Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.6.3.44 (P-glycoprotein)
 13,344 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

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

Single-step selection with vinblastine was performed in populations of the human sarcoma cell line MES-SA, to assess cellular mechanisms of resistance to the drug and mutation rates via fluctuation analysis. At a stringent selection with 20 nM vinblastine, resulting in 5-6 logs of cell killing, the mutation rate was 7 x 10(-7)per cell generation. Analysis of variance supported the hypothesis of spontaneous mutations conferring vinblastine resistance, rather than induction of adaptive response elements. Surviving clones displayed a stable multidrug resistance phenotype over a 3-month period. All propagated clones demonstrated high levels of resistance to vinblastine and paclitaxel, and lower cross-resistance to doxorubicin and etoposide. Activation of MDR 1 gene expression and P-glycoprotein function was demonstrable in all clones. No elevation was found in the expression of the mrp gene, the LRP-56 major vault protein and beta-tubulin isotypes (M40, beta4, 5beta, and beta9) in these mutants. We conclude that initial-step resistant mechanism in these vinblastine-selected mutants commonly arises from a stochastic mutation event with activation of the MDR 1 gene.
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PMID:MDR 1 activation is the predominant resistance mechanism selected by vinblastine in MES-SA cells. 1097 Jun 91

Laulimalide is a cytotoxic natural product that stabilizes microtubules. The compound enhances tubulin assembly, and laulimalide is quantitatively comparable to paclitaxel in its effects on the reaction. Laulimalide is also active in P-glycoprotein overexpressing cells, while isolaulimalide, a congener without the drug's epoxide moiety, was reported to have negligible cytotoxic and biochemical activity [Mooberry et al. (1999) Cancer Res. 59, 653-660]. We report here that laulimalide binds at a site on tubulin polymer that is distinct from the taxoid site. We found that laulimalide, while as active as paclitaxel, epothilone A, and eleutherobin in promoting the assembly of cold-stable microtubules, was unable to inhibit the binding of radiolabeled paclitaxel or of 7-O-[N-(2,7-difluoro-4'-fluoresceincarbonyl)-L-alanyl]paclitaxel, a fluorescent paclitaxel derivative, to tubulin. Confirming this observation, we demonstrated that microtubules formed in the presence of both laulimalide and paclitaxel contained near-molar quantities, relative to tubulin, of both drugs. Laulimalide was active against cell lines resistant to paclitaxel or epothilones A and B on the basis of mutations in the M40 human beta-tubulin gene. We also report that a laulimalide analogue lacking the epoxide moiety, while less active than laulimalide in biochemical and cellular systems, is probably more active than isolaulimalide. Further exploration of the role of the epoxide in the interaction of laulimalide with tubulin is therefore justified.
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PMID:The microtubule stabilizing agent laulimalide does not bind in the taxoid site, kills cells resistant to paclitaxel and epothilones, and may not require its epoxide moiety for activity. 1211 25

Hemiasterlins are sponge-derived tripeptides that inhibit cell growth by depolymerizing existing microtubules and inhibiting microtubule assembly. Since hemiasterlins are poor substrates for P-glycoprotein, they are attractive candidates for cancer therapy and have been undergoing clinical trials. The basis of resistance to a synthetic analogue of hemiasterlin, HTI-286 (HTI), was examined in cell populations derived from ovarian carcinoma (A2780/1A9) cells selected in HTI-286. 1A9-HTI-resistant cells (1A9-HTI(R) series) were 57-89-fold resistant to HTI. Cross-resistance (3-186-fold) was observed to other tubulin depolymerizing drugs, with collateral sensitivity (2-14-fold) to tubulin polymerizing agents. Evaluation of the percentage of polymerized and soluble tubulin in 1A9 parental and 1A9-HTI(R) cells corroborated the HTI cytotoxicity data. At 22 degrees C or 37 degrees C, in the absence of any drug, the percentage of polymerized microtubules for each of the 1A9-HTI(R) populations was greater than that in the 1A9 parental cells, consistent with more stable microtubules. Furthermore, microtubules in the 1A9-HTI(R) populations were also more resistant to depolymerization at 4 degrees C and had more acetylated and detyrosinated (Glu-tubulin) alpha-tubulin, all characteristic of more stable microtubules. The 1A9-HTI(R) cell populations exhibited either a single nucleotide change in the M40 beta-tubulin isotype, S172A, or in two cell populations where no beta-tubulin mutation was detected, mutations in the Kalpha-1 alpha-tubulin isotype, S165P and R221H in one resistant cell population and I384V in another. Unlike reports of mutations resulting in reduced drug affinity, the experimental data and location of mutations are consistent with resistance to HTI-286 mediated by microtubule-stabilizing mutations in beta- or alpha-tubulin.
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PMID:Tumor cells resistant to a microtubule-depolymerizing hemiasterlin analogue, HTI-286, have mutations in alpha- or beta-tubulin and increased microtubule stability. 1551 43

Resistance to paclitaxel-based therapy is frequently encountered in the clinic. The mechanisms of intrinsic or acquired paclitaxel resistance are not well understood. We sought to characterize the resistance mechanisms that develop upon chronic exposure of a cancer cell line to paclitaxel in the presence of the P-glycoprotein reversal agent, CL-347099. The epidermoid tumor line KB-3-1 was exposed to increasing concentrations of paclitaxel and 5 micromol/L CL-347099 for up to 1 year. Cells grown in 15 nmol/L paclitaxel plus CL-347099 (KB-15-PTX/099) developed 18-fold resistance to paclitaxel and were dependent upon paclitaxel for maximal growth. They grew well and retained resistance to paclitaxel when grown in athymic mice. Cross-resistance (3- to 5-fold) was observed in tissue culture to docetaxel, the novel taxane MAC-321, and epothilone B. Collateral sensitivity (approximately 3-fold) was observed to the depolymerizing agents vinblastine, dolastatin-10, and HTI-286. KB-15-PTX/099-resistant cells did not overexpress P-glycoprotein nor did they have an alteration of [14C]paclitaxel accumulation compared with parental cells. However, a novel point mutation (T to A) resulting in Asp26 to glutamate substitution in class I (M40) beta-tubulin was found. Based on an electron crystallography structure of Zn-stabilized tubulin sheets, the phenyl ring of C-3' NHCO-C6H5 of paclitaxel makes contact with Asp26 of beta-tubulin, suggesting a ligand-induced mutation. Optimized model complexes of paclitaxel, docetaxel, and MAC-321 in beta-tubulin show a novel hydrogen bonding pattern for the glutamate mutant and rationalize the observed resistance profiles. However, a mutation in the paclitaxel binding pocket does not explain the phenotype completely. KB-15-PTX/099 cells have impaired microtubule stability as determined by a reduced percentage of tubulin in microtubules and reflected by less acetylated tubulin. These results suggest that a mutation in tubulin might affect microtubule stability as well as drug binding and contribute to the observed resistance profile.
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PMID:Paclitaxel-resistant cells have a mutation in the paclitaxel-binding region of beta-tubulin (Asp26Glu) and less stable microtubules. 1650