Role of the retinoblastoma protein in cell cycle arrest mediated by a novel cell surface proliferation inhibitor

Abstract

A novel cell regulatory sialoglycopeptide (CeReS-18), purified from the cell surface of bovine cerebral cortex cells has been shown to be a potent and reversible inhibitor of proliferation of a wide array of fibroblasts as well as epithelial-like cells and nontransformed and transformed cells. To investigate the possible mechanisms by which CeReS-18 exerts its inhibitory action, the effect of the inhibitor on the posttranslational regulation of the retinoblastoma susceptibility gene product (RB), a tumor suppressor gene, has been examined. It is shown that CeReS-18 mediated cell cycle arrest of both human diploid fibroblasts (HSBP) and mouse fibroblasts (Swiss 3T3) results in the maintenance of the RB protein in the hypophosphorylated state, consistent with a late G1 arrest site. Although their normal nontransformed counterparts are sensitive to cell cycle arrest mediated by CeReS-18, cell lines lacking a functional RB protein, through either genetic mutation or DNA tumor virus oncoprotein interaction, are less sensitive. The refractory nature of these cells is shown to be independent of specific surface receptors for the inhibitor, and another tumor suppressor gene (p53) does not appear to be involved in the CeReS-18 inhibition of cell proliferation. The requirement for a functional RB protein product, in order for CeReS-18 to mediate cell cycle arrest, is discussed in light of regulatory events associated with density-dependent growth inhibition.

Calcium influences sensitivity to growth inhibition induced by a cell surface sialoglycopeptide

“…CeReS-18 binds to a specific cell surface receptor (Sharifi and Johnson, 1987), and binding to this receptor correlates with DNA and protein synthesis inhibition (Bascom et al, 1986). Although the mechanism of action of the CeReS-18 growth inhibitor has not yet been elucidated, several lines of evidence suggest that calcium regulation and the retinoblastoma protein (Rb) may play important roles (Sharifi et al, 1986a;Toole-Simms et al, 1991;Johnson et al, 1992;Enebo et al, 1994).…”

Purification and stability characterization of a cell regulatory sialoglgycopeptide inhibitor

“…However, the likelihood that contaminants have played a role in any of the cell cycle regulatory activities is minimal since the increased purity inhibitor displays even superior cell proliferation inhibition and the contaminants did not appear to influence other means of measuring biological activity [Bascom et al, 19861. The cell regulatory activities of CeReS is Ca2+ sensitive [Betz et al, 19941 and apparently requires a functional retinoblastoma gene product in mammalian cells for cell cycle arrest [Enebo et al, 1994;Johnson et al, 19921.…”

“…The biological activities of the CeReS glycopeptide have been studied in some detail [Johnson, 1994;Fattaey et al, 1991;Enebo et al, 1994;Betz et al, 19941. However, the likelihood that contaminants have played a role in any of the cell cycle regulatory activities is minimal since the increased purity inhibitor displays even superior cell proliferation inhibition and the contaminants did not appear to influence other means of measuring biological activity [Bascom et al, 19861.…”

“…However, the likelihood that contaminants have played a role in any of the cell cycle regulatory activities is minimal since the increased purity inhibitor displays even superior cell proliferation inhibition and the contaminants did not appear to influence other means of measuring biological activity [Bascom et al, 19861. The cell regulatory activities of CeReS is Ca2+ sensitive [Betz et al, 19941 and apparently requires a functional retinoblastoma gene product in mammalian cells for cell cycle arrest [Enebo et al, 1994;Johnson et al, 19921. The importance of the carbohydrate component in the biological activities of many glycoproteins is being elucidated [Kobata, 19921. Oligosaccharides have been found to perform important biological roles in cell-cell recognition, e.g., myelination of neurons [Ratner et al, 19861 and lymphocyte homing [Lasky et al, 1989;Coombe and Rider, 19891, hormonal activation [Amano and Kobata, 19931, protease resistance [Gu et al, 19891, and a number of cell proliferation regulatory roles including fertiliza-tion [Bleil and Wasserman, 19881, in vivo erythropoietin mediated stimulation [Narhi et al, 19911, and growth inhibition mediated by contactinhibin [Wieser et al, 19901.…”

“…Sharifi et al [1986b] reported the isolation of a glycopeptide released from intact bovine cerebral cortex tissue by mild proteolysis that displayed the characteristics of a membrane-associated cell proliferation inhibitor. Cell proliferation arrest appeared t o be cell cycle specific in GI [Fattaey et al, 19911 and when the retinoblastoma gene product was examined from exponentially growing cells treated with this inhibitor, the protein was found in its underphosphorylated state [Enebo et al, 1994;Johnson et al, 19921. CeReS has an unusually broad target cell range spanning from insect cells to human diploid fibroblasts and demonstrates its activity at 5 x 10-lo to 9 x lops M for most of the cells types tested [Johnson, 19941. When the sialoglycopeptide was initially characterized there were two activities associated with the inhibitory fraction, cell proliferation inhibition and a proteolytic activity [Sharifi et al, 1986al.…”