Time Dependency of a Critical Effect of EGTA on DNA Synthesis in Cultures of Swiss 3T3 Cells

Abstract

In a temporal analysis of the mitogenic response to serum, a critical period has been demonstrated just prior to the onset of replicative DNA synthesis during which transient calcium depletion blocks the subsequent entry of the cells into the S phase of the mitotic cycle. Transient washing of monolayer cultures of 3T3 cells with 2.5 mM EGTA between 6 and 8 h after serum-stimulated initiation of DNA synthesis was found to reduce cell-associated calcium levels and to inhibit thymidine incorporation, whereas similar treatment before (1–5 h) and after (8–9 h) had no detectable effect on either of these parameters when estimated after 21 h incubation. The effects during the chelation-sensitive period were reversed by the subsequent addition of fresh serum.

Calcium, cyclic AMP and protein kinase C ? partners in mitogenesis

“…However, as is the case in stage 1, driving protein kinase C into the cell membrane and activating it with saccharin or TPA can greatly reduce (but not completely eliminate) the need for external Ca 2+ [45, 180,251]. If the cells are confluent and serum factors (needed to maintain pools of labile components [299]) are in short supply, withdrawing Ca 2+ from the medium and stripping it from surface binding sites with the highly selective chelator EGTA for as little as 5 minutes causes the stage 2 build-up in cells such as Swiss albino 3T3 mouse cells to dissipate, which forces the cells to go back to the beginning where they must be stimulated with fresh serum to start all over again [300]. The same late lability of the stage 2 build-up has been found in serum-stimulated confluent BALB/c 3T3 mouse cells and T51B rat liver cells which, like hepatocytes in regenerating livers of TPTX rats [158[, 'deprogram' themselves within a couple of hours if enough external Ca 2+ is not on hand when they reach the end of stage 2 [45,283].…”

“…By releasing Ca 2+ from ER stores [31, 32, 34, 37] (Fig. 2), IP 3 would trigger the internal Ca :+ surge that produces the Ca 2+-CaM complexes and other Ca2+-dependent first events in cells such as BALB/c 3T3 and Swiss albino 3T3 mouse cells that do not rely on external Ca 2+ in stage 1 [283,300]. An opening of membrane channels would provide the Ca 2+ influx from the medium that is needed for the external CaZ+-dependent G~j--+G 1 transition of serum-stimulated T51B rat liver cells and WI-38 human fibroblasts [12,45,309].…”

“…Thus, G O cells can be activated by exposure to very high (5 to 15 mM) concentrations of external Ca 2+, or even better by the internal release of Ca :+ from ingested hydroxyapatite crystals [300,[310][311][312][313]. Stimulating protein kinase C with various DGs, teleocidin or TPA [44] also gives an adequate first signal that stimulates stage 1 cell cycle genes, the transcription of ribosomal RNA genes, and cell growth [54, 165,166,287,314] (Fig.…”

Du Temps Perdu a la Recherche

“…The problem of motility in relation to migration attracted my attention and with Rayya Damluji we examined the effects of calcium on adhesion (99, 100). Later, with Bryan Groves, I devised a chamber to measure cell‐to‐substratum adhesion by shear resistance (101).…”

Ca2+‐dependent cell surface protein phosphorylation may be involved in the initiation of DNA synthesis

“…plastic cells (Boynton et al, 1976a(Boynton et al, ,b, 1977(Boynton et al, , 1985aDamluji and Riley, 1979;Hazelton et al, 1979;Marceau and Swierenga, 1985;Whitfield et al, 1985). That this surface protein phosphorylation might participate in the GI-S transition is also indicated by the fact that both Swiss albino 3T3 mouse cells and regenerating rat liver cells activate surface protein kinases as they near the end of the G1 phase (MacManus and Mastro and Rozengurt, 1976).…”