Thesis (M.Sc. (Chemistry)) --University of Limpopo, 2007 / The biochemical mechanism of apoptosis induced by lithium remains unclear, although there is evidence suggesting the involvement of Bax and Bcl-2. Bcl-2 family of proteins play a critical role in the regulation of apoptosis in various tumour cell lines. This pathway may be altered in cancer cells. We have used calyculin-A (CL-A), an inhibitor of protein phosphatase 2A (PP2A), to investigate the mechanism by which lithium induces apoptosis in HL-60 cells. Previous studies in our laboratory established that lithium induces apoptosis of HL-60 cells at 10 mM and above; while CL-A induces apoptosis at 1 nM and above. The observed apoptotic effects were additive. These observations led to the hypothesis that lithium and CL-A exert their biological effects by acting on a similar target. It was, therefore, the aim of this study to establish whether lithium would also exert similar inhibitory effects on the apoptotic and cell cycle regulatory genes. We further aimed at delineating the effects of both lithium and CL-A on the expression profiles of apoptotic and cell cycle regulatory genes. In this study, HL-60 cells were treated with lithium, CL-A and the combination of both. This was followed by the assessment of cell proliferation and viability at specific time points, using Coulter Counter and trypan blue dye exclusion assay, respectively. Concentrations of lithium at 10 mM and 20 mM were found to inhibit cell proliferation and exerted modest effects on cell viability in a time- and dose-dependent manner. Likewise, CL-A inhibited cell proliferation and viability in a time- and dose-dependent fashion. The combination of lithium and CL-A showed additive inhibitory effects on the growth of HL-60 cells. Further, semi-quantitative RT-PCR analyses of apoptotic (bax and bcl-2) and cell cycle regulatory genes (cdc2 and cyclin-B1) were determined. Our data revealed an under-expression of bcl-2 mRNA and an up-regulation of bax mRNA in HL-60 cells treated with lithium, CL-A and the combination of both. In addition, the expression levels of cdc2 mRNA remained constant, while cyclin-B1 mRNA expression levels were up-regulated after 24 h in HL-60 cells that were treated with cytotoxic concentrations of lithium and CL-A alone. Furthermore, the combination of lithium and CL-A showed an up-regulation of cyclin-B1 mRNA while cdc2 mRNA levels remained constant in both treated and untreated HL-60 cells. To corroborate the RT-PCR data, we present evidence by Western blot analysis that Bcl-2 family of proteins and cell cycle regulatory genes indeed play a critical role in the regulation of apoptosis in HL-60 cells. Western blot analysis revealed a down-regulation of Bcl-2 under all treatment conditions. However, lithium and CL-A alone failed to show any detectable expression levels of both Bax and cyclin-B1 proteins. In contrast, the combination of both lithium and CL-A showed an up-regulation of Bax and Cdc2 proteins in HL-60 cells. These findings suggest that the molecular mechanism elicited by lithium, CL-A and the combination of both on the growth inhibition of HL-60 cells involves an aberrant expression of apoptotic and cell cycle regulatory genes. In addition, these observations may allude to a notion that both lithium and CL-A may be used and administered successfully as positive alternative anticancer drugs. / the National Research Foundation,and the University of Limpopo Research and Administration

Small heat shock proteins (hsp) have been implicated in mediation of classic preconditioning in the rabbit. Hsp27 is a terminal substrate of the p38 MAPK cascade. One and 2D gel electrophoresis and immunoblotting of cell fractions was used to determine p38 MAPK and hsp27 phosphorylation levels, respectively, during in vitro ischemia in control, calyculin A (Cal A)-treated (protein phosphatase inhibitor), SB203580-treated (p38MAPK inhibitor) and preconditioned (IPC) isolated adult rabbit cardiomyocytes. The dual phosphorylation of p38 MAPK was increased by early ischemia (30-60 min), after which there was a loss of total cytosolic p38 MAPK. The ischemic increase of p38 MAPK dual phosphorylation was enhanced by IPC. Cal A strongly activated dual phosphorylation of p38 MAPK in oxygenated cells and this was maintained into early ischemia. SB203580 inhibited the dual phosphorylation of p38 MAPK and attenuated the loss of total cytosolic p38 MAPK. In each protocol, ischemia translocated hsp27 from the cytosolic fraction to the cytoskeletal fraction at similar rates and extents. Hsp27 phosphorylation was quantitated as the fraction of diphosphorylated hsp27, based on IEF mobility shifts of hsp27 phosphorylation isoforms. In oxygenated control cells, cytosolic and cytoskeletal hsp27 was highly phosphorylated. After 90 min ischemia, cytoskeletal hsp27 was markedly dephosphorylaled. Cal A slightly increased control cytoskeletal hsp27 phosphorylation. During ischemic incubation, Cal A blocked ischemic dephosphorylation. SB203580 accelerated ischemic hsp27 dephosphorylation and injury. IPC insignificantly decreased the initial rate of ischemic dephosphorylation of hsp27, but not the extent of dephosphorylation in later ischemia. Phosphorylation is regulated by both kinase and phosphatase activities. IPC protection was not correlated with a significant increase in cytosolic or cytoskeletal hsp27 phosphorylation levels during prolonged (> 60-90 min) ischemia.

No / 1 Pharmacological inhibitors of protein kinase A (PKA) and protein phosphatases 1/2A were used to determine whether basal L-type Ca2+ current (ICa) observed in the absence of exogenous ß-adrenergic receptor stimulation is sustained by PKA-mediated phosphorylation. Amphotericin B was used to record whole-cell ICa in the perforated patch-clamp configuration. 2 Calyculin A and isoprenaline (both 1 ¿mol l¿1) increased basal ICa (P<0.05), whereas H-89 inhibited ICa in a concentration-dependent manner with an IC50 ~5 ¿mol l¿1. H-89 also inhibited the response to 1.0 ¿mol l¿1 isoprenaline, although relatively high concentrations (30 ¿mol l¿1) were required to achieve complete suppression of the response. 3 Double-pulse protocols were used to study the effects of 10 ¿mol l¿1 H-89 on time-dependent recovery of ICa from voltage-dependent inactivation as well as the steady-state gating of ICa. T0.5 (time for ICa to recover to 50% of the preinactivation amplitude) increased in the presence of H-89 (P<0.05) but was unaffected by calyculin A or isoprenaline. 4 Steady-state activation/inactivation properties of ICa were unaffected by 10 ¿mol l¿1 H-89 or 1 ¿mol l¿1 calyculin A, whereas isoprenaline caused a leftward shift in both curves so that V0.5 for activation and inactivation became more negative. 5 Data show that basal ICa is regulated by cAMP-PKA-mediated phosphorylation in the absence of externally applied ß-receptor agonists and that relatively high concentrations of H-89 are required to fully suppress the response to ß-adrenergic receptor stimulation, thereby limiting the value of H-89 as a useful tool in dissecting signalling pathways in intact myocytes.

Accumulation of osmotically active metabolites, which create an osmotic gradient estimated at ∼60 mOsM, and cell swelling are prominent features of ischemic myocardial cell death. This study tests the hypothesis that reduction of ischemic swelling by enhanced cell volume regulation is a key mechanism in the delay of ischemic myocardial cell death by ischemic preconditioning (IPC). Experimental protocols address whether: (i) IPC triggers a cell volume regulation mechanism that reduces cardiomyocyte swelling during subsequent index ischemia; (ii) this reduction in ischemic cell swelling is sufficient in magnitude to account for the IPC protection; (iii) the molecular mechanism that mediates IPC also mediates cell volume regulation. Two experimental models with rabbit ventricular myocytes were studied: freshly isolated pelleted myocytes and 48-h cultured myocytes. Myocytes were preconditioned either by distinct short simulated ischemia (SI)/simulated reperfusion protocols (IPC), or by subjecting myocytes to a pharmacological preconditioning (PPC) protocol (1 μM calyculin A, or 1 μM N6-2-(4-aminophenyl)ethyladenosine (APNEA), prior to subjecting them to either different durations of long SI or 30 min hypo-osmotic stress. Cell death (percent blue square myocytes) was monitored by trypan blue staining. Cell swelling was determined by either the bromododecane cell flotation assay (qualitative) or video/confocal microscopy (quantitative). Simulated ischemia induced myocyte swelling in both the models. In pelleted myocytes, IPC or PPC with either calyculin A or APNEA produced a marked reduction of ischemic cell swelling as determined by the cell floatation assay. In cultured myocytes, IPC substantially reduced ischemic cell swelling (P < 0.001). This IPC effect on ischemic cell swelling was related to an IPC and PPC (with APNEA) mediated triggering of cell volume regulatory decrease (RVD). IPC and APNEA also significantly (P < 0.001) reduced hypo-osmotic cell swelling. This IPC and APNEA effect was blocked by either adenosine receptor, PKC or Cl- channel inhibition. The osmolar equivalent for IPC protection approximated 50-60 mOsM, an osmotic gradient similar to the estimated ischemic osmotic load for preconditioned and non-preconditioned myocytes. The results suggest that cell volume regulation is a key mechanism that accounts for most of the IPC protection in cardiomyocytes.