R. H. Rixon

THE POSITIVE CONTROL OF CELL PROLIFERATION BY THE INTERPLAY OF CALCIUM IONS AND CYCLIC NUCLEOTIDES. A REVIEW

ConclusionCalcium, 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 ROLES OF CALCIUM AND CYCLIC AMP IN CELL PROLIFERATION

Besides activating eggs and triggering muscle contraction, calcium and the three hormones (calcitonin, la , 25(OHh vitamin D,, parathyroid hormone) controlling its level in the blood are important regulators of DNA synthesis and mitotic activity in the bone marrow, liver and thymus of the rat.’-’’ Calcium also controls the proliferation of nontumorigenic epithelial and mesenchymaUy derived cells in ~itro,’~-~~ but it has little or no influence on corresponding tumor cells.~7-m, 4 a. 25-28

CALCIUM, CYCLIC ADENOSINE 3',5'-MONOPHOSPHATE, AND THE CONTROL OF CELL PROLIFERATION: A REVIEW*

ConclusionBy manipulating the rats calcium balance, we have discovered that the calcium homeostatic system is a main regulator of cell proliferation in the bone marrow and thymus gland. Although the limits of the systems sphere of influence have yet to be completely defined, it is already known to include such diverse elements as chicken fibroblasts, liver parenchymal cells, and circulating small lymphocytes. Of even greater significance is the possibility that the ubiquitous cyclic AMP is calciums partner and may even be the ions intracellular agent for the control of cell proliferation. Thus, we now have a wide variety of possible explanations for diseases involving uncontrolled cell proliferation.

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.

An increase in calmodulin during growth of normal and cancerous liver in vivo

The calcium-dependent aspects of such diverse processes as contraction, secretion and nerve function seem to be mediated by the ubiquitous calcium receptor protein, calmodulin (CaM) ,[ l]. Another well-established, calcium-dependent process is cell proliferation both in vivo and in vitro [2]. Whether CaM is involved with this action of calcium remains unclear. However several lines of evidence suggest that this calcium receptor protein does have some function in the control of cell proliferation: (i) An increase in CaM has been found in a tumour cell line as the cells synchronously enter the DNA synthetic phase of the cell cycle [3]; (ii) CaM does have the ability to stimulate DNA synthesis in isolated liver cells [4]; (iii) Many cell lines when transformed by tumour viruses have increased amounts of CaM [5-71. This report submits evidence for the first time of a change in CaM during growth of normal liver, regenerating in vivo, and a greater content of CaM in many solid tumours of liver.

REGULATION OF PROLIFERATION OF NORMAL AND NEOPLASTIC RAT LIVER CELLS BY CALCIUM AND CYCLIC AMP

Calcium and cyclic AMP control the proliferation of nontumorigenic liver cells. These agents seem to be cogenerators of a signal to start synthesizing deoxyribonucleotides, the earliest of the DNA synthetic processes. By contrast, calcium has little or no effect on the proliferation of tumorigenic cells in vitro. Some possible reasons for this loss of control are presented, and the usefulness of this property as a tool for the detection of carcinogens is discussed.

Parathyroid hormone fragment [3-34] stimulates protein kinase C (PKC) activity in rat osteosarcoma and murine T-lymphoma cells.

The parathyroid hormone (PTH) fragment [1-34] strongly stimulated both adenylate cyclase and membrane-associated PKC activities in rat 17/2 osteosarcoma cells. By contrast, the PTH [3-34] fragment, which was unable to stimulate adenylate cyclase, remained a potent stimulator of membrane-associated PKC activity in these cells. Both PTH fragments also strongly stimulated membrane-PKC activity in cyc-S49T-lymphoma cells possessing a defective adenylate cyclase system. This ability of PTH [3-34] to stimulate membrane-associated PKC activity could explain the residual bioactivity of this fragment.

Restoration of severely depleted femoral trabecular bone in ovariectomized rats by parathyroid hormone-(1–34)

It is commonly believed that the parathyroid hormones (PTHs) main function in bone is to stimulate osteoclastic resorption. However, intermittent injections of small doses of PTH holoprotein, but more often its bioactive hPTH-(1–34) fragment, have been shown to stimulate bone growth in animals and humans through their ability to stimulate adenylyl cyclase and not their ability to independently activate a protein kinases-C stimulating mechanism. This anabolic action suggests that PTH might be an effective therapeutic for osteoporosis. If so, the hormone must be able to restore severely depleted trabecular bone, and the goal of this study was to find out if it can. To do this, we started a multiweek program of daily subcutaneous injections of 0.8 nmoles of hPTH-(1–34)/100 g body weight into rats at 4, 8, or 16 weeks after ovariectomy (OVX) and the increasingly severe selective loss of trabecular bone. These injections strongly stimulated femoral trabecular bone to grow and mineralize at the same rate regardless of how much of it had been lost before the injections were started. Thus, the progressively depleting trabecular bone in the femurs of OVX rats does not lose its anabolic responsiveness to PTH. This finding is another indication of the likelihood of small, adenylyl cyclase-stimulating PTH fragments being effective therapeutics for osteoporosis.

The control of liver regeneration by calcitonin, parathyroid hormone and 1α,25-dihydroxycholecalciferol

Removal of the thyroid in normocalcemic rats with functional parathyroid transplants was found to reduce the hepatocyte DNA synthetic activity which normally follows partial hepatectomy. This proliferative incapacitation of hepatocytes appeared to be due specifically to a calcitonin deficiency since it was overcome by a single injection of pure synthetic salmon calcitonin shortly after partial hepatectomy. Salmon calcitonin and bovine parathyroid hormone were equally able to reverse the similar proliferative incapacitation of hepatocytes in hypocalcemic rats which had both their parathyroid and thyroid glands removed one day before partial hepatectomy. However, these two hormones (individually or together) could not reverse the proliferative incapacity resulting from a more prolonged (3-day) exposure to the hypocalcemic conditions in thyroparathyroidectomized rats, but the proliferative incapacity could be reversed by simultaneous treatment with the vitamin D3 metabolite, 1 alpha,25-dihydroxycholecalciferol. We suggest that extracellular calcium ions are the actual regulators of this hormonally-controlled hepatocyte proliferative development and that parathyroid hormone and the vitamin D3 metabolite affect proliferation indirectly by determining the extracellular calcium concentration, while calcitonin directly, or indirectly, sensitizes hepatocytes to the action of calcium.

Parathyroid Hormone and Liver Regeneration

Summary Removal of the parathyroid glands in rats reduced the initiation of DNA synthesis and proliferation which normally occurs in the liver parenchymal cells remaining after partial hepatectomy. However, this effect was observed only when the parathyroid glands were removed 24 or more hours before partial hepatectomy. When the glands were removed 12 hr before the partial hepatectomy, there was no decrease in the ability of hepatocytes to initiate the regenerative process. These observations suggest that parathyroid hormone is needed to maintain the competence of liver parenchymal cells to proliferate when activated by partial hepatectomy, but is not involved in the initial activation process itself. The authors are grateful to Miss J. Bayliss and Mr. D. Gillan for their excellent technical assistance.