John P. MacManus

Global ischemia can cause DNA fragmentation indicative of apoptosis in rat brain

Laddered DNA indicative of apoptosis was observed in the CA1 layer of hippocampus and in dorsolateral striatum following a global cerebral ischemic insult produced by transient two vessel occlusion in rats. The extent of this DNA damage was proportional to the duration of the ischemic episode. Breaks in DNA were demonstrated in situ in sections from post-ischemic brain in neurons of the hippocampal CA1 which undergo selective neuronal death but not in other cell types. It is concluded that there is an apoptotic component to selective neuronal death following global ischemia in rat brain.

Gene Expression Induced by Cerebral Ischemia: An Apoptotic Perspective

The flow of new information on gene expression related to apoptosis has been relentless in the last several years. This has also been the case with respect to gene expression after cerebral ischemia. Many of genes associated with an apoptotic mode of cell death have now been studied in the context of experimental cerebral ischemia from the immediate early genes through modulating genes such as bcl-2 to genes in the final execution phase such as interleukin-1β converting enzyme (ICE)-related proteases. It was impossible to adequately cite all primary reports on these subjects. However, many excellent reviews have appeared in the last year, which together, cover all these areas of interest. In this review, we have elected to cite only reports published since January 1996 and use an extensive collection of reviews (indicated in italics) to guide the reader to the earlier literature. Our intent is to provide the reader with a timely and useful analysis of the current state of the art. It is hoped that this approach does not cause offense with our colleagues whose contributions before 1996 laid the foundation for much of this work.

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.

DNA damage consistent with apoptosis in transient focal ischaemic neocortex.

Transient focal ischaemia was produced in rat right neocortex by temporary middle cerebral artery occlusion. DNA damage was visualized in situ in cells of this right hemisphere but not in the contralateral hemisphere. The extracted damaged DNA exhibited laddered fragmentation which is indicative of apoptotic degradation. The amount of DNA damage was quantified by an end-labelling technique and shown to increase with the duration of the ischaemic insult. We conclude that the neurodegeneration resulting from focal ischaemia has an apoptotic component.

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.

Molecular mechanisms of cerebral ischemia-induced neuronal death

The mode of neuronal death caused by cerebral ischemia and reperfusion appears on the continuum between the poles of catastrophic necrosis and apoptosis: ischemic neurons exhibit many biochemical hallmarks of apoptosis but remain cytologically necrotic. The position on this continuum may be modulated by the severity of the ischemic insult. The ischemia-induced neuronal death is an active process (energy dependent) and is the result of activation of cascades of detrimental biochemical events that include perturbion of calcium homeostasis leading to increased excitotoxicity, malfunction of endoplasmic reticulum and mitochondria, elevation of oxidative stress causing DNA damage, alteration in proapoptotic gene expression, and activation of the effector cysteine proteases (caspases) and endonucleases leading to the final degradation of the genome. In spite of strong evidence showing that brain infarction can be reduced by inhibiting any one of the above biochemical events, such as targeting excitotoxicity, up-regulation of an antiapoptotic gene, or inhibition of a down-stream effector caspase, it is becoming clear that targeting a single gene or factor is not sufficient for stroke therapeutics. An effective neuroprotective therapy is likely to be a cocktail aimed at all of the above detrimental events evoked by cerebral ischemia and the success of such therapeutic intervention relies upon the complete elucidation of pathways and mechanisms of the cerebral ischemia-induced active neuronal death.

Increases in rat liver cyclic AMP concentrations prior to the initiation of DNA synthesis following partial hepatectomy or hormone infusion

Abstract In rat liver following partial hepatectomy an unprecedented biphasic increase in cyclic AMP concentration was observed. After a lag period of 1 1 2 hr , the cyclic AMP content rose to a peak at 2 1 2 hr . The cyclic AMP level fell to normal at 8 hr, and then rose again to a second peak at 12 hr. DNA synthesis started at 18 hr, by which time the cyclic AMP level was approaching control values. A biphasic increase in cyclic AMP concentration was also observed to precede the initiation of DNA synthesis in intact liver following the infusion of a mixture of tri-iodothyronine, amino acids, glucagon and heparin. The relationship between these biphasic changes in cyclic AMP levels and the initiation of DNA synthesis in liver is discussed.

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.