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Molecular Cancer Therapeutics Feb 2010The involvement of tubulin mutations as a cause of clinical drug resistance has been intensely debated in recent years. In the studies described here, we used...
The involvement of tubulin mutations as a cause of clinical drug resistance has been intensely debated in recent years. In the studies described here, we used transfection to test whether beta1-tubulin mutations and polymorphisms found in cancer patients are able to confer resistance to drugs that target microtubules. Three of four mutations (A185T, A248V, R306C, but not G437S) that we tested caused paclitaxel resistance, as indicated by the following observations: (a) essentially 100% of cells selected in paclitaxel contained transfected mutant tubulin; (b) paclitaxel resistance could be turned off using tetracycline to turn off transgene expression; (c) paclitaxel resistance increased as mutant tubulin production increased. All the paclitaxel resistance mutations disrupted microtubule assembly, conferred increased sensitivity to microtubule-disruptive drugs, and produced defects in mitosis. The results are consistent with a mechanism in which tubulin mutations alter microtubule stability in a way that counteracts drug action. These studies show that human tumor cells can acquire spontaneous mutations in beta1-tubulin that cause resistance to paclitaxel, and suggest that patients with some polymorphisms in beta1-tubulin may require higher drug concentrations for effective therapy.
Topics: Animals; Antineoplastic Agents, Phytogenic; CHO Cells; Cricetinae; Cricetulus; Demecolcine; Drug Resistance, Neoplasm; Epothilones; Humans; Mitosis; Mutation; Paclitaxel; Polymorphism, Genetic; Tubulin; Vinblastine
PubMed: 20103599
DOI: 10.1158/1535-7163.MCT-09-0674 -
The Journal of Biological Chemistry Jan 2003The human multidrug resistance P-glycoprotein (P-gp, ABCB1) uses ATP to transport many structurally diverse compounds out of the cell. It is an ABC transporter with two...
The human multidrug resistance P-glycoprotein (P-gp, ABCB1) uses ATP to transport many structurally diverse compounds out of the cell. It is an ABC transporter with two nucleotide-binding domains (NBDs) and two transmembrane domains (TMDs). Recently, we showed that the "LSGGQ" motif in one NBD ((531)LSGGQ(535) in NBD1; (1176)LSGGQ(1180) in NBD2) is adjacent to the "Walker A" sequence ((1070)GSSGCGKS(1077) in NBD2; (427)GNSGCGKS(434) in NBD1) in the other NBD (Loo, T. W., Bartlett, M. C., and Clarke, D. M. (2002) J. Biol. Chem. 277, 41303-41306). Drug substrates can stimulate or inhibit the ATPase activity of P-gp. Here, we report the effect of drug binding on cross-linking between the LSGGQ signature and Walker A sites (Cys(431)(NBD1)/C1176C(NBD2) and Cys(1074)(NBD2)/L531C(NBD1), respectively). Seven drug substrates (calcein-AM, demecolcine, cis(Z)-flupentixol, verapamil, cyclosporin A, Hoechst 33342, and trans(E)-flupentixol) were tested for their effect on oxidative cross-linking. Substrates that stimulated the ATPase activity of P-gp (calcein-AM, demecolcine, cis(Z)-flupentixol, and verapamil) increased the rate of cross-linking between Cys(431)(NBD1-Walker A)/C1176C(NBD2-LSGGQ) and between Cys(1074)(NBD2-Walker A)/L531C(NBD1-LSGGQ) when compared with cross-linking in the absence of drug substrate. By contrast, substrates that inhibited ATPase activity (cyclosporin A, Hoechst 33342, and trans(E)-flupentixol) decreased the rate of cross-linking. These results indicate that interaction between the LSGGQ motifs and Walker A sites must be essential for coupling drug binding to ATP hydrolysis. Drug binding in the transmembrane domains can induce long range conformational changes in the NBDs, such that compounds that stimulate or inhibit ATPase activity must decrease and increase, respectively, the distance between the Walker A and LSGGQ sequences.
Topics: ATP Binding Cassette Transporter, Subfamily B, Member 1; Adenosine Triphosphate; Amino Acid Sequence; Humans; Pharmaceutical Preparations; Protein Binding; Protein Conformation
PubMed: 12421806
DOI: 10.1074/jbc.M211307200 -
The Journal of Cell Biology Jul 1984Normal interphase PtK2 and A549 cells display long microtubules radiating from the microtubule-organizing center (MTOC) to the plasma membrane. Both MTOC and Golgi...
Normal interphase PtK2 and A549 cells display long microtubules radiating from the microtubule-organizing center (MTOC) to the plasma membrane. Both MTOC and Golgi apparatus are contained in the same perinuclear area. Treatment of cells with 1 microM colcemid for 2 h results in microtubule depolymerization and fragmentation of the Golgi apparatus into elements scattered throughout the cytoplasm. Both normal microtubules and the Golgi apparatus assemble again following removal of colcemid. Injection of the alpha, beta-nonhydrolyzable GTP analog, guanosine 5'(alpha, beta-methylene)diphosphate [pp(CH2)pG], into interphase cells growing in normal medium results in the formation of microtubule bundles resistant to colcemid and prevents the fragmentation of the Golgi apparatus. Injection of pp(CH2)pG into cells incubated with colcemid results in substitution of tubulin ribbons for microtubules and has no effect on the Golgi-derived elements scattered throughout the cytoplasm. Removal of colcemid 1 h after the injection of pp(CH2)pG results in polymerization of large numbers of short, single randomly oriented microtubules, whereas the Golgi apparatus remains fragmented. Treatment of cells with 10 microM taxol for 3 h results both in polymerization of microtubule bundles without relation to the MTOC in the cell periphery and fragmentation of the Golgi apparatus. The Golgi-derived fragments are present exclusively in regions of the peripheral cytoplasm enriched in microtubules. The codistribution of microtubules and Golgi elements can be reversed in taxol-treated cells by injection of a monoclonal (YL 1/2) antibody reacting specifically with the tyrosylated form of alpha-tubulin. Cells incubated with colcemid after treatment with taxol have large numbers of Golgi-derived elements in close association with colcemid-resistant microtubule bundles. Incubation of cells with 50 microM vinblastine for 90 min results in microtubule dissembly, formation of tubulin paracrystals, and fragmentation of the Golgi apparatus into elements without relation to the tubulin paracrystals.
Topics: Alkaloids; Animals; Antibodies, Monoclonal; Cell Line; Demecolcine; Golgi Apparatus; Humans; Interphase; Kidney; Lung Neoplasms; Microscopy, Fluorescence; Microtubules; Paclitaxel; Polymers; Rats; Tubulin; Vinblastine
PubMed: 6146626
DOI: 10.1083/jcb.99.1.113s -
The Journal of Biological Chemistry Jun 2003The human multidrug resistance P-glycoprotein (ABCB1) transports a broad range of structurally diverse compounds out of the cell. The transport cycle involves coupling...
The human multidrug resistance P-glycoprotein (ABCB1) transports a broad range of structurally diverse compounds out of the cell. The transport cycle involves coupling of drug binding in the transmembrane domains with ATP hydrolysis. Compounds such as verapamil stimulate ATPase activity. We used cysteine-scanning mutagenesis of the transmembrane segments and reaction with the thiol-reactive substrate analog of verapamil, methanethiosulfonate (MTS)-verapamil, to test whether it caused permanent activation of ATP hydrolysis. Here we report that one mutant, I306C(TM5) showed increased ATPase activity (8-fold higher than untreated) when treated with MTS-verapamil and isolated by nickel-chelate chromatography. Drug substrates that either enhance (calcein acetoxymethyl ester, demecolcine, and vinblastine) or inhibit (cyclosporin A and trans-(E)-flupentixol) ATPase activity of Cys-less or untreated mutant I306C P-glycoprotein did not affect the activity of MTS-verapamil-treated mutant I306C. Addition of dithiothreitol released the covalently attached verapamil, and ATPase activity returned to basal levels. Pretreatment with substrates such as cyclosporin A, demecolcine, verapamil, vinblastine, or colchicine prevented activation of mutant I306C by MTS-verapamil. The results suggest that MTS-verapamil reacts with I306C in a common drug-binding site. Covalent modification of I306C affects the long range linkage between the drug-binding site and the distal ATP-binding sites. This results in the permanent activation of ATP hydrolysis in the absence of transport. Trapping mutant I306C in a permanently activated state indicates that Ile-306 may be part of the signal to switch on ATP hydrolysis when the drug-binding site is occupied.
Topics: ATP Binding Cassette Transporter, Subfamily B, Member 1; Adenosine Triphosphatases; Adenosine Triphosphate; Binding Sites; Cell Line; Cysteine; Humans; Kidney; Mesylates; Mutagenesis; Protein Structure, Tertiary; Verapamil
PubMed: 12711602
DOI: 10.1074/jbc.C300154200 -
The Journal of Cell Biology Apr 2013The spindle assembly checkpoint (SAC) functions as a surveillance mechanism to detect chromosome misalignment and to delay anaphase until the errors are corrected. The...
The spindle assembly checkpoint (SAC) functions as a surveillance mechanism to detect chromosome misalignment and to delay anaphase until the errors are corrected. The SAC is thought to control mitosis and meiosis, including meiosis in mammalian eggs. However, it remains unknown if meiosis in the eggs of nonmammalian vertebrate species is also regulated by SAC. Using a novel karyotyping technique, we demonstrate that complete disruption of spindle microtubules in Xenopus laevis oocytes did not affect the bivalent-to-dyad transition at the time oocytes are undergoing anaphase I. These oocytes also acquired the ability to respond to parthenogenetic activation, which indicates proper metaphase II arrest. Similarly, oocytes exhibiting monopolar spindles, via inhibition of aurora B or Eg5 kinesin, underwent monopolar anaphase on time and without additional intervention. Therefore, the metaphase-to-anaphase transition in frog oocytes is not regulated by SAC.
Topics: Anaphase; Animals; Aurora Kinases; Cell Polarity; Chromosomes; Cyclin B; Cysteine; Demecolcine; Enzyme Activation; Female; Karyotyping; M Phase Cell Cycle Checkpoints; Meiosis; Metaphase; Microtubules; Nocodazole; Oocytes; Protein Serine-Threonine Kinases; Proteolysis; Spindle Apparatus; Time Factors; Xenopus; cdc42 GTP-Binding Protein
PubMed: 23569212
DOI: 10.1083/jcb.201211041 -
Molecular and Cellular Biology Aug 1999c-myc has been shown to regulate G(1)/S transition, but a role for c-myc in other phases of the cell cycle has not been identified. Exposure of cells to colcemid...
c-myc has been shown to regulate G(1)/S transition, but a role for c-myc in other phases of the cell cycle has not been identified. Exposure of cells to colcemid activates the mitotic spindle checkpoint and arrests cells transiently in metaphase. After prolonged colcemid exposure, the cells withdraw from mitosis and enter a G(1)-like state. In contrast to cells in G(1), colcemid-arrested cells have decreased G(1) cyclin-dependent kinase activity and show hypophosphorylation of the retinoblastoma protein. We have found that overexpression of c-myc causes colcemid-treated human and rodent cells to become either apoptotic or polyploid by replicating DNA without chromosomal segregation. Although c-myc-induced polyploidy is not inhibited by wild-type p53 in immortalized murine fibroblasts, overexpression of c-myc in primary fibroblasts resulted in massive apoptosis of colcemid-treated cells. We surmise that additional genes are altered in immortalized cells to suppress the apoptotic pathway and allow c-myc-overexpressing cells to progress forward in the presence of colcemid. Our results also suggest that c-myc induces DNA rereplication in this G(1)-like state by activating CDK2 activity. These observations indicate that activation of c-myc may contribute to the genomic instability commonly found in human cancers.
Topics: Animals; Apoptosis; Cell Cycle; Cell Line, Transformed; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinases; DNA Replication; Demecolcine; Fibroblasts; Gene Expression Regulation; Genes, Retinoblastoma; Genes, myc; Genes, p53; Genes, ras; Humans; Mitosis; Models, Biological; Neoplasms; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-myc; Rats; Recombinant Fusion Proteins; Spindle Apparatus
PubMed: 10409725
DOI: 10.1128/MCB.19.8.5339 -
Experimental Cell Research Jul 1995We have studied the effect of the cell synchronization agents compactin, ciclopirox olamine, mimosine, aphidicolin, ALLN, and colcemid on several parameters of cell...
We have studied the effect of the cell synchronization agents compactin, ciclopirox olamine, mimosine, aphidicolin, ALLN, and colcemid on several parameters of cell cycle progression in mitotically synchronized HeLa S3 cells. Using cell size and cyclin A and B levels as markers of cytoplasmic progression and DNA content as a measure of nuclear cell cycle position, we have examined coordination of cytoplasmic and nuclear events during induction synchrony. Each synchronizing agent was unique in its effect on the coordination of the cytoplasmic and nuclear cycle. Mimosine, aphidicolin, ALLN, and colcemid disrupted cell cycle integration while compactin and ciclopirox olamine did not. Continued net cell growth during cell cycle arrest was the most dramatic in aphidicolin-treated cells, which averaged a 60% increase in size. Mimosine, ALLN, and colcemid produced an increase in cell size of approximately 25%, and ciclopyrox olamine and compactin exerted a negligible effect. Cyclin A and B were found at mitotic (high) or G1 (low) levels, or in combination of high and low concentrations not correlated with DNA content in drug-treated cells. For example, treatment with mimosine, which arrests cells in G1 with 2C DNA, resulted in cyclin A accumulating to mitotic levels, whereas cyclin B remained at a low concentration, the first time this phenomenon has been observed. These results demonstrate that populations of synchronized cells obtained by different drug treatments are blocked at biochemically distinct cell cycle points not apparent by cytometric measurement of DNA content. Our results provide conclusive evidence that induced synchrony methods differ with respect to their impact on cell cycle organization and from the pattern seen with nonperturbing cell selection methods.
Topics: Aphidicolin; Cell Cycle; Cell Nucleus; Ciclopirox; Cyclins; Cytoplasm; DNA, Neoplasm; Demecolcine; HeLa Cells; Humans; Kinetics; Leupeptins; Mimosine; Protease Inhibitors; Pyridones; Time Factors
PubMed: 7628532
DOI: 10.1006/excr.1995.1216 -
Molecular Biology of the Cell Apr 1997It is well established that microtubules interact with intracellular membranes of eukaryotic cells. There is also evidence that tubulin, the major subunit of...
It is well established that microtubules interact with intracellular membranes of eukaryotic cells. There is also evidence that tubulin, the major subunit of microtubules, associates directly with membranes. In many cases, this association between tubulin and membranes involves hydrophobic interactions. However, neither primary sequence nor known posttranslational modifications of tubulin can account for such an interaction. The goal of this study was to determine the molecular nature of hydrophobic interactions between tubulin and membranes. Specifically, I sought to identify a posttranslational modification of tubulin that is found in membrane proteins but not in cytoplasmic proteins. One such modification is the covalent attachment of the long chain fatty acid palmitate. The possibility that tubulin is a substrate for palmitoylation was investigated. First, I found that tubulin was palmitoylated in resting platelets and that the level of palmitoylation of tubulin decreased upon activation of platelets with thrombin. Second, to obtain quantities of palmitoylated tubulin required for protein structure analysis, a cell-free system for palmitoylation of tubulin was developed and characterized. The substrates for palmitoylation were nonpolymerized tubulin and tubulin in microtubules assembled with the slowly hydrolyzable GTP analogue guanylyl-(alpha, beta)-methylene-diphosphonate. However, tubulin in Taxol-assembled microtubules was not a substrate for palmitoylation. Likewise, palmitoylation of tubulin in the cell-free system was specifically inhibited by the antimicrotubule drugs Colcemid, podophyllotoxin, nocodazole, and vinblastine. These experiments identify a previously unknown posttranslational modification of tubulin that can account for at least one type of hydrophobic interaction with intracellular membranes.
Topics: Animals; Blood Platelets; Brain Chemistry; Cell Membrane; Cell-Free System; Demecolcine; Guanosine Triphosphate; Humans; Male; Microtubules; Nocodazole; Paclitaxel; Palmitoyl Coenzyme A; Podophyllotoxin; Protein Processing, Post-Translational; Rats; Rats, Sprague-Dawley; Substrate Specificity; Swine; Thrombin; Tubulin; Vinblastine
PubMed: 9247643
DOI: 10.1091/mbc.8.4.621 -
Cell Proliferation Aug 2001Polyploid cells are made by DNA reduplication without cell division, however, it is not easy to establish polyploid mammalian cell lines. It is worth studying the...
Polyploid cells are made by DNA reduplication without cell division, however, it is not easy to establish polyploid mammalian cell lines. It is worth studying the difference in cell character between hyperploid and parent cell lines. Meth-A cells were polyploidized by demecolcine, K-252a, staurosporine and paclitaxel. The cell-cycle responses of highly polyploid Meth-A cells after the removal of the drugs were examined by flow cytometry (FCM). Meth-A cells were highly polyploidized by these drugs. The polyploid Meth-A cells gradually decreased in ploidy after the drug release. A tetraploid Meth-A cell line was established only from the demecolcine-induced polyploid Meth-A cells. The duration of G1, S and G2/M phases of the tetraploid cell line were mostly the same as those of the parent diploid cells, except that the G2/M phase was 1.5 h longer. The chromosome number of tetraploid Meth-A cell line was about twice of the diploidy. A tetraploid Meth-A cell line was established.
Topics: Animals; Antineoplastic Agents, Phytogenic; Carbazoles; Cell Count; Cell Cycle; Cell Division; DNA; Demecolcine; Enzyme Inhibitors; Flow Cytometry; Indole Alkaloids; Karyotyping; Mice; Paclitaxel; Ploidies; Sarcoma; Staurosporine; Tumor Cells, Cultured
PubMed: 11529879
DOI: 10.1046/j.1365-2184.2001.00204.x -
Molecular and Cellular Biology Mar 2007The mouse gene Recql is a member of the RecQ subfamily of DEx-H-containing DNA helicases. Five members of this family have been identified in both humans and mice, and...
The mouse gene Recql is a member of the RecQ subfamily of DEx-H-containing DNA helicases. Five members of this family have been identified in both humans and mice, and mutations in three of these, BLM, WRN, and RECQL4, are associated with human diseases and a cellular phenotype that includes genomic instability. To date, no human disease has been associated with mutations in RECQL and no cellular phenotype has been associated with its deficiency. To gain insight into the physiological function of RECQL, we disrupted Recql in mice. RECQL-deficient mice did not exhibit any apparent phenotypic differences compared to wild-type mice. Cytogenetic analyses of embryonic fibroblasts from the RECQL-deficient mice revealed aneuploidy, spontaneous chromosomal breakage, and frequent translocation events. In addition, the RECQL-deficient cells were hypersensitive to ionizing radiation, exhibited an increased load of DNA damage, and displayed elevated spontaneous sister chromatid exchanges. These results provide evidence that RECQL has a unique cellular role in the DNA repair processes required for genomic integrity. Genetic background, functional redundancy, and perhaps other factors may protect the unstressed mouse from the types of abnormalities that might be expected from the severe chromosomal aberrations detected at the cellular level.
Topics: Alleles; Animals; Cells, Cultured; Chromosomal Instability; DNA Damage; Demecolcine; Electroporation; Embryonic Stem Cells; Fibroblasts; Fluorescent Dyes; In Situ Hybridization, Fluorescence; Indoles; Male; Mice; Mice, Inbred C57BL; Mice, Inbred Strains; Mice, Knockout; RNA, Messenger; Radiation, Ionizing; RecQ Helicases; Sister Chromatid Exchange; Tissue Distribution
PubMed: 17158923
DOI: 10.1128/MCB.01620-06