宫颈癌细胞系及宫颈组织PTEN基因启动子区甲基化状态的检测与分析

目的:探讨宫颈癌细胞系及宫颈组织中PTEN基因启动子甲基化状态对其表达的影响。方法:选取体外培养的4种宫颈癌细胞系HeLa、Caski、C-33A、HT-3,以及18例宫颈癌组织和8例宫颈正常组织为研究对象。应用重亚硫酸盐测序PCR(bisulfite genomic sequencing PCR,BSP)联合TA克隆测序检测PTEN基因启动子甲基化状态;采用去甲基化药物5-氮杂-2'-脱氧胞苷(5-Aza-dC)处理体外培养的4种宫颈癌细胞系,RT-PCR法检测处理前后PTEN基因mRNA转录表达的差异,并与本课题组前期5-Aza-dC作用前后4种细胞系表达谱芯片PTEN基因的差异进行比较分析。结果:4种宫颈癌细胞系、宫颈癌组织及正常宫颈组织中PTEN基因启动子区均呈低甲基化状态;且宫颈癌组织与正常宫颈组织PTEN基因启动子甲基化水平无显著差异(T=34.5,P=0.720)。表达谱芯片中4种细胞系PTEN基因差异倍数均0.5且2。RT-PCR显示,5-Aza-dC处理前后4种细胞系中PTEN mRNA表达无显著差异(HeLa:t=0.384,P=0.738;Caski:t=0.073,P=0.949;C-33A:t=0.097,P=0.931;HT-3:t=0.073,P=0.542)。结论:宫颈癌组织及宫颈癌细胞系中未发现PTEN基因启动子区CpG岛的高甲基化,PTEN基因表达的差异与该基因启动子区甲基化无显著相关性。     

表达谱基因芯片筛选子宫颈癌甲基化差异基因的研究

目的筛选人乳头瘤病毒（HPV）阳性宫颈癌细胞系中由于HPV感染诱导沉默的特异抑癌基因,探讨HPV感染可能导致抑癌基因发生甲基化的机制。方法选取HPV阳性HeLa和Caski宫颈癌细胞系和HPV阴性C-33A和HT-3宫颈癌细胞系,分别经去甲基化药物5-氮杂-2′-脱氧胞苷（5-Aza～CdR,Aza）处理,采用Agilent人类全基因组表达谱芯片检测Aza处理前后细胞全基因组的表达水平,采用实时荧光定量PCR（RTqPCR）验证芯片结果,亚硫酸氢盐-基因组测序法（BGS）检测目的基因甲基化水平。结果①芯片结果显示：HPV阳性和阴性细胞系共721条基因表达存在差异。②用药后表达显著上调的基因：HeLa细胞系825条,Caski细胞系815条,c_33A细胞系1023条,HT-3细胞系1196条。HeLa和Caski细胞系共表达上调的基因为182条,剔除阴性细胞系共表达上调基因,筛选出阳性细胞系HPV相关表达上调基因97条,与阴性细胞系基因表达比较,差异有统计学意义（P〈0．01）,最终筛选出13条差异表达基因,其中具有功能者7条。③RT-qPCR结果：筛选基因在宫颈癌细胞系中的表达与芯片检测结果一致;HPV阳性宫颈癌细胞系中甲基化频率明显高于阴性细胞系,支持芯片结果。结论筛选出的7条新的潜在抑癌基因可能由于HPV感染诱导发生甲基化而沉默,为进一步探讨HPV诱导抑癌基因发生甲基化的机制提供实验基础。     

PTEN基因逆转卵巢上皮性癌细胞耐药的机制研究

目的通过检测PTEN基因在卵巢上皮性癌（卵巢癌）顺铂敏感细胞株0V2008及0V2008配对的顺铂耐药细胞株C13K中的表达,探讨转染PTEN基因能否逆转C13K细胞对顺铂的耐药及其相关机制。方法半定量RT-PCR技术和蛋白印迹法检测0V2008和C13K细胞中PTENmRNA和蛋白的表达。将野生型PTEN基因真核表达质粒在脂质体介导下转染C13K细胞,同时以转染空载体和未转染的C13K细胞作为对照,分别应用RT-PCR技术检测各组细胞PTENmRNA表达的变化,应用蛋白印迹法检测各组细胞PTEN、蛋白激酶B（AKT）及磷酸化AKT（p-AKT）蛋白表达的变化;四甲基偶氮唑蓝（M1Tr）比色法观察转染PTEN基因后C13K细胞对顺铂敏感性的变化,流式细胞仪分析顺铂作用后的细胞凋亡情况。结果（1）PTENmRNA在0V2008和C13K细胞中的表达水平分别为1．02±0.05和0．45±0.03,而0V2008、C13K细胞中PTEN蛋白的表达水平分别为1.02±0.07、0．55±0．03,两种细胞PTENmRNA和蛋白的表达水平分别比较,差异均有统计学意义（P〈0．05）。（2）PTEN基因转染48h后,C13K细胞中PTENmRNA、蛋白的表达水平分别为2.04±0．10和0．94±0．04,分别与转染空载体和未转染的C13K细胞比较,差异均有统计学意义（P〈0．01）;p-AKT蛋白的表达水平（0.94±0．07）较转染空载体（1．66±0．10）和未转染（1．68±0．14）的C13K细胞显著降低（P〈0．05）。（3）转染PTEN基因的C13K细胞对顺铂的半数抑制浓度（IC50）为（7.2±0．3）μmol／L,明显高于转染空载体和未转染的C13K细胞[分别为（12．7±0．4）、（13．0±0．3）μmol／L,P〈0,05]。（4）顺铂作用24h后,转染PTEN基因、转染空载体和未转染的C13K细胞的凋亡率分别为（41.7±0.9）％、（18．6±0．7）％和（15,3±0．8）％,前者明显高于后两者（P〈0．01）。结论PTEN基因在0V2008细胞中的表达明显高于C13K细胞。转染野生型PTEN基因能有效提高C13K细胞内PTEN基因的表达,并通过降低C13K细胞中AKT磷酸化的水平恢复C13K细胞对顺铂的敏感性。     

HeLa和SiHa细胞中Skp2、p27和C-myc的表达

目的:检测S期激酶相关蛋白2(Skp2)、C-myc、p27在宫颈癌HeLa及SiHa细胞系中的表达,明确这些指标在宫颈癌细胞系表达的意义。方法:通过细胞免疫组化、Westernblot、RT-PCR技术分析Skp2、p27和C-myc在蛋白和mRNA水平表达的差异。结果:免疫组化显示:He-La细胞中Skp2和C-myc表达强度高于SiHa细胞(P=0.032和P=0.026),而HeLa细胞中p27蛋白表达弱于SiHa细胞(P=0.035)。Westernblot显示:在HeLa细胞中Skp2和C-myc蛋白表达分别是SiHa细胞表达量的2.8倍和1.5倍;而SiHa细胞中的p27蛋白的表达水平是HeLa细胞中表达的2.7倍。RT-PCR结果显示:HeLa细胞中内源Skp2和C-myc的mRNA表达水平高于SiHa细胞(P=0.034和P=0.028),而SiHa细胞中的p27的mRNA表达水平高于HeLa细胞的表达水平(P=0.002)。结论:Skp2及C-myc在HeLa细胞中的表达水平高于SiHa细胞中的表达水平,而p27在HeLa细胞中的表达水平低于SiHa细胞中的表达水平。     

短发夹状RNA干扰对子宫颈癌细胞中Pin1基因表达及细胞增殖和凋亡的影响

目的研究短发夹状RNA(shRNA)干扰对宫颈癌细胞中Pin1基因表达及细胞增殖和凋亡的影响。方法构建靶向Pin1基因的shRNA真核表达质粒pSIREN-Pin1,在脂质体介导下转染人宫颈癌细胞系HeLa细胞(HeLa/p-shRNA组),同时以对照质粒pSIREN-Con(HeLa/p-Con组)和无血清培养基转染HeLa细胞(HeLa组)作为对照,分别应用RT-PCR技术及蛋白印迹法检测Pin1mRNA及蛋白表达水平,四甲基偶氮唑蓝(MTT)比色法和软琼脂细胞克隆实验检测细胞增殖状况,流式细胞仪分析细胞凋亡情况。结果转染后48h,HeLa/p-shRNA组Pin1mRNA及蛋白表达水平分别为0·19±0·05和0·33±0·14,HeLa/p-Con组分别为0·84±0·16和0·79±0·17,HeLa组分别为0·89±0·11和0·81±0·15,前组Pin1mRNA及蛋白表达水平分别与后两组比较,差异均有统计学意义(P<0·05);转染pSIREN-Pin1质粒后HeLa细胞中Pin1mRNA及蛋白表达抑制率分别为77%和58%。MTT比色法检测显示,HeLa/p-shRNA组细胞增殖率明显下降(P<0·05)。软琼脂克隆实验显示,HeLa/p-shRNA组细胞克隆小而稀疏,细胞克隆形成率为(12±3)%,明显低于HeLa/p-Con组的(20±5)%和HeLa组的(24±4)%(P<0·05)。流式细胞仪分析显示,HeLa/p-shRNA组细胞凋亡率为(24·3±5·7)%,明显高于HeLa/p-Con组的(5·0±1·4)%和HeLa组的(1·8±0·4)%(P<0·05)。结论shRNA干扰技术能有效抑制靶基因Pin1的表达,进而可抑制宫颈癌细胞增殖并诱导细胞凋亡增加,为宫颈癌的基因研究及治疗提供新思路。     

RNA干扰技术阻抑survivin基因表达对子宫颈癌细胞放射和化疗敏感性的影响

目的探讨利用RNA干扰(RNAi)技术阻抑survivin基因的表达,对宫颈癌HeLa细胞放射敏感性和化疗敏感性的影响。方法通过脂质体介导,将含survivin基因小分子干扰RNA的重组真核表达质粒pSilencer2．1-s2、阴性对照质粒pSilencer2．1-NC和空载质粒pSilencer2．1-U6 neo转染宫颈癌细胞系HeLa,获得HeLa-s2、HeLa-NC和HeLa-U6 neo细胞,同时设未转染的HeLa细胞为阴性对照。RT-PCR技术、蛋白印迹法分别检测survivin mRNA和蛋白的表达水平,并计算survivin mRNA和蛋白表达抑制率;激酶活性检测法测定波长在405 nm处的吸光度(A405)值,表示半胱氨酸天冬氨酸蛋白酶3(caspase-3)活性;流式细胞仪检测细胞凋亡率;平板克隆形成实验观察细胞的放射敏感性变化,以克隆形成率表示;四甲基偶氮唑蓝比色法检测细胞存活率并计算顺铂的50％抑制浓度(IC50)。结果与HeLa-NC、HeLa-U6 neo及未转染的HeLa细胞比较,HeLa-s2细胞survivin mRNA和蛋白表达水平明显下降,survivin mRNA和蛋白表达抑制率分别为(62．8±0．3)％和(60．1±0．5)％。HeLa-s2细胞的A405值为1．261±0．043,未转染的HeLa细胞的A405值为0．314±0．012,两者比较,差异有统计学意义(P<0．05)。HeLa-s2与未转染的HeLa细胞相比,细胞凋亡率明显升高(P<0．05),分别为(29．23±1．41)％和(2．74±0．32)％。不同照射剂量下,HeLa-s2与未转染的HeLa细胞分别比较,克隆形成率均明显降低(P<0．05)。在同一顺铂浓度下,HeLa-s2与未转染的HeLa细胞比较,细胞存活率显著降低(P<0．05),HeLa-s2细胞对顺铂的IC50值较未转染的HeLa细胞下降显著(P< 0．05),分别为(0．873±0．021)和(9．212±0．033)μg／ml。结论利用RNAi技术可阻抑HeLa细胞中survivin基因的表达,增强caspase-3活性,诱导细胞凋亡,显著提高细胞的放射敏感性和对顺铂的化疗敏感性。     

卵巢上皮性癌组织中p16INK4A基因表达缺陷的意义及其与甲基化的关系

目的研究抑癌基因p16INK4A在卵巢上皮性癌(卵巢癌)组织及细胞系中的表达变化,分析其表达变化与甲基化的关系。方法选取7种卵巢癌细胞系、18份卵巢癌组织和10份正常卵巢组织为研究对象。采用甲基化特异性PCR方法检测p16INK4A基因甲基化状态;RT-PCR技术检测p16INK4A基因的mRNA表达;蛋白印迹(western blot)法检测P16INK4A蛋白的表达。5-杂氮-2′-脱氧胞苷对p16INK4A基因甲基化的卵巢癌细胞进行去甲基化处理,再次进行p16INK4A基因的mRNA和蛋白表达的检测,以及p16INK4A基因甲基化的分析。检测5-杂氮-2′-脱氧胞苷处理前后卵巢癌细胞的生长情况;并将处理前后的细胞接种于裸鼠,观测肿瘤的体积、重量。结果3种卵巢癌细胞系(Anglne、SW626和OVCAR3细胞)、6份卵巢癌组织中存在p16INK4A基因甲基化,卵巢癌细胞和卵巢癌组织中的甲基化率分别为3/7和33%(6/18)。卵巢癌细胞系、卵巢癌组织和正常卵巢组织中,p16INK4A基因的mRNA相对含量的平均值分别为0·34±0·11、0·81±0·13、1·52±0·12,蛋白相对含量的平均值分别为0·56±0·14、1·32±0·12、2·09±0·11,卵巢癌细胞系、卵巢癌组织分别与正常卵巢组织相比,差异均有统计学意义(P<0·05)。有甲基化表现的卵巢癌细胞和组织中p16INK4A基因的mRNA和蛋白表达均下降。5-杂氮-2′-脱氧胞苷处理能使p16INK4A基因甲基化的卵巢癌细胞中的p16INK4A基因的mRNA和蛋白重新表达或表达增高。与去甲基化处理前比较,去甲基化处理后Anglne、SW626和OVCAR3细胞的生长速度均减慢;接种去甲基化处理的OVCAR3细胞的裸鼠中,肿瘤体积和重量明显减小,分别为(0·243±0·022)cm3、(0·035±0·004)g。结论p16INK4A基因的表达下降或缺失在卵巢癌的发生中起重要作用,DNA甲基化是其表达缺陷的原因,去甲基化处理可以恢复p16INK4A基因的表达并抑制卵巢癌细胞的增殖。     

子痫期胎盘组织差异表达基因PP2ACβ的初步研究

目的寻找并克隆子痫期胎盘组织高表达基因蛋白磷酸酶2A催化亚基β（PP2ACβ）,研究其mR-NA及蛋白水平的表达,探讨其在子痫期中的作用。方法标本取自2004年5月至2004年12月天津中心妇产科医院重度子痫期及正常孕妇各30例。应用荧光mRNA差异显示技术（FDD）发现并克隆子痫期胎盘组织差异表达的基因PP2ACβ,进一步应用半定量RT-PCR及免疫组化技术分析差异表达基因PP2ACβmRNA及其蛋白水平PP2A的表达。结果通过FDD发现PP2ACβ基因在子痫期胎盘组织中高表达,半定量RT-PCR证实PP2ACβmRNA在子痫期胎盘组织中的表达（0.888±0.104）高于正常胎盘组织（0.692±0.099）,差异有统计学意义（P〈0.05）,免疫组化验证其蛋白水平PP2A的表达（0.14±0.02）亦高于正常（0.12±0.02）,差异有统计学意义（P〈0.05）。结论FDD可有效用于筛选子痫期胎盘组织差异表达基因;PP2A可能通过促进胎盘滋养层细胞的凋亡及阻碍血管生成而参与子痫期的发病。     

GPR30在宫颈癌细胞生长中的作用及其机制研究

目的:体外研究雌激素膜受体GPR30对宫颈癌细胞生长的影响及其作用机制。方法:选择宫颈腺癌HeLa和宫颈鳞癌SiHa细胞株,分别用GPR30特异性激动剂G1和拮抗剂G15处理宫颈癌细胞株。RT-PCR、Western blot法检测处理前后宫颈癌HeLa与SiHa细胞中GPR30、TLR3 mRNA及其蛋白表达变化;MTT法检测G1、G15及Poly I:C处理对宫颈癌细胞生长的影响。结果:(1)HeLa细胞中GPR30表达量高于SiHa细胞。G1处理后HeLa、SiHa细胞中GPR30 mRNA及其蛋白表达水平增高,与对照组比较,差异有统计学意义(P0.05;P0.05);G15处理后,HeLa、SiHa细胞中GPR30 mRNA及其蛋白表达水平降低,与对照组比较,差异有统计学意义(P0.05;P0.05)。(2)HeLa、SiHa细胞中TLR3 mRNA表达量分别为(0.5327±0.05373)、(0.3526±0.05774),蛋白表达量分别为(0.3572±0.097039)、(0.5002±0.09718)。G1能降低He La、Si Ha细胞中TLR3mRNA及其蛋白表达水平,G1 10-6mol/L处理组与对照组比较差异有统计学意义(P均0.05)。G15能增高HeLa、SiHa细胞中TLR3 mRNA及其蛋白表达水平,G15 10-5mol/L处理组与对照组比较,差异有统计学意义(P均0.05),与Poly I:C处理组比较差异无统计学意义(P0.05)。(3)10-6mmol/L、10-5mmol/L G1分别处理后,宫颈癌HeLa、SiHa细胞生长增殖率分别为(16.68±5.86)%、(26.67±3.25)%及(14.99±6.43)%、(22.72±1.77)%,与空白对照组相比,差异有统计学意义(P均0.05)。10-6mmol/L、10-5mmol/L G15分别处理后,宫颈癌HeLa、SiHa细胞的生长抑制率分别为(21.09±2.32)%、(22.99±3.15)%及(15.86±6.49)%、(19.18±2.61)%,与空白对照组相比,差异有统计学意义(P均0.05)。结论:宫颈癌细胞中存在雌激素膜受体GPR30表达,宫颈腺癌细胞中GPR30表达量高于宫颈鳞癌细胞,体外调节GPR30表达可影响宫颈癌细胞生长。抑制GPR30表达可通过上调TLR3表达而抑制宫颈癌细胞生长,GPR30可能成为宫颈癌治疗的新靶点。     

NS-398对宫颈癌细胞系的增殖抑制作用及对survivin基因表达的影响

目的:研究选择性环氧合酶-2(COX-2)抑制剂NS-398对宫颈癌HeLa,SiHa细胞系的增殖、凋亡作用及其对凋亡抑制基因survivin表达的影响。方法:体外培养宫颈癌HeLa,SiHa细胞系,用四甲基偶氮唑蓝(MTT)比色法分析不同浓度的NS-398分别作用于HeLa,SiHa细胞系24h、48h后对细胞增殖的作用;流式细胞仪(FCM)检测对细胞凋亡的作用;RT-PCR分析对凋亡抑制基因survivin表达的影响。结果:MTT检测显示,NS-398可抑制宫颈癌HeLa,SiHa细胞系增殖,并有浓度时间依赖性,与对照组相比差异有统计学意义(P<0.05)。FCM检测提示,NS-398可诱导HeLa,SiHa细胞系凋亡,有浓度依赖性,与对照组相比差异有统计学意义(P<0.05)。RT-PCR分析表明,NS-398可抑制HeLa,SiHa细胞系凋亡抑制基因survivinmRNA表达,与对照组的差异有统计学意义(P<0.05)。结论:NS-398可抑制宫颈癌HeLa,SiHa细胞增殖,诱导凋亡,其机制与抑制凋亡抑制基因survivin mRNA表达有关,为宫颈癌治疗提供了新的靶点和理论依据。     

Recombinant adenovirus-p53 gene transfer and cell-specific growth suppression of human cervical cancer cells in vitro and in vivo

PURPOSE: We investigated the time-course expression patterns of p53 and E6 on cervical cancer cells to obtain a molecular level understanding of cell-dependent tumor growth suppression effects of recombinant adenovirus expressing p53 in vitro and in vivo. METHODS: Four human papillomavirus (HPV)-infected human cervical cancer cell lines (HPV 16-positive cells, CaSki and SiHa cells; and HPV 18-positive cells, HeLa and HeLaS3 cells) were used. Also, HPV negative C33A and HT3 cell line that has a mutation on p53 gene were used. After infection with AdCMVp53, the cell growth inhibition was studied via cell count assay, MTT assay, and Neutral red assay. After transfecting AdCMVp53 and AdCMVLacZ into the cancer cells-xenografted nude mice, antitumor effects were investigated for 1 month, respectively. RESULTS: For each cervical cancer cell, IC50 was as follows; CaSki (68.5 multiplicity of infection, or MOI), SiHa (43.5 MOI), HeLa (31 MOI), HeLaS3 (42 MOI), C33A (21 MOI), and HT3 (62 MOI). In particular, complete inhibition of cell growth was observed at 125 MOI in both CaSki and SiHa cells. However, the complete inhibition was detected at 62.5 MOI in HeLa and HeLaS3. In contrast, at these MOI, no suppression of cell growth was observed when cells were infected with recombinant adenovirus expressing beta-gal as a negative control. The levels of p53 protein were notably expressed in CaSki and HeLa more than in SiHa and HeLaS3 on days 2 and 4. However, the p53 was only detected in HeLaS3 on day 6. In contrast, p53 expression was continually maintained in C33A and HT3 during the same periods. After transfection AdCMVp53 into CaSki- and SiHa-xenografted nude mice, the size of tumor was remarkably decreased in SiHa cells as compared to AdCMVLacZ transfection. CONCLUSION: The adenovirus-mediated p53 gene transfection was done effectively in vitro and in vivo. Also, the antitumor effects were accomplished via differential role of p53-specific apoptotic cell death, which is dependent upon the cervical cancer cell line.     

Sensitivity to Fas-mediated apoptosis in high-risk HPV-positive human cervical cancer cells: relationship with Fas, caspase-8, and Bid

OBJECTIVE: Binding of Fas ligand or agonistic anti-Fas antibody to the death receptor Fas can activate a caspase-cascade resulting in apoptosis. In the present study, the functionality of the Fas pathway was studied in human cervical cancer cells with different HPV and p53 status. METHODS: HeLa (HPV-18 positive), CaSki, and SiHa (both HPV-16 positive) contain wild-type p53, while C33A (HPV negative) expresses mutant p53. Fas cell surface expression was determined by flow cytometry. Expression of proteins involved in the apoptotic pathway was analyzed by Western blotting and apoptosis was measured by acridine orange staining of nuclear chromatin. RESULTS: Despite high Fas membrane expression in the HPV-positive cells, CaSki was highly sensitive, HeLa slightly sensitive, and SiHa and C33A were resistant for agonistic anti-Fas antibody. Almost undetectable Fas membrane levels can explain the non-responsiveness of C33A for anti-Fas. Although interferon-gamma (IFNgamma) strongly and cisplatin to a lesser extend enhanced Fas membrane expression in all HPV-positive cells, sensitization to anti-Fas by IFNgamma or cisplatin was only observed in HeLa. Analysis of the Fas apoptotic pathway showed that anti-Fas treatment induced caspase-8 activation and concomitantly Bid cleavage, caspase-9 and caspase-3 activation, PARP cleavage and apoptosis in HeLa and CaSki. IFNgamma plus anti-Fas treatment, in contrast to anti-Fas alone, facilitated caspase-8 activation in HeLa and SiHa, while an increase in Bid cleavage, caspase-9 activation and apoptosis was only observed in HeLa. Apoptotic failure in SiHa (even in the presence of IFNgamma) was probably due to low caspase-8, almost undetectable Bid protein levels and therefore lack of caspase-9 activation. CONCLUSION: Sensitivity to anti-Fas depends on Fas, caspase-8, and Bid protein levels in cervical cancer cells. Additionally, IFNgamma and cisplatin can increase sensitivity to anti-Fas in a subset of HPV-positive cervical cancer cell lines by upregulation of Fas and caspase-8 expression without major changes in p53 levels.     

Sensitization of cervical cancer cell lines to low-dose radiation by retinoic acid does not require functional p53

OBJECTIVE: Current therapy for cervical cancer includes radiation therapy. Retinoic acid (RA) can increase the sensitivity of cervical cancer cell lines to radiation. The mechanism of this sensitization may not involve the p53 protein because the human papillomavirus (HPV) E6 protein, which is present in the majority of cervical cancers, promotes p53 degradation. The objective of this study was to determine if p53 is involved in the mechanism of RA radiosensitization. METHOD: The effects of radiation on cervical (SiHa, CC-1, and C33a) and vulvar (SW962) cancer cell lines under various experimental conditions were evaluated using clonogenic, Coulter Counter, electrophoretic mobility shift (EMSA) and a multi-probe RNase protection assay of p53-inducible genes. RESULTS: RA (5 microM 9-cis-RA) radiosensitized the SiHa and CC-1 cell lines that contain HPV-degraded p53, but did not radiosensitize the SW962 cell line, which is HPV negative and contains wild-type p53, nor the C33a cell line, which contains mutant p53 (R273C). Expression of mutant p53 (R273H) in SiHa cells increased the growth rate, but did not prevent RA-induced differentiation or radiosensitization at clinically relevant doses. Inhibition of p53 transactivation with pifithirin alpha did not prevent RA radiosensitization of SiHa at 5 Gy. RA repressed c-fos mRNA expression in control and irradiated SiHa cultures, but did not repress bcl-x(L), p53, GADD45, p21, bax, bcl-2, or mcl-1 mRNA expression. CONCLUSIONS: The mechanism of RA radiosensitization does not require functional p53 and may involve c-fos in cervical cancer cell lines.     

信号诱导增殖相关蛋白1和16、18型人乳头瘤病毒E6蛋白在宫颈癌中的表达及临床意义

目的:探讨信号诱导增殖相关蛋白1(SIPA1)和16、18型人乳头瘤病毒E6蛋白(HPV16/18E6)在宫颈癌中的表达及其与临床病理之间的关系。方法:Western blot检测宫颈癌C33A、HT3、SiHa、HeLa和CaSki细胞株中HPV16/18E6和SIPA1蛋白表达;免疫组织化学Envi-sion法检测正常宫颈组织(40例)和宫颈癌组织(174例)石蜡标本中HPV16/18E6和SIPA1蛋白;分析SIPA1和HPV16/18E6相互之间及其与宫颈癌盆腔淋巴结转移之间的关系。结果:宫颈癌细胞系中SIPA1与HPV16/18E6表达呈负相关。SIPA1在正常宫颈组织和宫颈癌组织中阳性率分别为87.5%和58.6%,差异有高度统计学意义(χ2=11.78,P=0.001);SIPA1蛋白在有和无盆腔淋巴结转移时阳性率分别为19.0%和71.2%,差异有高度统计学意义(χ2=21.45,P=0.000),且SIPA1阴性者发生淋巴结转移风险高于阳性者(OR=5.011,95%CI2.311～10.866,P<0.01)。HPV16/18E6在正常宫颈组织和宫颈癌组织中的阳性率分别为30.0%和79....     

Human nonmetastatic clone 23 type 1 gene suppresses migration of cervical cancer cells and enhances the migration inhibition of fungal immunomodulatory protein from Ganoderma tsugae

The authors investigate the effects of human nonmetastatic clone 23 type 1 (nm23-H1 ) gene and fungal immunomodulatory protein-Ganoderma tsugae (FIP-gts) on the metastatic potential of cervical cancer cells and assess whether nm23-H1 can influence the action of FIP-gts using cell migration and invasion assays and gelatin zymography. The nm23-H1 gene was stably transfected into Caski cells, which lacked nm23-H1 expression. The results show that nm23-H1 stably transfected Caski cells exhibit reduced cell migration but no change of cell invasion and matrix metalloproteinase (MMP)-2 and -9 activities. FIP-gts reduced cell migration in SiHa and nm23-H1 transfected Caski cells more significantly compared with Caski cells and reduced invasion in Caski and nm23-H1-transfected Caski cells, but it exerted no influence on MMP-2 and MMP-9 activities in them. Conclusively, the nm23-H1 gene suppresses cervical cancer cell migration but not invasion and activities of MMP-2 and MMP-9 and enhances the inhibition of FIP-gts upon migration.     

Cellular response to chemotherapy and radiation in cervical cancer

Effect of irradiation alone and irradiation after 5-fluorouracil (5-FU), paclitaxel, or cisplatin (CDDP) was investigated in human cervical cell lines (CaSki, ME180, SiHa, and C33A). High-risk human papillomavirus (HPV) (+) CaSki and SiHa cells were the most resistant to CDDP, 5-FU, and radiation treatments. Radiation and CDDP and 5-FU resulted in decreased survival of HPV 16 and 18 (+) cells, whereas addition of paclitaxel to radiation treatments decreased killing. Enhanced killing of ME180 cells containing HPV39 sequences was demonstrated with chemoradiotherapy with all agents. HPV(-) C33A was more sensitive to radiation than the other cell lines, and the addition of chemotherapeutic agents did not result in significant change in cytotoxicity. Expression of survivin was inversely proportional to cell sensitivity to CDDP, 5-FU, and radiation. Constitutive AKT levels are the lowest in cell lines that are the most resistant to CDDP, 5-FU, and radiation. These data provide correlation of response to combined therapeutic modalities with HPV status of cervical cancer and expression of survivin and AKT.