Leonard P. Kleine

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

SummaryEvidence is steadily mounting that the proto-oncogenes, whose products organize and start the programs that drive normal eukaryotic cells through their chromosome replication/mitosis cycles, are transiently stimulated by sequential signals from a multi-purpose, receptor-operated mechanism (consisting of internal surges of Ca2+ and bursts of protein kinase C activity resulting from phosphatidylinositol 4,5-bisphosphate breakdown and the opening of membrane Ca2+ channels induced by receptor-associated tyrosine-protein kinase activity) and bursts of cyclic AMP-dependent kinase activity. The bypassing or subversion of the receptor-operated Ca2+/phospholipid breakdown/protein kinase C signalling mechanism is probably the basis of the freeing of cell proliferation from external controls that characterizes all neoplastic transformations.

Ca2+/phospholipid-dependent protein kinase activity correlates to the ability of transformed liver cells to proliferate in Ca2+-deficient medium.

Extracellular Ca2+-deprivation inhibited the proliferation of normal T51B rat liver cells and reduced (2-4 fold) the amount of EDTA/EGTA-extractable protein kinase C activity. By contrast, preneoplastic and neoplastic T51B rat liver cells were able to proliferate in Ca2+-deficient medium and retained all of their extractable protein kinase C activity.

Involvement of the Ca2+/phospholipid-dependent protein kinase in the G1 transit of T51B rat liver epithelial cells

The G0----G1 and G1----S transitions (but not the intervening events) in the G1 phase of T51B rat liver epithelial cells in serum-stimulated confluent cultures required a high concentration of extracellular Ca2+ and were accompanied or immediately preceded by increases in the amount of EDTA-extractable protein kinase C, a Ca2+/phospholipid-dependent enzyme. Involvement of this Ca2+-dependent enzyme in the two Ca2+-dependent transitions was further indicated by the facts that 12-O-tetradecanoyl phorbol-13-acetate (TPA), a compound that stimulated protein kinase C from T51B cells even in the absence of Ca2+, enabled these cells to transit G1 in Ca2+-deficient medium, while a TPA analogue (4 alpha-phorbol-12, 13-didecanoate (4 alpha-PDD) that did not stimulate the enzyme in cell-free preparations did not promote G0----G1 or G1----S transit in Ca2+-deficient medium.

Relation between colony formation in calciumdashdeficient medium, colony formation in soft agar, and tumor formation by T51B rat liver cells

T51B rat liver cells and several carcinogen-treated, carcinogen + saccharin-treated, or spontaneously altered clones of T51B cells were tested for their abilities to form colonies in calcium-deficient medium and soft agar and to produce tumors in athymic nude mice. Most (10 out of 11) of the clones which were derived from colonies in calcium-deficient medium were unable to form colonies in soft agar and 8 out of 11 were non-tumorigenic. Conversely, 6 out of 9 clones derived from colonies in soft agar were unable to multiply significantly in calcium-deficient medium and 5 of these 6 clones were also non-tumorigenic. Two of these 9 soft agar-growing clones were tumorigenic, one of which also proliferated in calcium-deficient medium, and the other of which acquired the ability to proliferate in calcium-deficient medium after it became able to form tumors in athymic nude mice. Thus, T51B rat liver cells gain the ability to grow in calcium-deficient medium and soft agar independently during the process of neoplastic transformation and neither characteristic by itself reliably predicts tumorigenicity.

The epidermal growth factor mitogenic signal is modulated by protein kinase C in T51B rat liver cells

The regulation of cell proliferation involves a complex interplay between several signal transduction pathways. The effect of EGF on DNA synthesis in serum starved quiescent, synchronized T51B cells was investigated by [3H]thymidine incorporation and flow cytometry. 1 nM EGF or readdition of serum initiated G1 progression and entry into S phase by 18 h and DNA synthesis reached a maximum by 28 h. Low concentrations of EGF markedly stimulated DNA synthesis, but EGF was not as potent as readdition of serum. The effect of EGF on DNA synthesis was only partially blocked by the tyrosine inhibitors genistein and tyrphostin, suggesting that other signalling pathways play a role in EGF-stimulated mitogenesis. 1 nM EGF caused a rapid, transient increase in the activity of membrane-associated protein kinase C (PKC) followed by a longer sustained increase that continued into S phase. TPA (12-O-tetradecanoyl-phorbol-13-acetate) did not mimic EGF, rather it caused a slight stimulation of membrane-associated PKC activity within 1 h followed by a dramatic downregulation of PKC within 4 h. TPA was without effect on DNA synthesis alone, but when added along with EGF or serum TPA caused a significant enhancement of DNA synthesis. Pretreatment of quiescent, serum-deprived T51B cells with TPA reduced the basal level of DNA synthesis; however, under these conditions EGF became as potent a mitogen as serum. We hypothesize that EGF via activation of PKC regulates the activity of its receptor by switching from high affinity to low affinity states. Downregulation of PKC by long term treatment with TPA removes this regulation thus rendering T15B cells more sensitive to exogenous EGF.