cyclin-d1 and olomoucine

Herpes simplex encephalitis remains one of the most devastating intracranial infections despite available antiviral treatment, with sequelae secondary to a persistent inflammatory response. Recently, cyclin-dependent kinases (CDKs) have been found to act as cellular targets for antiviral drugs. However, the pharmacological effects of CDK inhibitors on glial cell function in herpes simplex encephalitis have not been elucidated. The aim of this work was to determine the influence of olomoucine on microglial activation during the inflammatory response after herpes simplex virus 1(HSV-1) infection.. Microglial cells were treated with various concentrations of olomoucine after HSV-1 infection. The expression change of cyclin D1 and myeloid cell leukemia 1 (Mcl-1) in microglia were detected by Western blot analysis. Flow cytometry was used to assess the apoptosis ratio of microglial cells among the groups of control, HSV-1 infected and olomoucine treated with or without zVAD-fmk. ELISA was adopted to analyse cytokines in the supernatant. We used semiquantitative reverse transcription polymerase chain reaction to detect HSV glycoprotein D gene.. The following are the results of this work: (1) olomoucine reduced HSV-1-induced proliferation associated cyclin D1 expression; (2) olomoucine also induced microglial cells apoptosis early at 24 hours post-infection and inhibited the release of proinflammatory cytokine and chemokine, including tumor necrosis factor alpha and monocyte chemoattractant protein 1; and (3) olomoucine-induced apoptosis was caspase-dependent, and it also reduced the antiapoptotic protein Mcl-1.. Our conclusion is that microglial cells are targets for olomoucine and that modulation of glial response and inflammation may be an appendant mechanism of CDK inhibitor-mediated neuroprotection in HSV-1 encephalitis.

Topics: Animals; Animals, Newborn; Apoptosis; Cell Count; Cell Proliferation; Cells, Cultured; Chemokine CCL2; Cyclin D1; Cyclin-Dependent Kinases; Dose-Response Relationship, Drug; Encephalitis; Encephalitis, Herpes Simplex; Enzyme Inhibitors; Herpesvirus 1, Human; Kinetin; Mice; Mice, Inbred BALB C; Microglia; Myeloid Cell Leukemia Sequence 1 Protein; Proto-Oncogene Proteins c-bcl-2; Rabbits; Tumor Necrosis Factor-alpha

The finding of nuclear translocation of cathepsin L and its ability to process the CDP/Cux transcription factor uncovers an important role of cathepsin L in control of cell cycle progression. As the expression of certain cell cycle regulators is associated with nigral neuronal death, the present study was sought to investigate if nuclear translocation of cathepsin L and expression of certain cyclins were induced in DA neurons by 6-hydroxydopamine (6-OHDA). The neuroprotective effects of the cell cycle inhibitor olomoucine against 6-OHDA-induced death of nigral neurons were examined. Using immunocytochemistry and real-time PCR we demonstrated that cyclin D1, cyclin B1 and proliferating cell nuclear antigen (PCNA) were aberrantly expressed in some dopaminergic neurons after 6-OHDA infusion. The nuclear translocation of cathepsin L and up-regulation of LC3, a protein involved in autophagy, were observed in nigral DA neurons. Olomoucine, a cyclin dependent kinase (CDK) inhibitor, reduced contralateral rotations and the loss of TH-positive neurons in substantia nigra induced by lesion with 6-OHDA. Pretreatment of rats or primary DA neurons with olomoucine resulted in a partial blockade of nuclear translocation of cathepsin L. Olomoucine also increased the expression of punctate LC3 immunoreactivity, indicating activation of autophagy. These findings suggest that olomoucine may exert neuroprotective effects through inhibiting cathepsin L nuclear translocation and activating autophagy.

Topics: Analysis of Variance; Animals; Autophagy; Cathepsin L; Cathepsins; Cell Count; Cell Cycle; Cell Death; Cells, Cultured; Cyclin B; Cyclin D1; Cysteine Endopeptidases; Enzyme Inhibitors; Fluorescent Antibody Technique; Kinetin; Male; Microtubule-Associated Proteins; Neurons; Oxidopamine; Parkinson Disease, Secondary; Proliferating Cell Nuclear Antigen; Protein Transport; Rats; Rats, Sprague-Dawley; Reverse Transcriptase Polymerase Chain Reaction; Substantia Nigra; Time Factors; Tyrosine 3-Monooxygenase; Up-Regulation

Glucocorticoids are anti-inflammatory steroids with important applications in the treatment of inflammatory diseases. Endogenous glucocorticoids are mainly produced by the adrenal glands, although there is increasing evidence for extra-adrenal sources. Recent findings show that intestinal crypt cells produce glucocorticoids, which contribute to the maintenance of intestinal immune homeostasis. Intestinal glucocorticoid synthesis is critically regulated by the transcription factor liver receptor homologue-1 (LRH-1). As expression of steroidogenic enzymes and LRH-1 is restricted to the proliferating cells of the crypts, we aimed to investigate the role of the cell cycle in the regulation of LRH-1 activity and intestinal glucocorticoid synthesis. We here show that either pharmacological or molecular modulation of cell cycle progression significantly inhibited expression of steroidogenic enzymes and synthesis of glucocorticoids in intestinal epithelial cells. Synchronization of intestinal epithelial cells in the cell cycle revealed that expression of steroidogenic enzymes is preferentially induced at the G(1)/S stage. Differentiation of immature intestinal epithelial cells to mature nonproliferating cells also resulted in reduced expression of steroidogenic enzymes. This cell cycle-related effect on intestinal steroidogenesis was found to be mediated through the regulation of LRH-1 transcriptional activity. This mechanism may restrict intestinal glucocorticoid synthesis to the proliferating cells of the crypts.

Topics: Animals; Aphidicolin; CDC2 Protein Kinase; Cell Cycle; Cell Differentiation; Cell Line; Cholesterol Side-Chain Cleavage Enzyme; Colforsin; Cyclin B; Cyclin B1; Cyclin D1; Doxorubicin; Glucocorticoids; Intestinal Mucosa; Kinetin; Mice; Nocodazole; Receptors, Cytoplasmic and Nuclear; Retinoblastoma Protein; Steroid 11-beta-Hydroxylase; Transfection

Astrogliosis occurs in a variety of neuropathological disorders and injuries, and excessive astrogliosis can be devastating to the recovery of neuronal function. In this study, we asked whether reactive astrogliosis can be suppressed in the lesion area by cell cycle inhibition and thus have therapeutic benefits. Reactive astrogliosis induced in either cultured astrocytes by hypoxia or scratch injury, or in a middle cerebral artery occlusion (MCAO) ischemia model were combined to address this issue. In the cultured astrocytes, hypoxia induced a cell cycle activation that was associated with upregulation of the proliferating cell nuclear marker (PCNA). Significantly, the cell cycle inhibitor, olomoucine, inhibited hypoxia-induced cell cycle activation by arresting the cells at G1/S and G2/M in a dose-dependent manner and also reversed hypoxia-induced upregulation of PCNA. Also in the cultured astrocytes, scratch injury induced reactive astrogliosis, such as hypertrophy and an increase in BrdU(+) astrocytes, both of which were ameliorated by olomoucine. In the MCAO ischemia mouse model, dense reactive glial fibrillary acidic protein and PCNA immunoreactivity were evident at the boundary zone of focal cerebral ischemia at days 7 and 30 after MCAO. We found that intraperitoneal olomoucine administration significantly inhibited these astrogliosis-associated changes. To demonstrate further that cell cycle regulation impacts on astrogliosis, cyclin D1 gene knockout mice (cyclin D1(-/-)) were subjected to ischemia, and we found that the percentage of Ki67-positive astrocytes in these mice was markedly reduced in the boundary zone. The number of apoptotic neurons and the lesion volume in cyclin D1(-/-) mice also decreased as compared to cyclin D1(+/+) and cyclin D1(+/-) mice at days 3, 7, and 30 after local cerebral ischemia. Together, these in vitro and in vivo results strongly suggest that astrogliosis can be significantly affected by cell cycle inhibition, which therefore emerges as a promising intervention to attenuate reactive glia-related damage to neuronal function in brain pathology.

Topics: Analysis of Variance; Animals; Astrocytes; Brain Injuries; Brain Ischemia; Cell Cycle; Cell Death; Cells, Cultured; Cyclin D1; Disease Models, Animal; Enzyme Inhibitors; Gliosis; Ki-67 Antigen; Kinetin; Male; Mice; Mice, Knockout; Middle Cerebral Artery; Rats; Rats, Wistar; Statistics, Nonparametric; Wounds and Injuries

Recent evidence suggests that apoptosis in post-mitotic neurons involves an aborted attempt of cells to re-enter the cell cycle which is characterized by increased expression of cyclins, such as cyclin D1, prior to death. However, such cyclins activation prior to apoptotic cell death remains controversial. Many neurological disorders are characterized by neuronal loss, particularly amyotrophic lateral sclerosis (ALS). ALS is a motoneuronal degenerative condition in which motoneuron loss could be due to an inappropriate return of these cells in the cell cycle. In the present study, we observed that deprivation of neurotrophic factor in purified motoneuron cultures induces an apoptotic pathway. After neurotrophic factor withdrawal, DAPI (4,6-diamidin-2-phenylindol dichlorohydrate) staining revealed the presence of nuclear condensation, DNA fragmentation, and perinuclear apoptotic body. Similarly, release of apoptotic microparticles and activation of caspases-3 and -9 were observed within the first hours following neurotrophic factor withdrawal. Next, we tested whether inhibition of cell cycle-related cyclin-dependent kinases (cdks) can prevent motoneuronal cell death. We showed that three cdk inhibitors, olomoucine, roscovitine and flavopiridol, suppress the death of motoneurons. Finally, we observed early increases in cyclin D1 and cyclin E expression after withdrawal of neurotrophic factors. These findings support the hypothesis that after removal of trophic support, post-mitotic neuronal cells die due to an attempt to re-enter the cell cycle in an uncoordinated and inappropriate manner.

Topics: Animals; Apoptosis; Cell Survival; Cells, Cultured; Cyclin D1; Cyclin E; Cyclin-Dependent Kinases; Flavonoids; Kinetin; Mice; Mitosis; Motor Neurons; Nerve Growth Factors; Piperidines; Protein Kinase Inhibitors; Purines; Roscovitine

After mild ischemic insults, many neurons undergo delayed neuronal death. Aberrant activation of the cell cycle machinery is thought to contribute to apoptosis in various conditions including ischemia. We demonstrate that loss of endogenous cyclin-dependent kinase (Cdk) inhibitor p16(INK4a) is an early and reliable indicator of delayed neuronal death in striatal neurons after mild cerebral ischemia in vivo. Loss of p27(Kip1), another Cdk inhibitor, precedes cell death in neocortical neurons subjected to oxygen-glucose deprivation in vitro. The loss of Cdk inhibitors is followed by upregulation of cyclin D1, activation of Cdk2, and subsequent cytoskeletal disintegration. Most neurons undergo cell death before entering S-phase, albeit a small number ( approximately 1%) do progress to the S-phase before their death. Treatment with Cdk inhibitors significantly reduces cell death in vitro. These results show that alteration of cell cycle regulatory mechanisms is a prelude to delayed neuronal death in focal cerebral ischemia and that pharmacological interventions aimed at neuroprotection may be usefully directed at cell cycle regulatory mechanisms.

Topics: Animals; Brain Ischemia; Bromodeoxyuridine; CDC2-CDC28 Kinases; Cell Cycle; Cell Cycle Proteins; Cell Death; Cell Hypoxia; Cells, Cultured; Cyclin D1; Cyclin-Dependent Kinase 2; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinase Inhibitor p16; Cyclin-Dependent Kinase Inhibitor p27; Cyclin-Dependent Kinases; Disease Models, Animal; Enzyme Inhibitors; Glucose; In Situ Nick-End Labeling; Kinetin; Mice; Mice, Inbred Strains; Microtubule-Associated Proteins; Neurons; Oxygen; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Purines; Rats; Rats, Wistar; Tumor Suppressor Proteins

The specificity and the temporal location of cell cycle arrest induced by the cyclin-dependent kinase (CDK) inhibitors olomoucine and roscovitine were investigated in normal human fibroblasts. Effects on the cell cycle were compared with those induced by the kinase inhibitor staurosporine, which arrests normal cells in early G1 phase by acting upstream of CDK2. Consistent with their in vitro activity, olomoucine and roscovitine, but not the related compound iso-olomoucine, induced a dose-dependent arrest in G1 phase. Following removal of CDK inhibitors, cells resumed cycle progression entering S phase with a kinetics faster than staurosporine-treated samples. Cellular levels of PCNA, cyclin D1, and cyclin E were not affected by the CDK inhibitors. In contrast, staurosporine significantly reduced the levels of these proteins, as determined by immunocytometry and Western blot analysis. Cyclin A was detectable only in some cells remaining in the G2 + M compartment of samples treated with CDK inhibitors, but not in samples treated with staurosporine. Significant reduction in the hyperphosphorylated forms of retinoblastoma protein was found in samples treated with CDK inhibitors, while only hypophosphorylated forms were observed in staurosporine-treated samples. Concomitantly, CDK2, but not CDK4, activity immunoprecipitated from cells treated with olomoucine or roscovitine was markedly inhibited. These results suggest that in normal cells, CDK2 kinase activity is the specific target of olomoucine and roscovitine.

Topics: CDC2-CDC28 Kinases; Cell Line; Cyclin B; Cyclin D1; Cyclin E; Cyclin-Dependent Kinase 2; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinases; DNA; Enzyme Inhibitors; Fibroblasts; G1 Phase; Humans; Kinetin; Phosphorylation; Proliferating Cell Nuclear Antigen; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Purines; Retinoblastoma Protein; Roscovitine