BAK基因过表达对胃癌细胞的诱导凋亡作用及其分子机制
郑丽端,华中科技大学同济医学院附属协和医院病理科 湖北省武汉市 430022
童强松,汪良,华中科技大学同济医学院附属协和医院外科 湖北省武汉市 430022
刘俊,钱伟, 华中科技大学同济医学院附属协和医院消化内科 湖北省武汉市 430022
郑丽端,女, 1973-10-11生,福建省南安市人,汉族. 1997年同济医科大学本科毕业,2001级华中科技大学同济医学院在读硕士研究生,主治医师. 主要从事消化系统疾病的分子病理研究.
项目负责人:童强松,430022, 湖北省武汉市,华中科技大学同济医学院附属协和医院外科. qs_tong@hotmail.com
电话:027-85726129
收稿日期:2003-11-22 接受日期:2003-12-22
Effects of BAK gene over-expression on apoptosis in gastric cancercells and its molecular mechanisms
Li-Duan Zheng, Qiang-Song Tong, Jun Liu, Liang Wang, Wei Qian
Li-Duan Zheng, Department of Pathology, Union Hospital of Tongji MedicalCollege, Huazhong University of Science and Technology, Wuhan 430022,Hubei Province, China
Qiang-Song Tong, Liang Wang, Department of Surgery, Union Hospital ofTongji Medical College, Huazhong University of Science and Technology,Wuhan 430022, Hubei Province, China
Jun Liu, Wei Qian, Department of Gastroenterology, Union Hospital ofTongji Medical College, Huazhong University of Science and Technology,Wuhan 430022, Hubei Province, China
Correspondence to: Dr. Qiang-Song Tong, Department of Surgery,Union Hospital of Tongji Medical College, Huazhong University of Scienceand Technology, Wuhan 430022, Hubei Province, China. qs_tong@hotmail.com
Received: 2003-11-22 Accepted:2003-12-22
AbstractAIM: To explore the apoptosis-inducing effects of extrinsic BAK genetransfer and its over-expression on gastric cancer cells and its molecularmechanisms.
METHODS: The eukaryotic expression for BAK gene was constructed andtransferred into gastric cancer MKN-45 cell line. After being transferredfor 1 to 5 days, cellular BAK gene expression was detected by RT-PCR andWestern blotting methods. The growth activities of cancer cells weredetected by cell count and MTT colorimetry. Cell cycle changes wereassayed by flow cytometry. Cellular apoptosis was assayed by electronicmicroscopy and in situ terminally labeled transferase technique (TUNEL).Cellular caspase-3 activities were observed by colorimetric method.
RESULTS: After being transferred for 1 to 5 days, cellular BAK mRNAand protein expression levels were significantly increased (P <0.01).In vitro growth of gastric cancer cells was inhibited by 11.6-35.3% (P<0.01). The cellular proliferation activities were decreased by10.2-32.4% (P <0.01), with cell cycle being blocked at G0/G1phase. Partial cancer cells presented the characteristic morphologicalchanges of apoptosis, with the apoptotic rates being 21.4% (P <0.01).The cellular caspase-3 activities were enhanced by 4.45 times (P <0.01).
CONCLUSION: Transfection of extrinsic BAK gene, resulting in itsover-expression, can significantly induce apoptosis of gastric cancerMKN-45 cells through activating caspase-3, which is a potential strategyfor gene therapy of gastric cancer.
Zheng LD, Tong QS, Liu J, Wang L, Qian W. Effects of BAK geneover-expression on apoptosis in gastric cancer cells and its molecularmechanisms. Shijie Huaren Xiaohua Zazhi 2004;12(5):1025-1029
摘要
目的:探讨转染外源性BAK基因及其过表达对胃癌细胞的诱导凋亡作用和分子机制.
方法:构建携带有BAK基因的真核表达载体并转染胃癌MKN-45细胞1-5d后，RT-PCR和WesternBlotting法检测BAK基因表达;细胞计数、MTT比色法检测癌细胞生长活性，流式细胞仪分析细胞周期时相改变，透射电镜、末端TdT酶标记技术检测癌细胞凋亡;比色法检测癌细胞内Caspase-3活性改变.
结果:转染1-5 d后，癌细胞BAKmRNA和蛋白表达水平显著增强(P<0.01)，癌细胞体外生长抑制11.6-35.3%(P <0.01)，增生活性抑制10.2-32.4%(P <0.01)，细胞周期出现G0/G1期阻滞，部分癌细胞出现胞体缩小、核固缩等凋亡形态学改变，凋亡率为21.4%(P<0.01); 癌细胞Caspase-3活性增强4.45倍(P<0.01).
结论:转染外源性BAK基因使其过表达能激活Caspase-3，显著诱导胃癌MKN-45细胞凋亡，有望成为胃癌治疗的新途径.
郑丽端,童强松, 刘俊,汪良, 钱伟.BAK基因过表达对胃癌细胞的诱导凋亡作用及其分子机制.世界华人消化杂志 2004;12(5):1025-1029
0 引言胃癌是我国常见的恶性肿瘤，在不同地区约居癌症发病数的第1-3位，且近年来发病率有上升趋势，但其晚期治疗效果仍不理想[1-7].利用基因转导技术促进肿瘤细胞凋亡，成为目前胃癌治疗研究的热点之一[8-14].BAK (Bcl-2 homologus antagonist/ killer) 基因是近年来发现的一种促凋亡基因，应用基因转导的手段将其导入肿瘤细胞已成功抑制了肺癌、乳腺癌等多种肿瘤的生长[15].我们通过构建携带BAKcDNA的真核表达载体，观察了BAK基因过表达对胃癌细胞的诱导凋亡作用及其分子机制，旨在寻求胃癌治疗的新途径.
1 材料和方法
1.1 材料 质粒pET-BAK由美国冷泉港分子生物学实验室PattyWendel博士惠赠，在其限制性酶切位点XhoI和Hind III间含有BAK基因cDNA(633 bp). 真核表达载体pcDNA3由本校生化教研室屈伸教授惠赠.限制性内切酶和DNA快速回收纯化试剂盒购自Promega公司，T4DNA连接酶购自Takara公司.碘化丙锭购自Sigma公司.原位末端转移酶标记(TUNEL)试剂盒购自武汉博士德公司，Caspase-3活性检测试剂盒购自Clontech公司.BAK基因真核表达载体的构建参见(SambrookJ，Russell DW.Molecular Cloning: A Laboratory Manual. 3rd Ed. Cold Spring HarborLaboratory Press 1998; 254-325). 限制性内切酶XhoI、Hind III双切质粒pET-BAK和pcDNA3，分别回收0.63kb的基因片段和5.4kb的线性化载体片段，T4DNA酶进行连接反应，将重组体命名为pcDNA-BAK，酶切鉴定.
1.2 方法 人胃癌细胞株MKN-45由本院中心实验室董继华教授惠赠.用含100 mL/L胎牛血清、100kU/L青霉素和100mg/L链霉素的RPMI1640培养基(Gibco )，在37°C，50mL/L CO2条件下培养.设置未转染对照组、pcDNA3转染组、pcDNA-BAK转染组，基因转染参照脂质体lipofectamine2000 (Gibco)说明书进行.BAK mRNA表达水平检测采用RT-PCR法.细胞总RNA的提取参照Trizal试剂盒(LifeTechnologies公司)说明书进行.逆转录反应后，加入下列引物进行PCR扩增:BAK (502 bp)上游 5’-CTGCCCTCTGCTTCTGA-3’，下游5’-CGTTCAGGATGGGACCA-3’; 同时以a-tubulin(295 bp) 上游 5’-CCCGTCTTCAGGGTCTCTTG-3’，下游5’-TTAAGGTAAGTGTAGGTTGGG-3’ 作为反应内参照.扩增产物经10g/L琼脂糖凝胶电泳分离，紫外灯下观察并拍照.凝胶图像分析仪(GelDoc 1000型，Bio-Rad公司)检测BAK与a-tubulin扩增片段灰度的比值.BAK蛋白表达水平检测采用WesternBlotting法，参照(SambrookJ，Russell DW.Molecular Cloning: A Laboratory Manual. 3rd Ed. Cold Spring HarborLaboratory Press 1998; 254-325)进行蛋白质提取、定量和分离.转膜后先后与10g/L 脱脂奶粉、鼠抗人BAKmAb (武汉博士德公司)、过氧化物酶偶联的山羊抗鼠IgG孵育，运用ECLWestern blotting kit显色.图像分析系统测定BAK蛋白条带密度.
1.2.1 细胞生长特征 接种2×105/LMKN-45细胞0.5mL入24孔培养板，每组5个复孔，转染后用RPMI1640完全培养基在37°C，50mL/LCO2条件下培养，每24h用1.25 g/L胰酶+0.1g/L EDTA消化细胞，台酚兰染色，于倒置显微镜下计数活细胞，每次计数重复3次，连续测定5d，绘制细胞生长曲线. 细胞增生活性检测采用MTT比色法.转染1、3、5d后，加入5g/L MTT (Clontech公司)20 mL/孔，继续培养4h，弃去上清，加DMSO100 mL/孔，振荡至结晶溶解，酶标仪570nm 波长处测定吸光度A570nm值.细胞增生抑制率(%)=(1-实验组平均吸光度A570nm值/对照组平均吸光度A570nm值)×100%. 细胞周期时相检测采用流式细胞仪法.上述三组癌细胞培养至3d，分别收集2×106细胞，PBS洗涤2次，700mL/L乙醇固定过夜，4°C保存，检测前用PBS洗涤，加入RNase(1 g/L) 200 mL，37°C水浴30min，用碘化丙锭(500mg/L) 800 mL进行染色，室温避光30min，流式细胞仪(BectonDickson公司)进行DNA含量分析，所用软件为CellQest.
1.2.2 细胞形态学检测 收集上述三组癌细胞，PBS洗涤1次，25mL/L戊二醛固定30min，PBS洗涤并悬浮细胞，常规包埋、切片，透射电镜观察并摄影.采用末端TdT酶标记技术(TUNEL)检测转染3d后各处理组的细胞凋亡.3 mL/L H2O2封闭30min，20 mg/L蛋白酶K消化液作用20min，TUNEL反应液37°C孵育60min，滴加过氧化物酶连接的抗体，37°C孵育30min，DAB显色，苏木素复染.以PBS替代TUNEL反应液作阴性对照.在光学显微镜下，凋亡细胞体积缩小，核固缩，染色质呈特异的棕黄色.细胞凋亡率(%)=(500个细胞中凋亡细胞数/500)×100%.
1.2.3 Caspase-3活性测定 分别收集2×105各处理组细胞，加入细胞裂解缓冲液50mL，冰上孵育10min; 12 000 r/min，4°C离心细胞裂解液3min，回收上清，每管依次50mL的2×反应缓冲液，1mmoL/L Caspase-3底物DEVD-pNA5 mL，37°C孵育1h，转移至96孔板中，用酶标仪测定波长405nm的吸光度值(A405nm)，表示Caspase-3的相对活性.
统计学处理 采用SPSS统计学软件进行数据分析.
2 结果将BAK基因片段正向插入到载体pcDNA3中，得到携带BAK基因的真核表达载体pcDNA-BAK(6.0 kb). 根据基因和载体的物理图谱，选取XhoI、Hind III单酶切和XhoI和Hind III双酶切该重组体，酶切产物经10g/L琼脂糖凝胶电泳，证实BAKcDNA已插入重组体pcDNA-BAK中(图1).RT-PCR产物电泳后，未转染对照组和空载体pcDNA3转染组均可见BAK(502 bp)和a-tubulin(295 bp) 条带，二者BAK/a-tubulin灰度比值分别为0.81，083，统计学分析差异无显著性意义(P>0.05). 外源性BAK基因转染3d后，BAK/ a-tubulin灰度比值为2.98，即BAKmRNA表达较未转染对照组增强3.67倍(P<0.01，图2).未转染对照组、pcDNA3转染组、pcDNA-BAK转染组细胞经WesternBlotting检测均可见Mr28 000的蛋白条带.经图像分析系统证实，pcDNA-BAK转染组的BAK蛋白条带灰度值为对照组、pcDNA3转染组的3.1倍(P<0.01)和3.2倍(P<0.01); 未转染对照组、pcDNA3转染组细胞BAK蛋白条带灰度值之间无显著性差异(P>0.05，图3).
图1(PDF)真核表达载体 pcDNA-BAK的酶切鉴定分析.1: pcDNA-BAK; 2: pcDNA-BAK / Xho I; 3: pcDNA-BAK / Hind III ; 4: pcDNA-BAK/Xho I+Hind III ; M: l DNA / Hind III + EcoR I.
图2(PDF)RT-PCR 检测各组癌细胞BAKmRNA表达水平.1: 未转染对照组;2: 空载体转染组;3: BAK转染组 M:PCR marker (100 bp, 200 bp,300 bp, 400 bp, 500 bp, 600 bp, 700 bp, 800 bp,900 bp, 1 000 bp).
图3(PDF)Western Blotting检测各组癌细胞BAK蛋白表达水平.1: BAK转染组;2: 空载体转染组;3: 未转染对照组.
2.1 细胞生长特征 未转染对照组MKN-45细胞体外生长活跃，经pcDNA-BAK转染1-5d后，癌细胞体外生长减慢，抑制率为11.6-35.3%(P<0.01); 空载体pcDNA3转染组癌细胞体外生长活性与未转染对照组比较差异无显著性意义(P>0.05，图4).细胞增生活性检测未转染对照组A570nm值随时间推移而递增，反映癌细胞体外增生活跃.pcDNA-BAK转染1-5d后，MKN-45细胞增生活性均不同程度地减弱，抑制率为10.2-32.4%(P<0.01). pcDNA3转染组与未转染对照组A570nm值之间的差异无显著性意义(P>0.05，图5).在DNA直方图上，pcDNA-BAK转染组癌细胞S期比例(36.6%)显著低于非转染对照组(55.1%)，细胞生长受阻于G0/G1期(比率分别为32.2%、12.5%).pcDNA3转染组与未转染对照组比较细胞周期时相分布无显著差异(P>0.05，图6).
图4(PDF)BAK基因过表达对胃癌MKN-45细胞体外生长活性的影响.
图5(PDF)BAK基因过表达对胃癌MKN-45细胞增生活性的抑制作用.
图6(PDF)BAK基因过表达诱导MKN-45细胞周期G0/G1期阻滞.A: 未转染对照组;B: pcDNA3转染组;C: pcDNA-BAK转染组.
2.2 细胞形态学 未转染对照组细胞生长旺盛，形态规则，以多边形居多.pcDNA-BAK转染后部分细胞体积缩小，形态不规则，圆形细胞比例增多.透射电镜观察见部分癌细胞呈现核固缩、染色质核膜下聚集、胞体缩小、胞膜完整等凋亡特征性改变(图7).未转染对照组胃癌MKN-45细胞凋亡率为4.7%.pcDNA-BAK转染3d后，细胞凋亡率为21.4%，二者比较差异有显著性意义(P<0.01); 空载体转染对癌细胞凋亡率无显著性影响(P>0.05，图8).
图7 透射电镜观测BAK基因过表达后胃癌细胞形态学改变.A: 未转染对照组;B: pcDNA-BAK转染组细胞染色质边集;C: pcDNA-BAK转染组细胞核固缩.
2.3 Caspase-3活性 未转染对照组MKN-45细胞A405nm值为0.055±0.008.经pcDNA-BAK转染3d后，癌细胞A405nm值为0.245±0.012(P <0.01)，较对照组增强4.45倍.pcDNA3转染组与未转染对照组比较A405nm值差异无显著性意义(P>0.05，图9).
图8 TUNEL技术检测BAK基因过表达后癌细胞凋亡(×200 ). A:未转染对照组未见明显的细胞凋亡;B: pcDNA-BAK转染组癌细胞胞体缩小、核黄染.
图9(PDF)BAK基因过表达后癌细胞Caspase-3活性检测.
3 讨论肿瘤的发生不但与细胞增生和分化异常有关，而且与细胞凋亡异常关系密切.Bcl-2家族包括凋亡抑制基因Bcl-2，Bcl-xl，ced-9和促凋亡基因Bax，Bad，Bcl-xs等[16-18]. BAK基因促进细胞凋亡的机制:BAK通过其BH3结构域与Bcl-2和Bcl-xl相互作用形成异源二聚体，从而抑制Bcl-2和Bcl-xl的抑凋亡活性，具有加速细胞凋亡作用[19]; BAK直接激活凋亡途径或作为细胞死亡过程中的组成部分被活化而促进细胞凋亡.有研究表明，BAK可通过与CD95和TNF死亡受体相似的途径活化而促进细胞凋亡[20].BAK基因表达的异常与肿瘤的发生、发展紧密相关.在胃癌的研究中，Kondoet al [21]发现BAK基因表达的阳性率与胃癌病理分级、临床分期负相关.许多肿瘤治疗药物(紫苏子醇、g-干扰素)作用机制均与上调BAK基因表达有关[22-24].最近，Pataer etal [15, 25]应用腺病毒作为载体，将BAK基因导入肺癌及乳腺癌细胞，获得了显著的抑瘤效应，为肿瘤基因治疗提供了新的思路.
我们通过分子克隆技术成功构建BAK基因的真核表达载体，观测了转导外源性BAK基因对胃癌MKN-45细胞体外生长的影响，结果表明:未转染对照组胃癌MKN-45细胞中存在BAK基因弱阳性表达，当脂质体介导BAK真核表达载体转染3d后，癌细胞BAKmRNA和蛋白水平均显著增强，说明转染外源性BAK基因能使其在MKN-45细胞中过表达;BAK过表达后癌细胞体外生长及增生活性显著减慢，细胞周期阻滞于G0/G1期;通过不同方法均观察到癌细胞凋亡现象:透射电镜观察到癌细胞凋亡特征性形态学改变，TUNEL进一步证实过表达BAK基因对胃癌细胞凋亡的促进作用，凋亡率为21.4%.
近年来认为细胞凋亡的发生最终可能涉及到一种依赖天门冬氨酸的半胱氨酸酶，称之为Caspase.至今为止，已有编码14种Caspase的cDNA被克隆，所有的Caspase都以非活性形式合成，然后以亚单位形式激活后释放.其中，Caspase-3是 Caspase酶联反应的终末因子之一.Bcl-2蛋白作为细胞凋亡的主要拮抗成分，能抑制线粒体膜的通透性和阻止细胞色素C的释放.在凋亡信号传导的过程中，Bcl-2作用在Caspase-3的上游，可通过阻断Caspase-3激活而发挥抑制细胞凋亡作用.本研究表明:过表达BAK基因能促使癌细胞Caspase-3活性增强，这可能与BAK基因可以拮抗Bcl-2生物学功能，减轻对Caspases级联效应的负性调控有关.随着研究的深入，我们相信:作为一种新的诱导凋亡基因，BAK在胃癌治疗方面必将展现广泛的研究及应用前景.
4 参考文献1 Gulmann C, Hegarty H, Grace A, Leader M, Patchett S, Kay E.Differences in proximal (cardia) versus distal (antral) gastric
carcinogenesis via the retinoblastomapathway. World J Gastroenterol 2004;10:17-21
2 Ding YB, Chen GY, Xia JG, Zang XW, Yang HY, Yang L, Liu YX.Correlation of tumor-positive ratio and number of
perigastric lymph nodes with prognosis ofpatients with surgically-removed gastric carcinoma. World J Gastroenterol
2004; 10:182-185
3 Yu Y, Zhang YC, Zhang WZ, Shen LS, Hertzog P, Wilson TJ, Xu DK.Ets1 as a marker of malignant potential in gastric
carcinoma. World J Gastroenterol 2003;9:2154-2159
4 Zhang J, Su XQ, Wu XJ, Liu YH, Wang H, Zong XN, Wang Y, Ji JF.Effect of body mass index on adenocarcinoma of gastric
cardia. World J Gastroenterol 2003;9:2658-2661
5 Guo HQ, Guan P, Shi HL, Zhang X, Zhou BS, Yuan Y. Prospectivecohort study of comprehensive prevention to gastric
cancer. World J Gastroenterol 2003;9:432-436
6 Shi XY, Zhao FZ, Dai X, Ma LS, Dong XY, Fang J. Effect ofjianpiyiwei capsule on gastric precancerous lesions in rats.
World J Gastroenterol 2002;8:608-612
7 Wu GH, Zhang YW, Wu ZH. Modulation of postoperative immune andinflammatory response by immune-enhancing
enteral diet in gastrointestinal cancerpatients. World J Gastroenterol 2001;7:357-362
8 Yang LQ, Fang DC, Wang RQ, Yang SM. Effect of NF-kappaB, survivin,Bcl-2 and Caspase3 on apoptosis of gastric cancer
cells induced by tumor necrosis factorrelated apoptosis inducing ligand. World J Gastroenterol 2004;10:22-25
9 Liu YB, Wei ZX, Li L, Li HS, Chen H, Li XW. Construction andanalysis of SSH cDNA library of human vascular endothelial
cells related to gastrocarcinoma. World JGastroenterol 2003;9:2419-2423
10 Zhang JH, Wan MX, Yuan JY, Pan BR. Construction and identificationof recombinant vectors carrying herpes simplex virus
thymidine kinase and cytokine genesexpressed in gastric carcinoma cell line SGC7901. World J Gastroenterol
2004;10:26-30
11 Fu YG, Qu YJ, Wu KC, Zhai HH, Liu ZG, Fan DM. Apoptosis-inducingeffect of recombinant Caspase-3 expressed by
constructed eukaryotic vector on gastriccancer cell line SGC7901. World J Gastroenterol 2003;9:1935-1939
12 Zhu XD, Lin GJ, Qian LP, Chen ZQ. Expression of survivin in humangastric carcinoma and gastric carcinoma model of
rats. World J Gastroenterol 2003;9:1435-1438
13 Li HL, Chen DD, Li XH, Zhang HW, Lu JH, Ren XD, Wang CC.JTE-522-induced apoptosis in human gastric adenocarinoma
cell line AGS cells by caspase activationaccompanying cytochrome C release, membrane translocation of Bax andloss
of mitochondrial membrane potential. WorldJ Gastroenterol 2002;8:217-223
14 Han ZY, Wu KC, He FT, Han QL, Nie YZ, Han Y, Liu XN, Zheng JY, XuMH, Lin T, Fan DM. Screening and identification of
mimotope of gastric cancer associatedantigen MGb1-Ag. World J Gastroenterol 2003;9:1920-1924
15 Pataer A, Fang B, Yu R, Kagawa S, Hunt KK, McDonnell TJ, Roth JA,Swisher SG. Adenoviral Bak overexpression mediates
caspase-dependent tumor killing. Cancer Res 2000;60: 788-792
16 Burlacu A. Regulation of apoptosis by Bcl-2 family proteins. J CellMol Med 2003;7:249-257
17 Sakudo A, Lee DC, Saeki K, Matsumoto Y, Itohara S, Onodera T. Tumornecrosis factor attenuates prion protein-deficient
neuronal cell death by increases inanti-apoptotic Bcl-2 family proteins. Biochem Biophys Res Commun
2003;310:725-729
18 Kochendoerfer SK, Krishnan N, Buckley DJ, Buckley AR. Prolactinregulation of Bcl-2 family members: increased expression
of bcl-xL but not mcl-1 or bad in Nb2-Tcells. J Endocrinol 2003;178:265-273
19 Ruffolo SC, Shore GC. Bcl-2 selectively interacts with theBID-induced open conformer of BAK, inhibiting BAK
auto-oligomerization. J Biol Chem 2003;278:25039-25045
20 Werner AB, de Vries E, Tait SW, Bontjer I, Borst J. Bcl-2 familymember Bfl-1/A1 sequesters truncated bid to inhibit is
collaboration with pro-apoptotic Bak or Bax.J Biol Chem 2002;277:22781-22788
21 Kondo S, Shinomura Y, Miyazaki Y, Kiyohara T, Tsutsui S, KitamuraS, Nagasawa Y, Nakahara M, Kanayama S,Matsuzawa Y. Mutations of the bak gene inhuman gastric and colorectal cancers. Cancer Res 2000;60:4328-4330
22 Shyu RY, Su HL, Yu JC, Jiang SY. Direct growth suppressive activityof interferon-alpha and-gamma on human gastric
cancer cells. J Surg Oncol 2000;75:122-130
23 Ray S, Almasan A. Apoptosis induction in prostate cancer cells andxenografts by combined treatment with Apo2
ligand/tumor necrosis factor-relatedapoptosis-inducing ligand and CPT-11. Cancer Res 2003;63:4713-4723
24 Zhou XM, Wong BC, Fan XM, Zhang HB, Lin MC, Kung HF, Fan DM, LamSK. Non-steroidal anti-inflammatory drugs induce
apoptosis in gastric cancer cells throughup-regulation of bax and bak. Carcinogenesis 2001;22:1393-1397
25 Pearson AS, Spitz FR, Swisher SG, Kataoka M, Sarkiss MG, Meyn RE,McDonnell TJ, Cristiano RJ, Roth JA. Up-regulation
of the proapoptotic mediators Bax and Bakafter adenovirus-mediated p53 gene transfer in lung cancer cells. ClinCancer
Res 2000;6:887-890, 百拇医药( 郑丽端, 童强松, 刘 俊, 汪 良, 钱 伟)
童强松,汪良,华中科技大学同济医学院附属协和医院外科 湖北省武汉市 430022
刘俊,钱伟, 华中科技大学同济医学院附属协和医院消化内科 湖北省武汉市 430022
郑丽端,女, 1973-10-11生,福建省南安市人,汉族. 1997年同济医科大学本科毕业,2001级华中科技大学同济医学院在读硕士研究生,主治医师. 主要从事消化系统疾病的分子病理研究.
项目负责人:童强松,430022, 湖北省武汉市,华中科技大学同济医学院附属协和医院外科. qs_tong@hotmail.com
电话:027-85726129
收稿日期:2003-11-22 接受日期:2003-12-22
Effects of BAK gene over-expression on apoptosis in gastric cancercells and its molecular mechanisms
Li-Duan Zheng, Qiang-Song Tong, Jun Liu, Liang Wang, Wei Qian
Li-Duan Zheng, Department of Pathology, Union Hospital of Tongji MedicalCollege, Huazhong University of Science and Technology, Wuhan 430022,Hubei Province, China
Qiang-Song Tong, Liang Wang, Department of Surgery, Union Hospital ofTongji Medical College, Huazhong University of Science and Technology,Wuhan 430022, Hubei Province, China
Jun Liu, Wei Qian, Department of Gastroenterology, Union Hospital ofTongji Medical College, Huazhong University of Science and Technology,Wuhan 430022, Hubei Province, China
Correspondence to: Dr. Qiang-Song Tong, Department of Surgery,Union Hospital of Tongji Medical College, Huazhong University of Scienceand Technology, Wuhan 430022, Hubei Province, China. qs_tong@hotmail.com
Received: 2003-11-22 Accepted:2003-12-22
AbstractAIM: To explore the apoptosis-inducing effects of extrinsic BAK genetransfer and its over-expression on gastric cancer cells and its molecularmechanisms.
METHODS: The eukaryotic expression for BAK gene was constructed andtransferred into gastric cancer MKN-45 cell line. After being transferredfor 1 to 5 days, cellular BAK gene expression was detected by RT-PCR andWestern blotting methods. The growth activities of cancer cells weredetected by cell count and MTT colorimetry. Cell cycle changes wereassayed by flow cytometry. Cellular apoptosis was assayed by electronicmicroscopy and in situ terminally labeled transferase technique (TUNEL).Cellular caspase-3 activities were observed by colorimetric method.
RESULTS: After being transferred for 1 to 5 days, cellular BAK mRNAand protein expression levels were significantly increased (P <0.01).In vitro growth of gastric cancer cells was inhibited by 11.6-35.3% (P<0.01). The cellular proliferation activities were decreased by10.2-32.4% (P <0.01), with cell cycle being blocked at G0/G1phase. Partial cancer cells presented the characteristic morphologicalchanges of apoptosis, with the apoptotic rates being 21.4% (P <0.01).The cellular caspase-3 activities were enhanced by 4.45 times (P <0.01).
CONCLUSION: Transfection of extrinsic BAK gene, resulting in itsover-expression, can significantly induce apoptosis of gastric cancerMKN-45 cells through activating caspase-3, which is a potential strategyfor gene therapy of gastric cancer.
Zheng LD, Tong QS, Liu J, Wang L, Qian W. Effects of BAK geneover-expression on apoptosis in gastric cancer cells and its molecularmechanisms. Shijie Huaren Xiaohua Zazhi 2004;12(5):1025-1029
摘要
目的:探讨转染外源性BAK基因及其过表达对胃癌细胞的诱导凋亡作用和分子机制.
方法:构建携带有BAK基因的真核表达载体并转染胃癌MKN-45细胞1-5d后，RT-PCR和WesternBlotting法检测BAK基因表达;细胞计数、MTT比色法检测癌细胞生长活性，流式细胞仪分析细胞周期时相改变，透射电镜、末端TdT酶标记技术检测癌细胞凋亡;比色法检测癌细胞内Caspase-3活性改变.
结果:转染1-5 d后，癌细胞BAKmRNA和蛋白表达水平显著增强(P<0.01)，癌细胞体外生长抑制11.6-35.3%(P <0.01)，增生活性抑制10.2-32.4%(P <0.01)，细胞周期出现G0/G1期阻滞，部分癌细胞出现胞体缩小、核固缩等凋亡形态学改变，凋亡率为21.4%(P<0.01); 癌细胞Caspase-3活性增强4.45倍(P<0.01).
结论:转染外源性BAK基因使其过表达能激活Caspase-3，显著诱导胃癌MKN-45细胞凋亡，有望成为胃癌治疗的新途径.
郑丽端,童强松, 刘俊,汪良, 钱伟.BAK基因过表达对胃癌细胞的诱导凋亡作用及其分子机制.世界华人消化杂志 2004;12(5):1025-1029
0 引言胃癌是我国常见的恶性肿瘤，在不同地区约居癌症发病数的第1-3位，且近年来发病率有上升趋势，但其晚期治疗效果仍不理想[1-7].利用基因转导技术促进肿瘤细胞凋亡，成为目前胃癌治疗研究的热点之一[8-14].BAK (Bcl-2 homologus antagonist/ killer) 基因是近年来发现的一种促凋亡基因，应用基因转导的手段将其导入肿瘤细胞已成功抑制了肺癌、乳腺癌等多种肿瘤的生长[15].我们通过构建携带BAKcDNA的真核表达载体，观察了BAK基因过表达对胃癌细胞的诱导凋亡作用及其分子机制，旨在寻求胃癌治疗的新途径.
1 材料和方法
1.1 材料 质粒pET-BAK由美国冷泉港分子生物学实验室PattyWendel博士惠赠，在其限制性酶切位点XhoI和Hind III间含有BAK基因cDNA(633 bp). 真核表达载体pcDNA3由本校生化教研室屈伸教授惠赠.限制性内切酶和DNA快速回收纯化试剂盒购自Promega公司，T4DNA连接酶购自Takara公司.碘化丙锭购自Sigma公司.原位末端转移酶标记(TUNEL)试剂盒购自武汉博士德公司，Caspase-3活性检测试剂盒购自Clontech公司.BAK基因真核表达载体的构建参见(SambrookJ，Russell DW.Molecular Cloning: A Laboratory Manual. 3rd Ed. Cold Spring HarborLaboratory Press 1998; 254-325). 限制性内切酶XhoI、Hind III双切质粒pET-BAK和pcDNA3，分别回收0.63kb的基因片段和5.4kb的线性化载体片段，T4DNA酶进行连接反应，将重组体命名为pcDNA-BAK，酶切鉴定.
1.2 方法 人胃癌细胞株MKN-45由本院中心实验室董继华教授惠赠.用含100 mL/L胎牛血清、100kU/L青霉素和100mg/L链霉素的RPMI1640培养基(Gibco )，在37°C，50mL/L CO2条件下培养.设置未转染对照组、pcDNA3转染组、pcDNA-BAK转染组，基因转染参照脂质体lipofectamine2000 (Gibco)说明书进行.BAK mRNA表达水平检测采用RT-PCR法.细胞总RNA的提取参照Trizal试剂盒(LifeTechnologies公司)说明书进行.逆转录反应后，加入下列引物进行PCR扩增:BAK (502 bp)上游 5’-CTGCCCTCTGCTTCTGA-3’，下游5’-CGTTCAGGATGGGACCA-3’; 同时以a-tubulin(295 bp) 上游 5’-CCCGTCTTCAGGGTCTCTTG-3’，下游5’-TTAAGGTAAGTGTAGGTTGGG-3’ 作为反应内参照.扩增产物经10g/L琼脂糖凝胶电泳分离，紫外灯下观察并拍照.凝胶图像分析仪(GelDoc 1000型，Bio-Rad公司)检测BAK与a-tubulin扩增片段灰度的比值.BAK蛋白表达水平检测采用WesternBlotting法，参照(SambrookJ，Russell DW.Molecular Cloning: A Laboratory Manual. 3rd Ed. Cold Spring HarborLaboratory Press 1998; 254-325)进行蛋白质提取、定量和分离.转膜后先后与10g/L 脱脂奶粉、鼠抗人BAKmAb (武汉博士德公司)、过氧化物酶偶联的山羊抗鼠IgG孵育，运用ECLWestern blotting kit显色.图像分析系统测定BAK蛋白条带密度.
1.2.1 细胞生长特征 接种2×105/LMKN-45细胞0.5mL入24孔培养板，每组5个复孔，转染后用RPMI1640完全培养基在37°C，50mL/LCO2条件下培养，每24h用1.25 g/L胰酶+0.1g/L EDTA消化细胞，台酚兰染色，于倒置显微镜下计数活细胞，每次计数重复3次，连续测定5d，绘制细胞生长曲线. 细胞增生活性检测采用MTT比色法.转染1、3、5d后，加入5g/L MTT (Clontech公司)20 mL/孔，继续培养4h，弃去上清，加DMSO100 mL/孔，振荡至结晶溶解，酶标仪570nm 波长处测定吸光度A570nm值.细胞增生抑制率(%)=(1-实验组平均吸光度A570nm值/对照组平均吸光度A570nm值)×100%. 细胞周期时相检测采用流式细胞仪法.上述三组癌细胞培养至3d，分别收集2×106细胞，PBS洗涤2次，700mL/L乙醇固定过夜，4°C保存，检测前用PBS洗涤，加入RNase(1 g/L) 200 mL，37°C水浴30min，用碘化丙锭(500mg/L) 800 mL进行染色，室温避光30min，流式细胞仪(BectonDickson公司)进行DNA含量分析，所用软件为CellQest.
1.2.2 细胞形态学检测 收集上述三组癌细胞，PBS洗涤1次，25mL/L戊二醛固定30min，PBS洗涤并悬浮细胞，常规包埋、切片，透射电镜观察并摄影.采用末端TdT酶标记技术(TUNEL)检测转染3d后各处理组的细胞凋亡.3 mL/L H2O2封闭30min，20 mg/L蛋白酶K消化液作用20min，TUNEL反应液37°C孵育60min，滴加过氧化物酶连接的抗体，37°C孵育30min，DAB显色，苏木素复染.以PBS替代TUNEL反应液作阴性对照.在光学显微镜下，凋亡细胞体积缩小，核固缩，染色质呈特异的棕黄色.细胞凋亡率(%)=(500个细胞中凋亡细胞数/500)×100%.
1.2.3 Caspase-3活性测定 分别收集2×105各处理组细胞，加入细胞裂解缓冲液50mL，冰上孵育10min; 12 000 r/min，4°C离心细胞裂解液3min，回收上清，每管依次50mL的2×反应缓冲液，1mmoL/L Caspase-3底物DEVD-pNA5 mL，37°C孵育1h，转移至96孔板中，用酶标仪测定波长405nm的吸光度值(A405nm)，表示Caspase-3的相对活性.
统计学处理 采用SPSS统计学软件进行数据分析.
2 结果将BAK基因片段正向插入到载体pcDNA3中，得到携带BAK基因的真核表达载体pcDNA-BAK(6.0 kb). 根据基因和载体的物理图谱，选取XhoI、Hind III单酶切和XhoI和Hind III双酶切该重组体，酶切产物经10g/L琼脂糖凝胶电泳，证实BAKcDNA已插入重组体pcDNA-BAK中(图1).RT-PCR产物电泳后，未转染对照组和空载体pcDNA3转染组均可见BAK(502 bp)和a-tubulin(295 bp) 条带，二者BAK/a-tubulin灰度比值分别为0.81，083，统计学分析差异无显著性意义(P>0.05). 外源性BAK基因转染3d后，BAK/ a-tubulin灰度比值为2.98，即BAKmRNA表达较未转染对照组增强3.67倍(P<0.01，图2).未转染对照组、pcDNA3转染组、pcDNA-BAK转染组细胞经WesternBlotting检测均可见Mr28 000的蛋白条带.经图像分析系统证实，pcDNA-BAK转染组的BAK蛋白条带灰度值为对照组、pcDNA3转染组的3.1倍(P<0.01)和3.2倍(P<0.01); 未转染对照组、pcDNA3转染组细胞BAK蛋白条带灰度值之间无显著性差异(P>0.05，图3).
图1(PDF)真核表达载体 pcDNA-BAK的酶切鉴定分析.1: pcDNA-BAK; 2: pcDNA-BAK / Xho I; 3: pcDNA-BAK / Hind III ; 4: pcDNA-BAK/Xho I+Hind III ; M: l DNA / Hind III + EcoR I.
图2(PDF)RT-PCR 检测各组癌细胞BAKmRNA表达水平.1: 未转染对照组;2: 空载体转染组;3: BAK转染组 M:PCR marker (100 bp, 200 bp,300 bp, 400 bp, 500 bp, 600 bp, 700 bp, 800 bp,900 bp, 1 000 bp).
图3(PDF)Western Blotting检测各组癌细胞BAK蛋白表达水平.1: BAK转染组;2: 空载体转染组;3: 未转染对照组.
2.1 细胞生长特征 未转染对照组MKN-45细胞体外生长活跃，经pcDNA-BAK转染1-5d后，癌细胞体外生长减慢，抑制率为11.6-35.3%(P<0.01); 空载体pcDNA3转染组癌细胞体外生长活性与未转染对照组比较差异无显著性意义(P>0.05，图4).细胞增生活性检测未转染对照组A570nm值随时间推移而递增，反映癌细胞体外增生活跃.pcDNA-BAK转染1-5d后，MKN-45细胞增生活性均不同程度地减弱，抑制率为10.2-32.4%(P<0.01). pcDNA3转染组与未转染对照组A570nm值之间的差异无显著性意义(P>0.05，图5).在DNA直方图上，pcDNA-BAK转染组癌细胞S期比例(36.6%)显著低于非转染对照组(55.1%)，细胞生长受阻于G0/G1期(比率分别为32.2%、12.5%).pcDNA3转染组与未转染对照组比较细胞周期时相分布无显著差异(P>0.05，图6).
图4(PDF)BAK基因过表达对胃癌MKN-45细胞体外生长活性的影响.
图5(PDF)BAK基因过表达对胃癌MKN-45细胞增生活性的抑制作用.
图6(PDF)BAK基因过表达诱导MKN-45细胞周期G0/G1期阻滞.A: 未转染对照组;B: pcDNA3转染组;C: pcDNA-BAK转染组.
2.2 细胞形态学 未转染对照组细胞生长旺盛，形态规则，以多边形居多.pcDNA-BAK转染后部分细胞体积缩小，形态不规则，圆形细胞比例增多.透射电镜观察见部分癌细胞呈现核固缩、染色质核膜下聚集、胞体缩小、胞膜完整等凋亡特征性改变(图7).未转染对照组胃癌MKN-45细胞凋亡率为4.7%.pcDNA-BAK转染3d后，细胞凋亡率为21.4%，二者比较差异有显著性意义(P<0.01); 空载体转染对癌细胞凋亡率无显著性影响(P>0.05，图8).
图7 透射电镜观测BAK基因过表达后胃癌细胞形态学改变.A: 未转染对照组;B: pcDNA-BAK转染组细胞染色质边集;C: pcDNA-BAK转染组细胞核固缩.
2.3 Caspase-3活性 未转染对照组MKN-45细胞A405nm值为0.055±0.008.经pcDNA-BAK转染3d后，癌细胞A405nm值为0.245±0.012(P <0.01)，较对照组增强4.45倍.pcDNA3转染组与未转染对照组比较A405nm值差异无显著性意义(P>0.05，图9).
图8 TUNEL技术检测BAK基因过表达后癌细胞凋亡(×200 ). A:未转染对照组未见明显的细胞凋亡;B: pcDNA-BAK转染组癌细胞胞体缩小、核黄染.
图9(PDF)BAK基因过表达后癌细胞Caspase-3活性检测.
3 讨论肿瘤的发生不但与细胞增生和分化异常有关，而且与细胞凋亡异常关系密切.Bcl-2家族包括凋亡抑制基因Bcl-2，Bcl-xl，ced-9和促凋亡基因Bax，Bad，Bcl-xs等[16-18]. BAK基因促进细胞凋亡的机制:BAK通过其BH3结构域与Bcl-2和Bcl-xl相互作用形成异源二聚体，从而抑制Bcl-2和Bcl-xl的抑凋亡活性，具有加速细胞凋亡作用[19]; BAK直接激活凋亡途径或作为细胞死亡过程中的组成部分被活化而促进细胞凋亡.有研究表明，BAK可通过与CD95和TNF死亡受体相似的途径活化而促进细胞凋亡[20].BAK基因表达的异常与肿瘤的发生、发展紧密相关.在胃癌的研究中，Kondoet al [21]发现BAK基因表达的阳性率与胃癌病理分级、临床分期负相关.许多肿瘤治疗药物(紫苏子醇、g-干扰素)作用机制均与上调BAK基因表达有关[22-24].最近，Pataer etal [15, 25]应用腺病毒作为载体，将BAK基因导入肺癌及乳腺癌细胞，获得了显著的抑瘤效应，为肿瘤基因治疗提供了新的思路.
我们通过分子克隆技术成功构建BAK基因的真核表达载体，观测了转导外源性BAK基因对胃癌MKN-45细胞体外生长的影响，结果表明:未转染对照组胃癌MKN-45细胞中存在BAK基因弱阳性表达，当脂质体介导BAK真核表达载体转染3d后，癌细胞BAKmRNA和蛋白水平均显著增强，说明转染外源性BAK基因能使其在MKN-45细胞中过表达;BAK过表达后癌细胞体外生长及增生活性显著减慢，细胞周期阻滞于G0/G1期;通过不同方法均观察到癌细胞凋亡现象:透射电镜观察到癌细胞凋亡特征性形态学改变，TUNEL进一步证实过表达BAK基因对胃癌细胞凋亡的促进作用，凋亡率为21.4%.
近年来认为细胞凋亡的发生最终可能涉及到一种依赖天门冬氨酸的半胱氨酸酶，称之为Caspase.至今为止，已有编码14种Caspase的cDNA被克隆，所有的Caspase都以非活性形式合成，然后以亚单位形式激活后释放.其中，Caspase-3是 Caspase酶联反应的终末因子之一.Bcl-2蛋白作为细胞凋亡的主要拮抗成分，能抑制线粒体膜的通透性和阻止细胞色素C的释放.在凋亡信号传导的过程中，Bcl-2作用在Caspase-3的上游，可通过阻断Caspase-3激活而发挥抑制细胞凋亡作用.本研究表明:过表达BAK基因能促使癌细胞Caspase-3活性增强，这可能与BAK基因可以拮抗Bcl-2生物学功能，减轻对Caspases级联效应的负性调控有关.随着研究的深入，我们相信:作为一种新的诱导凋亡基因，BAK在胃癌治疗方面必将展现广泛的研究及应用前景.
4 参考文献1 Gulmann C, Hegarty H, Grace A, Leader M, Patchett S, Kay E.Differences in proximal (cardia) versus distal (antral) gastric
carcinogenesis via the retinoblastomapathway. World J Gastroenterol 2004;10:17-21
2 Ding YB, Chen GY, Xia JG, Zang XW, Yang HY, Yang L, Liu YX.Correlation of tumor-positive ratio and number of
perigastric lymph nodes with prognosis ofpatients with surgically-removed gastric carcinoma. World J Gastroenterol
2004; 10:182-185
3 Yu Y, Zhang YC, Zhang WZ, Shen LS, Hertzog P, Wilson TJ, Xu DK.Ets1 as a marker of malignant potential in gastric
carcinoma. World J Gastroenterol 2003;9:2154-2159
4 Zhang J, Su XQ, Wu XJ, Liu YH, Wang H, Zong XN, Wang Y, Ji JF.Effect of body mass index on adenocarcinoma of gastric
cardia. World J Gastroenterol 2003;9:2658-2661
5 Guo HQ, Guan P, Shi HL, Zhang X, Zhou BS, Yuan Y. Prospectivecohort study of comprehensive prevention to gastric
cancer. World J Gastroenterol 2003;9:432-436
6 Shi XY, Zhao FZ, Dai X, Ma LS, Dong XY, Fang J. Effect ofjianpiyiwei capsule on gastric precancerous lesions in rats.
World J Gastroenterol 2002;8:608-612
7 Wu GH, Zhang YW, Wu ZH. Modulation of postoperative immune andinflammatory response by immune-enhancing
enteral diet in gastrointestinal cancerpatients. World J Gastroenterol 2001;7:357-362
8 Yang LQ, Fang DC, Wang RQ, Yang SM. Effect of NF-kappaB, survivin,Bcl-2 and Caspase3 on apoptosis of gastric cancer
cells induced by tumor necrosis factorrelated apoptosis inducing ligand. World J Gastroenterol 2004;10:22-25
9 Liu YB, Wei ZX, Li L, Li HS, Chen H, Li XW. Construction andanalysis of SSH cDNA library of human vascular endothelial
cells related to gastrocarcinoma. World JGastroenterol 2003;9:2419-2423
10 Zhang JH, Wan MX, Yuan JY, Pan BR. Construction and identificationof recombinant vectors carrying herpes simplex virus
thymidine kinase and cytokine genesexpressed in gastric carcinoma cell line SGC7901. World J Gastroenterol
2004;10:26-30
11 Fu YG, Qu YJ, Wu KC, Zhai HH, Liu ZG, Fan DM. Apoptosis-inducingeffect of recombinant Caspase-3 expressed by
constructed eukaryotic vector on gastriccancer cell line SGC7901. World J Gastroenterol 2003;9:1935-1939
12 Zhu XD, Lin GJ, Qian LP, Chen ZQ. Expression of survivin in humangastric carcinoma and gastric carcinoma model of
rats. World J Gastroenterol 2003;9:1435-1438
13 Li HL, Chen DD, Li XH, Zhang HW, Lu JH, Ren XD, Wang CC.JTE-522-induced apoptosis in human gastric adenocarinoma
cell line AGS cells by caspase activationaccompanying cytochrome C release, membrane translocation of Bax andloss
of mitochondrial membrane potential. WorldJ Gastroenterol 2002;8:217-223
14 Han ZY, Wu KC, He FT, Han QL, Nie YZ, Han Y, Liu XN, Zheng JY, XuMH, Lin T, Fan DM. Screening and identification of
mimotope of gastric cancer associatedantigen MGb1-Ag. World J Gastroenterol 2003;9:1920-1924
15 Pataer A, Fang B, Yu R, Kagawa S, Hunt KK, McDonnell TJ, Roth JA,Swisher SG. Adenoviral Bak overexpression mediates
caspase-dependent tumor killing. Cancer Res 2000;60: 788-792
16 Burlacu A. Regulation of apoptosis by Bcl-2 family proteins. J CellMol Med 2003;7:249-257
17 Sakudo A, Lee DC, Saeki K, Matsumoto Y, Itohara S, Onodera T. Tumornecrosis factor attenuates prion protein-deficient
neuronal cell death by increases inanti-apoptotic Bcl-2 family proteins. Biochem Biophys Res Commun
2003;310:725-729
18 Kochendoerfer SK, Krishnan N, Buckley DJ, Buckley AR. Prolactinregulation of Bcl-2 family members: increased expression
of bcl-xL but not mcl-1 or bad in Nb2-Tcells. J Endocrinol 2003;178:265-273
19 Ruffolo SC, Shore GC. Bcl-2 selectively interacts with theBID-induced open conformer of BAK, inhibiting BAK
auto-oligomerization. J Biol Chem 2003;278:25039-25045
20 Werner AB, de Vries E, Tait SW, Bontjer I, Borst J. Bcl-2 familymember Bfl-1/A1 sequesters truncated bid to inhibit is
collaboration with pro-apoptotic Bak or Bax.J Biol Chem 2002;277:22781-22788
21 Kondo S, Shinomura Y, Miyazaki Y, Kiyohara T, Tsutsui S, KitamuraS, Nagasawa Y, Nakahara M, Kanayama S,Matsuzawa Y. Mutations of the bak gene inhuman gastric and colorectal cancers. Cancer Res 2000;60:4328-4330
22 Shyu RY, Su HL, Yu JC, Jiang SY. Direct growth suppressive activityof interferon-alpha and-gamma on human gastric
cancer cells. J Surg Oncol 2000;75:122-130
23 Ray S, Almasan A. Apoptosis induction in prostate cancer cells andxenografts by combined treatment with Apo2
ligand/tumor necrosis factor-relatedapoptosis-inducing ligand and CPT-11. Cancer Res 2003;63:4713-4723
24 Zhou XM, Wong BC, Fan XM, Zhang HB, Lin MC, Kung HF, Fan DM, LamSK. Non-steroidal anti-inflammatory drugs induce
apoptosis in gastric cancer cells throughup-regulation of bax and bak. Carcinogenesis 2001;22:1393-1397
25 Pearson AS, Spitz FR, Swisher SG, Kataoka M, Sarkiss MG, Meyn RE,McDonnell TJ, Cristiano RJ, Roth JA. Up-regulation
of the proapoptotic mediators Bax and Bakafter adenovirus-mediated p53 gene transfer in lung cancer cells. ClinCancer
Res 2000;6:887-890, 百拇医药( 郑丽端, 童强松, 刘 俊, 汪 良, 钱 伟)
