Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.6.3.44 (P-glycoprotein)
13,344 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The pathogenic yeast, Candida albicans, is insensitive to the anti-mitotic drug, benomyl, and to the dihydrofolate reductase inhibitor, methotrexate. Genes responsible for the intrinsic drug resistance were sought by transforming Saccharomyces cerevisiae, a yeast sensitive to both drugs, with genomic C. albicans libraries and screening on benomyl or methotrexate. Restriction analysis of plasmids isolated from benomyl- and methotrexate-resistant colonies indicated that both phenotypes were encoded by the same DNA fragment. Sequence analysis showed that the fragments were nearly identical and contained a long open reading frame of 1694 bp (ORF1) and a small ORF of 446 bp (ORF2) within ORF1 on the opposite strand. By site-directed mutagenesis, it was shown that ORF1 encoded both phenotypes. The protein had no sequence similarity to any known proteins, including beta-tubulin, dihydrofolate reductase, and the P-glycoprotein of the multi-drug resistance family. The resistance gene was detected in several C. albicans strains and in C. stellatoidea by DNA hybridization and by the polymerase chain reaction.
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PMID:Analysis of a Candida albicans gene that encodes a novel mechanism for resistance to benomyl and methotrexate. 206 11

Mammalian cell lines often become multidrug-resistant to cytotoxic drugs by amplification and/or overexpression of the P-glycoprotein (Pgy) genes. However, several malignant cell lines seem to acquire low levels of drug resistance by non-P-glycoprotein mediated mechanisms. We report here on cytogenetical signs of non-Pgy gene amplification in murine SEWA cells during the early steps of selection in Colcemid (COL). In line TC13COL0.01, rare cells exhibited a homogeneously staining region (HSR) distally in chromosome 16. As the COL-concentration was raised the HSR-chromosome was retained and, in addition, the cells developed numerous double minutes (DMs). The DMs, but not the HSR, contained amplified Pgy genes. The HSR may correspond to amplified heat shock protein 70 (Hsp70) genes, detected by Southern analysis. A second low-level COL-resistant line, TC13D70.01, contained DMs but showed no amplification of Pgy, Hsp70, Hsp90, alpha- or beta-tubulin genes. In higher COL-concentration, P-glycoprotein mediated drug resistance was induced. In contrast to actinomycin D-resistant SEWA cells, in which higher amplification levels of Pgy1 than of Pgy2 are regularly present, the COL-resistant lines showed a preference for Pgy2 gene amplification. These results are in line with the suggestion that the murine Pgy1 and Pgy2 genes have overlapping but distinct drug specificities.
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PMID:Colcemid resistance in murine SEWA cells: non-Pgy gene amplification at low levels of resistance and preferential Pgy2 gene amplification at high levels of resistance. 755 81

A multidrug-resistant lymphoma cell line resistant to 1.0 microM vincristine (designated HOB1/VCR1.0) was established. The tubulins of parental and resistant cell lines were purified by ion-exchange chromatography. Two-dimensional polyacrylamide gel electrophoresis of tubulins showed a decrease in the basic component of beta-tubulin in the HOB1/VCR1.0 cell line; native isoelectric focusing of tubulins showed decreased expression of two more basic tubulin dimers in the same cell line. The [3H]vincristine-tubulin binding studies were performed by filtration and HPLC and displayed the tubulin of HOB1/VCR1.0 cells having a weaker binding affinity to vincristine than those of parental HOB1 and HOB1/VCR0.5 cells. The binding constant Ka of purified tubulin to vincristine, calculated from the slope of the Scatchard curve, for parental HOB1 cells was 5.6 x 10(6), and that for HOB1/VCR1.0 cells was 3.1 x 10(6). The Scatchard kinetics was also used to determine the binding ability of the purified tubulins to [3H]colcemid: the Kas for parental and HOB1/VCR1.0 cells were 3.9 x 10(5) and 2.0 x 10(5), respectively. The current study suggests that high-level resistant cells, HOB1/VCR1.0, tend to express fewer tubulin isoforms of stronger binding affinities to antimitotic agents; that is, they preserve weak drug-binding forms rather than produce additional species. This may be a mechanism for the cells to protect themselves from drug injury when the P-glycoprotein cannot efficiently pump out the agent of high concentration within the cells.
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PMID:Purification and characterization of tubulin from parental and vincristine-resistant HOB1 lymphoma cells. 778 33

Vincristine (VCR) accumulation in chronic lymphatic leukemia of B-cell origin (B-CLL) has recently been shown not to be inversely correlated to P-glycoprotein (PGP) levels. Therefore, we studied, in addition to PGP expression and accumulation of VCR, the cellular beta-tubulin content in quiescent and rhIL-2 activated B-CLL cells. VCR mediates cytotoxicity by binding to tubulin. Constitutive beta-tubulin levels in B-CLL cells varied considerably. Upon activation with rhIL-2, beta-tubulin expression increased significantly. Therefore, tubulin levels could be correlated over a wide range to VCR accumulation. When the PGP-mediated drug efflux was blocked by verapamil (VRP), tubulin levels correlated linearly to VCR accumulation. All B-CLL cases expressed PGP at different levels. There was no linear correlation between PGP expression and VCR accumulation. A modulation factor m was defined as a quotient of VCR accumulation in the presence and absence of VRP to define the extent by which VRP inhibited a steady-state accumulation of VCR. The factor allowed discrimination between B-CLLs expressing low versus high PGP, irrespective of the levels of tubulin. However, PGP and beta-tubulin levels together were predictive for VCR accumulation in steady state. There was no uniform-accumulation defect for VCR in B-cell CLL because beta-tubulin and PGP were expressed independently. Non PGP-mediated VCR transport seems to play a minor role in B-cell CLL. Leukemia-associated varying of cytoskeletal organization in B-cell CLL might be one reason for the diverse cellular responses to receptor-mediated signals.
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PMID:Beta-tubulin and P-glycoprotein: major determinants of vincristine accumulation in B-CLL cells. 855 99

The efficacy of taxol against a wide range of sensitive and refractory solid tumors has prompted extensive investigation into the factors that influence its cytotoxicity. Our preliminary observations indicated that taxol had a superior antitumor effect against human cells (Daudi, K562, 2008, 2008/C13*, 2780 and C70) compared with its effect against rodent cells (WS, WR, NIH3T3, and CHO). Although verapamil, an inhibitor of P-glycoprotein function, markedly increased the efficacy of taxol against the rodent cells (WS, WR, and CHO), the expression of P-glycoprotein was found only at low levels in the WR cells. In addition, levels of the multidrug resistance-associated protein (MRP), as assessed by reverse transcriptase-polymerase chain reaction analysis, were found to be higher in the human than in the rodent cells, although MRP mRNA was not detected by northern blotting. Transport studies indicated that the reduced sensitivity of the rodent cells to taxol was due to decreased intracellular taxol levels and reduced intracellular binding. However, no correlation was found between the intracellular binding of taxol and the intracellular levels of alpha- and beta-tubulin, or the intracellular concentration of polymerized tubulin. These studies were extended further by assessing the binding of taxol to semi-purified microtubule proteins from WS, CHO and 2008/C13* cells in vitro. The microtubule protein preparations from WS, CHO and 2008/C13* cells, which have a 50-fold difference in their sensitivity to taxol, were found to bind equal amounts of radiolabeled taxol, and this binding was inhibited (80%) in the presence of unlabeled taxol. These results lead us to propose the presence in the rodent cells of an alternative taxol transport system that is distinct from the P-glycoprotein and MRP systems.
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PMID:Species-specific differences in taxol transport and cytotoxicity against human and rodent tumor cells. Evidence for an alternate transport system. 857 97

Taxol-resistant clones from a human ovarian carcinoma cell line (2008) were selected by an initial exposure to 0.05 microM (2008/13) or 0.5 microM (2008/17) taxol. Thereafter, a series of clones with increasing taxol resistance were derived from the 2008/17 and 2008/13 cells by stepwise sequential exposure to increasing concentrations of taxol. The 2008/17 clones displayed a classical P-glycoprotein-mediated drug-resistance phenotype. In contrast, the 2008/13 clones followed the classical P-glycoprotein-mediated resistance phenotype until a 245-fold taxol-resistant clone (2008/13/2) was obtained, which was followed by a further increase in the degree of resistance but significant down-regulation of P-glycoprotein expression in the 252-fold taxol-resistant 2008/13/4 cells. This clone (2008/13/4) also accumulated significantly higher intracellular levels of taxol than those expressing the P-glycoprotein. No correlation between the expression of the multidrug resistance-associated protein and taxol resistance was observed. Verapamil increased the sensitivity of all drug-resistant clones to taxol, and this was probably related to the ability of verapamil to increase the intracellular concentration of taxol (except in the case of 2008/13/4 cells). The 2008/17 clones were highly cross-resistant to Adriamycin, etoposide, and vincristine. They also displayed a low level of cross-resistance to camptothecin but were not cross-resistant to cisplatin. The taxol-resistant 2008/13 clones displayed a similar pattern of cross-resistance for all drugs (except Adriamycin). The 2008/13 clones were only 2-to 4-fold cross-resistant Adriamycin. The levels of alpha-tubulin and beta-tubulin were similar in the parental 2008 and taxol-resistant 2008/13/4 cells. Furthermore, the in vitro binding of [3H]taxol to semipurified microtubule preparations derived from the parental 2008 and the taxol-resistant 2008/13/2 and 2008/13/4 cells was similar. These results show that in human ovarian carcinoma cells resistance to taxol can be acquired via as yet undescribed mechanisms.
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PMID:Acquisition of taxol resistance via P-glycoprotein- and non-P-glycoprotein-mediated mechanisms in human ovarian carcinoma cells. 910 96

Acquired resistance to paclitaxel can be mediated by P-glycoprotein or by alterations involving tubulin. We report two paclitaxel-resistant sublines derived from 1A9 human ovarian carcinoma cells. Single-step paclitaxel selection with verapamil yielded two clones that are resistant to paclitaxel and collaterally sensitive to vinblastine. The resistant sublines are not paclitaxel-dependent, and resistance remained stable after 3 years of drug-free culture. All cell lines accumulate [3H]paclitaxel equally, and no MDR-1 mRNA was detected by polymerase chain reaction following reverse transcription. Total tubulin content is similar, but the polymerized fraction increased in parental but not in resistant cells following the paclitaxel addition. Purified tubulin from parental cells demonstrated paclitaxel-driven increased polymerization, in contrast to resistant cell tubulin, which did not polymerize under identical conditions. In contrast, epothilone B, an agent to which the resistant cells retained sensitivity, increased assembly. Comparable expression of beta-tubulin isotypes was found in parental and resistant cells, with predominant expression of the M40 and beta2 isotypes. Sequence analysis demonstrated acquired mutations in the M40 isotype at nucleotide 810 (T --> G; Phe270 --> Val) in 1A9PTX10 cells and nucleotide 1092 (G --> A; Ala364 --> Thr) in 1A9PTX22 cells. These results identify residues beta270 and beta364 as important modulators of paclitaxel's interaction with tubulin.
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PMID:Paclitaxel-resistant human ovarian cancer cells have mutant beta-tubulins that exhibit impaired paclitaxel-driven polymerization. 920 30

The lactone-bearing polyhydroxylated alkatetraene (+)-discodermolide, which was isolated from the sponge Discodermia dissoluta, induces the polymerization of purified tubulin with and without microtubule-associated proteins or GTP, and the polymers formed are stable to cold and calcium. These effects are similar to those of paclitaxel (Taxol), but discodermolide is more potent. We confirmed that these properties represent hypernucleation phenomena; we obtained lower tubulin critical concentrations and shorter polymers with discodermolide than paclitaxel under a variety of reaction conditions. Furthermore, we demonstrated that discodermolide is a competitive inhibitor with [3H]paclitaxel in binding to tubulin polymer, with an apparent Ki value of 0.4 microM. Multidrug-resistant human colon and ovarian carcinoma cells overexpressing P-glycoprotein, which are 900- and 2800-fold resistant to paclitaxel, respectively, relative to the parental lines, retained significant sensitivity to discodermolide (25- and 89-fold more resistant relative to the parental lines). Ovarian carcinoma cells that are 20-30-fold more resistant to paclitaxel than the parental line on the basis of expression of altered beta-tubulin polypeptides retained nearly complete sensitivity to discodermolide. The effects of discodermolide on the reorganization of the microtubules of Potorous tridactylis kidney epithelial cells were examined at different times. Intracellular microtubules were reorganized into bundles in interphase cells much more rapidly after discodermolide treatment compared with paclitaxel treatment. A variety of spindle aberrations were observed after treatment with both drugs. The proportions of the different types of aberration were different for the two drugs and changed with the length of drug treatment.
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PMID:The microtubule-stabilizing agent discodermolide competitively inhibits the binding of paclitaxel (Taxol) to tubulin polymers, enhances tubulin nucleation reactions more potently than paclitaxel, and inhibits the growth of paclitaxel-resistant cells. 938 24

Resistance to antimitotic agents is caused by decreased accumulation, altered tubulin, altered microtubule-associated proteins and increased metabolism. Vinca alkaloids, paclitaxel and docetaxel are actively effluxed by P-glycoprotein and/or the MRP1. Decreased intracellular accumulation is one of the major determinants of resistance to antimitotic agents. Increased tubulin levels and a decreased polymerization ratio were observed in resistant cells. Increased acetylation of tubulin and altered intracellular distribution of tubulin were also observed in resistant cells; however, the relationship between the function of tubulin and resistance remains unclear. The expression of each beta-tubulin isotype (beta 1-beta 6) is altered in resistant cells, but the functional differences among the isotypes have not been clarified. Recent evidence has demonstrated the alteration of binding properties of antimitotic agents in resistant cells. Therefore, the altered expressions of tubulin isotypes and related molecules might influence the antimitotic action and adverse events by antimitotic agents. Taxanes are metabolized and inactivated by p450 isozymes, and this is related to drug-resistant to taxanes.
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PMID:Cytoskeletons and antimitotic agents developed in Japan. 1040 40

Beta(beta)-tubulin isotype variation has recently been implicated in the modulation of resistance to paclitaxel in human lung cancer cells and in primary human ovarian tumour samples. Whether alpha-tubulin is involved in drug resistance has not been reported. We have generated a paclitaxel-resistant cell line (H460/T800) from the sensitive human lung carcinoma parental cell line NCI-H460. The resistant cells are more than 1000-fold resistant to taxol and overexpress P-glycoprotein. Interestingly, H460/T800 cells also overexpress alpha- and beta-tubulin as detected by Western blot analysis. From Northern blot analysis, the mechanism of tubulin overexpression appears to be post-transcriptional. To understand whether alpha-tubulin plays a role in drug resistance, we transfected antisense human kalpha1 cDNA construct into the H460/T800 paclitaxel-resistant cells. The antisense clones displayed a reduced alpha-tubulin expression, and the cells were 45-51% more sensitive to paclitaxel and other known antimitotic drugs, compared with vector transfected controls. Complementary experiments of transfecting the sense kalpha1 cDNA into H460 cells conferred a 1.8- to 3.3-fold increase in the IC(50) of several antimitotic agents. Our study suggests that alpha-tubulin is one of the factors that contributes to drug resistance.
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PMID:Modulation of drug resistance by alpha-tubulin in paclitaxel-resistant human lung cancer cell lines. 1093 Aug 5


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