Protein Expression of BLM Gene and Its Apoptosis Sensitivity in Hematopoietic Tumor Cell Strains

Patients with Bloom syndrome （BS） show an immunodeficiency, an enhanced sister chromatid exchanges （SCEs）, a strong genetic instability and an increased predisposition to all. In order to investigate the differential expression of BLM protein in hematopoietic tumor cell strains and study the effects of BLM gene on ultraviolet （UV）- or hydroxyurea （HU）-induced apoptosis, Western blot was used to detect the expression of BLM protein in normal human bone marrow mononuclear cells and 4 kinds of hematopoietic tumor cell strains. The 4 kinds of hematopoietic tumor cells were exposed to UV light with a germicidal UV lamp or treated with 2 mmol/L hydroxyurea and the apoptotic rate was detected by using AnnexinV-FITC. The results showed that these tumor cells expressed BLM protein higher than the normal human bone marrow mononuclear cells （P〈0.01）. In the 4 hematopoietic tumor cells, BLM protein was all specially cleaved in response to UV- or HU-induced apoptosis. The increase of BLM protein expression may play an important role in the development of these tumors, and BLM proteolysis is likely to be a general feature of the apoptotic response.

Growth arrest-specific gene 2 suppresses hepatocarcinogenesis by intervention of cell cycle and p53-dependent apoptosis

BACKGROUND Growth arrest-specific gene 2(GAS2)plays a role in modulating in reversible growth arrest cell cycle,apoptosis,and cell survival.GAS2 protein is universally expressed in most normal tissues,particularly in the liver,but is depleted in some tumor tissues.However,the functional mechanisms of GAS2 in hepatocellular carcinoma(HCC)are not fully defined.AIM To investigate the function and mechanism of GAS2 in HCC.METHODS GAS2 expression in clinic liver and HCC specimens was analyzed by real-time PCR and western blotting.Cell proliferation was analyzed by counting,MTS,and colony formation assays.Cell cycle analysis was performed by flow cytometry.Cell apoptosis was investigated by Annexin V apoptosis assay and western blotting.RESULTS GAS2 protein expression was lower in HCC than in normal tissues.Overexpression of GAS2 inhibited the proliferation of HCC cells with wide-type p53,while knockdown of GAS2 promoted the proliferation of hepatocytes(P<0.05).Furthermore,GAS2 overexpression impeded the G1-to-S cell cycle transition and arrested more G1 cells,particularly the elevation of sub G1(P<0.01).Apoptosis induced by GAS2 was dependent on p53,which was increased by etoposide addition.The expression of p53 and apoptosis markers was further enhanced when GAS2 was upregulated,but became diminished upon downregulation of GAS2.In the clinic specimen,GAS2 was downregulated in more than 60%of HCCs.The average fold changes of GAS2 expression in tumor tissues were significantly lower than those in paired non-tumor tissues(P<0.05).CONCLUSION GAS2 plays a vital role in HCC cell proliferation and apoptosis,possibly by regulating the cell cycle and p53-dependent apoptosis pathway.

Involvement of gene expressions in apoptosis of vascularendothelial cells induced by rattlesnake venom

Formation of apoptotic bodies is a typical character ofaPoptotic cell death, but how the processes are controlledis not known. In this study, we compared two apoptosisinducing systems in vascular endothelial cells (VEC). Wefound that the formation of aPoptotic bodies during apop-tosis induced by rattlesnake venom, which is an unique andspecific aPoptosis inducer to vascular endotheliaI cells, wasmuch faster than that induced by deprivation of survivalfactors (aFGF and serum). When we blocked the synthesisof mRNAs in cells treated with rattlesnake venom by DRB(5, 6- dichloro- 1 -β- D- rib ofur anosylb enzimidazole ), an in-hibitor of transcription, the formation of aPoptotic bodieswas dramatically inhibited. We examined the expressionof Psa gene and found that its expression was much higherin apoptosis induced by rattlesnake venom than that inaPoptosis induced by deprivation of aFGF and serum. Ourresults suggest that gene expression is important and P53gene may play a major role in inducing the formation ofapoptotic bodies in VEC.

CLONING AND EXPRESSION OF A GENE ASS0CIATE WITH HL_(60)CELL APOPTOSIS INDUCED BY INHIBITIONOF POLYAMINE BIOSYNTHESIS

Objective: To clone the gene associated with apoptosis induced by an inhibitor of polyamine biosynthesis, a-difluoromethylornithime(DFMO). Methods: The differential sufbtraction sereening was used for gene cloning from cDNA library of HL60 cells treated by DFMO. Northern blot,morphological observation, FCM assays and ladder map of DNA electrophoresis were performed. Results: The transfectiong gene expression and activity of inducing apoptosis in the cell transfected from recombinant plasmid containing the cloned fragment df4 wasproved. Conclusion: It is suggest that df4 gene cloned in the study coul be a gene regulating apoptosis of HL60 cells.

Effect of Silencing LRIG3 Gene on the Proliferation and Apoptosis of Bladder Cancer T24 Cells

This study examined the effect of silencing LRIG3 expression on the proliferation and apoptosis of bladder cancer T24 cells and explored the role of LRIG3 in the tumorigenesis of bladder cancer. Bladder cancer T24 cells were routinely cultured and pSilencer plasmids were employed to construct LRIG3 eukaryotic expression vector of LRIG3-siRNA, i.e., pSilencer-LRIG3-siRNA. After confirmation, the vector was transfected into HEK293 cells to make a replication-deficient adenovirus, pAd-LRIG3-siRNA, which was then introduced into bladder cancer T24 cells. RT-PCR, Western- blotting were performed to detect the levels of LRIG3 mRNA and proteins. Cells number was determined by using MTT test. Hoechst33258 staining, transmission microscopy, flow cytometery were conducted to examine the cell apoptosis. Three groups included a blank control group, a negative control group （containing non-interfering plasmids） and a pAd-LRIG3-siRNA group. Our results showed that the recombinant pAd-LRIG3-siRNA was successfully transfected into the bladder cancer T24 cells. The siRNA formed by the transcription of the recombinant plasmids resulted in significantly reduced expressions of LRIG3 gene and protein and significantly decreased cell proliferation and growth in the pAd-LRIG3-siRNA group as compared with the control group （P0.01）. The siRNA also caused apoptotic changes of some cells, with the apoptosis rate being （17.69±0.75）%, which was significantly different from that of the control group （P0.01）. It was concluded that recombinant pAd-LRIG3-siRNA plasmids could effectively decrease the expression of LRIG3 mRNA and proteins and, to some extent, inhibit the proliferation and promote the apoptosis of bladder cancer T24 cells. Silencing LRIG3 gene might be a novel alternative for the treatment of bladder cancer.

Effects of Bcl-2 Gene Interference on the Apoptosis,Proliferation and Progesterone Secretion of Goose Follicular Granulosa Cells

Based on published sequences for chicken Bcl-2,three siRNAs（small interfering RNA）were designed,and expression vectors were constructed and transfected into goose granulosa cells cultured in vitro.Bcl-2 protein,apoptosis and proliferation of granulosa cells,48 h after the transf ection,were analyzed by flow cytometry,and progesterone（P）secreted into the culture medium was measured by radioimmunoassay.In addition,apoptosis and Bcl-2 protein level were assessed in untreated granulosa cells from the four largest preovulatory follicles（F1F4）,the smallest preovulatory follicles（SPF）,small yellow follicles（SYF）and atretic follicles.The highest level of Bcl-2 protein was observed in granulosa cells from SPF,and levels in cells from healthy follicles were significantly higher than those of atretic follicles（P【0.05）.Bcl-2 protein levels in cells subjected to RNAi were significantly lower than those of controls（P【0.05）,while apoptosis indices（AI）,proliferation indices（PI）and P secretion in the RNAi treatments were higher than those of controls（P【0.05）.

Influence of Ginkgo biloba extract on the proliferation,apoptosis of ACC-2 cell and Survivin gene expression in adenoid cystic carcinoma of lacrimal gland

Objective:To explore the influence of extract of Ginkgo biloba（EGB） on the proliferation, apoptosis of ACC-2 cell and Survivin gene expression in adenoid cystic carcinoma（ACC） of lacrimal gland.Methods:ACC-2 cell in human with ACC of lacrimal gland was in vitro cultured. MTT method was used for cell proliferation detection.Annexin V/PI double-staining flow cytometer was used to detect cell apoptosis and cell cycle.Survivin gene expression was analyzed by RT-PCR and Western blotting.Results:EGB had inhibitory effect on the proliferation of ACC-2 cell with significant dose-effect relationship,and there was statistical difference when compared with the control group（P【0.01）.The inhibitory concentration 50%(IC50) is 88 mg/L. The flow cytometer test indicated that CGB can gradually increase ACC-2 cell in G0-G1 stage and decrease it in G2-M and S stage.With the increase of dose,the apoptosis rate of ACC-2 cell was obviously increased（P【0.05 or P【0.01）.EGB had certain inhibitor)’ effect on Survivin gene expression of ACC-2 cell,and Survivin gene expression was decreased with the increasing of the EGB concentration（P【0.01）.Conclusions:EGB can effectively inhibit Survivin gene expression of ACC-2 cell in human with ACC of lacrimal gland,induce the apoptosis of ACC-2 cell and inhibit tumor cell proliferation.

EXPRESSION AND REVERSION OF DRUG RESISTANCE-AND APOPTOSIS-RELATED GENES OF A DDP-RESISTANT LUNG ADENOCARCINOMA CELL LINE

Objective: To investigate the co-expression of drug resistance- and apoptosis-related genes of cisplatin (CDDP)-selected lung adenocarcinoma cell line A 549 DDP for compared to the parental cell line A549, and reverse of drug resistance by antisense s-oligodeoxynucleotides (S-ODNs) of differentially expressed genes. Methods: Sense and antisense S-ODN were transferred into A 549 DDP cells by lipofectin. The expression of drug resistance and apoptosis related genes was examined by RT-PCR, immunocytochemistry and flow cytometry, respectively. Apoptostic cells were identified by DNA electrophoresis and terminal deoxynucleotidyl transferase (TdT)-mediated biotin dUTP nick end-labeling(TUNEL). Drug resistance of tumor cells was detected by a cell viability (MTT) assay. Results: The expression of bcl-2 was positive and that of multidrug resistance-associated protein (MRP) at mRNA and protein level was increased in A 549 DDP compared to A549 cells. MDR1, c-myc and topoisomeras II (TOPO II) were similarly co-expressed in two cell lines. Both cell lines were negative for c-erbB-2 expression. In A 549 DDP cells, the expression of bcl-2 and MRP was significantly inhibited by their respective antisense S-ODNs. Antisense S-ODNs could also decrease significantly drug resistance of A 549 DDP cells to CDDP by promoting cell apoptosis. Conclusion: Both intrinsic and acquired drug resistance were involved in co-expression of multiple MDR-related genes in lung adenocarcinoma. Cooperation of bcl-2 and MRP genes appeared to play an important action to confer the resistance of A 549 DDP cells to CDDP. Their antisense S-ODNs are responsible for the decrease of drug resistance of this cell line by promoting apoptosis.

Effects of Peptide Nucleic Acids against Ki-67 Gene on the Proliferation and Apoptosis of Human Renal Carcinoma Cell Line

To investigate the effects of anti-sense peptide nucleic acids （PNAs） targeting Ki-67 gene on modulation of the proliferation and apoptosis of human renal carcinoma cell lines, human renal carcinoma cell line 786-0 cells were treated with anti-sense PNAs at different concentrations （1.0 μmol/L, 2.0 μmol/L, 10.0 μmol/L）. The Ki-67 expression of 786-0 cells was detected by immunohistochemical technique and Western blot method respectively. The proliferation of 786-0 cells was studied by cell growth curves and ^3H-thymidine incorporation. The apoptosis of 786-0 cells was detected by TUNEL assay. The control groups were treated with anti-sense oligonucleotide （ASODNs） targeting Ki-67 gene. Our results showed that the Ki-67 expression of 786-0 cells treated with anti-sense PNAs （16.9±0.7） was significantly inhibited as compared with that of the control groups （28.6±0.4） （P〈0.01）. The Ki-67 protein rate of 786-0 cells treated with anti-sense PNAs （42.1 ±2.2） was significantly reduced when compared with that of the control groups （83.6± 1.4） （P〈0.01）. Proliferation of 786-0 cells treated with anti-sense PNAs （20.7 ± 1.5） was significantly inhibited as compared with that of the control groups （58.6± 1.4） （P〈0.01）. The apoptosis rate of 786-0 cells treated with anti-sense PNAs （28.7 ± 2.3） was significantly increased higher compared with that of the control groups （13.8 ±1.0） （P〈0.01）. From these finds we are led to conclude that anti-sense PNAs targeting Ki-67 gene have stronger effects on the inhibition of the proliferation and induction of apoptosis of human renal carcinoma cells than ASODNs targeting Ki-67 gene. The strategies using anti-sense PNAs targeting Ki-67 gene may be a promising approach for the treatment of renal cell carcinoma.

Neuronal nitric oxide synthase/reactive oxygen species pathway is involved in apoptosis and pyroptosis in epilepsy

Dysfunction of neuronal nitric oxide synthase contributes to neurotoxicity,which triggers cell death in various neuropathological diseases,including epilepsy.Studies have shown that inhibition of neuronal nitric oxide synthase activity increases the epilepsy threshold,that is,has an anticonvulsant effect.However,the exact role and potential mechanism of neuronal nitric oxide synthase in seizures are still unclear.In this study,we performed RNA sequencing,functional enrichment analysis,and weighted gene coexpression network analysis of the hippocampus of tremor rats,a rat model of genetic epilepsy.We found damaged hippocampal mitochondria and abnormal succinate dehydrogenase level and Na+-K+-ATPase activity.In addition,we used a pilocarpine-induced N2a cell model to mimic epileptic injury.After application of neuronal nitric oxide synthase inhibitor 7-nitroindazole,changes in malondialdehyde,lactate dehydrogenase and superoxide dismutase,which are associated with oxidative stress,were reversed,and the increase in reactive oxygen species level was reversed by 7-nitroindazole or reactive oxygen species inhibitor N-acetylcysteine.Application of 7-nitroindazole or N-acetylcysteine downregulated the expression of caspase-3 and cytochrome c and reversed the apoptosis of epileptic cells.Furthermore,7-nitroindazole or N-acetylcysteine downregulated the abnormally high expression of NLRP3,gasdermin-D,interleukin-1βand interleukin-18.This indicated that 7-nitroindazole and N-acetylcysteine each reversed epileptic cell death.Taken together,our findings suggest that the neuronal nitric oxide synthase/reactive oxygen species pathway is involved in pyroptosis of epileptic cells,and inhibiting neuronal nitric oxide synthase activity or its induced oxidative stress may play a neuroprotective role in epilepsy.

Killing effect of TNF-related apoptosis inducing ligand regulated by tetracycline on gastric cancer cell line NCI-N87

AIM: To clone the cDNA fragment of human TRAIL (TNF-related apoptosis inducing ligand) into a tetracycline-regulated gene expression system, the RevTet-On system, transduce expression vectors into a gastric carcinoma cell line-NCI-N87 and examine the effects of controlled expression of TRAIL in vitro on the gastric carcinoma cells. METHODS: The full-length cDNA of TRAIL was inserted into a vector under the control of the tetracycline-responsive element (TRE) to obtain the plasmid pRevTRE-TRAIL, which was transfected into a packaging cell line PT67. In addition, vector pRev-Tet On and pRevTRE were also transfected into PT67 separately. After hygromycin and G418 selection, the viral titer was determined. The medium containing retroviral vectors was collected and used to transduce a gastric carcinoma cell line NCI-N87. The resulting cell line NCI-N87-Tet On TRE-TRAIL and a control cell line, NCI-N87 Tet On-TRE, were established. TRAIL expression in the cell line was induced by incubating cells with doxycycline (Dox), which is a tetracycline analogue. The killing effect on gastric carcinoma cells was analyzed after induction. RESULTS: The recombinant plasmid pRev-TRE-TRAIL was constructed. After hygromycin or G418 selection, the producer cell lines PT67-TRE, PT67-TRE-TRAIL and PT67-Tet On were obtained,with titers of about 10(8)CFU.L(-1). By transducing NCI-N87 cells with retroviral vectors from these cell lines, stable cell lines NCI-N87-Tet-On TRE-TRAIL (NN3T) and control cell line NCI-N87-Tet-On-TRE (NN2T) were established. The growth curves of the selected cell lines were the same with the wild type NCI-N87. When Dox was added, cell death was obvious in the test groups (29%-77%), whereas no difference was observed in control and wild type cell lines. With the addition of a medium from the test group, human leukemia cell line Jurkat was activated till death (83%), indicating the secretion of active TRAIL proteins from the test cells to the medium. CONCLUSION: With the use of the RevTet-On system, a regulated expression system for TRAIL was constructed. Using this system, the selected killing effect of TRAIL on gastric carcinoma cell line NCI-N87 could be observed.

Apoptosis,proliferation and p53 gene expression of H.pylori associated gastric epithelial lesions

AIM: To study the relationship between Helicobacter pylori (H. pylori) and gastric carcinoma and its possible pathogenesis by H. pylori. METHODS: DNEL technique and immunohistochemical technique were used to study the state of apoptosis, proliferation and p53 gene expression. A total of 100 gastric mucosal biopsy specimens, including 20 normal mucosa, 30 H. pylori-negative and 30 H. pylori-positive gastric precancerous lesions along with 20 gastric carcinomas were studied. RESULTS: There were several apoptotic cells in the superficial epithelium and a few proliferative cells within the neck of gastric glands, and no p53 protein expression in normal mucosa. In gastric carcinoma, there were few apoptotic cells, while there were a large number of proliferative cells, and expression of p53 protein significantly was increased. In the phase of metaplasia, the apoptotic index (AI, 4.36%+/-1.95%), proliferative index (PI, 19.11%+/-6.79%) and positivity of p53 expression (46.7%) in H. pylori-positive group were higher than those in normal mucosa (P【0.01). AI in H. pylori-positive group was higher than that in H. pylori-negative group (3.81%+/-1.76%), PI in H. pylori-positive group was higher than that in H. pylori-negative group (12.25%+/-5.63%, P【0.01). In the phase of dysplasia, AI (2.31%+/-1.10%) in H. pylori-positive group was lower (3.05%+/-1.29%) than that in H. pylori-negative group, but PI (33.89%+/-11.65%) was significantly higher (22.09+/-8018%, P【0.01). In phases of metaplasia, dysplasia and gastric cancer in the H. pylori-positive group, AIs had an evidently graduall decreasing trend (P【0.01), while PIs had an evidently gradual increasing trend (P【0.05 or P【0.01), and there was also a trend of gradual increase in the expression of p53 gene. CONCLUSION: In the course of the formation of gastric carcinoma, proliferation of gastric mucosa can be greatly increased by H. pylori, and H. pylori can induce apoptosis in the phase of metaplasia, but in the phase of dysplasia H. pylori can inhibit cellular apoptosis. And H. pylori infection can strengthen the expression of mutated p53 gene.

Inhibition of human telomerase in MKN-45 cell line by antisense hTR expression vector induces cell apoptosis and growth arrest

AIM: To investigate the effects of antisense human telomerase RNA (hTR)on the biologic behavior of human gastric cancer cell line: MKN-45 by gene transfection and its potential role in the gene therapy of gastric cancer. METHODS: The hTR cDNA fragment was cloned from MKN-45 through RT-PCR and subcloned into eukaryotic expression vector (pEF6/V5-His-TOPO) in cis-direction or trans-direction by DNA recombinant methods. The constructed sense, antisense and empty vectors were transfected into MKN-45 cell lines separately by lipofectin-mediated DNA transfection technology. After drug selection, the expression of antisense hTR gene in stable transfectants and normal MKN-45 cells was detected by RT-PCR, the telomerase activity by TRAP, the apoptotic features by PI and Hoechst 33258 staining, the cell cycle distribution by flow cytometry and the population doubling time by cell counting. Comparison among the stable transfectants and normal MKN-45 cells was made. RESULTS: The sense, antisense hTR eukaryotic expression vectors and empty vector were successfully constructed and proved to be the same as original design by restriction endonuclease analysis and sequencing. Then, they were successfully transfected into MKN-45 cell lines separately with lipofectin. The expression of antisense hTR gene was only detected in MKN-45 cells stably transfected with antisense hTR vector (named as MKN-45-ahTR) but not in the control cells. In MKN-45-ahTR, the telomerase activity was inhibited by 75%, the apoptotic rate was increased to 25.3%, the percentage of cells in the G0/G1 phase was increased to 65%, the proliferation index was decreased to 35% and the population doubling time was prolonged to 35.3 hours. However, the telomerase activity, the apoptotic rate, the distribution of cell cycle, the proliferation index and the population doubling time were not different among the control cells. CONCLUSION: Antisense hTR can significantly inhibit telomerase activity and proliferation of MKN-45 cells and induce cell apoptosis. Antisense gene therapy based on telomerase inhibition can be a potential therapeutic approach to the treatment of gastric cancer.

Inhibitory effect of Polo-like kinase 1 depletion on mitosis and apoptosis of gastric cancer cells

AIM： Polo-like kinase 1 （PLK1） serine/threonine kinase plays a vital role in multiple phases of mitosis in gastric cancer cells. To investigate the effect of PLK1 depletion on mitosis and apoptosis of gastric cancer cells. METHODS： PLK1 expression was blocked by small RNA interference（siRNA）. The expression levels of PLK1, cdc2, cyclin B and caspase 3 were detected by Western blotting. Then, PLK1 depletion, cdc2 activity, cell proliferation, cell cycle phase distribution, mitotic spindle structure, and the rate of apoptosis of the PLK1 knockdown cells were observed. RESULTS： PLK1 gene knockdown was associated with increased cyclin B expression, increased cdc2 activity （but not with the expression levels）, accumulation of gastric cancer cells at G2/M, improper mitotic spindle formation, delayed chromosome separation and delayed or arrested cytokinesis. Moreover, PLK1 depletion in gastric cancer cells was associated with decreased proliferation, attenuated pro-caspase 3 levels and increased apoptosis. CONCLUSION： Blockage of to decreased mitosis or even PLK1 expression may lead apoptosis in gastric cancer cells, indicating that PLK1 may be a valuable therapeutic target for gastric cancer.

Simvastatin inhibits apoptosis of endothelial cells induced by sepsis through upregulating the expression of Bcl-2 and downregulating Bax

BACKGROUND: Many studies have showed that apoptosis of endothelial cells plays a curial role in the progress of sepsis. But the role of simvastatin in apoptosis of endothelial cells induced by sepsis is not clear. The present study aimed to investigate the role of simvastatin in apoptosis of endothelial cells induced by sepsis and its mechanism.METHODS: Human umbilical vein endothelial cells(HUVECs) were randomly divided into three groups: control group, sepsis serum intervention group(sepsis group) and simvastatin+sepsis serum intervention group(simvastatin group). After 24-hour incubation with corresponding culture medium, the relative growth rate of HUVECS in different groups was detected by MTT assay; the apoptosis of HUVECs was detected by Hoechst33258 assay and fl ow cytometry; and the expression of the Bcl-2 and Bax genes of HUVECs was detected by PCR.RESULTS: Compared with the sepsis group, HUVECs in the simvastatin group had a higher relative growth rate. Apoptotic HUVECs decreased significantly in the simvastatin group in comparison with the sepsis group. Expression of the Bcl-2 gene in HUVECs decreased obviously, but the expression of the Bax gene increased obviously after 24-hour incubation with sepsis serum; however, the expression of the Bcl-2 and Bax genes was just the opposite in the simvastatin group.CONCLUSIONS: Our study suggests that simvastatin can inhibit apoptosis of endothelial cells induced by sepsis through upregulating the expression of Bcl-2 and downregulating Bax. It may be one of the mechanisms for simvastatin to treat sepsis.

Induction of SiHa Cells Apoptosis by Nanometer Realgar Suspension and Its Mechanism

The effects of nanometer realgar uterine cervix cancer cell line SiHa cells and suspension on proliferation and apoptosis of human oncogenic genes HPV16E6/E7 were investigated. A ＂micro-jet effiux＂ strategy was used for the preparation of nanometer realgar suspension. SiHa cells were treated with nanometer Realgar suspension in various concentrations （6.25, 12.5, 25 and 50 mg/L） for different durations （12, 24, 48 and 72 h）. The inhibitive effect of nanometer realgar suspension on growth of SiHa cells was detected by MTT method. Special morphological changes of apoptosis were observed by transmission electron microscopy （TEM） and DNA fragments electrophoresis. The apoptotic rate was quantified by flow cytometry （FCM）. The expression of HPV 16E6/E7 mRNA and protein was assayed by RT-PCR and Western blot respectively. The results showed after being treated with 25--50 mg/L nanometer realgar suspension for 48 h, the survival rate of SiHa cells was decreased, and apoptotic rate markedly increased in a time- and concentration-dependent manner. TEM and DNA electrophoresis revealed the special morphological changes of apoptosis. The apoptotic rate of SiHa cells treated with nanometer realgar suspension was significantly higher than in the control group （P〈0.01）, and G0/G1 phase arrest appeared following treatment with nanometer realgar suspension in 25 and 50 mg/L for 48 h. RT-PCR and Western blot assay indicated that nanometer realgar suspension reduced the HPV16E6/E7 gene expression. Nanometer realgar suspension could inhibit the proliferation and induce apoptosis of SiHa cells. The mechanism may be related to the down-regulation of the HPV16E6/E7 gene expression.

Bcl-2 over-expression and activation of protein kinase C suppress the Trail-induced apoptosis in Jurkat T cells

Trail, a tumor necrosis factor-related apoptosis-inducing ligand, is a novel potent endogenous activator of the cell death pathway through the activation of cell surface death receptors Trail-R1 and Trail-R2. Its role, like FasL in activation-induced cell death (AICD), has been demonstrated in immune system. However the mechanism of Trail induced apoptosis remains unclear. In this report, the recombinant Trail protein was expressed and purified. The apoptosis-inducing activity and the regulation mechanism of recombinant Trail on Jurkat T cells were explored in vitro. Trypan blue exclusion assay demonstrated that the recombinant Trail protein actively killed Jurkat T cells in a dose-dependent manner. Trail-induced apoptosis in Jurkat T cells were remarkably reduced by Bcl-2 over expression in Bcl-2 gene transfected cells. Treatment with PMA (phorbol 12-myristate 13-acetate), a PKC activator, suppressed Trail-induced apoptosis in Jurkat T cells. The inhibition of apoptosis by PMA was abolished by pretreatment with Bis, a PKC inhibitor. Taken together, it was suggested that Bcl-2 over-expression and PMA activated PKC actively down-regulated the Trail-mediated apoptosis in Jurkat T cell.

Dual effects of 8-Br-cAMP on differentiation and apoptosis of human esophageal cancer cell line Eca-109

AIM: To investigate the effects of 8-Br-cAMP on differentiation and apoptosis of human esophageal cancer cell line Eca-109, and the related gene expression.METHODS: The cultured Eca-109 cells were divided into four groups: E1 group (co-cultured with 8-Br-cAMP for 24 h); E2 group (co-cultured with 8-Br-cAMP for 48 h); C1 group (treated without 8-Br-cAMP for 24 h); and C2 group (treated without 8-Br-cAMP for 48 h). The same concentration of cell suspension of each group was dropped separately onto the slides and nitrocellulose membranes (NCM). The biotin-labeled cDNA probes for c-myc, wild-type (wt) p53, bcl-2 and iNOS were prepared for in situ hybridization. The expressions of epidermal growth factor receptor (EGFR), p38 kinase, FAS, FasL and caspase-3 were detected using immunocytochemistry, and the NOS activity and the ratio of differentiated cells/proliferating cells were examined by cytochemistry. Immunocytochemistry, cytochemistry,and in situ hybridization were separately carried out on both slides and NCM specimens for each group. In addition, TUNEL was used to detect the cell apoptosis rate in each group.RESULTS: The apoptotic rate of E2 group was significantly higher compared to E1 group, while there was no difference in the ratio of differentiated cells/ proliferating cells between E1 and E2 groups. The signals of wt p53 and iNOS were markedly stronger, while the signals of c-myc and EGFR were obviously weaker in E1 group than those in C1 group (P＜0.05). Moreover, the signals of wt p53, iNOS, p38 kinase, caspase-3 and NOS activity were significantly stronger, whereas, the signals of bcl-2, c-myc and Fas/FasL were markedly weaker in E2 group than those in C2 group (P＜0.05). CONCLUSION: The differentiation and apoptosis of human esophageal cancer cell Eca-109 can be induced after 24- and 48-h treatment with 8-BrcAMP, respectively. Upregulation of wt p53, iNOS and downregulation of c-myc may be associated with differentiation and apoptosis of Eca-109 cells.Furthermore, upregulation of FasL, p38 kinase and caspase-3 as well as downregulation of bcl-2, and Fas may be involved in the apoptosis of Eca-109 cells.

SUMO-1 Enhancing the p53-induced HepG2 Cell Apoptosis

In order to investigate the effect of small ubiquitin-like modifier-1 (SUMO-1) on the p53-induced HepG2 cell apoptosis, HepG2 cells were transfected by recombinant plasmids as pwtp53, pMDM2 and pSUMO-1 respectively. Western blot was employed to detect the protein expression of the transfected recombinant plasmids and the rate of apoptosis was measured by flow cytometry. The results showed that in cells transfected with pwtp53 and pwtp53+pSUMO-1, the apoptosis rate was (16.79±1.62) % and (18.15±1.36) % respectively, while transfected with pwtp53+pMDM2, the rate was decreased to (5.17±1.23) %. The apoptosis rate was (14.06±1.84) % in the cells transfected with pwtp53+pMDM2+pSUMO-1, significantly higher than that in the cells Transfected with pwtp53+pMDM2 (P<0.01). The apoptosis rates in the cells were all less than 2 % and had no significant difference among the groups. It was suggested that in the HepG2 cells, SUMO-1 can increase the apoptosis induced by wild-type p53 through binding to p53 protein, post-translational modification and inhibiting the p53 degradation by MDM2.

Relationship between Fas/ FasL expression and apoptosis of colon adenocarcinoma cell lines

INTRODUCTIONFas/ FasL system has been identified as a keymediator of apoptosis in tumor cells[1-4]. Theoccurrence and development of neoplasm are closelyrelated to apoptosis[5-7] Most chemotherapeuticdrugs kill cancer cells mainly by inducingapoptosis[8-14].'