细胞周期蛋白E同乳腺癌的预后及分子靶向治疗相关性的实验研究
摘要
目的乳腺癌是常见的女性恶性肿瘤,在欧洲及北美等发达地区,乳腺癌的发病率长期位列女性恶性肿瘤的第一位。随着经济的发展,我国大城市和沿海发达地区的发病率也开始呈现迅速上升的趋势,其发病率也已成为女性所有恶性肿瘤中的第一或第二位,严重威胁女性健康。目前在临床上治疗乳腺癌的手段主要有手术治疗、化疗和内分泌治疗,分子靶向治疗刚刚崭露头角,但是相较于传统的治疗手段,它已经显示出巨大的潜在价值。所以,寻找能够对乳腺癌进行早期诊断,或能准确判断其预后,以及能为分子治疗提供新靶点的基因,对于乳腺癌基因治疗的发展和推广就显得尤为重要。目前在分子肿瘤学领域的热点之一就是对肿瘤细胞周期的研究,细胞周期蛋白家族(Cyclins)自然而然就进入了广大研究者的视野,而其中的细胞周期蛋白E(Cyclin E)作为一个已经确定的癌基因,作为细胞周期中G1/S期的关键调节蛋白,其研究价值正日渐浮出水面。近年来,很多国内外乳腺癌疾病的研究者都注意到了Cyclin E,尤其是低分子量剪切型Cyclin E(LMW Cyclin E)同乳腺癌预后之间的密切关系。本课题正是从Cyclin E在乳腺癌组织中的表达水平入手,总结Cyclin E的表达同乳腺癌患者预后之间的关系。接着以RNAi为干预手段,设计可以抑制Cyclin E表达水平的实验工具,在体内及体外水平分别抑制乳腺癌中Cyclin E的表达,进而分析Cyclin E对肿瘤生物学行为的影响并试图挖掘Cyclin E作为一个乳腺癌基因治疗新靶点的潜在价值。
方法收集华中科技大学同济医学院附属协和医院病理科2000-2003年部分乳腺病检蜡块,结合其相应的临床资料,按国际抗癌学会(UICC)及美国癌症联合会(AJCC)建议的TNM分期法(第六版)对其进行临床分期。其中乳腺良性肿瘤18例,恶性肿瘤(乳腺癌)80例,平均年龄50岁,所有患者于手术前未接受任何抗癌治疗。用免疫组织化学法检测上述研究对象中Cyclin E蛋白的表达,以及C-erbB-2(HER-2/neu)、nm23-H1、actin(肌动蛋白)的表达。根据Cyclin E cDNA序列设计引物。再依照载体法合成siRNA的原则选择2段靶序列,设计并合成Cyclin E基因的siRNA片段,退火后于pGENSIL-1载体连接,构建Cyclin E基因的siRNA真核表达载体。再用该载体瞬时转染人乳腺癌细胞株MCF-7,检测转染前后对照组及转染组中Cyclin E mRNA的表达与蛋白水平的表达,CCK-8法检测siRNA对细胞的抑制效应,流式细胞术检测细胞周期。分别用对照组及转染组MCF-7细胞建立裸鼠移植瘤模型,绘制肿瘤生长曲线。取一部分荷瘤裸鼠模型以siRNA进行肿瘤局部注射,模拟基因治疗,并绘制肿瘤生长曲线。
结果Cyclin E在良、恶性肿瘤组织中过表达的差异性有极显著的统计学意义(χ2=13.29,P<0.01),即在良性乳腺肿瘤中Cyclin E的过表达率很低,而在乳腺癌组织中Cyclin E的过表达率较高;而在乳腺癌的不同组织类型中Cyclin E过表达的差异性没有统计学意义(χ2=0.1099,P>0.05)。在临床分期较早的乳腺癌组织中Cyclin E的过表达率较低,而在临床分期较晚的乳腺癌组织中Cyclin E的过表达率较高,且随着临床分期的进展,Cyclin E的过表达率也相应地升高。ER阳性的乳腺癌组织中Cyclin E的过表达率较ER阴性者为低,其Cyclin E过表达率的差异具有显著的统计学意义(χ2=4.1112,P<0.05)。在PR阳性或阴性的乳腺癌组织中,Cyclin E阳性表达率的差异性没有显著的统计学意义(χ2=1.3267,P>0.05)。在C-erbB-2(HER-2/neu)呈阳性表达乳腺癌组织中,Cyclin E的阳性表达率较高;而在C-erbB-2(HER-2/neu)呈阴性表达的乳腺癌组织中,Cyclin E的阳性表达率较低,这一阳性表达率的差异性有着显著的统计学意义(χ2=4.0635,P<0.05)。在nm23-H1表达呈阳性和阴性的乳腺癌组织中,Cyclin E的阳性表达率的差异性没有显著的统计学意义(χ2=1.6737,P>0.05)。actin呈阴性表达或是呈不连续分布即恶性度高的乳腺癌组织同actin呈阳性表达且分布连续的恶性度低的乳腺癌组织相比,其Cyclin E的阳性表达率的差异有着显著的统计学意义(χ2=4.2092,P<0.05)。成功地设计并合成了序列ctrl,CCNE-1和CCNE-2,其测序结果与我们设计合成的靶向Cyclin E的反义链完全一致,并将上述目的基因正确地插入pGENESIL-1载体,成功构建重组质粒。转染24 h后的转染效率约50%。转染48h后实验组细胞内Cyclin E的mRNA RT-PCR产量明显低于空白对照及阴性对照组。siRNA作用于细胞后,Western Blot检测显示Cyclin E蛋白水平的表达有显著降低。经化疗药物处理后,转染后细胞的抑制率较未转染细胞有明显上升,且这一差异有统计学意义。转染siRNA后,乳腺癌细胞被大量阻滞于G1期。动物实验方面,对照组裸鼠移植瘤生长速度较转染组快,瘤体体积也较大。空白对照组和生理盐水对照组中裸鼠移植瘤的生长较siRNA局部注射治疗组迅速,肿瘤体积也较大。
结论在一定程度上,Cyclin E的过表达可以反映乳腺的良、恶性病变的差异,Cyclin E的表达与乳腺癌临床分期密切相关,即由Ⅰ期至Ⅳ期阳性表达率逐渐升高,或可将其作为一个判断乳腺肿瘤预后的分期性指标。抑制乳腺癌细胞中Cyclin E的表达可以显著抑制肿瘤细胞的生长,提高肿瘤对化疗的敏感性,抑制肿瘤细胞的成瘤能力,Cyclin E siRNA对肿瘤生长亦具有抑制作用。Cyclin E具有成为乳腺癌基因治疗新靶点的潜在价值。
Objective Breast cancer is a common women malicious disease. In Europe and North America, the incidence of breast cancer has been the first place of all the women malicious diseases for many years. As the development of economy, the incidence of breast cancer began to raise quickly in some developed littoral area of China, it has already been the first or second place of all the cancers of women. Breast cancer seriously threatened women health. At present, the major clinical treatments to breast cancer are surgery, chemical therapy and endocrine therapy. Gene therapy has emerged recently, it showed tremendous potential ability to treat breast cancer. So, it is very important to find some new genes which can early diagnose breast cancer, or predict the prognosis of breast cancer, or provide a target for gene therapy. Lately, the hottest spot in molecular oncology is the research of cell cycle, and Cyclins are the most popular gene family in this field. As a oncogene and a gatekeeper gene of G1/s phase in cell cycle, Cyclin E, especially LMW Cyclin E has been noticed for the strong relationship to breast caner prognosis by many researchers. This study measured the expression of Cyclin E in breast cancer, conclude the relationship between Cyclin E expression and breast cancer prognosis. Then we designed siRNA sequences according to RNAi technology to inhibit the expression of Cyclin E in vivo and in vitro, and analysed the influence of Cyclin E to breast cancer's biology behavior, and finally discovered the potential value of Cyclin E as a new taget to gene therapy.
Methods Tumor tissues were obtained from the pathology department of Huazhong University of Science and Technology Tongji Medical College Union Hospital. Each patient had received a diagnosis of breast cancer according to the TNM method which is recommended by UICC and AJCC between 2000 and 2003. Among them there are 18 cases of benign tumor and 80 cases of breast cancer. All patients have not accepted any treatment before surgery. We examined the expression of Cyclin E, HER-2/neu, nm23-H1 and actin by immunohistochemical methods. We designed and synthesized two pairs of siRNA according to the Cyclin E cDNA sequence in Genebank, then inserted them into pGENESIL-1 plasmids respectively. The recombinants (named pGENESIL/Cyclin E-1 and pGENESIL /Cyclin E-1) were sequenced and identified. We transfected the vector into MCF-7 cell line, RT-PCR and Western blot were performanced to examine the expression of Cyclin E in mRNA and protein level respectively. Cell cycle was measured by FCM. And after being treated with chemotherapy drugs, the MCF-7 growth and proliferation were examined by CCK-8 assay. The nude mice bearing human breast cancer were prepared with MCF-7 and transfected MCF-7 and Cyclin E siRNA was injected to nude mice bearing human breast cancer. The growth of tumor was observed.
Results The over expression rate of Cyclin E in malignant tissues is obviously higher than that in benign tumor tissues, P<0.01. The over expression of Cyclin E in later stage of disease is higher than that in early stage of disease, P<0.05.The expression of Cyclin E in ER positive tissues is lower than that in ER negative tissues, P<0.05. The expression of Cyclin E in PR positive tissues and in PR negative tissues has no significant different, P>0.05. The expression of Cyclin E in HER-2/neu positive tissues is higher than that in HER-2/neu negative tissues, P<0.05. And the expression of Cyclin E in ER, PR and HER-2/neu all positive tissues is much higher, P<0.01. The expression of Cyclin E in nm23-H1 positive tissues and in nm23-H1 negative tissues has no significant different, P>0.05. The expression of Cyclin E in actin positive and continuous distribution tissues is lower than that in actin negative or uncontinuous distribution tissues, P<0.05. Cyclin E siRNA eukaryotic expression vectors were succesfully constructed. Sequence analysis of inserted fragments revealed the same sequences as synthesized siRNA oligonucleotides. RT-PCR and Western blot showed the expression of Cyclin E was significantly lower after MCF-7 was transfected. After being treated by chemical drugs, the growth and proliferation of transfected MCF-7 was lower then that of non-tranfected cells, and there was a significant difference. And its cell cycle was arrested at G1 phase. The tumor forming ability of transfected MCF-7 was lower than that of non-transfected MCF-7. Cyclin E siRNA could inhibit the growth of tumor beared by nude mice.
Conclusions To some extend, the over expression of Cyclin E can reflect the difference between benign and malicious breast diseases. The level of Cyclin E expression has a strong relationship with the clinical phases of breast cancer. The expression of Cyclin E can be a division index of breast cancer prognosis. Inhibition of Cyclin E can obviously depress cancer cells' proliferation, raise their sensibility to chemical therapy and restrain their tumor forming ability. Cyclin E siRNA can be used to inhibit the growth of breast cancer cells. Cyclin E has the potential to be a new target for gene therapy of breast cancer.
方法收集华中科技大学同济医学院附属协和医院病理科2000-2003年部分乳腺病检蜡块,结合其相应的临床资料,按国际抗癌学会(UICC)及美国癌症联合会(AJCC)建议的TNM分期法(第六版)对其进行临床分期。其中乳腺良性肿瘤18例,恶性肿瘤(乳腺癌)80例,平均年龄50岁,所有患者于手术前未接受任何抗癌治疗。用免疫组织化学法检测上述研究对象中Cyclin E蛋白的表达,以及C-erbB-2(HER-2/neu)、nm23-H1、actin(肌动蛋白)的表达。根据Cyclin E cDNA序列设计引物。再依照载体法合成siRNA的原则选择2段靶序列,设计并合成Cyclin E基因的siRNA片段,退火后于pGENSIL-1载体连接,构建Cyclin E基因的siRNA真核表达载体。再用该载体瞬时转染人乳腺癌细胞株MCF-7,检测转染前后对照组及转染组中Cyclin E mRNA的表达与蛋白水平的表达,CCK-8法检测siRNA对细胞的抑制效应,流式细胞术检测细胞周期。分别用对照组及转染组MCF-7细胞建立裸鼠移植瘤模型,绘制肿瘤生长曲线。取一部分荷瘤裸鼠模型以siRNA进行肿瘤局部注射,模拟基因治疗,并绘制肿瘤生长曲线。
结果Cyclin E在良、恶性肿瘤组织中过表达的差异性有极显著的统计学意义(χ2=13.29,P<0.01),即在良性乳腺肿瘤中Cyclin E的过表达率很低,而在乳腺癌组织中Cyclin E的过表达率较高;而在乳腺癌的不同组织类型中Cyclin E过表达的差异性没有统计学意义(χ2=0.1099,P>0.05)。在临床分期较早的乳腺癌组织中Cyclin E的过表达率较低,而在临床分期较晚的乳腺癌组织中Cyclin E的过表达率较高,且随着临床分期的进展,Cyclin E的过表达率也相应地升高。ER阳性的乳腺癌组织中Cyclin E的过表达率较ER阴性者为低,其Cyclin E过表达率的差异具有显著的统计学意义(χ2=4.1112,P<0.05)。在PR阳性或阴性的乳腺癌组织中,Cyclin E阳性表达率的差异性没有显著的统计学意义(χ2=1.3267,P>0.05)。在C-erbB-2(HER-2/neu)呈阳性表达乳腺癌组织中,Cyclin E的阳性表达率较高;而在C-erbB-2(HER-2/neu)呈阴性表达的乳腺癌组织中,Cyclin E的阳性表达率较低,这一阳性表达率的差异性有着显著的统计学意义(χ2=4.0635,P<0.05)。在nm23-H1表达呈阳性和阴性的乳腺癌组织中,Cyclin E的阳性表达率的差异性没有显著的统计学意义(χ2=1.6737,P>0.05)。actin呈阴性表达或是呈不连续分布即恶性度高的乳腺癌组织同actin呈阳性表达且分布连续的恶性度低的乳腺癌组织相比,其Cyclin E的阳性表达率的差异有着显著的统计学意义(χ2=4.2092,P<0.05)。成功地设计并合成了序列ctrl,CCNE-1和CCNE-2,其测序结果与我们设计合成的靶向Cyclin E的反义链完全一致,并将上述目的基因正确地插入pGENESIL-1载体,成功构建重组质粒。转染24 h后的转染效率约50%。转染48h后实验组细胞内Cyclin E的mRNA RT-PCR产量明显低于空白对照及阴性对照组。siRNA作用于细胞后,Western Blot检测显示Cyclin E蛋白水平的表达有显著降低。经化疗药物处理后,转染后细胞的抑制率较未转染细胞有明显上升,且这一差异有统计学意义。转染siRNA后,乳腺癌细胞被大量阻滞于G1期。动物实验方面,对照组裸鼠移植瘤生长速度较转染组快,瘤体体积也较大。空白对照组和生理盐水对照组中裸鼠移植瘤的生长较siRNA局部注射治疗组迅速,肿瘤体积也较大。
结论在一定程度上,Cyclin E的过表达可以反映乳腺的良、恶性病变的差异,Cyclin E的表达与乳腺癌临床分期密切相关,即由Ⅰ期至Ⅳ期阳性表达率逐渐升高,或可将其作为一个判断乳腺肿瘤预后的分期性指标。抑制乳腺癌细胞中Cyclin E的表达可以显著抑制肿瘤细胞的生长,提高肿瘤对化疗的敏感性,抑制肿瘤细胞的成瘤能力,Cyclin E siRNA对肿瘤生长亦具有抑制作用。Cyclin E具有成为乳腺癌基因治疗新靶点的潜在价值。
Objective Breast cancer is a common women malicious disease. In Europe and North America, the incidence of breast cancer has been the first place of all the women malicious diseases for many years. As the development of economy, the incidence of breast cancer began to raise quickly in some developed littoral area of China, it has already been the first or second place of all the cancers of women. Breast cancer seriously threatened women health. At present, the major clinical treatments to breast cancer are surgery, chemical therapy and endocrine therapy. Gene therapy has emerged recently, it showed tremendous potential ability to treat breast cancer. So, it is very important to find some new genes which can early diagnose breast cancer, or predict the prognosis of breast cancer, or provide a target for gene therapy. Lately, the hottest spot in molecular oncology is the research of cell cycle, and Cyclins are the most popular gene family in this field. As a oncogene and a gatekeeper gene of G1/s phase in cell cycle, Cyclin E, especially LMW Cyclin E has been noticed for the strong relationship to breast caner prognosis by many researchers. This study measured the expression of Cyclin E in breast cancer, conclude the relationship between Cyclin E expression and breast cancer prognosis. Then we designed siRNA sequences according to RNAi technology to inhibit the expression of Cyclin E in vivo and in vitro, and analysed the influence of Cyclin E to breast cancer's biology behavior, and finally discovered the potential value of Cyclin E as a new taget to gene therapy.
Methods Tumor tissues were obtained from the pathology department of Huazhong University of Science and Technology Tongji Medical College Union Hospital. Each patient had received a diagnosis of breast cancer according to the TNM method which is recommended by UICC and AJCC between 2000 and 2003. Among them there are 18 cases of benign tumor and 80 cases of breast cancer. All patients have not accepted any treatment before surgery. We examined the expression of Cyclin E, HER-2/neu, nm23-H1 and actin by immunohistochemical methods. We designed and synthesized two pairs of siRNA according to the Cyclin E cDNA sequence in Genebank, then inserted them into pGENESIL-1 plasmids respectively. The recombinants (named pGENESIL/Cyclin E-1 and pGENESIL /Cyclin E-1) were sequenced and identified. We transfected the vector into MCF-7 cell line, RT-PCR and Western blot were performanced to examine the expression of Cyclin E in mRNA and protein level respectively. Cell cycle was measured by FCM. And after being treated with chemotherapy drugs, the MCF-7 growth and proliferation were examined by CCK-8 assay. The nude mice bearing human breast cancer were prepared with MCF-7 and transfected MCF-7 and Cyclin E siRNA was injected to nude mice bearing human breast cancer. The growth of tumor was observed.
Results The over expression rate of Cyclin E in malignant tissues is obviously higher than that in benign tumor tissues, P<0.01. The over expression of Cyclin E in later stage of disease is higher than that in early stage of disease, P<0.05.The expression of Cyclin E in ER positive tissues is lower than that in ER negative tissues, P<0.05. The expression of Cyclin E in PR positive tissues and in PR negative tissues has no significant different, P>0.05. The expression of Cyclin E in HER-2/neu positive tissues is higher than that in HER-2/neu negative tissues, P<0.05. And the expression of Cyclin E in ER, PR and HER-2/neu all positive tissues is much higher, P<0.01. The expression of Cyclin E in nm23-H1 positive tissues and in nm23-H1 negative tissues has no significant different, P>0.05. The expression of Cyclin E in actin positive and continuous distribution tissues is lower than that in actin negative or uncontinuous distribution tissues, P<0.05. Cyclin E siRNA eukaryotic expression vectors were succesfully constructed. Sequence analysis of inserted fragments revealed the same sequences as synthesized siRNA oligonucleotides. RT-PCR and Western blot showed the expression of Cyclin E was significantly lower after MCF-7 was transfected. After being treated by chemical drugs, the growth and proliferation of transfected MCF-7 was lower then that of non-tranfected cells, and there was a significant difference. And its cell cycle was arrested at G1 phase. The tumor forming ability of transfected MCF-7 was lower than that of non-transfected MCF-7. Cyclin E siRNA could inhibit the growth of tumor beared by nude mice.
Conclusions To some extend, the over expression of Cyclin E can reflect the difference between benign and malicious breast diseases. The level of Cyclin E expression has a strong relationship with the clinical phases of breast cancer. The expression of Cyclin E can be a division index of breast cancer prognosis. Inhibition of Cyclin E can obviously depress cancer cells' proliferation, raise their sensibility to chemical therapy and restrain their tumor forming ability. Cyclin E siRNA can be used to inhibit the growth of breast cancer cells. Cyclin E has the potential to be a new target for gene therapy of breast cancer.
引文
1. Wu Zhengyan, Zhen Linlin, Fan Ping. Mutation Analysis in the BRCA1 Gene in Chinese Breast Cancer Families. The Chinese-German Journal of Clinical Oncology, September 2003, Vol.2, No.3, 153-155
2. Sutherland R L, Musgrove E A. Cyclin E and prognosis in patients with breast cancer.N Engl J Med, 2002, 347: 1546
3. Wu Zhengyan, Zheng Wei. Expression of EGFR, C-erbB-2 Protein in Breast Cancer and Their Relation with Micrometastasis in Bone Marrow. The Chinese-German Journal of Clinical Oncology, june 2003, Vol.2, No.2, 95-98
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1. WU Z Y, ZHEN L L, FAN P. Mutation Analysis in the BRCA1 gene in chinese breast cancer Families[J]. The Chinese-German Journal of Clinical Oncology, 2003, 2:153-155.
2. MARTIN M. Molecular biology of breast cancer[J]. Clin Transl Oncol. 2006, 8:7-14.
3. SUTHERLAND R L, MUSGROVE E A. Cyclin E and prognosis in patients with breast cancer[J]. N Engl J Med, 2002, 347: 1546.
4.何文山,黄韬.细胞周期蛋白E及其与乳腺癌预后关系的研究近况[J].医学分子生物学杂志, 2004 , 1: 292-294.
5.何文山,黄韬,王海久. Cyclin E在乳腺癌组织中的表达及其与乳腺癌预后的关系[J].华中科技大学学报(医学版), 2004, 33:304-307.
6. KEYOMARSI K, OLEARY N, MOLNAR G, et al. Cyclin E, a potential prognostic marker for breast cancer[J]. Cancer Res, 1994; 54:380-385.
7. MUMBERG D, HAAS K., M?r?y T, et al. Uncoupling of DNA replication and cell cycle progression by human cyclin E[J]. Oncogene, 1996, 13: 2493-2497.
8. KEYOMARSI K, TUCKER S L, BUCHHOLZ T A, et al. Cyclin E and survival in patients with breast cancer[J]. N Engl J Med, 2002, 347: 1566-1575.
9. MORRIS KV. Therapeutic potential of siRNA-mediated transcriptional gene silencing[J]. Biotechniques, 2006, Suppl: 7-13.
1. Sutherland RL, Musgrove EA. Cyclins and breast cancer. J Mammary Gland Biol Neoplasia. 2004 Jan;9(1):95-104.
2.何文山,黄韬,王海久. Cyclin E在乳腺癌组织中的表达及其与乳腺癌预后的关系.华中科技大学学报(医学版),2004,33(3):304-307.
3. HE Wenshan, HUANG Tao, and WANG Haijiu. Expression of Cyclin E and Its Relationship with the Prognosis of Patients With Breast Cancer. The Chinese-German Journal of Clinical Oncology,2006,5(4):275-278.
4. Keyomarsi K, O’leary N, Molnar G, Lees E, Fingert HJ,Pardee AB. Cyclin E, a potential prognostic marker for breast cancer. Cancer Res 1994;54:380-5.
5. Mumberg D, Haas K, Moroy T, et al. Uncoupling of DNA replication and cell cycle progression by human cyclin E.Oncogene, 1996, 13: 2493-2497.
6. Desmedt C, Ouriaghli FE, Durbecq V, Soree A, Colozza MA, Azambuja E, Paesmans M, Larsimont D, Buyse M, Harris A, Piccart M, Martiat P, Sotiriou C. Impact of cyclins E, neutrophil elastase and proteinase 3 expression levels on clinical outcome in primary breast cancer patients. Int J Cancer. 2006 Dec 1;119(11):2539-45.
7. Keyomarsi K, Tucker S L, Buchholz T A, Callister M, Ding Y, Hortobagyi G N,Bedrosian I, Knickerbocker C, Toyofuku W, Lowe M, Herliczek T W, Bacus S S.Cyclin E and survival in patients with breast cancer. N Engl J Med, 2002, 347: 1566
8. Potemski P, Kusinska R, Watala C, Pluciennik E, Bednarek AK, Kordek R. Cyclin E expression in breast cancer correlates with negative steroid receptor status, HER2 expression, tumor grade and proliferation. J Exp Clin Cancer Res. 2006 Mar;25(1):59-64.
9. Lopez-Beltran A, MacLennan GT, Montironi R. Cyclin E as molecular marker in the management of breast cancer: a review. Anal Quant Cytol Histol. 2006 Apr;28(2):111-4.
10. Yan Geng, Qunyan Yu, Wendy Whoriskey, Fred Dick, Kenneth Y. Tsai, Heide L. Ford,Debajit K. Biswas, Arthur B. Pardee, Bruno Amati, Tyler Jacks, Andrea Richardson, Nicholas Dyson, and Piotr Sicinski. Expression of cyclins E1 and E2 during mouse development and in neoplasia. PNAS. 2001 November;98(23):13138-13143.
11. Lents NH, Baldassare JJ. CDK2 and cyclin E knockout mice: lessons from breast cancer. Trends Endocrinol Metab. 2004 Jan-Feb;15(1):1-3.
12. Morris KV. Therapeutic potential of siRNA-mediated transcriptional gene silencing. Biotechniques. 2006 Apr; Suppl:7-13.
13. Berglund P, Landberg G. Cyclin e overexpression reduces infiltrative growth in breast cancer: yet another link between proliferation control and tumor invasion. Cell Cycle. 2006 Mar;5(6):606-9. Epub 2006 Mar 15.
14. Berglund P, Stighall M, Jirstrom K, Borgquist S, Sjolander A, Hedenfalk I, Landberg G. Cyclin E overexpression obstructs infiltrative behavior in breast cancer: a novel role reflected in the growth pattern of medullary breast cancers. Cancer Res. 2005 Nov 1;65(21):9727-34.
15. Porter PL, Malone KE, Heagerty PJ, et al. Expression of cell-cycle regulators p27Kip1 and cyclinE, alone and incombination, correlate with survival in young breast cancer patients[J]. Nat Med, 1997, 3(2):222-225.
16. Donnellan R, Kleinschmidt I, Chetty R. Cyclin E immunoexpression in breast ductal carcinoma: pathologic and prognostic implications. Hum Pathol. 2001; 32:89-94.
17. Akli S, Keyomarsi K. Low-molecular-weight cyclin E: the missing link between biology and clinical outcome. Breast Cancer Res. 2004;6(5):188-91. Epub 2004 Jul 7.
18. Spruck C, Sun D, Fiegl H, Marth C, Mueller-Holzner E, Goebel G, Widschwendter M, Reed SI. Detection of low molecular weight derivatives of cyclin E1 is a function of cyclin E1 protein levels in breast cancer. Cancer Res. 2006 Jul 15;66(14):7355-60.
1. Hinds PW, Mittnacht S, Dulic V, et al. Regulation of retinoblastoms protein functions by ectopic of human cyclins. Cell, 1992, 70:993-1006.
2. Koff A,Giordano A,Desai D,et al.Formation and activation of a cyclin E-cdk2 complex during the G1 phase of the human cell cycle.Science,1992;257:1689-1694.
3. Koff A, Cross F, Fisher A, et al. Hu man cyclin E, a new cyclin that interactswith two members of the CDC2 gene family[J]. Cell, 1991, 66 (6): 1217 -1228.
4. Lew DJ, Dulic V, Reed SI. Isolation of three novel human cyclins by rescue of G1cyclin (cln) function in yeast. Cell, 1991, 66:1197-1206.
5. Keyomarsi K, Pardee AB. Redundant cyclin overexpression and gene amplification in breast cancer cell. Proc Natl Acad Sci U S A 1993;90:1112-6
6. Keyomarsi K, O’leary N, Molnar G, Lees E, Fingert HJ,Pardee AB. Cyclin E, a potential prognostic marker for breast cancer. Cancer Res 1994;54:380-5.
7. Keyomarsi K, Herliczek TW. The role of Cyclin E in cell proliferation, development and cancer. In: Meijer L, Guidert S, Philippe M, eds. Progress in cell cycle research. Vol. 3. New York: Plenum Press, 1997:171-91.
8. Geng Y. Eaton E N. Picon M, et al. Regulation of cyclin E transcription byE2Fs and retinoblastoma protein[J]. Oncogene. 1996,12(6): 1173-1180.
9. Hartwell L, Kastsan MB. Cell cycle control and cancer. Science, 1994, 266:1820-1828.
10. Resnitzky D, Gossen M, Bujard H, Reed SI. Acceleration of the G1/S phase transition by expression of cyclin D1 and E with an induction system. Mol Cell Biol, 1994, 14:1669-1679.
11. Wimmel A, Lucibello FC, Sewing A, et al. Inducible acceleration of G1 progressi on through tetracycline-regulated expression of human cyclin E. Oncogene 1994; 9:995-997.
12. Kwon TK, Nordin AA. Overexpression of cyclin E and cyclin dependent kinase inhibitor p27kip1 Effect on cell cycle regulation in Hela cell. Biochemical and Biophysical Research Communications, 1997, 238:534-538.
13. Charles J. Sherr Cancer Cell Cycles[J]. Science, 1996, 274:1672-1677.
14. Ikezawa K, Ohtsubo M, Norwood TH, et al.Role of cyclin E and cyclin E-dependent kinase in mitogenic stimulation by cementum-derived growth factor in human fibroblasts[J]. FASEB J, 1998, 12(12):1233-1239.
15. Geng Y, Eaton En, Picon M, et al. Regulation of Cyclin E transcription by E2Fs and retinoblastoma a protein[j]. Ocogene, 1996, 12(6):1173-1180.
16. Leng X, Connell-Crow ley L, Goodrich D, et al. S-Phase entry upon ectopicexpressionof G1 cyclin-dependent kinase in the absence of retinoblastoma a protein phosphorylation[J]. Curr Biol, 1997, 7(9):709-7712.
17. Mumberg D, Haas K, Moroy T, et al. Uncoupling of DNA replication and cell cycle progression by human cyclin E. Oncogene, 1996, 13: 2493-2497..
18. ScottKA, WalkerRA. Lack of cyclinE immuno reactivity in non-malignant-breast and association with proliferation in breast cancer[J]. Br J Cancer, 1997, 76(10):1288-1292.
19. Porter PL, Malone KE, Heagerty PJ, et al. Expression of cell-cycle regulators p27Kip1 and cyclinE, alone and incombination, correlate with survival in young breast cancer patients[J]. Nat Med, 1997, 3(2):222-225.
20. Porter D, Zhang N, Danes C, et al. Tumor-specific proteolytic processing of cyclin E generates hyperactive low-molecular-weight forms. Mol Cell Boil. 2001; 21:6254-69
21. Neilsen Nh, Arnerlov C, Emdin SO and Landberg G. Cyclin E overexpression, a negative prognostic factor in breast cancer with strong correlation to oestrogen receptor status. Br J Cancer, 1996, 74:874-880.
22. Ford HL, Kabingu En, Bump EA, Mutter GL and Pardee Ab. Abrogation of the G2 cell cycle checkpoint associated with overexpression of HSIX1: a possible mechanism of breast carcinogenesis. Proc Natl Acad Sci USA, 1998, 95:12608-12613.
23. Keyomarsi K, Conte D, Toyofuku W and Fox MP. Deregulation of cyclin E in breast cancer. Oncogene, 1995, 11:941-950.
24. Wang XD, Rosales JL, Magliocco A, Gnanakumar R, Lee KY. Cyclin E in breast tumors is cleaved into its low molecular weight forms by calpain. Oncogene. 2003 Feb 6;22(5):769-74.
25. Donnellan R, Kleinschmidt I, Chetty R. Cyclin E immunoexpression in breast ductal carcinoma: pathologic and prognostic implications. Hum Pathol. 2001; 32:89-94.
26. Keyomarsi K, Tucker SL, Buchholz TA, Callister M, Ding Y, Hortobagyi GN,Bedrosian I, Knickerbocker C, Toyofuku W, Lowe M, Herliczek TW, Bacus SS. Cyclin E and survival in patients with breast cancer. N Engl J Med. 2002 Nov14;347(20):1566-75.
27. Han S, Park K, Bae BN, Kim KH, Kim HJ, Kim YD, Kim HY. Prognostic implication of cyclin E expression and its relationship with cyclin D1 and p27Kip1 expression on tissue microarrays of node negative breast cancer. J Surg Oncol. 2003 Aug;83(4):241-7.
28. Rudolph P, Kuhling H, Alm P, Ferno M, Baldetorp B, Olsson H, Parwaresch R. Differential prognostic impact of the cyclins E and B in premenopausal and postmenopausal women with lymph node-negative breast cancer. Int J Cancer. 2003 Jul 10;105(5):674-80.
29. Span PN, Tjan-Heijnen VC, Manders P, Beex LV, Sweep CG. Cyclin-E is a strong predictor of endocrine therapy failure in human breastcancer. Oncogene. 2003 Jul 31;22(31):4898-904.
30. Christov K, Ikui A, Shilkaitis A, Green A, Yao R, You M, Grubbs C, Steele V, Lubet R, Weinstein IB. Cell proliferation, apoptosis, and expression of cyclin D1 and cyclin E as potential biomarkers in tamoxifen-treated mammary tumors. Breast Cancer Res Treat. 2003 Feb;77(3):253-64.
31. Early Breast Cancer Trialists’Collaborative Group. Polychemotherapy for early breast cancer: an overview of the randomised trials. Lancet 1998;352:930-42.
32. Early Breast Cancer Trialists'Collaborative Group. Tamoxifen for early breast cancer: an overview of the randomised trials. Lancet 1998;351:1451-67.
2. Sutherland R L, Musgrove E A. Cyclin E and prognosis in patients with breast cancer.N Engl J Med, 2002, 347: 1546
3. Wu Zhengyan, Zheng Wei. Expression of EGFR, C-erbB-2 Protein in Breast Cancer and Their Relation with Micrometastasis in Bone Marrow. The Chinese-German Journal of Clinical Oncology, june 2003, Vol.2, No.2, 95-98
4. Porter P L, Malone K E, Heagerty P J, et al. Expression of cell-cycle regulators p27Kip1 and cyclinE, alone and incombination, correlate with survival in young breast cancer patients[J]. Nat Med, 1997, 3: 222
5. Donnellan R, Kleinschmidt I, Chetty R. Cyclin E immunoexpression in breast ductal carcinoma: pathologic and prognostic implications. Hum Pathol, 2001, 32:89
6. Keyomarsi K, Tucker S L, Buchholz T A, Callister M, Ding Y, Hortobagyi G N, Bedrosian I, Knickerbocker C, Toyofuku W, Lowe M, Herliczek T W, Bacus S S. Cyclin E and survival in patients with breast cancer. N Engl J Med, 2002, 347: 1566
7. Han S, Park K, Bae B N, Kim K H, Kim H J, Kim Y D, Kim H Y. Prognostic implication of cyclin E expression and its relationship with cyclin D1 and p27Kip1 expression on tissue microarrays of node negative breast cancer. J Surg Oncol, 2003, 83: 244
8. Rudolph P, Kuhling H, Alm P, Ferno M, Baldetorp B, Olsson H, Parwaresch R. Differential prognostic impact of the cyclins E and B in premenopausal and postmenopausal women with lymph node-negative breast cancer. Int J Cancer, 2003, 105: 674
9. Span P N, Tjan-Heijnen V C, Manders P, Beex L V, Sweep C G.Cyclin-E is a strong predictor of endocrine therapy failure in human breast cancer.Oncogene, 2003, 22:4898
10. Christov K, Ikui A, Shilkaitis A, Green A, Yao R, You M, Grubbs C, Steele V, Lubet R, Weinstein I B. Cell proliferation, apoptosis, and expression of cyclin D1 and cyclin E as potential biomarkers in tamoxifen-treated mammary tumors. Breast Cancer Res Treat, 2003, 77: 253
1. WU Z Y, ZHEN L L, FAN P. Mutation Analysis in the BRCA1 gene in chinese breast cancer Families[J]. The Chinese-German Journal of Clinical Oncology, 2003, 2:153-155.
2. MARTIN M. Molecular biology of breast cancer[J]. Clin Transl Oncol. 2006, 8:7-14.
3. SUTHERLAND R L, MUSGROVE E A. Cyclin E and prognosis in patients with breast cancer[J]. N Engl J Med, 2002, 347: 1546.
4.何文山,黄韬.细胞周期蛋白E及其与乳腺癌预后关系的研究近况[J].医学分子生物学杂志, 2004 , 1: 292-294.
5.何文山,黄韬,王海久. Cyclin E在乳腺癌组织中的表达及其与乳腺癌预后的关系[J].华中科技大学学报(医学版), 2004, 33:304-307.
6. KEYOMARSI K, OLEARY N, MOLNAR G, et al. Cyclin E, a potential prognostic marker for breast cancer[J]. Cancer Res, 1994; 54:380-385.
7. MUMBERG D, HAAS K., M?r?y T, et al. Uncoupling of DNA replication and cell cycle progression by human cyclin E[J]. Oncogene, 1996, 13: 2493-2497.
8. KEYOMARSI K, TUCKER S L, BUCHHOLZ T A, et al. Cyclin E and survival in patients with breast cancer[J]. N Engl J Med, 2002, 347: 1566-1575.
9. MORRIS KV. Therapeutic potential of siRNA-mediated transcriptional gene silencing[J]. Biotechniques, 2006, Suppl: 7-13.
1. Sutherland RL, Musgrove EA. Cyclins and breast cancer. J Mammary Gland Biol Neoplasia. 2004 Jan;9(1):95-104.
2.何文山,黄韬,王海久. Cyclin E在乳腺癌组织中的表达及其与乳腺癌预后的关系.华中科技大学学报(医学版),2004,33(3):304-307.
3. HE Wenshan, HUANG Tao, and WANG Haijiu. Expression of Cyclin E and Its Relationship with the Prognosis of Patients With Breast Cancer. The Chinese-German Journal of Clinical Oncology,2006,5(4):275-278.
4. Keyomarsi K, O’leary N, Molnar G, Lees E, Fingert HJ,Pardee AB. Cyclin E, a potential prognostic marker for breast cancer. Cancer Res 1994;54:380-5.
5. Mumberg D, Haas K, Moroy T, et al. Uncoupling of DNA replication and cell cycle progression by human cyclin E.Oncogene, 1996, 13: 2493-2497.
6. Desmedt C, Ouriaghli FE, Durbecq V, Soree A, Colozza MA, Azambuja E, Paesmans M, Larsimont D, Buyse M, Harris A, Piccart M, Martiat P, Sotiriou C. Impact of cyclins E, neutrophil elastase and proteinase 3 expression levels on clinical outcome in primary breast cancer patients. Int J Cancer. 2006 Dec 1;119(11):2539-45.
7. Keyomarsi K, Tucker S L, Buchholz T A, Callister M, Ding Y, Hortobagyi G N,Bedrosian I, Knickerbocker C, Toyofuku W, Lowe M, Herliczek T W, Bacus S S.Cyclin E and survival in patients with breast cancer. N Engl J Med, 2002, 347: 1566
8. Potemski P, Kusinska R, Watala C, Pluciennik E, Bednarek AK, Kordek R. Cyclin E expression in breast cancer correlates with negative steroid receptor status, HER2 expression, tumor grade and proliferation. J Exp Clin Cancer Res. 2006 Mar;25(1):59-64.
9. Lopez-Beltran A, MacLennan GT, Montironi R. Cyclin E as molecular marker in the management of breast cancer: a review. Anal Quant Cytol Histol. 2006 Apr;28(2):111-4.
10. Yan Geng, Qunyan Yu, Wendy Whoriskey, Fred Dick, Kenneth Y. Tsai, Heide L. Ford,Debajit K. Biswas, Arthur B. Pardee, Bruno Amati, Tyler Jacks, Andrea Richardson, Nicholas Dyson, and Piotr Sicinski. Expression of cyclins E1 and E2 during mouse development and in neoplasia. PNAS. 2001 November;98(23):13138-13143.
11. Lents NH, Baldassare JJ. CDK2 and cyclin E knockout mice: lessons from breast cancer. Trends Endocrinol Metab. 2004 Jan-Feb;15(1):1-3.
12. Morris KV. Therapeutic potential of siRNA-mediated transcriptional gene silencing. Biotechniques. 2006 Apr; Suppl:7-13.
13. Berglund P, Landberg G. Cyclin e overexpression reduces infiltrative growth in breast cancer: yet another link between proliferation control and tumor invasion. Cell Cycle. 2006 Mar;5(6):606-9. Epub 2006 Mar 15.
14. Berglund P, Stighall M, Jirstrom K, Borgquist S, Sjolander A, Hedenfalk I, Landberg G. Cyclin E overexpression obstructs infiltrative behavior in breast cancer: a novel role reflected in the growth pattern of medullary breast cancers. Cancer Res. 2005 Nov 1;65(21):9727-34.
15. Porter PL, Malone KE, Heagerty PJ, et al. Expression of cell-cycle regulators p27Kip1 and cyclinE, alone and incombination, correlate with survival in young breast cancer patients[J]. Nat Med, 1997, 3(2):222-225.
16. Donnellan R, Kleinschmidt I, Chetty R. Cyclin E immunoexpression in breast ductal carcinoma: pathologic and prognostic implications. Hum Pathol. 2001; 32:89-94.
17. Akli S, Keyomarsi K. Low-molecular-weight cyclin E: the missing link between biology and clinical outcome. Breast Cancer Res. 2004;6(5):188-91. Epub 2004 Jul 7.
18. Spruck C, Sun D, Fiegl H, Marth C, Mueller-Holzner E, Goebel G, Widschwendter M, Reed SI. Detection of low molecular weight derivatives of cyclin E1 is a function of cyclin E1 protein levels in breast cancer. Cancer Res. 2006 Jul 15;66(14):7355-60.
1. Hinds PW, Mittnacht S, Dulic V, et al. Regulation of retinoblastoms protein functions by ectopic of human cyclins. Cell, 1992, 70:993-1006.
2. Koff A,Giordano A,Desai D,et al.Formation and activation of a cyclin E-cdk2 complex during the G1 phase of the human cell cycle.Science,1992;257:1689-1694.
3. Koff A, Cross F, Fisher A, et al. Hu man cyclin E, a new cyclin that interactswith two members of the CDC2 gene family[J]. Cell, 1991, 66 (6): 1217 -1228.
4. Lew DJ, Dulic V, Reed SI. Isolation of three novel human cyclins by rescue of G1cyclin (cln) function in yeast. Cell, 1991, 66:1197-1206.
5. Keyomarsi K, Pardee AB. Redundant cyclin overexpression and gene amplification in breast cancer cell. Proc Natl Acad Sci U S A 1993;90:1112-6
6. Keyomarsi K, O’leary N, Molnar G, Lees E, Fingert HJ,Pardee AB. Cyclin E, a potential prognostic marker for breast cancer. Cancer Res 1994;54:380-5.
7. Keyomarsi K, Herliczek TW. The role of Cyclin E in cell proliferation, development and cancer. In: Meijer L, Guidert S, Philippe M, eds. Progress in cell cycle research. Vol. 3. New York: Plenum Press, 1997:171-91.
8. Geng Y. Eaton E N. Picon M, et al. Regulation of cyclin E transcription byE2Fs and retinoblastoma protein[J]. Oncogene. 1996,12(6): 1173-1180.
9. Hartwell L, Kastsan MB. Cell cycle control and cancer. Science, 1994, 266:1820-1828.
10. Resnitzky D, Gossen M, Bujard H, Reed SI. Acceleration of the G1/S phase transition by expression of cyclin D1 and E with an induction system. Mol Cell Biol, 1994, 14:1669-1679.
11. Wimmel A, Lucibello FC, Sewing A, et al. Inducible acceleration of G1 progressi on through tetracycline-regulated expression of human cyclin E. Oncogene 1994; 9:995-997.
12. Kwon TK, Nordin AA. Overexpression of cyclin E and cyclin dependent kinase inhibitor p27kip1 Effect on cell cycle regulation in Hela cell. Biochemical and Biophysical Research Communications, 1997, 238:534-538.
13. Charles J. Sherr Cancer Cell Cycles[J]. Science, 1996, 274:1672-1677.
14. Ikezawa K, Ohtsubo M, Norwood TH, et al.Role of cyclin E and cyclin E-dependent kinase in mitogenic stimulation by cementum-derived growth factor in human fibroblasts[J]. FASEB J, 1998, 12(12):1233-1239.
15. Geng Y, Eaton En, Picon M, et al. Regulation of Cyclin E transcription by E2Fs and retinoblastoma a protein[j]. Ocogene, 1996, 12(6):1173-1180.
16. Leng X, Connell-Crow ley L, Goodrich D, et al. S-Phase entry upon ectopicexpressionof G1 cyclin-dependent kinase in the absence of retinoblastoma a protein phosphorylation[J]. Curr Biol, 1997, 7(9):709-7712.
17. Mumberg D, Haas K, Moroy T, et al. Uncoupling of DNA replication and cell cycle progression by human cyclin E. Oncogene, 1996, 13: 2493-2497..
18. ScottKA, WalkerRA. Lack of cyclinE immuno reactivity in non-malignant-breast and association with proliferation in breast cancer[J]. Br J Cancer, 1997, 76(10):1288-1292.
19. Porter PL, Malone KE, Heagerty PJ, et al. Expression of cell-cycle regulators p27Kip1 and cyclinE, alone and incombination, correlate with survival in young breast cancer patients[J]. Nat Med, 1997, 3(2):222-225.
20. Porter D, Zhang N, Danes C, et al. Tumor-specific proteolytic processing of cyclin E generates hyperactive low-molecular-weight forms. Mol Cell Boil. 2001; 21:6254-69
21. Neilsen Nh, Arnerlov C, Emdin SO and Landberg G. Cyclin E overexpression, a negative prognostic factor in breast cancer with strong correlation to oestrogen receptor status. Br J Cancer, 1996, 74:874-880.
22. Ford HL, Kabingu En, Bump EA, Mutter GL and Pardee Ab. Abrogation of the G2 cell cycle checkpoint associated with overexpression of HSIX1: a possible mechanism of breast carcinogenesis. Proc Natl Acad Sci USA, 1998, 95:12608-12613.
23. Keyomarsi K, Conte D, Toyofuku W and Fox MP. Deregulation of cyclin E in breast cancer. Oncogene, 1995, 11:941-950.
24. Wang XD, Rosales JL, Magliocco A, Gnanakumar R, Lee KY. Cyclin E in breast tumors is cleaved into its low molecular weight forms by calpain. Oncogene. 2003 Feb 6;22(5):769-74.
25. Donnellan R, Kleinschmidt I, Chetty R. Cyclin E immunoexpression in breast ductal carcinoma: pathologic and prognostic implications. Hum Pathol. 2001; 32:89-94.
26. Keyomarsi K, Tucker SL, Buchholz TA, Callister M, Ding Y, Hortobagyi GN,Bedrosian I, Knickerbocker C, Toyofuku W, Lowe M, Herliczek TW, Bacus SS. Cyclin E and survival in patients with breast cancer. N Engl J Med. 2002 Nov14;347(20):1566-75.
27. Han S, Park K, Bae BN, Kim KH, Kim HJ, Kim YD, Kim HY. Prognostic implication of cyclin E expression and its relationship with cyclin D1 and p27Kip1 expression on tissue microarrays of node negative breast cancer. J Surg Oncol. 2003 Aug;83(4):241-7.
28. Rudolph P, Kuhling H, Alm P, Ferno M, Baldetorp B, Olsson H, Parwaresch R. Differential prognostic impact of the cyclins E and B in premenopausal and postmenopausal women with lymph node-negative breast cancer. Int J Cancer. 2003 Jul 10;105(5):674-80.
29. Span PN, Tjan-Heijnen VC, Manders P, Beex LV, Sweep CG. Cyclin-E is a strong predictor of endocrine therapy failure in human breastcancer. Oncogene. 2003 Jul 31;22(31):4898-904.
30. Christov K, Ikui A, Shilkaitis A, Green A, Yao R, You M, Grubbs C, Steele V, Lubet R, Weinstein IB. Cell proliferation, apoptosis, and expression of cyclin D1 and cyclin E as potential biomarkers in tamoxifen-treated mammary tumors. Breast Cancer Res Treat. 2003 Feb;77(3):253-64.
31. Early Breast Cancer Trialists’Collaborative Group. Polychemotherapy for early breast cancer: an overview of the randomised trials. Lancet 1998;352:930-42.
32. Early Breast Cancer Trialists'Collaborative Group. Tamoxifen for early breast cancer: an overview of the randomised trials. Lancet 1998;351:1451-67.