Accompanying protein alterations in malignant cells with a microtubule-polymerizing drug-resistance phenotype and a primary resistance mechanism.
Microtubules (MTs) are cytoskeletal components whose structural integrity is mandatory for the execution of many basic cell functions. Utilizing parental and drug-resistant ovarian carcinoma cell lines that have acquired point mutations in beta-tubulin and p53, we studied the level of expression and modification of proteins involved in apoptosis and MT integrity. Extending previous results, we demonstrated phosphorylation of pro-survival Bcl-x(L) in an epothilone-A resistant cell line, correlating it with drug sensitivity to tubulin-active compounds. Furthermore, Mcl-1 protein turned over more rapidly following exposure to tubulin-modifying agents, the stability of Mcl-1 protein paralleling the drug sensitivity profile of the paclitaxel or epothilone-A resistant cell lines. The observed decreases in Mcl-1 were not a consequence of G(2)M arrest, as determined by flow cytometry analysis, which showed prominent levels of Mcl-1 in the absence of any drug treatment in populations enriched in mitotic cells. We also observed that a paclitaxel-resistant cell line expressed Bax at a much lower level than the sensitive parental line [A2780(1A9)], consistent with its mutant p53 status. MT-associated protein-4 (MAP4), whose phosphorylation during specific phases of the cell cycle reduces its MT-polymerizing and -stabilizing capabilities, was phosphorylated in response to drug challenge without a change in expression. Phosphorylation of MAP4 correlated with sensitivity to tubulin-binding drugs and with a dissociation from MTs. We propose that the tubulin mutations, which result in a compromised paclitaxel:tubulin or epothilone:tubulin interaction and paclitaxel or epothilone resistance, indirectly inhibit downstream events that lead to cell death, and this, in turn, may contribute to the drug-resistance phenotype
Poruchynsky
M S
MS
NIH, The National Cancer Institute Center for Cancer Research, Bldg. 10, Rm. 12N226, 9000 Rockville Pike, Bethesda, MD 20892-1910, USA. mporuch@helix.nih.gov
Giannakakou
P
P
Ward
Y
Y
Bulinski
J C
JC
Telford
W G
WG
Robey
R W
RW
Fojo
T
T
D000972:Antineoplastic Agents, Phytogenic / Q000494:pharmacology; D019008:Drug Resistance, Neoplasm / Q000235:genetics*; D005260:Female; D005455:Fluorescent Antibody Technique; D006801:Humans; D008869:Microtubule-Associated Proteins / Q000378:metabolism; D008870:Microtubules; D064549:Myeloid Cell Leukemia Sequence 1 Protein; D009363:Neoplasm Proteins / Q000378:metabolism*; D010051:Ovarian Neoplasms / Q000473:pathology; D017239:Paclitaxel / Q000494:pharmacology*; D010641:Phenotype; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism*; D014404:Tubulin / Q000187:drug effects / Q000378:metabolism; D014407:Tumor Cells, Cultured; D014747:Vinblastine / Q000494:pharmacology; D051028:bcl-2-Associated X Protein; D051020:bcl-X Protein
D000972:Antineoplastic Agents, Phytogenic; C494826:BAX protein, human; C494811:BCL2L1 protein, human; D008869:Microtubule-Associated Proteins; D064549:Myeloid Cell Leukemia Sequence 1 Protein; D009363:Neoplasm Proteins; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D014404:Tubulin; D051028:bcl-2-Associated X Protein; D051020:bcl-X Protein; D014747:Vinblastine; D017239:Paclitaxel