Cloning Of Basic Fibroblast Growth Factor And Its Influence On The Growth Of HeLa Cells And The Expression Of Angiogenin

Cancer of the uterine cervix is the second most common cancer among women in the world, and the most common gynecological malignancy in P.R. China. Angiogenesis stimulates tumor growth, invasion and metastasis, and therefore is a crucial step in tumorigenesis. Further, evidence of increased neovascularization has been shown to be a negative prognostic indicator in many solid tumors, including cervical cancer. Several growth factors may be involved in the angiogenic process, including angiogenin and basic fibroblast growth factor (bFGF).Basic fibroblast growth factor (bFGF), a member of a group of heparin-binding multifunctional polypeptides, is one of the most potent angiogenic factors. It is also involved in proliferation and differentiation of a variety of normal and malignant cells and tissues. Many malignant tumors showed an increase in bFGF expression. Angiogenin has been shown to play a role in tumor angiogenesis. Its expression is upregulated in many types of cancers. Angiogenin undergoes nuclear translocation in endothelial cells, which has been shown to be necessary for angiogenesis. Tsuji et al have shown that nuclear translocation of angiogenin occurred in HeLa cells regardless of the cell confluence status, and that angiogenin stimulated rRNA synthesis in HeLa nuclei.The role of individual angiogenic growth factors in the formation of new blood vessels and in the progression of tumor growth has been extensively investigated. The expression of angiogenin and bFGF is especially associated with a poor prognosis. Nevertheless, little is known about how these factors coordinately regulate tumor angiogenesis and how they react on the targeted inhibition of a single factor. Kishimoto et al have observed that angiogenin antisense HeLa transfectants and tumors in athymic mice actually expressed a higher amount of bFGF than the controlcells and tumors in athymic mice. Song et al demonstrated that the angiogenin sense transfectants actually expressed a lower amount of bFGF, and the angiogenin antisense transfectants expressed a higher amount of bFGF than A375 cells. To further understand the biological role of angiogenin and bFGF, we investigated the influence of bFGF on angiogenin ggene expression and angiogenin secretion in HeLa cell lines.We cloned bFGF and constructed the expression plasmid of bFGF. We transfected the bFGF gene in the sense and ntisense orientation into HeLa cells, and obtained stable bFGF underexpressing and overexpressing transfectants. In our experiments, we demonstrated that inhibition of bFGF gene and protein expression in the bFGF antisense transfectants induced increased protein expression of angiogenin. In contrast, in the bFGF sense transfectants the expression of angiogenin decreased. Delivery of recombinant angiogenin into transfected and control cells led to increased proliferation in the bFGF antisense transfectants and the control cells. However, the cell proliferation had no change in the bFGF sense transfectants. In conclusion, Furthermore, endogenous bFGF affects the expression of angiogenin in HeLa cells.