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Epothilones are a new class of natural and potent antineoplastic agents that stabilize microtubules. Although 12,13-epoxide derivatives are potent antiproliferative agents, the activities of the corresponding 12,13-olefin analogs are significantly decreased. These data were confirmed for two new analogs, 6-propyl-EpoB (pEB) and 6-propyl-EpoD (pED), in comparison with the natural compounds EpoB͞EpoD, by using human A431, MCF7, and MDR1-overexpressing NCI͞Adr cells. By using tritiated pEB͞pED, compound uptake, release, and nuclear accumulation were investigated in A431 and NCI͞Adr cells. In these cells, epothilones can principally be recognized and exported by Verapamil-sensitive efflux pumps, which are not identical to MDR1. The degree of export depends on the structure, olefin vs. epoxide-analog, and also on the intracellular drug concentration. The accumulation of pED used at 3.5 or 70 nM, respectively, was increased in the presence of 10 M Verapamil in both cell lines 2-to 8-fold. In contrast, the intracellular levels of pEB were affected by Verapamil only at 3.5 nM pEB in NCI͞Adr (2-fold) and not in A431 cells. In addition, strong nuclear accumulation was observed for pEB (40 -50%) but not paclitaxel or pED (5-15%) in both cell lines. Our study suggests that differences in growth inhibitory efficacy between epoxide and olefin analogs may be based on different mechanisms of drug accumulation and subcellular distribution. Epothilones are a new class of natural products and potential antineoplastic agents. Although they have no structural similarities to taxanes, they exert cellular effects similarly to paclitaxel (Taxol). Thus, epothilones bind to tubulin and cause hyperstabilization of microtubules with subsequent mitotic arrest and apoptotic cell death (1-3). The molecular mechanisms by which epothilones and paclitaxel induce apoptosis remain to be elucidated.It has been demonstrated that epothilones are generally superior to paclitaxel in their ability to inhibit the proliferation of human cancer cell lines that are resistant to commonly used anticancer agents, including paclitaxel. The best understood mechanism of resistance to cytotoxic drugs, including antimicrotubule agents is drug export by multidrug-resistant pglycoprotein (MDR1) (4). Although taxanes are substrates for MDR1, epothilones are not, and thus MDR1-positive tumor cells remain sensitive to epothilones (1, 2, 5-9).The 12,13-epoxide moiety of epothilone A (EpoA) and B (EpoB) is dispensable for tubulin͞microtubule-related effects in vitro, because the corresponding olefin analogs (EpoC and EpoD), as inducers of tubulin polymerization, are equally potent to EpoA and EpoB (7,8,10). Formal removal of epoxide oxygen in EpoB, thus leading to EpoD, does cause a significant decrease (ϳ10-to 30-fold) in antiproliferative activity (9). Furthermore, exposure of tumor cells to EpoB for a 4-h period produces virtually the same growth inhibitory effect as a continuous 3-day exposure, whereas for EpoD, exposure times more than 4 h were required to produce ef...

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To identify viral myc proteins, we have prepared myc‐specific antibodies: (i) against a synthetic peptide corresponding to the nine carboxy‐terminal amino acids of the viral myc (C9); (ii) against a bacterially expressed viral myc protein obtained by inserting the SalI‐BamHI fragment of the viral MC29 DNA clone in the expression vector pPLc24. Both antisera recognize a protein of 55 000 mol. wt., p55v‐myc, in MH2‐ and OK10‐transformed fibroblasts. The protein is located in the nucleus, as shown by indirect immunofluorescence and cell fractionation. Antibodies against the C9 peptide were used to purify the p55v‐myc by immunoaffinity column purification (3000‐fold) from OK10‐ and MH2‐transformed fibroblasts. p55v‐myc binds to double‐stranded DNA in vitro as does p110gag‐myc. DNA binding in vitro is inhibited by the immunoglobulin fraction of antibodies against the bacterially expressed myc protein. Furthermore, a synthetic peptide consisting of 16 amino acids (C16) was used to isolate specific immunoglobulins which also inhibit DNA binding in vitro. OK10 codes, in addition to p55v‐myc, for a p200gag‐pol‐myc polyprotein. The majority of this protein is located in the cytoplasm (79%). The purified protein binds to single‐stranded RNA in vitro, unlike other gag‐myc or myc proteins.

The localization of the transformation‐specific proteins was analyzed in quail embryo fibroblast cell lines transformed by wild‐type avian myelocytomatosis virus MC29 and by three of its deletion mutants, Q10A, Q10C, and Q10H, with altered transforming capacities, and in a chicken fibroblast cell line transformed by the avian erythroblastosis virus (AEV). These viruses code for polyproteins consisting of part of the gag gene and of a transformation‐specific region, myc for MC29 and erb A for AEV. Analysis by indirect immunofluorescence using monoclonal antibodies against p19, the N‐terminal region of the polyprotein, showed that the gag‐myc proteins in cells transformed by the wild‐type MC29 as well as by the three deletion mutants are located in the nucleus. In contrast, cells transformed by AEV, which express the gag‐erb A protein, give rise to cytoplasmic fluorescence. Fractionation of cells into nuclear and cytoplasmic fractions and analysis by immunoprecipitation and gel electrophoresis confirmed these results. About 60% of the gag‐myc proteins of wild‐type as well as of mutant origin were found in the nucleus, while 90% of the gag‐erb A protein was present in the cytoplasm. Also, pulse‐chase analysis indicated that the gag‐myc protein rapidly accumulates in the nucleus in just 30 min. Further, it was shown that the wild‐type and also mutant gag‐myc proteins are associated with isolated chromatin. Association to chromatin was also observed for the gag‐myc protein from MC29‐transformed bone marrow cells, which are believed to be the target cells for MC29 virus in vivo.