance in Chinese hamster lung cells.

Plasma membrane preparations from Chinese hamster lung cells, which are resistant to the antitumor agent Adriamycin, were analyzed using fluorescence polarization of the membrane lipid probe trans-parinaric acid. The results of these studies reveal that membranes from several drug-resistant isolates have a substantial decrease in lipid structural order relative to membranes from drug-sensitive cells. Additional studies have shown that certain isolates are unstable and undergo a sequential phenotypic reversion after continuous passage in culture. Thus, we have identified cells which have reverted for membrane lipid physical changes but which still remain highly resistant to Adriamycin. At later passages, these cells are found to revert to drug sensitivity. These results indicate that an alteration of plasma membrane lipid structural order is not an essential component of the Adriamycin-resistant phenotype. However, in certain isolates, drug resistance and changes in membrane physical properties are both associated with an unstable genetic element.

Animals↗
PubMed1983

Identification and characterization of a plasma membrane phosphoprotein which is present in Chinese hamster lung cells resistant to adriamycin.

Studies have been carried out to analyze the phosphoprotein composition of plasma membranes from Chinese hamster lung cells resistant to the action of adriamycin. Gel electrophoretic analysis of [32Pi]-labeled proteins revealed that plasma membranes from resistant cells contain a phosphoprotein of 180,000 molecular weight (P180) which is not detected in drug sensitive cells. Protein P180 can also be identified after phosphorylation of resistant plasma membranes in an in vitro protein kinase system. Pulse-chase experiments indicated that the P180 was metabolically active and underwent cycles of phosphorylation and dephosphorylation in the cell. Additional studies showed that, in the presence of N-ethylmaleimide (NEM), there was a major increase in the uptake of adriamycin in resistant cells. A similar effect was observed with KCN but not with sodium azide. When resistant cells were grown in the presence of [32Pi] and then incubated in the presence of NEM, there was a considerable increase in the phosphorylation of P180. In contrast, many other plasma membrane proteins were dephosphorylated under these incubation conditions. The results suggest the possibility that, as P180 was hyperphosphorylated, the protein was inactivated and this contributed to the ability of resistant cells to accumulate adriamycin.

Animals↗
PubMed1983

Evidence that adriamycin resistance in Chinese hamster lung cells is regulated by phosphorylation of a plasma membrane glycoprotein.

Incubation of adriamycin resistant Chinese hamster lung cells with low levels of N-ethylmaleimide (NEM) results in a major increase in the cellular accumulation of drug. When resistant cells are prelabeled with [32Pi] and thereafter treated with NEM there also occurs a selective superphosphorylation of an 180K plasma membrane glycoprotein (P-180). This phosphorylation reaction occurs at both serine and threonine residues. In similar experiments with drug sensitive cells only minor levels of this protein can be detected. Detailed studies have established that in cells which have reverted to drug sensitivity there is a parallel loss in the presence of phosphorylated P-180. Also in cells which have undergone partial reversion to drug sensitivity there is a correlation between levels of superphosphorylated P-180 and adriamycin resistance. These results provide evidence that adriamycin resistance is dependent on the presence of P-180. The results also suggest that the biological activity of this protein is highly regulated by phosphorylation and that in the superphosphorylated state P-180 is inactive and under these conditions the resistant cell is converted to a drug sensitive phenotype.

Animals↗
PubMed1982

Adriamycin-induced compaction of isolated chromatin.

Adriamycin and certain other agents such as daunomycin, rubidazone and 1,4-dihydroxyl-5,8 bis [[2-[(2-hydroxyethyl)amino]ethyl]amino]-9,10-anthracenedione (DHAQ) were capable of inducing a considerable increase in the sedimentation rate of isolated chromatin. In contrast, N-trifluoroacetyl-adriamycin-14-valerate (AD-32) was without effect in this system. Electron microscope studies of chromatin incubated with adriamycin, rubidazone and DHAQ showed that all of these drugs were capable of inducing the formation of highly condensed DNA structures. When chromatin was treated with 0.4 M NaCl, its ability to serve as a substrate for adriamycin-induced compaction was abolished. However, the salt-treated chromatin could still be condensed by DHAQ and rubidazone. The results of this study, therefore, show that a variety of closely related antitumor agents are capable of condensing isolated chromatin. However, certain of these compounds had distinct substrate requirements for the compaction reaction.

Animals↗