Summary To investigate the role of ,B-tubulin isotype composition in resistance to paclitaxel, an anti-microtubule agent, human prostate carcinoma (DU-145) cells were intermittently exposed to increasing concentrations of paclitaxel. Cells that were selected and maintained at 10 nm paclitaxel (Pac-1 0) were fivefold resistant to the drug. Pac-1 0 cells accumulated radiolabelled paclitaxel to the same extent as DU-1 45 cells and were negative for MDR-1. Analysis of Pac-10 and DU-145 cells by flow cytometry showed similar cell cycle patterns. Immunofluorescent staining revealed an overall increase of a-and 1-tubulin levels in Pac-10 cells compared with DU-145 cells. Examination of 3-tubulin isotype composition revealed a significant increase in pill isotype in the resistant cells, both by immunofluorescence and by western blot analysis. Reverse transcription-polymerase chain reaction (RT-PCR) analysis of the isotypes confirmed the increase observed for the ,,, by exhibiting ninefold higher ,,, mRNA levels and also showed fivefold increase of the Plv. transcript. In addition, analysis of paclitaxel-resistant cells that were selected at increasing levels of the drug (Pac 2, 4, 6, 8 and 10) exhibited a positive correlation between increasing P,,, levels and increasing resistance to paclitaxel. Increased expression of specific ,B-tubulin isotypes and subsequent incorporation into microtubules may alter cellular microtubule dynamics, providing a defence against the anti-microtubule effects of paclitaxel and other tubulin-binding drugs.Paclitaxel has gained considerable attention in cancer therapy in recent years and is successfully used in treating a variety of tumours, including those of the breast, ovary and lung. In treatment of prostate cancer, paclitaxel is inactive when used as a single agent (Roth et al, 1993). However, in combination with estramustine, another anti-microtubule agent, paclitaxel has significant activity against hormone refractory prostate cancer (Hudes et al, 1995).Despite its preclinical and clinical success, the exact mechanism of action of paclitaxel is not known. At low concentrations, paclitaxel blocks mitosis by kinetic stabilization of spindle microtubules (Jordan et al, 1993). Paclitaxel differs from the other anti-microtubule agents such as vinblastine and colchicine by causing microtubule polymerization instead of depolymerization.The c4p-tubulin heterodimer is the major component of microtubules. Most of the anti-microtubule agents, including paclitaxel, vinblastine, colchicine and estramustine bind to 3-tubulin. Paclitaxel binding sites on 3-tubulin were identified at the N-terminal 31 amino acids and at residues 217-231 of the protein . These two binding sites are part of the colchicine binding site and are highly conserved among species.Both a-and ,-tubulins are encoded by multigene families and exist as several isotypes in cells. 3-Tubulin exists as six isotypes that are evolutionarily conserved across species and differ from each other predominantly at the carboxy terminus. Sever...

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In the past several years many laboratories have studied the significance of α and β tubulin proteins in antimicrotubule drug resistance. These studies have extended to several antimicrotubule agents in various carcinoma cell lines. Early studies showed mutations and alterations in the steady state soluble and polymerized α and β tubulin fractions in drug resistant cell lines Cabral et al, 1986;Minotti et al, 1991). Although the existence of different α and β tubulin isotypes and their tissue specific expression was reported in the past, only recently demonstrated were the effects of individual β tubulin isotypes on overall microtubule functions including assembly, dynamics, drug sensitivity and drug binding. Banerjee et al (1990) have shown that β III isotype depleted tubulin assembles into microtubules at a faster rate than unfractioned tubulin. These microtubules are also more sensitive to paclitaxel-induced assembly compared to unfractioned tubulin (Lu and Luduena, 1993). Subsequent studies revealed alterations in the expression of specific β tubulin isotypes as result of antimicrotubule drug resistance (Haber et al, 1995;Ranganathan et al, 1996;1998a;1998b;Kavallaris et al, 1997;Kavallaris et al, 1999). In addition, combination studies have shown that cyclosporin A enhances paclitaxel efficacy in lung carcinoma cell lines by modulating β tubulin isotype composition (Ross and Antoniono, 1999). Recently, Kavallaris et al, (1999) have shown that antisense oligonucleotides to β III isotype sensitized the drug resistant cells to paclitaxel. In addition, mutations in β I isotype were reported in paclitaxel resistant human ovarian carcinoma cells (Giannakakou et al, 1997), breast carcinoma cells (Wiesen and Horwitz, 2000) and non-small-cell lung cancer patients (Monzo et al, 1999) with tumours that were unresponsive to paclitaxel therapy.Antimicrotubule drug resistance associated changes in tubulin isotypes prompted several investigators to determine the contribution of individual isotypes by transfecting cells and examining their antimicrotubule drug response. Wu et al (1998) have shown that transfection of β IVa isotype into human leukemic cell line failed to confer resistance to paclitaxel. Blade et al (1999) have over-expressed rodent β I , β II or β IVb isotypes in CHO cells and shown that these isotypes do not confer resistance to paclitaxel. Our previous work demonstrated increases in β III and β IVa isotypes in human prostate carcinoma cells that were made resistant to estramustine or paclitaxel (Ranganathan et al, 1996(Ranganathan et al, , 1998a. In addition, acute exposures to these agents resulted in elevations of β III levels. To further understand the function of β III isotype in antimicrotubule drug response, parental DU145 human carcinoma cells were transfected with the human β III cDNA. The results presented herein indicate that regulation of tubulin in cells is complex and that attempts to increase levels of a single isotype may lead to compensatory changes in the expression of other isotypes. Sum...

All solid malignancies share characteristic traits, including unlimited cellular proliferation, evasion of immune regulation, and the propensity to metastasize. The authors have previously described that a subnuclear structure, the perinucleolar compartment (PNC), is associated with the metastatic phenotype in solid tumor cancer cells. The percentage of cancer cells that contain PNCs (PNC prevalence) is indicative of the malignancy of a tumor both in vitro and in vivo, and thus PNC prevalence is a marker that reflects metastatic capability in a population of tumor cells. Although the function of the PNC remains to be determined, the PNC is highly enriched with small RNAs and RNA binding proteins. The initial chemical biology studies using a set of anticancer drugs that disassemble PNCs revealed a direct association of the structure with DNA. Therefore, PNC prevalence reduction as a phenotypic marker can be used to identify compounds that target cellular processes required for PNC maintenance and hence used to elucidate the nature of the PNC function. Here the authors report the development of an automated high-content screening assay that is capable of detecting PNC prevalence in prostate cancer cells (PC-3M) stably expressing a green fluorescent protein (GFP)-fusion protein that localizes to the PNC. The assay was optimized using known PNC-reducing drugs and non-PNC-reducing cytotoxic drugs. After optimization, the fidelity of the assay was probed with a collection of 8284 compounds and was shown to be robust and capable of detecting known and novel PNC-reducing compounds, making it the first reported high-content phenotypic screen for small changes in nuclear structure.

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