Description of paclitaxel resistance-associated genes in ovarian and breast cancer cell lines.

PURPOSE: To identify genes involved in the paclitaxel resistance phenotype. METHODS: High-density Affymetrix HG-U95Av2 microarrays were used to quantify gene expression in the resulting cell lines, SKOV-3TR, OVCAR8TR and MCF-7TR, and their drug-sensitive parental lines, SKOV-3, OVCAR8 and MCF-7. RESULTS: Three paclitaxel-resistant human ovarian and breast cancer cell lines were established. We identified 790 (SKOV-3TR), 689 (OVCAR8TR) and 964 (MCF-7TR) transcripts that were more than twofold overexpressed relative to their expression in the corresponding parental cell line. A comparison of these transcripts identified eight genes that were significantly overexpressed in all three drug-resistant daughter cell lines. These genes included MDR1, a gene often implicated in both in vitro and in vivo resistance to multiple chemotherapeutics, including paclitaxel. The remaining seven genes have not been previously associated with resistance to paclitaxel in human cancer. Furthermore, we identified 815 (SKOV-3TR), 430 (OVCAR8TR) and 332 (MCF-7TR) transcripts that were more than twofold decreased relative to their expression in the corresponding parental cell line. Comparison of these transcripts identified three genes that were significantly underexpressed in all three drug-resistant cell lines, none of which have been previously associated with paclitaxel resistance. CONCLUSIONS: Our results confirm that the paclitaxel resistance phenotype is associated with a large number of transcriptional changes. In addition, acquired paclitaxel resistance was associated with distinct transcriptional changes in each of the cell lines studied, suggesting that paclitaxel resistance is a complex phenotype that can arise through multiple mechanisms.

Overexpression of MAGE/GAGE genes in paclitaxel/doxorubicin-resistant human cancer cell lines.

Previous studies directed at identifying paclitaxel resistance genes in a paclitaxel-resistant subclone of the human ovarian cancer cell line SKOV-3 identified a novel cancer testis antigen, Taxol resistance-associated gene 3 (TRAG-3). Because investigation suggested that TRAG-3, located on chromosome Xq28, does not directly participate in the paclitaxel-resistant phenotype, it was hypothesized that TRAG-3 might be linked to a neighboring gene that is directly involved in the drug-resistant phenotype, or alternatively, overexpression of TRAG-3 might be attributable to coregulation with other cancer testis antigens. To distinguish between these two hypotheses, expression of the genes that flank TRAG-3 was evaluated, namely the Centrin 2 gene and several members of the MAGE gene cluster. Northern analysis demonstrates overexpression of MAGE2 but not Centrin 2. Extension of this analysis to other neighboring and non-neighboring representative cancer testis antigens reveals overexpression of MAGE3, MAGE6, MAGE11, and MAGE12, as well as GAGE-2, GAGE-4, GAGE-5, GAGE-6, and GAGE-7 (clustered on Xp11) in SKOV-3(TR), as compared with SKOV-3. In addition, Affymetrix-based analysis of gene expression in SKOV-3 subclones with variable paclitaxel resistance demonstrates MAGE gene overexpression occurs early in the development of the paclitaxel-resistant phenotype, whereas GAGE gene overexpression occurs somewhat later. Evaluation of additional breast and ovarian cancer cell lines reveals MAGE/GAGE overexpression in both paclitaxel- and doxorubicin-resistant cell lines, whereas gemcitabine-resistant subclones of several ovarian cancer cell lines, including SKOV-3(GR), reveals no change in MAGE/GAGE expression. To determine whether MAGE gene overexpression contributes directly to the drug-resistant phenotype, MAGE2 or MAGE6, cDNA was introduced into the paclitaxel-sensitive human ovarian cancer cell line OVCAR8. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide cytotoxicity analysis of both MAGE2 and MAGE6 transfectants demonstrates a 4-fold increase in resistance to paclitaxel and 2-fold increase in resistance to doxorubicin but not to other drugs, such as topotecan and cisplatin, through a nonmultidrug resistance-1 mechanism. MAGE2 or MAGE6 overexpression also induces a growth advantage in OVCAR8-transfected cells. These studies suggest that the in vitro acquisition of paclitaxel and doxorubicin resistance can be associated with increased expression of a variety of both neighboring and non-neighboring cancer testis antigens genes. This does not appear to be a consequence of random genetic instability or genomic amplification of the X chromosome. These antigens, because of limited expression in normal tissues, may be suitable targets for immunotherapy and novel therapeutic strategies in the treatment of chemotherapy-resistant epithelial tumors.

Molecular description of evolving paclitaxel resistance in the SKOV-3 human ovarian carcinoma cell line.

Ovarian cancer is currently the most lethal gynecological malignancy in the United States. Although effective therapies exist, the acquisition of multidrug resistance within persisting tumor cells renders curative therapies elusive for the majority of women with ovarian cancer. In an attempt to better define the evolution of paclitaxel resistance, three SKOV-3 sublines were selected during successive rounds of exposure to increasing paclitaxel concentrations. The sublines were selected to represent early (0.003 micro M), intermediate (0.03 micro M), and late (0.3 micro M) paclitaxel resistance. RNA from these cell lines, SKOV-3(0.003TR), SKOV-3(0.03TR), and SKOV-3(0.3TR), as well as the parent cell line SKOV-3, was analyzed by cDNA array to evaluate transcript expression profiles. Arrays were performed using Affymetrix HG-U95Av2 arrays, which contain probes for approximately 9600 known human genes. Signal intensities were calculated by Microarray Suite 5.0 (Affymetrix, Santa Clara, CA). Expression patterns were analyzed by Affymetrix Data Mining Tool 3.0 with filtering of expression patterns for fold change in expression (maximum divided by minimum expression value/gene) and for variation of expression (maximum minus minimum expression value/gene). This analysis dismissed approximately 11,000 of approximately 12,000 expression patterns. The remaining approximately 1000 expression patterns were normalized and segregated into 20 partitions of a self-organizing map (SOM). The resulting SOM discriminates between genes, which are differentially expressed in early versus intermediate versus late paclitaxel resistance. For example, multidrug resistance 1 transcript expression is not elevated in SKOV-3(0.003TR) as compared with parental SKOV-3 but demonstrates elevated expression in SKOV-3(0.03TR) and SKOV-3(0.3TR). In contrast, SOM analysis demonstrates early (SKOV-3(0.003TR)) transcriptional changes in a wide variety of genes, including gene families involved in cell growth/maintenance, cell structure, signal transduction, and inflammatory response. The use of array analysis with SOMs in sublines with progressive paclitaxel resistance can successfully define an evolution of resistance. Such an analysis may be useful at defining candidate gene families involved in the early-drug resistance phenotype.

beta tubulin mutations are rare in human ovarian carcinoma.

BACKGROUND: The interaction between paclitaxel and its target, beta tubulin, is essential for effective cytotoxicity. Alterations or mutation of beta tubulin have the potential to alter paclitaxel binding and confer a drug resistant phenotype. MATERIALS AND METHODS: Twenty-nine paired tumor samples from women with ovarian cancer were examined to evaluate the incidence of exon four mutations in tumors with evolving paclitaxel resistance. Tissue was dissected from five-micron paraffin slices and analyzed for mutations in exon four of human beta tubulin by PCR-SSCP. Nested PCR primers generated three partially overlapping or neighboring fragments corresponding to exon four of beta tubulin. 32P labeled PCR fragments were then subjected to SSCP analysis polyacrylamide gel electrophoresis. RESULTS: PCR-SSCP analysis demonstrated no mutations in the twenty-nine paired tumor samples studied. CONCLUSION: This result suggests that mutations within exon four of human beta tubulin are rare in newly-diagnosed and recurrent paclitaxel resistant human ovarian cancer.

cDNA Technologies and their application to drug resistance research: power, potential and problems.

The effectiveness of systemic chemotherapy is dramatically limited by both intrinsic and acquired drug resistance. Several new technologies have been developed over the last decade to more rapidly identify underlying genetic alterations that impart a drug-resistant phenotype. Techniques such as cDNA-based subtraction technologies, SAGE analysis, and most recently cDNA array and high-density micro-array technologies have rapidly expanded our ability to detect changes in RNA transcription in drug-resistant tumors. These technologies are currently being applied to generate a large number of new hypotheses regarding the regulatory molecules underlying the drug-resistance phenotype. These techniques suggest that there is a large number of transcriptional changes which occur in the drug-resistance phenotype and future work will need to focus on dissecting which of these transcriptional changes are central to the drug resistance phenotype, both in vitro, and more importantly within clinical neoplasia. Copyright 2000 Harcourt Publishers Ltd.