Effect of transient cyclic AMP elevation on DNA synthesis in rat hepatocytes at G1 phase

: Maintaining high levels of intracellular cyclic AMP (cAMP) is known to inhibit the growth of various proliferating cells including hepatocytes. We show here that transient (30 min) elevations of cAMP induced by addition of 8-bromo-cAMP (1 mmol/L) to rat hepatocytes in primary culture at three time points (12h, 16h and 20h) after seeding stimulated DNA synthesis. Sustained levels of cAMP stimulated DNA synthesis to a lesser degree at a lower concentration (1 μmol/L), but inhibited it at concentrations higher than 100μmol/L. We also determined cyclin-dependent kinase 2 (cdk2) activity in the hepatocytes during this incubation period. The transient addition of 8-bromo-cAMP at the late G1 phase increased cdk2 activity. This suggests that transient cAMP elevation in hepatocytes at the late G1 phase has a growth stimulation effect. Up-regulation of cdk2 activity may have a role in this process.     

Recombinant human prothrombin kringle-2 induces bovine capillary endothelial cell cycle arrest at G0–G1 phase through inhibition of cyclin D1/CDK4 complex: Modulation of reactive oxygen species generation and up-regulation of cyclin-dependent kinase inhibitors

Prothrombin is a plasma glycoprotein involved in blood coagulation and, as we have previously reported, prothrombin kringles inhibit BCE (bovine capillary endothelial) cell proliferation. To reveal the mechanism, we investigated the influence of rk-2 (recombinant human prothrombin kringle-2) on the BCE cell cycle progression and ROS (reactive oxygen species) generation using FACS (fluorescence-activated cell sorter) analysis. Cell cycle analysis showed a decrease of G₁ phase cells in cells treated with bFGF (basic fibroblast growth factor) and an increase in cells treated with rk-2, as compared with the control cells. But, the portion of the S phase was reversed. In Western blot analysis, bFGF induced cytoplasmic translocation of p21^Waf1/Cip1 and p27^Kip1 and phosphorylation of p27^Kip1 but rk-2 treatment inhibited translocation of p21^Waf1/Cip1 and p27^Kip1 from nucleus to cytoplasm and phosphorylation of p27^Kip1. Also, rk-2 induced up-regulation of p53 and nuclear p21^Waf1/Cip1 and inhibited the cyclin D1/CDK4 (cyclin-dependent kinase 4) complex. The ROS level of rk-2-treated BCE cells was increased 2-fold when compared with the control, but treatment with NAC (N-Acetyl-L-cysteine), an anti-oxidant, decreased ROS generation about 55% as compared with the rk-2 treatment. NAC treatment also restored cell cycle progression inhibited by rk-2 and down-regulated p53 and nuclear p21^Waf1/Cip1 expression induced by rk-2.These data suggest that rk-2 induces the BCE cell cycle arrest at G₀–G₁ phase through inhibition of the cyclin D1/CDK4 complex caused by increase of ROS generation and nuclear cyclin-dependent kinase inhibitors.     

Identification of High-Risk Human Papillomavirus DNA,p16, and E6/E7 Oncoproteins in Laryngeal and Hypopharyngeal Squamous Cell Carcinomas

Human papillomavirus (HPV) was proven to play a significant role in cancer development in the oropharynx. However, its role in the development of laryngeal (LSCC) and hypopharyngeal squamous cell carcinoma (HPSCC) remains to be clarified. High-risk HPV (HR-HPV) viral proteins E6 and E7 are considered to be pertinent to HPV-related carcinogenesis. Hence, our aim was to estimate LSCC and HPSCC for HR-HPV DNA, p16, and E6/E7 oncoprotein status by using molecular virology and immunohistochemistry methods. The prevalence of HPV16 infection was 22/41 (53.7%) and 20/31 (64.5%) for LSCC and HPSCC, accordingly. The majority of HPV16+ tumor samples were stage III or IV. In most samples, the presence of either HPV16 E6 or HPV16 E7 viral protein in dysplastic or tumor cells was confirmed using immunohistochemistry. Our results suggest a high prevalence of HPV16 as a primary HR-HPV type in LSCC and HPSCC. The lack of HPV E6/E7 oncoproteins in some tumor samples may suggest either the absence of viral integration or the presence of other mechanisms of tumorigenesis. The utilization of p16 IHC as a surrogate marker of HR-HPV infection is impractical in LSCC and HPSCC.     

新生小鼠胰岛增殖及口袋蛋白家族、E2F转录因子表达变化

目的 观察新生小鼠胰岛细胞、细胞周期和视网膜母细胞瘤蛋白（Rb）、视网膜母细胞瘤样蛋白1（p107）、视网膜母细胞瘤样蛋白2（p130）及E2F转录因子的变化特点,探讨其与胰岛细胞增殖的关系.方法 采用免疫组化法检测出生第1、3和8周小鼠胰岛β/α细胞量、单个细胞面积和细胞数量的变化.采用胶原酶消化法分离纯化小鼠胰岛,以流式细胞术检测细胞周期,实时定量PCR及蛋白印迹技术检测Rb、p107、p130和E2F转录因子mRNA和蛋白表达.结果 （1）小鼠3周和8周胰岛β/α细胞量显著高于1周（P＜0.05）;单个β/α细胞面积在3周时无明显差异,8周时扩大1.7倍（P＜0.01）;而β/α细胞数量在3周增加约3.5/3.7倍（P＜0.01）,8周与3周无明显差异.（2）1周和3周时G0/G1期细胞比8周明显减少（81.3±1.2、82.8±3.3比92.6±1.3,F=6.5,P＜0.05）,而G2/M期显著增多（13.4±1.4、12.2±1.8比5.5±0.5,F=6.3,P＜0.05）,细胞增殖指数明显增加（P＜0.05）.（3）8周时Rb mRNA表达明显低于1周和3周（P＜0.01）;3周和8周时p107 mRNA显著低于1周（P＜0.05）,而p130 mRNA显著高于1周（P＜0.05）,E2F-1、E2F-2 mRNA均显著低于1周（均P＜0.01）;8周时E2F-3 mRNA显著低于1周（P＜0.05）;而E2F-4、E2F-5 mRNA在3周和8周时表达增加.（4）8周时Rb和p107蛋白表达均比1周明显降低,p130表达显著增高,3周和8周时E2F1表达显著降低（均P＜0.05）.结论 新生小鼠胰岛细胞量扩增明显,以细胞数量增加为主,细胞增殖显著.Rb、p107、p130及其相关E2F转录因子的表达存在动态变化,可能参与细胞增殖的调控.     

PI3K抑制剂LY294002对人晶状体上皮细胞E2F-1、cyclinE蛋白表达的影响

目的探讨PI3K抑制剂LY294002对人晶状体上皮细胞（LECs）增殖及E2F-1、细胞周期素E（cyclinE）蛋白表达的影响。方法人LECs常规培养,分为实验组和对照组,实验组加入PI3K抑制剂LY294002（25μmol/L）,对照组加入同体积DMSO,两组分别培养36 h后,MTT比色法测定细胞增殖状况,Western blot法检测细胞中E2F-1及cyclinE蛋白表达。结果实验组细胞增殖受到明显抑制,与对照组比较差异有统计学意义（P〈0.05）,人LECs中E2F-1及cyclinE蛋白表达降低（P〈0.05）。结论 PI3K抑制剂LY294002可抑制人LECs的增殖,可能与下调E2F-1及cyclinE蛋白的表达水平相关。     

Mechanisms of Cancer Cell Killing by the Adenovirus E4orf4 Protein

During adenovirus (Ad) replication the Ad E4orf4 protein regulates progression from the early to the late phase of infection. However, when E4orf4 is expressed alone outside the context of the virus it induces a non-canonical mode of programmed cell death, which feeds into known cell death pathways such as apoptosis or necrosis, depending on the cell line tested. E4orf4-induced cell death has many interesting and unique features including a higher susceptibility of cancer cells to E4orf4-induced cell killing compared with normal cells, caspase-independence, a high degree of evolutionary conservation of the signaling pathways, a link to perturbations of the cell cycle, and involvement of two distinct cell death programs, in the nucleus and in the cytoplasm. Several E4orf4-interacting proteins including its major partners, protein phosphatase 2A (PP2A) and Src family kinases, contribute to induction of cell death. The various features of E4orf4-induced cell killing as well as studies to decipher the underlying mechanisms are described here. Many explanations for the cancer specificity of E4orf4-induced cell death have been proposed, but a full understanding of the reasons for the different susceptibility of cancer and normal cells to killing by E4orf4 will require a more detailed analysis of the complex E4orf4 signaling network. An improved understanding of the mechanisms involved in this unique mode of programmed cell death may aid in design of novel E4orf4-based cancer therapeutics.     

Early pregnancy induced expression of prostaglandin E2 receptors EP2 and EP4 in the ovine endometrium and regulated by interferon tau through multiple cell signaling pathways

Prostaglandin E2 (PGE₂) plays pleiotropic roles at fetal-maternal interface during establishment of pregnancy. The objectives of the study were to: (i) determine regulation of PGE2 receptors EP1, EP2, EP3, and EP4 in the endometrium during the estrous cycle and early pregnancy; and (ii) understand endometrial epithelial and stromal cell-specific hormonal regulation of EP2 and EP4 in sheep. Results indicate that: (i) early pregnancy induces expression of EP2 and EP4 but not EP1 and EP3 proteins in the endometrium on days 12-16 compared to that of estrous cycle; (ii) intrauterine infusion of interferon tau (IFNT) increases expression of EP2 and EP4 proteins in endometrium; and (iii) IFNT activates distinct epithelial and stromal cell-specific JAK, EGFR, ERK1/2, AKT, or JNK signaling module to regulate expression of EP2 and EP4 proteins in the ovine endometrium. Our results indicate a role for EP2 and EP4-mediated PGE₂ signaling in endometrial functions and establishment of pregnancy in ruminants.     

Revisiting the evolution of gonadotropin-releasing hormones and their receptors in vertebrates: secrets hidden in genomes

Gonadotropin-releasing hormone (GnRH) and its G protein-coupled receptor, GnRHR, play a pivotal role in the control of reproduction in vertebrates. To date, many GnRH and GnRHR genes have been identified in a large variety of vertebrate species using conventional biochemical and molecular biological tools in combination with bioinformatic tools. Phylogenetic approaches, primarily based on amino acid sequence identity, make it possible to classify these multiple GnRHs and GnRHRs into several lineages. Four vertebrate GnRH lineages GnRH1, GnRH2, GnRH3, and GnRH4 (for lamprey) are well established. Four vertebrate GnRHR lineages have also been proposed—three for nonmammalian GnRHRs and mammalian GnRHR2 as well as one for mammalian GnRHR1. However, these phylogenetic analyses cannot fully explain the evolutionary origins of each lineage and the relationships among the lineages. Rapid and vast accumulation of genome sequence information for many vertebrate species, together with advances in bioinformatic tools, has allowed large-scale genome comparison to explore the origin and relationship of gene families of interest. The present review discusses the evolutionary mechanism of vertebrate GnRHs and GnRHRs based on extensive genome comparison. In this article, we focus only on vertebrate genomes because of the difficulty in comparing invertebrate and vertebrate genomes due to their marked divergence.