110 results on '"Gottesman M"'
Search Results
2. Synthesis, processing, and localization of human Lon protease.
- Author
-
Wang, N, primary, Maurizi, M R, additional, Emmert-Buck, L, additional, and Gottesman, M M, additional
- Published
- 1994
- Full Text
- View/download PDF
3. Targeted disruption of the mouse mdr1b gene reveals that steroid hormones enhance mdr gene expression.
- Author
-
Altuvia, S, primary, Stein, W D, additional, Goldenberg, S, additional, Kane, S E, additional, Pastan, I, additional, and Gottesman, M M, additional
- Published
- 1993
- Full Text
- View/download PDF
4. Cyclic AMP responses are suppressed in mammalian cells expressing the yeast low Km cAMP-phosphodiesterase gene.
- Author
-
Van Lookeren Campagne, M M, primary, Wu, E, additional, Fleischmann, R D, additional, Gottesman, M M, additional, Chason, K W, additional, and Kessin, R H, additional
- Published
- 1990
- Full Text
- View/download PDF
5. Photosensitized labeling of a functional multidrug transporter in living drug-resistant tumor cells.
- Author
-
Raviv, Y, primary, Pollard, H B, additional, Bruggemann, E P, additional, Pastan, I, additional, and Gottesman, M M, additional
- Published
- 1990
- Full Text
- View/download PDF
6. Heat shock and arsenite increase expression of the multidrug resistance (MDR1) gene in human renal carcinoma cells.
- Author
-
Chin, K V, primary, Tanaka, S, additional, Darlington, G, additional, Pastan, I, additional, and Gottesman, M M, additional
- Published
- 1990
- Full Text
- View/download PDF
7. Genomic organization of the human multidrug resistance (MDR1) gene and origin of P-glycoproteins.
- Author
-
Chen, C J, primary, Clark, D, additional, Ueda, K, additional, Pastan, I, additional, Gottesman, M M, additional, and Roninson, I B, additional
- Published
- 1990
- Full Text
- View/download PDF
8. The localization and activity of cAMP-dependent protein kinase affect cell cycle progression in thyroid cells.
- Author
-
Feliciello, A, Gallo, A, Mele, E, Porcellini, A, Troncone, G, Garbi, C, Gottesman, M E, and Avvedimento, E V
- Abstract
cAMP signals are received and transmitted by multiple isoforms of cAMP-dependent protein kinases (PKAs), typically determined by their specific regulatory subunits. We describe changes in the cAMP signal transduction pathway during cell cycle progression in synchronized rat thyroid cells. Both PKA type II (PKAII) localization and nuclear cAMP signaling are significantly modified during G(0) and G(1)-S transitions. G(1) is characterized by PKA activation and amplified cAMP signal transduction. This is associated with a decrease in the concentration of RI and RII regulatory subunits and enhanced anchoring of PKAII to the Golgi-centrosome region. Just prior to S, the cAMP pathway is depressed. Up-regulation of the pathway by exogenous cAMP in G(1) inhibited the subsequent decay of the Cdk inhibitor p27 and delayed the onset of S phase. Forced translocation of endogenous PKAII to the cytosol down-regulated cAMP signaling, advancing the timing of p27 decay and inducing premature exit from G(1). These data indicate that membrane-bound PKA amplifies the transduction of cAMP signals in G(1) and that the length of G(1) is influenced by cAMP-PKA.
- Published
- 2000
9. Mechanism of action of human P-glycoprotein ATPase activity. Photochemical cleavage during a catalytic transition state using orthovanadate reveals cross-talk between the two ATP sites.
- Author
-
Hrycyna, C A, Ramachandra, M, Ambudkar, S V, Ko, Y H, Pedersen, P L, Pastan, I, and Gottesman, M M
- Abstract
Human P-glycoprotein (P-gp), an ATP-dependent efflux pump responsible for cross-resistance of human cancers to a variety of lipophilic compounds, is composed of two homologous halves, each containing six transmembrane domains and an ATP-binding/utilization domain. To determine whether each site can hydrolyze ATP simultaneously, we used an orthovanadate (Vi)-induced ADP-trapping technique (P-gp.MgADP.Vi). In analogy with other ATPases, a photochemical peptide bond cleavage reaction occurs within the Walker A nucleotide binding domain consensus sequence (GX4GK(T/S)) when the molecule is trapped with Vi in an inhibited catalytic transition state (P-gp.MgADP.Vi) and incubated in the presence of ultraviolet light. Upon reconstitution into proteoliposomes, histidine-tagged purified P-gp from baculovirus-infected insect cells had drug-stimulated ATPase activity. Reconstituted P-gp was incubated with either ATP or 8-azido-ATP in the presence or absence of Vi under ultraviolet (365 nm) light on ice for 60 min. The resultant products were separated by SDS-polyacrylamide gel electrophoresis and subjected to immunoblotting with seven different human P-gp-specific antibodies covering the entire length of the molecule. Little to no degradation of P-gp was observed in the absence of Vi. In the presence of Vi, products of approximately 28, 47, 94, and 110 kDa were obtained, consistent with predicted molecular weights from cleavage at either of the ATP sites but not both sites. An additional Vi-dependent cleavage site was detected at or near the trypsin site in the linker region of P-gp. These results suggest that both the amino- and carboxyl-terminal ATP sites can hydrolyze ATP. However, there is no evidence that ATP can be hydrolyzed simultaneously by both sites.
- Published
- 1998
10. The v-Ki-Ras oncogene alters cAMP nuclear signaling by regulating the location and the expression of cAMP-dependent protein kinase IIbeta.
- Author
-
Feliciello, A, Giuliano, P, Porcellini, A, Garbi, C, Obici, S, Mele, E, Angotti, E, Grieco, D, Amabile, G, Cassano, S, Li, Y, Musti, A M, Rubin, C S, Gottesman, M E, and Avvedimento, E V
- Abstract
The v-Ki-Ras oncoprotein dedifferentiates thyroid cells and inhibits nuclear accumulation of the catalytic subunit of cAMP-dependent protein kinase. After activation of v-Ras or protein kinase C, the regulatory subunit of type II protein kinase A, RIIbeta, translocates from the membranes to the cytosol. RIIbeta mRNA and protein were eventually depleted. These effects were mimicked by expressing AKAP45, a truncated version of the RII anchor protein, AKAP75. Because AKAP45 lacks membrane targeting domains, it induces the translocation of PKAII to the cytoplasm. Expression of AKAP45 markedly decreased thyroglobulin mRNA levels and inhibited accumulation of C-PKA in the nucleus. Our results suggest that: 1) The localization of PKAII influences cAMP signaling to the nucleus; 2) Ras alters the localization and the expression of PKAII; 3) Translocation of PKAII to the cytoplasm reduces nuclear C-PKA accumulation, resulting in decreased expression of cAMP-dependent genes, including RIIbeta, TSH receptor, and thyroglobulin. The loss of RIIbeta permanently down-regulates thyroid-specific gene expression.
- Published
- 1996
11. The multidrug transporter, a double-edged sword.
- Author
-
Gottesman, M M and Pastan, I
- Published
- 1988
- Full Text
- View/download PDF
12. Membrane localization of cAMP-dependent protein kinase amplifies cAMP signaling to the nucleus in PC12 cells.
- Author
-
Cassano, S, Gallo, A, Buccigrossi, V, Porcellini, A, Cerillo, R, Gottesman, M E, and Avvedimento, E V
- Abstract
The A126 cell line, in contrast to its PC12 parent, does not differentiate, accumulate nuclear cAMP-dependent protein kinase A (PKA) catalytic subunit, or transcribe cAMP-dependent promoters in response to cAMP. Total PKA is reduced by 50% and is partly resistant to cAMP-induced dissociation in vivo. Unlike PC12, where PKAII is membrane-associated, PKAII is exclusively cytosolic in A126. Cotransfection with the RII anchor protein (AKAP75) and the PKA catalytic subunit (C-PKA) restored cAMP-induced transcription to levels found in PC12. These data indicate that membrane-bound PKAII amplifies cAMP signaling to the nucleus and suggest that cAMP-mediated responses are specified by the type and cellular localization of the PKA isoform.
- Published
- 1996
13. The major excreted protein of transformed fibroblasts is an activable acid-protease.
- Author
-
Gal, S and Gottesman, M M
- Abstract
Malignant transformation of mouse cells by a variety of agents or treatment with the tumor promoter 12-O-tetradecanoylphorbol 13-acetate or platelet-derived growth factor results in increased synthesis and secretion of a 39,000-dalton protein termed major excreted protein (MEP). We report here that secreted MEP is an acid-activable protease. The secreted precursor form of the protease is auto-activated at low pH and is able to digest a variety of proteins, including the extracellular matrix proteins fibronectin, collagen, and laminin. MEP protease activity has pH optimum of 3.3-3.6 and is temperature- and concentration-dependent. The activity is inhibited by sulfhydryl protease inhibitors such as leupeptin and iodoacetic acid and not by metallo-, seryl-, or carboxyprotease inhibitors. The MEP-derived protease has characteristics distinct from the cathepsins previously reported and thus may be a new acid-protease of mouse cells.
- Published
- 1986
- Full Text
- View/download PDF
14. Expression of a multidrug resistance-adenosine deaminase fusion gene
- Author
-
Germann, U A, Gottesman, M M, and Pastan, I
- Abstract
A novel fusion gene has been created in which the expression of a dominant selectable marker, the human multidrug resistance gene, is directly linked to the expression of human adenosine deaminase cDNA. The chimeric gene was inserted between the long terminal repeats of a Harvey murine sarcoma virus expression vector and used to transfect drug-sensitive human KB carcinoma cells. Transfectants were selected in increasing concentrations of colchicine and found to contain multiple copies of the intact fusion gene, which is stably and efficiently expressed. A membrane-associated 210-kDa human P-glycoprotein-adenosine deaminase fusion protein is synthesized which retains function of the multidrug transporter and also exhibits adenosine deaminase activity. The data indicate that the human multidrug resistance gene may be used as a dominant selectable marker to introduce other genes in the form of gene fusions into cultured cells.
- Published
- 1989
- Full Text
- View/download PDF
15. Cyclic AMP treatment of Rous sarcoma virus-transformed Chinese hamster ovary cells increases phosphorylation of pp60src and increases pp60src kinase activity.
- Author
-
Roth, C W, Richert, N D, Pastan, I, and Gottesman, M M
- Abstract
Treatment of growing Rous sarcoma virus-transformed Chinese hamster ovary cells with the cyclic AMP analog 8-bromo-cyclic adenosine 3‘,5‘-monophosphate (8-bromo-cyclic AMP) stimulates the incorporation of 32Pi into the viral transforming protein pp60src. Based on one-dimensional and two-dimensional peptide analysis and phosphoamino acid analysis, the increase is on a single phosphoserine residue at the NH2 terminus of the protein. The phosphate incorporation increases during the first 4 h of treatment. The pp60src kinase activity in extracts of cells treated with 8-bromo-cyclic AMP was stimulated about 2- to 3-fold. This stimulation of kinase activity increased during the first 3 h of treatment with 1 mM 8-bromo-cAMP and the activity was increased in both the soluble and particulate fraction of the cells. These results suggest that cyclic AMP can modulate the activity of pp60src in transformed cells.
- Published
- 1983
- Full Text
- View/download PDF
16. DNA-mediated gene transfer of a mutant regulatory subunit of cAMP-dependent protein kinase.
- Author
-
Abraham, I, Brill, S, Hyde, J, Fleischmann, R, Chapman, M, and Gottesman, M M
- Abstract
We have used DNA-mediated gene transfer of genomic DNA to introduce into wild-type Chinese hamster ovary (CHO) cells a mutant gene that confers resistance to the growth inhibitory effect of cAMP. This dominant mutation in CHO cell line 10248 is responsible for an alteration in the RI subunit (RI*) of the type I cAMP-dependent protein kinase (Singh, T. J., Hochman, J., Verna, R., Chapman, M., Abraham, I., Pastan, I.H., and Gottesman, M.M. (1985) J. Biol. Chem. 260, 13927-13933). The transformant 11564 which was studied in detail, has the same characteristics as the original mutant 10248 including continued growth in medium containing 8-Br-cAMP, an increase in the Ka for cAMP activation of the kinase, a greatly reduced amount of type II protein kinase activity, an altered incorporation of the photoaffinity label 8-N3[32P]cAMP into the RI* subunit of PKI, and an absence of cAMP-dependent phosphorylation of a Mr = 52,000 protein in intact cells. In addition, analysis of the DNA of the transformant indicates the presence of an increased amount of DNA for the RI gene. These results are consistent with the transfer of a mutant gene for the RI* subunit of the cAMP-dependent protein kinase and its phenotypic expression in the transformant and also support the hypothesis that the mutation responsible for the defect in cell line 10248 is due to an alteration in the gene for RI.
- Published
- 1985
- Full Text
- View/download PDF
17. Characterization of a cyclic AMP-resistant Chinese hamster ovary cell mutant containing both wild-type and mutant species of type I regulatory subunit of cyclic AMP-dependent protein kinase.
- Author
-
Singh, T J, Hochman, J, Verna, R, Chapman, M, Abraham, I, Pastan, I H, and Gottesman, M M
- Abstract
We have characterized a cyclic AMP-resistant Chinese hamster ovary (CHO) cell mutant in which one of two major species of type I regulatory subunit (RI) of cyclic AMP-dependent protein kinase is altered. Wild-type CHO cell extracts contain two cyclic AMP-dependent protein kinase activities. As shown by DEAE-cellulose chromatography, there is a peak of type I protein kinase activity in mutant extracts, but the type II protein kinase activity is considerably reduced even though free type II regulatory subunit (RII) is present. The type I kinase from the mutant has an altered RI (RI*) whose KD for the binding of 8-N3[32P] cAMP (KD = 1.3 X 10(-5) M) is increased by more than 200-fold compared to RI from the wild-type enzyme (KD = 5.5 X 10(-8) M). No differences were found between the catalytic subunits from the wild-type and mutant type I kinases. A large portion of RI in mutant and wild-type extracts is present in the free form. The RI* derived from mutant type I protein kinase shows altered labeling by 8-N3[32P]cAMP (KD = 1.3 X 10(-5) M) whereas the free RI from the mutant is labeled normally by the photoaffinity label (KD = 7.2 X 10(-8) M), suggesting that the RI* which binds to the catalytic subunit is functionally different from the free form of RI. The decreased amount of type II kinase activity in the mutant appears to be due to competition of RI* with RII for binding to the catalytic subunit. Translation of mRNA from wild-type CHO cells results in the synthesis of two different charge forms of RI, providing biochemical confirmation of two different species of RI in CHO cells. Additional biochemical evidence based on isoelectric focusing behavior of 8-N3[32P]cAMP-labeled RI species and [35S]methionine-labeled RI from mutant and wild-type extracts confirms the charge heterogeneity of RI species in CHO cells. These genetic and biochemical data taken together are consistent with the conclusion that there are at least two different species of RI present in CHO cells and that one of these species is altered in the mutant analyzed in this work.
- Published
- 1985
- Full Text
- View/download PDF
18. Cloning and expression of the gene for the major excreted protein of transformed mouse fibroblasts. A secreted lysosomal protease regulated by transformation.
- Author
-
Troen, B R, Ascherman, D, Atlas, D, and Gottesman, M M
- Abstract
The major excreted protein (MEP) of mouse fibroblast cells is the 39,000 Mr precursor to a lysosomal acid protease (cathepsin L) induced by malignant transformation, growth factors, and tumor promoters. We have cloned and characterized the gene for MEP from NIH-3T3 cells. This cosmid clone (pcosMMEP), containing the unique 12,000-base pair mouse MEP gene, has been transfected into monkey kidney (CV-1) cells and human epidermoid carcinoma (A431) cells. The stable A4MEP transfectants produce mouse MEP that is an active cathepsin which is secreted, glycosylated, and processed intracellularly to lower molecular weight forms as in the wild-type NIH-3T3 cells. The CVMEP cells (nontransformed phenotype) produce quantities of mouse MEP similar to that found in NIH-3T3 cells, whereas the A4MEP cells (transformed phenotype) produce greater amounts of MEP similar to the levels seen in Kirsten virus-transformed NIH-3T3 cells. The MEP mRNAs from both mouse cells and stably transfected human cells are the same size and have the same single major site for initiation of transcription, indicating that the cloned mouse MEP promoter is active in transfected cells.
- Published
- 1988
- Full Text
- View/download PDF
19. Two different regions of Phosphoglycoprotein are photoaffinity-labeled by azidopine*
- Author
-
Bruggemann, E P, Germann, U A, Gottesman, M M, and Pastan, I
- Abstract
Cells that express P-glycoprotein are resistant to many unrelated anticancer drugs. All evidence suggests that P-glycoprotein is a plasma membrane protein that confers multidrug resistance by actively transporting these cytotoxic drugs out of cells. The objective of our work is to locate drug binding sites on P-glycoprotein. Azidopine is a photoaffinity drug analog that specifically labels P-glycoprotein. To determine the region of P-glycoprotein that binds azidopine, we labeled P-glycoprotein with azidopine and digested the labeled protein into fragments. We then identified the labeled fragments with specific antibodies. We have determined that azidopine labels two different regions of P-glycoprotein: one region is in the amino half of P-glycoprotein, and the other is in the carboxyl half of the protein. Our results suggest that P-glycoprotein contains either two binding sites for azidopine or a single site formed by the two homologous halves of the protein.
- Published
- 1989
- Full Text
- View/download PDF
20. The predominant secreted protein of transformed murine fibroblasts carries the lysosomal mannose 6-phosphate recognition marker.
- Author
-
Sahagian, G G and Gottesman, M M
- Abstract
We have found that the major excreted protein (MEP) of transformed mouse fibroblasts, a phosphoglycoprotein of Mr = 35,000, carries the mannose 6-phosphate recognition marker. MEP secreted by Kirsten virus-transformed NIH 3T3 cells binds to a purified preparation of lysosomal enzyme phosphomannosyl receptor, and this binding is specifically inhibited by mannose 6-phosphate. 32Pi introduced into MEP by metabolic labeling of intact cells is exclusively associated with asparagine-linked oligosaccharides as indicated by sensitivity to endohexosaminidase H. Labeling studies utilizing [2-3H]mannose indicate that approximately one-fifth of the mannose residues of MEP are phosphorylated. Comparative studies of the synthesis, secretion, and uptake of MEP and the lysosomal enzyme beta-galactosidase indicate that MEP made by Kirsten virus-transformed NIH 3T3 cells is not handled in the same manner as are other lysosomal enzymes. MEP may be an unusual lysosomal protein, or both.
- Published
- 1982
- Full Text
- View/download PDF
21. The human multidrug resistance (mdr1) gene. cDNA cloning and transcription initiation.
- Author
-
Ueda, K, Clark, D P, Chen, C J, Roninson, I B, Gottesman, M M, and Pastan, I
- Abstract
Multidrug resistance in human KB carcinoma cells selected for resistance to colchicine, vinblastine, or adriamycin results from overexpression, and frequently amplification, of a specific gene (mdr1). Overlapping cDNA clones representing a complete 4.7-kilobase mdr1 transcript have been obtained from multidrug-resistant KB cells. Primer extension and S1 nuclease protection experiments show that two transcripts initiate 136 and 140 bases upstream from the first ATG codon in all human multidrug-resistant cell lines. The mdr1 gene is expressed in human normal kidney cells and HepG2 liver cells as a poly(A)+ RNA which starts from the same sites. Less prominent transcripts were found to initiate 155-180 bases upstream from the first ATG codon in vinblastine- or adriamycin-selected cell lines and 480-630 bases upstream in colchicine-selected cell lines. Southern hybridization analyses with different portions of a full-length cDNA indicate that the human mdr1 gene encompasses at least 70 kilobases of DNA amplified in all highly multidrug-resistant cell lines.
- Published
- 1987
- Full Text
- View/download PDF
22. Increased vinblastine binding to membrane vesicles from multidrug-resistant KB cells.
- Author
-
Cornwell, M M, Gottesman, M M, and Pastan, I H
- Abstract
Human KB carcinoma cells resistant to high levels of colchicine, vinblastine, vincristine, adriamycin, and actinomycin D exhibit reduced accumulation of these structurally unrelated chemotherapeutic agents (Akiyama, S.-I., Fojo, A., Hanover, J. A., Pastan, I., and Gottesman, M. M. (1985) Somatic Cell Mol. Genet. 11, 117-126; Fojo, A., Akiyama, S.-I., Gottesman, M. M., and Pastan, I. (1985) Cancer Res. 45, 3002-3007). To examine the mechanism of reduced drug accumulation in these cells, we measured [3H]vinblastine ([3H]VBL) binding to membrane vesicles made from drug-sensitive (KB-3-1), drug-resistant (KB-C4), and revertant (KB-R1) cells. Membrane vesicles from KB-C4 cells bound up to 8-fold more [3H]VBL than vesicles from the parental KB-3-1 or revertant KB-R1 cell lines. No difference in binding of [3H]dexamethasone, to which the cells are equally sensitive, was observed. The difference in [3H]VBL binding by vesicles from resistant and sensitive cells was eliminated by the addition of 10 micrograms/ml verapamil, which is known to reverse the multidrug-resistance phenotype. Drug binding by KB-C4 vesicles was osmotically insensitive, temperature-dependent, and trypsin-sensitive. Binding of [3H]VBL by KB-C4 vesicles was inhibited by vinblastine, vincristine, and daunomycin (in decreasing order). Dexamethasone at 100 microM, colchicine at 100 microM, and actinomycin D at 100 microM did not significantly inhibit [3H]VBL accumulation. No significant differences in tubulin content were detected among vesicles from sensitive and resistant cells. These data demonstrate that membrane vesicles from multiply drug-resistant cells bind increased amounts of vinblastine.
- Published
- 1986
- Full Text
- View/download PDF
23. Multiple drug-resistant human KB carcinoma cells independently selected for high-level resistance to colchicine, adriamycin, or vinblastine show changes in expression of specific proteins.
- Author
-
Shen, D W, Cardarelli, C, Hwang, J, Cornwell, M, Richert, N, Ishii, S, Pastan, I, and Gottesman, M M
- Abstract
We have established four cell lines derived from the human KB carcinoma cell line which express high-level multiple drug resistance. One of these lines was selected for resistance to colchicine, one was selected for resistance to colchicine in the presence of the tumor promoter, mezerein, one for resistance to vinblastine, and one for resistance to adriamycin. All of these cell lines are cross-resistant to the other selective agents. The development of multidrug resistance in these cultured human carcinoma cells is associated with a limited number of specific protein alterations revealed by high resolution two-dimensional gel electrophoresis and Western blot analysis. These protein alterations in multidrug-resistant lines include the decreased prevalence of members of a family of proteins of molecular mass 70,000 to 80,000 daltons, pI 4.8-5.0, the increased synthesis of a protein of molecular mass 21,000 daltons, pI 5.0, in the colchicine-resistant cell lines only, and the increased expression of a 170,000-dalton protein in membrane preparations from all of the resistant cells. The loss of the 70,000- to 80,000-dalton proteins in the multidrug-resistant lines, which can also be demonstrated by immunoprecipitation of these proteins with specific antisera, is associated with a loss of translatable mRNA for these proteins. These studies suggest that only a limited number of protein changes occur in multidrug-resistant cell lines.
- Published
- 1986
- Full Text
- View/download PDF
24. Altered drug-stimulated ATPase activity in mutants of the human multidrug resistance protein.
- Author
-
Müller, M, Bakos, E, Welker, E, Váradi, A, Germann, U A, Gottesman, M M, Morse, B S, Roninson, I B, and Sarkadi, B
- Abstract
The characteristics of P-glycoprotein (MDR1), an ATP-dependent drug extrusion pump responsible for the multidrug resistance of human cancer, were investigated in an in vitro expression system. The wild-type and several mutants of the human MDR1 cDNA were engineered into recombinant baculoviruses and the mutant proteins were expressed in Sf9 insect cells. In isolated cell membrane preparations of the virus-infected cells the MDR1-dependent drug-stimulated ATPase activity, and 8-azido-ATP binding to the MDR1 protein were studied. We found that when lysines 433 and/or 1076 were replaced by methionines in the ATP-binding domains, all these mutations abolished drug-stimulated ATPase activity independent of the MgATP concentrations applied. Photoaffinity labeling with 8-azido-ATP showed that the double lysine mutant had a decreased ATP-binding affinity. In the MDR1 mutant containing a Gly185 to Val replacement we found no significant alteration in the maximum activity of the MDR1-ATPase or in its activation by verapamil and vinblastine, and this mutation did not modify the MgATP affinity or the 8-azido-ATP binding of the transporter either. However, the Gly185 to Val mutation significantly increased the stimulation of the MDR1-ATPase by colchicine and etoposide, while slightly decreasing its stimulation by vincristine. These shifts closely correspond to the effects of this mutation on the drug-resistance profile, as observed in tumor cells. These data indicate that the Sf9-baculovirus expression system for MDR1 provides an efficient tool for examining structure-function relationships and molecular characteristics of this clinically important enzyme.
- Published
- 1996
25. Characterization of phosphorylation-defective mutants of human P-glycoprotein expressed in mammalian cells.
- Author
-
Germann, U A, Chambers, T C, Ambudkar, S V, Licht, T, Cardarelli, C O, Pastan, I, and Gottesman, M M
- Abstract
To assess the role of phosphorylation of the human multidrug resistance MDR1 gene product P-glycoprotein for its drug transport activity, phosphorylation sites within its linker region were subjected to mutational analysis. We constructed a 5A mutant, in which serines at positions 661, 667, 671, 675, and 683 were replaced by nonphosphorylatable alanine residues, and a 5D mutant carrying aspartic acid residues at the respective positions to mimic permanently phosphorylated serine residues. Transfection studies revealed that both mutants were targeted properly to the cell surface and conferred multidrug resistance by diminishing drug accumulation. In contrast to wild-type P-glycoprotein, the overexpressed 5A and the 5D mutants exhibited no detectable levels of phosphorylation, either in vivo following metabolic labeling of cells with [32P]orthophosphate or in vitro in phosphorylation assays with protein kinase C, cAMP-dependent protein kinase, or a P-glyco-protein-specific protein kinase purified from multidrug-resistant KB-V1 cells. These results reconfirm that the major P-glycoprotein phosphorylation sites are located within the linker region. Furthermore, the first direct evidence is provided that phosphorylation/dephosphorylation mechanisms do not play an essential role in the establishment of the multidrug resistance phenotype mediated by human P-glycoprotein.
- Published
- 1996
26. Studies on the metabolism of retinol and retinol-binding protein in transthyretin-deficient mice produced by homologous recombination.
- Author
-
Wei, S, Episkopou, V, Piantedosi, R, Maeda, S, Shimada, K, Gottesman, M E, and Blaner, W S
- Abstract
Tissue needs for retinoids are believed to be satisfied through the delivery in the circulation of retinol by its specific plasma transport protein, retinol-binding protein (RBP), which circulates as a 1-to-1 protein complex with transthyretin (TTR). The binding of RBP to TTR is thought to prevent filtration of retinol-RBP in the kidney and to play a role in secretion of RBP from hepatocytes. Recently a strain of mice (TTR-) that totally lacks immunoreactive TTR was produced by targeted mutagenesis. We have explored the effects of TTR deficiency on retinol and RBP metabolism in this mutant strain. In pooled plasma from the TTR- mice retinol levels averaged 6% of those of wild type animals. Similarly, plasma RBP in the TTR- mice was found to be 5% of wild type levels. Hepatic retinol and retinyl ester levels were similar for mutant and wild type mice, suggesting that the mutation affects neither the uptake nor storage of dietary retinol. Levels of retinol and retinyl esters in testis, kidney, spleen, and eye cups from TTR- mice were normal. Plasma all-trans-retinoic acid levels for the TTR- mice were 2.3-fold higher than those of wild type (425 versus 190 ng/dl). Kidney RBP levels were similar for the mutant and wild type mice and we were unable to detect intact RBP in urine from TTR- mice. Hepatic RBP levels in the TTR- mice were 60% higher than those of wild type mice (39.8 versus 25.0 micrograms of RBP/g of tissue). These data may suggest that there is a partial blockage in RBP secretion from TTR- hepatocytes that leads to lessened plasma levels of retinol-RBP.
- Published
- 1995
27. Isolation and sequence of the promoter region of the human multidrug-resistance (P-glycoprotein) gene.
- Author
-
Ueda, K, Pastan, I, and Gottesman, M M
- Abstract
Intrinsic and acquired multidrug resistance is an important problem in cancer therapy. Multidrug resistance results from overexpression of the MDR 1 gene, which encodes a drug-efflux pump called P-glycoprotein. We have isolated a 1-kilobase genomic fragment containing the major transcription initiation sites for the human MDR 1 gene. Ribonuclease protection experiments using this fragment indicate that normal human adrenal, colon, and liver cells, the human hepatoma cell line HepG2, and vinblastine-selected human KB multidrug-resistant cells initiate transcription of the MDR 1 gene at the same site within this fragment. The 0.43-kilobase region upstream from the major transcription initiation site linked to the chloramphenicol acetyltransferase gene showed promoter activity in CV-1 monkey kidney cells and in human KB cells. The putative promoter region has a consensus CAAT box and two GC box-like sequences, but no TATA sequence. This identification and isolation of promoter sequences for the MDR 1 gene will permit studies on how expression of this gene is regulated in normal human tissues and cancers.
- Published
- 1987
- Full Text
- View/download PDF
28. Transepithelial Transport of Drugs by the Multidrug Transporter in Cultured Madin-Darby Canine Kidney Cell Epithelia
- Author
-
Horio, M, Chin, K V, Currier, S J, Goldenberg, S, Williams, C, Pastan, I, Gottesman, M M, and Handler, J
- Abstract
We studied transepithelial transport of 3H-labeled hydrophobic cationic drugs in epithelia formed by wild-type and by drug-resistant Madin-Darby canine kidney (MDCk) cells that had been infected with a retrovirus carrying the multidrug-resistance (MDR1) cDNA which encodes the P-glycoprotein. P-glycoprotein is an ATP consuming plasma membrane multidrug transporter responsible for the efflux of cytotoxic chemotherapeutic drugs from resistant cancer cells. Wild-type MDCK cells have small amounts of P-glycoprotein detected by immunoprecipitation. Net transepithelial transport across wild-type MDCK epithelia was demonstrated. Basal to apical flux of 100 nMvinblastine was about six times higher than apical to basal flux. Addition of unlabeled vinblastine reduced basal to apical flux of tracer and increased apical to basal flux of tracer, a pattern expected if there is a saturable pump that extrudes vinblastine at the apical plasma membrane. Daunomycin, vincristine, and actinomycin D were also actively transported and at 20 εMthese agents inhibited transport of vinblastine, suggesting that wild-type MDCK cells have a common transporter for all these drugs. Vinblastine transport was also inhibited by 20 εMverapamil, which inhibits the multidrug transporter and reverses multidrug-resistance in non-polarized cells. Net transepithelial transport of all these cytotoxic drugs and of verapamil was much higher in epithelia formed by MDCK cells infected with a human MDR1 virus (MDR-MDCK) which is expressed on the apical surface of MDR-MDCK monolayers. Because the transport of these cytotoxic drugs and verapamil is increased in MDR-MDCK epithelia compared to wild-type MDCK epithelia, transport in both these cell populations can be attributed to P-glycoprotein. These results are consistent with a role for P-glycoprotein in multidrug secretory transport across the epithelium of the proximal tubule since P-glycoprotein is normally expressed on the apical membrane of proximal tubule cells.
- Published
- 1989
- Full Text
- View/download PDF
29. Expression of a kinase anchor protein 121 is regulated by hormones in thyroid and testicular germ cells.
- Author
-
Feliciello, A, Rubin, C S, Avvedimento, E V, and Gottesman, M E
- Abstract
Distinct A Kinase Anchor Proteins (AKAPs) immobilize and concentrate protein kinase A II (PKAII) isoforms at specific intracellular locations. AKAP121 binds and targets PKAIIalpha to the cytoplasmic surface of mitochondria. Mechanisms that control expression of this mitochondrial AKAP are unknown. We have cloned cDNA for rat AKAP121 and show that AKAP121 protein expression is regulated by thyroid stimulating hormone (TSH) and cAMP. Differentiated thyroid cells (TL5) accumulate AKAP121 upon incubation with TSH or a cAMP analog. Levels of total and newly synthesized AKAP121 mRNA also increased after treatment. AKAP121 mRNA accumulated in the presence of cycloheximide, suggesting that transcription of the anchor protein gene is directly controlled by cAMP and PKA. AKAP121 is induced with similar kinetics when an unrelated, spermatocyte-derived cell line (GC-2) is incubated with 8-chlorophenylthio-cAMP. Thus, AKAP121 concentration may be controlled by hormones that activate adenylate cyclase. This mode of regulation could provide a general mechanism for (a) enhancing the sensitivity of distal organelles to cAMP and (b) shifting the focus of cAMP-mediated signaling from cytoplasm to organelles.
- Published
- 1998
30. Deletion and Insertion Mutants of the Multidrug Transporter
- Author
-
Currier, S J, Ueda, K, Willingham, M C, Pastan, I, and Gottesman, M M
- Abstract
The multidrug transporter is a 170,000-dalton membrane glycoprotein which confers multidrug resistance through its activity as an ATP-dependent efflux pump for hydrophobic, cytotoxic drugs. To determine the essential structural components of this complex membrane transporter we have altered an MDR1 cDNA in an expression vector by deletion and insertion mutations. The structure of the transporter deduced from its amino acid sequence suggests that it consists of two homologous, perhaps functionally autonomous, halves each with six transmembrane segments and a cytoplasmic ATP-binding domain. However, several carboxyl-terminal deletions, one involving 53 amino acids, the second removing 253 amino acids, and an internal deletion within the carboxyl-terminal half of the molecule, totally eliminate the ability of the mutant transporter to confer drug resistance. An internal deletion of the amino-terminal half, which removed residues 140–229, is also nonfunctional. Small carboxylterminal deletions of up to 23 amino acids leave a functional transporter, although the removal of 23 COOH-terminal amino acids reduces its ability to confer colchicine resistance. Insertions of 4 amino acids in a transmembrane domain, and in one of the two ATP-binding regions, have no effect on activity. These studies define some of the limits of allowable deletions and insertions in the MDR1 gene, and demonstrate the requirement for two intact halves of the molecule for a functional multidrug transporter.
- Published
- 1989
- Full Text
- View/download PDF
31. Phosphorylation of the 10-nm filament protein from Chinese hamster ovary cells.
- Author
-
Cabral, F and Gottesman, M M
- Published
- 1979
- Full Text
- View/download PDF
32. A mutation affecting the catalytic subunit of cyclic AMP-dependent protein kinase in CHO cells.
- Author
-
Evain, D, Gottesman, M, Pastan, I, and Anderson, W B
- Published
- 1979
- Full Text
- View/download PDF
33. Selective effects of MgCl2 and temperature on the initiation of transcription at lac, gal, and lambda promoters.
- Author
-
Nakanishi, S, primary, Adhya, S, additional, Gottesman, M, additional, and Pastan, I, additional
- Published
- 1975
- Full Text
- View/download PDF
34. Regulation of Galactokinase Synthesis by Cyclic Adenosine 3',5'-Monophosphate in Cell-free Extracts of Escherichia coli
- Author
-
Parks, John S., primary, Gottesman, M., additional, Perlman, Robert L., additional, and Pastan, Ira, additional
- Published
- 1971
- Full Text
- View/download PDF
35. Protein-tyrosine Phosphatase PTPD1 Regulates Focal Adhesion Kinase Autophosphorylation and Cell Migration
- Author
-
Emma Villa-Moruzzi, Luca Lignitto, Max E. Gottesman, Enrico V. Avvedimento, Annalisa Carlucci, Chiara Gedressi, Corrado Garbi, Antonio Feliciello, Luigi Nezi, Carlucci, A, Gedressi, C, Lignitto, L, Nezi, L, Villa Moruzzi, E, Avvedimento, VITTORIO ENRICO, Gottesman, M, Garbi, Corrado, and Feliciello, Antonio
- Subjects
PTK2 ,Protein tyrosine phosphatase ,Biology ,Models, Biological ,Biochemistry ,Catalysis ,Focal adhesion ,Mice ,Cell Adhesion ,Animals ,Humans ,Phosphorylation ,Cell adhesion ,Molecular Biology ,src ,Cytoskeleton ,Glutathione Transferase ,FAK ,FERM domain ,Autophosphorylation ,Cell Biology ,PTPD1 ,Protein Tyrosine Phosphatases, Non-Receptor ,Actins ,Cell biology ,motility ,Focal Adhesion Protein-Tyrosine Kinases ,NIH 3T3 Cells ,Tyrosine kinase ,Signal Transduction ,Proto-oncogene tyrosine-protein kinase Src - Abstract
PTPD1 is a cytosolic nonreceptor tyrosine phosphatase and a positive regulator of the Src-epidermal growth factor transduction pathway. We show that PTPD1 localizes along actin filaments and at adhesion plaques. PTPD1 forms a stable complex via distinct molecular modules with actin, Src tyrosine kinase, and focal adhesion kinase (FAK), a scaffold protein kinase enriched at adhesion plaques. Overexpression of PTPD1 promoted cell scattering and migration, short hairpin RNA-mediated silencing of endogenous PTPD1, or expression of PTPD1 mutants lacking either catalytic activity (PTPD1(C1108S)) or the FERM domain (PTPD1(Delta1-325)) significantly reduced cell motility. PTPD1 and Src catalytic activities were both required for epidermal growth factor-induced FAK autophosphorylation at its active site and for downstream propagation of ERK1/2 signaling. Our findings demonstrate that PTPD1 is a component of a multivalent scaffold complex nucleated by FAK at specific intracellular sites. By modulating Src-FAK signaling at adhesion sites, PTPD1 promotes the cytoskeleton events that induce cell adhesion and migration.
- Published
- 2008
36. Essential Role of A-kinase Anchor Protein 121 for cAMP Signaling to Mitochondria
- Author
-
Antonio Feliciello, Tiziana de Cristofaro, Enrico V. Avvedimento, Max E. Gottesman, Stelio Varrone, Adele Affaitati, Michael D. Ginsberg, Luca Cardone, Annalisa Carlucci, Affaitati, A., Cardone, L., Carlucci, A., DE CRISTOFARO, T., Ginsberg, M. D., Varrone, Stelio, GOTTESMAM M. E, ., Avvedimento, V. E., Feliciello, Antonio, Varrone, S., Gottesman, M. E., and Avvedimento, VITTORIO ENRICO
- Subjects
A Kinase Anchor Proteins ,Apoptosis ,Biology ,Mitochondrion ,PC12 Cells ,Biochemistry ,Cyclic AMP ,Animals ,Phosphorylation ,Protein kinase A ,Molecular Biology ,Adaptor Proteins, Signal Transducing ,DNA Primers ,Base Sequence ,Cytochrome c ,Signal transducing adaptor protein ,Cell Biology ,Cyclic AMP-Dependent Protein Kinases ,Mitochondria ,Rats ,Cell biology ,Enzyme Activation ,Cytoplasm ,Caspases ,biology.protein ,Signal transduction ,Carrier Proteins ,Signal Transduction - Abstract
A-Kinase anchor proteins (AKAPs) immobilize and concentrate protein kinase A (PKA) isoforms at specific subcellular compartments. Intracellular targeting of PKA holoenzyme elicits rapid and efficient phosphorylation of target proteins, thereby increasing sensitivity of downstream effectors to cAMP action. AKAP121 targets PKA to the cytoplasmic surface of mitochondria. Here we show that conditional expression of AKAP121 in PC12 cells selectively enhances cAMP.PKA signaling to mitochondria. AKAP121 induction stimulates PKA-dependent phosphorylation of the proapoptotic protein BAD at Ser(155), inhibits release of cytochrome c from mitochondria, and protects cells from apoptosis. An AKAP121 derivative mutant that localizes on mitochondria but does not bind PKA down-regulates PKA signaling to the mitochondria and promotes apoptosis. These findings indicate that PKA anchored by AKAP121 transduces cAMP signals to the mitochondria, and it may play an important role in mitochondrial physiology.
- Published
- 2003
37. Protein-tyrosine phosphatase PTPD1 regulates focal adhesion kinase autophosphorylation and cell migration.
- Author
-
Carlucci A, Gedressi C, Lignitto L, Nezi L, Villa-Moruzzi E, Avvedimento EV, Gottesman M, Garbi C, and Feliciello A
- Subjects
- Actins chemistry, Animals, Catalysis, Cell Adhesion, Cytoskeleton metabolism, Focal Adhesion Protein-Tyrosine Kinases metabolism, Glutathione Transferase metabolism, Humans, Mice, Models, Biological, NIH 3T3 Cells, Phosphorylation, Signal Transduction, Protein Tyrosine Phosphatases, Non-Receptor metabolism
- Abstract
PTPD1 is a cytosolic nonreceptor tyrosine phosphatase and a positive regulator of the Src-epidermal growth factor transduction pathway. We show that PTPD1 localizes along actin filaments and at adhesion plaques. PTPD1 forms a stable complex via distinct molecular modules with actin, Src tyrosine kinase, and focal adhesion kinase (FAK), a scaffold protein kinase enriched at adhesion plaques. Overexpression of PTPD1 promoted cell scattering and migration, short hairpin RNA-mediated silencing of endogenous PTPD1, or expression of PTPD1 mutants lacking either catalytic activity (PTPD1(C1108S)) or the FERM domain (PTPD1(Delta1-325)) significantly reduced cell motility. PTPD1 and Src catalytic activities were both required for epidermal growth factor-induced FAK autophosphorylation at its active site and for downstream propagation of ERK1/2 signaling. Our findings demonstrate that PTPD1 is a component of a multivalent scaffold complex nucleated by FAK at specific intracellular sites. By modulating Src-FAK signaling at adhesion sites, PTPD1 promotes the cytoskeleton events that induce cell adhesion and migration.
- Published
- 2008
- Full Text
- View/download PDF
38. Biochemical basis for depressed serum retinol levels in transthyretin-deficient mice.
- Author
-
van Bennekum AM, Wei S, Gamble MV, Vogel S, Piantedosi R, Gottesman M, Episkopou V, and Blaner WS
- Subjects
- Animals, Cells, Cultured, Female, Hepatocytes cytology, Humans, Kinetics, Liver cytology, Male, Mice, Mice, Inbred Strains, Mice, Knockout, Prealbumin deficiency, Prealbumin genetics, Retinol-Binding Proteins, Plasma, Sex Characteristics, Hepatocytes metabolism, Liver metabolism, Prealbumin metabolism, Retinol-Binding Proteins genetics, Retinol-Binding Proteins metabolism, Vitamin A blood
- Abstract
Transthyretin (TTR) acts physiologically in the transport of retinol in the circulation. We previously reported the generation and partial characterization of TTR-deficient (TTR(-)) mice. TTR(-) mice have very low circulating levels of retinol and its specific transport protein, retinol-binding protein (RBP). We have examined the biochemical basis for the low plasma retinol-RBP levels. Cultured primary hepatocytes isolated from wild type (WT) and TTR(-) mice accumulated RBP in their media to an identical degree, suggesting that RBP was being secreted from the hepatocytes at the same rate. In vivo experiments support this conclusion. For the first 11 h after complete nephrectomy, the levels retinol and RBP rose in the circulations of WT and TTR(-) mice at nearly identical rates. However, human retinol-RBP injected intravenously was more rapidly cleared from the circulation (t(12) = 0.5 h for TTR(-) versus t(12) >6 h for WT) and accumulated faster in the kidneys of TTR(-) compared with WT mice. The rate of infiltration of the retinol-RBP complex from the circulation to tissue interstitial fluids was identical in both strains. Taken together, these data indicate that low circulating retinol-RBP levels in TTR(-) mice arise from increased renal filtration of the retinol-RBP complex.
- Published
- 2001
- Full Text
- View/download PDF
39. Characterization of a new member of the fatty acid-binding protein family that binds all-trans-retinol.
- Author
-
Vogel S, Mendelsohn CL, Mertz JR, Piantedosi R, Waldburger C, Gottesman ME, and Blaner WS
- Subjects
- Adipose Tissue metabolism, Amino Acid Sequence, Animals, Cell Line, Cloning, Molecular, DNA, Complementary, Escherichia coli, Gene Expression Regulation, Developmental, Heart embryology, Heart growth & development, In Situ Hybridization, Kinetics, Mice, Molecular Sequence Data, Muscle Development, Muscle, Skeletal embryology, Muscle, Skeletal growth & development, Muscle, Skeletal metabolism, Myocardium metabolism, Recombinant Proteins metabolism, Retinol-Binding Proteins chemistry, Retinol-Binding Proteins metabolism, Retinol-Binding Proteins, Cellular, Reverse Transcriptase Polymerase Chain Reaction, Sequence Alignment, Sequence Homology, Amino Acid, Spodoptera, Substrate Specificity, Transfection, Retinol-Binding Proteins genetics, Vitamin A metabolism
- Abstract
Cellular retinol-binding protein, type I (CRBP-I) and type II (CRBP-II) are the only members of the fatty acid-binding protein (FABP) family that process intracellular retinol. Heart and skeletal muscle take up postprandial retinol but express little or no CRBP-I or CRBP-II. We have identified an intracellular retinol-binding protein in these tissues. The 134-amino acid protein is encoded by a cDNA that is expressed primarily in heart, muscle and adipose tissue. It shares 57 and 56% sequence identity with CRBP-I and CRBP-II, respectively, but less than 40% with other members of the FABP family. In situ hybridization demonstrates that the protein is expressed at least as early as day 10 in developing heart and muscle tissue of the embryonic mouse. Fluorescence titrations of purified recombinant protein with retinol isomers indicates binding to all-trans-, 13-cis-, and 9-cis-retinol, with respective K(d) values of 109, 83, and 130 nm. Retinoic acids (all-trans-, 13-cis-, and 9-cis-), retinals (all-trans-, 13-cis-, and 9-cis-), fatty acids (laurate, myristate, palmitate, oleate, linoleate, arachidonate, and docosahexanoate), or fatty alcohols (palmityl, petrosenlinyl, and ricinolenyl) fail to bind. The distinct tissue expression pattern and binding specificity suggest that we have identified a novel FABP family member, cellular retinol-binding protein, type III.
- Published
- 2001
- Full Text
- View/download PDF
40. Inhibition of DnaK autophosphorylation by heat shock proteins and polypeptide substrates.
- Author
-
Panagiotidis CA, Burkholder WF, Gaitanaris GA, Gragerov A, Gottesman ME, and Silverstein SJ
- Subjects
- Escherichia coli genetics, Genes, Bacterial, HSP40 Heat-Shock Proteins, Heat-Shock Proteins biosynthesis, Heat-Shock Proteins isolation & purification, Kinetics, Phosphoproteins isolation & purification, Phosphoproteins metabolism, Phosphorylation, Plasmids, Polymerase Chain Reaction methods, Recombinant Proteins biosynthesis, Recombinant Proteins isolation & purification, Recombinant Proteins metabolism, Adenosine Triphosphatases metabolism, Bacterial Proteins metabolism, Escherichia coli metabolism, Escherichia coli Proteins, HSP70 Heat-Shock Proteins, Heat-Shock Proteins metabolism
- Abstract
DnaK, the Hsp70 of Escherichia coli, autophosphorylates in vitro. Of the two heat shock proteins that interact with DnaK, GrpE inhibits DnaK phosphorylation, whereas DnaJ has no effect on the reaction. Three synthetic peptides are shown to inhibit DnaK phosphorylation. The potency of a given peptide correlates with its affinity for the DnaK protein. A truncated DnaK that lacks the carboxyl-terminal peptide-binding domain autophosphorylates; this reaction is resistant to the inhibitory peptides. Phosphorylation of the truncated DnaK is still inhibited by GrpE, indicating that the GrpE-binding site resides in the DnaK amino-terminal domain. Thus, DnaK phosphorylation is regulated in vitro, and possibly in vivo, by physiologically relevant substrates and cofactors.
- Published
- 1994
41. Fluorescent cellular indicators are extruded by the multidrug resistance protein.
- Author
-
Homolya L, Holló Z, Germann UA, Pastan I, Gottesman MM, and Sarkadi B
- Subjects
- 3T3 Cells, ATP Binding Cassette Transporter, Subfamily B, Member 1, Animals, Biological Transport, Carrier Proteins genetics, Cell Line, Esters metabolism, Humans, Membrane Glycoproteins genetics, Mice, Carrier Proteins metabolism, Drug Resistance, Fluorescent Dyes metabolism, Membrane Glycoproteins metabolism
- Abstract
In this report we show that NIH-3T3 mouse fibroblasts stably expressing the human multidrug transporter (MDR1 or P-glycoprotein), in contrast to the control NIH-3T3 cells, actively extrude the hydrophobic acetoxymethyl ester (AM) derivatives used for cellular loading of various fluorescent calcium and pH indicators. This dye extrusion is blocked by competing substrates and inhibitors of the multidrug transporters, e.g. by verapamil, vincristine, sodium orthovanadate, oligomycin, and a monoclonal anti-MDR1 antibody. The hydrophilic free acid forms of the indicators are not exported by MDR1. We also demonstrate that in isolated cell membranes the MDR1-ATPase, similar to that by known substrates of the transporter, is stimulated by the AM derivatives of fluorescent dyes whereas the free acid forms of the dyes are without effect. Since (i) the AM derivatives of the fluorescent indicators rapidly permeate the cell membrane and are readily cleaved by high activity and large capacity cytoplasmic esterases and (ii) the free acid forms are not substrates for export by MDR1, the observations above suggest that dye extrusion by MDR1 may occur without a cytoplasmic appearance of the AM compounds. These data also call attention to the possible interaction of widely used hydrophobic fluorescent indicators with MDR1 and offer an efficient detection of MDR1-expressing tumor cells as well as a screening method for examining drug interactions with the multidrug transporter.
- Published
- 1993
42. Cloning, genomic organization, and chromosomal localization of human cathepsin L.
- Author
-
Chauhan SS, Popescu NC, Ray D, Fleischmann R, Gottesman MM, and Troen BR
- Subjects
- Amino Acid Sequence, Animals, Base Sequence, Cathepsin L, Chromosome Banding, Chromosome Mapping, Cloning, Molecular, Cysteine Endopeptidases, Exons, Gene Expression Regulation, Enzymologic, Genetic Vectors, Humans, Introns, KB Cells, Karyotyping, Lymphocytes enzymology, Mice, Molecular Sequence Data, Oligodeoxyribonucleotides, Polymerase Chain Reaction, RNA, Messenger biosynthesis, Restriction Mapping, Sequence Homology, Nucleic Acid, Tumor Cells, Cultured, Cathepsins genetics, Chromosomes, Human, Pair 9, Endopeptidases, Isoenzymes genetics
- Abstract
Cathepsin L is a lysosomal cysteine protease whose expression and secretion is induced by malignant transformation, growth factors, and tumor promoters. Many human tumors express high levels of cathepsin L, which is a broad spectrum protease with potent elastase and collagenase activities. Two published human cathepsin L cDNA sequences differ only in their 5'-untranslated regions. In this study, we demonstrate the concurrent expression of two distinct human cathepsin L mRNAs (hCATL-A and hCATL-B) in adenocarcinoma, hepatoma, and renal cancer cell lines. Cloning of the human cathepsin L gene by polymerase chain reaction amplification of genomic DNA and subsequent sequencing reveals that hCATL-A and hCATL-B mRNAs are encoded by a single gene. The 3' end of the first intron contains the 5' portion of hCATL-B and is contiguous to the second exon of the gene. These data suggest either the possibility of alternative splicing or the presence of a second promoter within the first intron of the hCATL gene. We mapped the hCATL gene to chromosome 9q21-22. Sequencing of both the mouse and human cathepsin L genes demonstrates almost complete conservation of exon and intron position, but significant divergence in intron structure, possibly reflecting differences in regulation of expression of the mouse and human cathepsin L genes.
- Published
- 1993
43. Identification of residues in the first cytoplasmic loop of P-glycoprotein involved in the function of chimeric human MDR1-MDR2 transporters.
- Author
-
Currier SJ, Kane SE, Willingham MC, Cardarelli CO, Pastan I, and Gottesman MM
- Subjects
- 3T3 Cells, ATP Binding Cassette Transporter, Subfamily B, Member 1, Amino Acid Sequence, Animals, Base Sequence, Humans, Membrane Glycoproteins metabolism, Mice, Molecular Sequence Data, Oligodeoxyribonucleotides, Recombinant Fusion Proteins chemistry, Recombinant Fusion Proteins metabolism, Restriction Mapping, Sequence Homology, Amino Acid, Transfection, Azides metabolism, Dihydropyridines metabolism, Drug Resistance genetics, Membrane Glycoproteins chemistry, Membrane Glycoproteins genetics, Protein Structure, Secondary
- Abstract
The human MDR1 gene encodes the multidrug transporter (P-glycoprotein), a multidrug efflux pump. The highly homologous MDR2 gene product does not appear to be a functional multidrug pump. We have constructed a chimeric protein in which the first intracytoplasmic loop and the third and fourth transmembrane domains of the MDR1 protein were replaced by the analogous region of MDR2. Substitution of the MDR2 sequences encompassing amino acid residues 140 to 229 resulted in 17 amino acid changes, 10 in the intracytoplasmic loop (amino acids 141-188) and 7 in the transmembrane regions. This chimeric protein was expressed on the surface of NIH 3T3 cells where it bound [3H]azidopine but did not confer drug resistance. When only 4 residues, 165, 166, 168, and 169, were changed back to MDR1 amino acids, a functional drug transporter was recovered. When residues 165, 166, 168, and 169 from MDR2 were substituted into a functional MDR1 cDNA, the resulting construction was not able to confer drug resistance. These results indicate that the major functional differences between MDR1 and MDR2 in this region of P-glycoprotein reside in a small segment of the first intracytoplasmic loop. We also independently analyzed the effect of replacing Asn183 of MDR1 with Ser which occurs in MDR2. Substitution of Ser at position 183 in combination with Val at position 185 in P-glycoprotein resulted in a relative increase in resistance to actinomycin D, vinblastine, and doxorubicin in transfected NIH 3T3 cells. These results emphasize the importance of the first intracytoplasmic loop in P-glycoprotein in determining function and relative drug specificity of the transporter.
- Published
- 1992
44. Characterization of the azidopine and vinblastine binding site of P-glycoprotein.
- Author
-
Bruggemann EP, Currier SJ, Gottesman MM, and Pastan I
- Subjects
- ATP Binding Cassette Transporter, Subfamily B, Member 1, Animals, Binding Sites, Cell Line, Cell Membrane metabolism, Colchicine pharmacology, Cyanogen Bromide, Humans, KB Cells, Kinetics, Molecular Weight, Peptide Fragments isolation & purification, Transfection, Trypsin, Verapamil pharmacology, Vinblastine pharmacology, Vincristine pharmacology, Azides metabolism, Dihydropyridines metabolism, Drug Resistance physiology, Membrane Glycoproteins metabolism, Vinblastine metabolism
- Abstract
To determine the number of drug binding sites that exist on the multidrug transporter, P-glycoprotein, we used azidopine, a dihydropyridine photoaffinity compound that reverses multidrug resistance and labels P-glycoprotein. Azidopine labels P-glycoprotein in two distinct locations: one labeled site is within the amino half of P-glycoprotein between amino acid residues 198 and 440, and the other site is within the carboxy half of the protein. Vinblastine is a cytotoxic drug that is used in cancer chemotherapy and is a substrate for transport by P-glycoprotein. We found that vinblastine inhibits azidopine labeling to approximately the same extent at each labeled site on P-glycoprotein. Because several studies have shown that amino acid residue 185 of P-glycoprotein plays a critical role in some aspects of drug binding and transport, we also studied the effect that amino acid residue 185 has on azidopine labeling. These studies show that azidopine labels both sites equivalently in both wild-type (G185) and mutant (V185) P-glycoproteins. We conclude from our results that the two halves of P-glycoprotein approach each other to form a single binding site for these drugs.
- Published
- 1992
45. Decreased catalytic subunit mRNA levels and altered catalytic subunit mRNA structure in a cAMP-resistant Chinese hamster ovary cell line.
- Author
-
Howard P, Day KH, Kim KE, Richardson J, Thomas J, Abraham I, Fleischmann RD, Gottesman MM, and Maurer RA
- Subjects
- Amino Acid Sequence, Animals, Base Sequence, Blotting, Southern, Catalysis, Cell Line, Chromatography, Gel, Cricetinae, Cricetulus, DNA analysis, DNA genetics, Molecular Sequence Data, Mutation, Nucleic Acid Conformation, Polymerase Chain Reaction, Protein Kinase Inhibitors, Protein Kinases metabolism, RNA, Messenger analysis, Transfection, Cyclic AMP metabolism, RNA, Messenger chemistry
- Abstract
The mechanisms responsible for decreased levels of cAMP-dependent protein kinase activity in a mutant Chinese hamster ovary cell line have been examined. The cAMP-resistant Chinese hamster ovary 10260 cell line was found to possess only 20% of the cAMP-dependent protein kinase activity found in wild-type cells. The presence of decreased concentrations of the catalytic subunit in these cells was confirmed through binding studies using a radiolabeled, heat-stable inhibitor of the kinase. Cloned Chinese hamster ovary catalytic subunit cDNAs were isolated, characterized, and used as hybridization probes to examine the relative concentrations of catalytic subunit mRNAs in the wild-type and 10260 cell lines. A 40-50% decrease in the concentration of the mRNA for the C alpha isozyme of the catalytic subunit was observed in 10260 cells, as compared with wild-type. This decrease in catalytic subunit mRNA concentration probably accounts for a portion of the decreased kinase activity in the mutant cells. Further analysis of C alpha mRNA by polymerase chain reaction confirmed the decreased expression of C alpha mRNA in 10260 cells and further demonstrated the presence of two different species of C alpha mRNA in the 10260 cells. One species of C alpha cDNAs was indistinguishable from the wild-type cDNA, but the other species was shorter. Nucleotide sequence analysis of the amplified cDNAs led to the identification of a 191-base pair deletion in the shorter cDNA. Gene transfer studies using wild-type and 10260 C alpha cDNAs demonstrated that the longer cDNA from the 10260 cells produced wild-type activity, but the shorter cDNA was inactive. These studies suggest that at least two alterations in gene expression are responsible for decreased cAMP-dependent protein kinase activity in the 10260 cell line. One alteration results in an approximately 2-fold decrease in the concentrations of C alpha mRNA in the cells. The other change produces two species of C alpha mRNA; one of the C alpha mRNAs does not encode an active kinase.
- Published
- 1991
46. Mechanism of cyclic AMP effect on nutrient transport in Chinese hamster ovary cells. A genetic approach.
- Author
-
LeCam A, Gottesman MM, and Pastan I
- Subjects
- 8-Bromo Cyclic Adenosine Monophosphate, Aminoisobutyric Acids metabolism, Animals, Biological Transport drug effects, Cell Line, Cricetinae, Cricetulus, Drug Resistance, Female, Kinetics, Ovary, Protein Kinases metabolism, Amino Acids metabolism, Cyclic AMP analogs & derivatives, Cyclic AMP pharmacology, Deoxy Sugars metabolism, Deoxyglucose metabolism, Methylglucosides metabolism, Methylglycosides metabolism
- Abstract
The effect of 8-bromo cyclic adenosine 3':5'-monophosphate (8-Br-cAMP) on sugar and amino acid transport was investigated in wild-type Chinese hamster ovary (CHO) cells and in mutants selected for resistance to cAMP inhibition of cell growth. In wild type cells, both 3-O-methyl-D-glucose and alpha-aminoisobutyric acid transport were decreased in cells treated for 24 h with 8-Br-cAMP; kinetic analysis indicated that a decrease in Vmax, without a significant change in Km, accounted for the lower transport capacity of 8-Br-cAMP treated cells. Among the different transport systems contributing to amino acid entry, "alanine" preferring transport system (system A) appeared to be specifically affected. The sensitivity of transport processes to 8-Br-cAMP was tested in three cAMP-resistant cell lines. When tested for their capacity to phosphorylate histones in crude extracts, one strain had apparently normal amounts of protein kinase activity, one strain had a decreased enzyme sensitivity to cAMP, and one strain had little or no enzyme activity. In all three mutants, no effect of 8-Br-cAMP on 3-O-methyl glucose and alpha-aminoisobutyric acid transport could be observed, regardless of the level of cAMP-dependent protein kinase activity. These data do not indicate whether the effect of cAMP on nutrient transport in CHO cells is the cause or consequence of growth inhibition. However, they support the conclusion that, in CHO cells, the presence of a normally functioning cAMP-dependent protein kinase appears to be necessary but may not be sufficient to observe the effects of cAMP on nutrient transport as well as cell shape and cell growth.
- Published
- 1980
47. Two different regions of P-glycoprotein [corrected] are photoaffinity-labeled by azidopine.
- Author
-
Bruggemann EP, Germann UA, Gottesman MM, and Pastan I
- Subjects
- ATP Binding Cassette Transporter, Subfamily B, Member 1, Blood Proteins metabolism, Epitopes analysis, Humans, Immune Sera, KB Cells, Membrane Glycoproteins immunology, Membrane Glycoproteins isolation & purification, Peptide Fragments isolation & purification, Serine Endopeptidases, Trypsin, Affinity Labels metabolism, Azides metabolism, Dihydropyridines metabolism, Membrane Glycoproteins metabolism
- Abstract
Cells that express P-glycoprotein are resistant to many unrelated anticancer drugs. All evidence suggests that P-glycoprotein is a plasma membrane protein that confers multidrug resistance by actively transporting these cytotoxic drugs out of cells. The objective of our work is to locate drug binding sites on P-glycoprotein. Azidopine is a photoaffinity drug analog that specifically labels P-glycoprotein. To determine the region of P-glycoprotein that binds azidopine, we labeled P-glycoprotein with azidopine and digested the labeled protein into fragments. We then identified the labeled fragments with specific antibodies. We have determined that azidopine labels two different regions of P-glycoprotein: one region is in the amino half of P-glycoprotein, and the other is in the carboxyl half of the protein. Our results suggest that P-glycoprotein contains either two binding sites for azidopine or a single site formed by the two homologous halves of the protein.
- Published
- 1989
48. Cyclic AMP-dependent phosphorylation in intact cells and in cell-free extracts from Chinese hamster ovary cells. Studies with wild type and cyclic AMP-resistant mutants.
- Author
-
LeCam A, Nicolas JC, Singh TJ, Cabral F, Pastan I, and Gottesman MM
- Subjects
- Animals, Cell Line, Cricetinae, Cricetulus, Drug Resistance, Female, Macromolecular Substances, Molecular Weight, Ovary, Phosphoproteins isolation & purification, Phosphorylation, Proteins metabolism, Cyclic AMP pharmacology, Mutation, Protein Kinases metabolism
- Published
- 1981
49. Activation of transcription at specific promoters by glycerol.
- Author
-
Nakanishi S, Adhya S, Gottesman M, and Pastan I
- Subjects
- Bacteriophages metabolism, Binding Sites, Cyclic AMP pharmacology, DNA, Bacterial metabolism, DNA-Directed RNA Polymerases isolation & purification, Dimethyl Sulfoxide pharmacology, Escherichia coli metabolism, Ethylenes pharmacology, Genes, Glycols pharmacology, Mutation, Nucleic Acid Hybridization, Potassium Chloride pharmacology, Propylene Glycols pharmacology, Stimulation, Chemical, Sucrose pharmacology, Temperature, Templates, Genetic, Tritium, Glycerol pharmacology, Operon, RNA, Bacterial biosynthesis, Transcription, Genetic
- Published
- 1974
50. Intermediate filaments from Chinese hamster ovary cells contain a single protein. Comparison with more complex systems from baby hamster kidney and mouse epidermal cells.
- Author
-
Cabral F, Gottesman MM, Zimmerman SB, and Steinert PM
- Subjects
- Animals, Cell Line, Cricetinae, Cricetulus, Electrophoresis, Polyacrylamide Gel, Female, Macromolecular Substances, Microscopy, Electron, Molecular Weight, Organ Specificity, Peptide Fragments, Protein Conformation, Trypsin, Kidney analysis, Ovary analysis, Proteins analysis, Skin analysis
- Abstract
Intermediate filaments (IF) were prepared under identical conditions from Chinese hamster ovary (CHO), baby hamster kidney (BHK), and primary mouse epidermal cells. CHO and BHK cells each have a major IF protein (Mr = 55,000); these proteins comigrate on two-dimensional gels. BHK cells have an additional protein constituent (Mr = 54,000) which is absent from CHO. The IF preparation from the epidermal cells was considerably more complex, containing at least five proteins with molecular weights between 50,000 and 70,000, none of which has a mobility similar to the CHO protein. If assembled in vitro from crude CHO IF preparations were morphologically indistinguishable from filaments assembled from other cell types. Analysis of these in vitro polymerized filaments from CHO cells using circular dichroism and wide angle x-ray diffraction revealed that the CHO filaments were structurally similar to filaments prepared from BHK or epidermal cells. The Mr = 55,000 proteins from CHO and BHK cells were found to have identical one- and two-dimensional peptide maps, demonstrating that their amino acid sequences must be very similar. These studies indicate that CHO cells possess the simplest system thus far described for studying the assembly of intermediate filaments.
- Published
- 1981
Catalog
Discovery Service for Jio Institute Digital Library
For full access to our library's resources, please sign in.