Mutations at Leucine 215 of β-Tubulin Affect Paclitaxel Sensitivity by Two Distinct Mechanisms

Wang, Yaqing; Yin, Shanghua; Blade, Kristie; Cooper, George; Menick, Donald R.; Cabral, Fernando

doi:10.1021/bi051207d

Cited by 31 publications

(26 citation statements)

References 29 publications

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“…Furthermore, only recently, L215I substitution has been evidenced to enhance the binding or effectiveness of paclitaxel by affecting H6-H7 loop of beta-tubulin in microtubule assembly. 28 In the present study, a non tumorigenic breast cell line and 3 breast cancer cell lines with different sensitivity to paclitaxel and already deeply biologically characterized in our laboratory, 29 have been used. Missense mutations occurred only in MCF-7 and SK-BR-3 cell lines and the presence of mutations in the same regions of tumor cell lines with different paclitaxel-chemosensitivity suggested that tubulin alterations could be unrelated to the drug response.…”

Section: Discussionmentioning

confidence: 99%

Cytoskeleton and paclitaxel sensitivity in breast cancer: The role of β‐tubulins

Tommasi¹,

Mangia²,

Lacalamita³

et al. 2007

Intl Journal of Cancer

139

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The antineoplastic effect of paclitaxel is mainly related to its ability to bind the b subunit of tubulin, thus preventing tubulin chain depolarization and inducing apoptosis. The relevance of the Class I b-tubulin characteristics have also been confirmed in the clinical setting where mutations of paclitaxel-binding site of b-tubulin Class I have been related to paclitaxel resistance in non small cell lung and ovarian cancers. In the present study, we verified the hypothesis of a relationship between molecular alterations of b-tubulin Class I and paclitaxel sensitivity in a panel of breast cell lines with different drug IC 50 . The Class I b-tubulin gene cDNA has been sequenced detecting heterozygous missense mutations (exon 1 and 4) only in MCF-7 and SK-BR-3 lines. Furthermore, the expression (at both mRNA and protein level) of the different isotypes have been analyzed demonstrating an association between low cell sensitivity to paclitaxel and Class III b-tubulin expression increasing. Antisense oligonucleotide (ODN) experiments confirmed that the inhibition of Class III b-tubulin could at least partially increase paclitaxel-chemosensitivity. The hypothesis of a relationship between b-tubulin tumor expression and paclitaxel clinical response has been finally verified in a series of 92 advanced breast cancer patients treated with a first line paclitaxel-based chemotherapy. Thirty-five percent (95% CI: 45-31) of patients with high Class III b-tubulin expression showed a disease progression vs. only 7% of patients with low expression (35% vs. 7%, p < 0.002). Our study suggests that Class III b-tubulin tumor expression could be considered a predictive biomarker of paclitaxel-clinical resistance for breast cancer patients. ' 2007 Wiley-Liss, Inc.

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Section: Discussionmentioning

confidence: 99%

Cytoskeleton and paclitaxel sensitivity in breast cancer: The role of β‐tubulins

Tommasi¹,

Mangia²,

Lacalamita³

et al. 2007

Intl Journal of Cancer

139

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“…These mutations seem to act by altering the assembly of microtubules (25). More recent studies have further shown that an L215I mutation in h-tubulin increases sensitivity to paclitaxel, most likely by increasing the binding affinity for the drug (37). Thus, a variety of structural, biochemical, and genetic studies have identified this region as having important significance in microtubule assembly, drug action, and drug resistance.…”

Section: Discussionmentioning

confidence: 99%

Amino acid substitutions at proline 220 of β-tubulin confer resistance to paclitaxel and colcemid

Yin

Cabral

Veeraraghavan³

2007

Molecular Cancer Therapeutics

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Chinese hamster ovary cells selected for resistance to paclitaxel have a high incidence of mutations affecting L215, L217, and L228 in the H6/H7 loop region of B1-tubulin. To determine whether other mutations in this loop are also capable of conferring resistance to drugs that affect microtubule assembly, saturation mutagenesis of the highly conserved P220 codon in B1-tubulin cDNA was carried out. Transfection of a mixed pool of plasmids encoding all possible amino acid substitutions at P220 followed by selection in paclitaxel produced cell lines containing P220L and P220V substitutions. Similar selections in colcemid, on the other hand, yielded cell lines with P220C, P220S, and P220T substitutions. Site-directed mutagenesis and retransfection confirmed that these mutations were responsible for drug resistance. Expression of tubulin containing the P220L and P220V mutations reduced microtubule assembly, conferred resistance to paclitaxel and epothilone A, but increased sensitivity to colcemid and vinblastine. In contrast, tubulin with the P220C, P220S, and P220T mutations increased microtubule assembly, conferred resistance to colcemid and vinblastine, but increased sensitivity to paclitaxel and epothilone A. The results are consistent with molecular modeling studies and support a drug resistance mechanism based on changes in microtubule assembly that counteract the effects of drug treatment. These studies show for the first time that different substitutions at the same amino acid residue in B1-tubulin can confer cellular resistance to either microtubule-stabilizing or microtubule-destabilizing drugs. [Mol Cancer Ther 2007; 6(10):2798 -806]

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“…41 Drug development has focused on new drugs that are resistant to the function of this transporter, 42 and perhaps synthesis of MCAK inhibitors would alleviate this problem. In vitro studies have also found that mutations in tubulin subunits cause cell lines to have conferred a resistance to paclitaxel 43,44 and vinblastine. 45,46 MCAK may function at a site distinct from paclitaxel and vinblastine on the MT, and so its inhibitory effects may not be altered by these mutations, an idea that remains to be tested in the laboratory.…”

confidence: 99%

Effects of anti-microtubule agents on microtubule organization in cells lacking the kinesin-13 MCAK

Hedrick¹,

Stout²,

Walczak³

2008

Cell Cycle

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Dynamic microtubules are necessary for proper mitotic spindle assembly and chromosome segregation during mitosis. Members of the kinesin superfamily of molecular motor proteins are important to spindle function. Of particular interest is the Kinesin-13 family member MCAK, which acts to regulate microtubule dynamics during spindle assembly and to ensure proper attachments of chromosomes to spindle microtubules. The unique ability of MCAK to regulate microtubule dynamics makes it a potential target for development of new drugs that alter spindle function. Here, we knocked down MCAK via RNAi in normal and malignant cell lines and found that the two tested malignant cell lines were acutely sensitive to MCAK knockdown, while the tested normal cells were less sensitive. In addition, we looked at the effect of combining MCAK knockdown and drug treatment with paclitaxel or vinblastine to identify spindle assembly defects. We found that MCAK knockdown increased the morphological defects of the microtubule cytoskeleton in HeLa cells caused by anti-microtubule drugs. Our studies support the idea that MCAK would be a good target for new chemotherapeutic development and may be particularly useful in combination therapies with currently available anti-microtubule agents.

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Mutations at Leucine 215 of β-Tubulin Affect Paclitaxel Sensitivity by Two Distinct Mechanisms

Cited by 31 publications

References 29 publications

Cytoskeleton and paclitaxel sensitivity in breast cancer: The role of β‐tubulins

Cytoskeleton and paclitaxel sensitivity in breast cancer: The role of β‐tubulins

Amino acid substitutions at proline 220 of β-tubulin confer resistance to paclitaxel and colcemid

Effects of anti-microtubule agents on microtubule organization in cells lacking the kinesin-13 MCAK

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