Calcium content and distribution as a function of growth and transformation in the mouse 3T3 cell

Abstract

Total Ca content and that fraction of Ca sensitive to removal by the chelator ethylene glycol-bis(/~-aminoethyl ether) N,N,N',N'-tetraacetate (EGTA) have been investigated in the mouse 3T3 cell as a function of growth stage, transformation with SV40 virus, and serum levels of the media. Cells were allowed to grow through several doublings in media containing 45Ca. The cellular content of 45Ca was used to assess total cell Ca. That fraction of 45Ca removed by EGTA was presumed to represent primarily surface-localized Ca. The data are expressed on a per cell volume basis to compensate for size differences as a function of growth stage and transformation. During exponential growth phase, the 3T3 cell contains 525 pmol Ca//zl cell volume. Of this, approx. 457 pmol//zl is not removable by EGTA and, presumably, is cytoplasmically located. This value is in close agreement with previous studies on the HeLa cell (470 pmol Ca/~l cell water after removal of the surface Ca). The low level of EGTA-removable Ca present in the 3T3 cell during early exponential growth (68 pmol Ca//zl cell volume) increases progressively with increasing cell density, and upon quiescence it is sevenfold greater. In contrast, SV40-transformed 3T3 cells growing exponentially possess total levels of Ca which are approximately two-thirds the levels of the normal 3T3 cell. However, their EGTA-sensitive Ca is not significantly different from that of exponentially growing, normal 3T3 cells. As the transformed cells continue to grow at high density, their total Ca and their sensitivity to EGTA do not change, in contrast to the normal 3T3 cell. Thus, an increase in Ca associated with the cell surface appears to be correlated with growth inhibition. This has been investigated further by regulating growth of the normal and transformed cell with alterations in the serum levels of the media. In 4% calf serum the normal cell is stopped from continued proliferation. Growth stoppage under these conditions is characterized by a nearly fourfold increase in EGTA-removable Ca, similar to the increase observed upon quiescence in depleted 10% serum. Similar treatment of the transformed cell does not reduce its growth rate, nor does it significantly alter Ca distribution. However, at 0.5% medium serum levels, the SV40 3T3 growth rate is substantially reduced and, under these conditions, EGTA-removable Ca increases twofold.KEY WORDS calcium 9 mouse 3T3 cells Calcium ion is required for continued cellular protransformation calcium transport liferation of a wide variety of cell types (for re-

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

“…In preliminary experiments a range of different washing regimens were examined for cells exposed to radio-calcium. These showed a near exponen tial decrease in cell-associated calcium when cultures were washed successively with 0.9% saline and that in some cases up to ten washes were necessary to reach a basal level of radioactivity in the washes which is in agreement with previous findings (3,6). Approxima tely 96% of the residual cell-associated calcium was removed by 3 subsequent 0.1 M lanthanum chloride washes, 89% by washing the cultures with EGTA (2.5 mM at pH 7.2) and 78% by trypsinising the cells.…”

“…Approxima tely 96% of the residual cell-associated calcium was removed by 3 subsequent 0.1 M lanthanum chloride washes, 89% by washing the cultures with EGTA (2.5 mM at pH 7.2) and 78% by trypsinising the cells. These results imply that a significant proportion of the cell-associated calcium is external as has been previously demonstrated (3,4,6). The washing schedule that was finally employed in the present study consisted of two saline washes in which the wells were rinsed in a stream of 0.9% saline, allowed to stand in 1 ml/well of saline for 10 min, rinsed again and given a second wash (1 ml/well) for 3 -5 min.…”

“…Several authors (1)(2)(3) have reported that the serum-induced proliferation of quiescent 3T3 cells is characterised by alterations in the amount of calcium associated with the cells suggesting that serum stimulation of growth may be mediated in part through changes in the concentrations or localization of calcium. In this communication we demonstrate a phase in the initiation of cell proliferation during which the cells are sensitive to external calcium levels which implies that cell-associated calcium acts as a cofactor in the effect of serum.…”

Restoration of normal appearance, growth behavior, and calcium content to transformed 3T3 cells by magnesium deprivation.

“…shown to be substantially lower than that of nontransformed cells (10). It has also been shown that addition ofinsulin to chicken embryo cells or of fresh medium to 3T3 cells releases more -than half the surface-bound Ca2+ of these cells into the medium (11,12).…”

Activation of high levels of endogenous phospholipase A2 in cultured cells.

“…EDTA has been shown (24) to rapidly remove (half-life, 1.5 min) cell surface Ca2+ from HeLa cells, whereas a second larger pool, presumed to be cytoplasmic Ca2+, is removed by EDTA much more slowly (half-life, 31 min). Because chelating agents have been shown to have similar effects on the Ca2+ content of 3T3 cells (25), addition of 10 mM EDTA to the culture medium (which contains 1.8 mM CaCl2) would be expected to inhibit primarily cell surface PLase.…”