cyclin G1和p16蛋白在子宫平滑肌肿瘤中的表达及意义

目的:探讨cyc lin G1和p16蛋白在子宫平滑肌肿瘤(USMTs)中的表达及临床意义。方法:应用免疫组织化学方法检测35例良性子宫平滑肌瘤(UL),18例子宫平滑肌肉瘤(LMS)及22例交界性子宫平滑肌瘤(BLM):包括13例富于细胞型子宫平滑肌瘤(CL)、4例奇异型子宫平滑肌瘤(BL)及5例恶性潜能未定型子宫平滑肌瘤(UMP)的cyc lin G1和p16蛋白的表达。结果:cyc lin G1和p16蛋白在LMS组的阳性表达率和表达强度均显著高于UL组及BLM组(P<0.05);在UL组与BLM组,cyc lin G1和p16蛋白阳性表达率和表达强度均无明显差异(P>0.05)。结论:cyc lin G1和p16蛋白的异常表达与LMS的发生发展密切相关,细胞周期调控失衡可能是LMS的发病机制之一。

p16负向调控通路与辐射诱导的G_1期阻滞

辐射诱导细胞发生G1期阻滞,其分子调控机制尚不十分清楚。近期文献报道,独立于p53基因之外的p16-Cyclins-CDKs(细胞周期素依赖性激酶)细胞周期负向调控通路在紫外线和电离辐射照后发生改变,提示此通路可能在辐射诱导的G1期阻滞中发挥至关重要的作用。

1-Chloromethyl-6,7-dimethoxy-3,4-dihydro-1H-isoquinoline-2-sulfonic acid amide, a derivative of tetrahydroisoquinoline, induces granulocytic differentiation of the human leukemic HL-60 cells via G0/G1 phase arrest

Tetrahydroisoquinolines are known to have various biological effects, including antitumor activity. This study investigated the effect of 1-chloromethyl-6, 7-dimethoxy-3, 4-dihydro-1H-isoquinoline-2-sulfonic acid amide (CDST), a newly synthesized anticancer agent, on cellular differentiation and proliferation in HL-60 cells. Differentiation and proliferation of HL-60 cells were determined through expression of CD11b and CD14 surface antigens using flow cytometry and nitroblue tetrazolium (NBT) assay, and through analysis of cell cycle using propidium iodide staining, western blot analysis and immunoprecipitation, respectively. CDST induced the differentiation of HL-60, as shown by increased expression of differentiation surface antigen CD11b (but no significant change in CD14 expression) and increased NBT-reducing functional activity. DNA flow cytometry analysis indicated that CDST markedly induced a G0/G1 phase arrest of HL-60 cells. Subsequently, we examined the expre-ssion of G0/G1 phase cell cycle-related proteins, including cyclin-dependent kinases (CDKs), cyclins and cyclin dependent kinase inhibitors (CKIs), during the differentiation of HL-60. The levels of CDK2, CDK6, cyclin E and cyclin A were decreased, whereas steady-state levels of CDK4 and cyclin D1 were unaffected. The expression of the p27Kip1 was markedly increased by CDST, but not p21WAF1/Cip1. Moreover, CDST markedly enhanced the binding of p27Kip1 with CDK2 and CDK6, resulting in the reduced activity of both kinases. Taken together, these results demonstrate that CDST is capable of inducing cellular differentiation and growth inhibition through p27Kip1 protein-related G0/G1 phase arrest in HL-60 cells.

整体照射后胸腺细胞和脾细胞p16基因转录和蛋白表达的变化

研究X射线全身照射对小鼠胸腺细胞和脾细胞p16基因转录及蛋白表达的影响。采用Northern blot检测p16基因转录水平的变化;采用流式细胞术检测蛋白表达的变化。时程结果表明,2.0Gy照射后4-24h,胸腺细胞p16mRNA水平明显增高,8-48h P16蛋白表达显著增高(p<0.05-p<0.01);照射后4-8h脾细胞p16mRNA水平明显增高,24h P16蛋白表达显著增高(p<0.05)。量效结果表明,0.5-6.0Gy照射后,胸腺细胞p16mRNA水平呈剂量依赖性增高,P16蛋白表达在1.0-4.0Gy组明显高于假照射组(p<0.05-p<0.01);脾细胞p16mRNA水平亦增高,但增幅远低于胸腺细胞,P16蛋白表达在1.0-4.0Gy组明显高于假照射组(p<0.05-p<0.01)。X射线全身照射可诱导胸腺细胞和脾细胞p16基因转录及蛋白表达增高。p16表达参与整体照射诱导细胞G1期阻滞的分子调控。

在鼻咽癌细胞中EB病毒LMP1调控G1/S检测点重要相关蛋白的转录

目的在Tet-on-LMP1 HNE2鼻咽癌细胞中探讨EB病毒编码的潜伏膜蛋白1(LMP1)调控p16和cyclin D1的转录.方法采用Western blot方法、荧光素酶报道系统和RT-PCR方法检测在DOX诱导下不同时间点p16和cyclin D1的启动子活性和转录.结果LMP1可下调p16启动子活性和mRNA表达;同时上调cyclin D1启动子活性和mRNA表达.结论在Tet-on-HNE2鼻咽癌细胞中EB病毒LMP1在转录水平调控G1/S检测点重要相关蛋白p16和cyclin D1的表达.

p^(21)/waf1调节血管紧张素Ⅱ的抗增生作用

为了研究血管紧张素 (Ang )的抗内皮细胞增生作用及其分子生物学调节机制 ,将健康胎儿脐静脉内皮细胞培养至第 3代 ,用 Ang 培养并在不同时间计数细胞密度。用 TUNEL 或 EL ISA方法证明 Ang 或 CGP42 112 A诱导内皮细胞凋亡。用 RT- PCR和 Western Blot检测 p2 1 / waf1m RNA和 P2 1 蛋白的表达并测定条带的光密度。结果表明 ,在 Ang 培养后不同时间 ,细胞密度比对照减少 30 %左右 (P<0 .0 1)。Ang 作用后可以诱导典型的凋亡 ,阳性率极显著高于阴性对照组并具有剂量依赖性。Ang 组或 CGP42 112 A组的 p2 1 / waf1m RNA表达分别是对照组的 (4 73.6 9± 39.97) %和 (391.5 8± 48.2 4) %(P<0 .0 1)。Ang 组的 P2 1 蛋白表达比对照组极显著地增加并具有时间依赖性。认为 Ang 具有抗内皮细胞增生作用 ,其细胞内机制是同时诱导凋亡和 p2 1 / waf1m RNA与 P2 1 蛋白的高表达 ,使细胞发生 G1 期阻滞 ,增殖受抑。

Wound Healing Is a First Response in a Cancerous Pathway: Hyperplasia Developments to 4n Cell Cycling in Dysplasia Linked to Rb-Inactivation

In a series of publications, the hypothesis of a special-type of endo-polyploidy, marked by 4-chromatid chromosomes (diplochromosomes), in the initiation of tumorigenesis has been presented from in vitro experiments. This review uses cellular happenings in benign pre-neoplasia to substantiate this idea, which appears to be linked to the wound-healing process of injured tissue. Rarer association between a wound healing process and a cancer occurrence has long been known. The wound healing multi-program-system involved a phase of tetraploidy that showed diplochromosomes. The hypothesis is that the inflammatory phase may not always be sufficient in getting rid of dead and damaged cells (by apoptosis and autophagy), such that cells with genomic damage (DNA breakage) may survive by genomic repair associated with change to diplochromosomal tetraploidy. In vitro data have shown division of these cells to be an orderly, mechanistic two-step, meiotic-like system, resulting in only two types of progeny cells: 4n/4C/G1 and 2n/2C/G1 pseudo-diploid cells with hyperplastic-like growth-morphology. In vivo damage to tissues can be from many sources for example, physical, toxic environment or from a disease as in Barrett’s esophagus (BE) with acid reflux into the esophagus. For this condition, it is acknowledged that damage of the esophagus lining is a pre-condition to hyperplastic lesions of pre-neoplasia. These initial lesions were from “diploid” propagating cells and, 4n cells with G2 genomic content (no mitosis) accumulated in these lesions before a change to dysplasia. Cell cycle kinetics put these 4n cells in G1, which with S-phase entry would lead to asymmetric tetraploid mitoses, characteristic for dysplastic lesions. This change in hyperplasia to dysplasia is the root-essential condition for a potential progression of pre-neoplasia to cancer. In BE the hyperplastic lesion showed increasing gains of cells with inactivated p53 and p16[ink4a] genes, which destroyed the retinoblastoma (Rb) protein-control over S-phase entry from G1. Rb-protein is a key controller of cycling advancement from G1 (also for normal cells), and is frequently inactivated in tumor cells. Thus in BE, 4n/4C/G1 cells with mutated p53 and p16[ink4a] genes gained cycling ability to tetraploid aneuploid cell cycles, which constituted the change from hyperplasia to dysplastic lesions. In general, such lesions have high predictive value for a cancerous change. Proliferation rates of pre-neoplasia and progression have been shown to be increased by a component of the wound healing program.

外源性p16基因对人原发性肝癌细胞生物学行为的影响及其机制

目的:探讨导入外源性p16cDNA真核表达载体对人原发性肝癌细胞系SMMC- 772 1生物学行为的影响及其分子机制。方法:构建外源性p16基因真核表达载体并转染SMMC- 772 1细胞,经G4 18抗性筛选和扩增,得到稳定的表达株,并经RT -PCR及免疫细胞化学鉴定。通过生长曲线,流式细胞术,免疫细胞化学及Western -Blot等实验方法,对转基因后肿瘤细胞生物学行为及细胞周期调控因子CDK4 ,CyclinD1及pRb进行观察。结果:转染外源性p16基因的SMMC- 772 1细胞有外源性p16基因的整合及表达,细胞生长速度明显减慢,倍增时间明显延长,G1期细胞明显多于转基因前(P <0 .0 5 ) ;免疫细胞化学显示外源性p16表达可下调CDK4及cylinD1的表达(P <0 .0 5 ) ,Western -Blot提示转染外源性p16基因后细胞中磷酸化pRb表达明显降低。结论:外源性p16基因导入人肝癌细胞株SMMC -772 1中可稳定表达并使细胞停滞在G1期,抑制细胞生长,其机制可能为下调CDK4、cyclinD1的表达和抑制pRb磷酸化有关。

Mechanism of p53 downstream effectors p21 and Gadd45 in DNA damage surveillance

Both p21( WAF1/CIP1) and Gadd45 were activated in a p53-dependent manner in MCF-7 cells after being exposed to ionizing radiation. In order to investigate their roles in DNA damage surveillance, p21as/MCF-7 cells stably transfected by p21 antisense expression plasmid pC-WAFl-AS and Gadd45as/MCF-7 stably transfected by Gadd45 antisense expression plasmid pCMVas45 were established. It was observed that G1 arrest induced by radiation was significantly reduced in Gadd45as/MCF-7 cells as well as in p21as/MCF-7 cells. Repair of radiation damaged report gene greatly reduced in Gadd45asMCF-7 and p21as/MCF-7 cells. Apoptosis significantly increased in p21as/MCF-7 after exposure to radiation. These results suggest that both p21 and Gadd45 support cellular survival by taking roles in G1 arrest and DNA repair, furthermore, p21 protects cells from death by inhibiting apoptosis after exposure to ionizing radiation.

蛋氨酸脑啡肽对神经母细胞瘤细胞生长周期的影响及其机制研究

目的:研究蛋氨酸脑啡肽(Met-ENK)对神经母细胞瘤细胞SH-SY5Y的生长抑制作用及细胞周期的抑制机制。方法:采用MTT法检测细胞生长抑制率;流式细胞术检测Met-ENK作用后肿瘤细胞周期变化;Real-time PCR法检测Met-ENK作用后细胞内P21,P53基因转录量变化;Western blot法检测细胞内P21,P16,磷酸Rb蛋白表达量的变化情况。结果:MTT法结果显示,Met-ENK对SH-SY5Y细胞有生长抑制作用,在Met-ENK浓度为10-5mol·L-1时抑制率为40%;流式细胞术证实Met-ENK作用后大多数细胞被抑制于细胞周期的G0/G1期,该期细胞数由63.97%变为87.73%;P21的基因转录量是用药前的3倍,P53基因转录量是用药前的5倍;P21,P16蛋白表达量明显增加,磷酸Rb蛋白表达量明显降低。结论:Met-ENK可以通过上调细胞周期蛋白依靠性激酶抑制因子P21及抑癌基因P53的转录和蛋白表达量,下调Rb蛋白的磷酸化水平将SHSY5Y细胞的增殖抑制于生长周期的G0/G1期,表明Met-ENK的G0/G1期特异性周期抑瘤作用可以通过调节该途径产生。

氯乙烯对肝细胞周期G_1/S关卡相关蛋白表达影响

目的探讨氯乙烯(VCM)对细胞周期G1/S关卡功能影响及可能的机制。方法将人正常肝细胞HL-7702暴露于VCM气体(体积分数为0.8%、2.5%、7.5%、15.0%和30.0%)和空气(对照组)48 h后用流式细胞仪检测HL-7702细胞周期;Western blot测定细胞中G1/S关卡相关蛋白表达水平。结果染毒48 h后,在<7.5%剂量时,HL-7702细胞G0/G1期细胞百分数随染毒剂量增加呈上升趋势,与对照组[(73.35±1.56)%]比较,7.5%剂量组G0/G1期细胞百分比[(78.52±4.46)%]升高,差异有统计学意义(P<0.01);与对照组[(12.85±0.02)%]比较,2.5%剂量组S期细胞百分比[(10.97±0.17)%]下降,差异有统计学意义(P<0.05);细胞周期蛋白依赖性激酶(CDK 4)和P16蛋白表达量均呈上升趋势,与对照组比较,7.5%剂量组HL-7702细胞CDK 4、P16表达[分别为(1.43±0.51)、(0.80±0.30)]均升高,差异均有统计学意义(均P<0.05)。结论低剂量VCM染毒可使肝细胞发生G1期阻滞,高剂量时G1期阻滞作用消失;其机制主要与上调P16、CDK 4蛋白表达有关。

低浓度丰加霉素对人白血病K562细胞集落形成抑制作用的机制研究

目的初步探讨低浓度丰加霉素对人白血病K562细胞集落形成抑制作用的机制。方法甲基纤维素集落形成实验检测低浓度丰加霉素对人白血病K562细胞集落形成能力的影响；CCK-8法检测低浓度丰加霉素对K562细胞的生长抑制率；AnnexinV／PI双染流式细胞仪检测低浓度丰加霉素作用下的K562细胞凋亡率；PI单染流式细胞仪检测药物作用后细胞的周期分布改变；Western免疫印迹和实时定量PCR检测周期相关分子表达水平变化。结果低浓度丰加霉素对人白血病K562细胞具有较强的集落形成抑制作用；可明显抑制K562细胞的生长，呈时间一剂量依赖性；尽管短时间（48h）的药物处理仅出现轻度的细胞凋亡和周期阻滞，但10nmol/L和30nmol/L的丰加霉素长时间（7d）作用后，K562细胞G0／G1期比例分别是（62．3±1．7）％和（76．9±0．7）％，与对照组（38．9±1．1）％相比差异具有高度统计学意义（P〈0．01）；低浓度丰加霉素长时间作用后诱导K562细胞周期相关分子P16蛋白水平和转录水平的高表达。结论丰加霉素在低浓度，长时间作用于人白血病K562细胞后，具有较强的集落形成抑制和生长抑制作用，此作用可能与诱导细胞周期相关分子p16高表达，导致细胞G0／G1期阻滞有关。