James F. Whitfield

The regulation of cell proliferation by calcium and cyclic AMP.

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.

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

Calmodulin stimulates DNA synthesis by rat liver cells

Abstract Incubation in low (0.02 mM)-calcium medium prevented T51B rat liver cells from initiating DNA synthesis. Raising the calcium concentration in the medium from 0.02 to 1.25 mM caused these arrested cells to initiate DNA synthesis 1–2 hours later. The possibility of this rapid DNA-synthetic response to calcium addition being mediated through Ca-calmodulin complexes was suggested by the following observations: It was blocked by the putative Ca-calmodulin blockers chlorpromazine and trifluoperazine; the trifluoperazine-inhibited cells were stimulated by purified rat calmodulin; and purified rat calmodulin itself (10 −7 to 10 −6 moles/l) mimicked calcium action, unless the already low ionic calcium concentration in the calcium-deficient medium was reduced further by adding the specific calcium chelator EGTA.

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.

Ca2+–calmodulin and protein kinase Cs: a hypothetical synthesis of their conflicting convergences on shared substrate domains

Evidence is accumulating that suggests that Ca2+-calmodulin (Ca2+-CaM) and the protein kinase Cs (PKCs) obstruct each others actions because of the embedding of PKC phosphorylation sites in CaM or Ca2+-CaM-binding domains of a growing number of crucial substrates in neurons (and other cells). These substrates include the CaM storage proteins (neurogranin, neuromodulin), the membrane-associated MARCKS (myristoylated alanine-rich C-kinase substrate) protein, the NMDA receptor RI subunit and the autoinhibitory domain of the plasma membrane Ca2+ pump. In this review, the emerging data are woven into a hypothetical picture of the conflicting, timing-dependent convergence of two major signalers on neuronal functions.

Stimulation of DNA synthesis and mitotic activity of thymic lymphocytes by cyclic adenosine 3′5′-monophosphate

Abstract Physiological levels (10 −8 to 10 −6 M) of the cyclic nucleotide, adenosine 3′5′-monophosphate (cyclic AMP), were shown to promote deoxyribonucleic acid synthesis and mitotic activity in suspension cultures of rat thymic lymphocytes. Higher concentrations of cyclic AMP inhibited cell growth. It is concluded that cyclic AMP plays a central role in the control of cell division in thymocyte populations and could be the intracellular mediator of the mitogenic actions of a wide variety of hormones on this type of cell.

The direct measurement of protein kinase C (PKC) activity in isolated membranes using a selective peptide substrate

A protein kinase C (PKC)-selective peptide substrate was used to develop a method for measuring PKC activity directly and quantitatively in isolated cell membranes without prior detergent extraction and reconstitution of the enzyme with phosphatidylserine and TPA in the presence of excess Ca2+. This simple and rapid method can reliably measure changes in membrane-associated PKC activity induced by various bioactive compounds such as hormones and growth factors. Also, this method, which measures PKC activity in its native membrane-associated state, has the advantage of being able to distinguish between active and inactive PKC associated with cell membranes.

The effect of prostaglandins on cyclic AMP production and cell proliferation in thymic lymphocytes

Summary Prostaglandin A 1 and E 1 , but not F 1 α or F 2 α , stimulate thymic lymphocyte adenylate cyclase with resultant increases in the intracellular level of cyclic AMP and the rate of cell proliferation. A possible mechanism of action of the prostaglandins on adenylate cyclase and a role for these compounds in the stimulation of cell proliferation following injury will be discussed.

Small bone-building fragments of parathyroid hormone: new therapeutic agents for osteoporosis

The brittle, fracture-prone bones of an osteoporotic postmenopausal woman are the products of an excessive uncompensated resorption of trabecular bone by osteoclasts. Osteoporosis is currently treated with the osteoclast suppressors calcitonin, bisphosphonates, or oestrogen, which stop further bone resorption without stimulating new bone growth. Here, James Whitfield and Paul Morley review the growing evidence that small adenylate cyclase-stimulating fragments of the parathyroid hormone are promising therapeutic agents for osteoporosis that potently stimulate osteoblasts to make mechanically strong or supranormally strong bone.