CREB1 regulates glucose transport of glioma cell line U87 by targeting GLUT1

Chemical modification of chitosan is a promising method for the improvement of biological activity. In this study, chitosan-caffeic acid (CCA) was prepared and its in vitro hepatoprotective ability against hydrogen peroxide-induced hepatic damage in liver cells was evaluated. Treatment with CCA (50–400 µg/mL) did not show cytotoxicity and also significantly (p < 0.05) recovered cell viability against 650 µM hydrogen peroxide-induced hepatotoxicity. CCA treatment attenuated reactive oxygen species generation and lipid peroxidation in addition to increasing cellular glutathione level in cultured hepatocytes. To validate the underlying mechanism, antioxidant and phase II detoxifying enzyme expressions, which are mediated by NF-E2-related factor 2 (Nrf2) activation, were analyzed and CCA treatment was found to increase the expression of superoxide dismutase-1 (SOD-1), glutathione reductase (GR), heme oxygenase-1 (HO-1), and NAD(P)H:quinine oxidoreductase 1 (NQO1). CCA treatment resulted in increased Nrf2 nuclear translocation. The phosphorylation of extracellular regulated kinase (ERK), c-Jun NH₂-terminal kinase (JNK), and p38 mitogen-activated protein kinase (MAPK) by CCA treatment contributed to Nrf2 activation. Pharmacological blockade of ERK, JNK, and p38 MAPK revealed that SP600125 (JNK inhibitor) and PD98059 (ERK inhibitor) treatment reduced Nrf2 translocation into the nucleus while SB203580 (p38 inhibitor) exhibited weak inhibition. Collectively, CCA protects liver cells against hydrogen peroxide-induced injury and this ability is attributed to the induction of antioxidants and phase II detoxifying enzymes that are mediated by Nrf2 translocation via JNK/ERK signaling.     

Phosphatidylinositol 3-kinase activity regulates alpha -thrombin-stimulated G1 progression by its effect on cyclin D1 expression and cyclin-dependent kinase 4 activity

In this study, we present evidence that PI 3-kinase is required for alpha-thrombin-stimulated DNA synthesis in Chinese hamster embryonic fibroblasts (IIC9 cells). Previous results from our laboratory demonstrate that the mitogen-activated protein kinase (extracellular signal-regulated kinase (ERK)) pathway controls transit through G(1) phase of the cell cycle by regulating the induction of cyclin D1 mRNA levels and cyclin dependent kinase 4 (CDK4)-cyclin D1 activity. In IIC9 cells, PI 3-kinase activation also is an important controller of the expression of cyclin D1 protein and CDK4-cyclin D1 activity. Pretreatment of IIC9 cells with the selective PI 3-kinase inhibitor, LY294002 blocks the alpha-thrombin-stimulated increase in cyclin D1 protein and CDK4 activity. However, LY294002 does not affect alpha-thrombin-induced cyclin D1 steady state message levels, indicating that PI 3-kinase acts independent of the ERK pathway. Interestingly, expression of a dominant-negative Ras significantly decreased both alpha-thrombin-stimulated ERK and PI 3-kinase activities. These data clearly demonstrate that the alpha-thrombin-induced Ras activation coordinately regulates ERK and PI 3-kinase activities, both of which are required for expression of cyclin D1 protein and progression through G(1).     

Interplay between MEK-ERK signaling, cyclin D1, and cyclin-dependent kinase 5 regulates cell cycle reentry and apoptosis of neurons

In response to neurotoxic signals, postmitotic neurons make attempts to reenter the cell cycle, which results in their death. Although several cell cycle proteins have been implicated in cell cycle-related neuronal apoptosis (CRNA), the molecular mechanisms that underlie this important event are poorly understood. Here, we demonstrate that neurotoxic agents such as β-amyloid peptide cause aberrant activation of mitogen-activated kinase kinase (MEK)-extracellular signal-regulated kinase (ERK) signaling, which promotes the entry of neurons into the cell cycle, resulting in their apoptosis. The MEK-ERK pathway regulates CRNA by elevating the levels of cyclin D1. The increase in cyclin D1 attenuates the activation of cyclin-dependent kinase 5 (cdk5) by its neuronal activator p35. The inhibition of p35-cdk5 activity results in enhanced MEK-ERK signaling, leading to CRNA. These studies highlight how neurotoxic signals reprogram and alter the neuronal signaling machinery to promote their entry into the cell cycle, which eventually leads to neuronal cell death.     

PI3K通路抑制剂LY294002对人脑胶质瘤细胞株U87细胞衰老和凋亡的影响

目的:PI3K/Akt信号通路是与胶质瘤发生发展密切相关的核心通路之一,LY294002是该通路的特异性抑制剂。本研究通过探讨PI3K通路抑制剂LY294002对U87胶质瘤细胞系细胞衰老及凋亡的影响,从而为胶质瘤患者治疗的新策略奠定理论基础。方法:将体外培养的人脑胶质瘤U87细胞株分为DMSO处理的对照组和LY294002(100μM)处理的实验组,采用β-半乳糖苷酶染色和流式细胞术的方法,分别检测并比较两组肿瘤细胞衰老和凋亡的情况。结果:LY294002处理组U87胶质瘤细胞的衰老指数(32.20±4.46%)显著高于DMSO对照组(3.40±1.61%,t=6.254,P0.001)。另外,与DMSO对照组相比,凋亡蛋白caspase-3mRNA的表达在LY294002处理组胶质瘤细胞中显著上调(t=8.923,P0.05)。LY294002处理组肿瘤细胞的凋亡指数(80.10±4.832%)明显高于DMSO对照组(4.260±1.073%,t=8.923,P0.05)。结论:LY294002既能够诱导肿瘤细胞衰老,又能够诱导肿瘤细胞凋亡,然而其诱导胶质瘤细胞凋亡的能力占据主导地位,为其发挥抗胶质瘤效应的主要途径。另外,在LY294002的持续作用下,部分衰老的肿瘤细胞或许会发生凋亡。这些结论为为临床增强胶质瘤患者的联合化疗奠定了理论基础。     

Differential expression of cyclin G2, cyclin-dependent kinase inhibitor 2C and peripheral myelin protein 22 genes during adipogenesis

Increase of fat cells (FCs) in adipose tissue is attributed to proliferation of preadipocytes or immature adipocytes in the early stage, as well as adipogenic differentiation in the later stage of adipose development. Although both events are involved in the FC increase, they are contrary to each other, because the former requires cell cycle activity, whereas the latter requires cell cycle withdrawal. Therefore, appropriate regulation of cell cycle inhibition is critical to adipogenesis. In order to explore the important cell cycle inhibitors and study their expression in adipogenesis, we adopted a strategy combining the Gene Expression Omnibus (GEO) database available on the NCBI website and the results of quantitative real-time PCR (qPCR) data in porcine adipose tissue. Three cell cycle inhibitors – cyclin G2 (CCNG2), cyclin-dependent kinase inhibitor 2C (CDKN2C) and peripheral myelin protein (PMP22) – were selected for study because they are relatively highly expressed in adipose tissue compared with muscle, heart, lung, liver and kidney in humans and mice based on two GEO DataSets (GDS596 and GDS3142). In the latter analysis, they were found to be more highly expressed in differentiating/ed preadipocytes than in undifferentiated preadipocytes in human and mice as shown respectively by GDS2366 and GDS2743. In addition, GDS2659 also suggested increasing expression of the three cell cycle inhibitors during differentiation of 3T3-L1 cells. Further study with qPCR in Landrace pigs did not confirm the high expression of these genes in adipose tissue compared with other tissues in market-age pigs, but confirmed higher expression of these genes in FCs than in the stromal vascular fraction, as well as increasing expression of these genes during in vitro adipogenic differentiation and in vivo development of adipose tissue. Moreover, the relatively high expression of CCNG2 in adipose tissue of market-age pigs and increasing expression during development of adipose tissue was also confirmed at the protein level by western blot analysis. Based on the analysis of the GEO DataSets and results of qPCR and Western blotting we conclude that all three cell cycle inhibitors may inhibit adipocyte proliferation, but promote adipocyte differentiation and hold a differentiated state by inducing and maintaining cell cycle inhibition. Therefore, their expression in adipose tissue is positively correlated with age and mature FC number. By regulating the expression of these genes, we may be able to control FC number, and, thus, reduce excessive fat tissue in animals and humans.     

Inhibition of cell division by the human cytomegalovirus IE86 protein: role of the p53 pathway or cyclin-dependent kinase 1/cyclin B1

The human cytomegalovirus (HCMV) IE86 protein induces the human fibroblast cell cycle from G(0)/G(1) to G(1)/S, where cell cycle progression stops. Cells with a wild-type, mutated, or null p53 or cells with null p21 protein were transduced with replication-deficient adenoviruses expressing HCMV IE86 protein or cellular p53 or p21. Even though S-phase genes were activated in a p53 wild-type cell, IE86 protein also induced phospho-Ser(15) p53 and p21 independent of p14ARF but dependent on ATM kinase. These cells did not enter the S phase. In human p53 mutant, p53 null, or p21 null cells, IE86 protein did not up-regulate p21, cellular DNA synthesis was not inhibited, but cell division was inhibited. Cells accumulated in the G(2)/M phase, and there was increased cyclin-dependent kinase 1/cyclin B1 activity. Although the HCMV IE86 protein increases cellular E2F activity, it also blocks cell division in both p53(+/+) and p53(-/-) cells.     

Inhibitor of cyclin-dependent kinase (CDK) interacting with cyclin A1 (INCA1) regulates proliferation and is repressed by oncogenic signaling

The cell cycle is driven by the kinase activity of cyclin·cyclin-dependent kinase (CDK) complexes, which is negatively regulated by CDK inhibitor proteins. Recently, we identified INCA1 as an interaction partner and a substrate of cyclin A1 in complex with CDK2. On a functional level, we identified a novel cyclin-binding site in the INCA1 protein. INCA1 inhibited CDK2 activity and cell proliferation. The inhibitory effects depended on the cyclin-interacting domain. Mitogenic and oncogenic signals suppressed INCA1 expression, whereas it was induced by cell cycle arrest. We established a deletional mouse model that showed increased CDK2 activity in spleen with altered spleen architecture in Inca1(-/-) mice. Inca1(-/-) embryonic fibroblasts showed an increase in the fraction of S-phase cells. Furthermore, blasts from acute lymphoid leukemia and acute myeloid leukemia patients expressed significantly reduced INCA1 levels highlighting its relevance for growth control in vivo. Taken together, this study identifies a novel CDK inhibitor with reduced expression in acute myeloid and lymphoid leukemia. The molecular events that control the cell cycle occur in a sequential process to ensure a tight regulation, which is important for the survival of a cell and includes the detection and repair of genetic damage and the prevention of uncontrolled cell division.     

A dominant-negative cyclin D1 mutant prevents nuclear import of cyclin-dependent kinase 4 (CDK4) and its phosphorylation by CDK-activating kinase.

Cyclins contain two characteristic cyclin folds, each consisting of five alpha-helical bundles, which are connected to one another by a short linker peptide. The first repeat makes direct contact with cyclin-dependent kinase (CDK) subunits in assembled holoenzyme complexes, whereas the second does not contribute directly to the CDK interface. Although threonine 156 in mouse cyclin D1 is predicted to lie at the carboxyl terminus of the linker peptide that separates the two cyclin folds and is buried within the cyclin subunit, mutation of this residue to alanine has profound effects on the behavior of the derived cyclin D1-CDK4 complexes. CDK4 in complexes with mutant cyclin D1 (T156A or T156E but not T156S) is not phosphorylated by recombinant CDK-activating kinase (CAK) in vitro, fails to undergo activating T-loop phosphorylation in vivo, and remains catalytically inactive and unable to phosphorylate the retinoblastoma protein. Moreover, when it is ectopically overexpressed in mammalian cells, cyclin D1 (T156A) assembles with CDK4 in the cytoplasm but is not imported into the cell nucleus. CAK phosphorylation is not required for nuclear transport of cyclin D1-CDK4 complexes, because complexes containing wild-type cyclin D1 and a CDK4 (T172A) mutant lacking the CAK phosphorylation site are efficiently imported. In contrast, enforced overexpression of the CDK inhibitor p21Cip1 together with mutant cyclin D1 (T156A)-CDK4 complexes enhanced their nuclear localization. These results suggest that cyclin D1 (T156A or T156E) forms abortive complexes with CDK4 that prevent recognition by CAK and by other cellular factors that are required for their nuclear localization. These properties enable ectopically overexpressed cyclin D1 (T156A), or a more stable T156A/T286A double mutant that is resistant to ubiquitination, to compete with endogenous cyclin D1 in mammalian cells, thereby mobilizing CDK4 into cytoplasmic, catalytically inactive complexes and dominantly inhibiting the ability of transfected NIH 3T3 fibroblasts to enter S phase.     

Regulation of type II collagen expression by cyclin-dependent kinase 6, cyclin D1, and p21 in articular chondrocytes

This study examined whether cell cycle regulatory proteins, such as cyclin-dependent kinases (CDKs), cyclins, and CDK inhibitors, regulate type II collagen expression and mediate interlukin-1 (IL-1beta)-induced suppression of type II collagen expression in articular chondrocytes. IL-1beta inhibited type II collagen expression, but activated CDK6. Ectopic expression of CDK2 did not alter type II collagen expression. However, overexpression of CDK6 inhibited type II collagen expression, whereas inhibition of CDK6 activity blocked IL-1beta-induced suppression of type II collagen expression. IL-1beta upregulated the expression of cyclin D1, which is known to activate CDK6. In turn, overexpression of cyclin D1 suppressed type II collagen expression. In contrast to cyclin D1, IL-1beta triggered down-regulation of the CDK inhibitor, p21. Overexpression of p21 blocked IL-1beta- or CDK6-induced suppression of type II collagen expression. Our results collectively indicate that CDK6/cyclin D1/p21 complex regulates type II collagen expression in articular chondrocytes.     

Cell type-specific induction of cyclin D and cyclin-dependent kinase inhibitor p27(kip1) expression by estrogen in rat endometrium

Cyclins, cyclin-dependent kinases (CDKs) and the CDK inhibitor p27(kip1) are known to be involved in the regulation of G(1)/S phase transition by estrogen in the rodent endometrium. Little is known, however, of the cell-specific location and regulation of these proteins during this process, or the way they mediate the differential effect of estrogen in the epithelium and stroma of the endometrium. Here we studied the cell-specific regulation of D-type cyclin (D(1-3)), of cyclin A and E, of CDK(2) and p27(kip1) by 17beta-estradiol in the endometrium of ovariectomized rats. Time-course changes in these proteins in the endometrium of ovariectomized rats were examined by immunohistochemistry at 2, 4, 8, 12, 20, 28 and 32 h after estrogen stimulation. The expression of proliferation cell nuclear antigen (PCNA) was also studied as a marker of proliferating cells. As expected from previous studies, all the proteins investigated were up-regulated by estrogen, with peak times from 8 to 32 h. The induction of cyclin D(1) is predominant in the glandular epithelium, whereas cyclin D(3) increases mainly in the luminal epithelium. The up-regulation of p27(kip1) is restricted to stromal cells with a 'gradient-like' expression pattern, in which the sub-epithelial (functional) layer showed stronger staining than the basal layer. The differential regulation of cyclins and p27(kip1) in the epithelium and stroma of the endometrium appear indicative of distinct actions of estrogen in different cell types in the uterus, as D-type cyclins mediate the proliferative effect of estrogen in epithelial cells while p27(kip1) might help prevent the same effect in the stroma.     

Ringo/cyclin-dependent kinase and mitogen-activated protein kinase signaling pathways regulate the activity of the cell fate determinant Musashi to promote cell cycle re-entry in Xenopus oocytes

Cell cycle re-entry during vertebrate oocyte maturation is mediated through translational activation of select target mRNAs, culminating in the activation of mitogen-activated protein kinase and cyclin B/cyclin-dependent kinase (CDK) signaling. The temporal order of targeted mRNA translation is crucial for cell cycle progression and is determined by the timing of activation of distinct mRNA-binding proteins. We have previously shown in oocytes from Xenopus laevis that the mRNA-binding protein Musashi targets translational activation of early class mRNAs including the mRNA encoding the Mos proto-oncogene. However, the molecular mechanism by which Musashi function is activated is unknown. We report here that activation of Musashi1 is mediated by Ringo/CDK signaling, revealing a novel role for early Ringo/CDK function. Interestingly, Musashi1 activation is subsequently sustained through mitogen-activated protein kinase signaling, the downstream effector of Mos mRNA translation, thus establishing a positive feedback loop to amplify Musashi function. The identified regulatory sites are present in mammalian Musashi proteins, and our data suggest that phosphorylation may represent an evolutionarily conserved mechanism to control Musashi-dependent target mRNA translation.     

Transfection of 2,6 and 2,3-sialyltransferase genes and GlcNAc-transferase genes into human glioma cell line U-373 MG affects glycoconjugate expression and enhances cell death

Human glioma cell line U-373 MG expresses CMP-NeuAc : Galbeta1,3GlcNAc alpha2,3-sialyltransferase [EC No. 2.4.99.6] (alpha2,3ST), UDP-GlcNAc : beta-d-mannoside beta1,6-N-acetylglucosaminyltransferase V [EC 2.4.1.155] (GnT-V) and UDP-GlcNAc3: beta-d-mannoside beta1,4-N-acetylglucosaminyltransferase III [EC 2.4.1.144] (GnT-III) but not CMP-NeuAc : Galbeta1,4GlcNAc alpha2,6-sialyltransferase [EC 2.4.99.1] (alpha2,6ST) under normal culture conditions. We have previously shown that transfection of the alpha2,6ST gene into U-373 cells replaced alpha2,3-linked sialic acids with alpha2,6 sialic acids, resulting in a marked inhibition of glioma cell invasivity and a significant reduction in adhesivity. We now show that U-373 cells, which are typically highly resistant to cell death induced by chemotherapeutic agents (< 10% death in 18 h), become more sensitive to apoptosis following overexpression of these four glycoprotein glycosyltransferases. U-373 cell viability showed a three-fold decrease (from 20 to 60% cell death) following treatment with staurosporine, C2-ceramide or etoposide, when either alpha2,6ST and GnT-V genes were stably overexpressed. Even glycosyltransferases typically raised in cancer cells, such as alpha2,3ST and GnT-III, were able to decrease viability two-fold (from 20 to 40% cell death) following stable overexpression. The increased susceptibility of glycosyltransferase-transfected U-373 cells to pro-apoptotic drugs was associated with increased ceramide levels in Rafts, increased caspase-3 activity and increased DNA fragmentation. In contrast, the same glycosyltransferase overexpression protected U-373 cells against a different class of apoptotic drugs, namely the phosphatidylinositol 3-kinase inhibitor LY294002. Thus altered surface protein glycosylation of a human glioblastoma cell line can lead to lowered resistance to chemotherapeutic agents.     

Arginine vasopressin inhibition of cyclin D1 gene expression blocks the cell cycle and cell proliferation in the mouse Y1 adrenocortical tumor cell line

Arginine vasopressin (AVP) is a nonapeptide long known as an endocrine and paracrine regulator of important systemic functions, namely, vasoconstriction, gluconeogenesis, corticosteroidogenesis, and excretion of water and urea. Here we report, for the first time, that AVP specifically inhibits expression of the cyclin D1 gene, leading to cell cycle blockage and halting cell proliferation. In G0/G1-arrested mouse Y1 adrenocortical tumor cells, maintained in serum-free medium (SFM), AVP mimics FGF2, promoting rapid ERK1/2 activation (5 min) followed by c-Fos protein induction (2 h). PKC inhibitor Go6983 and PI3K inhibitors wortmannin and LY294002 all inhibit ERK1/2 activation by AVP, but not by FGF2. Thus, AVP and FGF2 concur to activate ERK1/2 by different regulatory pathways. However, AVP is not a mitogenic factor for Y1 cells. On the contrary, AVP strongly antagonizes FGF2 late induction (2-5 h) of the cyclin D1 gene, down-regulating both cyclin D1 mRNA and protein. AVP inhibition of cyclin D1 expression is sufficient to block G1 phase progression and cell entry into the S phase, monitored by BrdU nuclear labeling. In addition, AVP completely inhibits proliferation of Y1 cells in 10% fetal calf serum (10% FCS) medium. On the other hand, ectopic expression of the cyclin D1 protein renders Y1 cells resistant to AVP for both entry into the S phase in SFM and continuous proliferation in 10% FCS medium. In conclusion, inhibition of cyclin D1 expression by AVP is an efficient mechanism of cell cycle blockage and consequent proliferation inhibition in Y1 adrenocortical cells.     

Ectopic expression of cyclin D1 impairs the proliferation and enhances the apoptosis of a murine lymphoid cell line

Cyclin D1, a key regulator of the cell cycle, acts as an oncogene when over-expressed in several types of cancer. In some B-chronic lymphoproliferative disorders, the over-expression of cyclin D1 protein is thought to confer a proliferative phenotype. We have generated BaF3 pro-B cell derivatives in which cyclin D1 can be induced rapidly and reversibly in a dose-dependent manner by the hormone muristerone A. When non-expressing clones displayed the same proliferative capacity as the parental cell line, in the sub-clones, a moderate induction of cyclin D1 lengthened the proliferation rate. The over-expression of cyclin D1 had the same effects on cell proliferation but also led ultimately to cell death by apoptosis. The induction of cyclin D1 in growth factor-deprived cells as well as in anticancer drug-treated cells also reinforced the magnitude of apoptosis. Thus, the expression of cyclin D1 in lymphoid cells does not confer a proliferative advantage but rather alters the response of cells towards apoptotic stimuli in a p53-independent manner.     

Effect of red and blue light on the timing of cyclin-dependent kinase activity and the timing of cell division in Chlamydomonas reinhardtii.

In this study, we describe the effect of red and blue light on the timing of cell division, DNA synthesis, and activity and presence of cyclin-dependent kinases (CDKs), in synchronous cultures of the unicellular green alga Chlamydomonas reinhardtii. Cell division and DNA synthesis were found to occur later in cells grown in blue or white light, than in red light. CDK-like activity, measured using a histone H1 kinase assay, correspondingly occurred later in cultures that were grown in blue light compared to cultures grown in red light. The amount of CDK-like proteins, as detected using an antibody against the PSTAIRE motif, showed a maximum during the division phase. We conclude that the mechanism that causes the delay in the timing of cell division in blue light has its action before DNA replication takes place and also precedes the increase in CDK-like activity.