monorden and Cell-Transformation--Viral

Angiogenesis consists of endothelial cell proliferation, migration and tube formation. It is useful to investigate endothelial cell behavior using immortalized endothelial cell lines. We characterized cell growth property, growth factor dependency and response to angioinhibitory drugs; TNP-470, staurosporine, radicicol and genistein, using human umbilical vein endothelial cells (HUVECs) immortalized by human papilloma virus (HPV)-16 E6-E7, named HUVECs/E6-E7, and HUVECs/E6-E7 transformed by v-Ki-ras gene, named HUVECs/E6-E7/ras. The dependency to vascular endothelial cell growth factor (VEGF) and basic fibroblast growth factor (bFGF) for cell proliferation decreased in HUVECs/E6-E7, but were restored in HUVECs/E6-E7/ras. Flow cytometric analysis demonstrated that a VEGF receptor KDR/flk-1 was down-regulated in HUVECs/E6-E7 but not in HUVECs/E6-E7/ras. Expression of another VEGF receptor flt-1 was consistent in all cells including HUVECs, HUVECs/E6-E7 and HUVECs/E6-E7/ras. According to the analysis of the angioinhibitory drugs, HUVECs/E6-E7 was obviously resistant to TNP-470, but HUVECs/E6-E7/ras showed similar response compared to HUVECs which suggests that v-Ki-ras signaling pathway is associated with VEGF receptor expression and make HUVECs/E6-E7 sensitive to TNP-470 by modulating the signal transduction cascade. In conclusion, HPV-16 E6-E7 and v-Ki-ras genes have unique growth properties and these immortalized cells are useful for investigating signal transduction pathways of endothelial cells, and for screening of angioinhibitory drugs.

Topics: Angiogenesis Inhibitors; Cell Division; Cell Line, Transformed; Cell Transformation, Viral; Cyclohexanes; Cytokines; Drug Screening Assays, Antitumor; Endothelium, Vascular; Enzyme Inhibitors; Flow Cytometry; Gene Expression Regulation, Neoplastic; Genes, ras; Genistein; Humans; Lactones; Macrolides; Neoplasm Proteins; Neovascularization, Pathologic; O-(Chloroacetylcarbamoyl)fumagillol; Oncogene Protein p21(ras); Oncogene Proteins, Viral; Papillomaviridae; Papillomavirus E7 Proteins; Receptor Protein-Tyrosine Kinases; Receptors, Growth Factor; Receptors, Vascular Endothelial Growth Factor; Repressor Proteins; Sesquiterpenes; Signal Transduction; Staurosporine; Umbilical Veins

In addition to its classic role in the cellular stress response, heat shock protein 90 (Hsp90) plays a critical but less well appreciated role in regulating signal transduction pathways that control cell growth and survival under basal, nonstress conditions. Over the past 5 years, the antitumor antibiotics geldanamycin and radicicol have become recognized as selective Hsp90-binding agents (HBA) with a novel ability to alter the activity of many of the receptors, kinases, and transcription factors involved in these cancer-associated pathways. As a consequence of their interaction with Hsp90, however, these agents also induce a marked cellular heat shock response. To study the mechanism of this response and assess its relevance to the anticancer action of the HBA, we verified that the compounds could activate a reporter construct containing consensus binding sites for heat shock factor 1 (HSF1), the major transcriptional regulator of the vertebrate heat shock response. We then used transformed fibroblasts derived from HSF1 knock-out mice to show that unlike conventional chemotherapeutics, HBA increased the synthesis and cellular levels of heat shock proteins in an HSF1-dependent manner. Compared with transformed fibroblasts derived from wild-type mice, HSF1 knock-out cells were significantly more sensitive to the cytotoxic effects of HBA but not to doxorubicin or cisplatin. Consistent with these in vitro data, we found that systemic administration of an HBA led to marked increases in the level of Hsp72 in both normal mouse tissues and human tumor xenografts. We conclude that HBA are useful probes for studying molecular mechanisms regulating the heat shock response both in cells and in whole animals. Moreover, induction of the heat shock response by HBA will be an important consideration in the clinical application of these drugs, both in terms of modulating their cytotoxic activity as well as monitoring their biological activity in individual patients.

Topics: 3T3 Cells; Animals; Antibiotics, Antineoplastic; Benzoquinones; Cell Transformation, Viral; DNA-Binding Proteins; Heat Shock Transcription Factors; Heat-Shock Response; HSP90 Heat-Shock Proteins; Humans; Lactams, Macrocyclic; Lactones; Macrolides; Mice; Mice, Inbred BALB C; Mice, Knockout; Mice, SCID; Papillomaviridae; Quinones; Rifabutin; Transcription Factors; Transcriptional Activation; Xenograft Model Antitumor Assays