Screening of five specific cell cycle inhibitors using a T Cell lymphoma cell line synchrony/release assay

To obtain different cell populations at specific cell cycle stages, we used a cell culture synchronization protocol. Effects of five different cell cycle inhibitors acting throughout the cell cycle were examined by DNA flow cytometric analysis of a synchrony/release lymphoma cell line (CEM). The screening synchronized protocol showed that staurosporine, mimosine and aphidicolin are reversible G1 phase inhibitors that act at different times. Staurosporine acted in early G1, exhibited the strongest cytotoxic effect, and induced apoptosis. Mimosine and aphidicolin acted in late G1 and at the G1/S boundary, respectively. Hydroxyurea arrested CEM cells in early S phase, but later than the aphidicolin arrest point. Nocodazole synchronized CEM cells in M phase. All the inhibitors examined in this study can be used to synchronize cells at different phases of the cell cycle and were reversible with little toxicity except for staurosporine which is highly toxic. Because the regulatory mechanism of the cell cycle is disrupted by their effects on protein synthesis, however, these drugs must be used with caution.     

Long noncoding RNA HOXD-AS2 regulates cell cycle to promote glioma progression

Now, numerous exciting findings have been found that long noncoding RNAs (lncRNAs) play a vital role in cancer malignant progression. However, their potential involvement in glioma is not well understood. Here, we performed a high-throughput microarray to detect the lncRNA expression profiles between glioma cell lines and normal astrocyte cell lines. HOXD-AS2 was increased in glioma cells and it was associated with glioma grade and poor prognosis. Loss of HOXD-AS2 can inhibit glioma cell growth by inducing cell-cycle G1 arrest in vitro. The proliferation of glioma was inhibited followed by knocking down the expression of HOXD-AS2 not only in subcutaneous injection model but also in orthotopic implantation model. These findings indicate that HOXD-AS2 promotes the glioma progression and may serve as a potential target for glioma diagnosis and therapy.     

Enhancement of glutamate uptake mediates the neuroprotection exerted by activating group II or III metabotropic glutamate receptors on astrocytes

We investigated whether the activation of astroglial group II and III metabotropic glutamate receptors (mGluRs) could exert neuroprotective effects and whether the neuroprotection was related to glutamate uptake. Our results showed that the activation of astroglial group II or III mGluRs exerted neuroprotection against 1-methyl-4-phenylpyridinium (MPP+) astroglial conditioned medium-induced neurotoxicity in midbrain neuron cultures. Furthermore, MPP+ decreased glutamate uptake of primary astrocytes and C6 glioma cells, which was recovered by activating group II or III mGluRs. Specific group II or III mGluRs antagonists completely abolished the neuroprotective effects and the enhancement of glutamate uptake of their respective agonists. Our results showed that the primary cultured rat astrocytes and C6 glioma cells expressed receptor proteins for group II mGluR2/3, group III mGluR4, mGluR6 and mGluR7. C6 glioma cells expressed mRNA for group II mGluR3, group III mGluR4, mGluR6, mGluR7 and mGluR8. In conclusion, we confirmed that the activation of astroglial mGluRs exerted neuroprotection, and demonstrated that the mechanism underlying this protective role was at least partially related to the enhancement of glutamate uptake.     

人细胞周期相关激酶启动子的克隆和初步分析

克隆人的细胞周期相关激酶(CCRK)基因启动子,分析与其表达有关的转录调控因子。通过巢式PCR方法,从人的基因组总DNA中分离出CCRK基因5′端非翻译区大小为1072bp的片段。这段片段的3′端起始点在第一个外显子里第一个翻译密码子ATG( 1)上游128bp处。以1072bp为模板,对启动子进行5′端删除分析,分别扩增951bp(-1072／-128)、564bp(-692／-128)、313bp(-441／-128)、127bp(-239／-128)的片段,与pGL3-Basic荧光素酶报告载体重组构建,人工加上克隆位点,5′端带有KpnI、3′端带有XhoI位点。瞬时转染恶性神经胶质瘤细胞株U373,通过双荧光素酶活性进行分析。对分离出的1072bp的片段进行测序,结果与GenBank(Accession AF035013)的序列比较,同源序列达到98％。双荧光素酶活性分析564bp片段有最强的活性,313bp片段为有活性的最小片段。本研究为进一步研究分析CCRK的核心启动子和与其相关的转录因子奠定基础。     

Cell cycle regulation of astrocytes by extracellular nucleotides and fibroblast growth factor-2

Extracellular ATP enhances the mitogenic activity of fibroblast growth factor-2 (FGF2) in astrocytes, but the molecular mechanism underlying this synergistic interaction is not known. To determine whether the potentiating effect of extracellular ATP involves cell cycle control mechanisms, we have measured the expression of cyclins that are induced in different phases of the cell cycle in primary cultures of rat cortical astrocytes. We found that ATP potentiated the ability of FGF2 to stimulate expression of cyclin D1, a regulator of cell cycle entry, as well as cyclin A, a regulator of DNA replication. Because FGF2 and P2 purinergic receptors are coupled to extracellular signal regulated protein kinase (ERK), a key member of a signaling cascade that regulates proliferation, we also investigated the role of ERK in regulating cyclin expression induced by FGF2 and ATP. We found that the potentiating effect of ATP on cyclin expression was significantly reduced by U0126, an inhibitor of MEK, the upstream activator of ERK. P2 receptor agonist studies revealed that UTP enhanced FGF2-induced cyclin expression and mitogenesis whereas 2-methylthioADP was ineffective. By contrast, 2′,3′-O-(4-benzoyl)-benzoyl-ATP markedly inhibited FGF2-induced mitogenesis. Consistent with opposing effects of P2Y and P2X receptors on mitogenesis, UTP stimulated a transient activation of ERK whereas BzATP stimulated a more sustained ERK signal. These findings suggest that signaling by P2Y receptors, most likely of the purine/pyrimidine subtype, enhance the ability of FGF2 to stimulate entry into a new cell cycle, as well as DNA replication, by an ERK-dependent mechanism, whereas signaling by P2X receptors, possibly the P2X7 subtype, inhibits FGF2-induced mitogenesis in astrocytes. Interactions between P2Y, P2X and polypeptide growth factor signaling pathways may have important implications for CNS development as well as injury and repair.     

Regulation of morphological and functional properties of astrocytes by hydrogen peroxide

Effects of hydrogen peroxide on morphological characteristics, proliferation index, and menadione-dependent lucigenin-enhanced chemiluminescence of C6 glioma cells were studied. It was established that H₂O₂ at 5 × 10^?7?1 × 10^?8 M concentrations acted as a regulator of morphological and functional properties of astrocytes, inducing their reactivation, which is manifested as cell body hypertrophy and an increase of proliferative activity and menadione-induced production of superoxide anion radicals (O ₂ ^?? ). Cytodestructive action of hydrogen peroxide at a concentration higher than 1 × 10^?6 M on C6 glioma cells shows itself as a decrease of their proliferation index and the ability to generate O ₂ ^?? under the effect of menadione. Use of lipopolysaccharide B as a functional stimulator has shown that H₂O₂ modifies signaling pathways leading to an increase of mitotic activity of C6 glioma cells and decreases the yield of lucigenin-dependent chemiluminescence of astrocytes under the action of menadione to the level of control values.     

Endogenous cytokinin oscillations control cell cycle progression of tobacco BY-2 cells

The significance of cytokinins for the progression of the cell cycle is well known. Cytokinins contribute to the control of the expression of D-cyclins and other cell cycle genes, but knowledge as to how they affect the progression of the cell cycle is still limited. Highly synchronized tobacco BY-2 cells with clearly defined cell cycle stages were employed to determine cytokinin patterns in detail throughout the entire cycle. Concentrations of trans-zeatin, and of some other cytokinins, oscillated during the course of the cell cycle, increasing substantially at all four phase transitions and decreasing again to a minimum value during the course of each subsequent phase. Addition of exogenous cytokinins or inhibition of cytokinin biosynthesis promoted the progression of the cell cycle when the effects of these manipulations intensified the endogenous fluctuations, whereas the progression of the cycle was retarded when the amplitude of the fluctuations was decreased. The results show that the attainment of low concentrations of cytokinins is as important as the transient increases in concentration for a controlled progression from one phase of the cell cycle to the next. Cytokinin oxidase/dehydrogenase activity also showed fluctuations during the course of the cell cycle, the timing of which could at least partly explain oscillations of cytokinin levels. The activities of the enzyme were sufficient to account for the rates of cytokinin disappearance observed subsequent to a phase transition.     

Characterization of the effects of butyric acid on cell proliferation, cell cycle distribution and apoptosis

We examined concentration-dependent changes in cell cycle distribution and cell cycle-related proteins induced by butyric acid. Butyric acid enhanced or suppressed the proliferation of Jurkat human T lymphocytes depending on concentration. A low concentration of butyric acid induced a massive increase in the number of cells in S and G2/M phases, whereas a high concentration significantly increased the accumulation of cells in G2/M phase, suppressed the accumulation of cells in G0/G1 and S phases, and induced apoptosis that cell cycle-related protein expression in Jurkat cells treated with high levels of butyric acid caused a marked decrease in cyclin A, cyclin E, cyclin-dependent kinase 2 (CDK2), CDK4 and CDK6 protein levels in G0/G1 and S phases, with apoptosis induction, and a decrease in cyclin B, Cdc25c and p27KIP1 protein levels, as well as an increase in p21CIP1/WAF1 protein level, in the G2/M phase. Taken together, our results indicate that butyric acid has bimodal effects on cell proliferation and survival. The inhibition of cell growth followed by the increase in apoptosis induced by high levels of butyric acid were related to an increase in cell death in G0/G1 and S phases, as well as G2/M arrest of cells. Finally, these results were further substantiated by the expression profile of butyric acid-treated Jurkat cells obtained by means of cDNA array.     

Establishment and characterisation of a rapidly dividing diploid cell suspension culture of Arabidopsis thaliana suitable for cell cycle synchronisation

Polyploid plants often have altered gene expression, biochemistry, and metabolism compared to their diploid predecessors. Therefore cultured diploid cells have distinct benefits over cultured polyploid cells for the study of gene regulation and metabolism of the parent plant. Here we report methods for establishing and maintaining a rapidly dividing diploid Arabidopsis thaliana cell suspension culture, and subsequent cell cycle synchronisation. Rapid growth of homogeneous cell populations was achieved after 3 months of initiation of cultures from leaf calluses. The cells were grown in the dark on an orbital shaker (110 rpm, 50 mm orbit) at 24 °C. Continued maintenance of the culture required the use of late-exponential stage cells for subculture at weekly intervals using careful subculturing techniques to achieve accurate biomass transfer. Cell cycle synchronisation was achieved following sucrose starvation, phosphate starvation, hydroxyurea treatment, aphidicolin treatment, and a combination of phosphate starvation and aphidicolin treatment. Inhibition of the cell cycle and accumulation of cells in specific phases was monitored by microscopy to determine the metaphase/anaphase index, and by flow cytometry. The cell cycle was partially and reversibly blocked by sucrose or phosphate starvation and by hydroxyurea (2.5 mM) treatment. A complete block at G1/S interphase was achieved after aphidicolin treatment or phosphate starvation combined with aphidicolin treatment. Release from the aphidicolin block achieved ca. 78% cell cycle synchronisation in the cell population. Endoreduplication was evident after release from the block in all treatments but after one cycle (24 h) the cells returned to the diploid state. This diploid culture is currently being used in our laboratory for the genetic analysis of cell death.     

Abnormal cell cycle regulation in primary human uveal melanoma cultures

Uveal malignant melanoma is the most frequent primary intraocular tumor in adult humans. The cellular events leading to neoplasic transformation of normal uveal melanocytes are not well known when compared to other cancers. In this study, we investigated the role of G1 and G1/S regulatory proteins of the cell cycle in human uveal melanoma (UM) primary cell cultures, since these proteins are common targets in tumor development. Further, freshly established and characterized tumor cells are a better model for in vitro studies when compared to cell lines established long ago. Human primary cell cultures from eight different UM were established, as well as one primary culture from rhesus uveal normal melanocytes (UNM). Primary human UM cultures were characterized by a low establishment and growing rate. From four successful cultures, three showed a high expression of cyclin D1, cyclin E, p16NK4A, and p27KIP1 with no variations in cyclin A, cyclin-dependent kinase 2 (CDK2), and CDK4. Interestingly, in one of the cultured tumors, tumor suppressor protein retinoblastoma (Rb) did not bind E2F despite the fact that Rb was found in its hypophosphorylated form. No mutations in either RB1 or the Rb-binding pocket of E2F-1 were detected. Furthermore, we identified seven proteins co-immunoprecipitating with Rb in this tumor, including Lamin A/C and six proteins not previously reported to bind Rb: Hsc70, high mobility group protein 1 (HMG-1), hnRPN, glyceraldehyde 3 phosphate dehydrogenase (G3PDH), EF-1, and EF-2. Our results indicate that the overexpression of cyclins D1/E and CDKIs p16 and p27, together with a deregulation of the Rb/E2F pathway, may be implicated in the development of human UM.     

Opposed effects of lithium on the MEK-ERK pathway in neural cells: inhibition in astrocytes and stimulation in neurons by GSK3 independent mechanisms

Lithium is widely used in the treatment of bipolar disorder, but despite its proven therapeutic efficacy, the molecular mechanisms of action are not fully understood. The present study was undertaken to explore lithium effects of the MEK/ERK cascade of protein kinases in astrocytes and neurons. In asynchronously proliferating rat cortical astrocytes, lithium decreased time- and dose-dependently the phosphorylation of MEK and ERK, with 1 mM concentrations achieving 60 and 50% inhibition of ERK and MEK, respectively, after a 7-day exposure. Lithium also inhibited [3H]thymidine incorporation into DNA and induced a G2/M cell cycle arrest. In serum-deprived, quiescent astrocytes, pre-exposure to lithium resulted in the inhibition of cell cycle re-entry as stimulated by the mitogen endothelin-1: under this experimental setting, lithium did not affect the rapid, peak phosphorylation of MEK taking place after 3-5 min, but was effective in inhibiting the long-term, sustained phosphorylation of MEK. Lithium inhibition of the astrocyte MEK/ERK pathway was independent of inositol depletion. Further, compound SB216763 inhibited Tau phosphorylation at Ser396 and stabilized cytosolic beta-catenin, consistent with the inhibition of glycogen synthase kinase-3 beta (GSK-3 beta), but failed to reproduce lithium effects on MEK and ERK phosphorylation and cell cycle arrest. In cerebellar granule neurons, millimolar concentrations of lithium enhanced MEK and ERK phosphorylation in a concentration-dependent manner, again through an inositol and GSK-3 beta independent mechanism. These opposing effects in astrocytes and neurons make lithium treatment a promising strategy to favour neural repair and reduce reactive gliosis after traumatic injury.     

Ivermectin inhibits the growth of glioma cells by inducing cell cycle arrest and apoptosis in vitro and in vivo

Glioma, the most predominant primary malignant brain tumor, remains uncured due to the absence of effective treatments. Hence, it is imperative to develop successful therapeutic agents. This study aimed to explore the antitumor effects and mechanisms of ivermectin (IVM) in glioma cells in vitro and in vivo. The effects of IVM on cell viability, cell cycle arrest, apoptosis rate, and morphological characteristics were determined respectively by MTT assay/colony formation assay, flow cytometry, and transmission electron microscope. In addition, the expression levels of cycle-related and apoptosis-associated proteins were individually examined by Western blot analysis. Moreover, cell proliferation and apoptosis analyses were carried out by TUNEL, Ki-67, cleaved caspase-3, and cleaved caspase-9 immunostaining assay. Our results demonstrated that IVM has a potential dosage-dependent inhibition effect on the apoptosis rate of glioma cells. Meanwhile, the results also revealed that IVM induced apoptosis by increasing caspase-3 and caspase-9 activity, upregulating the expressions of p53 and Bax, downregulating Bcl-2, activating cleaved caspase-3 and cleaved caspase-9, and blocking cell cycle in G0/G1 phase by downregulating levels of CDK2, CDK4, CDK6, cyclin D1, and cyclin E. These findings suggest that IVM has an inhibition effect on the proliferation of glioma cells by triggering cell cycle arrest and inducing cell apoptosis in vitro and in vivo, and probably represents promising agent for treating glioma.     

In vitro effects of ecdysterone on the spermatogonial cell cycle in Locusta

The effect of ecdysterone on specific phases of the cell cycle of Locusta migratoria migratorioides spermatogonia was assayed in vitro. An increase in labeling index was noted, indicating a decrease in duration of the G₁ phase. On addition of the hormone to adapted organs in vitro, spermatogonial mitotic index increases rapidly, then declines to a basal level within a time period which approximates that of the G₂ phase. Such a response indicates a removal of a G₂/M inhibition by the hormone.     

Effects of cyclic AMP and butyrate on cell cycle,DNA, RNA,and purine synthesis of cultured astrocytes

Dibutyryl cyclic monophosphate (dBcAMP) has been shown to inhibit growth, and alter the morphology of astrocytes. However, the potential contribution of its hydrolytic product, butyrate, in inducing some of the changes that have been attributed to dBcAMP, is not clear. DNA, RNA, and purine synthesis were therefore studied in primary astrocyte cultures after 24 hours of exposure to varying concentrations of butyrate, dBcAMP, and agents that increase intracellular cAMP levels. Progression of cells through cell cycle was also studied by flow cytometry. Dibutyryl cAMP partially arrested cells in Go/G1 phase of cell cycle while sodium butyrate increased the percentage population of cells in G2/M phase. DNA synthesis and de novo purine synthesis were inhibited after treatment with dBcAMP, sodium butyrate, and various drugs that increase intracellular cAMP levels. RNA synthesis was increased with cAMP but was not affected by sodium butyrate. Our study shows that at millimolar concentrations, butyrate is capable of altering the cell cycle and inhibiting DNA synthesis in primary astrocyte cultures, in a manner that is similar although not identical to the effects of dBcAMP.     

Differential regulation of survivin by p53 contributes to cell cycle dependent apoptosis

Recent studies indicate that cell-cycle checkpoints are tightly correlated with the regulation of apoptosis, in which p53 plays an important role. Our present works show that the expression of E6/E7 oncogenes of human papillomavirus in HeLa cells is inhibited in the presence of anti-tumor reagent tripchlorolide (TC), which results in the up-regulation of p53 in HeLa cells. Interestingly, under the same TC-treatment, the cells at the early S-phase are more susceptible to apoptosis than those at the middle S-phase although p53 protein is stabilized to the same level in both situations. Significant difference is exhibited between the two specified expression profiles. Further analysis demonstrates that anti-apoptotic gene survivin is up-regulated by p53 in the TC-treated middle-S cells, whereas it is down-regulated by p53 in the TC-treated early-S cells. Taken together, the present study indicates that the differential p53-regulated expression of survivin at different stages of the cell cycle results in different cellular outputs under the same apoptosis-inducer.     

蛋白激酶C与细胞周期

近年的研究表明,PKC涉及到细胞的周期调节。在酵母细胞和哺乳动物细胞均发现PKC参与细胞周期调控,从而提示PKC可能在进化上是一种保守的细胞周期调节子。一般认为PKC在两个点上对细胞周期起作用,即G1期和G2期到M期的过渡期（G2／M）。在G1期,PKC分别在早G1期和晚G1期作用有所不同,主要作用表现在使细胞停留在G1期的中末阶段,这一过程,主要涉及到抑制肿瘤抑制因子－成视网膜细胞瘤（Rb)蛋白的磷酸化。PKC的主要作用是降低周期素依赖激酶CDK2的活性、降低周期素E和A的表达和增加周期素依赖的周期抑制蛋白p21^WAF1和p27^KIP1的表达;在G2／M期,PKC对细胞周期的调节主要与Cdc2(CDK1)的活性抑制有关。     

泛素化途径与细胞周期的关系

泛素化途径(the ubiquitin pathway)是一种有高度选择性的蛋白水解途径,是细胞周期调控的基础。本文主要论述了依赖SCF(skp-cullin-F-boxprotein)和APC／C(anaphase-promoting complexor cyclosome)的两种泛素化途径对细胞周期不同时期的调控作用及其研究进展。     

Functional and molecular identification of sodium-coupled dicarboxylate transporters in rat primary cultured cerebrocortical astrocytes and neurons

Na+-coupled carboxylate transporters (NaCs) mediate the uptake of tricarboxylic acid cycle intermediates in mammalian tissues. Of these transporters, NaC3 (formerly known as Na+-coupled dicarboxylate transporter 3, NaDC3/SDCT2) and NaC2 (formerly known as Na+-coupled citrate transporter, NaCT) have been shown to be expressed in brain. There is, however, little information available on the precise distribution and function of both transporters in the CNS. In the present study, we investigated the functional characteristics of Na+-dependent succinate and citrate transport in primary cultures of astrocytes and neurons from rat cerebral cortex. Uptake of succinate was Na+ dependent, Li+ sensitive and saturable with a Michaelis constant (Kt) value of 28.4 microM in rat astrocytes. Na+ activation kinetics revealed that the Na+ to succinate stoichiometry was 3:1 and the concentration of Na+ necessary for half-maximal transport was 53 mM. Although uptake of citrate in astrocytes was also Na+ dependent and saturable, its Kt value was significantly higher (approximately 1.2 mM) than that of succinate. Unlabeled succinate (2 mM) inhibited Na+-dependent [14C]succinate (18 microM) and [14C]citrate (4.5 microM) transport completely, whereas unlabeled citrate inhibited Na+-dependent [14C]succinate uptake more weakly. Interestingly, N-acetyl-L-aspartate, which is the second most abundant amino acid in the nervous system, also completely inhibited Na+-dependent succinate transport in rat astrocytes. The inhibition constant (Ki) for the inhibition of [14C]succinate uptake by unlabeled succinate, N-acetyl-L-aspartate and citrate was 15.9, 155 and 764 microM respectively. In primary cultures of neurons, uptake of citrate was also Na+ dependent and saturable with a Kt value of 16.2 microM, which was different from that observed in astrocytes, suggesting that different Na+-dependent citrate transport systems are expressed in neurons and astrocytes. RT-PCR and immunocytochemistry revealed that NaC3 and NaC2 are expressed in cerebrocortical astrocytes and neurons respectively. These results are in good agreement with our previous reports on the brain distribution pattern of NaC2 and NaC3 mRNA using in situ hybridization. This is the first report of the differential expression of different NaCs in astrocytes and neurons. These transporters might play important roles in the trafficking of tricarboxylic acid cycle intermediates and related metabolites between glia and neurons.