Gene expression profiling of epothilone Aresistant cells

Peter Atadja, Yan Yan-Neale, Harry Towbin, Frank Buxton and Dalia Cohen Functional Genomics, Novartis Corporation, Summit, NJ 07901, USA

Abstract. In the current study, we isolated sublines of the human breast adenocarcinoma cell line MDA 435 that exhibited increasing resistance to epothilone A, a microtubule-stabilizing cytotoxic agent. The resistant cells did not express P glycoprotein or multidrug resistance-associated protein (MRP) which are known mediators of multidrug resistance (MDR). Two groups of epothilone A-resistant cells were selected: cells which exhibited low resistance to both epothilone A and Taxol, and cells which exhibit low resistance to Taxol but high resistance to epothilone A. cDNA microarrays of epothilone A-resistant and Taxol-resistant cells were utilized to further characterize epothilone A resistance. Hierarchical clustering of genes according to their levels of expression indicated that the majority of genes which were highly expressed in epothilone A-resistant cells but not in taxol-resistant MDR cells encode known interferon-inducible proteins. Genes whose expression increased with increasing epothilone A resistance include microtubule-associated GTPases, cytoskeletal proteins, cell signalling proteins and a drug metabolising enzyme. The majority of the genes that were repressed in both epothilone A- and Taxol-resistant cells encode proteins regulating cellular growth signalling mechanisms.

2002 Mechanisms of drug resistance in epilepsy: lessons from oncology. Wiley, Chichester (Novartis Foundation Symposium 243) p 119-136

A major obstacle to the effective treatment of cancer is the intrinsic or acquired resistance to chemotherapeutic agents. One form of drug resistance, known as multidrug resistance (MDR), is a phenomenon whereby cells that acquire resistance to certain cytotoxic drugs generally demonstrate cross-resistance to other, sometimes structurally and functionally unrelated drugs. Several mechanisms that mediate MDR have been identified. P glycoprotein (Pgp) is a known mediator of MDR. However, failure to completely sensitize some drug resistant tumours to chemotherapy using Pgp-reversing agents suggests the existence of multiple mechanisms of cancer drug resistance. Selecting resistant cells to non-Pgp substrates would reveal novel mechanisms of resistance.

Epothilones A and B are macrolide polyketides that were isolated in a screening programme for antifungal metabolites from myxobacteria and were found to exhibit cytotoxic activity (Bollag et al 1995). Epothilones, like Taxol, have the ability to arrest cells in mitosis, bind directly to tubulin and microtubules, cause formation of bundles of intracellular microtubules in non-mitotic cells and induce the formation of hyperstable tubulin polymers (Bollag et al 1995, Ojima et al 1999, Muhlradt & Sasse 1997, Kowalski et al 1997). However, in contrast to Taxol, epothilones A and B do not appear to be substrates for Pgp and they retain high toxicity against Pgp-expressing multidrug-resistant cancer cell lines (Bollag et al 1995). Since the epothilones are not Pgp substrates, generation and characterization of epothilone-resistant cells may be helpful for identifying novel drug resistance mechanisms.

In the present study, we generated sublines of the breast adenocarcinoma cell line MDA-MB-435 that exhibit increasing resistance to epothilone A. Epothilone A-resistant cell lines did not express the major drug efflux proteins Pgp or multidrug resistance-associated protein (MRP), and exhibited a very limited cross resistance with Taxol and epothilone B. As a first step to elucidate the mechanism of resistance to epothilone A, we analysed gene expression changes in epothilone A-resistant cell lines. Potential mediators of epothilone A resistance have been identified.

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