核因子κB在精氨酸血管升压素诱导大鼠心肌成纤维细胞一氧化氮合成中的作用

本文探讨了精氨酸血管升压素(AVP)刺激下体外培养的大鼠心肌成纤维细胞(CFs)内一氧化氮(NO)含量、一氧化氮合酶(NOS)活性、诱导型一氧化氮合酶基因表达的变化及其与核因子κB(NF-κB)的关系。用胰酶消化法分离培养Sprague Dawley仔鼠的CFs,分别采用硝酸还原酶法、分光光度法、逆转录-聚合酶链式反应(RT-PCR)、免疫荧光-共聚焦显微镜和蛋白质印迹检测AVP干预下CFs的NO含量、NOS活性、iNOS mRNA表达和NF-κB的活化。结果显示,AVP浓度依赖性(0．001—0．1μmol／L)地增加CFs的NO含量,提高NOS活性,增加iNOS mRNA表达;AVP能够活化NF—κB,使其由细胞浆转位于细胞核;NF-κB特异性抑制剂吡咯啉烷二甲基硫脲(PDTC)能够抑制AVP诱导的CFs NO含量增加、NOS活性提高和iNOS mRNA表达增加。上述结果提示,AVP干预下CFs iNOS mRNA表达增加、NOS活性增高、NO合成增多可能通过NF-κB激活途径,NF-κB激活参与心肌纤维化的发生和发展。     

DNA损伤反应中细胞周期检查点蛋白p27~(Kip1)表达及其调控机制

为了研究DNA损伤反应中p2 7Kip1的表达及其调控机制 ,应用免疫印迹的实验结果表明 :10Gy 60 Coγ射线照射后 3h ,HeLa细胞中p2 7Kip1蛋白水平开始下降并持续到 2 4h ,进而失去它对CDKs的抑制功能 .Northern印迹结果显示 ,电离辐射 (IR)对p2 7Kip1mRNA表达水平无明显影响 ,说明电离辐射诱导p2 7Kip1表达水平的降低主要与蛋白质降解相关 ,但其具体的调控机制还不清楚 .已知在G1—S期p2 7Kip1蛋白的降低主要依赖细胞周期蛋白E Cdk2激酶将其磷酸化后的泛素化蛋白酶体途径 (ubiquitin proteasomepathway) .酶动力学研究结果揭示 :电离辐射后细胞周期蛋白E Cdk2激酶活性增高 ,12h细胞周期蛋白E Cdk2激酶活性达到最大 .当在照前用细胞周期蛋白E Cdk2抑制剂olomoucine (10 μmol L)抑制细胞周期蛋白E Cdk2激酶活性时 ,p2 7Kip1蛋白表达水平增加 .此外 ,还观察到电离辐射可诱导p2 7Kip1泛素化水平的增高 ,而在使用蛋白酶体抑制剂MG 132 (5 μmol L)处理HeLa细胞后 ,可抑制辐射诱导p2 7Kip1蛋白水平的下调 .研究结果提示 :泛素化蛋白酶体途径参与了辐射诱导P2 7Kip1蛋白表达下调的降解机制 .     

异钩藤碱调控ERK/p27^(Kip1)信号通路改善博莱霉素诱导的小鼠肺纤维化CSCD

基于细胞外调节蛋白激酶1/2(ERK1/2)、p27^(Kip1)信号通路探究异钩藤碱(isorhynchophylline,IRN)对博莱霉素(bleomycin,BLM)诱导的小鼠肺纤维化(pulmonary fibrosis,PF)的作用及机制。C57BL/6J小鼠48只,随机正常组、BLM组、BLM+IRN(10、20 mg/kg)两个剂量组,每组12只。气管注射BLM(5000 U/kg)诱导PF小鼠模型,造模后连续灌胃给药21天。HE和Masson染色观察肺组织病理变化及胶原沉积情况。免疫组化检测肺组织α-平滑肌肌动蛋白(alpha-smooth muscle actin,α-SMA)的表达。体外培养小鼠原代肺成纤维细胞,实验设对照组、转化生长因子-β1(transforming growth factor-beta 1,TGF-β1)(10 ng/mL)组和TGF-β1+IRN(5、10、20μmol/L)三个剂量组。EdU掺入法和流式细胞术检测细胞增殖,Transwell观察细胞的迁移能力。RT-qPCR检测肺组织或肺成纤维细胞TGF-β1、collagen I和α-SMA mRNA的表达。Western blot检测肺组织和(或)肺成纤维细胞TGF-β1、collagen I、α-SMA、p-ERK1/2,p27^(Kip1)、CDK2和Cyclin E1的蛋白水平。动物实验结果显示,与BLM组相比,不同剂量IRN均能明显减轻肺组织结构的损伤、降低炎症细胞的浸润和胶原的沉积;此外,IRN不同程度地降低肺组织TGF-β1、collagen I和α-SMA mRNA和蛋白的表达;同时,IRN还抑制了肺组织ERK1/2的磷酸化、上调p27^(Kip1)和下调CDK2和Cyclin E1的蛋白表达。细胞实验结果显示,与TGF-β1组相比,不同剂量IRN能够明显抑制TGF-β1诱导的肺成纤维细胞增殖、显著降低细胞迁移能力;明显降低TGF-β1诱导的collagen I和α-SMA mRNA和蛋白的表达,同时降低ERK1/2的磷酸化水平、上调p27^(Kip1)和下调CDK2和Cyclin E1的蛋白表达。以上结果表明IRN可能通过抑制ERK1/2信号通路、上调p27^(Kip1)的表达而抑制了肺成纤维细胞向肌成纤维细胞的转化,从而减轻了BLM诱导的PF。     

阿托伐他汀对甲状旁腺素致心肌肥大的保护作用

目的:观察阿托伐他汀对大鼠甲状旁腺素1-34(rPTH1-34)诱导的新生大鼠心室肌细胞肥大的抑制作用及丝裂素活化蛋白激酶激酶1/细胞外信号调节激酶1/2(MKK1/ERK1/2)表达的变化,分析阿托伐他汀抑制心肌肥大的可能机制。方法:以原代培养的新生Wsitar大鼠心室肌细胞为研究对象,3H-亮氨酸掺入率检测细胞蛋白合成速率,BCA法测定单个细胞蛋白含量,RT-PCR检测心房利钠尿肽mRNA(ANPmRNA)的表达,Western blot方法测定p-MKK1、p-ERK1/2蛋白的表达。结果:1.10-7mmol/LrPTH1-34孵育24h可增加体外培养的心肌细胞蛋白合成速率和ANPmRNA表达,使p-MKK1及p-ERK1/2蛋白表达升高(P<0.05)。2.10-5mmol/L阿托伐他汀能抑制心肌细胞蛋白合成速率及单细胞蛋白含量的增加(P<0.05),且对正常心肌细胞没有影响(P>0.05)。3.10-5mmol/L阿托伐他汀能抑制p-MKK1、p-ERK1/2的表达(P<0.05)。结论:阿托伐他汀能抑制PTH诱导的心肌细胞肥大的发生,可能与抑制MKK1/ERK1/2的活性有关。     

IL-1β对精氨酸升压素诱导的大鼠心肌成纤维细胞INOS-NO系统活性的影响

目的：探讨白细胞介素-1β（IL-1β）对精氨酸升压素（AVP）诱导下大鼠心肌成纤维细胞（CFs）诱导型一氧化氮合酶（iNOS）-一氧化氮（NO）系统活性的影响。方法：胰酶消化法分离培养SD仔鼠CFs,硝酸还原酶法、分光光度法和逆转录-聚合酶链式反应（RT—PCR）分别测定不同浓度IL-1β与AVP协同作用下CFs的NO含量、NOS活性和iNOSmRNA表达。结果：AVP诱导下CFs iNOSmRNA表达、NOS活性和NO合成均显著增加（P〈0.05）。一定浓度范围内IL-1β与AVP协同作用,剂量依赖性地增加AVP对CFs iNOS-NO系统活性的提高作用,其中AVP＋3ng／ml和AVP＋5ng／ml IL-1β组的iNOS mRNA表达、NOS活性和NO合成均显著高于AVP组（P〈0。05）,但IL-1β浓度增加至5ng／ml时,CFs的iNOSmRNA表达、NOS活性和NO合成不再继续升高,反而有所下降。结论：在一定浓度范围内IL-1β可与AVP协同提高CFs iNOS-NO系统活性。     

ERK活化对哮喘大鼠气道重塑及CyclinD1表达的影响

目的探讨细胞外信号调节蛋白激酶（ERK）对哮喘大鼠气道重塑及CyclinD1表达的作用。方法原代培养大鼠的平滑肌细胞（ASMCs）,给予ERK激动剂表皮生长因子EGF和抑制剂PD98059干预ASMCs生长,依处理方式不同分为5组：（1）正常对照组（2）哮喘对照组;（3）E组：EGF20 ng/mL;（4）P＋E组,PD98059 10μmol/L1 h后添加EGF 20 ng/mL;（5）PD组,PD98059 10μmol/L。采用四甲基偶氮唑盐（MTT）法检测气道平滑肌细胞（ASMCs）增殖能力,流式细胞术（FCM）测定细胞周期和cyclinD1的蛋白含量,RT-PCR方法检测cyclinD1mRNA表达水平。结果（1）与哮喘对照组比较,E组ASMCs S＋G2/M期比例、吸光度A值、cyclinD1蛋白阳性表达率和cyclinD1 mRNA的A值均显著升高,PD组均显著降低（P〈0.05）。P＋E组与哮喘对照在此4项指标上比较无明显差异。（2）哮喘（对照组、E组、PD组和P＋E组）组与正常对照组,其S＋G2/M期比例、吸光度A值、cyclinD1蛋白和cyclinD1 mRNA的表达均显著增高（P〈0.05）。结论ERK活性促进哮喘大鼠ASMCs的增殖,增加cyclinD1在哮喘平滑肌细胞中的表达,导致气道重塑的形成,提示ERK可能对CyclinD1的表达具有调节作用。     

大鼠内侧前额叶皮质内ERK1/2 活化参与脊髓损伤后抑郁情绪

目的:观察细胞外信号调节激酶1/2(ERK1/2)的活化在脊髓损伤引起抑郁中的作用。方法:应用Western blot和行为药理学方法,观察脊髓损伤后(SCI)大鼠内侧前额叶皮质内(mPFC)ERK1/2及磷酸化-ERK1/2(p-ERK1/2)的表达情况及ERK1/2磷酸化抑制剂U0126对抑郁样行为的影响。结果:脊髓损伤后的第2天到第8周,SCI模型大鼠的BBB评分均显著低于假手术组,差异具有统计学意义(p0.05)。脊髓损伤后8周-12周,SCI模型大鼠强迫游泳不动时间与假手术组相比明显缩短,mPFC内pERK1/2蛋白表达水平明显升高,总ERK 1/2的蛋白水平则未见组间差异,而给予U0126的大鼠的不动时间与给药之前相比明显延长增加,mPFC内pERK1/2蛋白表达水平较SCI模型大鼠明显降低,差异均具有统计学意义(P0.05)。结论:内侧前额叶皮质内ERK1/2的激活参与了脊髓损伤后引起的突触可塑性,在相关的抑郁样行为的产生中发挥了重要的作用。     

卡铂通过抑制ERK1/2通路诱导人卵巢癌细胞S和G0/G1期阻滞

卡铂(carboplatin, CBP)是一种抗肿瘤活性较强的化疗药物, 通过诱导细胞周期阻滞抑制肿瘤细胞生长, 但其诱导细胞周期阻滞的报告不甚一致. 本研究探索卡铂对卵巢癌HO-8910细胞生长及细胞周期进程的影响. MTS结果显示, 卡铂以浓度和时间依赖方式抑制卵巢癌HO-8910细胞生长, 联合使用ERK1/2通路抑制剂PD98059可使卡铂抗卵巢癌细胞增殖作用增强. 采用Giemsa染色法观察到, 卡铂与PD98059单用或联用均能致卵巢癌细胞发生明显的形态学变化. 流式细胞术检测细胞周期发现, 随卡铂浓度的增高, S期阻滞作用增强; 抑制ERK1/2通路可拮抗卡铂对HO-8910细胞S期阻滞作用, 增加G1期阻滞作用, 而对G2/M期细胞影响不明显. Western印迹结果显示, 随卡铂浓度的增高, p-ERK1/2、Cdc2(Y15)和p Cdc2(T161)的表达逐渐升高, Cyclin E1和Cyclin B1的表达逐渐降低; 抑制ERK1/2通路可将卡铂上调,p-ERK1/2和p-Cdc2(T161)的作用反转为下调作用, 上调Cdc2(Y15)的表达受阻, 抑制Cyclin B1的下调作用, 促进Cyclin E1的下调作用. 本研究结果提示, 卡铂通过抑制ERK1/2激活, 诱导人卵巢癌HO-8910细胞S和G1期阻滞, 抑制卵巢癌细胞生长.     

ERK-VEG FMMP-9信号通路与直肠癌细胞增殖和血管新生的关联

为了探讨ERK-VEGFMMP-9信号通路与直肠癌细胞增殖和血管新生的关联,本研究以不同浓度(10～100μmol/L)的丙泊酚处理HT-29细胞0、48 h、72 h,研究丙泊酚对HT-29细胞中ERK、MMP-9和VEGF的影响,并利用Western blotting法观察HT-29细胞中ERK1/2、MMP-9和VEGF的表达量与信号通路之间的关系。观察丙泊酚处理后对HT-29细胞增殖能力、血管生成能力、细胞侵袭能力的影响。研究结果表明,在细胞中,VEGF与MMP-9蛋白的表达,均随着丙泊酌剂量的增加而呈现逐渐减少的趋势。与对照组相比,50μmol/L组与100μmol/L组降低(p0.05);细胞内VEGF和MMP-9蛋白的表达量随药物处理时间的增加而逐渐降低,与0 h相比,48 h组和72h组降低(p0.05)。细胞中pERK与ERK的比率随着丙泊酌剂量的增加而逐渐降低。与对照组相比,50μmol/L组与100μmol/L组降低(p0.05),pERK与ERK的比率随着用药时间的增加而逐渐降低。与0相比,48 h组与72 h组降低(p0.05)。与0剂量相比,丙泊酚加PMA组MMP-9蛋白的表达量显著升高,50μmol/L组和100μmol/L组增加(p0.05),丙泊酚加PMA组MMP-9蛋白的表达量随着时间的增加而明显的增加,与0h相比,48 h和72 h增加(p0.05)。与0组相比,丙泊酚加PMA组VEGP的表达量明显增加,50μmol/L组和100μmol/L组增加(p0.05),丙泊酚加PMA组VEGP的表达量随时间增加而增加,与Oh相比,48h和72h显著增加(p0.05)。25μmol/L组、50μmol/L组、100μmol/L组Eca109细胞的增殖力、细胞中CAM的新生血管的生成、细胞的侵袭力与对照组相比均显著增加(p0.05),48 h组、72 h组Eca109细胞的增殖力、细胞中CAM的新生血管的生成、细胞的侵袭力与对照组相比均显著增加(p0.05)。丙泊酚通过ERK-VEGF/MMP-9的信号的调制,从而抑制人类的HT-29细胞的增殖、侵袭的体外和血管的生成。     

卡铂通过抑制ERK1/2通路诱导人卵巢癌细胞S和G_1期阻滞

卡铂(carboplatin,CBP)是一种抗肿瘤活性较强的化疗药物,通过诱导细胞周期阻滞抑制肿瘤细胞生长,但其诱导细胞周期阻滞的报告不甚一致.本研究探索卡铂对卵巢癌HO-8910细胞生长及细胞周期进程的影响.MTS结果显示,卡铂以浓度和时间依赖方式抑制卵巢癌HO-8910细胞生长,联合使用ERK1/2通路抑制剂PD98059可使卡铂抗卵巢癌细胞增殖作用增强.采用Giemsa染色法观察到,卡铂与PD98059单用或联用均能致卵巢癌细胞发生明显的形态学变化.流式细胞术检测细胞周期发现,随卡铂浓度的增高,S期阻滞作用增强;抑制ERK1/2通路可拮抗卡铂对HO-8910细胞S期阻滞作用,增加G1期阻滞作用,而对G2/M期细胞影响不明显.Western印迹结果显示,随卡铂浓度的增高,p-ERK1/2、Cdc2(Y15)和p-Cdc2(T161)的表达逐渐升高,Cyclin E1和Cyclin B1的表达逐渐降低;抑制ERK1/2通路可将卡铂上调,p-ERK1/2和p-Cdc2(T161)的作用反转为下调作用,上调Cdc2(Y15)的表达受阻,抑制Cyclin B1的下调作用,促进Cyclin E1的下调作用.本研究结果提示,卡铂通过抑制ERK1/2激活,诱导人卵巢癌HO-8910细胞S和G1期阻滞,抑制卵巢癌细胞生长.     

Involvement of ERK and AKT signaling in the growth effect of arginine vasopressin on adult rat cardiac fibroblast and the modulation by simvastatin

Arginine vasopressin (AVP) has been shown to directly induce neonatal rat cardiac fibroblasts (CFs) proliferation, a major component involved in cardiac hypertrophy. Herein, we explored whether AVP is also a growth factor for adult rat CFs and, if so, whether the growth effect could be inhibited by simvastatin, a 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor. AVP significantly increased DNA synthesis in adult rat CFs by 73.5 +/- 5.1% (P < or = 0.05), an effect inhibited by V1 receptor antagonist, d(CH(2))(5)[Tyr(2)(Me), Arg(8)]-vasopressin. AVP also activated extracellular signal-regulated kinase 1/2 (ERK1/2) as assessed by MBP phosphotransferase activity (5.1 +/- 0.6 fold over basal level, P < or = 0.05) and Western blot analysis, and effects were mimicked by protein kinase C (PKC) activator, phorbol 12-myristate 13-acetate (PMA), but abolished by inhibiting cellular PKC through chronic PMA incubation. In addition, AVP induced PKC activation (27.2 +/- 3.8% from a basal value of 9.3 +/- 0.7%, P < or = 0.05). AVP-induced increase in DNA synthesis could be attenuated by the specific inhibitors of ERK1/2 (PD98059), PI3K (LY294002), and AKT (1L-6-hydroxymethyl-chiro-inositol 2-(R)-2-O-methyl-3-O-octadecylcarbonate, HIMO). Simvastatin inhibited the effects of AVP on DNA synthesis, ERK1/2, and PKC activation in a dose-dependent manner. Phosphatidylinositol-3-kinase (PI3K)-dependent AKT activation induced by AVP was also inhibited by simvastatin. The effects of simvastatin on ERK1/2, PKC, and AKT activation and DNA synthesis could be reversed by mevalonate. These results support a growth-inducing effect of AVP on adult rat CFs through ERK and AKT signalings and the growth effect could be attenuated by simvastatin via inhibiting these two pathways.     

Docosahexaenoic acid inhibits cancer cell growth via p27Kip1, CDK2, ERK1/ERK2, and retinoblastoma phosphorylation

Docosahexaenoic acid (DHA), a PUFA of the n-3 family, inhibited the growth of FM3A mouse mammary cancer cells by arresting their progression from the late-G(1) to the S phase of the cell cycle. DHA upregulated p27(Kip1) levels by inhibiting phosphorylation of mitogen-activated protein (MAP) kinases, i.e., ERK1/ERK2. Indeed, inhibition of ERK1/ERK2 phosphorylation by DHA, U0126 [chemical MAPK extracellularly signal-regulated kinase kinase (MEK) inhibitor], and MEK(SA) (cells expressing dominant negative constructs of MEK) resulted in the accumulation of p27(Kip1). MAP kinase (MAPK) inhibition by DHA did not increase p27(Kip1) mRNA levels. Rather, this fatty acid stabilized p27(Kip1) contents and inhibited MAPK-dependent proteasomal degradation of this protein. DHA also diminished cyclin E phosphorylation, cyclin-dependent kinase-2 (CDK2) activity, and phosphorylation of retinoblastoma protein in these cells. Our study shows that DHA arrests cell growth by modulating the phosphorylation of cell cycle-related proteins.     

Arginine vasopressin controls p27 protein expression by PKC activation and irreversibly inhibits the proliferation of K-Ras-dependent mouse Y1 adrenocortical malignant cells

The neurohypophyseal hormone arginine vasopressin (AVP) is a classic mitogen in many cells. In K-Ras-dependent mouse Y1 adrenocortical malignant cells, AVP elicits antagonistic responses such as the activation of the PKC and the ERK1/2 mitogenic pathways to down-regulate cyclin D1 gene expression, which induces senescence-associated β-galactosidase (SA-βGal) and leads to cell cycle arrest. Here, we report that in the metabolic background of Y1 cells, PKC activation either by AVP or by PMA inhibits the PI3K/Akt pathway and stabilises the p27^Kip1 protein even in the presence of the mitogen fibroblast growth factor 2 (FGF2). These results suggest that p27^Kip1 is a critical signalling node in the mechanisms underlying the survival of the Y1 cells. In Y1 cells that transiently express wild-type p27^Kip1, AVP caused a severe reduction in cell survival, as shown by clonogenic assays. However, AVP promoted the survival of Y1 cells transiently expressing mutant p27-S10A or mutant p27-T187A, which cannot be phosphorylated at Ser10 and Thr187, respectively. In addition, PKC activation by PMA mimics the toxic effect caused by AVP in Y1 cells, and inhibition of PKC completely abolishes the effects caused by both PMA and AVP in clonogenic assays. The vulnerability of Y1 cells during PKC activation is a phenotype conditioned upon K-ras oncogene amplification because K-Ras down-regulation with an inducible form of the dominant-negative mutant H-RasN17 has resulted in Y1 cells that are resistant to AVP's deleterious effects. These data show that the survival destabilisation of K-Ras-dependent Y1 malignant cells by AVP requires large quantities of the p27^Kip1 protein as well as phosphorylation of the p27^Kip1 protein at both Ser10 and Thr187.     

Protein kinase Cdelta inhibition of S-phase transition in capillary endothelial cells involves the cyclin-dependent kinase inhibitor p27(Kip1).

Distinct protein kinase C (PKC) isoforms differentially regulate cellular proliferation in rat microvascular endothelial cells (EC). Overexpression of PKCalpha has little effect on proliferation, whereas PKCdelta slows endothelial cell proliferation and induces S-phase arrest. Analyses were performed on EC overexpressing PKCalpha (PKCalphaEC) or PKCdelta (PKCdeltaEC) to determine the role of specific cell cycle regulatory proteins in the PKCdelta-induced cell cycle arrest. Serum-induced stimulation of cyclins D1, E, and A-associated kinase activity was delayed by 12 h in the PKCdeltaEC line in association with S-phase arrest. However, the protein levels for cyclins D1, E, and A were similar. Nuclear accumulation of cyclin D1 protein in response to serum was also delayed in PKCdeltaEC. In the PKCdeltaEC line, serum induced p27(Kip1) but not p16(Ink4a) or p21(Cip1). Serum did not affect p27(Kip1) levels in the control vascular endothelial cell line. Immunoprecipitation-Western blotting analysis of p27(Kip1) showed serum stimulation of the vascular endothelial cell line resulted in increased amounts of cyclin D1 bound to p27(Kip1). In the PKCdeltaEC line, serum did not increase the amount of cyclin D1 bound to p27(Kip1). Transfection of full-length p27(Kip1) antisense into the PCKdeltaEC line reversed the S-phase arrest and resulted in normal cell cycle progression, suggesting a critical role for p27(Kip1) in the PKCdelta-mediated S-phase arrest.     

Arginine vasopressin controls p27(Kip1) protein expression by PKC activation and irreversibly inhibits the proliferation of K-Ras-dependent mouse Y1 adrenocortical malignant cells

The neurohypophyseal hormone arginine vasopressin (AVP) is a classic mitogen in many cells. In K-Ras-dependent mouse Y1 adrenocortical malignant cells, AVP elicits antagonistic responses such as the activation of the PKC and the ERK1/2 mitogenic pathways to down-regulate cyclin D1 gene expression, which induces senescence-associated β-galactosidase (SA-βGal) and leads to cell cycle arrest. Here, we report that in the metabolic background of Y1 cells, PKC activation either by AVP or by PMA inhibits the PI3K/Akt pathway and stabilises the p27(Kip1) protein even in the presence of the mitogen fibroblast growth factor 2 (FGF2). These results suggest that p27(Kip1) is a critical signalling node in the mechanisms underlying the survival of the Y1 cells. In Y1 cells that transiently express wild-type p27(Kip1), AVP caused a severe reduction in cell survival, as shown by clonogenic assays. However, AVP promoted the survival of Y1 cells transiently expressing mutant p27-S10A or mutant p27-T187A, which cannot be phosphorylated at Ser10 and Thr187, respectively. In addition, PKC activation by PMA mimics the toxic effect caused by AVP in Y1 cells, and inhibition of PKC completely abolishes the effects caused by both PMA and AVP in clonogenic assays. The vulnerability of Y1 cells during PKC activation is a phenotype conditioned upon K-ras oncogene amplification because K-Ras down-regulation with an inducible form of the dominant-negative mutant H-RasN17 has resulted in Y1 cells that are resistant to AVP's deleterious effects. These data show that the survival destabilisation of K-Ras-dependent Y1 malignant cells by AVP requires large quantities of the p27(Kip1) protein as well as phosphorylation of the p27(Kip1) protein at both Ser10 and Thr187.     

Blockade of the extracellular signal-regulated kinase pathway induces marked G1 cell cycle arrest and apoptosis in tumor cells in which the pathway is constitutively activated: up-regulation of p27(Kip1)

Constitutive activation of the ERK pathway is associated with the neoplastic phenotype of a relatively large number of human tumor cells. Blockade of the ERK pathway by treatment with PD98059, a specific inhibitor of mitogen-activated protein (MAP) kinase/ERK kinase (MEK), completely suppressed the growth of tumor cells in which the pathway is constitutively activated (RPMI-SE and HT1080 cells). Consistent with its prominent antiproliferative effect, PD98059 induced a remarkable G(1) cell cycle arrest, followed by a modest apoptotic response, in these tumor cells. Selective up-regulation of p27(Kip1) was observed after PD98059 treatment of RPMI-SE and HT1080 cells. Overexpression in RPMI-SE cells of either a kinase-negative form of MEK1 or wild-type MAP kinase phosphatase-3 also induced up-regulation of p27(Kip1). The up-regulation of p27(Kip1) correlated with increased association of p27(Kip1) with cyclin E-cyclin-dependent kinase (CDK) 2 complexes, a concomitant inhibition of cyclin E-CDK2 kinase activity, and a consequent decrease in the phosphorylation state of retinoblastoma protein, which would culminate in the marked G(1) cell cycle arrest observed in these tumor cells. These results suggest that the complete growth suppression that follows specific blockade of the ERK pathway in tumor cells in which the pathway is constitutively activated is mediated by up-regulation of p27(Kip1).     

Differential regulation of the cyclin-dependent kinase inhibitors p21(Cip1) and p27(Kip1) by phosphorylation directed by the cyclin encoded by Murine Herpesvirus 68

Members of the gamma2-herpesvirus family encode cyclin-like proteins that have the ability to deregulate mammalian cell cycle control. Here we report the key features of the viral cyclin encoded by Murine Herpesvirus 68, M cyclin. M cyclin preferentially associated with and activated cdk2; the M cyclin/cdk2 holoenzyme displayed a strong reliance on phosphorylation of the cdk T loop for activity. cdk2 associated with M cyclin exhibited substantial resistance to the cdk inhibitor proteins p21(Cip) and p27(Kip). Furthermore, M cyclin directed cdk2 to phosphorylate p27(Kip1) on threonine 187 (T187) and cellular expression of M cyclin led to down-regulation of p27(Kip1) and the partial subversion of the associated G1 arrest. Mutation of T187 to a non-phosphorylatable alanine rendered the p27(Kip1)-imposed G1 arrest resistant to M cyclin expression. Unlike the related K cyclin, M cyclin was unable to circumvent the G1 arrest associated with p21(Cip1) and was unable to direct its associated catalytic subunit to phosphorylate this cdk inhibitor. These results imply that M cyclin has properties that are distinct from other viral cyclins and that M cyclin expression alone is insufficient for S phase entry.     

Retinoic acid-mediated G1 arrest is associated with induction of p27(Kip1) and inhibition of cyclin-dependent kinase 3 in human lung squamous carcinoma CH27 cells

Retinoids are promising agents for the prevention and treatment of several human malignancies including lung cancer. In this study, the effect of retinoic acid (RA) on cell growth and the mechanism of growth modulation were examined in human lung squamous carcinoma CH27 cells. Here we report that RA mediated the dose- and time-dependent growth arrest in G1 phase, accompanied by the up-regulation of p27(Kip1) and the down-regulation of the cyclin-dependent kinase 3 (Cdk3) and p21(CIP1/Waf1) proteins. Furthermore, RA-induced growth arrest of CH27 cells was also associated with increased retinoic acid receptor beta (RARbeta) and reduced c-Myc expression. However, RA had no effect on the levels of cyclins A, D1, D3, E, or H, or on Cdk2, Cdk4, Cdk5, CDk6, Cdk7, p16(Ink4A), p15(Ink4B), p53, or pRb proteins in CH27 cells. Evaluation of the kinase activity of cyclin-Cdk complexes showed that RA increases p27(Kip1) expression in CH27 cells leading to markedly reduced cyclin A/Cdk2 kinase activity and slightly reduced cyclin E/Cdk2 kinase activity, with no effect on cyclin D/Cdk4 and cyclin D/Cdk6 activities. Moreover, coincident with the decrease in kinase activity was a drastic increase in cyclin A-bound p27(Kip1). These results suggest that increases in the levels of p27(Kip1) and its binding to cyclin A, as well as reduction of Cdk3 protein expression, are strong candidates for the cell cycle regulator that prevents the entry into the S phase in RA-treated CH27 cells, with prolongation of G1 phase and inhibition of DNA synthesis.     

The role of cyclin-dependent kinase inhibitor p27Kip1 in anti-HER2 antibody-induced G1 cell cycle arrest and tumor growth inhibition

Cyclin-dependent kinase (CDK) inhibitor p27Kip1 binds to the cyclin E.CDK2 complex and plays a major role in controlling cell cycle and cell growth. Our group and others have reported that anti-HER2 monoclonal antibodies exert inhibitory effects on HER2-overexpressing breast cancers through G1 cell cycle arrest associated with induction of p27Kip1 and reduction of CDK2. The role of p27Kip1 in anti-HER2 antibody-induced cell cycle arrest and growth inhibition is, however, still uncertain. Here we have provided several lines of evidence supporting a critical role for p27Kip1 in the anti-HER2 antibody-induced G1 cell cycle arrest and tumor growth inhibition. Induction of p27Kip1 and G1 growth arrest by anti-HER2 antibody, murine 4D5, or humanized trastuzumab (Herceptin) are concentration-dependent, time-dependent, irreversible, and long-lasting. The magnitude of G1 cell cycle arrest induced by trastuzumab or 4D5 is well correlated with the level of p27Kip1 protein induced. Up-regulation of p27Kip1 and G1 growth arrest could no longer be removed with as little as 14 h of treatment with trastuzumab. Anti-HER2 antibody-induced p27Kip1 protein, G1 arrest, and growth inhibition persist at least 5 days after a single treatment. The magnitude of growth inhibition of breast cancer cells induced by anti-HER2 antibody closely parallels the level of p27Kip1 induced. Induced expression of exogenous p27Kip1 results in a p27Kip1 level-dependent G1 cell cycle arrest and growth inhibition similar to that obtained with anti-HER2 antibodies. Reducing p27Kip1 expression using p27Kip1 small interfering RNA blocks anti-HER2 antibody-induced p27Kip1 up-regulation and G1 arrest. Treatment with anti-HER2 antibody significantly increases the half-life of p27Kip1 protein. Inhibition of ubiquitin-proteasome pathway, but not inhibition of calpain and caspase activities, up-regulates p27Kip1 protein to a degree comparable with that obtained with anti-HER2 antibodies. We have further demonstrated that anti-HER2 antibody significantly decreases threonine phosphorylation of p27Kip1 protein at position 187 (Thr-187) and increases serine phosphorylation of p27Kip1 protein at position 10 (Ser-10). Expression of S10A and T187A mutant p27Kip1 protein increases the fraction of cells in G1 and reduces a further antibody-induced G1 arrest. Consequently, p27Kip1 plays an important role in the anti-HER2 antibody-induced G1 cell cycle arrest and tumor growth inhibition through post-translational regulation. Regulation of the phosphorylation of p27Kip1 protein is one of the post-translational mechanisms by which anti-HER2 antibody upregulates the protein.