细胞周期调控相关蛋白在子宫内膜癌的表达及意义

目的　探讨与细胞周期G1→S调控点相关的P185 ,rasP2 1,P5 3,P16 ,P2 1,Rb ,nm2 3H1蛋白在子宫内膜癌组织中的表达及意义。②方法　应用免疫组化 (LSAB)法检测上述蛋白在 2 1例正常子宫内膜、19例子宫内膜上皮内瘤样病变 (EIN)及 45例子宫内膜癌中的表达。③结果　P185 ,rasP2 1,P5 3蛋白表达率由正常内膜、EIN至内膜癌逐渐升高 ,P16 ,P2 1,Rb ,nm2 3H1结果相反。在子宫内膜癌中 ,P185与rasP2 1呈正相关 (r=0 .36 3,P <0 .0 5 ) ,与P5 3,P2 1呈负相关 (r=- 0 .44 8,- 0 .30 3,P <0 .0 5 ) ,rasP2 1,P5 3与P2 1均呈负相关 (r=- 0 .36 5 ,- 0 .32 2 ,P <0 .0 5 ) ,P16 ,P2 1与Rb呈正相关 (r=0 .36 1,0 .44 1,P <0 .0 5 )。P185单因素分析为一预后保护性因素 (χ2 =5 .86 ,P <0 .0 5 ) ;P5 3的表达与临床各参数均有关 ,与ER ,PR的表达呈负相关性 ,单因素及多因素分析表明 ,P5 3异常表达均提示预后差 (χ2 =2 1.39,P <0 .0 5 )。④结论　与细胞周期G1→S调控相关的P185 ,rasP2 1,P5 3,P16 ,P2 1,Rb ,nm2 3H1蛋白异常表达均参与子宫内膜癌发生、发展 ,且其中部分基因相互关联 ;P5 3可作为独立预后因素 ,其阳性表达提示预后差。     

Mechanism of action of a tyrphostin, 3,4-dihydroxy-α-cyanothiocinnamamide, in breast cancer cell growth inhibition involves the suppression of cyclin B1 and the functional activity of cyclin B1/p34cdc2 complex

Tyrphostins are a group of compounds specifically targeted for the inhibition of tyrosine phosphorylation in signal transduction pathways. We studied the effects of a tyrphostin, 3,4-dihydroxy--cyanothiocinnamamide (tyrphostin-47), on hormone-responsive MCF-7 and hormone-unresponsive MCF-7-5C cell growth by DNA analysis for a period of 10 days. The growth of both cell lines was inhibited by this drug at 50 and 100 µM concentrations. Flow cytometric analysis showed that tyrphostin treatment caused a significant delay in the progression of MCF-7 cells through Gl and S phases of the cell cycle. The level of cyclin B1, a component of the mitosis promoting factor (MPF), was reduced by 90% in the presence of 100 µM tyrphostin. The other component of MPF, p34cdc2 kinase, was not affected; however, its functional activity was dramatically reduced, as determined by histone H1 phosphorylation assay. In contrast, G1 cyclins (D1 and E) and tyrosine kinase activity were not markedly affected by tyrphostin-47, as determined by Western immunoblot detection with specific antibodies. Our results suggest that a possible mechanism of tyrphostin action in breast cancer cells might involve the suppression of cyclin B1 and inhibition of the functional activity of cyclin B1/p34cdc2 complex. Our data indicate that the cell cycle machinery might be a target for developing novel drugs for breast cancer.     

视黄酸依赖反义cyclin D1对HL-60细胞增殖和分化的影响及其机制

目的 :探讨视黄酸 (retinoicacid ,RA)及其诱导的反义 (anti sense ,AS)cyclinD1对肿瘤细胞的协同抑制增殖与诱导分化效应及其机制。方法 :通过构建的含有视黄酸反应元件 (retinoicacidresponseelement ,RARE)的反义cyclinD1RNA表达载体 pCI neo/RARE3 TK/AscyclinD1,使反义cyclinD1的表达可受RA诱导调控。以脂质体转染HL 60白血病细胞后 ,运用生长曲线、MTT比色法和流式细胞仪分析检测细胞增殖功能 ,NBT还原实验和透射电镜观测细胞分化状况 ,western blot检测p16基因蛋白表达水平。结果 :反义cy clinD1转染和未转染的HL 60细胞经RA处理后 ,生长减慢 ,细胞被阻滞在G0 /G1期 ,分化能力明显增强 ,p16基因蛋白表达增加 ,其中 ,以反义cyclinD1转染细胞经RA处理后变化更为明显。结论 :RA及其诱导的反义cyclinD1可协同抑制HL 60细胞增殖和诱导细胞分化成熟 ,上调p16基因表达可能是其分子机制之一。     

Induction of cell-cycle arrest by all-trans retinoic acid in mouse embryonic palatal mesenchymal (MEPM) cells.

all-trans retinoic acid (atRA), the oxidative metabolite of vitamin A, is essential for normal embryonic development. Also, high levels of atRA are teratogenic in many species and can effectively induce cleft palate in the mouse. Most cleft palate resulted from the failed fusion of secondary palate shelves, and maintenance of the normal cell proliferation is important in this process of shelf growth. To clarify the mechanism by which atRA causes cleft palate, we investigated the effect of atRA on proliferation activity and cell cycle distribution in mouse embryonic palatal mesenchymal (MEPM) cells. atRA inhibited the growth of MEPM cells by inducing apoptosis in a dose-dependent manner. atRA also caused a G1 block in the cell cycle with an increase in the proportion of cells in G0/G1 and a decrease in the proportion of cells in S phase, as determined by flow cytometry. We next investigated the effects of atRA on molecules that regulate the G1 to S phase transition. These studies demonstrated that atRA inhibited expression of cyclins D and E at the protein level. Furthermore, atRA treatment reduced phosphorylated Rb and decreased cdk2 and cdk4 kinase activity. These data suggest that atRA had antiproliferative activity by modulating G1/S cell cycle regulators and by inhibition of Rb phosphorylation in MEPM cells, which might account for the pathogenesis of cleft palate induced by retinoic acid.     

γ-Rays-generated ROS induce apoptosis via mitochondrial and cell cycle alteration in smooth muscle cells

Abstract

Purpose: γ-rays (IR) cause an increase in intracellular calcium [Ca²⁺], alters contractility and triggers apoptosis via the activation of protein kinase C in intestinal guinea pig smooth muscle cells. The present study investigated the role of the mitochondria in these processes and characterized proteins involved in IR-induced apoptosis.

Materials and methods: Intestinal smooth muscle cells were exposed to 10–50 Gy from a ⁶⁰Co γ-source. Reactive oxygen species (ROS) levels were measured by colourimetry with a fluorescente probe. Protein expression was analyzed by immunoblotting and immunofluorescence.

Results: Apoptosis was inhibited by glutathione, possible by inhibiting the generation or scavenging ROS. Apoptosis was mediated by the mitochondria releasing cytochrome c leading to caspase 3 activation. IR increased the expression of the cyclins A, B2 and E and led to unbalanced cellular growth in an absorption dose-dependent manner. However, radiation did not induce alterations in the mitochondrial ultrastructure or in transmembrane electric potential. In contrast, IR increased the nuclear expression of cytoplasmic proteins and cyclins A and E.

Conclusion: Smooth muscle cells subjected to IR undergo mitochondrial-mediated apoptosis that involves oncoproteins activation and preserves mitochondrial structure. IR also cause alterations in the expression and localization of both pro- and anti-apoptotic proteins.     

Selective modulation of the cyclin B/CDK1 and cyclin D/CDK4 complexes during in vitro human megakaryocyte development

Mammalian megakaryocyte development is characterized by a progressive accumulation of cells exhibiting a polylobated nucleus with a polyploid DNA content. In this study human megakaryocytes were obtained from CD34+ haemopoietic progenitors by in vitro liquid culture in the presence of 100 ng/ml of recombinant thrombopoietin (TPO). Ultrastructural examination of polyploid megakaryocytes showed the presence of a large number of centrioles, the breakdown of the nuclear envelope, and the progressive chromatin condensation, all aspects characteristic of mitosis. At both indirect immunofluorescence and Western blot analyses, cyclin B and its related cyclin-dependent kinase (CDK)1, which forms the mitosis promoting factor (MPF), showed an increased expression in maturating megakaryoblasts and megakaryocytes (day 8 of culture) with respect to freshly isolated CD34+ progenitors. This expression tended to decline in fully developed megakaryocytes (day 15 of culture). The amount of cyclin D and of the related CDK4, governing the G1 phase of the cell cycle, increased during megakaryocyte development, maintaining high levels of expression also in mature megakaryocytes. These results indicate that megakaryocyte polyploidization depends on a true, although incomplete, mitotic process, and that cyclin D/CDK4 probably plays a crucial role throughout megakaryocytopoiesis.     

Adapter protein connections: The MRL and Grb7 protein families

Insulin-like growth factor-1 receptor (IGF-1R) has been shown to be important for melanoma cell growth and survival. In this study we first show, using immunohistochemistry, that progression from benign nevi to malignant melanoma is paralleled by an increased expression of IGF-1R and a down-regulation of the cyclin-dependent kinase inhibitor p27^Kip1. Even though the expression of p27^Kip1 was drastically reduced compared to benign tumors, detectable amounts of it could be assayed by Western blotting in cultured melanoma cells. To analyze whether there is a causative relationship between the IGF-1 pathway and p27^Kip1 expression, melanoma cells were treated with αIR-3, an antibody blocking the IGF-1 binding to IGF-1R, or Tunicamycin, which inhibits the translocation of IGF-1R to the cell surface. From these studies we could conclude that the overall expression of p27^Kip1 is independent of the IGF-1 pathway. In contrast, the association of p27^Kip1 with the different cyclins was drastically affected. Both TM and αIR-3 decreased the binding of p27^Kip1 to cyclin D1, whose expression was drastically reduced. On the other hand there was an increased binding of p27^Kip1 to cyclin E and cyclin A. This redistribution of p27^Kip1 may be a mechanism for growth arrest and induction of apoptosis following interruption of the IGF-1 pathway in melanoma cells.     

细胞周期素G1在大肠癌中的表达及意义

【目的】探讨细胞周期素G1（Cyclin G2）在大肠癌纽织中的表达及其意义。【方法】应用免疫纽化SP法检测31例正常大肠组织、70例大肠癌组织及相应癌旁组织中的Cyclin G1蛋白的表达。【结果】正常大肠组织未发现Cyclin G1表达；大肠癌组织中可见明显的胞核棕黄色阳性表达，相应的癌旁组织表达很少（P〈0．05）；Cyclin G1在大肠癌中的表达与肿瘤的Dukes分期相关。【结论】Cyclin G1可能参与大肠癌的发生和发展过程，有利于病情评估，并可能成为大肠癌癌基因治疗的新靶点。     

HPV16/18在膀胱癌中表达及其与bFGF、cyclinD1和cyclinE的相关关系

目的探讨人乳头瘤病毒(HPV)16/18与膀胱癌发病之间关系及其作用机制.方法应用免疫组化方法检测78例膀胱癌标本和11例正常膀胱组织中HPV16/18、碱性成纤维细胞生长因子(bFGF)、细胞周期蛋白(cyclin)D1和cyclinE的表达及相关关系.结果膀胱癌中HPV16/18阳性率为65.4%,显著高于正常的27.3%;而且与肿瘤病理分级、分期相关,但与肿瘤复发无相关.膀胱癌中HPV16/18与bFGF及cyclinD1表达之间有显著相关性,但与cyclinE表达之间无显著相关性.结论HPV16/18感染参与膀胱癌发病过程,而且通过多途径发挥作用.     

2001年诺贝尔生理学或医学奖获得者及其研究成果简介

介绍 3位英美科学家因发现调控细胞周期的关键分子 ,获 2 0 0 1年诺贝尔生理学或医学奖。这一基本发现对于全面了解细胞的生长过程具有重大意义 ,多数生物医学研究课题将因之受益 ,并会在许多不同领域得到应用。从长远来看 ,有可能为癌症治疗开辟新的途径。