WI-38 and SV40WI-38 cells have been synchronized using centrifugal elutriation . This technique allows for the rapid harvesting of early G, phase cells from exponentially growing populations of both the normal and transformed cell. Using these cells, as well as WI-38 cells synchronized by serum deprivation, we have examined the effects of extracellular Ca and Mg levels on the progression of cells through GI phase . A differential sensitivity to both Ca and Mg deprivation is observed between normal and transformed cells. The WI-38 cell requires higher levels of both ions for traversal of G, phase and for continued proliferation as compared to the transformed cell. The temporal nature of the Ca and Mg requirements for the WI-38 cell has been examined during G, phase . Ca is strictly required during early and late G, phase, but not necessarily throughout mid-G, .

The effect of serum stimulation on unidirectional and net K flux and their relationship to the initiation of DNA synthesis has been investigated in mouse 3T3 fibroblasts. increase on a per volume basis. This increase peaks a t four to five hours and then declines to initial levels a t 10 to 14 hours. Populations of quiescent cells given 20% serum plus 0.5 mM ouabain simultaneously are totally blocked from entering S phase, as determined by the appearance of 3H-thymidine labeled nuclei.However, if the ouabain is removed after six hours these cells then undergo the same changes in unidirectional K influx and content as serum stimulated cells with entrance into S phase retarded by five to six hours. If ouabain is added to serum stimulated cells at six hours, after the increase in K transport and K content have occurred, entrance into S phase is not entirely blocked. In cells stimulated with serum and 0.5 mM dBcAMP plus 1 mM theophylline simultaneously, entrance into S phase is greatly reduced as compared to serum stimulation only. However, the early and late changes in K flux and K content are not substantially altered. This indicates that the K transport events associated with G I and early S phase are not directly regulated by changes in CAMP levels which follow serum stimulation.Cultured mouse 3T3 fibroblasts exist as actively proliferating cells or as quiescent cells in the early G , phase of the cell cycle.

Serum stimulation of quiescent 3T3 cells returns the cells to a proliferative state. Changes in Ca content, transport and distribution during the transition through G1 and S phase have been investigated following serum stimulation of these cells. 45 Ca exchange data indicate at least two kinetically defined cellular compartments for Ca; a rapidly exchanging component presumably representing surface Ca which is removable by EGTA and a slowly exchanging component presumably representing cytoplasmically located Ca. Previous studies (Tupper and Zorgniotti, '77) indicate that the approach to quiescence in the 3T3 cells is characterized by a large increase in the surface Ca component. The present data demonstrate that this component is rapidly lost following serum stimulation. Furthermore, the serum induces an 8-fold increase in Ca influx into the cytoplasmic compartment and a reduction in the unidirectional efflux rate coefficient for Ca. The increased Ca uptake peaks at approximately six hours (mid G1) and is accompanied by a parallel increase in cellular Ca. Prior to entrance of the cells into S phase (10-12 hours), Ca uptake declines. This is followed by a slower decline in cytoplasmic Ca levels. Simultaneous addition to fresh serum plus 0.5 mM dibutryl cAMP inhibits the entrance of the cells into S phase. Under these conditions the loss of surface Ca is not blocked. However, the presence of 0.5 mM dibutyryl cAMP inhibits the increase in Ca uptake and, in turn, diminishes the increase in cellular Ca following serum stimulation. In contrast, a low level of dibutyryl cAMP (0.1 mM) enhances progression through G1 phase but also reduces both Ca uptake and Ca content of the cells. The data suggest that the serum induced changes in Ca content and transport are linked to intracellular cyclic nucleotide levels and progression through G1 phase and that extracellular cAMP elevating agents may enhance of inhibit these interactions in a concentration dependent manner.

The components of unidirectional K influx and efflux have been investigated in the 3T3 cell and the SV40 transformed 3T3 cell in expontntial and stationary growth phase. Over the cell densities used for transport experiments the 3T3 cell goes from exponential growth to density dependent inhibition of growth (4 X 10(4) to 4 X 10(5) cell cm-2) whereas the SV40 3T3 maintains exponential or near exponential growth (4 X 10(4) to 1 X 10(6) cell cm-2). In agreement with previous observations, volume per cell and mg protein per cell decrease with increasing cell density. Thus, transport measurements have been expressed on a per volume basis. Total unidirectional K influx and efflux in the 3T3 cell is approximately double that of the SV40 3T3 cell at all cell densities investigated. Both cell types have similar volumes initially and show similar decreases with increasing cell density. Thus, in this clone of the 3T3 cell SV40 transformation specifically decreases unidirectional K flux. The magnitude of the total K flux does not change substantially for either cell line during transition from sparse to dense cultures. However, the components of the K transport undergo distinct changes. Both cell lines possess a ouabain sensitive component of K influx, presumably representing the active inward K pump. Both also possess components of K influx and efflux sensitive to furosemide. The data suggest this component represents a one-for-one K exchange mechanism. The fraction of K influx mediated by the ouabain sensitive component is reduced to one half its value when exponential versus density inhibited 3T3 cells are compared (63% versus 31% of total influx). No comparable drop occurs in the SV40 3T3 cell at equivalent cell densities (64% versus 56% of total influx). Thus, the pump mediated component of K influx would appear to be correlated with growth. In contrast, the furosemide sensitive component represents approximately 20% of the total unidirectional K influx and efflux in both cell lines in sparse culture. At high cell densities, where growth inhibition occurs in the 3T3 cell but not the SV40 3T3, the furosemide sensitive component doubles in both cell lines. Thus, the apparent K-K exchange mechanism is density dependent rather than growth dependent.

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