The extracellular calcium ion regulates a late stage of pre-replicative development of non-neoplastic cells through a calcium, cyclic AMP and cyclic AMP-dependent protein kinase mechanism. This calcium-cyclic AMP regulatory system is somehow altered in neoplastic cells because they need 50-100-fold less extracellular calcium to proliferate than non-neoplastic cells. We will test a wide variety of non-neoplastic and neoplastic cells for their proliferative calcium requirements and correlate these requirements with their ability to grow in soft agar and to form tumors in athymic nude mice. We will exploit this difference between non-neoplastic and neoplastic cells by developing an assay for carcinogenic agents, in which transformed cells are selected by incubation in medium containing only 0.02 mM calcium. The blockage of non-neoplastic cells proliferation in low-calcium medium can be overcome by calcium or a variety of other agents which all seem to function by causing a brief cyclic AMP surge. Tumor-promoting agents (such as TPA, phenobarbitol and saccharin) can also replace calcium and just as rapidly (within one hour) stimulate DNA synthesis. This system suggests a rapid (48-72-hour) in vitro assay for tumor promoters. Experiments will also be performed to determine how tumor promoters affect the calcium, cyclic AMP, cyclic AMP-dependent protein kinase system.