Fumonisin B(1) alters sphingolipid metabolism and tumor necrosis factor alpha expression in heart and lung of mice

Fumonisin B(1) (FB(1)), produced by Fusarium verticillioides, is a common contaminant in foods and feeds. Increase in tissue free sphingoid bases resulting from the inhibition of ceramide synthase is a biomarker of fumonisin exposure. Tumor necrosis factor alpha (TNFalpha) is induced in liver in response to FB(1) treatment. This study determined whether fumonisin B(1) caused increases in free sphingoid bases and altered the expression of TNFalpha in heart and lung, organs that are not targets of FB(1) toxicity, of male and female mice treated with 5-daily subcutaneous injection of 2.25 mg/kg FB(1). A significant increase in free sphingoid bases was observed in both heart and lung of FB(1)-exposed mice. The magnitude of increases in free sphingoid bases in both organs of female mice was much higher than that in males. The expression of TNFalpha was increased by FB(1) treatment in the lung of male mice and in the heart of female mice, whereas the expression of interferon gamma was unaltered. Results suggest that both sphingolipid accumulation and TNFalpha induction are observed in the tissues of mice that are not associated with FB(1) toxicity.     

The mycotoxin fumonisin B1 transiently activates nuclear factor-κB, tumor necrosis factor α and caspase 3 via protein kinase Cα-dependent pathway in porcine renal epithelial cells

Fumonisin B1 (FB1) is a toxic mycotoxin produced by Fusarium verticillioides, predominantly present in corn. The principal biochemical responses of FB1 involve disruption of sphingolipid metabolism from the inhibition of ceramide synthesis leading to accumulation of free sphingoid bases, particularly sphinganine. The ability of FB1 to modulate signal transduction pathways plays a role in its toxicity. We recently reported that FB1 selectively and transiently activates protein kinase Calpha (PKCalpha) in porcine renal epithelial cells (LLC-PK1). The aim of current study was to investigate the effect of PKCalpha activation by FB1 on NF-kappaB activation and subsequently on TNFalpha gene expression and caspase 3 induction in LLC-PK1 cells. FB1 (1 micromol/L for 5 min) transiently activated PKCalpha and increased nuclear translocation of NF-kappaB, followed by their down-regulation at later time points. Preincubating the cells with the PKC inhibitor, calphostin C, prevented the activation of NF-kappaB by FB1. TNFalpha mRNA expression was increased after 15 min exposure to FB1 or the PKC activator, phorbol 12-myristate 13-acetate. In addition, an increase in caspase 3 activity was observed after addition of FB1 for 1 h. Calphostin C prevented both the FB1-induced increase in TNFalpha expression and caspase 3 activation. In summary, we hereby demonstrate that the FB1 activation of NF-kappaB and sequential induction of TNFalpha expression resulting in the subsequent increase in caspase 3 activity are all dependent on PKCalpha stimulation in LLC-PK1 cells.     

Tumor necrosis factor alpha-mediated activation of c-Jun NH(2)-terminal kinase as a mechanism for fumonisin B(1) induced apoptosis in murine primary hepatocytes

Fumonisin B(1) is a mycotoxin produced by Fusarium verticillioides, frequently associated with corn. It produces species-specific and organ-specific toxicity, including equine leukoencephalomalacia, porcine pulmonary edema, and hepatic or renal damage in most animal species. Fumonisin B(1) perturbs sphingolipid metabolism by inhibiting ceramide synthase. Our previous studies indicated that fumonisin B(1) caused localized activation of cytokines in liver produced by macrophages and other cell types that modulate fumonisin B(1) induced hepatic apoptosis in mice. The role of tumor necrosis factor alpha (TNFalpha) in fumonisin B(1) mediated hepatocyte apoptosis has been established; not much is known about the downstream events leading to apoptosis. In the current study, fumonisin B(1) induced apoptosis in primary culture of liver cells. In consistence with previous reports, fumonisin B(1) caused accumulation of sphingoid bases and led to increase in TNFalpha expression. Phosphorylated and total c-Jun NH(2)-terminal kinase (JNK) activities were increased after 24 h fumonisin B(1) treatment. JNK inhibitor (SP600125) and anti-TNFalpha reduced the apoptosis induced by fumonisin B(1). The role of JNK signaling in fumonisin B(1) induced apoptosis is downstream of TNFalpha production, as fumonisin B(1)-mediated activation of JNK was reduced by the presence of anti-TNFalpha in the medium, whereas the presence of JNK inhibitor did not change the fumonisin B(1) induced TNFalpha expression. Results of this study imply that generation of fumonisin B(1) induced TNFalpha results in modulation of mitogen activated protein kinases, particularly of JNK, and provides a possible mechanism for apoptosis in murine hepatocytes.     

A role for protein phosphatase-2A in p38 mitogen-activated protein kinase-mediated regulation of the c-Jun NH(2)-terminal kinase pathway in human neutrophils

Human neutrophil accumulation in inflammatory foci is essential for the effective control of microbial infections. Although exposure of neutrophils to cytokines such as tumor necrosis factor-alpha (TNFalpha), generated at sites of inflammation, leads to activation of MAPK pathways, mechanisms responsible for the fine regulation of specific MAPK modules remain unknown. We have previously demonstrated activation of a TNFalpha-mediated JNK pathway module, leading to apoptosis in adherent human neutrophils (Avdi, N. J., Nick, J. A., Whitlock, B. B., Billstrom, M. A., Henson, P. M., Johnson, G. L., and Worthen, G. S. (2001) J. Biol. Chem. 276, 2189-2199). Herein, evidence is presented linking regulation of the JNK pathway to p38 MAPK and the Ser/Thr protein phosphatase-2A (PP2A). Inhibition of p38 MAPK by SB 203580 and M 39 resulted in significant augmentation of TNFalpha-induced JNK and MKK4 (but not MKK7 or MEKK1) activation, whereas prior exposure to a p38-activating agent (platelet-activating factor) diminished the TNFalpha-induced JNK response. TNFalpha-induced apoptosis was also greatly enhanced upon p38 inhibition. Studies with a reconstituted cell-free system indicated the absence of a direct inhibitory effect of p38 MAPK on the JNK module. Neutrophil exposure to the Ser/Thr phosphatase inhibitors okadaic acid and calyculin A induced JNK activation. Increased phosphatase activity following TNFalpha stimulation was shown to be PP2A-associated and p38-dependent. Furthermore, PP2A-induced dephosphorylation of MKK4 resulted in its inactivation. Thus, in neutrophils, p38 MAPK, through a PP2A-mediated mechanism, regulates the JNK pathway, thus determining the extent and nature of subsequent responses such as apoptosis.     

BCL-2 is phosphorylated and inactivated by an ASK1/Jun N-terminal protein kinase pathway normally activated at G(2)/M

Multiple signal transduction pathways are capable of modifying BCL-2 family members to reset susceptibility to apoptosis. We used two-dimensional peptide mapping and sequencing to identify three residues (Ser70, Ser87, and Thr69) within the unstructured loop of BCL-2 that were phosphorylated in response to microtubule-damaging agents, which also arrest cells at G(2)/M. Changing these sites to alanine conferred more antiapoptotic activity on BCL-2 following physiologic death signals as well as paclitaxel, indicating that phosphorylation is inactivating. An examination of cycling cells enriched by elutriation for distinct phases of the cell cycle revealed that BCL-2 was phosphorylated at the G(2)/M phase of the cell cycle. G(2)/M-phase cells proved more susceptible to death signals, and phosphorylation of BCL-2 appeared to be responsible, as a Ser70Ala substitution restored resistance to apoptosis. We noted that ASK1 and JNK1 were normally activated at G(2)/M phase, and JNK was capable of phosphorylating BCL-2. Expression of a series of wild-type and dominant-negative kinases indicated an ASK1/Jun N-terminal protein kinase 1 (JNK1) pathway phosphorylated BCL-2 in vivo. Moreover, the combination of dominant negative ASK1, (dnASK1), dnMKK7, and dnJNK1 inhibited paclitaxel-induced BCL-2 phosphorylation. Thus, stress response kinases phosphorylate BCL-2 during cell cycle progression as a normal physiologic process to inactivate BCL-2 at G(2)/M.     

Novel synergistic mechanism for sst2 somatostatin and TNFalpha receptors to induce apoptosis: crosstalk between NF-kappaB and JNK pathways

Somatostatin is a multifunctional hormone that modulates cell proliferation, differentiation and apoptosis. Mechanisms for somatostatin-induced apoptosis are at present mostly unsolved. Therefore, we investigated whether somatostatin receptor subtype 2 (sst2) induces apoptosis in the nontransformed murine fibroblastic NIH3T3 cells. Somatostatin receptor subtype 2 expression induced an executioner caspase-mediated apoptosis through a tyrosine phosphatase SHP-1 (Src homology domain phosphatase-1)-dependent stimulation of nuclear factor kappa B (NF-kappaB) activity and subsequent inhibition of the mitogen-activated protein kinase JNK. Tumor necrosis factor alpha (TNFalpha) stimulated both NF-kappaB and c-Jun NH2-terminal kinase (JNK) activities, which had opposite action on cell survival. Importantly, sst2 sensitized NIH3T3 cells to TNFalpha-induced apoptosis by (1) upregulating TNFalpha receptor protein expression, and sensitizing to TNFalpha-induced caspase-8 activation; (2) enhancing TNFalpha-mediated activation of NF-kappaB, resulting in JNK inhibition and subsequent executioner caspase activation and cell death. We have here unraveled a novel signaling mechanism for a G protein-coupled receptor, which directly triggers apoptosis and crosstalks with a death receptor to enhance death ligand-induced apoptosis.     

NF-kappaB activation prevents apoptotic oxidative stress via an increase of both thioredoxin and MnSOD levels in TNFalpha-treated Ewing sarcoma cells

Repression of activation of c-Jun N-terminal kinase (JNK) participates in the anti-apoptotic effect of nuclear factor-kappaB (NF-kappaB) in TNFalpha-treated Ewing sarcoma cells. As oxidative stress is one of the most prominent activators of JNK, we investigated the relationship between TNFalpha-induced NF-kappaB activation and the control of oxidative stress. Inhibition of NF-kappaB activation resulted in an increase in TNFalpha-induced ROS production, lipid peroxidation and protein oxidation. Those ROS and lipid peroxides were both involved in TNFalpha-induced apoptosis, whereas only ROS elevation triggered sustained JNK activation. TNFalpha increased the level of two antioxidant enzymes, thioredoxin and manganese superoxide dismutase by an NF-kappaB-dependent mechanism. Inhibition of expression or activity of these enzymes sensitized cells to TNFalpha-induced apoptosis, indicating their functional role in protection from cell death. Thus, agents that inhibit activities of these enzymes may prove helpful in the treatment of Ewing tumors.     

TNFalpha-mediated cell death is independent of cdc25A

Tumor necrosis factor (TNFs) have been shown to be synthesized by ovarian carcinomas, and may therefore affect tumor cells in an autocrine manner. Therefore, we investigated the effects of recombinant TNFs on ovarian carcinoma cells N.1 and examined expression of the proto-oncogenes c-myc and cdc25A which are known to play a prominent role in apoptosis. TNFalpha elicited apoptosis in N.1 cells within 72 h which was shown by typical morphological changes, DNA fragmentation and signature type cleavage of poly(ADP-ribose) polymerase into a 89 kDa proteolytic peptide. TNFalpha-induced apoptosis was accompanied by constitutive c-Myc expression, although the mRNA level of phosphatase cdc25A was suppressed within 24 h of TNFalpha treatment and the protein level decreased after 48 h. Cdc25A tyrosine phosphatase is an activator of the cdk2-cyclin E complex which allows for cell cycle progression. As expected, we found TNFalpha-mediated Cdc25A down-regulation to inhibit Cdk2 activity. Cdc25A suppression was related to TNFalpha-induced apoptosis but not to a TNFalpha-induced G0 arrest because cyclin D1 expression was unaffected and the gene gas6 (growth arrest specific 6) was not induced. Arresting cells by treatment with genistein prevented TNFalpha-triggered apoptosis and inhibited c-myc expression. TNFalpha-induced apoptosis is not accompanied by cell cycle arrest which may be due to constitutive c-Myc expression, although Cdc25A and Cdk2 activity is also down-regulated. High c-Myc and low Cdc25A activity might present conflicting signals to the cell cycle machinery which are incompatible with cell survival.     

The E3 ubiquitin ligase itch couples JNK activation to TNFalpha-induced cell death by inducing c-FLIP(L) turnover

The proinflammatory cytokine tumor necrosis factor (TNF) alpha signals both cell survival and death. The biological outcome of TNFalpha treatment is determined by the balance between NF-kappaB and Jun kinase (JNK) signaling; NF-kappaB promotes survival, whereas JNK enhances cell death. Critically, identity of a JNK substrate that promotes TNFalpha-induced apoptosis has been outstanding. Here we show that TNFalpha-mediated JNK activation accelerates turnover of the NF-kappaB-induced antiapoptotic protein c-FLIP, an inhibitor of caspase-8. This is not due to direct c-FLIP phosphorylation but depends on JNK-mediated phosphorylation and activation of the E3 ubiquitin ligase Itch, which specifically ubiquitinates c-FLIP and induces its proteasomal degradation. JNK1 or Itch deficiency or treatment with a JNK inhibitor renders mice resistant in three distinct models of TNFalpha-induced acute liver failure, and cells from these mice do not display inducible c-FLIP(L) ubiquitination and degradation. Thus, JNK antagonizes NF-kappaB during TNFalpha signaling by promoting the proteasomal elimination of c-FLIP(L).     

Human immunodeficiency virus type 1 Vpr induces cell cycle arrest at the G(1) phase and apoptosis via disruption of mitochondrial function in rodent cells

Vpr of human immunodeficiency virus type 1 causes cell cycle arrest at the G(2)/M phase and induces apoptosis after G(2)/M arrest in primate cells. We have reported previously that Vpr also induces apoptosis independently of G(2)/M arrest in human HeLa cells. By contrast, Vpr does not induce G(2)/M arrest in rodent cells, but it retards cell growth. To clarify the relationship between cell cycle arrest and apoptosis, we expressed Vpr endogenously in rodent cells and investigated cell cycle profiles and apoptosis. We show here that Vpr induces cell cycle arrest at the G(1) phase and apoptosis in rodent cells. Vpr increased the activity of caspase-3 and caspase-9, but not of caspase-8. Moreover, Vpr-induced apoptosis could be inhibited by inhibitors of caspase-3 and caspase-9, but not by inhibitor of caspase-8. We also showed that Vpr induces the release of cytochrome c from mitochondria into the cytosol and disrupts the mitochondrial transmembrane potential. Finally, we showed that apoptosis occurred in HeLa cells through an identical pathway. These results suggest that disruption of mitochondrial functions by Vpr induces apoptosis via cell cycle arrest at G(1), but that apoptosis is independent of G(2)/M arrest. Furthermore, it appears that Vpr acts species-specifically with respect to induction of cell cycle arrest but not of apoptosis.     

Elevated fibroblast growth factor-2 increases tumor necrosis factor-alpha induced endothelial cell death in high glucose

Glucose and tumor necrosis factor-alpha (TNFalpha) concentrations are elevated in diabetes. Both of these factors correlate with diabetic vasculopathy and endothelial cell apoptosis, yet their combined effects have not been measured. We have previously shown that the angiogenic growth factor fibroblast growth factor-2 (FGF-2), which is generally protective against endothelial cell death, is similarly elevated in high glucose conditions. We therefore investigated the effect of TNFalpha on endothelial cell death under normal and elevated glucose conditions, with a particular focus on FGF-2. Porcine aortic endothelial cells were cultured in 5 and 30 mM glucose and stimulated with TNFalpha, together with FGF-2 or a neutralizing FGF-2 antibody. Cell death was measured via cell counts or an annexin apoptotic assay, and cell cycle phase was determined by propidium iodide labeling. TNFalpha-induced endothelial cell death increased for cells in high glucose, and cell death was enhanced with increasing FGF-2 exposure and negated by a neutralizing FGF-2 antibody. Endothelial cells were most susceptible to TNFalpha-induced cell death when stimulated with FGF-2 18 h prior to TNFalpha, corresponding to cell entry into S phase of the proliferative cycle. The FGF-2 associated increase in TNFalpha-induced cell death was negated by blocking cell entry into S phase. Endothelial cell release of FGF-2 in high glucose leads to cell cycle progression, which makes cells more susceptible to TNFalpha-induced cell death. These data suggest that growth factor outcomes in high glucose depend on secondary mediators such as cytokines and stimulation cell cycle timing.     

Sphingoid bases and ceramide induce apoptosis in HT-29 and HCT-116 human colon cancer cells

Complex dietary sphingolipids such as sphingomyelin and glycosphingolipids have been reported to inhibit development of colon cancer. This protective role may be the result of turnover to bioactive metabolites including sphingoid bases (sphingosine and sphinganine) and ceramide, which inhibit proliferation and stimulate apoptosis. The purpose of the present study was to investigate the effects of sphingoid bases and ceramides on the growth, death, and cell cycle of HT-29 and HCT-116 human colon cancer cells. The importance of the 4,5-trans double bond present in both sphingosine and C(2)-ceramide (a short chain analog of ceramide) was evaluated by comparing the effects of these lipids with those of sphinganine and C(2)-dihydroceramide (a short chain analog of dihydroceramide), which lack this structural feature. Sphingosine, sphinganine, and C(2)-ceramide inhibited growth and caused death of colon cancer cells in time- and concentration-dependent manners, whereas C(2)-dihydroceramide had no effect. These findings suggest that the 4,5-trans double bond is necessary for the inhibitory effects of C(2)-ceramide, but not for sphingoid bases. Evaluation of cellular morphology via fluorescence microscopy and quantitation of fragmented low-molecular weight DNA using the diphenylamine assay demonstrated that sphingoid bases and C(2)-ceramide cause chromatin and nuclear condensation as well as fragmentation of DNA, suggesting these lipids kill colon cancer cells by inducing apoptosis. Flow cytometric analyses confirmed that sphingoid bases and C(2)-ceramide increased the number of cells in the A(0) peak indicative of apoptosis and demonstrated that sphingoid bases arrest the cell cycle at G(2)/M phase and cause accumulation in the S phase. These findings establish that sphingoid bases and ceramide induce apoptosis in colon cancer cells and implicate them as potential mediators of the protective role of more complex dietary sphingolipids in colon carcinogenesis.     

Differential effect of heme oxygenase-1 in endothelial and smooth muscle cell cycle progression

Arterial remodeling in response to pathological insult is a complex process that depends in part on the balance between vascular cell apoptosis and proliferation. Studies in experimental models suggest that HO-1 mediates neointimal formation while limiting lumen stenosing, indicating a differential effect on vascular endothelial (EC) and smooth muscle cells (SMC). We investigated the effect of HO-1 expression on cell cycle progression in EC and SMC. The addition of SnMP (10 microM), an inhibitor of HO activity, to EC or SMC for 24h, resulted in significant abnormalities in DNA distribution and cell cycle progression compared to cells treated with the HO-1 inducers, heme (10 microM) or SnCl(2) (10 microM). SnMP increased G(1) phase and decreased S and G(2)/M phases in EC while heme or SnCl(2) decreased G(1) phase, but increased S and G(2)/M phases (p<0.05). Opposite effects were obtained in SMC. SnMP decreased G(1) phase and increased S and G(2)/M phases while heme or SnCl(2) increased G(1) phase but decreased S and G(2)/M phases (p<0.05). Our data demonstrate that HO-1 regulates the cell cycle in a cell-specific manner; it increases EC but decreases SMC cycle progression. The mechanisms underlying the HO-1 cell-specific effect on cell cycle progression within the vascular wall are yet to be explored. Nevertheless, these findings suggest that cell-specific targeting of HO-1 expression may provide a novel therapeutic strategy for the treatment of cardiovascular diseases.     

Phospholipase D protects ECV304 cells against TNFalpha-induced apoptosis

Tumor necrosis factor alpha (TNFalpha), a pleiotropic cytokine, activates both apoptotic and pro-survival signals depending on the cell model. Using ECV304 cells, which can be made TNFalpha-sensitive by cycloheximide (CHX) co-treatment, we evaluated the potential roles of ceramide and phospholipase D (PLD) in TNFalpha-induced apoptosis. TNFalpha/CHX induced a robust increase in ceramide levels after 16 h of treatment when cell death was maximal. PLD activity was increased at early time point (1h) whereas both PLD activity and PLD1 protein were strongly decreased after 24h. TNFalpha/CHX-induced cell death was significantly lowered by exogenous bacterial PLD and phoshatidic acid, and in cells overexpressing PLD1. Conversely, cells depleted in PLD proteins by small interference RNA (siRNA) treatment exhibited higher susceptibility to apoptosis. These results show that PLD exerts a protective role against TNFalpha-induced cell death.     

Evaluation of sphinganine and sphingosine as human breast cancer chemotherapeutic and chemopreventive agents

No comparative study of the effects of sphingolipid metabolites on proliferation and differentiation in normal human breast epithelial cells versus stem cells and tumorigenic cells has been reported. The purpose of this study was to evaluate the chemotherapeutic and chemopreventive potential of sphingoid bases (sphingosine and sphinganine) using a novel cell culture system of normal human breast epithelial cells (HBEC) developed from breast tissues of healthy women obtained during reduction mammoplasty (Type I HBEC with stem cell characteristics and Type II HBEC with basal epithelial cell phenotypes) and transformed tumorigenic Type I HBEC. The results show that sphinganine inhibited the growth and induced apoptosis of transformed tumorigenic Type I HBEC more potently than sphingosine (IC(50) for sphinganine 4 microM; sphingosine 6.4 microM). Both sphinganine and sphingosine at high concentrations (8-10 lM) arrested the cell cycle at G(2)/M. Sphinganine inhibited the growth and caused death of Type I HBEC more strongly than sphingosine. In comparison, Type II HBEC (normal differentiated cells) were less sensitive to the growth-inhibitory effects of sphingoid bases than Type I HBEC (stem cells) or transformed tumorigenic Type I HBEC, suggesting that sphingoid bases may serve as chemotherapeutic agents. At concentrations (0.05, 0.1, and 0.5 microM) that are below the growth-inhibitory range, sphingoid bases induced differentiation of Type I HBEC to Type II HBEC, as detected morphologically and via expression of a tumor suppressor protein, maspin, which is a marker of Type II HBEC. Thus, sphingoid bases may function as chemotherapeutic as well as chemopreventive agents by preferentially inhibiting cancer cells and eliminating stem cells from which most breast cancer cells arise.     

A novel human phosphatidylethanolamine-binding protein resists tumor necrosis factor alpha-induced apoptosis by inhibiting mitogen-activated protein kinase pathway activation and phosphatidylethanolamine externalization

The phosphatidylethanolamine (PE)-binding proteins (PEBPs) are an evolutionarily conserved family of proteins with pivotal biological functions. Here we describe the cloning and functional characterization of a novel family member, human phosphatidylethanolamine-binding protein 4 (hPEBP4). hPEBP4 is expressed in most human tissues and highly expressed in tumor cells. Its expression in tumor cells is further enhanced upon tumor necrosis factor (TNF) alpha treatment, whereas hPEBP4 normally co-localizes with lysosomes, TNFalpha stimulation triggers its transfer to the cell membrane, where it binds to Raf-1 and MEK1. L929 cells overexpressing hPEBP4 are resistant to both TNFalpha-induced ERK1/2, MEK1, and JNK activation and TNFalpha-mediated apoptosis. Co-precipitation and in vitro protein binding assay demonstrated that hPEBP4 interacts with Raf-1 and MEK1. A truncated form of hPEBP4, lacking the PE-binding domain, maintains lysosomal co-localization but has no effect on cellular responses to TNFalpha. Given that MCF-7 breast cancer cells expressed hPEBP4 at a high level, small interfering RNA was used to silence the expression of hPEBP4. We demonstrated that down-regulation of hPEBP4 expression sensitizes MCF-7 breast cancer cells to TNFalpha-induced apoptosis. hPEBP4 appears to promote cellular resistance to TNF-induced apoptosis by inhibiting activation of the Raf-1/MEK/ERK pathway, JNK, and PE externalization, and the conserved region of PE-binding domain appears to play a vital role in this biological activity of hPEBP4.     

Roles for basal and stimulated p21(Cip-1/WAF1/MDA6) expression and mitogen-activated protein kinase signaling in radiation-induced cell cycle checkpoint control in carcinoma cells

We investigated the role of the cdk inhibitor protein p21(Cip-1/WAF1/MDA6) (p21) in the ability of MAPK pathway inhibition to enhance radiation-induced apoptosis in A431 squamous carcinoma cells. In carcinoma cells, ionizing radiation (2 Gy) caused both primary (0-10 min) and secondary (90-240 min) activations of the MAPK pathway. Radiation induced p21 protein expression in A431 cells within 6 h via secondary activation of the MAPK pathway. Within 6 h, radiation weakly enhanced the proportion of cells in G(1) that were p21 and MAPK dependent, whereas the elevation of cells present in G(2)/M at this time was independent of either p21 expression or MAPK inhibition. Inhibition of the MAPK pathway increased the proportion of irradiated cells in G(2)/M phase 24-48 h after irradiation and enhanced radiation-induced apoptosis. This correlated with elevated Cdc2 tyrosine 15 phosphorylation, decreased Cdc2 activity, and decreased Cdc25C protein levels. Caffeine treatment or removal of MEK1/2 inhibitors from cells 6 h after irradiation reduced the proportion of cells present in G(2)/M phase at 24 h and abolished the ability of MAPK inhibition to potentiate radiation-induced apoptosis. These data argue that MAPK signaling plays an important role in the progression/release of cells through G(2)/M phase after radiation exposure and that an impairment of this progression/release enhances radiation-induced apoptosis. Surprisingly, the ability of irradiation/MAPK inhibition to increase the proportion of cells in G(2)/M at 24 h was found to be dependent on basal p21 expression. Transient inhibition of basal p21 expression increased the control level of apoptosis as well as the abilities of both radiation and MEK1/2 inhibitors to cause apoptosis. In addition, loss of basal p21 expression significantly reduced the capacity of MAPK inhibition to potentiate radiation-induced apoptosis. Collectively, our data argue that MAPK signaling and p21 can regulate cell cycle checkpoint control in carcinoma cells at the G(1)/S transition shortly after exposure to radiation. In contrast, inhibition of MAPK increases the proportion of irradiated cells in G(2)/M, and basal expression of p21 is required to maintain this effect. Our data suggest that basal and radiation-stimulated p21 may play different roles in regulating cell cycle progression that affect cell survival after radiation exposure.     

Furano-1,2-naphthoquinone inhibits EGFR signaling associated with G2/M cell cycle arrest and apoptosis in A549 cells

Furano-1,2-naphthoquinone (FNQ), prepared from 2-hydroxy-1,4-naphthoquinone and chloroacetaldehyde in an efficient one-pot reaction, exhibits an anti-carcinogenic effect. FNQ exerted anti-proliferative activity with the G(2)/M cell cycle arrest and apoptosis in A549 cells. FNQ-induced G(2)/M arrest was correlated with a marked decrease in the expression levels of cyclin A and cyclin B, and their activating partner cyclin-dependent kinases (Cdk) 1 and 2 with concomitant induction of p53, p21, and p27. FNQ-induced apoptosis was accompanied with Bax up-regulation and the down-regulation of Bcl-2, X-linked inhibitor of apoptosis (XIAP), and survivin, resulting in cytochrome c release and sequential activation of caspase-9 and caspase-3. Western blot analysis revealed that FNQ suppressed EGFR phosphorylation and JAK2, STAT3, and STAT5 activation, but increased in activation of p38 MAPK and c-Jun NH2-terminal kinase (JNK) stress signal. The combined treatment of FNQ with AG1478 (a specific EGFR inhibitor) significantly enhanced the G(2)/M arrest and apoptosis, and also led to up-regulation in Bax, p53, p21, p27, release of mitochondrial cytochrome c, and down-regulation of Bcl-2, XIAP, survivin, cyclin A, cyclin B, Cdk1, and Cdk2 in A549 cells. These findings suggest that FNQ-mediated cytotoxicity of A549 cell related with the G(2)/M cell cycle arrest and apoptosis via inactivation of EGFR-mediated signaling pathway.     

Apoptosis and cell cycle progression in an acidic environment after irradiation

Apoptosis and cell cycle progression in HL60 cells irradiated in an acidic environment were investigated. Apoptosis was determined by TUNEL staining, PARP cleavage, DNA fragmentation, and flow cytometry. The majority of the apoptosis that occurred in HL60 cells after 4 Gy irradiation took place after G(2)/M-phase arrest. When irradiated with 12 Gy, a fraction of the cells underwent apoptosis in G(1) and S phases while the rest of the cells underwent apoptosis in G(2)/M phase. The apoptosis caused by 4 and 12 Gy irradiation was transiently suppressed in medium at pH 7.1 or lower. An acidic environment was found to perturb progression of irradiated cells through the cell cycle, including progression through G(2)/ M phase. Thus it was concluded that the suppression of apoptosis in the cells after 4-12 Gy irradiation in acidic medium was due at least in part to a delay in cell cycle progression, particularly the prolongation of G(2)/M-phase arrest. Irradiation with 20 Gy indiscriminately caused apoptosis in all cell cycle phases, i.e. G(1), S and G(2)/M phases, rapidly in neutral pH medium and relatively slowly in acidic pH medium. The delay in apoptosis in acidic medium after 20 Gy irradiation appeared to result from mechanisms other than prolonged G(2)/ M-phase arrest.