Induction of G1 arrest by down-regulation of cyclin D3 in T cell hybridomas

The relationship between activation-induced growth inhibition and regulation of the cell cycle progression was investigated in T cell hybridomas by studying the function of the cell cycle-regulating genes such as G1 cyclins and their associated kinases. Activation of T cell hybridomas by anti-T cell receptor antibody induces growth arrest at G1 phase of the cell cycle and subsequently results in activation-driven cell death. Rapid reduction of both messenger RNA and protein level of the cyclin D3 is accompanied by growth arrest upon activation. Although the residual cyclin D3 protein forms a complex with cdk4 protein, cyclin D3-dependent kinase activity is severely impaired. Stable transfectants engineered to express cyclin D3 override the growth arrest upon activation. These results imply that the activation signal through T cell receptor induces the down-regulation of cyclin D3 expression and cyclin D3-dependent kinase activity, leading to growth arrest in G1 phase of the cell cycle in T cells.     

T-independent and T-dependent B lymphoblasts: helper T cells prime for interleukin 2-induced growth and secretion of immunoglobulins that utilize downstream heavy chains

Resting B cells enlarge, enter the cell cycle, and change their surface phenotype when activated via the surface immunoglobulin (Ig) receptor, but subsequent cell growth and antibody production is relatively limited. To identify stimuli that might prime B cells for enhanced function in vitro, we have compared the effects of anti-Ig with helper T (Th) cells on the formation of B lymphoblasts and the subsequent ability of the blasts to grow and secrete Ig. The B blasts first were induced by either anti-Ig, anti-Ig plus T cell-derived lymphokines, or alloreactive T blasts. Each population of B blasts showed enhanced expression of cell surface adhesion molecules, interleukin 2 receptor (IL-2R) p55, and MHC products, as well as decreased expression of IgD. The allo-activated B blasts were distinctive in expressing low levels of Thy-1 and increased reactivity with peanut agglutinin, a marker of germinal center B blasts in situ. The function of the different populations of B blasts was also different. Whereas anti-Ig or anti-Ig plus lymphokines primed for enhanced responses to lipopolysaccharide (LPS), the B blasts induced by Th cells were insensitive to LPS. B lymphoblasts that had been activated in the presence of helper factors or Th cells responded vigorously to recombinant IL-2 with growth and Ig secretion, and this response was enhanced in the presence of anti-Ig. The B blasts activated directly by Th cells, but not by anti-Ig plus lymphokines, were primed to secrete high levels of IgG1 and IgA. Therefore, the phenotype and function of a B lymphoblast depends upon the manner in which it is primed. When primed by Th cells, IL-2 proves to be the predominant mediator of clonal expansion and antibody secretion.     

Molecular targets for apigenin-induced cell cycle arrest and apoptosis in prostate cancer cell xenograft

Apigenin (4',5,7-trihydroxyflavone) is a promising chemopreventive agent abundantly present in fruits and vegetables that has been shown to promote cell cycle arrest and apoptosis in various malignant cell lines. To determine whether pharmacologic intervention with apigenin has a direct growth inhibitory effect on human prostate tumors implanted in athymic nude mice, we examined cell cycle regulatory molecules as precise molecular targets of apigenin action. Apigenin feeding by gavage to these mice at doses of 20 and 50 microg/mouse/d in 0.2 mL of a vehicle containing 0.5% methyl cellulose and 0.025% Tween 20 resulted in significant decreases in tumor volume and mass of androgen-sensitive 22Rv1 and androgen-insensitive PC-3-implanted cells. Oral intake of apigenin resulted in dose-dependent (a) increase in the protein expression of WAF1/p21, KIP1/p27, INK4a/p16, and INK4c/p18; (b) down-modulation of the protein expression of cyclins D1, D2, and E; and cyclin-dependent kinases (cdk), cdk2, cdk4, and cdk6; (c) decrease in retinoblastoma phosphorylation at serine 780; (d) increase in the binding of cyclin D1 toward WAF1/p21 and KIP1/p27; and (e) decrease in the binding of cyclin E toward cdk2 in both types of tumors. In addition, apigenin feeding resulted in stabilization of p53 by phosphorylation at serine 15 in 22Rv1 tumors, which seems to exhibit p53-dependent growth inhibitory responses. Apigenin intake by these mice also resulted in induction of apoptosis, which positively correlated with serum and tumor apigenin levels. Taken together, this is the first systematic in vivo study showing the involvement of cell cycle regulatory proteins as potential molecular targets of apigenin.     

Interleukin 5 and interleukin 2 cooperate with interleukin 4 to induce IgG1 secretion from anti-Ig-treated B cells

We have analyzed requirements for IL-4-induced secretion of IgG1 from anti-Ig-activated B cells. Activated B cell blasts prepared by culture of high density B cells with anti-Ig failed to secrete IgG1 upon subsequent culture with LPS and IL-4. However, IL-4 markedly suppressed IgM secretion in the same cultures. Addition of a mixture of T cell-derived lymphokines or rIL-5 to LPS-stimulated anti-Ig blasts restored IL-4-stimulated IgG1 secretion; rIL-2 further enhanced the response to IL-4 + rIL-5. These results suggest that IL-4, IL-5, and IL-2 cooperate in the regulation of B lymphocyte Ig isotype expression.     

Lymphoma models for B cell activation and tolerance. III. Cell cycle dependence for negative signalling of WEHI-231 B lymphoma cells by anti-mu

WEHI-231 B lymphoma cells have proven to be a useful model for the regulation of growth of normal B cells by anti-Ig reagents. We previously reported that the growth of these lymphoma cells is inhibited by heterologous or monoclonal anti-mu or anti-kappa reagents. Such cells cease to incorporate thymidine within 24-48 h of exposure to anti-Ig reagents, but are not adversely affected by antibodies directed at either class I or class II histocompatibility antigens. In fact, cell cycle analysis revealed that anti-mu causes a block in the transition of these cells from G1 to S phase. To further study the mechanism of growth inhibition, we have purified lymphoma cells in G1 by centrifugal elutriation, or enriched WEHI-231 cells at the G1/S interface by treatment with hydroxyurea, and followed their progression through the cell cycle in the presence or absence of anti-mu. Our data show that WEHI-231 B lymphoma cells receive a negative signal early in G1, since delayed addition of anti-mu (to late G1 cells) leads to no alteration in cell cycle progression at 24 h, and exposure to anti-mu during S does not alter progress through DNA synthesis and mitosis. Moreover, exposure to anti-mu for only 2 h prevents purified G1 cells from entering their first S phase. The nature of the relevant processes in early G1 is discussed in terms of models of B cell activation and tolerance induction.     

ERK和P38信号转导途径对慢性髓系白血病细胞周期的调控作用

本研究探讨ERK和P38信号转导途径对慢性髓系白血病（CML）细胞周期的调控作用。以RT-PCR、Western blot和FCM方法分别检测CML患者白血病细胞和K562细胞中ERK、p38、cyclin D2、cyclin E、p27的mRNA表达及蛋白表达（其中ERK和P38为磷酸化ERK和磷酸化P38）及细胞周期分布,并分析其相关关系。结果表明：CML患者白血病细胞和K562细胞中ERK、P38、cyclin D2、cyclin E的mRNA表达和蛋白表达增高,P27的表达降低,且cyclin D2蛋白表达与cyclin E、ERK和P38蛋白表达呈正相关（P〈0.01）,与P27蛋白表达呈负相关（P〈0.01）。G0/G1期细胞减少,S期细胞增多,与对照组相比有显著性差异。结论：CML中P38、ERK mRNA表达和活性增加,激活下游的cyclin D2、cyclin E和P27等细胞周期调控因子,致使G0/G1期缩短,细胞快速通过G1/S转换点进入S期,加速细胞周期进程和细胞增殖,导致CML的发生。     

丙戊酸钠对Molt-4细胞增殖的影响及其作用途径探讨

目的 观察丙戊酸钠(VPA)对人急性T细胞白血病细胞系Molt-4细胞增殖和细胞周期的影响,并探讨其对Molt-4细胞p15INK4B基因甲基化状态的影响.方法 不同浓度VPA作用Molt-4细胞后,用MTr法检测细胞增殖抑制率,流式细胞术检测细胞周期的变化,半巢式甲基化特异PCR(MSP)法检测p15INK4B基因甲基化状态,RT-PCR方法检测p15基因、DNA甲基转移酶DNMT-1、DNMT3A、DNMrT3B mRNA的表达.结果 VPA可明显抑制细胞增殖,影响Molt-4细胞周期分布,5.0mmol/L VPA作用Molt-4细胞48 h,G0/G1期细胞为(66.87±3.31)%,S期细胞为(8.47±2.56)%,与未加VPA组相比,S期细胞明显下降,G0/G1期细胞明显升高(P＜0.05).经VPA作用后Molt-4细胞p15基因甲基化程度明显下降,p15基因表达水平明显增加,DNMT-1、DNMT3B mRNA表达水平下降,以DNMT-1下降较明显.结论 VPA可能通过抑制甲基转移酶DNMT-1和(或)DNMT3B表达使p15INK4B基因去甲基化,使p15基因表达上调,从而抑制Molt-4细胞增殖,使细胞周期阻滞于G0/G1期.     

Distinct features of dendritic cells and anti-Ig activated B cells as stimulators of the primary mixed leukocyte reaction

Highly enriched populations of B lymphoblasts have been isolated after culture with anti-Ig-Sepharose and compared with dendritic cells as stimulators of CD4+ T cells in the murine MLR. The two populations clearly differed in phenotype; anti-Ig blasts were FcR+, B220+, 33D1-, while dendritic cells were FcR-, B220-, 33D1+. However, as MLR stimulators, they shared many common features. Both cells (a) expressed comparable levels of class II MHC products; (b) independently stimulated the primary MLR and the production of several T derived lymphokines including IL-2 and IL-4; and (c) were comparable in stimulating freshly sensitized T cells. However, the relative potencies of dendritic cells and anti-Ig blasts as primary MLR stimulators varied in a strain-dependent fashion. Only anti-Ig blasts could stimulate across an Mls barrier, being at least 100 times more active in stimulating Mls-mismatched, MHC-matched T cells, relative to syngeneic T cells. In contrast, dendritic cells were 10-30 times more potent than anti-Ig blasts when stimulating across an MHC barrier and were likewise more effective in binding MHC-disparate T cells to form the clusters in which the MLR was generated. Dendritic cell-T cell clustering was resistant to anti-LFA-1 mAb, while B blast-T cell clustering was totally blocked. Thus, anti-Ig B lymphoblasts and dendritic cells, two cell types which differ markedly in phenotype, also differ in efficiency and mechanism for initiating responses in allogeneic T cells.     

Inhibition of growth and induction of apoptosis in human cancer cell lines by an ethyl acetate fraction from shiitake mushrooms

OBJECTIVE: Shiitake (Lentinus edodes) mushrooms have been reported to have cancer-preventing properties. However, little research has been conducted verifying the antitumor activities of "mycochemicals" in shiitake mushrooms. In this study, potential roles of an ethyl acetate fraction from shiitake mushrooms were investigated by in vitro bioassays. DESIGN: The activities of an ethyl acetate fraction were evaluated by [3-(4,5-dimethylthiazol-yl)-2,5-diphenyltetrazolium bromide] (MTT), apoptosis bioassay, cell cycle analysis, and Western blot analysis using two human breast carcinoma cell lines (MDA-MB-453 and MCF-7), one human nonmalignant breast epithelial cell line (MCF-10F), and two myeloma cell lines (RPMI-8226 and IM-9). RESULTS: Concentration-dependent antiproliferative effects of the fraction were observed in all cell lines using the MTT assay. Approximately 50 mg/L concentration of the fraction induced apoptosis in 50% of the population of four human tumor cell lines and the fraction-induced apoptosis may have been mediated through the pro-apoptotic bax protein which was up-regulated. Cell cycle analysis revealed that the fraction induced cell cycle arrest by significant decrease of S phase, which was associated with the induction of cdk inhibitors p21 and the suppression of cdk4 and cyclin D1 activity. Compared to malignant tumor cells, nonmalignant cells were less sensitive to the fraction for the suppression of cell growth and regulation of bax, p21, cyclin D1, and cdk4 expression. A 51% antiproliferative effect occurred at the highest concentration of the fraction (800 mg/L). CONCLUSIONS: These data suggest that inhibition of growth in tumor cells by "mycochemicals" in shiitake mushrooms may result from induction of apoptosis.     

丁酸钠联合全反式维甲酸对MDS细胞株SKM-1的诱导分化作用及其机制初步探讨

本研究探讨丁酸钠(NaB)抑制MDS细胞株SKM-1细胞生长、诱导其分化的分子机制，并研究其与全反式维甲酸(ATRA)的协同作用。用台盼蓝拒染实验观察药物对细胞生长曲线的影响；四氮唑盐还原试验和细胞表面分化抗原检测观察药物对细胞的分化作用；流式细胞术分析细胞周期；RT-PCR检察D型细胞周期蛋白、CDK和P21在mRNA水平的表达。结果表明：NaB和(或)ATRA均可抑制SKM-1细胞的生长，诱导细胞分化，将细胞周期阻滞于G0／G1期；ATRA下调CDK6、CDK4、cyclin D3和cyclin D1 mRNA的水平；NaB下调CDK2、cyclin D2和cyclin D1 mRNA的水平；两药联用下调CDK6、CDK4、CDK2、cyclin D1、cyclin D2和cyclin D3 mRNA的水平；ATRA和(或)NaB均上调P21 mRNA的水平。结论:NaB诱导SKM-1的分化可能是通过上调P21 mRNA的水平和抑制cycLin D-CDK复合体的形成完成的，NaB与ATRA对SKM-1细胞株的分化有协同作用。     

IL-7 promotes T cell proliferation through destabilization of p27Kip1

Interleukin (IL)-7 is required for survival and homeostatic proliferation of T lymphocytes. The survival effect of IL-7 is primarily through regulation of Bcl-2 family members; however, the proliferative mechanism is unclear. It has not been determined whether the IL-7 receptor actually delivers a proliferative signal or whether, by promoting survival, proliferation results from signals other than the IL-7 receptor. We show that in an IL-7-dependent T cell line, cells protected from apoptosis nevertheless underwent cell cycle arrest after IL-7 withdrawal. This arrest was accompanied by up-regulation of the cyclin-dependent kinase inhibitor p27Kip1 through a posttranslational mechanism. Overexpression of p27Kip1 induced G1 arrest in the presence of IL-7, whereas knockdown of p27Kip1 by small interfering RNA promoted S phase entry after IL-7 withdrawal. CD4 or CD8 T cells transferred into IL-7-deficient hosts underwent G1 arrest, whereas 27Kip1-deficient T cells underwent proliferation. We observed that IL-7 withdrawal activated protein kinase C (PKC)theta and that inhibition of PKCtheta with a pharmacological inhibitor completely blocked the rise of p27Kip1 and rescued cells from G1 arrest. The conventional pathway to breakdown of p27Kip1 is mediated by S phase kinase-associated protein 2; however, our evidence suggests that PKCtheta acts via a distinct, unknown pathway inducing G1 arrest after IL-7 withdrawal from T cells. Hence, IL-7 maintains T cell proliferation through a novel pathway of p27Kip1 regulation.     

P27基因对人喉癌细胞抑制作用的研究

１Materialsandmethods１．１CelllinesandvectorHumanlaryngealcarcinomacelllineHep－２weregrowninDulbeccomodifiedEaglemedium．Thep２７eu－karyoticexpressionvectorpCMV－p２７wasagiftfromDrLijinge．１．２DNAtransfectionDNAtransfectionbyLipofectamineac－cordingtothemanufacturer｀sinstructions（Gibco）．１．３AnalysisofcellcyclebyflowcytometryCellstransfectedwithpCMV－p２７werecollectedbytrypsinization，fixedwith７０％ethanol．After４８hours，cellswerecollected，resuspendedin１mloflysisbuffe…     

凝血酶促进结肠癌SW480细胞生长的研究

目的探讨凝血酶对结肠癌SW480细胞增殖及细胞周期的影响。方法将20 nmol/L、100 nmol/L、500nmol/L的凝血酶作用于SW480细胞48 h,以未处理组（0 nmol/L）作为对照。CCK-8（cell counting kit-8）法检测细胞增殖情况;流式细胞术检测细胞周期变化;RT-PCR检测蛋白酶活化受体-4（protease-activated receptor-4,PAR-4）的mRNA表达;Western blotting检测NF-κB p50蛋白表达。结果在20-500 nmol/L浓度范围内,凝血酶可剂量依赖性的促进SW480细胞的增殖;细胞周期显示G0/G1期细胞百分数减少,S期及G2/M期细胞百分数增加,表明凝血酶可诱导细胞从G0/G1期向G2/M期发展,从而促进细胞增殖;随着凝血酶浓度的增加,PAR-4 mRNA及NF-κB p50蛋白含量也有不同程度的增加。结论在20-500 nmol/L浓度范围内,凝血酶可促进结肠癌SW480细胞的增殖,加速细胞周期进程;其作用可经PAR-4介导,并与NF-κB信号通路活化有关。     

亚砷酸钠选择性诱导G2+M期NB4细胞凋亡

目的探讨亚砷酸钠诱导ＮＢ４细胞凋亡的作用机制。方法采用流式细胞术、ＤＮＡ电泳和免疫印迹法研究亚砷酸钠对ＮＢ４细胞的作用。结果①亚砷酸钠作用的ＮＢ４细胞先阻滞在Ｇ２＋Ｍ期,而后发生凋亡;②ｄＵＴＰＦＩＴＣ特异性地标记Ｇ２＋Ｍ期ＮＢ４细胞,而且发生在Ｇ２＋Ｍ期阻滞后;③亚砷酸钠处理ＮＢ４细胞,周期蛋白Ａ、Ｅ、Ｄ１、Ｄ２和Ｄ３无明显变化,周期蛋白Ｂ１表达随作用时间的增加而增加;④流式细胞仪分析显示在亚砷酸钠处理的早期（１６ｈ）,有部分Ｓ期细胞表达周期蛋白Ｂ１,晚期大部分高表达周期蛋白Ｂ１的细胞处于Ｇ２＋Ｍ期,有少量亚二倍体细胞高表达周期蛋白Ｂ１。结论结果提示亚砷酸钠选择性诱导Ｇ２＋Ｍ期ＮＢ４细胞凋亡时,伴周期蛋白Ｂ１表达的升高。     

Preincubation with endothelial cell monolayers increases gene transfer efficiency into human bone marrow CD34(+)CD38(-) progenitor cells

Retroviral gene transfer studies targeting bone marrow CD34(+)CD38(-) stem cells have been disappointing because of the rarity of these cells, their G(0) cell cycle status, and their low or absent expression of surface retroviral receptors. In this study, we examined whether preincubation of bone marrow CD34(+)CD38(-) stem cells with a hematopoietically supportive porcine microvascular endothelial cell line (PMVECs) could impact the cell cycle status and expression of retroviral receptors in pluripotent CD34+CD38- cells and the efficiency of gene transfer into these primitive target cells. PMVEC coculture supplemented with GM-CSF + IL-3 + IL-6 + SCF + Flt-3 ligand induced >93% of the CD34(+)CD38(-) population to enter the G(1) or G(2)/S/M phase while increasing this population from 1.4% on day 0 to 6.5% of the total population by day 5. Liquid cultures supplemented with the identical cytokines induced 73% of the CD34(+)CD38(-) population into cell cycle but did not maintain cells with the CD34(+)CD38(-) phenotype over time. We found no significant increase in the levels of AmphoR or GaLVR mRNA in PMVEC-expanded CD34(+)CD38(-) cells after coculture. Despite this, the efficiency of gene transfer using either amphotropic vector (PA317) or GaLV vector (PG13) was significantly greater in PMVEC-expanded CD34(+)CD38(-) cells (11.4 +/- 5.6 and 10.9 +/- 5.2%, respectively) than in either steady state bone marrow CD34(+)CD38(-) cells (0.6 +/- 1.7 and 0.2 +/- 0.6%, respectively; p < 0.01 and p < 0.01) or liquid culture-expanded CD34(+)CD38(-) cells (1.4 +/- 3.5 and 0.0%, respectively; p < 0.01 and p < 0.01). Since PMVEC coculture induces a high level of cell cycling in human bone marrow CD34(+)CD38(-) cells and expands hematopoietic cells capable of in vivo repopulation, this system offers potential advantages for application in clinical gene therapy protocols.     

EBV立刻早期蛋白Zta基因对Daudi细胞周期的影响及其机制研究

目的 探讨EBV立刻早期蛋白Zta基因对Daudi细胞周期的影响及其可能机制.方法 构建Zta基因真核表达载体,通过电穿孔将该载体转染Daudi细胞,用流式细胞术检测细胞周期的变化,用蛋白印迹试验检测细胞周期蛋白p21、Rb、E2F-1的表达.结果 成功构建了 Zta基因表达载体,转染Zta基因可抑制Daudi细胞的增殖,并促进Daudi细胞由G0/G11期[(30.0±3.4)％)]向S期[(47.7±1.1)％]的转化.同时转染Zta基因可下调Rb的表达、上调E2F-1、p21的表达.结论 转染Zta基因使Daudi细胞周期由Go/G1期向S期转变,其机制可能与Rb的表达下降、E2F-1和p21表达上调有关.