o-(chloroacetylcarbamoyl)fumagillol 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

Hemangiomas represent the most frequent tumors of infancy. However, the pathogenesis of these tumors is still largely unknown, and current treatment of juvenile hemangiomas remains unsatisfactory. Here we present a novel animal model to study proliferating hemangiomas and to evaluate the effect of angiostatic compounds on their growth. Intraperitoneal (i.p.) infection of 4-day-old rats with murine polyomavirus resulted in the development of multiple cutaneous, intramuscular (i.m.), and cerebral hemangiomas with 100% frequency. Histological examination of the brain revealed the formation of immature lesions as soon as 4 days postinfection (p.i.). The subsequent exponential growth of the hemangiomas, both in number and size, was associated with severe hemorrhage and anemia. The cerebral, cutaneous, and i.m. lesions consisted of blood-filled cysts, histologically similar to human cavernous hemangiomas and stained positive for proliferating cell nuclear antigen, urokinase-type plasminogen activator, and vascular endothelial growth factor. Mature cerebral hemangiomas also expressed von Willebrand factor. Cerebral lesions caused death of the untreated animals within 19.2 +/- 1.1 days p.i. Remarkably fewer and smaller hemangiomas developed in animals that had been treated s.c. with the angiogenesis inhibitor TNP-470. Accordingly, TNP-470 (50 mg/kg), administered twice a week from 3 days p.i., significantly delayed tumor-associated mortality [mean day of death, 28.2 +/- 3.3 (P < 0.001)]. Even if therapy was initiated when cerebral hemangiomas were already macroscopically visible (i.e., 9 days p.i.), a significant delay in hemangioma-associated mortality was observed. Also, the IFN-inducer polyinosinic-polycytidylic acid caused a delay of 9 days (P < 0.005) in tumor-associated mortality when administered i.p. at 5 mg/kg, twice a week, starting at day 3 p.i. The model described here may be useful for investigating (a) the angiogenic mechanism(s) underlying hemangioma progression; and (b) the effect of anti-angiogenic compounds on vascular tumor growth.

Topics: Animals; Animals, Newborn; Antibiotics, Antineoplastic; Body Weight; Brain Neoplasms; Cell Transformation, Viral; Cyclohexanes; Disease Models, Animal; Disease Progression; Endothelium, Vascular; Female; Hemangioma; Humans; Infant; Infant, Newborn; Injections, Subcutaneous; Interferon Inducers; Male; Neovascularization, Pathologic; O-(Chloroacetylcarbamoyl)fumagillol; Poly I-C; Polyomavirus; Polyomavirus Infections; Rats; Sesquiterpenes; Sex Factors; Tumor Virus Infections; Viral Load