Overexpression of bcl-2 protects hepatoma cell line HCC-9204 from ethanol-induced apoptosis

Chinese Medical Journal 2002 115(1): 8-11Overexpression of bcl-2 protects hepatoma cell line HCC-9204 fromethanol-induced apoptosisYANG Lianjun杨连君, SI Xiaohui司晓辉 and WANG Wenliang王文亮Keywordsbcl-2. carcinoma, hepatocellular. transfection gene expressionObjective To investigate the effect of overexpression of bcl-2 on ethanol-induced apoptosis of primaryhepatocellular carcinoma( HCC ) cellsMethods The retrovirus expression vector pDOR-SB containing human bcl-2 CDNA was introduced into ahuman HCC cell line HCC-9204 by liposome-mediated transfection. pDOR-transfected and non-transfectedHCC-9204 cells were used as controls. The expression of Bcl-2 protein by transfected HCC-9204 cells wasdetected by the immunohistochemical method. Then the cells were cloned with the limited dilution methodcontinually until a monoclonal cell strain whose positive rate of Bcl-2 protein was 100%detected by flowcytometry was obtained. The killing rates of cells were detected by Methabenzthiazuron assay aftertreatment of 6%ethanol for 6 h. The extent of apoptosis was analyzed by transferase-mediated dUTPend labeling( TUNEL staining and flow cytometryResults Most of the pDOR-SB-transfected cells demonstrated Bcl-2 positive signals while no signal wasfound in the controls. The positive rate of Bcl-2 protein detected by flow cytometry in the obtainedmonoclonal cell strain, which was named HcC-bcl2, was 100% after the cells had been cloned 3 timescontinually. The killing rate, TUNEL index and the scale of sub-G, apoptotic peak in HCC-bcl2 cells were allsignificantly lower than those in the control cellsConclusion Overexpression of Bcl-2 protein suppresses ethanol-induced apoptosis of the HCC cell lineHcC-9204Chin Med J2002: 115(1): 8-11which are studied extensively at present. I Accumulones University )was introduced into a human HCC cell lineThe bcl-2 gene family is a group of apoptosis-relatedHCC-9204 cells established by our department ) byreports show that there is a high-level expression of bcl-2 in Liposome-mediated cell transfection kit LipofectAMINEmany tumor tissues and the transfection of the bcl-2 gene GibcoBRL, USA ) Three days later, the cells werecan protect a variety of cells from apoptosis inducer entransferred to RPMI-1640 medium containing 500 mg/Ldifferent factors 2-6rimary hepatocellular carcinomG-418( GibcoBRL, USA) and maintained for 2-3 wk.TheHCC )is a very common malignant tumor in China. 78 clones that survived G-418 selection were isolated andThere are specific characteristics in the expression of bcl-2 expanded. The cells cultured on coverslips were subjected toin HCC.9 In the present study a retrovirus expression normal immunohistochemical staining procedures provided byvector containing human bcl-2 cdNa was introduced into a ABC kit( Vector, USA ) Mouse anti-human Bcl-2HCC cell line HCC-9204 by gene transfection tomonoclonal antibody( mAb, Oncogene, USA )was used asregulatory effect of bcl-2 overexpression on ethanol-induced the primary antibody and substitution of PBs for Bcl-2 mAbapoptosis in HCC cellsDepart:YH中国煤化工 Medical University,ximn710032METHODSResearchCNMHGNinth People s HospitalShanghai Second Medical University, Shanghai 200011, ChinaCell transfection, selection and identificationSi XH)The purified pDOR-SB retrovirus expression vector Correspondence to: Dr. Yang Lianjun, Department of Pathology1-2 cdNA provided by Dr. Zhu Feng, Fourth Military Medical University, Xi' an 710032, China( Tel: 029-DepariochemistryForthMilitaryMedical3374541-115.Email:yangsi@sina.comChinese Medical Journal 2002; 11( 1): 8-11was used as negative control. The pDOR-SB-transfectedcells were cloned by limited dilution method continually untilall the cells were Bcl-2 positive. These cells were namedHCC-bcl2 cells. The cloned cells were subjected to indirectimmunofluorescence staining. Mouse anti-human bcl-2 mAbwas used as the primary antibody and FITC-labeled sheepanti-mouse IgG mAb( Oncogene, USA )was used as thesecondary antibody. The substitution of PBS for Bcl-2 mAwas used as negative control. Then, approximately 10 x 10cells were detected by flow cytometry for each group. ThepDOR-transfected vector only and non-transfected HCC9204 cells were used as controlsApoptosis induction and detectionThe pDOR-SB-transfected, pDOR-transfected and nontransfected HCC-9204 cells at log phase were respectivelyultured with RPMI-1640 medium containing 6%0 ethanol for6 hours. The following assays were performedO Methabenzthiazuron assay was carried out according tonormal procedures and 10 wells were used for each group ofcells. Absorbance(a) was measured at 490 nm withvme-linked immunosorbent assay detector. Then thekilling rates were calculated according to the followingformula: Killing rate %)=( 1-A4go-test/A49o-controlX 100%0. Finally the data was statistically analyzed byStudents t test; @2 Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling( Tunel )staining wasperformed according to the protocol of the TUNEL kit1000Boehringer Mannheim Germany ) substitution of PBS forFL1TUNEL staining solution was used as negative control. Theells were then observed under fluorescence microscopeThree hundred cells were counted, and the TUNel indexwas expressed as the number of positive cells/the totalnumber of cells. The data was statistically analyzed with the1. Expression of Bcl-2 protein detected by flow cytometryx test; B DNA content of the cells was measured with flowafter the cells had been cloned for 3 times. non-transfected HCC-ometry after they were stained with9204 cells (A) pDOR-SB-transfected cells( B ) FLl: intensitynce:B: measure scale of the positive ratesRESULTSApoptosis induction and detectionMethabenzthiazuron assay demonstrated that the killing rateof HCC-bcl2 cells(0. 519+0.053 ) was significantly lowerCell transfection, selection and identificationthan those of the pDOR-transfected cells(0. 338+0.052)Immunohistochemical staining showed that most HCC-bcl2and non-transfected cells(0. 366+0.046)after 6 h of 6%cells displayed brown Bcl-2 positive signals within ther Pcnns The TUNEL index of HCCtoplasts. No Bcl-2 staining was found in pDORthanoltransfected, non-transfected HCC-9204, or the negative bcl2 cell中国煤化工lower than those of thecontrol. After the cells had been cloned for 3 timespDOR-traCNMH Gd non-transfected cellscontinually the positive rate of bcl-2 expression detected by(0.613, P<0.01 ) No positive signal was found in theflow cytometry in the pDOR-SB-transfected cells was 100%, negative control. The scale of the sub-Gi peak in the HCCcompared with 1.4% in non-transfected HCC-9204 cells bcl2 cells was evidently lower than that in the pDORFg.1)transfected and non-transfected HCC-9204 cells( Fig. 2)Chinese Medical Journal 2002 115(1): 8-11Nevertheless, most studies have demonstrated that HCCtissues do notpositive rate of Bel2 protein. Moreover sometimes the positive rate of Bcl-2 inHCC tissues was lower than that in the non-tumor livertissues immediately adjacent to HCC tissues. 9Themechanism of this phenomenon is still unclear. There maybe specific characteristics of the regulation of Bcl-2 in HCCSome other studies have shown that transfection of bcl-2can suppress apoptosis in HCC cells induced by anti-FamAb or TGF-B. b The hepatocytes of the bcl-2 geneknockout mice have a tendency to undergo apoptosis becauseof their decreased anti-oxygenation abilitAll these326496128160192224256observations indicate that bcl-2 can suppress apoptosis inHCC cells induced by certain factors. There have been fewhepatocytes or HCC cells from apoptosis induced by otherThe transfected HCC-9204 cell strain which overexpressesBcl-2 protein was established successfully in this study. Itprovided a basis for better understanding the regulatory effectof Bcl-2 on HCC cell apoptosis induced by different factorsIt is suggested that low-concentrated ethanol is able toinduce many cell types including hepatocytes and HCCcells, to undergo apoptosis. 3 Previous studies havedemonstrated that 6% ethanol for 6 h is able to induceNDA contentobvious apoptosis in HCC-9204 cells. 4 The results ofMethabenzthiazTUNEL and dNa content assays in thisexperiment showed that overexpression of Bcl-2 protein canobviously suppress 6% ethanol-induced apoptosis in HCCFig. 2. The DNA content of the cells treated with 6% ethanol9204 cells. The present study suggested an involvement offor 6 h, detected by flow cytometry. non-transfected HCC-9204bcl-2 in protecting HCC cells from ethanol-inducedcells(A ) pDOR-SB-transfected cells(BapoptosiDe la Coste et al show that transfection of bcl-2 geneDISCUSSIONcannot suppress apoptosis in hepatocytes but transfection ofthe bel-xL gene a homologous analog of bcl-2, can protectStudying the regulation of apoptosis-related geneshepatocytes from apoptosis under certain conditions. Thisimportant to reveal the physiological and pathological indicates that though sometimes the bcl-2 gene does not takeprocesses of embryogenesis oncogenesis and immunologicalapoptotic regulation of hepatocytes or HCC cellstolerance10 bcl-2 is an anti-apoptosis gene that is the bel-2 gene family may still play a major role in apoptosisoriginally found in follicle B lymphoma. The Bcl-2 protein is in hepatocytes and HCC cells. Bcl-2 participates in thelocated in the membranes of mitochondria or endoplasmic control of apoptosis by forming dimers with Bax which is areticulum, or surrounds nuclearproapoptotic gene in the bcl-2 gene family. It is suggestedbcl-2 gene can protect a variety of cell types from apoptosis that when the expression of bcl-2 is stronger, Bcl-2 and Baxinduced by many factors, such as chemotherapeutics, form hetasis, whereas Bax formsp53 genes and lead to prolonged survival time of cells 24-6 homodimradiation rdeficiency of growth factors c-myc and中国煤化工the expression of baxCNMH Gt of Bcl-2 and Bax inHowever, it has been shown that transfection of the bcl-2in hepatocytes and HCC cells needgene cannot suppress apoptosis in some cells suggesting further investigationthe different regulatory effects of bcl-2 on apoptosis indifferent cellThe expression of Bcl-2 protein in mostREFERENCESunor tssger than that in the tissues of origin. 3 1. Chen Y, Zhou J, Yue B, et al. 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