Serum Withdrawal-induced Apoptosis Research Articles

Protective effects of olanzapine and haloperidol on serum withdrawal-induced apoptosis in SH-SY5Y cells

Purpose Recent clinical studies have suggested that treatment with second generation antipsychotic drugs such as olanzapine may prevent progressive alterations of brain structure in patients with schizophrenia. However, the molecular mechanisms underlying these different effects remain to be determined. We investigated the mechanisms of action of olanzapine and haloperidol, on serum withdrawal apoptosis in human neuroblastoma SH-SY5Y cells. Methods SH-SY5Y cells were cultured with olanzapine and haloperidol in medium with or without serum. We determined the effects of the drugs on cell viability against serum withdrawal by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay. Additionally, to explore the drugs' actions, Western blot was performed to examine the expression of key genes involved in GSK-3β-mediated signaling, notably GSK-3β, β-catenin, and Bcl-2. Results SH-SY5Y cells suffered about a 38% loss in cell number under serum-free conditions for 48 h. Olanzapine (10–200 μM) up to 100 μM significantly attenuated serum withdrawal-induced cell loss ( p < 0.01), and a dose of 100 μM also increased cell viability ( p < 0.05). In contrast, haloperidol (0.01–10 μM) did not affect cell viability but exacerbated cell death at 10 μM under serum-free conditions ( p < 0.01). Western blot analysis showed that olanzapine, but not haloperidol, prevented the serum withdrawal-induced decrease in levels of neuroprotective proteins such as p-GSK-3β, β-catenin, and Bcl-2 ( p < 0.01), whereas haloperidol robustly reduced the levels of these proteins at a 10 μM dose in serum-starved cells ( p < 0.05). Moreover, olanzapine alone significantly increased phosphorylation of GSK-3β under normal conditions ( p < 0.05). Conclusions This study showed that olanzapine may have neuroprotective effects, whereas haloperidol was apparently neurotoxic. The actions of signaling systems associated with GSK-3β may be key targets for olanzapine and haloperidol, but their effects are distinct. These differences suggest different therapeutic effects of first and second generation antipsychotic drugs in patients with schizophrenia.

Insulin-like growth factor-I activation of Akt survival cascade in neuronal cells requires the presence of its cognate receptor in caveolae

Insulin-like growth factor-I (IGF-I) plays important roles in survival of neurons. Caveolae, cholesterol-rich microdomains of plasma membrane, act as platforms for some neurotrophic factors. In this study, we examined a possible role of caveolae in IGF-I signal transduction in pheochromocytoma PC12 cells. IGF-I treatment attenuated serum withdrawal-induced apoptosis, which was reversed by treatment with methyl-β-cyclodextrin (CD) that removes cholesterol from plasma membrane. Immunocytochemical and subcellular fractionation analyses revealed that IGF-I receptor (IGF-IR) was colocalized with caveolin-1, a major protein component in caveolae, and that CD treatment reduced IGF-IR contents in caveolae. Consistent with these findings, IGF-I phosphorylation of insulin receptor substrate-1 and Akt was impaired, and cholesterol supply restored the IGF-I action. Furthermore, experiments using small interfering RNA revealed that the reduction of caveolin-1 expression impaired the IGF-I action. In addition, the colocalization of IGF-IR with caveolin-1, and the caveolae-dependent IGF-I action were duplicated in primary culture of rat cerebellar granule neurons. These results demonstrate that the presence of IGF-IR in caveolae is required for the neuroprotective action of IGF-I.

The neuroprotective effect of Activin A and B: implication for neurodegenerative diseases

Activin is a member of the transforming growth factor-beta superfamily which comprises a growing list of multifunctional proteins that function as modulators of cell proliferation, differentiation, hormone secretion and neuronal survival. This study examined the neuroprotective effect of both Activin A and B in serum withdrawal and oxidative stress apoptotic cellular models and investigated the expression of pro- and anti-apoptotic proteins, which may account for the mechanism of Activin-induced neuroprotection. Here, we report that recombinant Activin A and B are neuroprotective against serum deprivation- and toxin- [either the parkinsonism-inducing neurotoxin, 6-hydroxydopamine (6-OHDA) or the peroxynitrite donor, 3-(4-morpholinyl) sydnonimine hydrochloride (SIN-1)] induced neuronal death in human SH-SY5Y neuroblastoma cells. Furthermore, we demonstrate for the first time that transient transfection with Activin betaA or betaB significantly protect SH-SY5Y and rat pheochromocytoma PC12 cells against serum withdrawal-induced apoptosis. This survival effect is mediated by the Bcl-2 family members and involves inhibition of caspase-3 activation; reduction of cleaved poly-ADP ribose polymerase and phosphorylated H2A.X protein levels and elevation of tyrosine hydroxylase expression. These results indicate that both Activin-A and -B share the potential to induce neuroprotective activity and thus may have positive impact on aging and neurodegenerative diseases to retard the accelerated rate of neuronal degeneration.

Serum Withdrawal‐Induced Apoptosis in ZrchI Prion Protein (PrP) Gene‐Deficient Neuronal Cell Line Is Suppressed by PrP, Independent of Doppel

Previous studies have shown that cellular prion protein (PrP(C)) plays anti-apoptotic and antioxidative role against cell death induced by serum-deprivation (SDP) in an immortalized prion protein gene-deficient neuronal cell line derived from Rikn prion protein (PrP) gene-deficient (Prnp(-/-)) mice, which ectopically produce excess Doppel (Dpl) (PrP-like glycoprotein). To investigate whether PrP(C) inhibits apoptotic neuronal cell death without Dpl, an immortalized cell line was established from the brain of ZrchI Prnp(-/-) mice, which do not show ectopic expression of Dpl. The results using a ZrchI neuronal Prnp(-/-) cell line (NpL2) showed that PrP(C) potently inhibited SDP-induced apoptotic cell death. Furthermore, PrP(C) expression enhanced the superoxide dismutase (SOD) activity in NpL2 cells. These results indicate that Dpl production did not affect anti-apoptotic and anti-oxidative functions of PrP, suggesting that PrP(C) may be directly correlated with protection against oxidative stress.

Bone Marrow Stroma-Produced TGF-beta1 Confers Chemoresistance of Leukemic Cells.

TGF-beta1 is secreted at high levels by bone marrow stromal cells and osteoblasts, however its role in leukemia/stroma interactions is unknown. We investigated the effects of TGF-beta1 on survival and proliferation of human monoblastic U937 cells. First, the production of TGF-beta1 by bone marrow-derived mesenchymal stem cells (MSCs) and by U937 cells was analyzed by ELISA. U937 cells secreted approximately 0.1 ng/ml of TGF-beta, while TGF-beta1 level was over 20-fold higher in MSC conditioned medium (2.1±1.7 ng/ml at 72 hours). When U937 cells were co-cultured with MSC, TGFbeta concentration was 2.2±0.2 ng/ml. Hence, we selected 2 ng/ml concentration of recombinant TGF-beta to examine the effects on survival and proliferation of U937 cells exposed to chemotherapy or serum starvation. TGF-beta1 significantly reduced Ara-C or serum withdrawal-induced apoptosis of U937 cells in particular when U937 cells were co-cultured with MSCs (AnnexinV(+)%;U937 alone, control 34.5±8.4, TGF-beta1 18.4±4.5, Ara-C 88.6±3.0, Ara-C/TGF-beta1 60.4±8.0, p=0.04; U937 co-cultured with MSC, control 19.4±2.8, TGF-beta1 3.5±1.0, Ara-C 69.0±3.6, Ara-C+TGF-beta1 24.9±3.3, p=0.01). Treatment with rhTGF-beta1 also enhanced percentage of cells in G0/1 cell cycle phase hence promoting quiescence (G0/G1 phase; control 50.1±6.7, TGF-beta1 63.8±5.5, p=0.04). Most importantly, inhibition of TGF-beta1 by neutralizing antibody (TGF-NA) significantly diminished the pro-survival effects of rhTGF-beta1 in serum-deprived U937 cells co-cultured with MSC (AnnexinV(+)%; U937 alone, control 27.9±9.0, TGF-beta1 20.1±3.3, TGF-NA 21.8±2.2, TGF-beta1+ TGF-NA 18.9±4.1; U937 co-cultured with MSC, control 22.2±1.9, TGF-beta1 10.9±2.5, TGF-NA 29.6±5.1, TGF-beta1+ TGF-NA 20.2±0.9, p<0.001). Dissection of molecular mechanisms demonstrated that TGF-beta1 induced anti-apoptotic Bcl-2 protein expression in U937 cells. Furthermore, concomitant exposure of U937 cells to rhTGF-beta1 in MSC co-cultures results in activation of AKT signaling and pro-survival phosphorylation of Bcl-2, implicating their role in anti-apoptotic effects of TGF-beta1. Taken together, these findings demonstrate that TGF-beta1 secreted by BM stroma cells promotes survival of U937 cells and confers chemoresistance of leukemic cells within the bone marrow microenvironment. Since small molecule inhibitors of TGF-beta signaling are undergoing final steps of pre-clinical development, this approach may represent a viable strategy to enhance efficacy of chemotherapy in AML.

Cycloheximide Protects HepG2 Cells from Serum Withdrawal-Induced Apoptosis by Decreasing p53 and Phosphorylated p53 Levels

Cycloheximide (CHX), an inhibitor of protein synthesis, has been reported to prevent cell death in a wide variety of cell types and produced by different apoptotic stimuli. However, the mechanisms by which CHX protects cells from apoptosis are still unclear. In this study, we investigated whether p53 plays a role in the protection by CHX against serum withdrawal-induced apoptosis. Deprivation of serum from the culture medium causes apoptosis in HepG2 cells, and CHX dramatically protects cells from death. p53, p21, and Bax protein levels were elevated, and cell cycle arrest was produced after serum withdrawal. CHX abolished this elevation of p53, p21, and Bax as well as the cell cycle arrest induced by serum deprivation. The p53 inhibitor pifithrin-alpha protects HepG2 cells against apoptosis induced by serum withdrawal. HepG2 cells expressing a dominant negative form of mutant p53 and Hep3B cells lacking p53 were resistant to serum withdrawal-induced apoptosis. Lowering of p53 by small interfering RNA protects HepG2 cells from serum withdrawal-induced apoptosis. p53 phosphorylation was induced by serum withdrawal and other chemotherapeutic reagents such as actinomycin D, doxorubicin, and etoposide. CHX decreases the levels of phosphorylated p53 (pp53) even in the presence of a proteasome inhibitor, which maintains the total p53 levels, whereas it does not affect the dephosphorylation of pp53. These results suggest the possibility that kinases that phosphorylate p53 might be affected by CHX administration. In summary, CHX protects HepG2 cells from serum withdrawal-induced apoptosis through inhibiting the synthesis of p53 and the phosphorylation of p53.

Wnt Proteins Prevent Apoptosis of Both Uncommitted Osteoblast Progenitors and Differentiated Osteoblasts by β-Catenin-dependent and -independent Signaling Cascades Involving Src/ERK and Phosphatidylinositol 3-Kinase/AKT

Genetic studies in humans and mice have revealed an important role of the Wnt signaling pathway in the regulation of bone mass, resulting from potent effects on the control of osteoblast progenitor proliferation, commitment, differentiation, and perhaps osteoblast apoptosis. To establish the linkage between Wnts and osteoblast survival and to elucidate the molecular pathways that link the two, we have utilized three cell models: the uncommitted bipotential C2C12 cells, the pre-osteoblastic cell line MC3T3-E1, and bone marrow-derived OB-6 osteoblasts. Serum withdrawal-induced apoptosis was prevented by the canonical Wnts (Wnt3a and Wnt1) and the noncanonical Wnt5a in all cell types. Wnt3a induced LRP5-independent transient phosphorylation and nuclear accumulation of ERKs and phosphorylation of Src and Akt. The anti-apoptotic effect of Wnt3a was abrogated by inhibitors of canonical Wnt signaling, as well as by inhibitors of MEK, Src, phosphatidylinositol 3-kinase (PI3K), or Akt kinases, or by the addition of cycloheximide to the culture medium. Wnt3a-induced phosphorylation of GSK-3beta and downstream activation of beta-catenin-mediated transcription required ERK, PI3K, and Akt signaling. Wnt3a increased the expression of the anti-apoptotic protein Bcl-2 in an ERK-dependent manner. Beta-catenin-mediated transcription was permissive for the anti-apoptotic actions of Wnt1 and Wnt3a but was dispensable for the anti-apoptotic action of Wnt5a. However, Src, ERKs, PI3K, and Akt kinases were required for the anti-apoptotic effects of Wnt5a. These results demonstrate for the first time that Wnt proteins, irrespective of their ability to stimulate canonical Wnt signaling, prolong the survival of osteoblasts and uncommitted osteoblast progenitors via activation of the Src/ERK and PI3K/Akt signaling cascades.

Increased Expression of Mcl-1 Is Required for Protection against Serum Starvation in Phosphatase and Tensin Homologue on Chromosome 10 Null Mouse Embryonic Fibroblasts, but Repression of Bim Is Favored in Human Glioblastomas

Inactivating mutations in the tumor suppressor gene phosphatase and tensin homologue on chromosome 10 (PTEN) result in elevated levels of phosphatidylinositol (3,4,5)-trisphosphate, activation of protein kinase B (PKB), and protection against apoptotic insults such as withdrawal of survival factors. Protection may arise through the inhibition of the pro-apoptotic protein Bim, which is normally repressed by a PKB-dependent mechanism. Here we show that PTEN-/- immortalized mouse embryonic fibroblasts (MEFs) exhibit elevated PKB phosphorylation and are resistant to serum withdrawal-induced death, but exhibit normal Bim expression following withdrawal of serum. In contrast, expression of Mcl-1, a prosurvival member of the Bcl-2 family, was elevated in PTEN-/- MEFs. Transient or stable overexpression of Mcl-1 in PTEN+/- MEFs conferred resistance to serum withdrawal, whereas ablating expression of Mcl-1 in PTEN-/- MEFs, using RNA interference, abolished their resistance to serum withdrawal-induced apoptosis. To determine if Mcl-1 is selected for overexpression in human tumors we examined human glioblastoma cell lines but found that loss of PTEN had no effect on Mcl-1 expression. In contrast, two of three PTEN-/- glioblastoma cell lines exhibited low expression of Bim, which was refractory to serum withdrawal. These results indicate that the resistance of PTEN-/- MEFs to serum withdrawal is largely due to the up-regulation of Mcl-1 but that loss of PTEN in tumor cell lines is more complex and may favor de-regulation of different apoptotic regulators such as Bim.

Insulin-like growth factor-I induces the phosphorylation and nuclear exclusion of forkhead transcription factors in human neuroblastoma cells

Akt-mediated phosphorylation of forkhead transcription factors is linked to growth factor-stimulated cell survival. We investigated whether the survival activity of insulin-like growth factor-I (IGF-I) in SH-SY5Y human neuroblastoma (NBL) cells is associated with phosphorylation and/or localization changes in forkhead proteins. IGF-I induced phosphorylation of Erks (p42/p44), FKHR (FOXO1a) (Ser 253), FKHRL1 (FOXO3a) (Ser 256), and Akt (Ser 473). PI3-K inhibitor, LY294002, reduced IGF-I-stimulated phosphorylation of FKHR, FKHRL1, and Akt, but did not affect Erk phosphorylation. Using a GFP-FKHR construct, FKHR imported into the nucleus during growth factor withdrawal-induced apoptosis. In addition, IGF-I rescue from serum withdrawal-induced apoptosis is associated with a rapid export of GFP-FKHR into the cytoplasm. Leptomycin B, an inhibitor of Crm1-mediated nuclear export, decreased the level of FKHRL1 phosphorylation in the presence of IGF-I in vector and FKHR overexpressing cells, but had no effect on the phosphorylation status of FKHR. In addition, leptomycin B prevented IGF-I stimulated nuclear export of GFP-FKHR. These studies show IGF-I phosphorylation of FKHR and FKHRL1 via a PI3-K-dependent pathway in NBL cells.

Novel Neuroprotective Mechanism of Action of Rasagiline Is Associated with Its Propargyl Moiety: Interaction of Bcl-2 Family Members with PKC Pathway

Our studies have provided new insights into the biological mechanism of neuroprotection of the anti-Parkinson drug, rasagiline [N-propargyl-(1R)-aminoindan], involving the association of Bcl-2 family proteins with protein kinase C (PKC) pathway. In a model of serum withdrawal-induced apoptosis of rat pheochromocytoma PC12 cells, rasagiline and its propargyl moiety, N-propargylamine, decreased cell death via multiple neuroprotective pathways that include the stimulation of PKC phosphorylation; upregulation of PKCepsilon mRNA; induction of Bcl-X(L), Bcl-w, and brain-derived neurotrophic factor (BDNF) mRNAs; and downregulation of PKCgamma, Bad, and Bax mRNAs. Moreover, these drugs inhibited the cleavage and activation of pro-caspase-3 and poly(ADP-ribose) polymerase (PARP), while PKC inhibitor, GF109203X, reversed these actions. In addition, rasagiline decreased serum-free-induced levels of the important regulator of cell death, Bad, which was also blocked by GF109203X, indicating the involvement of PKC-dependent cell survival activity of rasagiline. Structure activity studies have established that N-propargylamine is essential for the novel neuroprotective and the neuronal cell survival activity of rasagiline since this moiety itself revealed similar protective effects and mechanisms of action. These results have led us to develop several multifunctional neuroprotective drugs containing the propargyl moiety and iron-chelating property for the treatment and/or prevention of neurodegenerative diseases.

Irs–2 Mediates the Antiapoptotic Effect of Insulin in Neonatal Hepatocytes

To assess the role of insulin action and inaction in the liver, immortalized hepatocyte cell lines have been generated from insulin receptor substrate (IRS)-2(-/-) and wild-type mice. Using this model, we have recently demonstrated that the lack of IRS-2 in neonatal hepatocytes resulted in insulin resistance. In the current study, we show that immortalized neonatal hepatocytes undergo apoptosis on serum withdrawal, with caspase-3 activation and DNA laddering occurring earlier in the absence of IRS-2. Insulin rescued wild-type hepatocytes from serum withdrawal-induced caspase-3 activation and DNA fragmentation in a dose-dependent manner, but it failed to rescue hepatocytes lacking IRS-2. In IRS-2(-/-) cells, insulin failed to phosphorylate Bad. Furthermore, in these cells, insulin was unable to translocate Foxo1 from the nucleus to the cytosol. Adenoviral infection of wild-type cells with constitutively active Foxo1 (ADA) induced caspase-8 and caspase-3 activities, proapoptotic gene expression, DNA laddering and apoptosis. Dominant negative Foxo1 regulated the whole pathway in an opposite manner. Prolonged insulin treatment (24 hours) increased expression of antiapoptotic genes (Bcl-xL), downregulated proapoptotic genes (Bim and nuclear Foxo1), and decreased caspase-3 activity in wild-type hepatocytes but not in IRS-2(-/-) cells. Infection of IRS-2(-/-) hepatocytes with adenovirus encoding IRS-2 reconstituted phosphatidylinositol 3-kinase (PI 3-kinase)/Akt/Foxo1 signaling, restored pro- and antiapoptotic gene expression, and decreased caspase-3 activity in response to insulin, thereby blocking apoptosis. In conclusion, IRS-2 signaling is specifically required through PIP3 generation to mediate the survival effects of insulin. Epidermal growth factor, via PIP3/Akt/Foxo1 phosphorylation, was able to rescue IRS-2(-/-) hepatocytes from serum withdrawal-induced apoptosis, modulating pro- and anti-apoptotic gene expression and downregulating caspase-3 activity.

Phorbol ester-induced cell death in PC-12 cells overexpressing Bcl-2 is dependent on the time at which cells are treated.

The aim of the present study was to investigate the involvement of PKC in Bcl-2 protection against serum withdrawal-induced apoptosis in PC-12 cells. Human Bcl-2 was overexpressed in PC-12 cells and was found to totally inhibit serum withdrawal-induced apoptosis. 12-O-tetradecanoylphorbol-13-acetate (TPA) could induce cell death in PC-12 cells that overexpressed Bcl-2, implicating protein kinase C (PKC) in Bcl-2 protection. However, TPA-induced cell death did not involve caspase-3 activation or DNA degradation, suggesting that Bcl-2 was still inhibiting these processes and that TPA was mediating cell death either downstream of Bcl-2 or via independent signalling pathways. High cytosolic and particulate protein levels of PKC delta were correlated with TPA-induced cell death suggesting that PKC delta positively regulated this cell death. However, substantial down-regulation of PKC by prolonged exposure to TPA did not reduce the frequency of TPA-induced cell death, raising the possibility that PKC delta did not regulate cell death alone. Surprisingly, TPA-induced cell death was dependent on the time at which cells were treated, suggesting that cells were changing with time. Supporting this idea, the cytosolic and particulate protein levels of PKC delta and were found to change with time, and may account for the time-dependent manner in which TPA induced cell death. This is the first report to show that sensitivity to drug induced cell death in a cultured cell line changes with time. Experimental and theoretical evidence suggests that many cellular constituents exhibit temporal variations, oscillations or rhythms due to feedback regulation. We believe that investigation of these temporal changes, how they alter cell function with time and how they might be manipulated in single cells as well as across cellular populations is paramount in furthering our understanding of cellular behaviour.

A Role for Caspase-1 in Serum Withdrawal-Induced Apoptosis of Endothelial Cells

Mouse lung endothelial cells (MLEC) and HUVEC were used under serum withdrawal (SW) conditions as a model of endothelial cell (EC) apoptosis. Apoptosis was quantified by time-lapse video microscopy. Mouse lung ECs from caspase-1−/− mice had significantly reduced rates of SW-induced apoptosis compared with wild-type mice, specifically implicating caspase-1 in proapoptotic signaling in ECs. SW conditions induced HUVEC apoptosis with concomitant activation of caspase-1. Further studies demonstrated that the caspase-1 inhibitors z-VAD and z-YVAD significantly reduced the rate of SW-induced HUVEC apoptosis. HUVEC, when transfected with caspase-1, showed a highly significant increase in apoptosis. SW was associated with increases in reactive oxygen species production that were significantly inhibited by the antioxidant N-acetyl-l-cysteine, although rates of apoptosis and caspase-1 activation were unaffected. These results demonstrate the involvement of caspase-1 in SW-induced EC apoptosis, independently of reactive oxygen species production.

Brain-Derived Neurotrophic Factor Protection of Cortical Neurons from Serum Withdrawal-Induced Apoptosis Is Inhibited by cAMP

Programmed cell death plays an important role both during the development of the CNS and in its homeostasis throughout adulthood. A complex balance between cell death- and survival-inducing signals determines the fate of individual neurons. Intracellular cAMP is thought to regulate neuronal survival, and previous studies have shown that the survival of retinal ganglion cells by brain-derived neurotrophic factor (BDNF) is dependent on cAMP. Here we report the surprising observation that cAMP attenuates the ability of BDNF to rescue cortical neurons from apoptosis after serum deprivation, a process mediated via the phosphatidylinositol 3 (PI3)-kinase signal transduction cascade. Depolarization by KCl, which increases cAMP in cortical neurons, also attenuates BDNF protection against serum withdrawal. Our data indicate that cAMP antagonizes neurotrophin protection from serum withdrawal by inhibiting the PI3-kinase signal transduction cascade. This study indicates that cAMP may inhibit some forms of neurotrophin-mediated neuronal survival and suggests that a number of PI3-kinase-regulated processes in neurons may be inhibited by cAMP.

Characteristics of binding of insulin-like growth factor (IGF)-I and IGF-II analogues to the type 1 IGF receptor determined by BIAcore analysis.

Insulin-like growth factor (IGF) binding to the type 1 IGF receptor (IGF1R) elicits mitogenic effects, promotion of differentiation and protection from apoptosis. This study has systematically measured IGF1R binding affinities of IGF-I, IGF-II and 14 IGF analogues to a recombinant high-affinity form of the IGF1R using BIAcore technology. The analogues assessed could be divided into two groups: (a) those designed to investigate binding of IGF-binding protein, which exhibited IGF1R-binding affinities similar to those of IGF-I or IGF-II; (b) those generated to probe IGF1R interactions with greatly reduced IGF1R-binding affinities. The relative binding affinities of IGF-I analogues and IGF-I for the IGF1R determined by BIAcore analysis agreed closely with existing data from receptor-binding assays using cells or tissue membranes, demonstrating that BIAcore technology is a powerful tool for measuring affinities of IGFs for IGF1R. In parallel studies, IGF1R-binding affinities were related to ability to protect against serum withdrawal-induced apoptosis in three different assays including Hoechst 33258 staining, cell survival, and DNA fragmentation assays using the rat pheochromocytoma cell line, PC12. In this model system, IGF-I and IGF-II at low nanomolar concentrations are able to prevent apoptosis completely. We conclude that ability to protect against apoptosis is directly related to ability to bind the IGF1R.

Eligibility for Lipid-Lowering Drug Therapy in Primary Prevention

In May 2001, the Adult Treatment Panel III (ATP III) of the National Cholesterol Education Program issued revised guidelines for diagnosing and treating high blood cholesterol.1 These guidelines represent a major advance in risk assessment. They were preceded by the ATP I guidelines (1988), which focused on primary prevention of coronary heart disease (CHD), and by the ATP II guidelines (1993),2 which discussed primary and secondary prevention. In the ATP II and ATP III guidelines, low-density lipoprotein cholesterol (LDL-C) is the primary target of risk-reduction therapy. See p 152 The ATP II recommendations were based on epidemiological, preclinical, and incomplete clinical trial evidence. Within a few years after their publication, the results of 5 large-scale trials of 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors (ie, “statins”) were reported. Two of these (West of Scotland Coronary Prevention Study [WOSCOPS] and Air Force/Texas Coronary Atherosclerosis Prevention Study [AFCAPS/TexCAPS]) were primary-prevention trials.3,4 The remaining 3 focused on secondary prevention.5-7 Based on these epic trials and other laboratory, epidemiological, and clinical evidence, the ATP III guidelines were developed (Tables 1 and 2⇓). View this table: Table 1103484. ATP III Guidelines: New Features View this table: Table 2103484. ATP III Guidelines: Global Risk Assessment The major statin trials and resulting meta-analyses provided a firm foundation for a truly evidence-based approach to guideline revision. Involving 5 different populations with widely varying absolute risk levels and using 3 different statins (simvastatin, pravastatin, lovastatin), they produced remarkably consistent reductions of 24% to 37% in relative risk for major CHD events. In WOSCOPS, 6595 men with moderate hypercholesterolemia (LDL-C=192 mg/dL±SD) received pravastatin 40 mg/day for up to 4.9 years. Results indicated a 31% relative decrease in major CHD events, with a 26% reduction in LDL-C levels. AFCAPS/TexCAPS evaluated lovastatin 20 to 40 mg/day for up to 5.2 years in 6605 generally healthy men and women with …

Role of mitochondria in serum withdrawal-induced apoptosis of immortalized neuronal precursors

The intracellular mechanisms controlling apoptosis in immature neurons are still largely unknown. Taking immortalized hippocampal neuronal precursors (mouse cell line HN9.10e) as a model, we have analyzed the cellular events associated to apoptosis induced by serum deprivation. We observed translocation of Bax from cytosol to mitochondria after 1 h of serum withdrawal followed, 2 h later, by cytochrome c release from mitochondria. These events occurred without mitochondrial membrane potential loss nor mitochondrial calcium raise. As calcium is implicated in several cell death pathways, we analyzed intracellular calcium levels after longer periods of serum deprivation. After 6 h, an increase of cytosolic Ca 2+ was detected in HN9.10e cells loaded with the Ca 2+ indicator Fluo3-AM. This increase of calcium preceded morphological signs of apoptosis such as cell shrinkage and nuclear fragmentation, and was followed by a more pronounced raise that persisted until the terminal phases of the apoptotic process. Cells serum-deprived for 4 h and then grown in complete medium for 20 h fully recovered viability. Summarizing, in HN9.10e cells, calcium deregulation occurs in the late phases of apoptosis; earlier events involve translocation of Bax, release of cytochrome c, and maintenance of mitochondrial functionality. This allows an enlargement of the temporal window in which commitment to death is reversible.

Caspase-mediated Cleavage of the Ca2+/Calmodulin-dependent Protein Kinase-like Kinase Facilitates Neuronal Apoptosis

This study was designed to identify the role of a recently identified Ca(2+)/calmodulin-dependent protein kinase (CaMK)-like kinase (CaMKLK) in neuronal apoptosis. For this purpose, we studied proteolytic cleavage of CaMKLK by caspases in vitro and in neuronal NG108 cells. In addition, we have investigated the effect of overexpression of wild type and mutant CaMKLK proteins on staurosporine- and serum deprivation-induced apoptosis of NG108 cells. We found that CaMKLK is a substrate for caspase-3 and -8, both in vitro and in NG108 cells during staurosporine- and serum withdrawal-induced apoptosis. Substitution of an aspartic acid residue at position 62 in an asparagine residue within a putative caspase cleavage site completely blocked cleavage of CaMKLK, strongly indicating that (59)DEND(62) is the caspase recognition site. Overexpression of an Asp(62) --> Asn CaMKLK mutant protected NG108 cells from staurosporine-induced apoptosis to a similar extent as Bcl-x(L). In contrast, overexpression of wild type CaMKLK did not lead to protection. Moreover, microinjection of Asp(62) --> Asn CaMKLK protected NG108 cells from serum deprivation-induced apoptosis, while overexpression of a caspase-generated noncatalytic N-terminal CaMKLK fragment exacerbated apoptosis. Together, our data suggest that cleavage of CaMKLK and generation of the noncatalytic N-terminal domain of CaMKLK facilitate neuronal apoptosis.

Cytokine response gene 8 (CR8) regulates the cell cycle G1-S phase transition and promotes cellular survival.

Cellular proliferation and survival are modulated by the expression of specific genes. Cytokine response gene 8 (CR8), which was originally cloned as an IL-2-induced gene in human T lymphocytes, encodes a basic helix--loop--helix (bHLH) transcription factor. The CR8 gene product is highly conserved among human, mouse and rat, and contains sequence motifs that distinguish it from other bHLH families. The CR8 gene is ubiquitously expressed, and CR8 gene expression is induced by both growth-promoting as well as growth-inhibitory stimuli. As bHLH proteins have been found to regulate both the G1-S phase cell cycle transition, as well as cellular survival, the effects of CR8 on these processes were investigated. Ectopic CR8 expression in asynchronous U2OS cell cultures reduces the percentage of cells in the cell cycle S phase, and also slows the entry of G1-synchronized cells into S phase. The prolonged G1 interval correlates with impaired elevation of cyclin E protein and prolonged p21 protein expression in G1. CR8 expression also protects U2OS cells from serum-withdrawal induced apoptosis. These results indicate that CR8 is an important modulator of both the G1-S phase cell cycle transition, and cellular survival.

Sphingosine kinase expression regulates apoptosis and caspase activation in PC12 cells.

Sphingosine-1-phosphate (SPP), a bioactive sphingolipid metabolite, suppresses apoptosis of many types of cells, including rat pheochromocytoma PC12 cells. Elucidating the molecular mechanism of action of SPP is complicated by many factors, including uptake and metabolism, as well as activation of specific G-protein-coupled SPP receptors, known as the endothelial differentiation gene-1 (EDG-1) family. In this study, we overexpressed type 1 sphingosine kinase (SPHK1), the enzyme that converts sphingosine to SPP, in order to examine more directly the role of intracellularly generated SPP in neuronal survival. Enforced expression of SPHK1 in PC12 cells resulted in significant increases in kinase activity, with corresponding increases in intracellular SPP levels and concomitant decreases in both sphingosine and ceramide, and marked suppression of apoptosis induced by trophic factor withdrawal or by C(2)-ceramide. NGF, which protects PC12 cells from serum withdrawal-induced apoptosis, also stimulated SPHK1 activity. Surprisingly, overexpression of SPHK1 had no effect on activation of two known NGF-stimulated survival pathways, extracellular signal regulated kinase ERK 1/2 and Akt. However, trophic withdrawal-induced activation of the stress activated protein kinase, c-Jun amino terminal kinase (SAPK/JNK), and activation of the executionary caspases 2, 3 and 7, were markedly suppressed. Moreover, this abrogation of caspase activation, which was prevented by the SPHK inhibitor N,N-dimethylsphingosine, was not affected by pertussis toxin treatment, indicating that the cytoprotective effect was likely not mediated by binding of SPP to cell surface G(i)-coupled SPP receptors. In agreement, there was no detectable release of SPP into the culture medium, even after substantially increasing cellular SPP levels by NGF or sphingosine treatment. In contrast to PC12 cells, C6 astroglioma cells secreted SPP, suggesting that SPP might be one of a multitude of known neurotrophic factors produced and secreted by glial cells. Collectively, our results indicate that SPHK/SPP may play an important role in neuronal survival by regulating activation of SAPKs and caspases.