Invertebrate Iridovirus Modulation of Apoptosis

Programmed cell death （apoptosis） is a key host response to virus infection. Viruses that can modulate host apoptotic responses are likely to gain important opportunities for transmission. Here we review recent studies that demonstrate that particles of Invertebrate iridescent virus 6 （IIV-6） （Iridoviridae, genus Iridovirus）, or an IIV-6 virion protein extract, are capable of inducing apoptosis in lepidopteran and coleopteran cells, at concentrations 1000-fold lower than that required to shut-off host macromolecular synthesis. Induction of apoptosis depends on endocytosis of one or more heat-sensitive virion component（s）. Studies with a JNK inhibitor （SP600125） indicated that the JNK signaling pathway is significantly involved in apoptosis in IIV-6 infections of Choristoneurafumiferana cells. The genome of IIV-6 codes for an inhibitor of apoptosis iap gene （193R） that encodes a protein of 208 aa with 15% identity and 28% similarity in its amino acid sequence to IAP-3 from Cydia pomonella ganulovirus （CpGV）. Transcription of IIV-6 iap did not require prior DNA or protein synthesis, indicating that it is an immediate-early class gene. Transient expression and gene knockdown studies have confirmed the functional nature of the IIV-6 lap gene. We present a tentative model for IIV-6 induction and inhibition of apoptosis in insect cells and discuss the potential applications of these findings in insect pest control.

Expression of X-linked Inhibitor of Apoptosis Protein and Its Effect on Chemotherapeutic Sensitivity of Bladder Carcinoma

The expression of X-linked inhibitor of apoptosis protein (XIAP) gene and its effect on chemotherapeutic sensitivity in bladder carcinoma was explored. By using immunohistochemistry, the expression of XIAP was detected in 47 bladder carcinomas and 5 normal bladder tissues. The XIAP gene was transfected into bladder cancer cell line T24 by liposome and the positive clone was screened by G418. Cellular XIAP mRNA level was detected by RT-PCR. Low-dose mitocycin C was administered to induce the apoptosis of T24 cells. The in vitro growth of bladder carcinoma cells was analyzed by MTT colorimetry, and the apoptosis rate was assayed by TUNEL methods. It was found XIAP was moderately expressed in bladder carcinomas with the the positive rate being 78.73% (37/47), but the positive rate was not correlated with carcinoma stages and grades (P<0.05). XIAP mRNA level in transfected T24 cells was significantly increased by 3.8 times as compared with that in the cells not transfected with XIAP. After treatment with low-dose mitomycin C (0.005 and 0.05 mg/mL), the growth rate in XIAP no-transfected control group was increased by (11.60±0.25)% and (16.51±0.87)% (P<0.05), and the apoptosis rate was decreased by (10.1±0.2)% and (11.9±0.2%) (P<0.05) respectively as compared with XIAP transfected group. It was concluded that XIAP was expressed in most of bladder carcimoma samples. Overexpression of XIAP in T24 could significantly reduce the MMC-induced apoptosis of bladder carcinoma, suggesting its effect on the chemothera- peutic sensitivity of T24 cells.

EFFECTS OF ANTI-PML/RARα OR ANTI-PML ANTISENCE ON THE GROWTH, DIFFERENTIATION AND APOPTOSISOF NB4 CELL LINES

Objectire To investigate the ellects of anti - PML/RARx or anti - PML antisence on the growth,dtherentiation and apoptosis of NB4 cell lines. Methods Wright’s stain for cell morphology, flow cytometry andDNA gel electronphoresis for cell apoptosis, methylcellulose assays for leukemic colony forming unit andtrypan - blue exclusion for cell counts. Results Both the start cordon region of the PML or PML - RARx mRNA(STAS) and the fusion point region of the long type PML - RARx mRNA (FUAS) could inhibit cell growth. Cellsbecame partially differentiated at 5d of treatment, and FUAS - treated cells showed more significant differentiationthan STAS- treated cells. Morphology of typical apoptosis could be seen at 7, 9d incubation with antisenceoligodeoxynucleotides (AS). In contrast, no cell growth inhibition, no morphology changes were seen in Sen or Rantreated cells compared with untreated cells. The number of acute myelocytic leukemia colony forming unit(AML - CFU) markedly decreased in STAS and FUAS treated cells. Cell DNA content analyzed by flow cytometryshowed the typical profile of apoptotic cells, in which pre - G1 peak appear before G1 peak at 7,9d of treatment withSTAS or FUAS. Conclusion Anti - PML/RARx or anti- PML antisence inhibit the cell growth, inducedifferentiation and differentiated cell apoptosis of NB4 cells.

Acceleration of apoptosis by transfection of bak gene in multi-drug resistant (MDR) bladder cancer cellsI

Objective To observe the effects of bak gene on killing MDR bladder cancer cells and to study its mechanisms. Methods Bak gene was transfected into MDR bladder cancer cells by liposome. The mRNA of bak and bcl-2 were detected by in situ hybridization. The protein of bak and bcl-2 were detected by SABC immunohistochemistry. The growth rate of human bladder cancer cells was studied by constructing the growth curve, cell apoptosis being observed by flow cytometry, and the outline of cells observed by fluorescence stain. Results The expression of bak mRNA was positive in EJ/bak cells (64% ,P【0.05).Bak protein expression of EJ/bak cells was positive (60 % ) and bcl-2 protein expression was de creased (P【0.05). The growth of MDR bladder cancer cells was significantly inhibited by 32% after bak gene was transfected (P 【 0. 05 ). Apoptosis cells increased significantly. The apoptosis rate was 35 %. Apoptotic bodies can be found in these cells on fluorescence stain. Conclusion Bak gene could inhibit the growth

Relationship between expression and distribution of cyclooxygenase-2 and bcl-2 in human gastric adenocarcinoma

AIM: To explore expression and distribution features of COX-2 and bcl-2 in human gastric adenocarcinoma tissues and to study its biological significance.METHODS: Totally 36 human gastric carcinoma samples were enrolled in this study (cardiac adenocarcinoma 16 cases, distal gastric adenocarcinoma 20 cases). The expressions of COX-2 and bcl-2 in cancerous tissues and corresponding para-cancerous tissues were investigated by immunohistochemistry using COX-2 polyclonal antibody and bcl-2 monoclonal antibody. The normal gastric mucosa tissues were used as control.RESULTS: The expressions of COX-2 and bcl-2 in gastric carcinoma were significantly higher than that in the paracancerous tissues (77.8% vs 47.2%, P＜0.01, 80.56% vs 58.33%, P＜0.05). The expression of COX-2 in cardiac adenocarcinoma was remarkably higher than that in the distal gastric carcinoma (93.8% vs 65.0%, P＜0.01). The expression of COX-2 was mainly localized in the cytoplasm of tumor cells and partly in the nucleus. There is a transition of the COX-2 cytoplasmic positivity to nucleic in tumor cells with the increase of gastric carcinoma pathological grade. Interstitial macrophages, fibroblasts and vascular endothelial cells also expressed COX-2. The tissues with higher expression of COX-2 also expressed high level of bcl-2 protein.CONCLUSION: Abnormal expression pattern of COX-2within the tissues of human gastric cancer is correlated with tumor location and lymph node metastasis. COX-2may regulate expression of apoptosis suppressor gene (bcl-2) through interaction of tumor cells and stromal cells and play an important role in the generation and development of tumors, which will be of great help in developing new methods for antitumor therapy.

Survivin isoforms and clinicopathological characteristics in colorectal adenocarcinomas using real-time qPCR

AIM:To investigate three isoforms of survivin in colorectal adenocarcinomas.METHODS:We used the LightCycler Technology(Roche),along with a common forward primer and reverse primers specific for the splice variants and two common hybridization probes labeled with fluorescein and LightCycler-Red fluorophore(LC-Red 640).Real time quantitative polymerase chain reaction(PCR) was performed on cDNAs from 52 tumor specimens from colorectal cancer patients and 10 unrelated normal colorectal tissues.In the patients group,carcinoembryonic antigen(CEA) and CA19-9 tumor markers were also measured immunochemically.RESULTS:Wild type survivin mRNA isoform was expressed in 48%of the 52 tumor samples,survivin-2b in 38%and survivin-ΔΕx3 in 29%,while no expression was found in normal tissues.The mRNA expression of wild type survivin presented a significant correlation with the expression of the ratio of survivin-2b,survivin-ΔΕx3,survivin-2b/wild type survivin and survivin-ΔΕx3/wild type survivin(P<0.001).The mRNA expression of wildsurvivin and survivin-ΔΕx3 was related with tumor size and invasion(P=0.006 and P<0.005,respectively).A significant difference was found between survivin-2b and morphologic cancer type.Also,the ratio of survivin-ΔEx3/ wild-survivin was significantly associated with prognosis.No association was observed between the three isoforms and grade,metastasis,Dukes stage and gender.The three isoforms were not correlated with CEA and CA19-9.CONCLUSION:Survivin isoforms may play a role in cell apoptosis and their quantification could provide information about clinical management of patients suffering from colorectal cancer.

Growth-inhibitory effects of MOB2 on human hepatic carcinoma cell line SMMC-7721

AIM:To investigate the growth-inhibiting and apoptosis-inducing effects of the gene MOB2 on human hepatic carcinoma cell line SMMC-7721.METHODS:The full-length cDNA of the MOB2 gene was amplified from human umbilical vein endothelial cells.The correct full-length MOB2 cDNA was subcloned into the eukaryotic expression vector pEGFP-C1.After lipofection of the MOB2 gene into cancer cells,the levels of MOB2 protein in the cancer cells were detected by immunoblotting.To transfect the recombined plasmid vector pEGFP-CI-MOB2 into SMMC-7721 cells,the cells were cultured in Dulbecco's Modified Eagle'sMedium with 10% fetal calf serum and glutamine,and then mixed with liposomes,Lipofectamine 2000 and the plasmid vector pEGFP-CI-MOB2.RESULTS:We observed the growth and proliferation of SMMC-7721 cells containing pEGFP-CI-MOB2 and analyzed their apoptosis and growth cycle phases by flow cytometry.We successfully transfected the recombined plasmid vector pEGFP-CI-MOB2 into SMMC-7721 cells and screened for a single clone cell containing MOB2.After transfection,MOB2 enhanced growth suppression,induced apoptosis,increased the ratio of G0/G1,significantly inhibited the advance of cell cycle phase,and arrested cells in G0/G1 phase.CONCLUSION:MOB2 overexpression induces apoptosis and inhibits the growth of human hepatic cancer cells,which may be useful in gene therapy for hepatic carcinoma.

MicroRNA-219 alleviates glutamate-induced neurotoxicity in cultured hippocampal neurons by targeting calmodulin-dependent protein kinase Ⅱ gamma

Septic encephalopathy is a frequent complication of sepsis,but there are few studies examining the role of micro RNAs（mi Rs） in its pathogenesis.In this study,a mi R-219 mimic was transfected into rat hippocampal neurons to model mi R-219 overexpression.A protective effect of mi R-219 was observed for glutamate-induced neurotoxicity of rat hippocampal neurons,and an underlying mechanism involving calmodulin-dependent protein kinase II γ（Ca MKIIγ） was demonstrated.mi R-219 and Ca MKIIγ m RNA expression induced by glutamate in hippocampal neurons was determined by quantitative real-time reverse transcription-polymerase chain reaction（q RT-PCR）.After neurons were transfected with mi R-219 mimic,effects on cell viability and apoptosis were measured by 3-（4,5-dimethylthiazolyl-2）-2,5-diphenyltetrazolium bromide（MTT） assay and flow cytometry.In addition,a luciferase reporter gene system was used to confirm Ca MKIIγ as a target gene of mi R-219.Western blot assay and rescue experiments were also utilized to detect Ca MKIIγ expression and further verify that mi R-219 in hippocampal neurons exerted its effect through regulation of Ca MKIIγ.MTT assay and q RT-PCR results revealed obvious decreases in cell viability and mi R-219 expression after glutamate stimulation,while Ca MKIIγ m RNA expression was increased.MTT,flow cytometry,and caspase-3 activity assays showed that mi R-219 overexpression could elevate glutamate-induced cell viability,and reduce cell apoptosis and caspase-3 activity.Moreover,luciferase Ca MKIIγ-reporter activity was remarkably decreased by co-transfection with mi R-219 mimic,and the results of a rescue experiment showed that Ca MKIIγ overexpression could reverse the biological effects of mi R-219.Collectively,these findings verify that mi R-219 expression was decreased in glutamate-induced neurons,Ca MKIIγ was a target gene of mi R-219,and mi R-219 alleviated glutamate-induced neuronal excitotoxicity by negatively controlling Ca MKIIγ expression.

CO-EXPRESSIONS OF SURVIVIN GENE, BCL-2 AND BAX PROTEINS IN OVARIAN CARCINOMA

Objective To characterize the cellular properties of ovarian cancer, we examined the correlation between the expression of apoptosis-related gene survivin and those of Bcl-2 and Bax proteins. Methods Expressions of survivin mRNA, and Bcl-2 and Bax proteins in 35 cases of ovarian carcinoma, 10 cases of borderline carcinoma, 10 cases of benign tumors and 10 cases of normal tissue were evaluated by reverse transcription polymer-ase chain reaction (RT-PCR) and immunohistochemistry SABC method, respectively. Results Expression of survivin gene was detected in a significantly greater proportion in ovarian carcinoma and borderline carcinoma than those in benign tumors and normal tissues. Although there was no relationship between expression of survivin gene and FIGO stage, histologic grade, pathological type and lymphatic metastasis, expressions of Bcl-2 and Bax proteins were positively and negatively correlated with that of survivin gene, respectively. Conclusion Survivin may play an important role in pathogenesis of ovarian carcinoma, with a synergistic role of apoptosis-related gene Bcl-2 protein and an antagonistic role of Bax protein in formation and progression of ovarian carcinoma.

Deletion analysis of SMN1 and NAIP genes in southern Chinese children with spinal muscular atrophy

Spinal muscular atrophy （SMA） is a disorder characterized by degeneration of lower motor neurons and occasionally bulbar motor neurons leading to progressive limb and trunk paralysis as well as muscular atrophy. Three types of SMA are recognized depending on the age of onset, the maximum muscular activity achieved, and survivorship： SMA1, SMA2, and SMA3. The survival of motor neuron （SMN） gene has been identified as an SMA determining gene, whereas the neuronal apoptosis inhibitory protein （NAlP） gene is considered to be a modifying factor of the severity of SMA. The main objective of this study was to analyze the deletion of SMN1 and NAIP genes in southern Chinese children with SMA. Here, polymerase chain reaction （PCR） combined with restriction fragment length polymorphism （RFLP） was performed to detect the deletion of both exon 7 and exon 8 of SMN1 and exon 5 of NAIP in 62 southern Chinese children with strongly suspected clinical symptoms of SMA. All the 32 SMA1 patients and 76% （13/17） of SMA2 patients showed homozygous deletions for exon 7 and exon 8, and all the 13 SMA3 patients showed single deletion of SMNI exon 7 along with 24% （4/17） of SMA2 patients. Eleven out of 32 （34%） SMA1 patients showed NAIP deletion, and none of SMA2 and SMA3 patients was found to have NA1P deletion. The findings of homozygous deletions ofexon 7 and/or exon 8 ofSMN1 gene confirmed the diagnosis of SMA, and suggested that the deletion ofSMN1 exon 7 is a major cause of SMA in southern Chinese children, and that the NAIP gene may be a modifying factor for disease severity of SMAI. The molecular diagnosis system based on PCR-RFLP analysis can conveniently be applied in the clinical testing, genetic counseling, prenatal diagnosis and preimplantation genetic diagnosis of SMA.

Effect of adenovirus-mediated p27 gene expression on the proliferation and apoptosis of HL-60 and Raji cell lines

Background p27 is an essential mediator of cell cycle control,which plays a key negative role in the proliferation and tumorigenesis of certain cell types. Here, we designed this study to explore the possible effects of p27 on the proliferation and apoptosis of HL-60 and Raji cell lines.Methods HL-60 and Raji cells were transfected with p27 via an adenovirus-mediated approach. The efficiency of Adp27 infection and the expression of p27 mRNA and protein were evaluated by X-gal staining, RT-PCR, and flow cytometry. The proliferation and apoptosis of HL-60 and Raji cells were estimated by means of trypan blue staining, MTT assay, Annexin V/PI, and DNA ladder electrophoresis. Results The infection efficiencies in HL-60 and Raji cells were 40.3% and 32.0%, respectively. RT-PCR and flow cytometry showed that there was significant expression of p27 mRNA and protein in HL-60 and Raji cells infected with Adp27; on the other hand, uninfected HL-60 cells showed faint traces of p27 mRNA and protein and Raji cells showed nearly no signs of p27 mRNA and protein. As demonstrated by a cell growth curve and by an MTT assay, strong time-dependent proliferation inhibition was apparent in HL-60 and Raji cells infected by Adp27. After 72 hours of infection, the Annexin V+/PI- apoptotic cell rates in HL-60 and Raji cell lines were 46.9% and 35.7%, respectively, significantly higher than in the control groups (4.7% and 5.6%, respectively). Typical DNA ladder bands were detectable in HL-60 and Raji cells after 48 hours of Adp27 infection. Conclusions Adenoviral vector-mediated p27 gene transfection of HL-60 and Raji cells leads to the inhibition of cellular proliferation and the promotion of cell apoptosis. This technique may provide an approach to gene therapy for leukemia or lymphoma.

Study on Intervenient Effect of Angelica Sinensis Polysaccharide on Immunological Liver Injury and Its Mechanism in Mice

Objective: To study the changes of expressions of nitric oxide synthase (NOS) of the constitutive type (cNOS) and inducible type (iNOS), the apoptosis related genes bax and bcl 2, as well as the tumor necrosis factor (TNF α) in immunological liver injury (ILI) and to explore the preventive effects of Angelica sinensis polysaccharide (ASP) on ILI and its mechanism in mice.Methods: ILI model mice induced by intraperitoneal injection of lipo polysaccharide (LPS) and BCG vaccine were treated with ASP of different doses (30mg/kg, 60mg/kg) by gastrogavage every day for 7 days. The serum alanine transaminase (ALT) and glutathione S transferase (GST) activities and NO content in the liver were detected; the expressions of cNOS, iNOS, bcl 2, bax were assessed with immuno histochemical method, and the TNF α mRNA expression in the liver was observed by reverse transcriptase polymerase chain reaction (RT PCR).Results: Compared with the normal mice , the NO production and ALT, GST levels were raised significantly in the model mice, the TNF α mRNA expression was also raised significantly. But no obvious changes of cNOS was found. Small dose ASP (30mg/kg) could reduce NO production and ALT, GST levels in model mice by 19.5%, 23.7% and 40.0% respectively, decrease the expression of iNOS and bax by 48.3%, and 26.4%, and increase the expression of cNOS, bcl 2 by 66.9% and 337.3%, respectively, but it could not reduce the TNF α mRNA expression in the liver. Large dose of ASP (60mg/kg) was not more effective than that of small dose.Conclusion: Changes of NO production and TNF α mRNA may play an important role in ILI. The mechanism of ASP in intervening ILI may be through modulation on cNOS, iNOS, bax, bcl 2 expression to block the damage of BCG vaccine and LPS on hepatocytes.

Effects of anti-HPV16E6-ribozyme on phenotype and gene expression of a cervical cancer cell line

To investigate the effects of anti HPV16E6 ribozyme (HRz) on phenotype and gene expression of a cervical cancer cell line Methods HRz was designed by computer programs HRz’s activity was identified by cleavage experiments in vitro HRz and empty eukaryotic plasmids were transfected into CaSKi cells with lipofectin, then renamed CaSKi R and CaSKi P, respectively The expression of ribozyme in transfected cells was observed by RNA dot blot The amounts of E6 mRNA in three kinds of cells lines were detected by Northern blot Cell growth curves and soft agar forming ability were studied The ability of each cell line to form tumors was assessed in nude mice Apoptosis rates and expression of c myc, bcl 2, p53 and Fas were detected by flow cytometry (FCM) Antigens of tumor cells, HLA 1, HLA 2, B7 1 and B7 2 were also detected NK, LAK, and CD 3AK cells were induced Their cytotoxicities were detected in CaSKi R, CaSKi P, and CaSKi cells Results In vitro cleavage reaction demonstrated that HRz could cleave HPV16E6 mRNA in a site specific manner HRz could be expressed stably in transfected CaSKi cells Northern blot analysis showed that E6 mRNA levels were lower in CaSKi R than in CaSKi The growth rate of CaSKi R was slower than those of CaSKi and CaSKi P The soft agar forming rate of CaSKi R was lower compared with those of CaSKi and CaSKi P cells The ability of CaSKi R to form tumors in nude mice was also poor The apoptosis rate of CaSKi R cells was much higher than those of CaSKi and CaSKi P HRz could reduce the expression of E6, c myc and bcl 2 proteins, and increase the expression of p53 as well HRz could increase the expression of HLA 2, B7 1 and B7 2 antigens The cytotoxicity of NK, LAK and CD 3AK cells was much higher in CaSKi R than in CaSKi P and CaSKi cells Conclusion HRz not only reverses the malignant phenotype of CaSKi cells partially, but also induces apoptosis in the cells, and increases sensitivity of CaSKi cells to immune cells