Biomedical subjects

M M Gottesman

Publications and source records attributed to M M Gottesman.

At least 253 records · Page 14Linked to original sources

PubMed1986

Depolymerization of microtubules alters membrane potential and affects the motional freedom of membrane proteins.

Two independent lines of evidence were obtained indicating that microtubule depolymerization affects the functions and the physical state of membranes in intact Chinese hamster ovary cells. The first type of evidence was obtained by using the dye dihexyloxacarbocyanine iodide to measure membrane potential before and after treatment with several microtubule active agents. Microtubule depolymerization resulted in a decrease in cell fluorescence, whereas stabilization of microtubules with taxol resulted in an increase in cell fluorescence. These effects of the drugs were due to their interactions with microtubules and not to direct effects of the drugs on the plasma membranes for the following reasons: effects were time dependent and required entry into the cells as indicated by the lack of fluorescence change in a multi-drug-resistant mutant that does not accumulate antimicrotubule drugs and a colcemid-resistant tubulin mutant did not show these effects on cell fluorescence. Evidence for altered motional freedom of membrane proteins in the plasma membrane was obtained by using electron spin resonance analysis of maleimide spin probe labeled cells. This study showed that depolymerization of microtubules results in increased motional freedom of maleimide-labeled sulfhydryl group containing proteins. Taken together, these data argue that microtubules function in mammalian cells to regulate the physical state of membranes and modulate membrane potential generated across cell membranes.

Animals↗

PubMed1986

The major excreted protein (MEP) of transformed mouse cells and cathepsin L have similar protease specificity.

The major excreted protein of transformed mouse cells is an acid activable cysteine protease. In this paper, oxidized insulin B chain is shown to be a substrate for this protease. By isolation and analysis of the insulin B peptides generated by the protease, the bond specificity of this protease was determined. The bonds preferentially cleaved are glu13-ala14, leu17-val18, and tyr26-thr27. No obvious preference for a specific amino acid was found in these studies. The bond specificity of this cysteine protease for oxidized insulin B chain has been compared with that of other proteases, and it is the same as that reported for cathepsin L, suggesting that the major excreted protein and cathepsin L may be the same protein.

Amino Acid Sequence↗

PubMed1986

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.

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.

Animals↗

PubMed1986

Increased vinblastine binding to membrane vesicles from multidrug-resistant KB cells.

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.

Cell Membrane↗

PubMed1986

Human multidrug-resistant cell lines: increased mdr1 expression can precede gene amplification.

The development of simultaneous resistance to multiple structurally unrelated drugs is a major impediment to cancer chemotherapy. Multidrug resistance in human KB carcinoma cells selected in colchicine, vinblastine, or Adriamycin is associated with amplification of specific DNA sequences (the multidrug resistance locus, mdr1). During colchicine selection resistance is initially accompanied by elevated expression of a 4.5-kilobase mdr1 messenger RNA (mRNA) without amplification of the corresponding genomic sequences. During selection for increased levels of resistance, expression of this mRNA is increased simultaneously with amplification of mdr1 DNA. Increased expression and amplification of mdr1 sequences were also found in multidrug-resistant sublines of human leukemia and ovarian carcinoma cells. These results suggest that increased expression of mdr1 mRNA is a common mechanism for multidrug resistance in human cells. Activation of the mdr1 gene by mutations or epigenetic changes may precede its amplification during the development of resistance.

Animals↗

PubMed1986

The major excreted protein of transformed fibroblasts is an activable acid-protease.

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.

Animals↗

PubMed1986

Localization of multidrug resistance-associated DNA sequences to human chromosome 7.

Multidrug resistance in several human cell lines correlates with amplification or increased expression of two related DNA sequences, designated mdr1 and mdr2. These DNA sequences were used as probes for hybridization with DNA with a panel of human-mouse somatic cell hybrids and from individual human chromosomes separated by fluorescence-activated chromosome sorting. By these assays, both mdr1 and mdr2 sequences were localized to chromosome 7.

Animals↗

PubMed1986

Membrane vesicles from multidrug-resistant human cancer cells contain a specific 150- to 170-kDa protein detected by photoaffinity labeling.

Multiple drug resistance of tumor cells is a common problem in cancer therapy. We have demonstrated that membrane vesicles from highly multidrug-resistant human KB carcinoma cell lines exhibit increased specific and saturable binding of vinblastine. To identify the molecules that bind vinblastine, membrane vesicles from multidrug-resistant cells were exposed to two analogs of vinblastine, N-(p-azido-[3,5-3H]benzoyl)-N'-(beta-aminoethyl)vindesine and N-(p-azido-[3-125I]salicyl)-N'-(beta-aminoethyl)vindesine, that could be photoactivated. Our studies show the specific labeling of a 150- to 170-kDa protein in membrane vesicles from two independently selected multidrug-resistant KB cell lines, which was not seen in drug-sensitive parental or revertant cell lines. The labeling of the high molecular weight protein was inhibited in a dose-dependent manner by vinblastine with half-maximal inhibition at about 1 microM. Photolabeling was also inhibited by 100 microM vincristine or 100 microM verapamil but not by 100 microM colchicine or 100 microM dexamethasone. The data suggest that the 150- to 170-kDa protein may play an important role in the multidrug-resistance phenotype.

Affinity Labels↗

PubMed1986

Isolation of human mdr DNA sequences amplified in multidrug-resistant KB carcinoma cells.

The ability of tumor cells to develop simultaneous resistance to structurally different cytotoxic drugs constitutes a major problem in cancer chemotherapy. It was previously demonstrated that multidrug-resistant Chinese hamster cell lines contain an amplified, transcriptionally active DNA sequence designated mdr. This report presents evidence that multidrug-resistant sublines of human KB carcinoma cells, selected for resistance to either colchicine, vinblastine, or Adriamycin (doxorubicin), display amplification of two different DNA sequences homologous to the hamster mdr gene. Segments of the human mdr DNA sequences, designated mdr1 and mdr2, have been cloned. mdr1 sequences were amplified in all of the highly drug-resistant sublines and were expressed as a poly(A)+ RNA species of 4.5 kilobases that was detected in the resistant cells but not in the parental cell line. No expression of mdr2 sequences was detected. mdr2 sequences were coamplified with mdr1 in some of the multidrug-resistant sublines and, in two independently derived cell lines, underwent very similar rearrangements. The data suggest that the mdr1 gene is involved in multidrug resistance in human cells.

Carcinoma↗

PubMed1986

2,4-Dichlorobenzyl thiocyanate, an antimitotic agent that alters microtubule morphology.

A compound of simple structure, 2,4-dichlorobenzyl thiocyanate (DCBT), is an antimitotic agent with a number of unusual properties. The drug causes an extreme reorganization of microtubules in cells in culture. Most normal microtubules disappear, and remaining tubulin-containing structures appear to be bundled or aggregated. DCBT irreversibly inhibits in vitro polymerization of purified tubulin, but only after a prolonged preincubation of the protein with the drug. Binding of radiolabeled DCBT to tubulin similarly requires a long incubation time, with the reaction not being complete even after 6 hr at 37 degrees C. A specific interaction with tubulin is also shown by the crossresistance to DCBT of Colcemid-resistant cells with an altered beta-tubulin. A human KB carcinoma cell line and a Chinese hamster ovary cell line selected for crossresistance to multiple chemotherapeutic agents, including most antimitotic drugs, are sensitive to DCBT. Initial structure-function studies have demonstrated weak antimitotic and antitubulin activity with the parent compound benzyl thiocyanate. Chlorination at either position 2 or position 4 of the phenyl ring produces compounds of intermediate activity (4-chlorobenzyl thiocyanate is more active than 2-chlorobenzyl thiocyanate). The thiocyanate moiety appears to be essential for activity.

Cell Line↗

PubMed1986

The tumor promoter phorbol 12-myristate 13-acetate induces a program of altered gene expression similar to that induced by platelet-derived growth factor and transforming oncogenes.

Treatment of mouse NIH 3T3 cells with the phorbol ester tumor promoter, phorbol 12-myristate 13-acetate, results in altered transcription of several genes as measured in nuclear run-off experiments. The first set of genes, whose altered transcription occurs rapidly in the absence of protein synthesis, is typified by induction of c-myc and c-fos and decreased transcription of alpha 2 type I procollagen. This work demonstrates the existence of a second class of genes whose rapidly increased transcription requires prior protein synthesis, which is represented by the gene encoding a secreted lysosomal protein, MEP. Similar induction of MEP RNA is seen after treatment with platelet-derived growth factor or transformation with Kirsten sarcoma virus.

Actins↗

PubMed1986

Molecular genetic analysis of cAMP-dependent protein kinase.

Genetic evidence has been obtained indicating that the responsiveness of CHO cells to cAMP requires an intact cADepPK system. DNA carrying mutant RI and C subunit genes has been transferred to wild-type cells. Recipient cells carrying the DNA have been detected by selecting for expression of the phenotype for cAMP resistance. We are in the process of cloning a functional mutant RI subunit to use as a moveable genetic element that can confer cAMP resistance on recipient cells. This cloned gene should allow us to test hypotheses concerning the mechanism of cAMP regulation of transcription in mammalian cells.

Animals↗

PubMed1986

Multidrug resistance of DNA-mediated transformants is linked to transfer of the human mdr1 gene.

Mouse NIH 3T3 cells were transformed to multidrug resistance with high-molecular-weight DNA from multidrug-resistant human KB carcinoma cells. The patterns of cross resistance to colchicine, vinblastine, and doxorubicin hydrochloride (Adriamycin; Adria Laboratories Inc.) of the human donor cell line and mouse recipients were similar. The multidrug-resistant human donor cell line contains amplified sequences of the mdr1 gene which are expressed at high levels. Both primary and secondary NIH 3T3 transformants contained and expressed these amplified human mdr1 sequences. Amplification and expression of the human mdr1 sequences and amplification of cotransferred human Alu sequences in the mouse cells correlated with the degree of multidrug resistance. These data suggest that the mdr1 gene is likely to be responsible for multidrug resistance in cultured cells.

Animals↗

PubMed1986

Transfer and amplification of a mutant beta-tubulin gene results in colcemid dependence: use of the transformant to demonstrate regulation of beta-tubulin subunit levels by protein degradation.

Total genomic DNA from a temperature-sensitive, colcemid-resistant Chinese hamster ovary (CHO) cell mutant expressing an electrophoretic variant beta-tubulin was used to transform wild-type CHO cells to colcemid-resistant cells at 37 degrees C. Southern blot analysis of the transformant demonstrated the three- to fivefold amplification of one of many beta-tubulin sequences compared with that of the wild type or mutant, thereby identifying a functional tubulin gene in CHO cells. This amplification of one tubulin-coding sequence resulted in a threefold increase in two beta-tubulin mRNA species, suggesting that both species may be encoded by a single gene. Pulse-chase experiments showed that in the transformant, total beta-tubulin was synthesized and degraded faster than in the revertant or wild-type cells, so that the steady-state levels of beta-tubulin and alpha-tubulin were unchanged in the transformant compared with those of wild-type, mutant, or revertant cells. Increased ratios of mutant to wild-type beta-tubulin made the transformant dependent on microtubule-depolymerizing drugs for growth at 37 but not 34 degrees C and supersensitive to the microtubule-stabilizing drug taxol at 34 degrees C.

Animals↗

PubMed1986

The role of cAMP in regulating tumour cell growth.

Addition of cAMP to various cultured cell types has a dramatic effect on cell growth. Both positive and negative effects on growth have been demonstrated. Analysis of mutants with altered cAMP dependent protein kinases suggests that tumour cells do not require a functional endogenous cAMP system for normal cell cycling. Whether or not cAMP stimulates or inhibits cell growth depends on the cell type, the oncogene driving its growth, the dose of cAMP and the environment of the cell. The ras gene product does not appear to be a component of the adenylate cyclase system, and no other oncogenes have been shown to use cAMP as a second messenger. However, another class of oncogenes possesses a serine/threonine kinase activity analogous to that of cAMP dependent protein kinase. Several oncogene products and growth factor receptors are phosphorylated on serine residues, suggesting that some oncogene products, such as pp60src may be targets for the action of cAMP. The role of cAMP in tumour cell growth may be to modulate the activity of the oncogenes or their protein targets which control cell growth.

Adenylyl Cyclases↗

PubMed1986

Single cell analysis of daunomycin uptake and efflux in multidrug-resistant and -sensitive KB cells: effects of verapamil and other drugs.

The accumulation of daunomycin in drug-sensitive and multidrug-resistant human KB cells was examined using light microscopy to detect the inherent fluorescence of daunomycin. Intracellular accumulation of fluorescent drug occurred rapidly in parental KB cells and was markedly reduced in several multidrug-resistant mutants. The addition of verapamil, which reverses multidrug resistance, resulted in increased accumulation of daunomycin in resistant cells. In living cells, most of the daunomycin was found in the nucleus, but significant amounts were detected associated with the plasma membrane, in the cytoplasm, in organelles of the Golgi region, and in lysosomes. The nuclear fluorescence was measured using a photometer system, and the loss of daunomycin from the cells was determined under various conditions. When sensitive cells were allowed to accumulate daunomycin for 5 min at 37 degrees C and then placed in medium without the drug, daunomycin remained inside the nuclei for longer than 1 day. When resistant cells were loaded in the presence of verapamil and the verapamil was removed, the resistant cells lost daunomycin with a half-time of about 1 min. The continuous presence of verapamil markedly inhibited the loss of daunomycin from the cells. Similar results were obtained in separate experiments using [3H]daunomycin. Vinblastine, vincristine, and quinidine were also effective in stimulating daunomycin accumulation in multidrug-resistant cells and in preventing the loss of daunomycin from these resistant cells. This effect required half-maximal concentrations of 1-2 microM for verapamil, vinblastine, and quinidine. Ouabain, lanthanum, colchicine, amiloride, probenecid, and 1-beta-D-arabinofuranosylcytosine had no effect on this process. Quinine was effective at 10 microM and nifedipine was effective at 50 microM. Depletion of cellular adenosine triphosphate levels by preincubation of cells with azide and 2-deoxyglucose partially inhibited daunomycin loss from resistant cells. These single-cell measurements indicate that diminished daunomycin accumulation in multidrug-resistant cells results from accelerated energy-dependent efflux across the plasma membrane, and this efflux is inhibited by verapamil, quinidine, vincristine, and vinblastine.

Biological Transport↗

PubMed1985

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.

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.

Affinity Labels↗

PubMed1985

DNA-mediated gene transfer of a mutant regulatory subunit of cAMP-dependent protein kinase.

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.

8-Bromo Cyclic Adenosine Monophosphate↗