monorden and Adenocarcinoma

The modulation of DNA topology by topoisomerase II plays a crucial role during chromosome condensation and segregation in mitosis and has thus become a highly attractive target for chemotherapeutic drugs. However, these drugs are highly toxic, and so new approaches are required. One such strategy is to target topoisomerase II-interacting proteins. Here we report the identification of potential topoisomerase II-associated proteins using immunoprecipitation, followed by 1-D and 2-D gel electrophoresis and MALDI-TOF mass spectrometry. A total of 23 proteins were identified and, of these, 17 were further validated as topoisomerase IIalpha-associated proteins by coimmunoprecipitation and Western blot. Six of the interacting proteins were cellular chaperones, including 3 members of the heat shock protein-90 (Hsp90) family, and so the effect of Hsp90 modulation on the antitumor activity of topoisomerase II drugs was tested using the sulforhodamine B assay, clonogenic assays and a xenograft model. The Hsp90 inhibitors geldanamycin, 17-AAG (17-allylamino-17-demethoxygeldanamycin) and radicicol significantly enhanced the activity of the topoisomerase II poisons etoposide and mitoxantrone in vitro and in vivo. Thus, our method of identifying topoisomerase II-interacting proteins appears to be effective, and at least 1 novel topoisomerase IIalpha-associated protein, Hsp90, may represent a valid drug target in the context of topoisomerase II-directed chemotherapy.

Topics: Adenocarcinoma; Animals; Benzoquinones; Breast Neoplasms; Carcinoma, Adenosquamous; Colonic Neoplasms; DNA Topoisomerases, Type II; Electrophoresis, Gel, Two-Dimensional; Enzyme Inhibitors; Female; HSP90 Heat-Shock Proteins; Humans; Immunoprecipitation; Lactams, Macrocyclic; Lactones; Macrolides; Mice; Mice, Nude; Molecular Chaperones; Neoplasms; Protein Binding; Quinones; Rifabutin; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Transplantation, Heterologous; Tumor Cells, Cultured

Activation of Src, which has an intrinsic protein tyrosine kinase (PTK) activity, has been demonstrated in human solid tumors, such as colorectal and breast cancers. To investigate the role of activated Src in drug resistance, we evaluated the effect of v-src on the resistance to various anti-cancer drugs using v-src-transfected HAG-1 human gallbladder adenocarcinoma cells. Compared with parental or mock-transfected HAG-1 cells, v-src-transfected HAG/src3-1 cells showed a 3.5-fold resistance to cis-diamminedichloroplatinum (II) (CDDP) but not to doxorubicin, etoposide or 5-fluorouracil. By contrast, activated H-ras, which acts downstream of src, failed to induce resistance to either of these drugs. Furthermore, wortmannin, a phosphatidylinositol (PI) 3-kinase inhibitor, and H7, a protein kinase C (PKC) inhibitor, did not alter CDDP resistance. Evaluation of the kinetics of the removal of DNA interstrand cross-links (ICLs), measured by alkaline elution, showed a significant increase in this removal in HAG/src3-1 cells as compared with mock-transfected cells, though no differences were found in the formation of DNA ICLs between these cell lines. CDDP resistance in v-src-transfected cells was reversed, if not completely, by either herbimycin A or radicicol, specific inhibitors of Src-family PTKs, suggesting that Src tyrosine kinase activity induces CDDP resistance. Moreover, significant reduction in the repair of CDDP-induced DNA ICLs was observed upon treatment with radicicol. The intracellular glutathione content and mRNA expression of topoisomerase II and metallothionein were virtually identical between these cell lines, except for topoisomerase I mRNA. Our data strongly suggest that the ability of activated src, but not ras, to induce CDDP resistance is mediated by augmentation of DNA repair through Src to downstream signal-transduction pathways distinct from either the Ras, PI 3-kinase or PKC pathway.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Adenocarcinoma; Androstadienes; Benzoquinones; Cell Survival; Cisplatin; DNA Adducts; DNA Damage; DNA Repair; Drug Resistance, Neoplasm; Enzyme Inhibitors; Gallbladder Neoplasms; Genes, src; Humans; Lactams, Macrocyclic; Lactones; Macrolides; Oncogene Protein pp60(v-src); Phosphoinositide-3 Kinase Inhibitors; Protein-Tyrosine Kinases; Quinones; Recombinant Proteins; Rifabutin; Transfection; Tumor Cells, Cultured; Wortmannin

We have described a microplate colorimetric assay to quantitate anchorage-independent cell growth, using plates coated with an anti-adhesive polymer poly (2-hydroxyethyl methacrylate) (polyHEMA).We investigated whether this method could be applied to human cancer cells of epithelial origin. HAG-1, a non-tumorigenic human gall-bladder carcinoma cell line, and its pSVneo and c-H-ras transfectants, which are also non-tumorigenic, did not grow on a polyHEMA-surface. In contrast, the v-src transformant which produced tumors in nude mice and formed colonies in soft agar, was able to proliferate on the coated surface, suggesting that tumorigenicity of human cancer cells correlates with the ability to grow on a polyHEMA-coated surface. We report on the feasibility of this method as a screening system for inhibitors of oncogenic transformation. Herbimycin A and radicicol, which have been reported to block Src function, suppressed the growth of v-src-transformed NRK and HAG-1 cells on the non-adhesive polyHEMA-surface at concentrations significantly lower than on plastic. Differences in the inhibitory concentrations were not observed with KB cells, and cytotoxic agents such as adriamycin did not show any selectivity between the 2 surfaces. Growth of ras-transformed cells on the coated surface was selectively blocked by L-739,749, a farnesyltransferase inhibitor. The results demonstrate that compounds causing reversion of transformed cells to normal, hence, selectively inhibiting cell growth in anchorage-independent conditions, can be screened using this microtiter plate assay.

Topics: Adenocarcinoma; Animals; Benzoquinones; Cell Adhesion; Cell Division; Cell Line, Transformed; Drug Screening Assays, Antitumor; Humans; Lactams, Macrocyclic; Lactones; Macrolides; Methacrylates; Mice; Neoplasms; Oligopeptides; Protein-Tyrosine Kinases; Quinones; Rifabutin; Tumor Cells, Cultured; Urinary Bladder Neoplasms