全反式维甲酸影响肝癌细胞株BEL-7402增殖、凋亡与Cks表达的相关性研究
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摘要
背景与目的:
原发性肝癌(hepatocellular carcinoma,HCC)是世界上常见的恶性肿瘤之一,其恶性程度高,发展迅速,若治疗不及时或治疗方案选择不当,平均生存时间为半年。中国是HCC高发区,约占全球患者的55%,而厦门市同安区是中国两大肝细胞癌高发区之一,年发病率在十万分之三十以上。由于肝细胞癌早期不易发现,患者大多错失最佳手术时机。目前的常规化疗药物疗效不佳,致使肝细胞癌的治愈率较低。因此,研究HCC的发生发展机制,探明重要的相关基因,将为HCC的诊治提供新的思路,对攻克肝细胞癌意义重大。本课题以人肝癌细胞株BEL-7402为研究对象,通过检测全反式维甲酸对肝癌细胞增殖、凋亡以及Cks1、Cks2基因蛋白表达的影响,分析瘤细胞增殖、凋亡与Cks1、Cks2表达的相关性,初步探讨可能存在的作用机制,为全反式维甲酸治疗肝癌提供理论和实验依据,为Cks1、Cks2基因及蛋白进一步的实验研究积累经验。
方法:
1、MTT法测定ATRA对BEL-7402细胞增殖的影响,实验组加入相应量的ATRA至终浓度为(0.1,1,5,10,20)μmol/L,对照组只加相应量的RPMI 1640,分别于加药后继续培养24 h、48 h、72 h、96 h检测各组细胞和对照组及相邻实验组间增殖抑制率的差异。
2、流式细胞仪Annexin V/PI双染法检测ATRA对BEL-7402细胞周期及凋亡的影响,实验组加入相应量的ATRA至终浓度为10μmol/L,对照组加入1‰的无水乙醇,于加药后继续培养24 h、48 h、72 h检测各组细胞周期分布和凋亡的差异。
3、RT-PCR法检测ATRA对BEL-7402细胞Cksl、Cks2基因mRNA表达的影响,实验组加入相应量的ATRA至终浓度为10μmol/L,对照组加入1‰的无水乙醇,于加药后继续培养24 h、48 h、72 h检测各组细胞之间Cks1、Cks2基因mRNA表达的差异。
4、免疫细胞化学ABC法检测ATRA对BEL-7402细胞Cks1、Cks2、P27蛋白表达的影响,实验组加相应量的ATRA至终浓度为10μmol/L,对照组加入1‰的无水乙醇,于加药后继续培养24 h、48 h、72 h检测Cks1、Cks2、P27蛋白表达的变化,采用HSCORE积分法分析ATRA对Cks1、Cks2、P27蛋白表达的影响。
结果:
1、ATRA对BEL-7402细胞增殖具有明显的抑制作用,抑制效应呈现明显的时间、剂量依赖性。10μmol/L组ATRA作用24小时抑制率为17.45±1.93,96小时抑制率达到50.23±2.44。各组之间的吸光度A值和抑制率IR相比差异有显著统计学意义(P＜0.01)。
2、BEL-7402细胞经10μmol/L ATRA作用后,细胞周期各期细胞比例发生明显变化,随培养时间延长,G_0-G_1期细胞比例不断增高,细胞大量阻滞在G_0-G_1期,对照组G_0-G_1期细胞平均为59.87±1.07,ATRA作用72小时后升高到72.78±1.13,S期细胞比例相应下降,G_2/M期细胞比例各组之间差异无统计学意义(P＞0.05)。
3、10μmol/LATRA作用24小时引起部分BEL-7402细胞发生凋亡6.48±0.45,并随作用时间延长,细胞凋亡率不断增加,72小时凋亡率达到17.31±1.00,实验组和对照组及各时间组间比较差异有显著统计学意义(P＜0.01)。
4、ATRA对Cks1、Cks2基因mRNA表达无明显影响(P＞0.05)。
5、BEL-7402细胞随ATRA作用时间延长,Cks1、Cks2蛋白表达量呈减低趋势,Cks1组化染色HSCORE积分从1.88±0.06降低到1.47±0.06,Cks2组化染色HSCORE积分从1.58±0.09降低到1.39±0.03,而P27蛋白表达量不断增加,组化染色HSCORE积分从2.32±0.08升高到2.97±0.11,各组之间的HSCORE积分相比差异均有显著统计学意义(P＜0.01或P＜0.05)。
结论:
1、全反式维甲酸对BEL-7402绌胞具有明显的增殖抑制作用,并呈现一定的时间、剂量依赖性。
2、全反式维甲酸具有诱导BEL-7402细胞凋亡的作用。
3、全反式维甲酸不影响BEL-7402细胞Cks基因mRNA的表达,却能够在蛋白水平减低Cks蛋白的表达,增强P27蛋白的表达。
4、全反式维甲酸抑制BEL-7402细胞增殖,使大量细胞阻滞在G_0-G_1期,可能通过下调Cks1蛋白的表达,从而抑制P27蛋白的泛素化降解,使得P27蛋白在细胞内积聚,对细胞周期起负调控作用。
5、全反式维甲酸诱导BEL-7402细胞凋亡的分子机制可能与Cks2蛋白表达下调有关。
Background and Objective:
Hepatocellular carcinoma,HCC is one of the word's common malignant tumor with high malignancy,rapid development,average survival time only for six months if treatment is not timely or inappropriate.China is one of the regions of high incidence of liver cancer,accounting for about 55%of worldwide patients,and Tongan District, Xiamen City,is one of Chinese two major areas,which annual incidence rate is about 30/100000.Hepatocellular carcinoma is easy to miss the best timing of surgery because of difficulty to early detect.At present,the poor efficacy of conventional chemotherapy drugs resulted in lower cure rates.Therefore,studing the occurrence and development mechanism of HCC,verifying the important related genes,that will provide the new mentality to HCC diagnosis,means a great deal to conquer the HCC. This topic raked the person liver cancer BEL-7402 cell as the object,provided experimental and theoretical basis for clincal application with all-trans retinoic acid, piled up experience and data to further study of Cks1 and Cks2,through detecting effect of all-trans retinoic acid on liver cancer cell proliferation,apoptosis and Cks1, Cks2 expression,analysed the correlation between cell proliferation,apoptosis and Cks1,Cks2 expression,elementary studied the possible mechanisms that may exist.
Methods:
1.The method of MTT was used to examine the cell proliferation suppression activity of ATRA to cell BEL-7402.The experimental groups was cultured with ATRA in (0.1,1,5,10,20)μmol/L different concentration and control groups only adds the corresponding quantity RPMI 1640.After a further culture 24 h,48 h,72 h,96 h.The effect of different group cells' inhibition rate was evaluated with MTT method.
2.Cell cycle and apoptosis rate was detected by flow cytometry(FCM) with PI and Annexin V/PI double stainings.The experimental groups was cultured with ATRA in 20μmol/L concentration and control groups with 1‰anhydrous ethanol.The Cell cycle and apoptosis rate was detected by FCM after a further culture 24 h,48 h,72 h.
3.Cks1,Cks2 mRNA were detected by RT-PCR.The experimental groups was cultured with ATRA in 20μmol/L concentration and control groups with 1‰anhydrous ethanol.The differential expression of Cks1,Cks2 mRNA was detected by RT-PCR after a further culture 24 h,48 h,72 h.
4.The change of protein Cks1,Cks2,P27 expression was examined by immunocytochemistry.The experimental groups was cultured with ATRA in 20μmol/L concentration and control groups with 1%0 anhydrous ethanol.The results of protein Cks1,Cks2,P27 expression was analysed by HSCORE.
Results:
1.Cell BEL-7402 proliferation was significantly inhibited in time-dependent and dose-dependent manners after induced by ATRA.Inhibition rate of proliferation was 17.45±1.93 after 24 h culture and 50.23±2.44 after 96 h culture.The difference of absorbance A and inhibition rate were statistically significant among different groups (P＜0.01).
2.After induced by ATRA,G_0-G_1 phase ratio of BEL-7402 cells increased,and plenty of cells were blocked in G_0-G_1 phase,while S phase ratio decreased.G_0-G_1 phase ratio was 59.87±1.07 after 24 h culture and 72.78±1.13 after 72 h culture.There was no difference among experimental groups and control groups of G_2/M phase ratio(P＞0.05).
3.The apoptotic rates were increased gradually along with time of culture.The ratio of apoptosis was 6.48±0.45 after 24 h and 17.31±1.00 after 72 h.there was significantly difference between experimental groups,the control groups or different time goups(P＜0.01).
4.ATRA has no significant effect on the expression of Cks1,Cks2 mRNA(P＞0.05).
5.Expression of protein Cks1,Cks2 decreased after induced by ATRA.HSCORE of protein Cks1 decreased from 1.88±0.06 to 1.47±0.06 and protein Cks2 from 1.58± 0.09 to 1.39±0.03,while protein P27 increased from 2.32±0.08 to 2.9±0.11. There was significant difference among groups(P＜0.01 or P＜0.05).
Conclusions:
1.ATRA inhibits the proliferation of human hepatoma cell line BEL-7402 significantly in time-dependent and dose-dependent manners;
2.The apoptosis in BEL-7402 cells could be induced by ATRA;
3.ATRA has no significant effect on the expression of Cks1,Cks2 mRNA,but down-regulation of their protein and up-regulation protein P27.
4.ATRA caused the massive ceils to hinder in the G_0-G_1 phase,probably through the mechanism of down regulation of protein Cks1 and inhibition ubiquitination of the protein P27 degradation.The protein P27 accumulated in cells and played a negative regulation effect during the cell mitosis.
5.The molecular mechanism of inducing apoptosis of cell BEL-7402 may be related to down-regulation of protein Cks2.
原发性肝癌(hepatocellular carcinoma,HCC)是世界上常见的恶性肿瘤之一,其恶性程度高,发展迅速,若治疗不及时或治疗方案选择不当,平均生存时间为半年。中国是HCC高发区,约占全球患者的55%,而厦门市同安区是中国两大肝细胞癌高发区之一,年发病率在十万分之三十以上。由于肝细胞癌早期不易发现,患者大多错失最佳手术时机。目前的常规化疗药物疗效不佳,致使肝细胞癌的治愈率较低。因此,研究HCC的发生发展机制,探明重要的相关基因,将为HCC的诊治提供新的思路,对攻克肝细胞癌意义重大。本课题以人肝癌细胞株BEL-7402为研究对象,通过检测全反式维甲酸对肝癌细胞增殖、凋亡以及Cks1、Cks2基因蛋白表达的影响,分析瘤细胞增殖、凋亡与Cks1、Cks2表达的相关性,初步探讨可能存在的作用机制,为全反式维甲酸治疗肝癌提供理论和实验依据,为Cks1、Cks2基因及蛋白进一步的实验研究积累经验。
方法:
1、MTT法测定ATRA对BEL-7402细胞增殖的影响,实验组加入相应量的ATRA至终浓度为(0.1,1,5,10,20)μmol/L,对照组只加相应量的RPMI 1640,分别于加药后继续培养24 h、48 h、72 h、96 h检测各组细胞和对照组及相邻实验组间增殖抑制率的差异。
2、流式细胞仪Annexin V/PI双染法检测ATRA对BEL-7402细胞周期及凋亡的影响,实验组加入相应量的ATRA至终浓度为10μmol/L,对照组加入1‰的无水乙醇,于加药后继续培养24 h、48 h、72 h检测各组细胞周期分布和凋亡的差异。
3、RT-PCR法检测ATRA对BEL-7402细胞Cksl、Cks2基因mRNA表达的影响,实验组加入相应量的ATRA至终浓度为10μmol/L,对照组加入1‰的无水乙醇,于加药后继续培养24 h、48 h、72 h检测各组细胞之间Cks1、Cks2基因mRNA表达的差异。
4、免疫细胞化学ABC法检测ATRA对BEL-7402细胞Cks1、Cks2、P27蛋白表达的影响,实验组加相应量的ATRA至终浓度为10μmol/L,对照组加入1‰的无水乙醇,于加药后继续培养24 h、48 h、72 h检测Cks1、Cks2、P27蛋白表达的变化,采用HSCORE积分法分析ATRA对Cks1、Cks2、P27蛋白表达的影响。
结果:
1、ATRA对BEL-7402细胞增殖具有明显的抑制作用,抑制效应呈现明显的时间、剂量依赖性。10μmol/L组ATRA作用24小时抑制率为17.45±1.93,96小时抑制率达到50.23±2.44。各组之间的吸光度A值和抑制率IR相比差异有显著统计学意义(P＜0.01)。
2、BEL-7402细胞经10μmol/L ATRA作用后,细胞周期各期细胞比例发生明显变化,随培养时间延长,G_0-G_1期细胞比例不断增高,细胞大量阻滞在G_0-G_1期,对照组G_0-G_1期细胞平均为59.87±1.07,ATRA作用72小时后升高到72.78±1.13,S期细胞比例相应下降,G_2/M期细胞比例各组之间差异无统计学意义(P＞0.05)。
3、10μmol/LATRA作用24小时引起部分BEL-7402细胞发生凋亡6.48±0.45,并随作用时间延长,细胞凋亡率不断增加,72小时凋亡率达到17.31±1.00,实验组和对照组及各时间组间比较差异有显著统计学意义(P＜0.01)。
4、ATRA对Cks1、Cks2基因mRNA表达无明显影响(P＞0.05)。
5、BEL-7402细胞随ATRA作用时间延长,Cks1、Cks2蛋白表达量呈减低趋势,Cks1组化染色HSCORE积分从1.88±0.06降低到1.47±0.06,Cks2组化染色HSCORE积分从1.58±0.09降低到1.39±0.03,而P27蛋白表达量不断增加,组化染色HSCORE积分从2.32±0.08升高到2.97±0.11,各组之间的HSCORE积分相比差异均有显著统计学意义(P＜0.01或P＜0.05)。
结论:
1、全反式维甲酸对BEL-7402绌胞具有明显的增殖抑制作用,并呈现一定的时间、剂量依赖性。
2、全反式维甲酸具有诱导BEL-7402细胞凋亡的作用。
3、全反式维甲酸不影响BEL-7402细胞Cks基因mRNA的表达,却能够在蛋白水平减低Cks蛋白的表达,增强P27蛋白的表达。
4、全反式维甲酸抑制BEL-7402细胞增殖,使大量细胞阻滞在G_0-G_1期,可能通过下调Cks1蛋白的表达,从而抑制P27蛋白的泛素化降解,使得P27蛋白在细胞内积聚,对细胞周期起负调控作用。
5、全反式维甲酸诱导BEL-7402细胞凋亡的分子机制可能与Cks2蛋白表达下调有关。
Background and Objective:
Hepatocellular carcinoma,HCC is one of the word's common malignant tumor with high malignancy,rapid development,average survival time only for six months if treatment is not timely or inappropriate.China is one of the regions of high incidence of liver cancer,accounting for about 55%of worldwide patients,and Tongan District, Xiamen City,is one of Chinese two major areas,which annual incidence rate is about 30/100000.Hepatocellular carcinoma is easy to miss the best timing of surgery because of difficulty to early detect.At present,the poor efficacy of conventional chemotherapy drugs resulted in lower cure rates.Therefore,studing the occurrence and development mechanism of HCC,verifying the important related genes,that will provide the new mentality to HCC diagnosis,means a great deal to conquer the HCC. This topic raked the person liver cancer BEL-7402 cell as the object,provided experimental and theoretical basis for clincal application with all-trans retinoic acid, piled up experience and data to further study of Cks1 and Cks2,through detecting effect of all-trans retinoic acid on liver cancer cell proliferation,apoptosis and Cks1, Cks2 expression,analysed the correlation between cell proliferation,apoptosis and Cks1,Cks2 expression,elementary studied the possible mechanisms that may exist.
Methods:
1.The method of MTT was used to examine the cell proliferation suppression activity of ATRA to cell BEL-7402.The experimental groups was cultured with ATRA in (0.1,1,5,10,20)μmol/L different concentration and control groups only adds the corresponding quantity RPMI 1640.After a further culture 24 h,48 h,72 h,96 h.The effect of different group cells' inhibition rate was evaluated with MTT method.
2.Cell cycle and apoptosis rate was detected by flow cytometry(FCM) with PI and Annexin V/PI double stainings.The experimental groups was cultured with ATRA in 20μmol/L concentration and control groups with 1‰anhydrous ethanol.The Cell cycle and apoptosis rate was detected by FCM after a further culture 24 h,48 h,72 h.
3.Cks1,Cks2 mRNA were detected by RT-PCR.The experimental groups was cultured with ATRA in 20μmol/L concentration and control groups with 1‰anhydrous ethanol.The differential expression of Cks1,Cks2 mRNA was detected by RT-PCR after a further culture 24 h,48 h,72 h.
4.The change of protein Cks1,Cks2,P27 expression was examined by immunocytochemistry.The experimental groups was cultured with ATRA in 20μmol/L concentration and control groups with 1%0 anhydrous ethanol.The results of protein Cks1,Cks2,P27 expression was analysed by HSCORE.
Results:
1.Cell BEL-7402 proliferation was significantly inhibited in time-dependent and dose-dependent manners after induced by ATRA.Inhibition rate of proliferation was 17.45±1.93 after 24 h culture and 50.23±2.44 after 96 h culture.The difference of absorbance A and inhibition rate were statistically significant among different groups (P＜0.01).
2.After induced by ATRA,G_0-G_1 phase ratio of BEL-7402 cells increased,and plenty of cells were blocked in G_0-G_1 phase,while S phase ratio decreased.G_0-G_1 phase ratio was 59.87±1.07 after 24 h culture and 72.78±1.13 after 72 h culture.There was no difference among experimental groups and control groups of G_2/M phase ratio(P＞0.05).
3.The apoptotic rates were increased gradually along with time of culture.The ratio of apoptosis was 6.48±0.45 after 24 h and 17.31±1.00 after 72 h.there was significantly difference between experimental groups,the control groups or different time goups(P＜0.01).
4.ATRA has no significant effect on the expression of Cks1,Cks2 mRNA(P＞0.05).
5.Expression of protein Cks1,Cks2 decreased after induced by ATRA.HSCORE of protein Cks1 decreased from 1.88±0.06 to 1.47±0.06 and protein Cks2 from 1.58± 0.09 to 1.39±0.03,while protein P27 increased from 2.32±0.08 to 2.9±0.11. There was significant difference among groups(P＜0.01 or P＜0.05).
Conclusions:
1.ATRA inhibits the proliferation of human hepatoma cell line BEL-7402 significantly in time-dependent and dose-dependent manners;
2.The apoptosis in BEL-7402 cells could be induced by ATRA;
3.ATRA has no significant effect on the expression of Cks1,Cks2 mRNA,but down-regulation of their protein and up-regulation protein P27.
4.ATRA caused the massive ceils to hinder in the G_0-G_1 phase,probably through the mechanism of down regulation of protein Cks1 and inhibition ubiquitination of the protein P27 degradation.The protein P27 accumulated in cells and played a negative regulation effect during the cell mitosis.
5.The molecular mechanism of inducing apoptosis of cell BEL-7402 may be related to down-regulation of protein Cks2.
引文
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[19]方建林,郑传胜,冯敢生等.全反式维甲酸对Walker-256肝癌细胞分化及凋亡的诱导作用.世界华人消化杂志.2008,16(26):2929-2934.
[20]邵晨,晏伟,朱峰等.维甲酸对多种肿瘤细胞生长影响的实验研究.苏州大学学报.2003,23(2):162-165.
[21]Breitman TR,Selonick SE,Collins SJ.Induction of differentiation of the human promyelocytic leukemia cell line(HL-60) by retinoic acid.Proc Natl Acad Sci U S A.1980,77(5):2936-40.
[22]王振义,孙关林,叶裕春等.全反式维甲酸治疗急性早幼粒细胞白血病90例的临床研究.中华血液学杂志,1990,11(9):480.
[23]Clavio M,Ghiso A,Ghiggi C et al.Seventeen years of experience with ATRA-based therapy for acute promyelocytic leukaemia:Long-term follow-up of patients treated at S.Martino Hospital,Genoa.Oncol Rep.2009,21(4):1045-52.
[24]Maeda Y,Yamaguchi T,Miyatake J,et al.Possible therapy with ATRA for adult T-cell leukemia/lymphoma.Nippon Rinsho.2007,1:719-23.
[25]Nijsten TE,Stem RS.Oral retinoid use reduces cutaneous squamous cell carcinoma risk in patients with psoriasis treated with psoralen-UVA:a nested cohort study.J Am Acad Dermatol.2003,49(4):644-50.
[26]Klopper JP,Sharma V,Berenz A,Retinoid and thiazolidinedione therapies in melanoma:an analysis of differential response based on nuclear hormone receptor expression.Mol Cancer.2009,8:16.
[27]王鲁,汤钊猷,薛琼等.α-干扰素和全反式维甲酸对肝癌根治性切除术后复发转移的干预作用.中华实验外科杂志,2000年05期.
[28]Zancai P,Cariati R,Rizzo S,Boiocchi M,Dolcetti R.Retinoic acid-mediated growth arrest of EBV-immortalized B lymphocytes is associated with multiple changes in G1 regulatory proteins:p27Kip1 up-regulation is a relevant early event.Oncogene.1998,17(14):1827-36.
[29]Tosi P,Visani G,Gibellini D,et al.All-trans retinoic acid and induction of apoptosis in acute promyelocytic leukemia cells.Leuk Lymphoma.1994,14(5-6):503-7.
[30]Yu F,Yang G,Zhao Z et al.Apoptosis related protein 3,an ATRA-upregulated membrane protein arrests the cell cycle at GI/S phase by decreasing the expression of cyclin D1.Biochem Biophys Res Commun.2007,358(4):1041-6.
[31]Yu Z,Xing Y.atRA-induced apoptosis of mouse embryonic palate mesenchymal cells involves activation of MAPK pathway.Toxicol Appl Pharmacol.2006,215(1):57-63.
[32]Yu Z,Hart J,Lin J.Apoptosis induced by atRA in MEPM cells is mediated through activation of caspase and RAR.Zoxicol Sci.2006,89(2):504-9.
[33]彭安,黄炜,黄济群.维甲酸对Bel-7402人肝癌细胞的诱导分化作用.中国现代医学杂志.2000,10(2):15-16.
[34]Taetle R,Santos B D,Akamatsu K I,et al.Effect of all-trans retinoic acid and antireceptor antibodies on growth and programmed cell death of human myeloma cells.Clinical cancer research,1996,2:253-259.
[35]维甲酸对人肝癌细胞凋亡的影响及临床意义.李良平;张正;韩盛玺.医学科技,2002,02:4-6.
[36]全反式维甲酸联合奥曲肽对肝癌细胞-HepG_2增生和凋亡的影响叶红;但自力;唐望先.世界华人消化杂志,2004,10期.
[37]Delia D,Aiello A,Soligo D et al.bcl-2 proto-oncogene expression in normal and neoplastic human myeloid cells.Blood.1992,79(5):1291-8.
[38]Yonish-Rouach E,Grunwald D,Wilder S,et al.p53-mediated cell death:relationship to cell cycle control.Mol Cell Biol.1993,13(3):1415-23.
[39]Glozak MA,Rogers MB.Specific induction of apoptosis in P19 embryonal carcinoma cells by retinoic acid and BMP2 or BMP4.Dev Biol.1996,179(2):458-70.
[40]曾辉,赵相印,马雅銮等.维甲酸诱导HL-60细胞Fas蛋白表达.中华血液学杂志.1998,05:237-240.
[41]Ramakrishnan S,Eppenberger U,Mueller H,et al.Expression profile of the putative catalytic subunit of the telomerase gene.Cancer Res.1998,58(4):622-5.
[42]Lan Y,Zhang Y,Wang F,et al.Aberrant expression of Cks1 and Cks2 contributes to prostate tumorigenesis by promoting proliferation and inhibiting programmed cell death.Int J Cancer.2008,123(3):543-51.
[43]Kawakami K,Enokida H,Tachiwada T,et al.Identification of differentially expressed genes in human bladder cancer through genome-wide gene expression profiling.Oncol Rep 2006,16:521-31.
[44]Wong YF,Cheung TH,Tsao GS,et al.Genomewide gene expression profiling of cervical cancer in Hong Kong women by oligonucleotide microarray.Int J Canc.2006,118:2461-9.
[45]Chow LS,Lam CW,Chan SY,et al.Identification of RASSF1A modulated genes in nasopharyngeal carcinoma.Oncogene.2006,25:310-16.
[46]de Wit N J,Rijntjes J,Diepstra JH,et al.Analysis of differential gene expression in human melanocytic tumour lesions by custom made oligonucleotide arrays.Br J Canc.2005,92:2249-61.
[47]Uchikado Y,Inoue H,Haraguchi N,et al.Gene expression profiling of lymph node metastasis by oligomicroarray analysis using laser microdissection in esophageal squamous cell carcinoma.Int J Oncol.2006,29:1337-47.
[48]Polyak K,Kato J,Solomon M,et al.P27Kipl,a Cyclin-CDKinhibitor;links transforming growth factor-β and contact inhibition to cell cycle arrest.Genes Dey,1994,8:9-22.
[49]Pagano M,Tam SW,Theodoras AM,et al.Role of the ubiquitin-proteasome pathway in regulating abundance of the cyclin-dependent kinase inhibitor p27.Science,1995,269:682-685.
[50]Carrano AC,Eytan E,Hershko A,SKP2 is required for ubiquitin-mediated degradation of the CDK inhibitor p27.Nat Cell Biol.1999,1(4):193-9.
[51]Barnes A,Pinder SE,Bell JA,et al.Expression of p27Kip1 in breast cancer and its prognostic significance.J Pathol.2003,201:451-459.
[52]Migaldi M,Zunarelli E,Sgambato A,et al.p27Kip1 expression and survival in NO gastric carcinoma.Pathol Res Pract.2001,197:231-236.
[53]Kagawa Y,Yoshida K,Hirai T,et al.Significance of the expression of p27Kip1 in esophageal squamous cell carcinomas.Dis Esophagus.2000,13:179-184.
[54]Hirabayashi H,Ohta M,Tanaka H,et al.Prognostic significance of p27Kip1 expression in resected non-small cell lung cancers:analysis in combination with expressions of pl6INK4A,pRB,and p53.J Surg Oncol.2002,81:177-184.
[55]Dreher T,Zentgraf H,Abel U,et al.Reduction of PTEN and p27Kip1 expression correlattes with tumor grade in prostate cancer.Analysis in radical prostatectomyh specimens and needle biopsies.Virchows Arch.2004,444:509-517.
[1]Ganoth,Bornstein D,Ko G,et al.The cell-cycle regulatory protein Cks1 is required for SCF Skp2-mediated ubiquitinylation of p27.Nat Cell Biol,2001,3:321-324.
[2]Richardson HE,Stueland CS,Thomas J,et al.Human cDNAs encoding homologs of the small p34-Cdc28/Cdc2-associated protein of Saecharomyces cerevisiae and Schizosaccharomyces pombe.Genes,1990,4:1332-1344.
[3]Bourne Y,Watson MH,Arvai AS,et al.Crystal structure and mutational analysis of the Saccharomyces cerevisiae cell cycle regulatory protein Cks1:implications for domain swapping,anion binding and protein interactions.Structure,2000,8:841-850.
[4]Spruck C,Strohmaier H,Watson M.A CDK-independent function of mammalian Cksl:targeting of SCF(Skp2) to the CDK inhibitor p27Kipl.Mol Cell,2001,7:639-650.
[5]Hao B,Zheng N,Brenda A,et al.Structural Basis of the Cks1-Dependent Recognition of p27Kip1 by the SCFSkp2 Ubiquitin Ligase.Mol Cell,2005,20:9-19.
[6]Yao ZP,Zhou M,Sadie E,et al.Activation of Ubiquitin Ligase SCFSkp2 by Cks1:Insights from Hydrogen Exchange Mass Spectrometry.J.Mol.Biol,2006,363:673-686.
[7]Xu S,Abbasian M,Patel P,et al.Substrate Recognition and Ubiquitination of SCFSkp2/Cksl Ubiquitin-Protein Isopeptide Ligase.J.Mol.Biol,2007,282:15462-15470.
[8]Seeliger MA,Spichty M,Kelly SE,et al.Role of conformational heterogeneity in domain swapping and adapter function of the Cks proteins.J.Biol.Chem,2005,280:30448-30459.
[9]Bhatt KV,Hu R,Spofford LS,et al.Mutant B-RAF signaling and cyclin D1 regulate Cksl/S-phase kinase-associated protein 2-mediated degradation of p27Kip1 in human melanoma cells.Oncogene,2007,26:1056-1066.
[10]Wang C,Chen YJ,Douglas H,et al.Forkhead Box Ml Regulates the Transcriptional Network of Genes Essential for Mitotic Progression and Genes Encoding the SCF (Skp2-Cksl) Ubiquitin Ligase.Molecular & Cellular Biology,2005,25:10875-10894.
[11]Keller UB,Old JB,Dorsey FC.Myc targets Cksl to provoke the suppression of p27Kip1,proliferation and lymphomagenesis.EMBO,2007,26:2562-2574.
[12]Wang W,Jin J,Bellur S,et al.Negative regulation of SCFSkp2 ubiquitin ligase by TGF-β signaling.Oncogene,2004,23:1064-1075.
[13]Zancai P,Col JD,Piccinin S.Retinoic acid stabilizes p27Kip1 in EBV-immortalized lymphoblastoid B cell lines through enhanced proteasome-dependent degradation of the p45Skp2 and Cksl proteins.Oncogene,2005,24:2483-2494.
[14]Tsai YS,Chang HC,Chuang LY,et al.RNA Silencing of Cksl Induced G2/M Arrest and Apoptosis in Human Lung Cancer Cells.IUBMB Life,2005,57:583-589.
[15]Kitajima S,Kudo Y,Ogawa I,et al.Role of Cksl Over expression in Oral Squamous Cell Carcinomas Cooperation with Skp2 in Promoting p27 Degradation.Am J Pathol,2004,165:2147-2155.
[16]Slotky M,Shapira M,Izhak OB,et al.The Expression of the Ubiquitin Ligase Subunit Cksl in Human Breast Cancer.Breast Cancer Res,2005,7:737-744.
[2]苏成豪,牛建军,张一中等.厦门市乙型肝炎、肝癌流行趋势及其关系研究.旅行医学科学,2007,13(3):30-31.
[3]Altekruse SF,McGlynn KA,Reichman ME et al.Hepatocellular Carcinoma Incidence,Mortality,and Survival Trends in the United States From 1975 to 2005.J Ciin Oncol,2009,27(9):1485-91.
[4]Pagano M,Tam SW,Theodoras AM,et al.Role of the ubiquitin-proteasome pathway in regulating abundance of the cyclin-dependent kinase inhibitor p27.Science,1995,269:682-685.
[5]Ganoth,Bomstein D,Ko G,et al.The ceil-cycle regulatory protein Cksl is required for SC F(Skp2)-mediated ubiquitinylation of p27.Nat Cell Biol,2001,3:321-324.
[6]Spruck CH,Miguel MP,Smith AP et al.Requirement of Cks2 for the first metaphase/anaphase transition of mammalian meiosis.Science,2003,300(5619):647-50.
[7]Martinsson-Ahlzén HS,Liberal V,Grünenfelder B,et al.Cyclin-dependent kinase-associated proteins Cksl and Cks2 are essential during early embryogenesis and for cell cycle progression in somatic cells.Mol Cell Biol,2008,28(18):5698-709.
[8]Yongsheng Lan,Yongyou Zhang,Jianghua Wang,et al.Aberrant expression of Cksl and Cks2contributes to prostate tumorigenesis by promoting proliferation and inhibiting programmed cell death.Int.J.Cancer,2008,123:543 - 551.
[9]Masuda TA,Inoue H,Nishida K,et al.Cyclin-Dependent Kinase 1 Gene Expression Is Associated with Poor Prognosis in Gastric Carcinoma.Clin Cancer Res,2003,9(15):5693-5698.
[10]Inui N,Kitagawa K,Miwa S,et al.High expression of Cks1 in human non-small cell lung carcinomas.Biochem Biophys Res Commun,2003,303(3):978 - 984.
[11]Slotky M,Shapira M,Izhak OB,et al.The expression of the ubiquitin ligase subunit Cks1 in human breast cancer.Breast Cancer Res,2005,7(5):R737-R744.
[12]Shapira M,Ben-Izhak O,Linn S,et al.The Prognostic Impact of the Ubiquitin Ligase Subunits Skp2 And Cks I in Colorectal Carcinoma.Cancer,2005,103(7):1336-46.
[13]Kawakami K,Enokida H,Tachiwada T,et al.Links Increased SKP2 and CKS1 gene expression contributes to the progression of human urothelial carcinoma.J Urol.2007,178(1):301-307.
[14]Hattori T,Kitagawa k,Uchida C,et al.Cksl is degraded via the ubiquitin-proteasome pathway in a cell cycle-dependent manner.Genes to Cells,2003,8:889-896.
[15]Zancai P,Col JD,Piccinin S,et al.Retinoic acid stabilizes p27Kip1 in EBV-immortalized lymphoblastoid B cell lines through enhanced proteasome-dependent degradation of the p45Skp2and Cks1 proteins.Oncogene,2005,24:2483-2494.
[16]陈瑞铭,朱德厚,叶秀珍等.人体肝癌体外细胞株(BEL-7402)的建立及其特征.实验生物学报,1978,01:434-436.
[17]Harrington D J,lessey B A,Rai V,et al.Tenascin is differentially expressed in endometrium and endometriosis.J Pathol,1999,187(2):242-248.
[18]Chan-JF,DebraM,Sutkowski,etal.Effect of retinoic acid on the proliferation and secretory activity of androgen-responsive prostatic carcinoma.Cells J.TJ Urol.1993,149(5):1190-1194.
[19]方建林,郑传胜,冯敢生等.全反式维甲酸对Walker-256肝癌细胞分化及凋亡的诱导作用.世界华人消化杂志.2008,16(26):2929-2934.
[20]邵晨,晏伟,朱峰等.维甲酸对多种肿瘤细胞生长影响的实验研究.苏州大学学报.2003,23(2):162-165.
[21]Breitman TR,Selonick SE,Collins SJ.Induction of differentiation of the human promyelocytic leukemia cell line(HL-60) by retinoic acid.Proc Natl Acad Sci U S A.1980,77(5):2936-40.
[22]王振义,孙关林,叶裕春等.全反式维甲酸治疗急性早幼粒细胞白血病90例的临床研究.中华血液学杂志,1990,11(9):480.
[23]Clavio M,Ghiso A,Ghiggi C et al.Seventeen years of experience with ATRA-based therapy for acute promyelocytic leukaemia:Long-term follow-up of patients treated at S.Martino Hospital,Genoa.Oncol Rep.2009,21(4):1045-52.
[24]Maeda Y,Yamaguchi T,Miyatake J,et al.Possible therapy with ATRA for adult T-cell leukemia/lymphoma.Nippon Rinsho.2007,1:719-23.
[25]Nijsten TE,Stem RS.Oral retinoid use reduces cutaneous squamous cell carcinoma risk in patients with psoriasis treated with psoralen-UVA:a nested cohort study.J Am Acad Dermatol.2003,49(4):644-50.
[26]Klopper JP,Sharma V,Berenz A,Retinoid and thiazolidinedione therapies in melanoma:an analysis of differential response based on nuclear hormone receptor expression.Mol Cancer.2009,8:16.
[27]王鲁,汤钊猷,薛琼等.α-干扰素和全反式维甲酸对肝癌根治性切除术后复发转移的干预作用.中华实验外科杂志,2000年05期.
[28]Zancai P,Cariati R,Rizzo S,Boiocchi M,Dolcetti R.Retinoic acid-mediated growth arrest of EBV-immortalized B lymphocytes is associated with multiple changes in G1 regulatory proteins:p27Kip1 up-regulation is a relevant early event.Oncogene.1998,17(14):1827-36.
[29]Tosi P,Visani G,Gibellini D,et al.All-trans retinoic acid and induction of apoptosis in acute promyelocytic leukemia cells.Leuk Lymphoma.1994,14(5-6):503-7.
[30]Yu F,Yang G,Zhao Z et al.Apoptosis related protein 3,an ATRA-upregulated membrane protein arrests the cell cycle at GI/S phase by decreasing the expression of cyclin D1.Biochem Biophys Res Commun.2007,358(4):1041-6.
[31]Yu Z,Xing Y.atRA-induced apoptosis of mouse embryonic palate mesenchymal cells involves activation of MAPK pathway.Toxicol Appl Pharmacol.2006,215(1):57-63.
[32]Yu Z,Hart J,Lin J.Apoptosis induced by atRA in MEPM cells is mediated through activation of caspase and RAR.Zoxicol Sci.2006,89(2):504-9.
[33]彭安,黄炜,黄济群.维甲酸对Bel-7402人肝癌细胞的诱导分化作用.中国现代医学杂志.2000,10(2):15-16.
[34]Taetle R,Santos B D,Akamatsu K I,et al.Effect of all-trans retinoic acid and antireceptor antibodies on growth and programmed cell death of human myeloma cells.Clinical cancer research,1996,2:253-259.
[35]维甲酸对人肝癌细胞凋亡的影响及临床意义.李良平;张正;韩盛玺.医学科技,2002,02:4-6.
[36]全反式维甲酸联合奥曲肽对肝癌细胞-HepG_2增生和凋亡的影响叶红;但自力;唐望先.世界华人消化杂志,2004,10期.
[37]Delia D,Aiello A,Soligo D et al.bcl-2 proto-oncogene expression in normal and neoplastic human myeloid cells.Blood.1992,79(5):1291-8.
[38]Yonish-Rouach E,Grunwald D,Wilder S,et al.p53-mediated cell death:relationship to cell cycle control.Mol Cell Biol.1993,13(3):1415-23.
[39]Glozak MA,Rogers MB.Specific induction of apoptosis in P19 embryonal carcinoma cells by retinoic acid and BMP2 or BMP4.Dev Biol.1996,179(2):458-70.
[40]曾辉,赵相印,马雅銮等.维甲酸诱导HL-60细胞Fas蛋白表达.中华血液学杂志.1998,05:237-240.
[41]Ramakrishnan S,Eppenberger U,Mueller H,et al.Expression profile of the putative catalytic subunit of the telomerase gene.Cancer Res.1998,58(4):622-5.
[42]Lan Y,Zhang Y,Wang F,et al.Aberrant expression of Cks1 and Cks2 contributes to prostate tumorigenesis by promoting proliferation and inhibiting programmed cell death.Int J Cancer.2008,123(3):543-51.
[43]Kawakami K,Enokida H,Tachiwada T,et al.Identification of differentially expressed genes in human bladder cancer through genome-wide gene expression profiling.Oncol Rep 2006,16:521-31.
[44]Wong YF,Cheung TH,Tsao GS,et al.Genomewide gene expression profiling of cervical cancer in Hong Kong women by oligonucleotide microarray.Int J Canc.2006,118:2461-9.
[45]Chow LS,Lam CW,Chan SY,et al.Identification of RASSF1A modulated genes in nasopharyngeal carcinoma.Oncogene.2006,25:310-16.
[46]de Wit N J,Rijntjes J,Diepstra JH,et al.Analysis of differential gene expression in human melanocytic tumour lesions by custom made oligonucleotide arrays.Br J Canc.2005,92:2249-61.
[47]Uchikado Y,Inoue H,Haraguchi N,et al.Gene expression profiling of lymph node metastasis by oligomicroarray analysis using laser microdissection in esophageal squamous cell carcinoma.Int J Oncol.2006,29:1337-47.
[48]Polyak K,Kato J,Solomon M,et al.P27Kipl,a Cyclin-CDKinhibitor;links transforming growth factor-β and contact inhibition to cell cycle arrest.Genes Dey,1994,8:9-22.
[49]Pagano M,Tam SW,Theodoras AM,et al.Role of the ubiquitin-proteasome pathway in regulating abundance of the cyclin-dependent kinase inhibitor p27.Science,1995,269:682-685.
[50]Carrano AC,Eytan E,Hershko A,SKP2 is required for ubiquitin-mediated degradation of the CDK inhibitor p27.Nat Cell Biol.1999,1(4):193-9.
[51]Barnes A,Pinder SE,Bell JA,et al.Expression of p27Kip1 in breast cancer and its prognostic significance.J Pathol.2003,201:451-459.
[52]Migaldi M,Zunarelli E,Sgambato A,et al.p27Kip1 expression and survival in NO gastric carcinoma.Pathol Res Pract.2001,197:231-236.
[53]Kagawa Y,Yoshida K,Hirai T,et al.Significance of the expression of p27Kip1 in esophageal squamous cell carcinomas.Dis Esophagus.2000,13:179-184.
[54]Hirabayashi H,Ohta M,Tanaka H,et al.Prognostic significance of p27Kip1 expression in resected non-small cell lung cancers:analysis in combination with expressions of pl6INK4A,pRB,and p53.J Surg Oncol.2002,81:177-184.
[55]Dreher T,Zentgraf H,Abel U,et al.Reduction of PTEN and p27Kip1 expression correlattes with tumor grade in prostate cancer.Analysis in radical prostatectomyh specimens and needle biopsies.Virchows Arch.2004,444:509-517.
[1]Ganoth,Bornstein D,Ko G,et al.The cell-cycle regulatory protein Cks1 is required for SCF Skp2-mediated ubiquitinylation of p27.Nat Cell Biol,2001,3:321-324.
[2]Richardson HE,Stueland CS,Thomas J,et al.Human cDNAs encoding homologs of the small p34-Cdc28/Cdc2-associated protein of Saecharomyces cerevisiae and Schizosaccharomyces pombe.Genes,1990,4:1332-1344.
[3]Bourne Y,Watson MH,Arvai AS,et al.Crystal structure and mutational analysis of the Saccharomyces cerevisiae cell cycle regulatory protein Cks1:implications for domain swapping,anion binding and protein interactions.Structure,2000,8:841-850.
[4]Spruck C,Strohmaier H,Watson M.A CDK-independent function of mammalian Cksl:targeting of SCF(Skp2) to the CDK inhibitor p27Kipl.Mol Cell,2001,7:639-650.
[5]Hao B,Zheng N,Brenda A,et al.Structural Basis of the Cks1-Dependent Recognition of p27Kip1 by the SCFSkp2 Ubiquitin Ligase.Mol Cell,2005,20:9-19.
[6]Yao ZP,Zhou M,Sadie E,et al.Activation of Ubiquitin Ligase SCFSkp2 by Cks1:Insights from Hydrogen Exchange Mass Spectrometry.J.Mol.Biol,2006,363:673-686.
[7]Xu S,Abbasian M,Patel P,et al.Substrate Recognition and Ubiquitination of SCFSkp2/Cksl Ubiquitin-Protein Isopeptide Ligase.J.Mol.Biol,2007,282:15462-15470.
[8]Seeliger MA,Spichty M,Kelly SE,et al.Role of conformational heterogeneity in domain swapping and adapter function of the Cks proteins.J.Biol.Chem,2005,280:30448-30459.
[9]Bhatt KV,Hu R,Spofford LS,et al.Mutant B-RAF signaling and cyclin D1 regulate Cksl/S-phase kinase-associated protein 2-mediated degradation of p27Kip1 in human melanoma cells.Oncogene,2007,26:1056-1066.
[10]Wang C,Chen YJ,Douglas H,et al.Forkhead Box Ml Regulates the Transcriptional Network of Genes Essential for Mitotic Progression and Genes Encoding the SCF (Skp2-Cksl) Ubiquitin Ligase.Molecular & Cellular Biology,2005,25:10875-10894.
[11]Keller UB,Old JB,Dorsey FC.Myc targets Cksl to provoke the suppression of p27Kip1,proliferation and lymphomagenesis.EMBO,2007,26:2562-2574.
[12]Wang W,Jin J,Bellur S,et al.Negative regulation of SCFSkp2 ubiquitin ligase by TGF-β signaling.Oncogene,2004,23:1064-1075.
[13]Zancai P,Col JD,Piccinin S.Retinoic acid stabilizes p27Kip1 in EBV-immortalized lymphoblastoid B cell lines through enhanced proteasome-dependent degradation of the p45Skp2 and Cksl proteins.Oncogene,2005,24:2483-2494.
[14]Tsai YS,Chang HC,Chuang LY,et al.RNA Silencing of Cksl Induced G2/M Arrest and Apoptosis in Human Lung Cancer Cells.IUBMB Life,2005,57:583-589.
[15]Kitajima S,Kudo Y,Ogawa I,et al.Role of Cksl Over expression in Oral Squamous Cell Carcinomas Cooperation with Skp2 in Promoting p27 Degradation.Am J Pathol,2004,165:2147-2155.
[16]Slotky M,Shapira M,Izhak OB,et al.The Expression of the Ubiquitin Ligase Subunit Cksl in Human Breast Cancer.Breast Cancer Res,2005,7:737-744.