Extent Of Apoptosis Research Articles

Aclarubicin-induced apoptosis and necrosis in cells derived from human solid tumours

In the present study, we investigated the response of A549 (non-small cell lung-cancer), HepG2 (human hepatoma) and MCF-7 (human breast adenocarcinoma) cell lines to treatment with aclarubicin (ACL). The aim of this research was to compare the ability of ACL to induce apoptosis or necrosis in solid tumours. The mode of cell death induced by ACL was evaluated by flow-cytometry and fluorescence microscopy. We show that the drug induced both apoptosis and necrosis in the cells. Apoptotic cell death was associated with morphological changes, DNA fragmentation, changes in activity of poly(ADP-ribose)polymerase (PARP) and drug-mediated activation of caspase-3 and caspase-8. The occurrence of all these events was time-dependent. The extent of apoptosis was also dependent on the kind of cell line, the sensitivity to ACL and the intracellular drug content. This study demonstrates that the cells most sensitive to ACL, A549, accumulated a significantly higher level of the drug and were also more susceptible to apoptosis than the other cells. In contrast, the relatively less sensitive HepG2 and MCF-7 cell lines appeared more resistant to apoptosis induction. On the basis of these results, it seems that aclarubicin is able to induce apoptosis in human solid tumours.

Abstract 2550: B7-H3 induces paclitaxel resistance in human breast cancer cell lines

Abstract B7-H3 is previously known as an immunoregulatory protein with either stimulatory or inhibitory effects on the activation of T-cells. The correlation between high expression of B7-H3 and poor prognosis has been reported in many human tumor types and this has been ascribed to the putative immune-regulatory function of the protein. Previously, we reported results indicating a direct association between B7-H3 expression and metastasis, whereas in this study we examined the role of B7-H3 with respect to paclitaxel resistance in metastatic breast cancer cells as well as the underlying mechanisms. The results show that silencing B7-H3 increased the sensitivity to both MDA-MB-231 and MDA-MB-435 breast cancer cells to paclitaxel. This drug is known to exert its cell killing effect through induction of apoptosis, and our data confirmed that the silencing of B7-H3 sensitized breast cancer cells to paclitaxel-induced apoptosis. The extent of apoptosis was investigated by checking the amount of Annexin-5 stained cells, a marker for early stage apoptosis and for the amount of TUNEL positive cells, which reflects late stage apoptosis. To confirm the apoptotic response observed by the use of flow cytometry, we examined the cleavage of poly (ADP-ribose) polymerase (PARP), another marker of apoptosis, and found that MDA-MB-231 and MDA-MB-435 B7-H3 knockdown cells showed increased PARP cleavage compared to the vector control cells upon treatment with paclitaxel. We then studied whether the effects of B7-H3 on drug resistance could be related to molecules and signaling pathways known to be involved in the apoptotic response. Interestingly we discovered that the STAT3 pathway, also known to be involved in chemo-resistance, was affected by B7-H3, with markedly decreased phosphorylation of STAT3 Tyr705 in B7-H3 knockdown cells. Furthermore, two members of the IAP family of antiapoptotic proteins, survivin and Mcl-1 which are direct downstream targets of Stat3, were both repressed by the decrease in B7-H3 and Stat3 phosphorylation levels. The phosphorylation level of JAK2, which is a well-known upstream component of STAT3 signaling, was also significantly decreased in B7-H3 knockdown cells. Finally, in vivo experiments showed that in nude mice treated with paclitaxel, the growth rate of tumors in the mice injected s.c with MDA-MB-435 B7-H3 knockdown cells was significantly reduced in contrast to the growth rate in mice injected with MDA-MB-435 control cells. Taken together, our data demonstrates that in breast cancer cells B7-H3 expression is associated with antiapoptosis and paclitaxel resistance, at least partly through the STAT3 pathway The novel findings of this study have important implications for the design of new approaches targeting B7-H3 overexpressing breast cancers. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2550.

Abstract 2422: The trimeric engineered fusion protein with CD154 peptide mimetic (OmpC-CD154p), induced apoptotic signaling in B-NHL cell lines mediated by caspase 3 and caspase 8 activation

Abstract CD154 (CD40-ligand) is a critical transmembrane molecule with potent immune-stimulatory properties and is clinical applied in gene therapy for leukemia and lymphoma. The interaction between CD40, a member of the tumor necrosis factor superfamily, and its ligand CD154 is essential for the development of humoral and cellular immune responses. Selective inhibition or activation of this pathway forms the basis for the development of new therapeutics against immunologically-based diseases and malignancies. Engagement of CD40-CD154 induces activation and proliferation of B lymphocytes and triggers apoptosis of carcinoma and B lymphoma cells. Several clinical trials have been reported targeting CD40 in cancer patients using recombinant CD154, mAbs and gene therapy, which were well tolerated and resulted in objective tumor responses. In addition to these therapies, CD154 mimetics have been considered with the objective to augment and potentiate the direct cytotoxic anti-tumor activity and for better accessibility to tumor sites. This approach was developed by us and we hypothesized that the genetic engineering of a fusion protein containing a CD154 peptide mimetic additional to be trimeric may be advantageous in that it may have a better affinity to CD40 on B cell malignancies and trigger cell death. The fusion partner may be a carrier targeting other surface molecules expressed on the malignant cells. This hypothesis was tested by the development of a gene fusion of Salmonella typhi OmpC protein expressing the CD154 Trp140-Ser149 amino acid strand. This OmpC-CD154p fusion protein binds CD40 and triggers the CD40 expressing B cells. In this study, we demonstrate that OmpC-CD154p treatment inhibits cell growth and proliferation, on B-NHL cell lines Raji and Ramos. In addition, significant apoptosis was achieved and the extent of apoptosis was a function of the concentration used and time of incubation. The anti-tumor effect was specific as treatment with OmpC alone had no effect. OmpC-CD154p treatment induced activation of caspase 3 active and caspase 8. These findings establish the basis for the development of new fusion proteins specificity targeting the tumor cells directly and mechanism mediated by extrinsic pathway. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2422.

Paracrine factors secreted by endothelial progenitor cells prevent oxidative stress-induced apoptosis of mature endothelial cells

Endothelial progenitor cells (EPC) play a fundamental role in tissue regeneration and vascular repair. Current research suggests that EPC are more resistant to oxidative stress as compared to differentiated endothelial cells. Here we hypothesized that EPC not only possess the ability to protect themselves against oxidative stress but also confer this protection upon differentiated endothelial cells by release of paracrine factors. To test this hypothesis, HUVEC incubated with conditioned medium obtained from early EPC cultures (EPC-CM) were exposed to H2O2 to assess the accumulation of intracellular ROS, extent of apoptosis and endothelial cell functionality. Under oxidative stress conditions HUVEC treated with EPC-CM exhibited substantially lower levels of intracellular oxidative stress (0.2±0.02 vs. 0.4±0.03 relative fluorescence units, p<0.05) compared to control medium. Moreover, the incubation with EPC-CM elevated the expression level of antioxidant enzymes in HUVEC (catalase: 2.6±0.4; copper/zinc superoxide dismutase (Cu/ZnSOD): 1.6±0.1; manganese superoxide dismutase (MnSOD): 1.4±0.1-fold increase compared to control, all p<0.05). Furthermore, EPC-CM had the distinct potential to reverse the functional impairment of HUVEC as measured by their capability to form tubular structures in vitro. Finally, incubation of HUVEC with EPC-CM resulted in a significant reduction of apoptosis (0.34±0.01 vs. 1.52±0.12 relative fluorescence units, p<0.01) accompanied by an increased expression ratio of the anti/pro-apoptotic factors Bcl-2/Bax to 2.9±0.7-fold (compared to control, p<0.05). Most importantly, neutralization of selected cytokines such as VEGF, HGF, IL-8 and MMP-9 did not significantly reverse the cyto-protective effect of EPC-CM (p>0.05), suggesting that soluble factors secreted by EPC, possibly via broad synergistic actions, exert strong cyto-protective properties on differentiated endothelium through modulation of intracellular antioxidant defensive mechanisms and pro-survival signals.

Anti-Apoptotic Effect of Hyperbaric Oxygen Preconditioning on a Rat Model of Myocardial Infarction

In our previous study, it was indicated that hyperbaric oxygen preconditioning (HOP) could induce a preconditioning against myocardial infarction and promote neovascularization. In this study, attempts were made to investigate whether a modified short-term pre-exposure protocol could also induce cardioprotection, and its potential mechanisms. Adult male Sprague Dawley rats were divided into seven groups; group 1 was sham surgery (SHAM) and group 2 was pre-exposed to normal air (CTL), and the other groups to HOP 1, 6, 24, 48, and 72 h (H1, H6, H24, H48, H72 groups) before permanent ischemia. The infarct size was measured by triphenyltetrazolium chloride staining, and left ventricular function parameters were recorded. The extent of apoptosis was determined by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)-positive staining. Caspase-3 activity, Bcl-2, and Bax expression were also measured. Compared with CTL group, myocardial infarct size was significantly decreased as well as cardiac cell apoptosis in area at risk zones (AAR) in H48 group. Meanwhile, the activity of caspase 3 was reduced, the ratio of Bcl-2/Bax expression was up-regulated, and the heart function parameters, including left ventricular systolic pressure (LVSP), left ventricular diastolic pressure (LVEDP), +(dP/dt)(max), and -(dP/dt)(max) were also significantly improved after preconditioning in H48 group. The results indicate that short-term HOP could induce cardioprotection and may not last for more than 24 h. HOP prevents myocardium from permanent ischemia injury by suppression of apoptotic pathways.

Regulation of p53 isoform expression in renal cell carcinoma

Differential expression of p53 isoforms might participate in the marked resistance towards conventional chemotherapy of renal cell carcinomas (RCCs). Therefore, we analysed their differential expression and regulation in RCCs. RCCs expressed a more p53 activating isoform pattern during tumor initiation and progression, in vivo. In vitro, two cell lines exhibiting a similar sensitivity towards Topotecan-induced cell death revealed a similar induction of p53 target genes but strongly differed in their extent of apoptosis. Furthermore, they strongly differed in their basal expression patterns and differential regulation of the isoforms. In conclusion, our study examined for the first time the differential expression and regulation of all p53 isoforms in a tumor in vivo. Furthermore, novel results in our in vitro studies show that p53 isoforms are strongly differentially regulated by chemotherapy in RCCs and that expression and regulation of so-called "p53-target genes" are obviously at least in part regulated by other transcription factors. In addition, our original findings show that p53 isoform expression in RCC cell lines is of minor importance for sensitivity towards chemotherapy.

Depletion of BIRC6 leads to retarded bovine early embryonic development and blastocyst formation in vitro

Baculoviral inhibitors of apoptosis repeat-containing 6 (BIRC6) is believed to inhibit apoptosis by targeting key cell-death proteins. To understand its involvement during bovine preimplantation embryo development, two consecutive experiments were conducted by targeted knockdown of its mRNA and protein using RNA interference. In Experiment 1, the effect of BIRC6 knockdown during the early stages of preimplantation embryo development was assessed by injecting zygotes with long double-stranded RNA (ldsRNA) and short hairpin RNA (shRNA) against BIRC6 mRNA followed by in vitro culturing until 96 h post insemination (hpi). The results showed that in RNA-injected zygote groups, reduced levels of BIRC6 mRNA and protein were accompanied by an increase (P < 0.05) in the proportion of 2- and 4-cell and uncleaved embryos and a corresponding decrease (P < 0.05) in the number of 8-cell embryos. In Experiment 2, the effect of BIRC6 knockdown on blastocyst formation, blastocyst total cell number and the extent of apoptosis was investigated. Consequently, zygotes injected with ldsRNA and shRNA resulted in lower (P < 0.05) blastocyst formation and total blastocyst cell number. Moreover, the apoptotic cell ratio, CASPASE 3 and 7 activity, BAX to BCL-2 ratio and levels of SMAC and CASPASE 9 were higher in blastocysts derived from the ldsRNA and shRNA groups, suggesting increased apoptosis in those blastocysts. The results of this study reveal the importance of BIRC6 expression for embryo survival during bovine preimplantation embryo development. However, whether BIRC6 is essential for implantation and fetal development during bovine pregnancy needs further research.

Abstract A137: Chemotherapy molecule delivered by tumor-targeted salmonella enterica serovar typhimurium under the control of integrated modular gate

Abstract Several strictly or facultative anaerobic bacteria have attracted more and more attention in cancer therapeutics because of their targeting and accumulating abilities in solid tumors, and Salmonella enterica serovar Typhimurium is one of them. These bacteria were also adapted to deliver therapeutic factors and were applied alone or in combination with conventional therapeutics. The aim of this study is to test a new integrated gene expression system in S. Typhimurim SL1344, and the benefit of the new system in amplifying therapeutic molecule production during bacterium targeting and accumulating in solid tumor. We have constructed a synthetic genetic gate in S. Typhimurim SL1344 that integrates information from two promoters as inputs and actives a promoter output only when both input promoters are induced. The integration occurs via an interaction between L-arabinose concentration and bacterium cell density. When certain amount of L-arabinose were added in the system and bacterium population were higher enough, the tumor necrosis factor apoptosis inducing ligand(TRAIL) and fluorescent protein ZsGreen as the control were produced by bacteria. LS174T colon carcinoma cells were processed to form cylindroids between the bottom surface of a 96-well plate and the top surface of a set of polycarbonate cylindrical plugs attached to a polycarbonate lid to mimic solid tumor, and then cylindroid cultures were inoculated with S. typhimurium SL1344 carrying TRAIL or ZsGreen expression system. 0.2% (w/v) L-arabinose was added into the well after 12hr inoculation, and then washed out after 2hr induced. Extents of apoptosis in cylindroids were tested by caspase-3 activity assay. Results showed that the bacteria accumulated inside cylindroids indicated by ZsGreen expression, and the average normalized intensity of apoptotic cell was higher in the cylindroids inoculated by the bacteria carrying the TRAIL than those inoculated by the bacteria carrying the ZsGreen. It can be concluded that the constructed integrated modular gate could effectively control bacteria to produce functional chemotherapy molecule in solid tumor at desirable time and places. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A137.

Postconditioning and Controlled Reperfusion

Postconditioning and Controlled Reperfusion

Recipient NOD2/CARD15 status affects cellular infiltrates in human intestinal graft-versus-host disease

Nucleotide-binding oligomerization domain 2/caspase recruitment domain 15 (NOD2/CARD15) polymorphisms have been identified as risk factors of both Crohn's disease and graft-versus-host disease (GVHD) following allogeneic stem cell transplantation. However, the role of these receptors of innate immunity in the pathophysiology of gastrointestinal GVHD is still poorly defined. Immunohistological features of intestinal GVHD were analysed in gastrointestinal biopsies from 58 patients obtained at the time of first onset of intestinal symptoms. The observed changes were correlated with concomitant risk factors and the presence of polymorphisms within the pathogen recognition receptor gene NOD2/CARD15. Intestinal GVHD was associated with a stage-dependent decrease in CD4 T cell infiltrates and an increase in CD8 T cells in the lamina propria; CD8 infiltrates correlated with extent of apoptosis and consecutive epithelial proliferation. The presence of NOD2/CARD15 variants in the recipient was associated with a significant loss of CD4 T cells: in a semiquantitative analysis, the median CD4 score for patients with wild-type NOD2/CARD15 was 1.1 (range 3), but only 0.4 (range 2) for patients with variants (P = 0.002). This observation was independent from severity of GVHD in multivariate analyses and could not be explained by the loss of forkhead box P3(+) T cells. Our results suggest a loss of protective CD4 T cells in intestinal GVHD which is enhanced further by the presence of NOD2/CARD15 variants. Our study might help to identify more selective therapeutic strategies in the future.

GalNAc-T14 may be involved in regulating the apoptotic action of IGFBP-3

Insulin-like growth factor binding protein-3 (IGFBP-3) is known to induce apoptosis in an insulin-like growth factor (IGF)-dependent and IGF-independent manner, but the mechanism underlying the IGF-independent effects remains unclear. Polypeptide N-acetylgalactosaminyltransferase 14 (GalNAc-T14) is a novel IGFBP-3 binding partner. In this paper, small interference RNA (siRNA) targeting GalNAc-T14 was used to examine whether GalNAc-T14 affects the apoptotic action of IGFBP-3. Using semi-quantitative reverse-transcriptase polymerase chain reaction (RT-PCR) and western blot analysis, we determined that GalNAc-T14 expression was downregulated by the siRNA directed against GalNAc-T14. Apoptosis analysis of IGFBP-3-overexpressing cells treated with siRNA against GalNAc-T14 was performed to determine if GalNAc-T14 was specifically involved in IGFBP-3 signalling. The results, as determined by flow cytometric analysis and caspase-3 assay, showed that the extent of apoptosis induced by IGFBP-increased with RNA interference (RNAi) knockdown of GalNAc-T14. Our data suggest that GalNAc-T14 influences the apoptotic action of IGFBP-3 and might mediate the signalling pathway of IGFBP-3. Experiments to determine the role of GalNAc-T14 in the regulation of apoptosis induced by IGFBP-3 are under way.

Inhibition of H 2O 2-induced neuroblastoma cell cytotoxicity by a triazine derivative, AA3E2

Alzheimer's disease is the major cause of senile dementia with the hallmark of β-amyloid deposition in neurons. Although the main cause(s) of this deposition is not fully understood, however, the wealth of the present literature data supports the pivotal role of reactive oxygen and nitrogen species in both the initiation and progression of β-amyloid aggregation and deposition. In the present study, we were interested to evaluate the free-radical protecting effect of AA3E2, a triazine derivative with a β-amyloid-breaking activity, among SK-N-MC neuroblastoma cells exposed to hydrogen peroxide (H 2O 2) as an exogenous source of free radicals. Exposure of the cells to different doses of AA3E2 (1–16 µM) for 3 h followed by subsequent exposure to a single dose of H 2O 2 (mainly 150 µM) attenuated the extent of superoxide dismutase (SOD) and catalase (CAT) inhibition by H 2O 2, in a dose dependent manner. Furthermore, significant reduction was observed in the extent of cellular lactate dehydrogenase release, intracellular ROS and the extent of apoptosis among the cells pre-treated with AA3E2. Based on these data, an antioxidant mode of action is proposed for AA3E2 besides its previously β-amyloid-breaking activity.

Transfection of Rat Pancreatic Islet Tissue by Polymeric Gene Vectors

In vitro genetic modification has been regarded as one option to improve the viability and functionality of pancreatic islets when used for transplantation in patients with diabetes, either as naked islets or in a type of bioartificial pancreas. In this approach, vector safety and poor transfection efficiency are major concerns. In this study, the influence of in vitro transfection conditions on polyplexes constructed of polyethyleneimine (PEI) and plasmid DNA (pDNA) on the transfection efficiency was investigated by varying the transfection medium, the pDNA dose, and the amines of polycation/phosphates of pDNA (N/P) ratio. Ca2+-containing Krebs-Ringer-HEPES medium was more effective than RPMI 1640 medium by increasing transfection efficiency (2.5-fold). An increase in pDNA dose slightly reduced the transfection efficiency but had minimal influence on islet loss. However, the N/P ratio had a large effect on islet viability and transfection efficiency. For example, the PEI/pDNA ratio at N/P = 10 caused greater islet loss (56% vs. 28%) and 30-fold less transfection efficiency than at N/P = 5. Even under a set of best conditions selected from this study, mostly a fraction of cells located in the peripheral regions of an islet were transfected, and the viability and insulin secretion from the treated islets were not altered. However, it was found that the extent of apoptosis was noticeably higher (approximately 16%) than in untreated islets (approximately 2%). These results suggest that the gene delivery efficacy to isolated islets can be improved by manipulating the transfection conditions. Polymeric vectors will broaden the options for islet transfection, which is currently limited to viral vectors.

Long-Term Exposure to Zidovudine Delays Cell Cycle Progression, Induces Apoptosis, and Decreases Telomerase Activity in Human Hepatocytes

Zidovudine (3'-azido-3'-deoxythymidine; AZT), which is currently used in the treatment of acquired immunodeficiency syndrome, has been shown to have anticancer properties. In the present study, we examined the mechanisms contributing to increased sensitivity of cancer cells to the growth-inhibitory effects of AZT. This was accomplished by incubating a hepatoma cell line (HepG2) and a normal liver cell line (THLE2) with AZT in continuous culture for up to 4 weeks and evaluating the number of viable and necrotic cells, the induction of apoptosis, cell cycle alterations, and telomerase activity. In HepG2 cells, AZT (2-100 microM) caused significant dose-dependent decreases in the number of viable cells at exposures > 24 h. During a 1-week recover period, there was only a slight increase in the number of viable cells treated with AZT. The decrease in viable cells was associated with an induction of apoptosis, a decrease in telomerase activity, and S and G2/M phase arrest of the cell cycle. During the recovery period, the extent of apoptosis and telomerase activity returned to control levels, whereas the disruption of cell cycle progression persisted. Western blot analysis indicated that AZT caused a decrease in checkpoint kinase 1 (Chk1) and kinase 2 (Chk2) and an increase in phosphorylated Chk1 (Ser345) and Chk2 (Thr68). Similar effects, to lesser extent, were observed in THLE2 cells given much higher concentrations of AZT (50-2500 microM). These data show that HepG2 cells are much more sensitive than THLE2 cells to AZT. They also indicate that a combination of a delay of cell cycle progression, an induction of apoptosis, and a decrease in telomerase activity is contributing to the decrease in the number of viable cells from AZT treatment, and that checkpoint enzymes Chk1 and Chk2 may play an important role in the delay of cell cycle progression.

Protective effect of galactooligosaccharide-supplemented enteral nutrition on intestinal barrier function in rats with severe acute pancreatitis

Intestinal barrier dysfunction may play an important role in the secondary infectious complications associated with severe acute pancreatitis (SAP). We aimed to investigate the protective effect of enteral nutrition supplemented with prebiotics, namely, galactooligosaccharides (GOS), on intestinal barrier function in SAP rats. We randomly divided 60 Sprague-Dawley (SD) rats into 6 groups: sham operation 4-d and 7-d groups, SAP with standard enteral nutrition (EN) 4-d and 7-d groups, and SAP with GOS-supplemented EN (PRE-EN) 4-d and 7-d groups (n = 10 in each group). The intestinal barrier function was assessed on the basis of several aspects, including the number of colonic bifidobacteria and lactobacilli, sIgA concentration in intestinal mucus, extent of apoptosis in intestinal epithelial cells, and the protein expression and mRNA of the intestinal tight junction protein occludin. The results for groups at the same time points were compared with each other. We observed that the intestinal barrier function was impaired after the establishment of the SAP model, in comparison with the sham control group. In the PRE-EN groups, the number of fecal bifidobacteria, sIgA level in intestinal mucus, intestinal occludin mRNA level at both day 4 and day 7, and extent of intestinal epithelial apoptosis at day 7 were significantly higher than the corresponding values in the standard EN groups (P < 0.05). Supplementation of the prebiotic GOS in EN can significantly improve intestinal barrier function in SAP rats.

Inhibition of cell death results in hyperganglionosis: implications for enteric nervous system development

The enteric nervous system (ENS) is derived from vagal and sacral neural crest cells (NCC) that delaminate from the neural tube and undergo extensive migration and proliferation in order to colonize the entire length of the gut and differentiate into many millions of neurons and glial cells. Although apoptotic programmed cell death is an essential physiological process during development of the majority of the vertebrate nervous system, apoptosis within early ENS development has not been comprehensively investigated. The aim of this study was to determine the presence and extent of apoptosis within the vagal NCC population that gives rise to most of the ENS in the chick embryo. We demonstrated that apoptotic cells, as shown by terminal deoxynucleotidyl transferase biotin-dUTP nick end labelling and active caspase-3 immunoreactivity, are present within an electroporated green fluorescent protein (GFP) and human natural killer-1 (HNK-1) immunopositive NCC population migrating from the vagal region of the neural tube to the developing foregut. Inhibition of caspase activity in vagal NCC, by electroporation with a dominant-negative form of caspase-9, increased the number of vagal NCC available for ENS formation, as shown by 3-dimensional reconstruction of serial GFP or HNK-1 labelled sections, and resulted in hyperganglionosis within the proximal foregut, as shown by NADPH-diaphorase whole gut staining. These findings suggest that apoptotic cell death may be a normal process within the precursor pool of pre-enteric NCC that migrates to the gut, and as such it may play a role in the control of ENS formation.

Rickettsia rickettsiiInfection Protects Human Microvascular Endothelial Cells against Staurosporine‐Induced Apoptosis by a cIAP2‐Independent Mechanism

Manipulation of host cell death is an important determinant of the outcome of an infection. Here, we investigate whether Rickettsia rickettsii-infected host endothelial cells resist the effects of staurosporine, a potent inducer of apoptosis, and we explore the mechanisms underlying the anti-apoptotic effect of infection. Human microvascular endothelial cells infected with R. rickettsii for 24 or 48 h were challenged with staurosporine. The extent of apoptosis was evaluated with flow cytometry. mRNA and protein expression levels were determined by use of microarray or polymerase chain reaction and immunoblotting, respectively. Staurosporine-induced apoptosis in endothelial cells infected for 24 and 48 h was significantly reduced, compared with simultaneously treated uninfected cells. A microarray of human genes involved in apoptosis and polymerase chain reaction analyses revealed increased steady-state mRNA expression of cIAP2 (a member of the inhibitor-of-apoptosis family of proteins) at 24 h after infection. The levels of cIAP2 protein (+/-SD) in infected cells were 3.5 +/- 1.7 -fold and 2.3 +/- 1.2 -fold higher than that in uninfected control cells at 24 and 48 h after infection. Nucleofection of human-specific cIAP2-targeted siRNA resulted in inhibition of protein expression by > or = 50% but had no effect on infection-induced protection against apoptosis. R. rickettsii-induced expression of cIAP2 in host endothelial cells is likely not a major contributor to protection against staurosporine-induced cell death.

Bone morphogenetic protein-4 promotes induction of cardiomyocytes from human embryonic stem cells in serum-based embryoid body development

Cardiomyocytes derived from human embryonic stem (ES) cells are a potential source for cell-based therapy for heart diseases. We studied the effect of bone morphogenetic protein (BMP)-4 in the presence of fetal bovine serum (FBS) on cardiac induction from human H1 ES cells during embryoid body (EB) development. Suspension culture for 4 days with 20% FBS produced the best results for the differentiation of early mesoderm and cardiomyocytes. The addition of Noggin reduced the incidence of beating EBs from 23.6% to 5.3%, which indicated the involvement of BMP signaling in the spontaneous cardiac differentiation. In this condition, treatment with 12.5-25 ng/ml BMP-4 during the 4-day suspension optimally promoted the cardiomyocyte differentiation. The incidence of beating EBs at 25 ng/ml BMP-4 reached 95.8% on day 6 of expansion and then plateaued until day 20. In real-time PCR analysis, the cardiac development-related genes MESP1 and Nkx2.5 were upregulated in the EB outgrowths by 25 ng/ml BMP-4. The activation of BMP signaling in EBs was confirmed by the increase in the phosphorylation of Smad1/5/8 and by the nuclear localization of phospho-Smad1/5/8 and Smad4. The addition of 150 ng/ml Noggin considerably decreased the incidence of beating EBs and Nkx2.5 expression, and Noggin alone increased Nestin expression and neural differentiation in EB outgrowths. The cardiomyocytes induced by 25 ng/ml BMP-4 showed proper cell biological characteristics and a course of differentiation as judged from isoproterenol administration, gene expression, protein assay, immunoreactivity, and subcellular structures. No remarkable change in the extent of apoptosis and proliferation in the cardiomyocytes was observed by BMP-4 treatment. These findings showed that BMP-4 in combination with FBS at the appropriate time and concentrations significantly promotes cardiomyocyte induction from human ES cells.

Vasoactive intestinal peptide induces apoptosis in naïve but not in activated HuT 78 cells

Activation induced cell death (AICD) of T lymphocytes plays a vital role in the regulation of the immune response. Cellular resistance to apoptosis can contribute to cancer and autoimmune diseases. Vasoactive intestinal peptide receptor 1 and 2 (VPAC1/2) are G‐protein coupled receptors highly expressed in the immune system that bind vasoactive intestinal peptide (VIP). It has been previously shown that inducible VPAC2 inhibits apoptosis in Th2 polarized CD4 T cells. However, the effect of VPAC1 signaling is not well understood. Naïve and activated HuT 78 cells, a transformed human T lymphocyte cell line predominantly expressing VPAC1, were treated +/‐ VIP. The extent of apoptosis was measured using a histone fragmentation‐based ELISA. In activated cells, VIP had little effect on apoptosis (10‐6‐10‐10M), but caused a significant increase in apoptosis in naïve cells (10‐8M). VIP/VPAC1 signaling induces apoptosis in naïve T cells but has little effect on activated cells. Furthermore, it appears that the VPAC1 and VPAC2 receptors affect apoptosis in opposing ways, most likely due to subtle differences in intracellular signaling. Research supported by NIH‐K01 1K01DK064828 and COBRE 2P20RR05566.

The effects of melatonin on the proliferative activity of retinoblastoma cell line HXO-RB44

Objective To investigate inhibited effects of melatonin （MLT） on proliferative activity of retinoblastoma cell line HXO-RB44 and its related mechanism. Methods HXO-RB44 cells were treated by MLT of different concentration （10^-10, 10^-9 , 10^-8, 10^-7 mmol/L. Cell counting and tetrazolium dye-reduction assay （MTT） were used to determine the effect of MLT on the survival and proliferation of HXO-RB44 cells. Apoptotic nuclei were further analyzed by Hoechst-PI fluorescence staining. Flow cytometry was used to measure the fluorescent intensity of ROS, cell cycle distribution and apoptosis. Results 10^-6 mmol/L （or exceed） of MLT could inhibit the proliferation of HXO-RB44 cells in vitro while 10^-7 mmol/L （or below） of MLT couldn＇t. With the increase of MLT concentration from 10-^10 mmol/L to 10^-7 mmol/L, HXO-RB44 cells gradually increased the expression of ROS. Hoechst staining showed that 4, 8, 12 and 24 hours after the incubation with MLT, the nuclear pyknosis and nuclear fragmentation increased in HXO-RB44 cells. The extent of apoptosis was proportional to the concentrations of MLT. Flow cytometry revealed that with the increasing of MLT concentration, G0/G1 and G2/M phase cells increased, S phase cells decreased. The apoptotic rate was also increased. Conclusion 10.6 M of MLT could inhibit the proliferation of HXO-RB44 cells. This effect may relate to the increased ROS expression, cell cycle arrest at Go/G1 phase and apoptosis of HXO-RB44 cells. Key words: Retinoblastoma/therapy; Cell cycle/drug effects; Apoptosis; Melatonin