An essential component of the multistep process of tumorigenesis is the development of a new blood supply, angiogenesis. A strategy in inhibition of tumor formation and growth is to inhibit neovascularization. We have shown that the 16 kd n terminal fragment of prolactin (16K PRL) is a specific angiolytic factor in vitro. Both rat and recombinant human 16K PRL inhibits basal and bFGF or VEGF stimulated growth of bovine and human vascular endothelial cells. 16K PRL also inhibits organization of capillary endothelial cells into capillary like structures when cultured in collagen gels. Finally 16K PRL inhibits development of the microvasculature in the chick chorioallantoic membrane. We will determine if 16K PRL inhibits activation of proteases in capillary endothelial cells by BFGF. In the chick chorioallantoic membrane we will determine if 16K PRL also inhibits the angiogenic actions of bFGF and VEGF. We will determine if 16K PRL inhibits tumor growth and associated angiogenesis in two model systems. NIH 3T3 cells transformed by transfections with a signal peptide FGF expression vector or the HCT 16 human colon carcinoma cells will be transplanted subcutaneously into nude athymic mice. 16K PRL will be administered systemically or intratumorally and the growth and vascularization of tumor histologically evaluated. In another model lines of transgenic mice will be developed in which 16K hPRL expression is directed to beta cells of the pancreas. This will be accomplished by constructing a chimeric gene consisting of the insulin promoter/enhancer elements (In) upstream from the coding region for 16K hPRL. These mice will then be crossed with transgenic mice expressing SV40 T antigen in the beta cells of the pancreas (In-Tag). The In-Tag mice are well characterized and develop time dependent tumors of the pancreas. Frequency, rate of growth and vascularization of tumors will be evaluated. The angiolytic action of 16K PRL is mediated via a novel receptor which is not the prolactin or FGF receptors. We will attempt to clone the receptor by two approaches. (1) Expression cloning in COS cells. A bovine brain capillary endothelial cell (BBE) cDNA library in pcDNA1 vector will be expressed in COS cells and screened by autoradiographic analysis of I-125 16K PRL binding. (2) Homology to GH/PRL/Interleukin receptor family: PCR fragments obtained with primers to conserved region of the receptor family will be used to screen cDNA libraries of BBE or human umbilical vein endothelial cells.
| D'Angelo, G; Struman, I; Martial, J et al. (1995) Activation of mitogen-activated protein kinases by vascular endothelial growth factor and basic fibroblast growth factor in capillary endothelial cells is inhibited by the antiangiogenic factor 16-kDa N-terminal fragment of prolactin. Proc Natl Acad Sci U S A 92:6374-8 |
