| Cell apoptosis plays an importment role not only in carcinogenesis but also inchemotherapy and recurrence of tumor. It is the key of chemotherapy resistance andmetastasis of tumor that cancer cells repress apoptosis. The cell-intrinsic pathwaymediated by mitochondria triggers apoptosis is a main signaling pathway initiate theapoptotic program in mammalian cells. Proteins correlation with apoptosis such ascytochrome c, AIF(apoptosis inducing factor, AIF), EndoG, Bitl (Bcl-2 inhibitor oftranscription) and so on were released by mitochondria in response to apoptoticstimuli, for example, DNA damage, cell cycle arrest, detachment from theextracellular matrix(ECM), hypoxia and other types of severe cell distress. Theapoptosis mediated by AIF or Bitl is a caspase-independent cell death.AIF, localization on mitochondria, is a conservative flavin protein in evolvementand also a new apoptosis inducing protein. It can induce cell apoptosis and has anoxidoreductase function. Like AIF, Bitl also locates on mitochondria. BindingAES(amino-terminal enhancer of split, AES)in cytosol, it can mediate anoikisinduced by losting attachment from ECM. The nomal physiological function isunclear. Up to now, Only fewness reference reported the relationship between AIF orBitl and tumorgenesis. As for the relationship between AIF or Bitl and esophagealcancer, no report was seen.p63, a new member of the p53 family, has two promoters, which result in twodifferent types of protein products with opposite functions: the full-lenghtranscriptionally active TAp63 and the dominant-negativeâ–³Np63. Furthermore,TAp63 andâ–³Np63 can vary at its carboxyterminal end, as a result of alternativesplicing at the 3' portion of the mRNA, which yields isoforms such asα,βandγ,respectively. Some researchers indicated that elevated expression of p63 might playan important role on carcinogenesis of squamous cell carcinomas. Further study showed that the major isotype of p63 expressed in tumors of the esophagus wasâ–³Np63 not TAp63 and also indicated that elevated expression ofâ–³Np63 wasprobably an early event in esophageal squamous cell carcinoma, which played asignificant role in the development of the disease. TAp63 isoforms contain regions ofhomology to p53 and shares 60ï¼…sequence identity with the DNA-binding domain ofp53 so they can cause target genes of p53 to express such as p21, Bax and MDM2.TAp63γ, shows strong capability in this aspect and is able to induce cell cycle arrestand apoptosis. However, the mechanism is not clear. Accordingly, the expression ofAIF and Bitl in esophageal cancer as well as their roles on TAp63γ-induced cellapoptosis will be studied. It is helpful for us to understand the roles of AIF and Bitl inesophageal tumorgenesis and basal mechanism of TAp63γ-induced cell death.Meanwhile, this finding will provide experimental bases for the opinion that AIF andBitl will be used to kill cancer cell.Based on these reviews, therefore, this study are going to be performed as follows:1. To test expression of AIF or Bitl in esophageal cancer, dysplasia and nomalesophageal tissue by Immunohistochemistry. 2. To detect the expression of AIF, Bitland p63 isoforms in EC9706 and Ecal09, esophageal squamous cancer cell (ESCC)lines, with general molecular biology technologies. 3. To reseach effect on cell cycledistribution and apoptosis in EC9706 cells after TAp63γoverexpression witheucaryote transfection technology and detection technologies of cell apoptosis. 4. Tostudy the roles of AIF and Bitl in TAp63γ-induced cell death with molecular biologytechnologies such as laser confocal microscope, subcellular isolation, RNAinterference etc.Four parts are included in this study.Partâ… Study on expression of AIF and Bitl in esophageal squamous cell carcinoma, dysplasia and normal mucosaMethods1 Expressions of AIF and Bit1 in 49 cases of biopsy specimens including 19 casesof early esophageal cancer, 20 cases of dysplasia and 10 cases of normal mucosawere tested by Immunohistochemistry.2 Expressions of AIF and Bitl in 35 cases of surgery specimens including 35 casesof middle or advanced esophageal cancer and correspondence normal esophgeal mucosa were tested by Immunohistochemistry.3 Statistical analysis: All the data were analyzed by SPSS 13.0 program. The countinformation was calculated as the positive rate and tested by Chi-square. The levelof significant difference wasα=0.05.Results1 The overexpression rates of AIF were 84.21ï¼…(16/19),75ï¼…(15/20) and 40ï¼…(4/10 )in early esophageal cancer, dysplasia and normal, respectively. Significantdifference of AIF overexpression was found between early esophageal cancer andnormal (P＜0.05), however, no significant difference of AIF overexpression werefound between dysplasia and early esophageal cancer or normal(P＞0.05).2 The positive expression rates of Bitl were78.94ï¼…(15/19), 80ï¼…(16/20) and 40ï¼…(4/10)in early esophageal cancer, dysplasia and normal, respectively. Significantdifference of Bit1 positive expression was found between dysplasia and normal(P＜0.05), however, no significant difference of Bitl positive expression werefound between early esophageal cancer and dysplasia or normal (P＞0.05).3 The overexpression rates of AIF were 80ï¼…(28/35) and 45.71ï¼…(16/35)in middleor advanced esophageal cancer and normal esophageal mucosa, respectively.There were significant differences of AIF overexpression between middle oradvanced esophageal cancer and normal (P＜0.05). In esophageal cancers, theoverexpression rates of AIF were not correlated with clinical stage and gender.(P＞0.05)4 The positive expression rates of Bitl were 37.14ï¼…(13/35) and 40ï¼…(14/35) inmiddle or advanced esophageal cancer and normal esophageal mucosa,respectively. No significant difference of Bitl positive expression were foundbetween middle or advanced esophageal cancer and normal (P＞0.05). Inesophageal cancers, the positive expression of Bitl were correlated with clinicalstage (P＜0.05), but not with gender(P＞0.05). The positive expression of Bitl inearly esophageal cancer was higher than that in middle or advanced esophagealcancer(P＜0.05). Partâ…¡Expression of AIF and Bitl as well as expression isoform of p63 in esophageal squamous cancer cell linesMethods1 The expression of Bitl, AES, ANp63 and TAp63 in mRNA level wererespectively investigated in EC9706 and Eca109 cells with reversetranscription-polymerase chain reaction (RT-PCR).2 The expression of AIF and Bitl with protein level in the two ESCC cell lines weretested with immunocytochemistry and Western Blotting.Results1 Both Bitl and AES were expressed at mRNA level disclosed with the RT-PCR andit verified the sub-type of p63 in these two ESCC cells isâ–³Np63, but did not reveal any TAp63 expression in these cells.2 Immunocytochemically, positive protein expressions of AIF and Bitl wereobserved in the cytoplasm of the two ESCC cells. Compared toβ-actin (insidecontrol) and HeLa229(positive control) , expression of AIF protein with highlevel and Bitl protein with middle level were respectively detected by WesternBlotting method in these cells.Partâ…¢Effect on cell cycle distribution and apoptosis in EC9706 cells after TAp63γoverexpressionMethods1 EC9706 were transfected respectively with pcDNA3.1-TAp63γandpcDNA3.1-GFE a control plasmid, to monitor the transfection efficiency ofpcDNA3.1 in EC9706.2. After transfected with pcDNA3.1-TAp63γ, pcDNA3.1/Myc-His-TAp63γandpcDNA3.1 24h, EC9706 cells were collected and total RNA were isolated tomeasure the expression of TAp63γ, by RT-PCR.3 After transfected with pcDNA3.1/Myc-His-TAp63γ, pcDNA3.1-p53 andpcDNA3.1 24h, EC9706 cells were collected and total proteins were isolated.Western Blotting analyzed that expression of TAp63γwith protein level was tested with anti-histidine tag antibody after transfection. Expression of p53 proteinbefore and after transfection were also tested with anti-p53 antibody by westernBlotting method.4 After transfected with pcDNA3.1 -TAp63γand pcDNA3.1 24h and 48h, EC9706cells were analyzed with flow cytometry to detect distribution of cell cycle.5 To detect apoptosis of EC906 cells transfected respectively with pcDNA3.1,pcDNA3.1-TAp63γand pcDNA3.1-p53 24h with isolation of DNA fragmentmethod.6 To detect apoptosis of EC906 cells transfected respectively with pcDNA3.1,pcDNA3.1-TAp63γand pcDNA3.1-p53 24h with TUNEL method.7 To detect apoptosis rates of EC906 cells transfected respectively with pcDNA3.1and pcDNA3.1-TAp63γ24h using flow cytometry.Results1 pcDNA3.1-GFP was used to monitor efficiency in EC9706, demonstrating nearly90ï¼…transfection efficiency of pcDNA3.1 in EC9706.2 The expressions of TAp63γmRNA were detected in pcDNA3.1-TAp63γtransfection group and pcDNA3.1/Myc-His-TAp63γtransfection group but not ineither pcDNA3.1 transfection group or untreated group. It showed that TAp63γdidn't express in the ESCC cells. Compared to GAPDH, however, TAp63γmRNAexpressed with high level in the ESCC cells after transfected withpcDNA3.1-TAp63γor pcDNA3.1/Myc-His-TAp63γ24h.3 Before and after pcDNA3.1/Myc-His-TAp63γor pcDNA3.1-p53 transfection, aWestern blot with protein levels of TAp63γor p53 was performed. The resultsshowed that cells expressed highly TAp63γand p53 in protein levels aftertransfection, however, they didn't express any TAp63γor expressed p53 in a lowerlevel before transfection4 EC9706 cells transfected with pcDNA3.1-TAp63γ24h, compared to EC9706 cellstransfected with pcDNA3.1 24h, showed an increase in the percentage of cells inthe G0/G1 phase and a decrease in the percentage of cells in the S phase(P＜0.05).While EC9706 cells transfected with pcDNA3.1-TAp63γ48h, the results were more prominence(P＜0.05).5 Agarose gel electrophoresis revealed a DNA ladder, a typical feature of apoptosisin both the TAp63γ-transfected cells and the p53-transfected cells, but not in thepcDNA3.1-transfected cells.6 The number of apoptotic cells in pcDNA3.1 -TAp63γtransfection group increasedmarkedly compared to that in pcDNA3.1 transfection group (P＜0.05). It revealedthat TAp63γexpression could induce EC9706 cell apoptosis.7 The percentages of annexin v-positive cells were 1.37ï¼…in the negative controland 13.64ï¼…in the TAp63γ-transfected cells. Later stage apoptosis, revealed byboth annexinv and propidium iodide (PI) uptake (upper right quardrants), wasalso greater in the TAp63γ-transfected cells compared to that in the negativecontrol (9.63ï¼…versus 4.49ï¼…). Three independent experiments showed thatsignificant difference of the apoptosis percentage between in theTAp63γ-transfected cells and in the pcDNA3.1-transfected cells (P＜0.05),indicating that expression of TAp63γinduces apoptosis in EC9706 cells.Partâ…£Study on the roles of AIF and Bitl in TAp63γ-indueed cell apoptosisChapter 1 Study on translocations of AIF and Bitl in TAp63γ-induced cell apoptosisMethods1 Active form of caspase-3 was tested during TAp63γ-induced apoptosis usingWestern blotting method. Afetr Z-VAD-fmk treatment, EC9706 cells weretransfected with pcDNA3.1-TAp63γor pcDNA3.1, then apoptosis rates of themwere detected with flow cytometry.2 For the detection of endogeous AIF, the ceils were cotransfected withpcDNA3.1-TAp63γand pDsRed2-Mito and laser confocal microscopy andimmunofluorescence analysis were performed. For the detection of extrinsic-Bitlprotein, the cells were cotransfected with pEGFPN2-Bitl and pDsRed2-Mito. 24hlater, they were transfected with TAp63γ48h after cotransfected, the cells wereanalyzed under the laser confocal microscopy. 3 24h after transfected with pcDNA3.1, pcDNA3.1-TAp63γor pcDNA3.1-p53,respectively, the EC9706 cells were harvested. The nucleus, mitochondrial andcytosol fractions were extracted for Western blot assay to examine the subcellularlocation of AIF and Bitl.Results1 Active form of caspase-3 was found only in the TAp63γ-tranfected cells andinhibition activity of caspases could reduce TAp63γ-induced apoptosis,conforming that caspases were actived during TAp63γ-induced apoptosis and itdepended on caspase.2 In control transfection cells, green fluorescence (AIF/Bitl) and red fluorescence(mitochondria) were in complete superposition ,that is, the distribution ofAIF/Bitl was coincident with that of mitochondria. In TAp63γ-overexpress cells,however, green fluorescence and red fluorescence were not in completesuperposition, that is, AIF/Bitl distributed not only in mitochondria but also incytosol even in nuclei. It indicated that endogeous AIF was localized inmitochondria, released firstly into the cytoplasm and then nuclei in the cellsundergoing apoptosis after transfected with the pcDNA3.1-TAp63γ. It was alsoimplied that Bitl was located in the mitochondria, released from them later, andtranslocated only into the cytosol during the stage of TAp63γ-induced cellapoptosis. The negative control transfection with pcDNA3.1 did not show such atranslocation.3 AIF in both nucleus and cytosol proteins, interestingly, could be detected in thecells transfected with TAp63γor p53 expression vector, but not in the cellstransfected with pcDNA3.1 control. However, Bitl protein could only be seen inthe cytosol, but not in the nuclei of the ceils transfected with TAp63γor p53expression vector. These indicated that AIF translocated from mitochondria tocytosol and nuclei, while Bitl translocated from mitochondria to cytosol duringthe process of TAp63γ-induced cell apoptosis..Chapter 2 Constructing and identifying plasmid vector expressing small hairpin target RNAMethods1 According to reference, DNA templets of SiRNA were designed and synthesized.2 Construction ofplasmid vectors expressing SiRNA specifical target to AIF or Bitl using molecular biology technologies.3 Identifying recombine plasmids by digesting with BamHâ… +Hindâ…¢and sequencing.Results1 Recombine plasmids were digested by BamHâ… +Hindâ…¢and two specificalfragment were obtained on the site of 4224bp and 64bp, respectively, indicatingthat the target fragments had been ligated to vector pSilencer3.1-H1-neo.2 Recombine plasmids were sequenced by using Sanger method through M13 primers. Results showed that sequences of them were right.Chapter 3 Effect of down-regulation of AIF or that of Bitl proteins by RNA interference on TAp63γ-induced cell deathMethods1 pSilencer3.1-Hl-neo containing the siRNA templates were transfected intoEC9706 cells and the cells expressing stably siRNA were obtained after G418selection.2 Changes of expression with protein level of AIF and Bitl in the experimental andcontrol groups ofEC9706 cells were analyzed with Western blot.3 After RNA interference to AIF or Bitl, cell viability was detected by CCK-8.4 Effect on TAp63γ-induced apoptosis after RNA interference to AIF or Bitl withTUNEL method.5 Effect on the rates of TAp63γ-induced apoptosis after RNA interference.to AIF orBitl using flow cytometry.Results1 AIF and Bitl protein levels were markedly reduced in the cells transfected withcorrespondence recombinant pSilencer(P＜0.05), while they remained unchangedin the cells transfected with negative plasmid(P＞0.05), implying that AIF-siRNAand Bitl-siRNA down-regulated AIF and Bitl protein expression, respectively. 2 The A value had no significant different between cells transfected withAIF-siRNA or Bitl-siRNA and untreated cells at 24, 48 and 72h (P＞0.05),indicating that there wrer no effect on proliferations of cells down-regulation ofboth AIF and Bitl.3 The numbers of apoptotic cells in both pcDNA3.1-TAp63γtransfection group andAIF-siRNA treatment and pcDNA3.1-TAp63γtransfection group reducedmarkedly compared to that in pcDNA3.1-TAp63γtransfection group (P＜0.05),while it increased markedly compared to that in pcDNA3.1 transfection group(P＜0.05). It implied that down-regulation of AIF or Bit l could alleviate butnot completely inhibit TAp63γ-induced apoptosis.4 The percentages of annexin v-positive cells were 4.52ï¼…and 5.72ï¼…inpcDNA3.1-TAp63γtransfection group and AIF-siRNA treatment andpcDNA3.1-TAp63γtransfection group, respectively. Both of them reducedmarkedly compared to that in pcDNA3.1-TAp63γtransfection group (P＜0.05),while it increased markedly compared to that in pcDNA3.1 transfection group(P＜0.05), indicating that down-regulation of AIF or Bitl could alleviate but notcompletely inhibit TAp63γ-induced apoptosis. Furthermore, It also revealedTAp63γ-induced apoptosis depended on both AIF and Bitl.Conclusions1 AIF overexpression may play a role in esophageal tumorigenesis. AIFoverexpression are not correlated with clinical stage and gender.2 Bitl expression may be a early event in esophageal tumorigenesis and correlatedwith metastasis of cancer cell.3 There are both AIF expression with high level and Bitl expression with middlelevel in EC9706and Ecal09 cells, ESCC cell lines. The sub-type of p63 in thesetwo cells isâ–³Np63 but not TAp63. TAp63γexpression can inhibit theproliferation of EC9706 cell and induce cell apoptosis.4 TAp63γ-induced apoptosis are correlated with caspase. Inhibition activity ofcaspase can alleviate but not completely inhibit TAp63γ-induced apoptosis. 5 The plasmid vectors expressing siRNA specifical target to AIF or Bit1 have beensuccessfully constructed.6 AIF translocats from mitochondria to cytosol and nuclei, while Bit1 translocatsfrom mitochondria to cytosol during the process of TAp63γ-induced cell apoptosis.Down-regulation of AIF or Bit1 by RNA interference can alleviate but notcompletely inhibit TAp63γ-induced apoptosis.7 TAp63γ-induced apoptosis depends on caspase, AIF and Bit1.
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