人表皮生长因子受体显性负性突变体诱导胃癌细胞发生G0/G1期阻滞

 目的：探讨人表皮生长因子受体显性负性突变体（dominant negative epidermal growth factor receptor,DNEGFR）对胃癌细胞细胞周期的影响及其分子机制。方法：选用2株人胃癌细胞,分为如下6组：SGC-7901细胞未转染组（US组）、SGC-7901细胞pEGFP-N1质粒转染组(ES组)、SGC-7901细胞pEGFPN1-DNEGFR质粒转染组(DS组)、NCI-N87细胞未转染组（UN组)、NCI-N87细胞pEGFP-N1质粒转染组(EN组)和NCI-N87细胞pEGFPN1-DNEGFR质粒转染组(DN组)。采用流式细胞术检测细胞周期,Western blotting检测细胞周期素依赖性蛋白激酶2（CDK2）、cyclin D1、Ser9位点磷酸化糖原合成酶激酶3β［p-GSK-3β(Ser9)］、p21和p27蛋白水平。结果：转染pEGFPN1-DNEGFR质粒的人胃癌细胞株出现G0/G1期阻滞,CDK2、cyclin D1和p-GSK-3β（Ser9）蛋白水平降低,p21和p27蛋白水平则升高。结论：DNEGFR通过激活GSK-3β使cyclin D1蛋白水平降低,并降低CDK2蛋白水平,上调p21和p27蛋白水平,最终导致胃癌细胞发生G0/G1期阻滞。这一结果将为胃癌生物治疗研究提供新思路。     

Simvastatin inhibits cell growth and induces apoptosis and G0/G1 cell cycle arrest in hepatic cancer cells

Hepatocellular carcinoma (HCC) is one of major health concerns worldwide and one of leading causes of cancer death after lung and gastric cancers. Simvastatin is a cholesterol-lowering drug which inhibits 3-hydroxy-3-methylglutarylcoenzyme CoA (HMG-CoA) reductase. Simvastatin exhibits numerous pleiotropic effects including anti-cancer activity. Yet, the anticancer effects in HCC remain poorly characterized. Therefore, in this study, we investigated the effects of simvastatin on tumor cell growth, apoptosis and cell cycle. HepG2 and Huh7 cell lines were treated with simvastatin (32 and 64 μM) for different time periods. Tumor cell growth was assessed using MTT assay. Apoptosis and cell cycle analysis were also evaluated. Analysis of cell cycle proteins involved in simvastatin-induced manipulation was performed by Western blot and quantitative RT-PCR analyses. Simvastatin induced a reduction of tumor cell growth. In both cell lines, simvastatin induced apoptosis and impaired cell cycle progression as depicted by the greater rates of G0/G1-phase cells than the rates of S-phase cells. Protein expression levels of cell cycle regulating proteins CDK1, CDK2, CDK4, cyclin D1, cyclin E, p19 and p27 were markedly altered by simvastatin. Moreover, CDC2, CCND1 and CDCN2D mRNA expressions were also altered by drug treatment. Collectively, these results suggest that simvastatin induces apoptosis in tumor cells and its anti-proliferative activity was accompanied by inhibition of cyclin-dependent kinases and cyclins, whereas CDK inhibitors p19 and p27 were enhanced. These results may provide novel insights into simvastatin tumor-suppressive action.     

HCV core/gC1qR interaction arrests T cell cycle progression through stabilization of the cell cycle inhibitor p27Kip1

Hepatitis C virus (HCV) is efficient in the establishment of persistent infection. We have previously shown that HCV core protein inhibits T cell proliferation through its interaction with the complement receptor, gC1qR. Here we show that HCV core-induced inhibition of T cell proliferation involves a G(0)/G(1) cell cycle arrest, which is reversible upon addition of anti-gC1qR antibody. Correspondingly, the expression of cyclin-dependent kinases (Cdk) 2/4 and cyclin E/D, as well as subsequent phosphorylation of retinoblastoma (pRb), is reduced in core-treated T cells in response to mitogenic stimulation. Remarkably, degradation of p27(Kip1), a negative regulator of both Cdk4/cyclin D and Cdk2/cyclin E complexes, is significantly diminished in T cells treated with HCV core upon mitogenic stimulation. These data indicate that the stability of p27(Kip1) by HCV core is associated with blocking activated T cells for the G(1) to S phase transition and inhibiting T cell proliferation.     

Herpes Simplex Virus Gene Products Required for Viral Inhibition of Expression of G1-Phase Functions

HSV infection blocks G1 events in the cell cycle and arrests host cell growth in the G1 phase. To further define the mechanism of the effect and determine the viral gene product(s) responsible, we examined various mutant viruses for their effects on cell cycle regulatory proteins (pRb, cyclin D1, and cdk4) and on cell cycle progression into S phase. Unlike the wild-type virus, the ICP27 mutant virus was defective for blocking the phosphorylation of pRb proteins, and the normal pRb pattern was restored in cells infected with a rescued virus. The virion host shutoff (vhs) function, DNA replication, and late gene functions were not required for the virus-induced effects on pRb protein. BrdU incorporation in synchronized HSV-infected cells showed that ICP27 was required for blocking the cell cycle in the G1 phase. Furthermore, ICP27, ICP4, ICP0, and vhs were required for blocking the induction of the G1 cell cycle regulators cyclin D1 and cdk4 in HSV-infected cells. Both ICP27 and the vhs function contributed to the reduction of cyclin D1 mRNA levels in HSV-infected cells. These results provide evidence that HSV-1 ICP27 protein is essential for viral inhibition of G1-phase functions and that certain other HSV proteins are required for some of the viral effects on the cell cycle. Finally, these results show that HSV-1 ICP27 and vhs act jointly to reduce host mRNA levels in infected cells.     

Bone morphogenetic protein-2 induces hypophosphorylation of Rb protein and repression of E2F in androgen-treated LNCaP human prostate cancer cells

Bone morphogenetic protein-2 (BMP-2), a multifunctional member of the transforming growth factor (TGF)-beta, superfamily, has powerful osteoinductive effects and causes cell cycle arrest in a variety of transformed cell lines. We have observed BMP-2-induced inhibition of cell proliferation in an androgen-dependent human prostate cancer cell line (LNCaP). To investigate the mechanism of inhibition of androgen-dependent growth by BMP-2, we examined the effect of dihydrotestosterone (DHT) and/or BMP-2 on cell cycle-related proteins in LNCaP cells. BMP-2 decreased the phosphorylation of retinoblastoma (Rb) protein induced by treatment with DHT. DHT-induced expression of cyclin A and cyclin-dependent kinase 2 (CDK2) protein was also inhibited by co-treatment with BMP-2. Furthermore, BMP-2 induced expression of p21(WAF1/CIP1), a CDK inhibitor. These results indicate that changes in expression of these proteins lead to modulation of the phosphorylation state of Rb. DHT-induced E2F-1 protein and mRNA expressions was also inhibited by BMP-2, suggesting that BMP-2 inhibits DHT-induced growth of LNCaP cells through a decrease in E2F protein expression and suppression of E2F activity by hypophosphorylation of Rb.     

Tunicamycin enhances the anticellular activity of interferon by inhibiting G1/S phase progression in 3T3 cells.

We have shown earlier that the cell growth inhibitory activity of interferon (IFN) is significantly enhanced by tunicamycin (TM) (Maheshwari et al., Science 219, 1339-1341, 1983). In this report, we investigated various regulatory points of synergistic action between TM and IFN-alpha/beta that inhibit cell growth in NIH 3T3 cells. The MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) viability assays showed a dose-dependent increase in percentage inhibition of the cells when treated with either TM or IFN. When doses of TM and IFN that had no significant inhibition on cell viability were used in combination, there was a pronounced suppression of DNA synthesis (tritiated thymidine incorporation). Flow cytometry studies revealed that individual treatments with either IFN or TM that did not alter the cell cycle profile, when combined, resulted in an impaired cell cycle by inhibiting G1/S progression. The blockage of G1/S transition was associated with reduction of cyclin-dependent kinase (CDK4) activity. The mRNA (analyzed by ribonuclease protection assay) and protein levels (assayed by Western blotting) of cyclins D1, D3, and CDK4 were downregulated by combined treatment with IFN and TM. An increase in the expression of p27/kipl, an inhibitor of CDK4, was observed in cells that were treated with both IFN and TM. These studies suggest that insufficient formation of the active cyclin/CDK complex could possibly be deferring the cells from normal cycling and may be responsible for the ability of TM to enhance cell growth inhibition induced by IFN.     

HCV NS2 protein inhibits cell proliferation and induces cell cycle arrest in the S-phase in mammalian cells through down-regulation of cyclin A expression

Chronic hepatitis C virus (HCV) infection often leads to liver cancer. NS2 protein is a HCV hydrophobic transmembrane protein that associates with several cellular proteins in mammalian cells. In this report, we investigated the functions of NS2 protein by examining its effects on cell growth and cell cycle progression. Stable NS2-expressing HeLa and Vero cell lines were established by transfection of the cells with pcDNA3.1(-)-NS2 followed by selection of the transfected cells in the presence of G418. We found that the proliferation rates of both NS2-expressing cell lines were inhibited by 40-50% compared with the control cells that were transfected with pcDNA3.1(-) control vector. Cell cycle analysis of these NS2-expressing cell lines shows that the proportion of cells in the S-phase increased significantly compared to that of control cells that do not express NS2 protein, suggesting NS2 protein induces cell cycle arrest in the S-phase. Further studies showed that the induction of cell cycle arrest in the S-phase by NS2 protein is associated with the decrease of cyclin A level. In contrast, the expression of NS2 protein does not affect the levels of cyclin-dependent kinase CDK2, CDK4, cyclin D1, or cyclin E. Our results suggest that HCV NS2 protein inhibits cell growth and induces the cell cycle arrest in the S-phase through down-regulation of cyclin A expression, which may be beneficial to HCV viral replication. Our findings not only provide information in the understanding mechanism of HCV infection, but also provide guidance for the future development of potential therapeutics for the prevention and treatment of the viral infection.     

低分子量周期蛋白E及其与恶性肿瘤的关系

 周期蛋白 E（cycli n E）与周期蛋白依赖性激酶2（cyclin-dependent kinase2, CDK2）形成活性复合物,促进细胞周期G1/S转换。正常组织和细胞主要表达全长型周期蛋白E（full-length cyclin E,FL-E）,而多种肿瘤可大量表达低分子量周期蛋白E（low molecular weight cyclin E,LMW-E）。LMW-E的稳定性增加,LMW-E/CDk2激酶活性提高,引起基因组不稳定、细胞周期失调,并使乳腺癌产生内分泌治疗耐受等。LMW-E是评判乳腺癌等恶性肿瘤临床预后的重要生物标记物,也是肿瘤生物治疗极具潜力的分子药靶。     

Catalytic activities of G1 cyclin-dependent kinases and phosphorylation of retinoblastoma protein in mobilized peripheral blood CD34+ hematopoietic progenitor cells

Depending on the source of cells, the cell cycle status of hematopoietic stem and progenitor cells capable of repopulating the marrow of transplant recipients is controversial. In this study, using biochemical methods, the cell cycle status of mobilized CD34+ cells was analyzed. It was demonstrated in CD34+ cell extracts that there was high catalytic activity of G(1) cyclin-dependent kinases 4 and 6 (CDK4 and CDK6) but low activity of CDK2. This was in contrast to the resting reference cells that showed only minimal or no activity of these CDKs. Since at the G0-->G1-->S transition CDK4/6 and CDK2 sequentially phosphorylate the retinoblastoma protein (pRB), its phosphorylation status was analyzed. Previously, we showed that p110RB was unphosphorylated at serine (Ser)-608 in CD34+ cells, consistent with the ability to suppress cell growth. Here, it was established that this form of pRB was phosphorylated at Ser-780, Ser-795, and Ser-807/811 in CD34+ but not in resting reference cells. This result was therefore consistent with the presence of high CDK4/6 activities in CD34+ cells. Conversely, CDK2 activity was low and the pRB residues Ser-612 and threonine (Thr)-821, which are exclusively phosphorylated by CDK2 in conjunction with either cyclin E or A, were unphosphorylated in >90% of CD34+ cells. We therefore show for the first time the exact position of mobilized CD34+ cells within the cell cycle; that is, they do not reside in G0 but in early G1 phase and did not cross the restriction point into late G1 phase.     

人乳头状瘤病毒16型E6E7肿瘤性转化人永生化支气管上皮细胞

目的 探讨人乳头状瘤病毒（ＨＰＶ）１６型Ｅ６Ｅ７片段对人永生化支气管上皮细胞系ＴＲ细胞的作用。方法 将Ｅ６Ｅ７片段构建入逆转录病毒载体,导入ＴＲ细胞,观察生长特性和致瘤性的改变;并用免疫沉淀（ＩＰ）－Ｗｅｓｔｅｒｎ ｂｌｏｔ检测ｐ２７蛋白功能及ＦＡＫ、桩蛋白数量及磷酸化状况,结果 嘌呤霉素抗药性克隆ＴＲ／Ｅ６Ｅ７有Ｅ６Ｅ７的存在和稳定表达;ＴＲ／Ｅ６Ｅ７细胞系细胞生长加快,软琼脂集落形成能力增强,     

Aberrant expression of the Rb pathway proteins in soft tissue sarcomas.

Cell cycle regulation depends on a fine balance between cyclins, cyclin-dependent kinases (CDKs), and cyclin-dependent kinase inhibitors (CKIs) that block the cycle progression. Alterations of the cell cycle regulators are a common feature of many malignant tumors, and some have been shown to have prognostic significance. In this study, 152 cases of different types of soft tissue sarcomas were evaluated for alterations of cell cycle regulator proteins that control the cell cycle progression from G1 to S phase and govern the Rb pathway. Immunohistochemical stains for proteins Rb, E2F1, cyclin D1, CDK4, CDK6, p16, and p27 were carried out on tissue microarrays. The relationship between the expression of these proteins and the histologic grade of the sarcomas was assessed. Altered expression for Rb and p16 proteins was identified in 67.8% and 65.1% of the cases, respectively. Overexpression of E2F1, cyclin D1, CDK4, and CDK6 was detected in 50.7%, 24.3%, 92.1%, and 10.5%, respectively. Overexpression of E2F1 was associated with altered expression of Rb protein. Overexpression of cyclin D1, CDK4, and CDK6 showed an association with normal Rb expression. CDK6 expression revealed a positive correlation with the histologic grade of the sarcoma, and p27 expression was inversely correlated with sarcoma grade. These results suggest that alterations of the Rb pathway proteins are common in soft tissue sarcomas and may participate in their tumorigenesis. CDK6 and p27 showed correlation with the histologic grade of the sarcomas, suggesting that these proteins could be used as prognostic markers.     

Inhibition of CDK activity and PCNA-dependent DNA replication by p21 is blocked by interaction with the HPV-16 E7 oncoprotein

p21 inhibits cyclin-dependent kinase (CDK) activity and proliferating cell nuclear antigen (PCNA)-dependent DNA replication by binding to CDK/cyclin complexes and to PCNA through distinct domains. The human papillomavirus (HPV)-16 E7 oncoprotein (16E7) abrogated a DNA damage-induced cell cycle arrest in vivo, despite high levels of p21. Using cell lysates and purified proteins we show that 16E7 prevented p21 both from inhibiting CDK2/cyclin E activity and PCNA-dependent DNA replication, whereas the nononcogenic HPV-6 E7 had reduced effects. Inactivation of both inhibitory functions of p21 was attained through binding between 16E7 and sequences in the carboxy-terminal end of p21 that overlap with the PCNA-binding site and the second p21 cyclin-binding motif. These data imply that the carboxyl terminus of p21 simultaneously modulates both CDK activity and PCNA-dependent DNA replication and that a single protein, 16E7, can override this modulation to disrupt normal cell cycle control.     

丙型肝炎病毒核心蛋白对HepG2细胞生长周期的影响

目的: 构建丙型肝炎病毒核心蛋白(HCV-core-1b)真核重组质粒,获得稳定表达HCV-core-1b的HepG2细胞株,观察HCV-core-1b对HepG2细胞株生长周期及cyclin D1 和pRb/p130表达的影响,探讨丙型肝炎病毒慢性感染的可能机制。方法: 将HCV-core-1b亚克隆入pBabe-Flag-puro载体,获得重组质粒pBabe-Flag-HCV-core-1b;将重组质粒转染病毒包装细胞Pheonix 293T,筛选获得分泌HCV-core-1b的病毒包装细胞株。利用包装细胞产生的病毒上清感染靶细胞,筛选后获得稳定表达HCV-core-1b的HepG2细胞株,流式细胞仪检测靶细胞生长周期的变化,Western blotting检测cyclin D1 和pRb/p130蛋白的表达。结果: 基因测序确认HCV-core-1b亚型基因编码区完整无移位,与标签蛋白Flag形成融合蛋白。HepG2-HCV-core细胞株成功表达Flag-HCV-core-1b蛋白,并导致细胞cyclin D1 和 pRb/p130的水平下调,显著改变了HepG2细胞生长周期,使细胞阻滞在G₀/G₁期。结论: 成功构建了pBabe-Flag-HCV-core-1b真核表达质粒,获得稳定表达Flag-HCV-core-1b融合蛋白的HepG2细胞。由于HCV-core-1b蛋白的表达,下调了HepG2细胞 cyclin D1和pRb/p130的表达,显著抑制HepG2细胞生长周期。     

Epstein-barr virus can infect B-chronic lymphocytic leukemia cells but it does not orchestrate the cell cycle regulatory proteins

OBJECTIVES: To understand the mechanism for the refractoriness of B-chronic lymphocytic leukemia (B-CLL) cells for Epstein-Barr virus (EBV)-induced immortalization. STUDY DESIGN/METHODS: Cultures were initiated with EBV-infected tonsillar B and B-CLL cells. Expression of EBNA-2 and some of the key players regulating G1/S phase transition such as c-myc expression, phosphorylation of Rb protein, expression of G1 cyclins, and the cyclin-dependent kinase inhibitor p27 were followed. RESULTS: In line with earlier studies, EBV infection induced c-myc expression, pRb phosphorylation, D2 and D3 expression, and disappearance of p27 in normal B cells. In contrast, EBV-infected B-CLL cells remained resting and they did not express c-myc; cyclin D2, ppRb and cyclin D3 were seen only in occasional cells. Importantly, p27 expression was maintained. CONCLUSIONS: In B-CLL cells, the expression of the EBV-encoded nuclear proteins EBNAs is not followed by entrance to the cell cycle. Thus, the difference in the interaction of EBV-normal B cells and EBV-B-CLL cells is already apparent early after infection.     

三丁基过氧化氢诱导WI-38细胞衰老的细胞周期调控机制

目的：探讨三丁基过氧化氢(t-BHP)诱导WI-38细胞衰老的细胞周期调控机制。方法： 从30代开始，隔代用t-BHP作用WI-38细胞4次，每次1 h，诱导细胞衰老，从细胞超微结构、细胞周期分析和β-半乳糖苷酶细胞化学染色观察衰老细胞的特点，同时用Western blotting方法检测细胞周期调控蛋白CDK4、CDK2、cyclin D1、cyclin E 、p21和p16的表达程度。 结果：100 μmol/L t-BHP作用4次后，WI-38细胞出现衰老的特征，细胞增殖分裂停止，细胞体积增大、胞体变平、次级溶酶体增多，同时G1期细胞比例增加，β-半乳糖苷酶染色阳性细胞数增加，提示t-BHP能有效地诱导细胞衰老。t-BHP作用后CDK4、CDK2、cyclin E 表达下降，cyclin D1、p21和p16表达增加。 结论： t-BHP有诱导细胞衰老的作用，其机制可能与通过调节细胞周期调控分子的表达有关。     

沉默ClC-3氯通道基因对HeLa细胞周期分布的影响

目的:探讨沉默HeLa细胞的ClC-3氯通道基因后细胞周期分布的变化及其作用机制。方法:依照siRNA设计原则构建沉默ClC-3基因的ClC-3 siRNA并转染HeLa细胞;实验分为空白对照组(control组)、转染试剂对照组(Lipo组)、阴性对照组(negative siRNA组)和ClC-3 siRNA组。采用real-time PCR检测ClC-3 siRNA的沉默效率;流式细胞术检测细胞周期分布情况;Western blot检测ClC-3蛋白及相关细胞周期蛋白(cyclin)D1、细胞周期蛋白依赖激酶(cyclin-dependent kinase,CDK)4、CDK6、P21和P27等表达。结果:CIC-3 siRNA成功沉默HeLa细胞的ClC-3基因。和其它组相比,ClC-3 siRNA组的细胞周期被阻抑在G_0/G_1期。CIC-3 siRNA组的cyclin D1、CDK4和CDK6蛋白表达水平明显下降,P21和P27蛋白表达水平明显上升。结论:沉默HeLa细胞ClC-3氯通道基因可影响cyclin D1、CDK4、CDK6、P21和27蛋白的表达水平胆抑HeLa细胞周期停滞在G_0/G_1期。