Dependent Apoptotic Pathway Research Articles

The Coexistence of Aging and Obesity Additively Increase Cardiac Apoptosis

PURPOSE: Cardiac apoptosis were found in obesity, but the cardiac molecular mechanism in the aging obesity were not available. The purpose of this study was to evaluate whether the aging and obesity will additively influence the apoptotic pathways. METHODS: Eleven young lean (YL), 10 young obese (YO), 11 old lean (OL), and 11 old obese (OO) Zucker rats were investigated at 4 and 14 months old. The cardiac characteristics, apoptotic levels and their related pathways in the excised left ventricle form rats were measured by heart weight index, histological analysis, positive TUNEL assays, H&E stain, and Western blotting. RESULTS: Compared with YL group, the whole heart weight, the abnormal myocardial architecture, and TUNEL-positive apoptotic cells were significantly higher in YO, OL or OO groups. The activity of cardiac Fas receptor-dependent apoptotic pathway (Fas L, Fas, FADD, caspase-8, caspase-3), mitochondrial -dependent apoptotic pathway (Bad, cytosolic cytochrome c, caspase-9,, caspase-3) and transition from Fas- to mitochondrial-dependent apoptotic pathway (tBid) were significantly higher in YO, OL or OO groups than those in YL group. These pathways were higher in OO group than either YO or OL groups. CONCLUSIONS: The coexistence of aging and obesity additively increase cardiac Fas receptor and mitochondrial -dependent apoptosis. These findings may provide a possible apoptotic mechanism for developing heart failure in aging obesity.

Angiotensin II Type 1 Receptor–Activated Caspase-3 Through Ras/Mitogen-Activated Protein Kinase/Extracellular Signal-Regulated Kinase in the Rostral Ventrolateral Medulla Is Involved in Sympathoexcitation in Stroke-Prone Spontaneously Hypertensive Rats

In the rostral ventrolateral medulla (RVLM), angiotensin II-derived superoxide anions, which increase sympathetic nerve activity, induce a pressor response by activating the p38 mitogen-activated protein kinase (p38 MAPK) and extracellular signal-regulated kinase (ERK) pathway. The small G protein Ras mediates a caspase-3-dependent apoptotic pathway through p38 MAPK, ERK, and c-Jun N-terminal kinase. We hypothesized that angiotensin II type 1 receptors activate caspase-3 through the Ras/p38 MAPK/ERK/c-Jun N-terminal kinase pathway in the RVLM and that this pathway is involved in sympathoexcitation in stroke-prone spontaneously hypertensive rats (SHRSP), a model of human hypertension. The activities of Ras, p38 MAPK, ERK, and caspase-3 in the RVLM were significantly higher in SHRSP (14 to 16 weeks old) than in age-matched Wistar-Kyoto rats (WKY). The mitochondrial apoptotic proteins Bax and Bad in the RVLM were significantly increased in SHRSP compared with WKY. c-Jun N-terminal kinase activity did not differ between SHRSP and WKY. In SHRSP, intracerebroventricular infusion of a Ras inhibitor significantly reduced sympathetic nerve activity and improved baroreflex sensitivity, partially because of inhibition of the Ras/p38 MAPK/ERK, Bax, Bad, and caspase-3 pathway in the RVLM. Intracerebroventricular infusion of a caspase-3 inhibitor also inhibited sympathetic nerve activity and improved baroreflex sensitivity in SHRSP. Intracerebroventricular infusion of an angiotensin II type 1 receptor blocker in SHRSP partially inhibited the Ras/p38 MAPK/ERK, Bax, Bad, and caspase-3 pathway in the RVLM. These findings suggest that in SHRSP, angiotensin II type 1 receptor-activated caspase-3 acting through the Ras/p38 MAPK/ERK pathway in the RVLM might be involved in sympathoexcitation, which in turn plays a crucial role in the pathogenesis of hypertension.

Platycodon grandiflorumInduces Apoptosis in SKOV3 Human Ovarian Cancer Cells Through Mitochondrial-Dependent Pathway

Platycodon grandiflorum (Jacq.) A. DC., a Chinese food and medicine, has been used as expectorant traditionally. The present study aimed to investigate the effect of Platycodon grandiflorum extract (PGE) on SKOV3 ovarian cancer cells. 3-(4,5- dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide (MTT) assay was used to monitor cell numbers, Annexin-V/propidium iodide (PI) staining, RT-PCR and Western blot were used to examine cell apoptosis, caspases activation. Bcl-2 and Bax expressions and mitochondrial cytochrome c release. Our result showed that PGE-induced apoptosis was associated with activation of caspase-3, -8 and -9, down-regulation of Bcl-2, up-regulation of Bax and release of mitochondrial cytochrome c to cytosol. The data indicate that PGE may have anti-tumor effect mainly via caspase-3 and caspase-9 dependent apoptotic pathway.

Chamaecypanone C, a novel skeleton microtubule inhibitor, with anticancer activity by trigger caspase 8-Fas/FasL dependent apoptotic pathway in human cancer cells

Microtubule is a popular target for anticancer drugs. Chamaecypanone C, is a natural occurring novel skeleton compound isolated from the heartwood of Chamaecyparis obtusa var. formosana. The present study demonstrates that chamaecypanone C induced mitotic arrest through binding to the colchicine-binding site of tubulin, thus preventing tubulin polymerization. In addition, cytotoxic activity of chamaecypanone C in a variety of human tumor cell lines has been ascertained, with IC 50 values in nanomolar ranges. Flow cytometric analysis revealed that chamaecypanone C treated human KB cancer cells were arrested in G 2–M phases in a time-dependent manner before cell death occurred. Additional studies indicated that the effect of Chamaecypanone C on cell cycle arrest was associated with an increase in cyclin B1 levels and a mobility shift of Cdc2/Cdc25C. The changes in Cdc2 and Cdc25C coincided with the appearance of phosphoepitopes recognized by a marker of mitosis, MPM-2. Interestingly, this compound induced apoptotic cell death through caspase 8-Fas/FasL dependent pathway, instead of mitochondria/caspase 9-dependent pathway. Notably, several KB-derived multidrug resistant cancer cell lines overexpressing P-gp170/MDR and MRP were sensitive to Chamaecypanone C. Taken together, these findings indicated that Chamaecypanone C is a promising anticancer compound that has potential for management of various malignancies, particularly for patients with drug resistance.

Activation of NFκB dependent apoptotic pathway in pancreatic islet cells by hypoxia

Insulin producing β-cells are exposed to hypoxic stress in the early period after islet transplantation. Although it has been suggested that hypoxia leads to islet loss, the underlying mechanisms are unclear. In this study, the early transcriptional profiles of b cell response to hypoxia were determined by using the Affymetrix Murine Genome Array U74Av2 GeneChip (about 12,422 genes). In addition to the upregulation of genes associated with glycolysis, genes related to apoptosis and stress response were also upregulated. The downregulated genes on hypoxia are classified as transcription and inflammatory response. These findings were confirmed by TUNEL assay and real-time reverse transcription-PCR for mRNA of genes related to apoptosis. On the other hand, we have found that the transcription factor NFκB was activated by hypoxia in islet cells. The affected genes involved in the activated NFκB pathway were mainly categorized as apoptosis-related genes by quantitative real-time PCR array (84 genes). Our comprehensive analysis of transcriptional changes of islets by hypoxia may assist the development of strategies that protect islet grafts from early loss.

Time course of Newcastle disease virus-induced apoptotic pathways

Newcastle disease virus (NDV) causes economically significant Newcastle disease (ND) in almost all birds worldwide. Previous studies have shown that NDV induces caspase dependent apoptotic pathways in infected cells. In the present study, time course induction of apoptotic pathways in Vero cells is described. In NDV-infected cells, caspase-8 activity, percentage of cells showing TRAIL expression was higher at 24 h p.i. (post-infection) compared to 48 h p.i. In contrast, caspase-9 activity, efflux of cytochrome c, loss of mitochondrial membrane potential was higher at 48 h compared to 24 h p.i. The caspase-3 activity was high both times. Based on these results, it was concluded that at 24 h p.i., NDV induces apoptosis through extrinsic apoptotic pathway while at 48 h p.i. predominantly through intrinsic apoptotic pathway.

Induction of PUMA-α and down-regulation of PUMA-β expression is associated with benzo(a)pyrene-induced apoptosis in MCF-7 cells

Benzo(a)pyrene (BP) forms benzo(a)pyrene-7,8-dihydrodiol-9,10-epoxide (BPDE)-DNA adducts in human breast adenocarcinoma MCF-7 cells, leading to p53 protein induction and phosphorylation. Although BP-induced apoptosis in rodent cells is known, it is still unclear in human cells. Here we have analyzed the effects of BP on p53 related apoptotic proteins, cell cycle and cell death in MCF-7 cells. PUMA-protein (p53 up-regulated modulator of apoptosis) levels were changed after BP exposure so that PUMA-alpha protein was statistically significantly increased whereas PUMA-beta protein was statistically significantly decreased. PUMA-protein levels were also investigated in ZR-75-1 cells, where PUMA-alpha protein was statistically significantly increased. Cytochrome c, which is released from mitochondria during apoptosis to form the apoptosome, was increased in cytoplasmic fraction after BP exposure in MCF-7 cells. Increased apoptosis was also seen after 48 and 72 h BP exposure (2.5 and 5 microM). In addition, BP decreased dose dependently cell viability (2.5 and 5 microM) and increased ROS formation (1 and 10 microM). Our results suggest that PUMA-alpha protein is involved in BP-induced cell death most likely through a p53 dependent apoptotic pathway.

Therapeutic Effect Of Exercise Training On Cardiac Apoptosis In Streptozotocin-induced Diabetic Rats

Background: Cardiac apoptosis was found in diabetes but very limited information regarding the effects of exercise training on cardiac apoptosis in diabetes was available. The purpose of this study was to evaluate the therapeutic effects of exercise training on cardiac apoptotic pathways. METHODS: Eight Wistar rats and Streptozotocin-induced diabetic rats at 4 months of age were served as negative (Control) and positive (DM) control. Eight Streptozotocin-induced diabetic rats underwent running exercise on treatmill 1 hour daily, 5 sections per week, for 8 weeks (DM-EX). After exercise training or sedentary status, the excised hearts were measured by positive TUNEL assays and Western Blotting. RESULTS: Citrate synthase activity in skeletal muscle in DM-EX is significantly increased compared with sedentary group (Control and DM). Protein levels of mitochondria dependent pathway, including cytochrome c, activated caspase 9, and activated caspase 3 were increased in DM group compared with control. Protein levels of death receptor dependent pathway, including TNF-alpha, Fas ligand, Fas death receptors, Fas-associated death domain (FADD), activated caspase 8, and activated caspase 3 were increased in DM group compared with control. Exercise training decreased diabetes-induced cardiac TUNEL-positive apoptotic cells. Exercise training decreased protein levels of cytochrome c, activated caspase 9, activated caspase 3 (mitochondria pathway) and TNF-alpha, Fas ligand, Fas death receptors, FADD, activated caspase 8, and activated caspase 3 (Fas pathway) in DM-EX group compared with DM group. CONCLUSIONS: Exercise training suppressed diabetes-induced cardiac mitochondria and Fas receptor dependent apoptotic pathways. The findings may provide one of possible therapeutic approaches for preventing cardiac apoptosis in diabetes.

Mathematical model of a network of interaction between p53 and Bcl-2 during genotoxic-induced apoptosis

Ionizing radiation like UV light, gamma and X rays, can produce genotoxic damage in fibroblast cells. This injury can be reverted by activation of specific nuclear molecules. However, intense genotoxic damage induces the activation of the p53 dependent apoptotic pathway. Activated nuclear p53 has the role of a transcription factor that switches on the transcription of the Puma protein, which once released into the cytoplasm leads to the activation of a network of chemical processes that produces cell death. This network is built up with the chemical interaction between pro-apoptotic p53, Puma and Bax proteins and the anti-apoptotic Bcl2 and Bcl-x L proteins. In this work we present a mathematical model of this modular network under different regimes of Puma release into the cytoplasm. In this model we use a set of coupled nonlinear differential equations, which is solved by numerical methods, to determine the nature of the equilibrium points of the dynamical system. With this information we construct the phase portrait associated with Puma induced apoptosis and we found that once the genotoxic injury is produced, Bax levels continuously increase. In this work we propose that a possible mechanism for the control of apoptosis is the Bax level in the cytoplasm, i.e., Bax is continuously released into the cytoplasm, even in absence of Puma, according to the kinetic properties of the set of chemical interactions in which the Bcl2/Bax complex takes part. If the damage is reverted before the Bax level reaches a threshold value the apoptotic process is stopped, otherwise the process goes on until cell death.

Newcastle disease virus-induced cytopathic effect in infected cells is caused by apoptosis

The velogenic Newcastle disease virus (NDV) causes highly infectious and economically significant Newcastle disease (ND) in birds of various species. In cell culture NDV induces cytopathic effect (CPE) characterized by rounding, vacuolation, syncytia formation and cell death. Aside from cell to cell fusion caused by the F and HN glycoprotein of the virus molecular events leading to cell death are not known. In the current study, NDV-infected Vero cells, at 48 h p.i., showed nuclear condensation, cytoplasm blebbing, DNA fragmentation, and phosphatidylserine translocation to the cell surface. In addition, virus-infected cells demonstrated decreased DNA content and an increased Bax to Bcl-2 ratio, p53 level and caspase 3, 8, 9 expression compared to mock-infected cells. Based on these results, it was concluded that CPE in NDV-infected cells was caused by to the induction of apoptosis with the involvement of p53 and the Bax, dependent apoptotic pathways.

Mitochondrial Extrusion through the Cytoplasmic Vacuoles during Cell Death

Under various conditions, noxious stimuli damage mitochondria, resulting in mitochondrial fragmentation; however, the mechanisms by which fragmented mitochondria are eliminated from the cells remain largely unknown. Here we show that cytoplasmic vacuoles originating from the plasma membrane engulfed fragmented mitochondria and subsequently extruded them into the extracellular spaces in undergoing acute tumor necrosis factor alpha-induced cell death in a caspase-dependent fashion. Notably, upon fusion of the membrane encapsulating mitochondria to the plasma membrane, naked mitochondria were released into the extracellular spaces in an exocytotic manner. Mitochondrial extrusion was specific to tumor necrosis factor alpha-induced cell death, because a genotoxic stress-inducing agent such as cisplatin did not elicit mitochondrial extrusion. Moreover, intact actin and tubulin cytoskeletons were required for mitochondrial extrusion as well as membrane blebbing. Furthermore, fragmented mitochondria were engulfed by cytoplasmic vacuoles and extruded from hepatocytes of mice injected with anti-Fas antibody, suggesting that mitochondrial extrusion can be observed in vivo under pathological conditions. Mitochondria are eliminated during erythrocyte maturation under physiological conditions, and anti-mitochondrial antibody is detected in some autoimmune diseases. Thus, elucidating the mechanism underlying mitochondrial extrusion will open a novel avenue leading to better understanding of various diseases caused by mitochondrial malfunction as well as mitochondrial biology.

Genistein induces apoptosis in ovarian cancer cells via different molecular pathways depending on Breast Cancer Susceptibility gene-1 (BRCA1) status

It has been reported that Breast Cancer Susceptibility gene-1 & 2 (BRCA1 & 2 are potential molecular targets for chemoprevention by isoflavone genistein (4′ 5, 7-trihydroxy isoflavone), in breast and prostate cancer cells. It is also known that BRCA1 has inhibitory activity on estrogen receptor-α and genistein's action on cells is mainly through modulation of estrogen receptor activity. The action of genistein with respect to BRCA1 status in ovarian cancer cells has not been reported so far. Therefore in this study, we analyzed the action of genistein on BRCA1 antisense blocked (AS4) and unblocked (NEO) BG-1 ovarian cancer cells. We found that genistein induced comparable cytotoxic effect in both AS4 and NEO cells, but through different pathways. We found that genistein induces caspase 8 dependent apoptotic pathway in NEO cells. Genistein inhibits estrogen receptor-α and activates BARD1 in BRCA1 blocked cells and induces estrogen receptor-β and FAS in presence of BRCA1. It can be concluded that even though there is no difference in the extent of cell death or apoptosis, the molecular mechanism of action of genistein in inducing apoptosis is different in BRCA1 blocked and unblocked cells. This could partially explain the beneficial effects of genistein in both wild type and mutated BRCA1 estrogen receptor positive tumors.

Tumor-associated protein SPIK/TATI suppresses serine protease dependent cell apoptosis

Serine protease dependent cell apoptosis (SPDCA) is a recently described caspase independent innate apoptotic pathway. It differs from the traditional caspase dependent apoptotic pathway in that serine proteases, not caspases, are critical to the apoptotic process. The mechanism of SPDCA is still unclear and further investigation is needed to determine any role it may play in maintaining cellular homeostasis and development of disease. The current knowledge about this pathway is limited only to the inhibitory effects of some serine protease inhibitors. Synthetic agents such as pefabloc, AEBSF and TPCK can inhibit this apoptotic process in cultured cells. There is little known, however, about biologically active agents available in the cell which can inhibit SPDCA. Here, we show that over-expression of a cellular protein called serine protease inhibitor Kazal (SPIK/TATI/PSTI) results in a significant decrease in cell susceptibility to SPDCA, suggesting that SPIK is an apoptosis inhibitor suppressing this pathway of apoptosis. Previous work has associated SPIK and cancer development, indicating that this finding will help to open the doorway for further study on the mechanism of SPDCA and the role it may play in cancer development.

Fortilin binds Ca2+ and blocks Ca2+-dependent apoptosisin vivo

Fortilin, a 172-amino-acid polypeptide present both in the cytosol and nucleus, possesses potent anti-apoptotic activity. Although fortilin is known to bind Ca2+, the biochemistry and biological significance of such an interaction remains unknown. In the present study we report that fortilin must bind Ca2+ in order to protect cells against Ca2+-dependent apoptosis. Using a standard Ca2+-overlay assay, we first validated that full-length fortilin binds Ca2+ and showed that the N-terminus (amino acids 1-72) is required for its Ca2+-binding. We then used flow dialysis and CD spectropolarimetry assays to demonstrate that fortilin binds Ca2+ with a dissociation constant (Kd) of approx. 10 mM and that the binding of fortilin to Ca2+ induces a significant change in the secondary structure of fortilin. In order to evaluate the impact of the binding of fortilin to Ca2+ in vivo, we measured intracellular Ca2+ levels upon thapsigargin challenge and found that the lack of fortilin in the cell results in the exaggerated elevation of intracellular Ca2+ in the cell. We then tested various point mutants of fortilin for their Ca2+ binding and identified fortilin(E58A/E60A) to be a double-point mutant of fortilin lacking the ability of Ca2+-binding. We then found that wild-type fortilin, but not fortilin(E58A/E60A), protected cells against thapsigargin-induced apoptosis, suggesting that the binding of fortilin to Ca2+ is required for fortilin to protect cells against Ca2+-dependent apoptosis. Together, these results suggest that fortilin is an intracellular Ca2+ scavenger, protecting cells against Ca2+-dependent apoptosis by binding and sequestering Ca2+ from the downstream Ca2+-dependent apoptotic pathways.

Anti-proliferative and pro-apoptotic effect of Smilax glabra Roxb. extract on hepatoma cell lines

Smilax glabra Roxb. (SGR) is the root of a traditional Chinese herb, referred to as tu fu ling in Chinese medicine. It is an inexpensive traditional Chinese medicine commonly used for the treatment of liver diseases, and a few studies have indicated that SGR has anti-hepatocarcinogenic and anti-cancer growth activities. In the current study, raw SGR plant was extracted with Accelerate Solvent Extractor, and the molecular mechanism by which S. glabra Roxb. extract (SGRE) has an anti-proliferative effect on the human hepatoma cell lines, HepG2 and Hep3B, was determined. We showed that SGRE inhibited HepG2 and Hep3B cell growth by causing cell-cycle arrest at either S phase or S/G2 transition and induced apoptosis, as evidenced by a DNA fragmentation assay. SGRE-induced apoptosis by alternation of mitochondrial transmembrane depolarization, release of mitochondrial cytochrome c, activation of caspase-3, and cleavage of poly(ADP-ribose) polymerase. The SGRE-mediated mitochondria-caspase dependent apoptotic pathway also involved activation of p38, JNK, and ERK mitogen-activated protein kinase signaling. Isometric compounds of astilbin (flavonoids) and smilagenin (saponin) have been identified as the main chemical constituents in SGRE by HPLC-MS/MS. These results have identified, for the first time, the biological activity of SGRE in HepG2 and Hep3B cells and should lead to further development of SGR for liver disease therapy.

Natural triterpenoids from Cecropia lyratiloba are cytotoxic to both sensitive and multidrug resistant leukemia cell lines

The cytotoxicity of four triterpenoids, euscaphic acid ( 1), tormentic acid ( 2), 2α-acetyl tormentic acid ( 3), and 3β-acetyl tormentic acid ( 4), isolated from the roots of Cecropia lyratiloba (Moraceae) by countercurrent chromatography, was evaluated in vitro in sensitive and multidrug resistant leukemia cell lines. A structure/activity relationship analysis of the compounds was performed. Acetylation of compound 2 at C2 increased its activity by a factor of 2 while acetylation at C3 had a smaller effect. Compound 1 induces death by activation of caspase-3, dependent apoptotic pathway. Furthermore, the four triterpenoids were also active toward a multidrug resistant (MDR) leukemia cell line, overexpressing glycoprotein-P (P-gp). These results reveal the potential of the terpenoids as source for the development of new anti-neoplastic and anti-MDR drugs.

Cloning and characterization of a novel caspase-10 isoform that activates NF-κB activity

Caspase-10 (also known as Mch4 and FLICE2) is an initiator caspase in the death receptor (DR)-dependent apoptotic pathway. So far six splice variants (caspase-10a–f) have been identified. Here we describe a novel isoform of the caspase-10 family named caspase-10g that is widely expressed in normal human tissues and various cell lines. Caspase-10g consists of 247 amino acids and does not contain the large or small subunit. A caspase-10g-specific exon is present between exon 5 and exon 6, which results in a protein product truncated shortly after the death-effector domain (DED)-containing prodomain. We further show that overexpression of caspase-10g dramatically enhances NF-κB activity in a dose- and time-dependent manner. Moreover, caspase-10g, unlike the protease-active caspase-10a, only promotes slight apoptosis when overexpressed in mammalian cells and it has no effect on caspase-10a-mediated apoptosis. Taken together, these results suggest that caspase-10g, as a novel prodomain-only isoform of caspase-10, may play a regulatory role preferentially in the NF-κB pathways.

Synergistic Tumor Suppression by Coexpression of FUS1 and p53 Is Associated with Down-regulation of Murine Double Minute-2 and Activation of the Apoptotic Protease-Activating Factor 1–Dependent Apoptotic Pathway in Human Non–Small Cell Lung Cancer Cells

FUS1 is a novel tumor suppressor gene identified in human chromosome 3p21.3 region. Loss of expression and deficiency of posttranslational modification of FUS1 protein have been found in a majority of human lung cancers. Restoration of wild-type FUS1 in 3p21.3-deficient human lung cancer cells exhibited a potent tumor suppression function in vitro and in vivo. In this study, we evaluated the combined effects of FUS1 and tumor suppressor p53 on antitumor activity and explored the molecular mechanisms of their mutual actions in human non-small cell lung cancer (NSCLC) cells. We found that coexpression of FUS1 and p53 by N-[1-(2,3-dioleoyloxyl)propyl]-NNN-trimethylammoniummethyl sulfate:cholesterol nanoparticle-mediated gene transfer significantly and synergistically inhibited NSCLC cell growth and induced apoptosis in vitro. We also found that a systemic treatment with a combination of FUS1 and p53 nanoparticles synergistically suppressed the development and growth of tumors in a human H322 lung cancer orthotopic mouse model. Furthermore, we showed that the observed synergistic tumor suppression by FUS1 and p53 concurred with the FUS1-mediated down-regulation of murine double minute-2 (MDM2) expression, the accumulation and stabilization of p53 protein, as well as the activation of the apoptotic protease-activating factor 1 (Apaf-1)-dependent apoptotic pathway in human NSCLC cells. Our results therefore provide new insights into the molecular mechanism of FUS1-mediated tumor suppression activity and imply that a molecular therapy combining two or more functionally synergistic tumor suppressors may constitute a novel and effective strategy for cancer treatment.

Role of oxidative stress and apoptosis in cadmium induced thymic atrophy and splenomegaly in mice

Cadmium immunotoxicity in rodents is primarily characterized by marked thymic damage and splenomegaly. To understand the toxicity of Cd on lymphoid cells in vivo, a single dose of Cd as CdCl 2 (1.8 mg/kg, i.p.) was administered to male BALB/c mice and cytotoxicity (MTT assay), oxidative stress indicators (glutathione, reactive oxygen species) and apoptotic markers (mitochondrial membrane potential, caspase-3 activity, phosphatidylserine externalization, apoptotic DNA, intranucleosomal DNA fragmentation) were assessed in thymic and splenic single cell suspensions, at various time intervals. Lowering of body weight gain and cellularity and a loss in cell viability was seen in the Cd treated mice. The earliest significant increase in ROS at 18 h, followed by mitochondrial membrane depolarization, caspase-3 activation and GSH depletion at 24 h in spleen and later at 48 h in thymus, strongly implicate the possible involvement of ROS. A pronounced inhibition of cell proliferative response at 48 h and 72 h may also be linked to Cd induced apoptosis. The morphological alterations including thymic cortical cell depletion and an increase in red pulp with diminished white pulp in spleen were observed at 48 h and beyond. The splenic cells appeared more susceptible than thymus cells to the adverse effects of Cd. The present study, therefore, demonstrates potentiation of oxidative stress followed by mitochondrial-caspase dependent apoptotic pathway. This may, in part, be responsible for causing suppression of cell proliferative response, thymic atrophy and splenomegaly.

Dual Role of Pim-2 Depends on the Associated Proteome.

Pim-2, a Ser/Thr kinase, is a proto-oncogene originally identified as common proviral insertion sites in T and B cell lymphomas in mice. Deregulation of Pim-2 expression has been documented in several human malignancies, including leukemia, lymphoma, multiple myeloma and prostate cancer. In human non-Hodgkin's lymphomas and in chronic lymphocytic leukemia Pim-2 is up-regulated and its expression correlates with disease activity. Pim-2 promotes cell survival in response to a wide variety of proliferative signals. Pim-2 promotes cell survival by phosphorylation of Bad and Cot. The goal of this study was to clarify the significance of our new unanticipated aspect of Pim-2′s function, namely, that over-expression of Pim-2 in HeLa cells led to cell cycle arrest at G1 and to increased apoptosis. We found that the G1 arrest was associated with increased (T14/Y15) phosphorylation of CDK2, increased proteosomic degradation of CDC25A, and increased levels of the CDK inhibitor p57. In addition, we found increased E2F-1 levels, which suggested the usage of the E2F-1 dependent apoptotic pathway. Using dominant negative forms of either E2F-1 or p73, which were co-expressed with Pim-2 in HeLa cells, revealed significant rescue of the G1 arrest and apoptotic phenomena. Silencing of Pim-2 in these cells, via siRNA, reversed the G1 arrest and pro-apoptotic effects, and verified the Pim-2 dependent specificity. We conclude that Pim-2 might play a dual role. Our data suggest that under certain environmental circumstances and in various cell types, Pim-2 appears to increase cell survival by abrogating some pro-apoptotic substrates, but under different proteomic associations Pim-2 might favor G1 arrest and apoptosis.