Our investigation of signal transduction pathways and their relationship to cancer has grown in many ways over the last year. We have used CAI as a tool to further our studies of key Ca++-mediated pathways. The molecular dissection of CAI demonstrated that CAI inhibition of Ca++- mediated signaling events is concordant with inhibition of proliferation. Novel compounds were identified and characterized and a patent application was filed. Further studies have identified a role for Ca++ influx regulation of tyrosine phosphorylation of phospholipase C-gamma which produces inositol trisphosphate and internal calcium release. Use of CAI helped clarify that receptor-operated Ca++ influx mediated maintenance of the malignant phenotype of CHO cells. Biotinylated CAI (B-CAI) has been produced. Avidin-linked B-CAI and a CAI analog have been used to immunize rabbits. B-CAI and/or antisera will be used for expression cloning of the CAI binding sites and studies at the protein level. Subtraction analysis of gene products amplified in cells resistant to chronic CAI exposure has identified several candidate genes. Transcripts of 1.2 and 2.7 kb were expressed in A2058 resistant to 10, 20, and 30 micromoles CAI and not in wild type A2058. Clones identified using the cDNA subtraction probe are being characterized. CAI has been used as a tool to investigate Ca++-regulated gene expression. CAI inhibits IL-2 gene transcription through several transactivating proteins. It also stimulates and inhibits Ca++ and growth factor-mediated production of a viral enhancer, VL-30. We have shown that CAI is antiangiogenic in vitro and in vivo. Incubation of human umbilical vein endothelial cells with CAI inhibited proliferation, adhesion, motility, MMP-2 production, and tube formation, and CAI inhibited microvascular proliferation in chick chorioallantoic membrane assays. Studies are ongoing to identify the sites of this Ca++- mediated signal transduction inhibition of angiogenesis.
