demecolcine and Prostatic-Neoplasms

Recent studies have implicated heat shock proteins (HSP) and heat shock transcription factor 1 (HSF1) in tumor progression. We have examined the role of HSF1 in the malignant phenotype of PC-3 prostate carcinoma cells. We have developed a dominant negative construct of HSF1 that antagonizes transcription from HSP promoters and results in the depletion of intracellular HSP 70. Our studies indicate that expression of DN-HSF1 dramatically alters the DNA content of PC-3 cells (derived from p53 null prostatic carcinoma) and inhibits aneuploidy in these cells. This effect is due to prolonged expression of DN-HSF1, and transient expression of the dominant negative factor from an inducible promoter failed to cause the effect. Inhibition of aneuploidy in p53 null PC-3 cells by DN-HSF1 expression was recapitulated by expression within the cells of wild type p53. Furthermore, cells expressing DN-HSF1 showed a profound inhibition in the development of aneuploidy when exposed to chemical agents that disrupt the mitotic spindle and prevent progression through metaphase. Inhibition of aneuploidy in PC-3 cells expressing DN-HSF1 was associated with delayed breakdown of cyclin B1 compared with controls, consistent with a role for wild type HSF1 in the regulation of cyclin B1 degradation, a key step in the control of mitosis. Our experiments therefore demonstrate that HSF1 plays a functional role in cancer cells under nonstress conditions and influences cell cycle behavior and progression through mitosis and promotes the development of the aneuploid state.

Topics: Aneuploidy; Antineoplastic Agents, Phytogenic; Cell Cycle; Cell Division; Cell Line; Cell Line, Tumor; Cells, Cultured; Cyclin B; Cyclin B1; Demecolcine; DNA; DNA-Binding Proteins; Dose-Response Relationship, Drug; Electrophoresis, Polyacrylamide Gel; Genes, Dominant; Genes, p53; Genes, Reporter; Genetic Vectors; Heat Shock Transcription Factors; Heat-Shock Proteins; HSP72 Heat-Shock Proteins; Humans; Immunoblotting; Luciferases; Male; Mitosis; Mutation; Phenotype; Ploidies; Promoter Regions, Genetic; Prostatic Neoplasms; Protein Structure, Tertiary; Resting Phase, Cell Cycle; Spectrometry, Fluorescence; Transcription Factors; Transfection

The EWS/FLI-1 fusion gene is characteristic of most cases of Ewing's sarcoma and has been shown to be crucial for tumor transformation and cell growth. In this study we demonstrate a drastic down-regulation of the EWS/FLI-1 protein, and a growth arrest, following serum depletion of Ewing's sarcoma cells. This indicates that growth factor circuits may be involved in regulation of the fusion gene product. Of four different growth factors tested, basic fibroblast growth factor (bFGF) was found to be of particular significance. In fact, upon treatment of serum-depleted cells with bFGF, expression of the EWS/FLI-1 protein and growth of the Ewing's sarcoma cells were restored. In addition, a bFGF-neutralizing antibody, which was confirmed to inhibit FGF receptor (FGFR) phosphorylation, caused down-regulation of EWS/FLI-1. Experiments using specific cell cycle blockers (thymidine and colcemide) suggest that EWS/FLI-1 is directly linked to bFGF stimulation, and not indirectly to cell proliferation. We also demonstrated expression of FGFRs in several tumor samples of Ewing's sarcoma. Taken together, our data suggest that expression of FGFR is a common feature of Ewing's sarcoma and, in particular, that the bFGF pathway may be important for the maintenance of a malignant phenotype of Ewing's sarcoma cells through up-regulating the EWS/FLI-1 protein. Oncogene (2000) 19, 4298 - 4301

Topics: Adenocarcinoma; Antibodies, Monoclonal; Bone Neoplasms; Cell Cycle; Cell Division; Chromosomes, Human, Pair 11; Chromosomes, Human, Pair 22; Culture Media, Serum-Free; Demecolcine; Drug Synergism; Epidermal Growth Factor; Fibroblast Growth Factor 2; Fibroblasts; Gene Expression Regulation, Neoplastic; Humans; Insulin-Like Growth Factor I; Male; Neoplasm Proteins; Oncogene Proteins, Fusion; Platelet-Derived Growth Factor; Prostatic Neoplasms; Proto-Oncogene Protein c-fli-1; Receptors, Fibroblast Growth Factor; RNA-Binding Protein EWS; Sarcoma, Ewing; Thymidine; Transcription Factors; Translocation, Genetic; Tumor Cells, Cultured