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.     

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.     

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。     

A novel immunohistochemical method to estimate cell-cycle phase distribution in archival tissue: implications for the prediction of outcome in colorectal cancer

An immunohistochemical method for assessing cell-cycle phase distribution in colorectal resection specimens would enable phase data to be incorporated into diagnostic algorithms for the estimation of prognosis and response to adjuvant chemotherapy in colorectal cancer. In contrast to flow cytometry, an immunohistochemical method would also allow the phase distribution to be examined within morphologically heterogeneous regions of neoplasms. Paraffin sections of normal colon (n = 25), colonic adenoma (n = 15), and colonic adenocarcinoma (n = 30) were analysed by immunohistochemistry using antibodies against markers of cell-cycle entry, Mcm-2 and Ki67, and putative markers of the cell-cycle phase, cyclins D1 and E (putative markers of G1 phase), cyclin A (S phase), cytoplasmic cyclin B1 (G2 phase), and phosphohistone H3 (M phase). The phase specificity of each marker was assessed by examining the degree of co-expression of adjacent phase markers using double-antibody fluorescence confocal microscopy and by comparison with flow cytometric analysis performed on adjacent tissue sections. The S-phase specificity of detectable cyclin A was also assessed in combination with in situ DNA replication using fluorescence confocal microscopy. All cells expressing phase markers co-expressed Mcm-2. Adjacent phase markers were not significantly co-expressed, confirming the relative specificity of these markers in tissue sections of colon. Cell-cycle phase distribution, calculated by immunohistochemistry, compared well with phase analyses obtained by flow cytometry. No cells expressed cyclin A in the absence of active DNA replication. The S-phase labelling index, as defined by detectable cyclin A expression, showed a positive correlation with the Mcm-2 labelling index and increased in the progression from normal colon to adenocarcinoma. In conclusion, a combination of these cell-cycle phase markers can be used to calculate the distribution of cells throughout each phase of the cell cycle in colorectal tissue sections. Detectable cyclin A can be used as a surrogate marker of S phase and may be of value in predicting prognosis and response to adjuvant therapy.     

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.     

非霍奇金淋巴瘤p27Kip1、p21WAF1及cyclin E表达的意义

目的 :探讨在非霍奇金淋巴瘤 (NHL)中 p2 7Kip1及其同族抑癌基因 p2 1WAF1、细胞周期素E(cyclinE)的表达意义。 方法 :应用免疫组化S P法检测 40例NHL患者受累淋巴结中 p2 7Kip1、p2 1WAF1、cyclinE蛋白表达情况 ,同时结合临床病理资料进行分析。结果 :40例NHL中 ,p2 7、p2 1、cyclinE蛋白阳性表达分别为 2 0例 (72 5 % )、8例 (2 0 % )、2 6例 (6 5 % )。 3种蛋白在NHL中的阳性表达与患者的性别、年龄及免疫分型无明显相关性 (P >0 0 5 ) ,但均与组织分化程度明显相关 (P <0 0 5 )。p2 7与cy clinE的表达水平间存在显著负相关 (r =- 0 2 7,P <0 0 1)。结论 :NHL患者淋巴组织中存在较高比例的 p2 7、p2 1蛋白阴性表达及较高比例的cyclinE阳性表达 ,说明在NHL的发生发展过程中 ,p2 7及其相关因素cyclinE ,同族基因 p2 1蛋白水平的异常变化起着重要作用 ,可作为NHL诊断标记物及预后指标。     

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。     

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.     

细胞周期蛋白D1基因表达在心肌肥大形成中的时相性变化

目的：观察心肌肥大形成过程中细胞周期蛋白Ｄ１（ｃｙｃｌｉｎ Ｄ１）基因表达的时相性变化,并探讨ｃｙｃｌｉｎ Ｄ１在心肌肥大形成中的作用。方法：肛用腹主动脉缩窄法建立大鼠心肌肥大模型,设缩窄手术组及假手术对照组,观察两组大鼠手术后不同时间心脏左心室（含左心室游离与室间隔）质量与体质量比、左心室游离壁厚度及心肌细胞横径的变化,以确证模型的稳定建立;其后应用ＲＮＡ印迹法分析检测ｃｙｃｌｉｎ Ｄ１基因表达     

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.