p53基因对人胚肺成纤维细胞周期影响的研究

背景与目的:研究野生型和179位残基突变型p53基因在HELF细胞周期调控中的作用.探讨p53基因的179位残基突变对细胞生长的影响.材料与方法:用野生型p53(pcDNA3-wtp53)和179位残基突变的突变型p53(pcDNA3-mtp53)转染HELF细胞,观察细胞生长情况,绘制细胞生长曲线;用流式细胞仪分析细胞周期;用RT-PCR和Western blotting方法检测p53基因转染后HELF细胞周期相关基因mRNA和蛋白的表达.结果:野生型p53表达的上调使HELF细胞周期阻滞于G1期,细胞体积减小,并下调cyclin D3、cyclin E、Cdk2和Cdk4的表达,同时上调p21的表达.而179位残基突变的突变型p53表达的上调则促进细胞周期从G1期到S期的转换,同时细胞体积增大,上调cyclin A和Cdk4的表达.结论:p53的179位残基突变对于HELF细胞cyclin A和Cdk4的表达有诱导作用,并可能借此促进细胞周期进程.     

Cyclin E、CDK2和p21WAF1在食管上皮癌变过程中的表达及意义

目的探讨食管上皮癌变过程中细胞周期调控因子cyclin E、CDK2和p21WAF1的表达状况及其意义.方法应用免疫组化SP法和原位杂交方法分别检测48例食管癌组织、31例非典型增生组织和17例正常食管粘膜中cyclin E、CDK2和p21WAF1蛋白及mRNA表达.应用半定量RT-PCR和Western blot检测22例新鲜食管癌及相应癌旁组织的mRNA和蛋白表达.结果从食管正常粘膜、非典型增生组织到癌组织,cyclin E和CDK2蛋白和mRNA阳性表达率逐渐上升,差异具有统计学意义(P＜0.01或P＜0.05).食管癌组织中cyclin E、CDK2和p21WAF1蛋白及mRNA高表达,与癌旁组织或切缘正常食管粘膜有显著性差异(P＜0.01).cyclin E、CDK2和p21WAF1基因表达显著正相关(P＜0.01或P＜0.05).结论食管上皮癌变过程中,细胞周期相关基因cyclin E和CDK2表达逐渐增强.cyclin E基因表达异常是食管癌变过程中的早期事件.p21WAF1基因在食管癌中高表达,可能与细胞周期调控的反馈机制有关.     

细胞周期调控因子与癌发生的关系

近年来 ,有关细胞周期调控机制及其与恶性肿瘤发生的关系研究进展较快。细胞周期调控可分为G1期调控和非G1期调控。在G1期调控中 ,细胞周期蛋白依赖性激酶复合体cyclin CDK激活后 ,通过Rb蛋白和转录因子启动基因转录。p16、p2 1、p15等蛋白通过抑制cyclin CDK的活性而发挥作用。p5 3蛋白和mdm2蛋白协同调节细胞周期活动。其中任何一个环节的变化都可引起细胞周期失控而促使癌发生     

褪黑素对小鼠肝癌细胞增殖及p27Kip1、cyclin D1基因表达的影响

目的探讨褪黑素(melatonin MT)抑制H22小鼠肝癌细胞增殖的可能机制.方法通过体外细胞培养,分别采用不同剂量MT作用不同的时间,MTT显色技术检测细胞的增殖;RT-PCR检测MT作用4 d后,H22细胞中p27Kip1、cyclin D1mRNA的表达;免疫组化检测H22细胞中p27Kip1、cyclin D1蛋白的表达.结果MT具有抑制肝癌细胞的生长和增殖的作用,且具有时间依赖关系.MT诱导细胞凋亡的过程中,与对照组相比,MT药理剂量组H22细胞中的p27Kip1 mRNA及蛋白表达升高(P＜0.01);cyclin D1 mRNA及蛋白表达下降(P＜0.01).MT生理剂量组p27Kip1mRNA表达升高(P＜0.01),而p27Kip1蛋白表达水平与对照组相比差异无显著性(P＞0.05),cyclinD1 mRNA及蛋白表达均下降,差异元显著性(P＞0.05).结论MT抑制肝癌细胞的增殖可能与上调细胞周期抑制因子p27Kip1的表达,降低周期蛋白cyclin D1表达,进而延迟细胞周期的进程有关.     

沉默BRG1基因对人胶质瘤细胞增殖的影响及其机制北大核心CSCD

目的探讨小干扰RNA(siRNA)沉默胶质瘤细胞BRG1基因表达对其增殖的影响及其机制。方法化学合成BRG1 siRNA,脂质体介导转染胶质瘤U251和U87细胞。CCK-8细胞增殖实验检测细胞增殖,流式细胞仪分析细胞周期变化,Western blot检测BRG1、cyclin家族、CDK抑制剂蛋白表达水平。结果转染BRG1 siRNA可以有效减少胶质瘤U251和U87细胞BRG1表达,细胞增殖下降,G1期细胞增加。BRG1沉默后cyclin D1和cyclin B1表达降低,CDK抑制剂p21和p27没有明显变化。结论沉默胶质瘤细胞BRG1表达影响细胞周期蛋白使细胞停滞在G1期,最终抑制胶质瘤细胞增殖。     

沙利度胺对人宫颈癌Hela细胞周期的影响及其作用机制

目的:探讨沙利度胺对人宫颈癌Hela细胞周期的影响及其作用机制。方法:沙利度胺（50 μg/ml、100 μg/ml和200 μg/ml）处理人宫颈癌Hela细胞24 h后,运用CCK-8法检测细胞活力;采用流式细胞术检测细胞周期分布;采用蛋白免疫印迹法检测Cyclin E、p53、p-p53和GAPDH蛋白表达水平。结果:沙利度胺(50 μg/ml、100 μg/ml和200 μg/ml)作用Hela细胞24 h后,细胞活力显著降低,且呈浓度依赖性(P<0.05)。50 μg/ml、100 μg/ml和200 μg/ml沙利度胺作用24 h后,Hela细胞G0/G1期细胞数明显增加,S期细胞数显著减少(P<0.05)。此外,沙利度胺作用Hela细胞24 h可明显下调Cyclin E蛋白的表达(P<0.05)。沙利度胺处理Hela细胞24 h后,p-p53/p53比值明显增大(P<0.05)。结论:沙利度胺可能通过促进p53蛋白的活化从而抑制人宫颈癌细胞周期进程。     

人剪切修复基因XPD转染对人肝癌细胞SMMC-7721的生物学影响

目的 将人剪切修复基因XPD稳定转染人SMMC-7721肝癌细胞,观察转染后细胞内野生型p53、XPD、周期素依赖性蛋白激酶(CDK)7、c-myc等基因表达的变化以及对细胞生长的影响,探讨野生型XPD基因与p53、CDK7、c-myc的相互作用及细胞凋亡机制.方法 将表达绿色荧光蛋白并含有人类全长野生型XPD的pEGFP-N2-XPD重组体质粒稳定转染人SMMC-7721肝癌细胞中,选择培养基筛选单克隆稳定转染重组质粒的人SMMC-7721肝癌细胞(SMMC-7721-pEGFP-N2-XPD)和稳定转染空载质粒的人SMMC-7721肝癌细胞(SMMC.7721-pEGFP-N2),并将人SMMC-7721肝癌细胞作为空白对照,利用荧光显微镜观测绿色荧光蛋白表达,用逆转录-聚合酶链反应(RT-PCR)、Westem blot法检测转染XPD基因后细胞内XPD、p53、CDK7、c-myc的表达量变化,并用细胞增殖力检测(MTT)法及流式细胞仪分别检测细胞增殖及凋亡和细胞周期变化.结果 ①免疫荧光显微镜下,SMMC.7721.pEGFP.N2.XPD和SMMC-7721-pEGFP-N2细胞中观察到绿色荧光蛋白表达,说明pEGFP-N2-XPD重组质粒和pEGFP-N2空载质粒成功转染.②RT-PCR检测:SMMC-7721-pEGFP-N2-XPD中p53 mRNA、XPD mR-NA表达量与SMMC-7721-pEGFP-N2和SMMC-7721相比均明显增高(P<0.01),CDK7 mRNA、c-myc mRNA在SMMC-7721-pEGFP-N2-XPD的表达量比两对照组明显降低(P<0.01),而两对照组各个基因的表达没有明显差异(P>0.05).③Western blot检测:SMMC-7721-pEGFP-N2-XPD细胞的p53、XPD蛋白表达最较两对照组升高(P<0.01),CDK7、c-myc的蛋白相对表达量比两对照组降低(P<0.01),两对照组差异没有统计学意义(P>0.05).④M1Tr检测:SMMC-7721-pEGFP-N2-XPD的细胞增殖力较对照组减弱(P<0.05),两对照组筹异没有统计学意义(P>0.05).⑤流式细胞仪检测:SMMC-7721-pEGFP-N2-XPD细胞进入s期出现障碍,停滞在G1期的细胞增多.结论 将XPD成功稳定转染到人SMMC-7721肝癌细胞中,XPD、p53在转录和蛋白水平的表达明显升高,CDK7、c-myc表达明显降低,野生型XPD基因的过表达可能抑制CDK7、c-myc表达,改变细胞周期,并促进p53抑制肝癌细胞增长,促进细胞凋亡.     

苦参碱诱导人肝癌细胞分化、凋亡时对 G1细胞周期调节因子的调控

目的：观察苦参碱诱导人肝癌细胞分化、凋亡时,G1细胞周期调节因子的变化,探讨苦参碱对肿瘤细胞增殖的调控。方法：0、0．3、0．8、1．5g/L苦参碱作用HepG2细胞3天,免疫组化检测p53,Rb,p21,p27,p16,cyclinD1蛋白表达：原位杂交检测p53,cyclin D1mRNA的表达。真彩色图像分析对基因表达强弱进行定量。结果用Microsoft-Excel软件统计处理。结果：用药后细胞周期负调控因子p53,Rb,p21,p27,p16表达增强;正调控因子cyclinD1表达减弱。结论：苦参碱诱导人肝癌细胞HepG2分化、凋亡的机制可能与苦参碱上调了G1细胞周期负调节因子的表达,下调了G1期正调节因子cyclinD1表达有关。     

放线菌素D处理V79 细胞的条件培养液对旁观者细胞p53 mRNA和蛋白表达 及细胞周期的影响

目的： 观察放线菌素D (actinomycin D，ACTD)处理V79靶细胞获得的条件培养液 (conditioned medium，CM)对V79旁观者细胞p53 mRNA和蛋白表达及细胞周期的影响，以研究ACTD诱导旁观者效应发生的可能机制。方法：4.0 mg/L ACTD处理V79靶细胞1 h，PBS洗3次，加入新鲜培养液后开始计时，分别在第4、8、12和24 h获取靶细胞不同时段的CM。用不同时段CM培养正常V79细胞24 h后，分别采用RT-PCR检测旁观者细胞中p53的mRNA水平；免疫组化法检测p53蛋白的表达；流式细胞术测定旁观者细胞的细胞周期分布。结果：与对照组比较，p53 mRNA水平在4、8和24 h CM处理组均增加 (P<0.01)，在4~12 h CM时段，p53 mRNA水平随着时段的延后呈逐渐降低趋势，12 h组降至最低，与阴性对照的水平接近 (P>0.05)。各CM处理组p53蛋白平均灰度值和积分光密度，除12 h CM组外均较对照组显著增加 (P<0.05)。随着时段的延后(4~12 h)，p53蛋白表达逐渐减少，12 h组降至最低，与阴性对照间的差异无统计学意义 (P>0.05)，p53蛋白表达与p53 mRNA表达结果一致。不同时段CM处理组旁观者细胞G0/G1期比阴性对照组增多 (P<0.05)，4 h CM组增加最明显；各CM处理组S期细胞均较阴性对照组减少 (P<0.01)，但组间差异无统计学意义 (P>0.05)；而G2/M期细胞在4 h CM 组下降明显，与阴性对照间的差异有统计学意义 (P<0.01)；8~12 h逐渐增加，12 h组接近对照水平(P>0.05)；24 h又下降，与阴性对照间的差异有统计学意义(P<0.01)。结论：ACTD 诱导旁观者效应可能是通过靶细胞的CM影响旁观者细胞p53 mRNA和蛋白的表达，并影响细胞周期的进展而引起的，以4 h CM诱导的效应最强。     

miR-1236模拟物转染对前列腺癌细胞生长影响

目的 miR-1236为新发现的具有肿瘤抑制作用的RNA.本研究旨在探讨外源性miR-1236转染对前列腺癌细胞p21基因的激活作用及对其生长的影响.方法 根据对前列腺癌细胞的不同处理分为阴性对照组(转染dsControl)和实验组(转染miR-1236)2组.qRT-PCR检测p21、细胞周期依赖性激酶CDK4和CDK6 mRNA的表达变化.蛋白质印迹检测p21、CDK4和CDK6蛋白的表达变化.流式细胞术检测细胞周期分布.MTS法检测细胞活力,集落形成实验检测细胞增殖能力.结果 与dsControl组相比,转染miR-1236后2种细胞中p21 mRNA表达分别上调2.16(t=12.67,P<0.01)和2.26倍(t=10.93,P<0.01);CDK4 mRNA的表达分别下调0.56(t=6.617,P<0.01)和0.43倍(t=5.698,P<0.01);CDK6 mRNA的表达分别下调0.78(t=3.101,P<0.05)和0.71倍(t=3.841,P<0.01).蛋白质印迹检测结果与qRT-PCR结果相符.流式细胞术结果显示,转染miR-1236后,位于S期和G2/M期的细胞比例明显下降,位于G0/G1期的细胞比例明显增大.MTS法显示,与dsControl组相比,转染miR-12362种细胞活力明显下降.dsControl组DU145和PC-3细胞形成的集落数分别为241±37.64和254.33±38.18;miR-1236组DU145和PC-3细胞形成的集落数明显减少,分别为99.67±34.2(t=3.939,P<0.05)和81±35.55(t=4.354,P<0.05),提示细胞增殖能力降低.结论 外源性miR-1236能通过激活前列腺癌细胞中p21蛋白的表达并显著抑制其生长,可能成为前列腺癌基因靶向治疗的新靶点.     

1,4-苯醌致人胚肺成纤维细胞的凋亡效应与调控机制

目的:研究1,4-苯醌(1,4-benzoquinone,PBQ)对人胚肺成纤维细胞(human embryonic lung fibroblast,HELF)的凋亡效应与调控机制。方法:分别用不同浓度(10、20、40、60、80μmol/L)的PBQ处理HELF细胞24 h和40μmol/L PBQ处理HELF细胞24、48、72 h后,应用噻唑蓝(MTT)比色法检测PBQ对HELF细胞增殖的抑制作用,以PI单染法检测细胞周期的改变,用Annexin-Ⅴ/PI双染法检测细胞凋亡,以实时荧光PCR法检测Bax、Bcl-2、p53 mRNA表达水平的改变。结果:与对照组相比,不同PBQ染毒浓度下作用24 h后,随PBQ浓度增加,细胞相对增殖率显著下降(P0.05),G0/G 1期细胞下降(在40和60μmol/L时,P0.05),S期细胞增加(P0.05),以40μmol/L PBQ浓度作用于HELF细胞不同时间,随着染毒时间的增加其相对增殖率和S期细胞均下降(P0.05),而G0/G1期细胞随着染毒时间的增加呈上升趋势(P0.05);当染毒浓度大于20μmol/L时,染毒24 h可诱导HELF细胞产生凋亡,凋亡率随着染毒时间的增加而上升,与对照组相比差异具有统计学意义(P0.05);不同浓度的PBQ染毒24 h,细胞的Bax、Bcl-2、p53 mRNA表达水平均上调。40μmol/L PBQ作用不同时间后,Bax、p53 mRNA水平随着染毒时间的增加而上升,与对照组相比差异均具有统计学意义(P均0.05);Bcl-2随染毒时间增加而下降,48和72 h时与对照组相比差异具有统计学意义(P0.05);Bax/Bcl-2比值随染毒时间增加而增加,与对照组相比差异具有统计学意义(P0.05)。结论:PBQ能抑制HELF细胞增殖,影响HELF细胞周期分布,诱导细胞发生凋亡,其凋亡机制可能与Bax/Bcl-2比值的上升,及p53 mRNA表达的上调有关。     

Synthetic retinoid CD437 induces cell-dependent cycle arrest by differential regulation of cell cycle associated proteins

BACKGROUND: The synthetic retinoid 6-[3-(1-adamantyl)-4-hydroxyphenyl]-2-naphthalene (CD437) exhibits a wide spectrum antitumor activity through induction of cellular apoptosis and cell cycle arrest. We investigated the effects and mechanisms of CD437 on cell cycle arrest of gastric cancer cells. MATERIALS AND METHODS: The activities of CD437 on cell growth were analyzed by measuring total cellular DNA. The effects of CD437 on cell cycle phase distribution were analyzed using flow cytometry. The levels of cell cycle associated proteins were analyzed by Western blot. The activities of cyclin-dependent kinases (cdks) were analyzed by phosphorylation of the histone H1 protein. RESULTS: CD437 at concentrations between 0.1 and 10 microM profoundly suppressed the growth of all six gastric cancer cell lines. Growth suppression associated with induction of G0/G1 or G2/M arrest was cell-line-dependent. CD437 decreased levels of cdk inhibitor p21 in G2/M-arrested SC-M1 cells. However, CD437 increased p21 levels in G0/G1-arrested AGS cells. Total and activated cyclin-dependent kinases were differentially regulated by CD437 in AGS and SC-M1 cells. CD437 (1 microM) induced activity of cdk2- and p34cdc2-associated H1 kinase by 14.6- and 1.8-fold, respectively, in SC-M1 cells. In contrast, CD437 slightly increased (1.6-fold) the cdk2-associated H1 kinase activity in AGS cells. CONCLUSION: CD437 profoundly suppressed the growth of gastric cancer cells, which was associated with cell-dependent induction of G0/G1 or G2/M arrest. The differential regulation of p21 that leads to alteration in the activity of cdks may play a critical role in cell-line-dependent regulation of cell cycle arrest following treatment with CD437.     

Effects of calcitriol, seocalcitol, and medium-chain triglyceride on a canine transitional cell carcinoma cell line

BACKGROUND: Transitional cell carcinoma (TCC) in dogs is associated with high morbidity and mortality. Calcitriol and its analog seocalcitol, combined with medium-chain triglyceride (MCT), have potential for the treatment of this disease. MATERIALS AND METHODS: TCC cells were treated with calcitriol or seocalcitol, alone or combined with MCT. Cell growth, cell cycle kinetics, vitamin D receptor (VDR) localization and expression, and Bcl-2 expression were measured. RESULTS: Canine TCC expresses high levels of nuclear VDR. Furthermore, calcitriol and seocalcitol significantly inhibited cell growth and calcitriol caused G0/G1 cell cycle arrest. Bcl-2 expression was slightly decreased in cells treated with these compounds, although no significant changes in VDR expression were observed. MCT enhanced the growth inhibitory effect of both compounds. CONCLUSION: Calcitriol and seocalcitol inhibited TCC cell growth via induction of cell cycle arrest and MCT enhanced this effect. Therefore, calcitriol and seocalcitol with MCT may have therapeutic potential for canine bladder cancer.     

Sodium phenylacetate (NaPa) improves the TAM effect on glioblastoma experimental tumors by inducing cell growth arrest and apoptosis

BACKGROUND: Multiform glioblastomas represent the most aggressive brain tumors. Here, the cooperative effects of sodium phenylacetate (NaPa) and/or tamoxifen (TAM) on CNS1 and 9L glioblastoma cell lines in vitro and in an experimental animal tumor model were investigated. MATERIALS AND METHODS: The drug effects on cell cycle and apoptosis were investigated by flow cytometry. CNS1 cells were implanted subcutaneously in nude mice to form tumors which were then treated with NaPa, TAM or NaPa/TAM. RESULTS: A significant inhibitory effect of NaPa on the two glioma cell lines (LD50 of 10 mM) was observed. 10(-5) M of TAM inhibited approximately 35% of 9L cell growth, and 90% of CNS1 cell growth. When a combination of both drugs included 10(-9) M of TAM, inhibition of about 50% of 9L cell growth and 75% of CNS1 cell growth occurred. The NaPa/TAM combined treatment increased the number of G0/G1 arrested cells and apoptotic cells as compared to treatments with NaPa or TAM alone. Inhibition of CNS1 tumor growth were observed after a two week treatment with NaPa (32 mg/kg/day) or TAM (6 mg/kg/day). CONCLUSION: These results showed a synergistic effect between these two drugs on tumor cell proliferation, caused by cell cycle arrest in the G0/G1 phase and by induction of apoptosis.     

Benzo(a)pyrene metabolism by lymphocytes from normal individuals and individuals carrying the Mediterranean variant of glucose-6-phosphate dehydrogenase

In vitro growing human lymphocytes (HL) and fibroblasts, isolated from glucose-6-phosphate dehydrogenase (G6PD)-deficient subjects (Mediterranean variant), show a sharp decrease in this enzymatic activity and in NADPH:NADP+ ratio. These cells are less able than controls to hydroxylate benzo(a)pyrene (BaP) when tested in the absence of an exogenous NADPH-generating system. They exhibit great resistance to the toxic effect of BaP. G6PD-deficient fibroblasts are less prone than controls to in vitro transformation by BaP. To investigate whether this depends on a decreased production of active BaP metabolites and BaP:DNA adducts by G6PD-deficient cells, BaP metabolism was studied in G6PD-deficient HL cultured in vitro in the presence of mitogens and treated with BaP for 24 hr. HPLC profiles of organo- and water-soluble metabolites revealed that both types of benzo(a)anthracene (BaA)-induced HL produced: 4,5-, 7,8-, 9,10-diols, 1,3-, 3,6-quinones, 3-, 9-hydroxy and 2 peaks of more polar metabolites. There was a 25-76% decrease of organo- and water-soluble metabolites in the G6PD-deficient cells. When HL were incubated with 7,8-diol, the formation of metabolites mutagenic for Salmonella typhimurium (His-) was very low in G6PD-deficient cells. BaP:deoxyadenosine (dAde) and BaP:deoxyguanosine (dGua) adducts were identified after incubation of both types of HL with BaP. There was a 31-79% fall in adduct formation by G6PD-deficient cells. Our results indicate that G6PD-deficient human lymphocytes are less able to metabolize BaP than normal lymphocytes. We suggest that the NADPH pool is inadequate, in deficient cells, for active BaP metabolism.     

香加皮提取物抗肿瘤活性的研究

背景与目的： 研究中药香加皮提取物的抗肿瘤活性,探讨其抗肿瘤机制。 材料与方法： 采用噻唑蓝(3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyltetrazolium bromide, MTT)法观察香加皮醇提物和水提物对MCF-7、TE-13、QG56、SMMC7721、T24、Hela、K562等肿瘤细胞增殖的抑制作用,应用流式细胞术检测肿瘤细胞的周期变化和对凋亡的影响。 结果： 香加皮醇提物和水提物均能明显抑制多种肿瘤细胞的增殖,呈浓度依赖性;醇提物的抑瘤作用(IC50<10 μg/ml)强于水提物(IC50<40 μg/ml),并且差异具有统计学意义(P<0.05)。香加皮醇提物可将MCF-7细胞阻滞于G0/G1期,并呈时间依赖性地诱导MCF-7细胞发生凋亡。10 μg/ml香加皮醇提物作用MCF-7细胞24 h,与对照组相比,G0/G1期细胞显著增多,作用48 h,凋亡率从对照组的0.70％±0.13％升高到13.54％±2.12％,两者之间的差异有统计学意义(P<0.05)。 结论： 香加皮提取物对体外肿瘤细胞的增殖具有显著的抑制作用,醇提物强于水提物,其抗瘤机制可能与阻滞肿瘤细胞周期和诱导凋亡有关。     

American ginseng and breast cancer therapeutic agents synergistically inhibit MCF-7 breast cancer cell growth

BACKGROUND AND OBJECTIVES: American ginseng (Panax quinquefolius L.) purportedly alleviates menopause symptoms because of putative estrogenicity. METHODS: Using a standardized American ginseng (AG) extract in MCF-7 breast cancer cells, the objectives were to evaluate the ability of AG to induce the estrogen- regulated gene pS2 by Northern blot analysis, determine the effect on cell growth using the MTT assay, and evaluate the cell cycle effects by flow cytometry. RESULTS: AG and estradiol equivalently induced RNA expression of pS2. AG, in contrast to estradiol, caused a dose-dependent decrease in cell proliferation (P < 0.005). AG had no adverse effect on the cell cycle while estradiol significantly increased the proliferative phase (percent S-phase) and decreased the resting phase (G(0)-G(1) phase) (P < 0.005). Concurrent use of AG and breast cancer therapeutic agents resulted in a significant (P < 0.005) suppression of cell growth for most drugs evaluated. CONCLUSIONS: In vitro use of AG and breast cancer therapeutics synergistically inhibited cancer cell growth.     

Inhibitory targeting of checkpoint kinase signaling overrides radiation-induced cell cycle gene regulation: a therapeutic strategy in tumor cell radiosensitization?

BACKGROUND AND PURPOSE: The tumor cell defense response to ionizing radiation involves a temporary arrest at the cell cycle G(2) checkpoint, which is activated by a signaling cascade initiated by the ATM kinase response to DNA damage, ultimately leading to the outcome of further cell survival if the DNA is properly repaired. The inhibitory targeting of the checkpoint kinase signaling elicited by ATM may define a biologically based strategy to override the G(2) phase delay that prevents mitotic entry after DNA damage, thereby increasing the probability of mitotic cell death following exposure to ionizing radiation. MATERIALS AND METHODS: Breast carcinoma cell lines with intact or defective function of the tumor-suppressor protein BRCA1 were exposed to ionizing radiation in the absence or presence of a specific inhibitor (UCN-01) of the checkpoint kinase CHK1, and the response profiles of cell cycle distribution and G(2) phase regulatory factors, as well as the efficiency of clonogenic regrowth, were analyzed. RESULTS: The radiation-induced G(2) phase accumulation was preceded by a transient down-regulation of the G(2) phase-specific polo-like kinase-1 and cyclin B1, which required intact function of both BRCA1 and CHK1. The concomitant treatment with UCN-01 seemed to amplify the cytotoxic effect of ionizing radiation on clonogenic regrowth. CONCLUSION: The effector mechanism of DNA damage on cell cycle gene regulation signals through the checkpoint kinase network. Among molecular cell cycle-targeted drugs currently in pipeline for testing in early phase clinical trials, CHK1 inhibitors may have therapeutic potential as radiosensitizers.     

Increased cyclooxygenase-2 expression in human squamous cell carcinomas of the head and neck and inhibition of proliferation by nonsteroidal anti-inflammatory drugs

BACKGROUND: Cyclooxygenase-2 (COX-2) has been found to be up-regulated in several types of human cancers and its role in the carcinogenic process has been proposed The aim of this study was to examine the expression of COX-2 in human squamous cell carcinoma of the head and neck (SCCHN) and to find out the effects of COX-2 inhibitors on the growth of cultured cells. MATERIALS AND METHODS: We investigated the effect of indomethacin and NS-398 at various concentrations on the growth of SCCHN cell lines using cell proliferation assay, cell cycle analysis and quantification of apoptosis. RESULTS: Immunostaining revealed a significantly increased COX-2 expression in tumor tissues compared with normal controls (p<0.05). Western blotting analysis using a COX-2 antibody, indicated that seven SCCHN cell lines tested constitutively expressed COX-2 protein. Treatment of head and neck cancer cells with NS-398 (10-200 microM) or indomethacin (50-1000 microM) for 72 hours showed a significant dose-dependent inhibition of cell growth (p<0.01) and a significant increase in the number of cells in the G0/G1-phases of the cell cycle with a concomitant reduction at the S-phase in a dose-dependent manner (p<0.05). NS-398 was more effective in cell cycle arrest and growth inhibition than indomethacin (p<0.05) and induced significant apoptosis in two out of three SCCHN cell lines tested at the concentration of 100 microM. CONCLUSION: Our study showed that COX-2 could be a participant in carcinogenesis of SCCHN and that COX-2 inhibitors would be a potential tool for the treatment and prevention of SCCHN.