乳腺癌cyclin D1的扩增和表达与预后的关系

目的：检测５７例乳腺浸润导管癌组织中ｃｙｃｌｉｎ Ｄ１基因的扩增和表达，并结合肿瘤大小、组织学分级、腋窝淋巴结转移和ＥＲ的表达进行临床病理预后分析。方法：应用ＳＰ免疫组织化学及Ｓｏｕｔｈｅｒｎ杂交方法检测乳腺癌ｃｙｃｌｉｎ Ｄ１／ＣＣＮＤ１基因的表达及扩增。结果：显示５７例乳腺癌中４２例在瘤细胞核内有ｃｙｃｌｉｎ Ｄ１的表达。阳性率７３．６％，其中强阳性１１例，中阳性１４例，弱阳性１７例，阴性１５     

cyclinD1,Rb基因蛋白在乳腺癌中的表达

目的：探讨ｃｙｃｌｉｎＤ１基因及Ｒｂ基因的表达与乳腺癌发生发展的关系。方法：应用ＡＢＣ免疫组化法检测２４例良性乳腺组织及５８例乳腺癌中ｃｙｃｌｉｎＤ１及Ｒｂ蛋白表达。结果：乳腺癌中ｃｙｃｌｉｎＤ１过表达阳性率５８．６２％（３４／５８）显著高于良性乳腺组织中的１６．６７％（４／２４），Ｐ＜０．０５。ｃｙｃｌｉｎＤ１过表达出现于导管原位癌并持续于浸润、转移等进展过程中，与年龄、肿瘤大小、组织学类型及淋巴结状态无相关性，但与组织学分级负相关。乳腺癌中Ｒｂ蛋白表达阳性率为３６．２％（２１／５８），显著低于良性乳腺组织的７５％（１８／２４），Ｐ＜０．０５；未见Ｒｂ失表达与临床病理参数间存在相关性，Ｒｂ表达与ｃｙｃｌｉｎＤ１过表达呈正相关。结论：ｃｙｃｌｉｎＤ１过表达及Ｒｂ失表达是乳腺癌发生中的重要事件且前者是一早期分子事件；ｃｙｃｌｉｎＤ１过表达发挥作用可能部分依赖于Ｒｂ蛋白的存在；提示细胞周期调控异常参与乳腺癌的发生。     

周期蛋白E在细胞周期调节中的作用

ｃｙｃｌｉｎ Ｅ是Ｇ１期的周期蛋白,基因定位于１９ｑ１２－１３,参与细胞周期的调节及肿瘤的发生。ｃｙｃｌｉｎ Ｅ与ＣＤＫ２结合在Ｇ１期末发挥作用,促进细胞进入Ｓ期。ｃｙｃｌｉｎ Ｅ的过量表达通过对ｐＲＢ及其它低物的磷酸化、释放出转录因子进而启动ＤＮＡ的合成,加速细胞的Ｇ１期进程。ｐ２１、ｐ２７及ＴＧＦ－β通过对ｃｙｃｌｉｎ Ｅ－ＣＤＫ２活性的抑制而减缓Ｇ１期的进程。ｃｙｃｌｉｎ Ｅ是周期进程的基本     

细胞周期调节因子与肿瘤的发生和调控

细胞的转化和肿瘤发生受到多种细胞周期调节因子的影响。Ｃｙｃｌｉｎｓ和ＣＤＫｓ／Ｃｄｃ２作为细胞周期的正调节因子，促进细胞的生长和分裂，ＣＫIｓ作为负调节因子则抑制细胞生长和促进细胞分化。Ｃｙｃｌｉｎｓ、ＣＤＫｓ／Ｃｄｃ２与ＣＫＩｓ相互调节，相互控制，组成一个复杂的分子调节网。Ｃｙｃｌｉｎｓ过度表达或ＣＫＩｓ的功能性缺失或突变，有利于细胞的转化和肿瘤的发生发展。     

p27表达和DNA倍体在软骨肉瘤中的研究

目的：软骨肉瘤是常见的骨恶性肿瘤之一。其形态多变、生物学行为复杂，对软骨瘤和高分化软骨肉瘤，只凭病理组织学特征鉴别它们通常是很困难的，并且软骨肉瘤组织学分级与其生物学行为时常不一致。因此，需要更为客观可靠的指标来协助诊断，指导软骨肉瘤的分级和估计软骨肉瘤的预后。肿瘤细胞周期的研究是当前肿瘤研究热点之一，肿瘤细胞周期受一系列细胞周期调节因子控制，包括Ｇ１期细胞周期素（ｃｙｃｌｉｎｓ）和相应的细胞周期素依赖激酶（ｃｙｃｌｉｎ－ｄｅｐｅｎｄｅｎｔｋｉｎａｓｅｓ，ＣＤＫｓ），ＣＤＫｓ活性又受一系列细胞周期素依赖激酶抑制物（ｃｙｃｌｉｎ－ｄｅｐｅｎｄｅｎｔｋｉｎａｓｅｉｎｈｉｂｉｔｏｒｓ，ＣＤＫＩｓ）控制。ｐ２７是新近发现的ＣＤＫＩｓ家族之一，在细胞周期增殖和分化调控中起非常重要作用。ｐ２７蛋白主要抑制Ｇ１期ｃｙｃｌｉｎＥ－ＣＤＫ２复合物及ｃｙ－ｃｌｉｎＤ－ＣＤＫ４／６复合物活性，它过度表达可使细胞周期停滞在Ｇ１中期。软骨肉瘤ｐ２７表达国内外尚未见报道。检测软骨肉瘤中ｐ２７表达，从而探讨ｐ２７表达与软骨肉瘤组织分级和ＤＮＡ倍体的关系及其对软骨肉瘤的诊断和预后判断是否具有应用价值十分必要。方法：３６例经典软骨肉瘤未脱钙     

原癌基因CyclinD1在肺癌中表达的初步研究

目的：探讨ＣｙｃｌｉｎＤ１基因表达与肺癌发生发展关系。方法：应用免疫组化技术（ＳＰ法）检测ＣｙｃｌｉｎＤ１基因在２５例肺癌及４例癌旁正常肺组织中的表达。结果：ＣｙｃｌｉｎＤ１在肺癌中有较高表达，过表达阳性率２８％（７／２５例）；在４例癌旁正常肺组织中无表达；７例过表达者均为非小细胞肺癌；过表达与肿瘤体积呈正相关。结论：ＣｙｃｌｉｎＤ１基因过表达是肺癌尤其是非小细胞肺癌发生发展过程中的重要事件。     

CyclinB1和CyclinD3基因在类风湿关节炎滑膜组织中的表达

滑膜衬里层增厚是类风湿关节炎的重要病理学特征之一，但机制不清。本研究用核酸分子原位杂交技术检测反映细胞增殖状况的细胞周期蛋白基因ＣｙｃｌｉｎＢ１和ＣｙｃｌｉｎＤ３在１０例ＲＡ、３例骨关节炎及４例正常关节滑膜组织的ｍＲＮＡ表达水平。发现ＣｙｃｌｉｎＢ１ｔ ＣｙｃｌｉｎＤ３在ＲＡ滑膜衬里层表达明显高于对照组，提示滑膜细胞的原位增殖在ＲＡ滑膜衬里层增厚中可能起一定作用。     

反义周期蛋白B1基因转染HT29细胞后若干细胞增殖调控相关基因表达的变化

用基因重组技术将插入有反义周期蛋白Ｂ１（ｃｙｃｌｉｎＢ１）基因ｐＸＪ４１－ｎｅｏ质粒，转染人结肠腺癌ＨＴ２９，获得了ＨＴＢ１细胞株。ＨＴＢ１细胞显示出ｃｙｃｌｉｎＢ１ｍＲＮＡ明显的表达抑制。为了探讨ＨＴＢ１细胞生长变慢和它的增殖相关基因表达之间的关系，我们用异硫氰酸胍－酚一步法提取ＨＴＢ１和ＨＴ２９细胞的总ＲＮＡ，并用一些有关的基因探针进行杂交实验。其结果表明：ｃ－ｍｙｃ和ＣＤＫ４在ＨＴＢ１中的表达低于在ＨＴ２９中的表达；而另一方面，ｐ５３、Ｒｂ、ＴＧＦβ和ｃｙｃｌｉｎＤ３在ＨＴＢ１细胞中的表达高于对照组。而ｃｄｃ２基因表达二者维持相似水平。根据研究结果我们发现，转染有反义ｃｙｃｌｉｎＢ１基因的ＨＴＢ１细胞在一组正、负调节基因的协同作用下，导致细胞周期运转的变化。     

细胞周期分子调控与癌变

细胞周期蛋白（ｃｙｃｌｉｎ）、细胞周期蛋白依赖的蛋白激酶（ＣＤＫ）以及ＣＤＫ抑制蛋白（ＣＫＩ）三类分子的调控。细胞周期蛋白分别在细胞周期的不同时期程序性合成和降解，同时做为调节亚基对ＣＤＫ活性进行调节。ＣＤＫ的活性还受磷酸化状态的影响。ＣＫＩ能结合ＣＤＫ－ｃｙｃｌｉｎ复合物并抑制其活性，ＣＫＩ的失活可导致ＣＤＫ活性的异常。使细胞无限增殖。肿瘤的发生同细胞周期调控机制的改变密切相关。     

细胞周期分子调控与癌变

细胞周期蛋白（ｃｙｃｌｉｎ）、细胞周期蛋白依赖的蛋白激酶（ＣＤＫ）以及ＣＤＫ抑制蛋白（ＣＫＩ）三类分子的调控。细胞周期蛋白分别在细胞周期的不同时期程序性合成和降解，同时做为调节亚基对ＣＤＫ活性进行调节，ＣＤＫ的活性还受磷酸化状态的影响。ＣＫＩ能结合ＣＤＫ－ｃｙｃｌｉｎ复合物并抑制其活性，ＣＫＩ的失活可导致ＣＤＫ活性的异常，使细胞无限增殖。肿瘤的发生同细胞周期调控机制的改变密切相关。     

Cyclin E overexpression enhances cytokine-mediated apoptosis in MCF7 breast cancer cells

Cyclin E, the regulatory component of the cyclin E/cyclin-dependent kinase (CDK) complex, is required for proliferation and overexpression of this cyclin is associated with many types of human tumors. To elucidate the mechanism by which cyclin E overexpression promotes tumorigenesis, cyclin E was overexpressed in two breast cancer lines: MCF7 and T47D. Cells overexpressing cyclin E display a marked decrease in the expression of Bcl-2, an antiapoptotic protein, and increased levels of the proapoptotic proteins Bad and Bax. The levels of Bcl-X(L) and Mcl-1 remain unchanged. Since the homeostasis of pro- and antiapoptotic proteins was altered, we asked if cyclin E overexpression modifies responses to cytokines. MCF7 cyclin E overexpressing cells have an enhanced sensitivity to Fas, TRAIL, and TNF-alpha-induced apoptosis. T47D cells overexpressing cyclin E have a significant increase in TNF-alpha and TRAIL-induced apoptosis. In conclusion, our results provide a link between expression of cyclin E, deregulation of Bcl-2, and an altered response to cytokine-mediated apoptosis.     

Expression of cyclins and cyclin dependent kinases in human benign and malignant melanocytic lesions

AIMS: The regulation of cell proliferation is a key event in normal development, pathophysiological responses to injury, and tumorigenesis. The orderly progression of cells through the cell cycle depends on a finely tuned balance between the concentrations of activated cyclins and cyclin dependent kinases. This study was undertaken to compare the expression of cell cycle regulators in benign and malignant melanocytic lesions during tumour progression. METHODS: Immunohistochemistry was used to analyse 49 primary cutaneous malignant melanomas, 18 metastatic melanomas, and 12 histologically confirmed naevus cell naevi for their expression of cyclins (A, B1, D1, D2, D3, and E) and cyclin dependent kinases (CDK1, CDK2, and CDK4). RESULTS: Cyclin E and CDK2 had the highest expression patterns in human cutaneous melanomas and metastases and correlated positively with histological type and tumour stage. Cyclins B1, D2, and D3 had significantly increased expression in metastases, but normal or even decreased expression in primary melanomas. However, cyclins A and D1, and CDK1 and CDK4 were expressed very weakly in situ with no significant differences between naevi, melanomas, or metastases, and there was no correlation with histopathological staging. The specificity of recognition by the antibodies used was confirmed by western blotting on a panel of seven human melanoma cell lines. Cyclins A, B, and E were expressed by all seven, whereas cyclin D1 was detectable in six of seven and CDK2 and cdc2 were present in five of seven lines analysed. CONCLUSIONS: Taken together, this study demonstrated a significant increase of cyclin E and CDK2 expression during tumour progression in malignant melanomas.     

抑制cyclinD1表达对乳腺癌细胞增殖及cyclinE、CDK2和p21cip1转录的影响

目的　分析探讨人乳腺癌细胞中 cyclin D1的表达受到反义 RNA抑制后 ,细胞增殖能力的变化以及cyclin E,CDK2和 p2 1cip1 (cip1/ waf1/ sdi1)基因表达受到的影响。　方法　将表达 cyclin D1反义 RNA的重组质粒转入人乳腺癌细胞 ,由此抑制细胞中 cyclin D1的表达 ,然后分析细胞生长速率和各时相细胞分布比例的变化 ,并通过Northern blot分析有关基因的表达水平。　结果　 cyclin D1表达受到抑制的细胞与对照相比 ,细胞增殖速率明显下降 ,培养至第 6 d时 ,生长抑制率为 5 4%。细胞周期各时相的分布比例也有较大的变化 ,G1期细胞比例上升 ,而 S期及 G2 / M期细胞比例下降。cyclin E和 CDK2的 m RNA水平显示出有不同程度的下降 ,而 p2 1cip1的表达无明显变化。　结论　 cyclin D1表达的抑制可明显减弱人乳腺癌细胞的异常增殖 ,同时表明同为细胞周期调控基因的 cyclin E和CDK2的表达与 cyclin D1的表达密切相关 ,而 p2 1cip1的表达不受 cyclin D1的影响     

Overexpression and localization of cyclin D1 mRNA and antigen in esophageal cancer.

We examined the expression of cyclin D1 mRNA in two human carcinoma cell lines (A431 and TT) and 17 specimens of esophageal cancer with in situ hybridization. Cyclin D1 mRNA was overexpressed in the cytoplasm of cancer cells that showed cyclin D1 gene amplification by Southern blot hybridization. Cyclin D1 antigen was overexpressed in the nucleus of these cancer cells. The distribution of cyclin D1 mRNA-positive cells was similar to that of cyclin D1 antigen-positive cells in the cancer tissues. We then attempted to correlate overexpression of cyclin D1 antigen and prognosis, by using 55 formalin-fixed, paraffin-embedded specimens of esophageal cancer. The overall 5-year survival of patients with strongly staining tumors was significantly lower than that of patients with weakly or nonstaining tumors (7 versus 59%; P < 0.01). There was no significant correlation between cyclin D1 expression and other clinicopathological factors. These results suggest that cyclin D1 may play an important role in carcinogenesis and that cyclin D1 overexpression may be a useful prognostic factor in esophageal cancer.     

MicroRNA-25 regulates small cell lung cancer cell development and cell cycle through cyclin E2

Purpose: We intended to examine the underlying mechanism of microRNA-25 (miR-25) in regulating small cell lung cancer (SCLC). Methods: The miR-25 expression was measured by quantitative RT-PCR (qRT-PCR) in 5 SCLC cell lines and 9 human SCLC tissues. In SCLC cell line H510A cells, endogenous miR-25 was downregulated by stable transfection of antisense oligonucleotide of miR-25 (miR-25-as). Then the effects of miR-25 downregulation on SCLC growth, invasion and chemoresistance were assessed by MTT, migration and cisplatin assays, respectively. Furthermore, the effects of miR-25 downregulation on cancer cell cycle arrest, production of cell cycle proteins cyclin E2 and CDK2 were examined by cell cycle assay, western blot and luciferase assays, respectively. Finally, cyclin E2 was over-expressed in H510A cells to investigate its effect on miR-25 mediated SCLC regulation. Results: In both SCLC cells and human SCLC tumor tissues, miR-25 was overexpressed. Down-regulation of miR-25 in H510A cells significantly reduced cancer cell growth, invasive capability and resistance to cisplatin. Also, it induced G1 cell cycle arrest and downregulated cell cycle related proteins cyclin E2 and CDK2. Luciferase assay demonstrated cyclin E2 was directly targeted by miR-25. Overexpression of cyclin E2 in H510A cells reversed the cell cycle arrest and restored invasive capability impaired by miR-25 downregulation. Conclusions: Our study shows miR-25 is overexpressed in SCLC and acting as oncogenic regulator by regulating cyclin E2.     

Alterations of gene expression of RB pathway in Opisthorchis viverrini infection-induced cholangiocarcinoma

Opisthorchiasis has the significant relationship with the high prevalence of cholangiocarcinoma (CCA; a bile duct cancer) in the endemic areas in Southeast Asia. To reveal the molecular mechanism of the tumorigenesis induced by Opisthorchis viverrini infection, the present study investigated the kinetic expression of RB pathway genes, including RB1, p16^INK4, cyclin D1, and CDK4, during the development of opisthorchiasis-associated CCA in hamster model. The results of quantitative real-time polymerase chain reaction indicated that the expressions of RB1 and p16^INK4 were down-regulated during the development of CCA induced by infection plus N-nitrosodimethylamine treatment in a time-dependent manner. On the other hand, the expressions of cyclin D1 and CDK4 were up-regulated. The expression kinetics was corresponding to the pathological progression of the opisthorchiasis-associated CCA, revealed by histopathological observation. Moreover, the analysis of the expression of these genes in human opisthorchiasis-associated CCA cases showed the decreased expression of RB1 and p16^INK4 in 50% and 82.7% cases and overexpression of cyclin D1 and CDK4 in half cases, respectively. The results suggested that RB pathway is likely involved in the tumorigenesis of opisthorchiasis-induced CCA and proposed the potential application of some of these genes as biomarkers in predispose and molecular therapy of the parasite-associated cancer.     

IGFBP7对人乳腺癌MCF-7细胞增殖的影响及其机制

目的: 探究胰岛素样生长因子结合蛋白7(IGFBP7)对人乳腺癌MCF-7细胞增殖的影响及其分子生物学机制。方法: 将质粒pCMV6-IGFBP7转染MCF-7细胞,构建稳定表达IGFBP7的MCF-7细胞系;采用Western blotting检测IGFBP7在MCF-7细胞稳定转染子的表达;采用软琼脂培养克隆形成实验检测IGFBP7对MCF-7细胞克隆形成能力的影响;采用流式细胞术检测IGFBP7对MCF-7细胞周期的影响;采用Western blotting检测IGFBP7对MCF-7细胞细胞外信号调节激酶1/2(ERK1/2)、p-ERK1/2、细胞周期素D1(cyclin D1)、细胞周期素依赖性激酶4(CDK4)、cyclin E、CDK2、p21^CIP1/WAF1、p27^KIP1、p53、视网膜母细胞瘤蛋白(Rb)和p-Rb蛋白含量的影响。结果: (1)只有稳定转染质粒pCMV6-IGFBP7的MCF-7细胞表达IGFBP7。(2)IGFBP7能够显著降低MCF-7细胞的克隆形成率(P<0.01),阻止细胞从G₁期进入S 期,使其停滞于G₁期(P<0.01)。(3)IGFBP7能够显著抑制ERK1/2的磷酸化(P<0.01)。(4)IGFBP7能够下调cyclin D1和cyclin E蛋白表达(P<0.01),上调p27^KIP1、p21^CIP1/WAF1和p53蛋白表达(P<0.01),抑制Rb的磷酸化(P<0.01)。(5)MEK1/2阻断剂PD98059可部分模拟IGFBP7的肿瘤抑制效应。结论: (1) IGFBP7可通过下调cyclin D1和cyclin E蛋白表达,上调p27^KIP1、p21^CIP1/WAF1和p53蛋白表达,以及抑制Rb磷酸化发挥抗肿瘤作用;(2) IGFBP7对cyclin D1和p27^KIP1的调节可能与其抑制ERK1/2信号通路有关。