Taxol, an antimitotic, anticancer agent induces cell cycle arrest in the mitotic phase by binding to the beta-tubulin subunit and forming highly stable microtubule polymers that resist depolymerization. The predominant mechanisms associated with development of taxol-resistance are an overexpression of the P- glycoprotein (P-gp) and/or alterations in the cellular microtubules. However, we have established a 252-fold, taxol- resistant human ovarian carcinoma subline (2008/13/4) wherein, the degree of resistance could not be correlated with overexpression of P-gp, alterations in the alpha- and beta-tubulin isotypes or diseases in the drug binding affinity of the microtubules. Differential display analysis (DDA) thus far (employing 40% of the available primer pairs) has revealed the overexpression of sorcin, a calcium-binding protein and the alpha-i-1 subunit of the guanine nucleotide binding protein (G-alpha-i 1) in the 2008113/4 cells. Transfection of the parental 2008 cells with full-length sorcin cDNA induced a low level (3- to 5-fold) of taxol resistance. The association of this phenomenon with the taxol resistance phenotype in conjunction with the upregulation of the G-alpha-i-1 protein is under investigation. The objectives of this proposal are to identify the remaining differentially expressed gene(s) in the atypical taxol resistant cells by DDA. To ascertain their role in taxol-resistance, a full-length cDNA of the differentially expressed gene(s) will be transfected into the parental 2008 cells. FISH analysis will also be performed to analyze the amplification of other target genes on chromosome 7 at locus q21, since sorcin and G-alpha-i-1 reside at this locus. The transcription rate of the mdr1 gene in the 2008/13/4 cells will be assessed, to understand the basis of its dramatic downregulation in the 2008/13/4 cells. Then, immunological characterization of the atypical taxol resistance-associated protein (ATRAP) will be performed in human tissues. If antibodies for the candidate ATRAP are not available, then the recombinant protein will be synthesized in a prokaryotic host and polyclonal antibodies raised against them. The antibodies will then be employed immunohistochemically to localize ATRAP in the 2008/13/4 cells and then used to determine if the expression of a candidate ATRAP's is observed in human normal and tumor ovarian tissues prior to and during treatment with taxol. Characterization of ATRAP will help us in the future design and synthesis of newer taxol analogs that can circumvent primary drug resistance due to ATRAP.
| Parekh, Hemant K; Deng, Hong Bing; Choudhary, Khuram et al. (2002) Overexpression of sorcin, a calcium-binding protein, induces a low level of paclitaxel resistance in human ovarian and breast cancer cells. Biochem Pharmacol 63:1149-58 |
| Kumar, A; Soprano, D R; Parekh, H K (2001) Cross-resistance to the synthetic retinoid CD437 in a paclitaxel-resistant human ovarian carcinoma cell line is independent of the overexpression of retinoic acid receptor-gamma. Cancer Res 61:7552-5 |
