THE ROLE OF INOSITOL IN MAMMALIAN CELL GROWTH IN CULTURE

Abstract

The role of the membrane component, inositol, in the regulation of cell growth was investigated. Procedures were developed to deplete serum of inositol, resulting in a macromolecular serum fraction which replaced whole serum in determining the effects of inositol starvation on the growth and survival of cultured cell lines.;Some cell lines, such as CHO-Kl and L929, were fully independent of exogenous inositol for growth. Other cell lines, such as BALB/c 3T3 and MDCK, were unable to form colonies in inositol-free medium; in mass culture, they divided many times, but eventually lysed. This phenotype was different from that of cell lines such as BHK 21, which divided only 2 to 3 times before lysis.;The phenomenon of "inositolless death" was analyzed in greater detail in Ins('-)A, an inositol- and proline-requiring mutant of CHO-Kl cells. In inositolless medium, Ins('-)A cells completed 2 cell cycles and then lysed. When cell growth was prevented by contact inhibition or by proline starvation, cell death was also prevented. Prior to lysis, the inositol-starved Ins('-)A cells continued to synthesize RNA, protein and DNA at the same rate as cells in complete medium. Inositol starvation resulted in a reduced membrane content of phosphatidylinositol as well as in other alterations in phospholipid metabolism. Thus, "inositolless death" appears to be a direct consequence of unbalanced cell growth, in which RNA and protein syntheses continue in the absence of proper membrane biogenesis.;The dependence of "inositolless death" on the ongoing processes of cellular growth and metabolism, permitted the development of a mutant enrichment procedure. Mutations which resulted in cell cycle arrest or inhibition of macromolecular syntheses protected inositol-requiring cells from the lethal consequences of inositol starvation. This protection of cells from "inositolless death" should be useful for the selection of mutant phenotypes expressing conditional lethality. These findings suggest that inositol is essential for the growth and survival of cells in culture and that its utilization is not coupled to RNA and protein synthesis. The phenomenon of "inositolless death" provides a novel system for studies of the regulation of membrane biogenesis and its relationship to cellular growth control mechanisms.