Search

Between 32 degrees and 39 degrees, uninfected normal rat kidney (NRK) cells proliferated minimally, or not at all, in medium containing 0 to 0.01 mM extracellular free calcium. Transformation of NRK cells by wild-type avian sarcoma virus B77 enabled them to proliferate equally rapidly at all temperatures in high (1.25 mM)- or low (0.001 mM-calcium medium. Transformation by the temperature-sensitive LA-23 mutant of Rous sarcoma virus also enabled NRK cells to proliferate in low (0.001 mM)-calcium medium at 32 degrees or 37 degrees. However, the LA-23 NRK cells could not proliferate in low (0.001 mM)-calcium medium at a nonpermissive temperature such as 39 degrees, which prevents expression of other aspects of the transformed phenotype.

Calcium, in partnership with cyclic AMP, controls the proliferation of non-tumorigenic cells in vitro and in vivo. While it does not seem to be involved in the proliferative activation of cells such as hepatocytes (in vivo) or small lymphocytes (in vitro), it does control two later stages of prereplicative (G1) development. It must be one of the very many regulatory and permissive factors affecting early prereplicative development, because severe calcium deprivation reversibly arrests some types of cell early in the G1 phase of their growth-division cycle in vitro. However, calcium more specifically and much more often regulates a later (mid or late G1) stage of prereplicative development. Thus, regardless of its severity or the type of cell, calcium deprivation in vitro or in vivo reversibly stops proliferative development at that part of the G1 phase in which the cellular cyclic AMP content transiently rises and the synthesis of the four deoxyribonucleotides begins. The evidence points to calcium and the cyclic AMP surge being co-generators of the signal committing the cell to DNA synthesis. The evidence is best explained so far by the cyclic AMP surge causing a surge of calcium ions which combine with molecules of the multi-purpose, calcium-dependent, regulator protein calmodulin (CDR) somewhere between the cell surface and the cytosol. The resulting Ca-calmodulin complexes then stimulate many different (and possibly membrane-associated) enzymes such as protein kinases, one of which produces the DNA-synthetic initiator. Calcium has little or no influence on the proliferation of tumor cells. Some possible explanations of this very important loss of control are considered.

Calcium, cyclic AMP, and cyclic GMP do not seem to be involved in proliferative activation of postmitotic differentiated cells. Instead, they are intracycle regulators, and we propose the following working model of their control of the initiation of DNA synthesis. While a role for cyclic GMP cannot yet be defined, a brief postmitotic burst of its synthesis might serve to prevent certain activated cells (e.g. 3T3 mouse cells) from being diverted into a nonproliferating (but still activated) G0 state (Figs. 1 and 17). In a latter part of the G1 phase, something happens to stimulate briefly the synthesis of cyclic AMP which, in turn, drives calcium ions from the mitochondria into the cytosol to activate newly synthesized thymidylate synthetase (or other primed enzymic assemblies) (Fig. 1). Having "turned on" their target enzymes, the accumulated cyclic AMP is destroyed and the excess calcium ions are reaccumulated by the mitochondria to avoid interfering with succeeding reactions. This model predicts that persistent changes in cyclic AMP metabolism and the respiration-linked, calcium-accumulating (ion-buffering) activity of mitochondria may be responsible for the sustained growth of tumors.

The extracellular calcium ion is required for the proliferative activity of a wide variety of nonneoplastic epithelial cells both in vivo and in vitro. This dependence in vitro appears to be density-dependent with sparse cultures unable to proliferate in Ca2+-deficient medium while dense culture can proliferate despite the Ca2+ deficiency. Some epithelial cells have specific cell cycle transitions that require extracellular Ca2+ while most all neoplastic epithelial cells in culture are able to proliferate in Ca2+-deficient media unlike their nonneoplastic controls. The mechanisms of Ca2+ action in nonneoplastic cells involve calmodulin, cyclic AMP and phosphatidylinositol turnover while in neoplastic cells oncogene activation induces the aberrant production of protein products which interact with and deregulate the normal growth-regulatory processes apparently linked to extracellular Ca2+.

Cells from thigh muscles of fetal rats proliferate readily in vitro in medium containing homologous adult rat "plasma". As the donor animals mature, these cells become unable to make DNA and proliferate in "plasma" medium, but retain an ability to proliferate in medium containing fetal bovine serum (FBS). This age-related loss of the ability to proliferate in "plasma"-medium is due to an increasing need by the cells for an exogenous prereplicative promoter which is found in FBS and a crude preparation of bovine luteinizing hormone. Adult cells (and possibly fetal cells) also require a "cycle-completion" factor which is found in FBS and adult rat "plasma". The requirements for such external proliferative promoters is completely eliminated by neoplastic transformation in vivo, and neoplastic adult cells isolated from a nickel sulfide-induced mixed rhabdomyo-fibro-sarcoma can make DNA and proliferate in vitro in the complete absence of exogenous growth factors.

Conditionally tumorigenic BALB/3T3 mouse cells (which produce tumors in BALB/c mice only under special conditions) cannot sustain DNA synthesis and consequently stop proliferating in media containing low concentrations (0-0.02 mM) of physiologically available calcium. By contrast, cells that have been neoplastically transformed (tumorigenic in mice without special assistance) in vitro by different oncogens, can sustain DNA synthesis and proliferate in such calcium-deficient media. The possible importance for tumor growth of an ability to withstand calcium deprivation is examined. It is suggested that this property may prove to be a reliable indicator of neoplastic transformation.

Proliferatively inactive BALB/c 3T3 mouse cells in dense cultures initiate a growth-division cycle upon exposure to fresh calf serum in a low-calcium (0.01 mM) medium. If these calcium-deprived cells are not supplied with calcium sometime during the first 10 hours after serum stimulation, they will rapidly return to a proliferatively inactive state without initiating DNA synthesis. The prereplicative development of such stimulated calcium-deprived cells appears to stop at an advanced stage, because addition of calcium as late as 10 hours after serum exposure rapidly initiates DNA synthesis, and enables the culture's DNA-synthetic activity subsequently to reach its peak value at the same time as in control cultures.

The proliferative activity of diploid human WI-38 cells in sparse cultures depends on the extracellular concentration of free (or physiologically available) calcium, and cultivation in a medium having a calcium concentration of 0.1 mM or less gradually, but reversibly, arrest their proliferative development in the prereplicative (G1) phase of the cell cycle. Calcium's proliferative control of this cell type is eliminated by proliferative and morphological transformation by the oncogenic SV-40 virus, and the proliferative activity of SV-WI-38 cells in sparse cultures is unaffected by variation of the extracellular free calcium concentration between 0.00 and 1.25 mM.

In the presence of a normal (1.25 to 1.80 mM) calcium concentration, addition of fresh bovine calf serum or completely changing the medium induces proliferatively quiescent BALB/c 3T3 mouse cells in dense cultures to start a growth division cycle and initiate DNA synthesis about 12 hr later. Fresh, low-calcium (0.02 mM physiologically available) medium also causes cells to start a growth-division cycle. Howver, the development of such stimulated, calcium-deprived cells tops just before the expected time of initiation of DNA synthesis, which can then be rapidly induced by restoration of the normal calcium concentration. Simply raising the calcium concentration to nonphysiologically high levels (without otherwise altering the medium) can mimic the action of fresh serum or fresh whole medium by inducing some of the cells in proliferatively quiescent confluent concluent cultures to start a growth-division cycle and initiate DNA synthesis 22 hr later.