Loss of cyclin D1 in necrotic and apoptotic models of cortical neuronal degeneration

Recent evidence suggests that apoptosis in post-mitotic neurons involves an aborted attempt of cells to re-enter the cell cycle and it is characterized by increased expression of cyclins, such as cyclin D1, prior to death. Cyclin D1 increases to permit transition from growth phase (G0/G1) to synthesis phase (S) during normal development but there is controversy as to which of the cyclins are activated prior to apoptotic cell death. We looked at the expression of cyclin D1 in cortical neuronal cultures treated with either staurosporine to produce apoptotic death, or with glutamate, to produce a non-apoptotic death. Cyclin D1 immunoreactivity was observed in the cytoplasm and nucleus of virtually all neurons under control conditions. Following the addition of either staurosporine or glutamate, cyclin D1 immunoreactivity did not change within 4 h. The cyclin D1 immunoreactivity was lost by 6 h with the appearance of either staurosporine-induced fragmented nuclei or glutamate-induced pyknotic nuclei. These immunocytochemical observations were confirmed with immunoblot analysis. Therefore, cyclin D1 is not a reliable indicator of apoptosis in cortical neuronal cultures and should not be used as an indicator of apoptotic cell death.     

Methylmercury elicits rapid inhibition of cell proliferation in the developing brain and decreases cell cycle regulator, cyclin E

The developing brain is highly sensitive to methylmercury (MeHg). Still, the initial changes in cell proliferation that may contribute to long-term MeHg effects are largely undefined. Our previous studies with growth factors indicate that acute alterations of the G1/S-phase transition can permanently affect cell numbers and organ size. Therefore, we determined whether an environmental toxicant could also impact brain development with rapid (6-7h) effects on DNA synthesis and cell cycle machinery in neuronal precursors. In vivo studies in newborn rat hippocampus and cerebellum, two regions of postnatal neurogenesis, were followed by in vitro analysis of two precursor models, cortical and cerebellar cells, focusing on the proteins that regulate the G1/S transition. In postnatal day 7 (P7) pups, a single subcutaneous injection of MeHg (3microg/g) acutely (7h) decreased DNA synthesis in the hippocampus by 40% and produced long-term (2 weeks) reductions in total cell number, estimated by DNA quantification. Surprisingly, cerebellar granule cells were resistant to MeHg effects in vivo at comparable tissue concentrations, suggesting region-specific differences in precursor populations. In vitro, MeHg altered proliferation and cell viability, with DNA synthesis selectively inhibited at an early timepoint (6h) corresponding to our in vivo observations. Considering that G1/S regulators are targets of exogenous signals, we used a well-defined cortical cell model to examine MeHg effects on relevant cyclin-dependent kinases (CDK) and CDK inhibitors. At 6h, MeHg decreased by 75% levels of cyclin E, a cell cycle regulator with roles in proliferation and apoptosis, without altering p57, p27, or CDK2 nor levels of activated caspase 3. In aggregate, our observations identify the G1/S transition as an early target of MeHg toxicity and raise the possibility that cyclin E degradation contributes to both decreased proliferation and eventual cell death.     

Differential effects of cyclin-dependent kinase blockers upon cell death in the developing retina

Pharmacological blockers of cyclin-dependent kinases (CDKs) can inhibit cell cycle progression. Deferoxamine (DFO) and mimosine (MIMO) arrest cells reversibly at the G1/S transition and olomoucine (OLO) inhibits the cell cycle at both G1/S and G2/M. We investigated the effect of these drugs upon cell death in histotypical explants taken from the retina of neonatal rats. Degeneration of retinal ganglions cells (RGC) induced by axotomy was inhibited by OLO (100 microM) but not by DFO (up to 2 mM) or MIMO (up to 1 mM). On the other hand, after 1 day in vitro, all cell cycle inhibitors induced cell death in the neuroblastic layer (NBL) of the explants. DFO and MIMO induced cell death only of proliferating cells, identified either by their incorporation of bromodeoxyuridine or by immunolabeling the proliferating cell nuclear antigen. In turn, OLO induced cell death of both proliferating and post-mitotic cells. However, the post-mitotic cells were unlabeled with markers of retinal differentiation. Our results indicate that cyclin-dependent kinases are involved in the control of sensitivity to cell death in the retina, and that retinal cells present differentiation-dependent responses to modulation of CDK activity.     

Disruption of cell cycle kinetics and cyclin-dependent kinase system by ethanol in cultured cerebellar granule progenitors.

An in vitro model of neuronal precursors, primary culture of cerebellar granule progenitors (CGPs), was used to investigate the mechanisms underlying ethanol-induced cell cycle damage. The CGP cultures were generated from 3-day-old rats. Ethanol significantly inhibited the proliferation of the CGPs in culture. Analysis of cell cycle kinetics by a cumulative 5-bromo-2'-deoxyuridine (BrdU) labeling technique demonstrated that ethanol exposure increased the duration of the cell cycle and decreased the growth fraction (the cycling population). The duration of the S-phase and total cell cycle was significantly prolonged by ethanol exposure by 220% and 135%, respectively, while the growth fraction was decreased from 44% in the control groups to 22% in the ethanol-exposed cultures. Cyclin-dependent kinase 2 (Cdk2) is a key protein that regulates both the passage from G1 into S, and the S phase progression. The results from in vitro phosphorylation assay and Western blot demonstrated that ethanol dramatically down-regulated both the activity and the expression of Cdk2. In addition, ethanol significantly decreased the expression of Cyclin A and Cyclin D(2). Further studies using in situ TUNEL assay and DNA fragmentation ELISA showed that ethanol caused a delayed apoptosis, i.e. the ethanol-induced apoptosis was evident only after chronic exposure. On the other hand, ethanol did not affect the necrotic index. In conclusion, ethanol decreases the cycling pool of CGPs by inducing cell cycle delay and promoting apoptosis. Ethanol-mediated disturbance of the cyclin-dependent kinase system may be an important mechanism to account for cell cycle arrest in neuronal precursor cells.     

Temporal and Spatial Expression of Cyclin H in Rat Spinal Cord Injury

Cyclin H regulates cell cycle transitions; it always forms trimeric cyclin-dependent protein kinases (CDK)-activating kinase (CAK) complex with CDK7 and MAT1 that phosphorylates a threonine residue in the CDK2 T loop region. However, neither the expression nor function of cyclin H in the central nervous system (CNS) injury is still clear. Therefore, we studied cyclin H in a rat spinal cord contusion model. Injury markedly increased cyclin H protein expression throughout the thoracic spinal cord but did not increase CDK7. However, double immunofluorescent staining for proliferating cell nuclear antigen (PCNA) and cell markers revealed increases of cyclin H and CDK2 in proliferating microglia and astrocytes, and the co-immunoprecipitation studies shown that the associations of cyclin H with CDK2 were enhanced evidently after injury. Our data suggest that cyclin H may play a proliferative role in spinal cord injury (SCI).     

Up-regulation of cyclin D1 occurs in apoptosis of immature but not mature cerebellar granule neurons in culture.

Cerebellar granule neurons isolated from 7-day-old rats and cultured in normal medium undergo apoptosis, but remain healthy under depolarizing conditions with elevated K(+) (>==25 mM) or in the presence of brain-derived neurotrophic factor. Northern blot analysis showed that cyclin D1 mRNA was up-regulated in this apoptotic process. Both granule neurons and microglia were immunostained with anti-cyclin D1 antibodies, which is consistent with our previous finding that microglia become activated in response to neuronal cell death under these conditions. Only granule neurons, however, showed an enhanced expression of both mRNA and protein levels of cyclin D1 in the presence of aphidicolin that completely eliminated non-neuronal cells. The entire cell body of granule neurons became immunostained prior to cell shrinkage or nuclear condensation. Moreover, cell cycle blockers and an inhibitor of cyclin-dependent kinases suppressed both increased immunoreactivity and cell death, further substantiating the involvement of an abortive cell cycle in this process. In contrast, both levels of cyclin D1 remained unaltered in mature granule neurons undergoing apoptosis following combined serum withdrawal and low K(+) shift, suggesting developmental stage dependence of granule neuron apoptosis in vitro. This culture system is suitable for further analysis of the role of cyclin D1 in cell death.     

Antiproliferative action of neomycin is associated with inhibition of cyclin D1 activation in glioma cells

The progression of mammalian cells through G1 phase of the cell cycle is governed by the D-type cyclins (D1, D2, D3). These proteins are induced at the beginning of the G1 phase and associate with serine/threonine cyclin-dependent kinases to form holoenzymes. Overexpression of cyclin D1 in human cancers as well as in several cancer cell lines has been reported. Here, we employed mitotic selection to synchronize the C6 glioma cell cycle at the start of the G1 phase and assessed the effects of neomycin on cyclin D1 protein detection by immunocytochemical analysis. Cyclin D1 activation as well as cell proliferation were already significantly reduced after 3 h of incubation of the cells with neomycin. These findings suggested that the antiproliferative effects of neomycin in gliomas could be mediated by inhibition of the expression of cyclin D1 gene and support further consideration of therapeutic use of neomycin in a Phase I clinical study for patients with recurrent glioblastoma.     

Effects of prenatal exposure to ethanol on the cyclin-dependent kinase system in the developing rat cerebellum

Prenatal exposure to ethanol inhibits neurogenesis in the developing cerebellum. Cyclin-dependent kinases (CDKs) are a family of protein kinases that play multiple roles in the regulation of cell proliferation, differentiation and survival. The activity of CDKs is positively regulated by CDK activators, cyclins, and negatively regulated by CDK inhibitors (CDKIs). We hypothesize that impaired cerebellar development induced by gestational ethanol exposure is mediated by disruption of the CDK system. Pregnant rats were fed ad libitum with an ethanol-containing liquid diet (Et) or pair-fed an isocaloric control diet (Ct). Cerebella were collected from pups (postnatal day (P) 0 through P21) and examined for CDK, cyclin, or CDKI expression using a quantitative immunoblotting procedure. In Ct-treated rats, the expression of CDK2 and its activator, cyclin A, paralleled the pattern of granule cell proliferation. Prenatal ethanol exposure produced a significant down-regulation of CDK2/cyclin A expression. Although the amounts of CDK4/CDK6 and their activator, cyclin D2, did not oscillate during postnatal development, their expression in Et-treated pups was significantly (P<0.05) higher than in controls. The expression of a CDK inhibitor, p27(Kip), was inversely correlated to proliferation of cerebellar granule progenitors. Prenatal ethanol exposure caused the down-regulation of p27(Kip) between P0 and P21. Thus, prenatal exposure to ethanol disturbed the expression of cell cycle machineries in the postnatal cerebellum. This may account for the teratogenic effects of ethanol on the developing cerebellum.     

Immunohistochemical examination of the INK4 and Cip inhibitors in the rat neonatal cerebellum: cellular localization and the impact of protein calorie malnutrition

Expression of the cyclin-dependent kinase inhibitors (CKIs) has been linked to the inhibition of cellular proliferation and the induction of differentiation. Based on structure function analysis, two distinct families of CDKIs, the INK4 and the Cip/Kip family have been identified. The INK4 family member p16(Ink4), and the Cip/Kip protein p27(Kip1) have been implicated in normal development of the CNS and cerebellum. Recent studies have suggested a functional inter-dependence between the CKI and the abundance of cyclin D1 in orchestrating growth factor-induced cellular proliferation. The neonatal rat cerebellum undergoes proliferative growth and differentiation, localized to distinct topographical regions and cell types. The cell type and the temporal profile of CKI expression during postnatal cerebellar development had not been described. The current studies determined the specific cerebellar cell types in which the CKIs were expressed during post natal development by co-staining for cell-type specific markers. p16(Ink4a) and p27(Kip1) immunostaining was identified in both neurons and glial cells, increasing progressively between postnatal days 6 to 13 into adulthood. By contrast, neuronal and glial cell p21(Cip1) staining was prominent at days 6-11 and decreased thereafter. Cyclin D1 was expressed in the proliferating external granular cells, with occassional staining in the molecular, and internal granular layers. Dual immunostaining demonstrated cyclin D1 within cells expressing CKI (p16(Ink4a), p21(Cip1),p27(Kip1)). Cerebellar cellular growth arrest, induced by protein-calorie malnutrition, inhibited cyclin D1 protein levels without affecting CKI immunostaining suggesting CKI do not mediate the developmental arrest. These results demonstrate that the CKIs are induced by differentiation cues in specific cell types with distinct kinetics in the developing cerebellum in vivo.     

S-phase entry of oligodendrocyte lineage cells is associated with increased levels of p21Cip1

The mechanisms regulating the number of myelinating cells in the central nervous system are crucial for both normal development and repair in pathological conditions. Among relevant growth factors involved in this process, fibroblast growth factor-2 (FGF2) induces oligodendrocyte progenitors (OLPs) to proliferate and stimulates mature oligodendrocytes (OLs) to reenter the S-phase of the cell cycle. S-phase entry is modulated by the formation of complexes between cyclins and cyclin-dependent kinases (CDKs), on one hand, and by their interactions with cell cycle inhibitors (e.g., p18INK, p27Kip1, p21Cip1), on the other. Although the roles of cyclin E/CDK2 complexes and the inhibitor p27Kip1 have been extensively investigated relative to proliferation and differentiation in the OL lineage, less is known about the regulation of the formation of cyclin D1/CDK4 complexes and the role of p21Cip1 in these events. In this study, we show that the FGF2-mediated increase in bromodeoxyuridine (BrdU) incorporation into OL progenitors and mature OLs occurs concomitantly with increase in the levels of p21Cip1 and the formation of p21Cip1/cyclin D1/CDK4 ternary complexes. These complexes are functionally active is indicated by the ensuing FGF2-dependent hyperphosphorylation of the downstream target Rb. In untreated mature OLs that do not incorporate BrdU, the levels of p21Cip1 are low, and the level of the inhibitor p18INK is high. Furthermore, p18INK sequesters CDK2 into binary complexes, precluding the formation of p21Cip1/cyclin D1/CDK4 ternary complexes in these cells. Therefore, we propose that p21Cip1 is acting as a positive regulator, rather than an inhibitor, of cell cycle entry by favoring the assembly of active cyclin D1/CDK4 complexes.     

Cyclin E1 knockdown induces apoptosis in cancer cells

OBJECTIVES: Cyclin E1 is expressed during the late G1 phase of the cell cycle and mediates the initiation of DNA synthesis by activating cyclin-dependent kinases 2 (CDK2). Abnormally high levels of cyclin E1 expression have frequently been found in cancer cells. Here, we investigate the effect of cyclin E1 knockdown on cancer cells. METHODS: RNA interference, expressed from a DNA-based retroviral vector, was used to knockdown cyclin E1 in adenocarcinoma (HeLa), breast (MDA-MB-31) and glioblastoma (U-373-MG) cell lines and an explant from one glioma patient (GB-LP-2). RESULTS: We have obtained very efficient depletion of cyclin E1 protein (over 80%) and considerable apoptotic induction (50-70%) after 96 hours post-infection. The ability of U-373-MG cells to induce tumor growth in nude mice was also abolished after cyclin E1 knockdown. DISCUSSION: Our results indicate that retrovirus carrying the DNA to be transcribed into a short hairpin RNA (shRNA) against cyclin E1 could be used as a therapeutic agent for cancer therapy.     

Proliferation and Differentiation of Neural Stem Cells Are Selectively Regulated by Knockout of Cyclin D1

Although stem cells can proliferate and differentiate through the completion of cell cycle progression, little is known about the genes and molecular mechanisms controlling this process. Here, we investigated the effect of the inhibition of cell cycle by cyclin D1 gene knockout on proliferation and differentiation of neural stem cells (NSCs). Knockout of cyclin D1 induced the cultured neural stem cells arrested at the G0/G1 phase as detected by flow cytometry. Cyclin D1 knockout led to the apoptosis of NSCs and inhibited the differentiation into astrocytes without affecting the differentiation into neurons. We further demonstrated that a significant reduction of BrdU⁺ cells in the subgranular zone of the dentate gyrus and subventricular zone was found in cyclin D1 gene knockout (cyclin D1^−/−) mice compared with cyclin D1^+/+ and cyclin D1^+/− mice. These observations demonstrated that cyclin D1 plays essential roles in the proliferation and differentiation of neural stem cells.     

Neuronal apoptosis in the striatum of rats treated with 3-nitropropionic acid is not triggered by cell-cycle re-entry

Although terminally differentiated neurons lack the capacity to undergo cell division, they retain the capacity to reactivate the cell cycle. This reactivation, however, has been linked to the degeneration of neurons in many experimental models of neurodegenerative disease and in post-mortem brains of affected patients. Expression of markers of the G1 phase and apoptotic neurons has been detected in the striatal lesion of rats treated with 3-nitropropionic acid (3-NPA). Here we examined whether neuronal apoptosis induced by 3-NPA was mediated by the reactivation of the cell cycle. To this end, we studied whether TUNEL-positive neurons expressed the G1-phase markers cyclin-dependent kinase 4 (CDK4) and cyclin D (CyD). In addition, we also evaluated the neuronal expression of pRb and Ki67 antigens, both of which are involved in the regulation of cell-cycle progression. In 3-NPA-treated rats, CDK4 and CyD were not detected in TUNEL-positive neurons, but they were expressed in neurons in the core of the lesion, which were assumed to be in a more advanced stage of degeneration, since they had weaker NeuN staining and lacked Hoechst staining. In addition, injured neurons in the striatal lesion of 3-NPA-treated rats had lost the constitutive expression of pRb and Ki67 that we had detected in control animals. Taken together, these results indicate that neuronal apoptosis in the striatal lesion of 3-NPA-treated rats was not triggered by cell-cycle re-entry, and we conclude that expression of G1 markers may be considered an aberrant survival response, with no relation to the mechanisms of apoptosis.     

Src kinase inhibition decreases thrombin-induced injury and cell cycle re-entry in striatal neurons

Since Src kinase inhibitors decrease brain injury produced by intracerebral hemorrhage (ICH) and thrombin is activated following ICH, this study determined whether Src kinase inhibitors decrease thrombin-induced brain injury. Thrombin injections into adult rat striatum produced focal infarction and motor deficits. The Src kinase inhibitor PP2 decreased thrombin-induced Src activation, infarction in striatum and motor deficits in vivo. Thrombin applied to cultured post-mitotic striatal neurons caused: injury to axons and dendrites; many TUNEL positive neuronal nuclei; and re-entry into the cell cycle as manifested by cyclin D1 expression, induction of several other cell cycle genes and cyclin-dependent kinase 4 activation. PP2 dose-dependently attenuated thrombin-induced injury to the cultured neurons; and attenuated thrombin-induced neuronal cell cycle re-entry. These results are consistent with the hypotheses that Src kinase inhibitors decrease injury produced by ICH by decreasing thrombin activation of Src kinases and, at least in part, by decreasing Src induced cell cycle re-entry.     

7蛋白酶体抑制剂对星形胶质细胞周期素D1和周期素依赖性激酶4表达的影响

目的：研究蛋白酶体抑制对体外培养的星形胶质细胞周期素Dl（cyclinD1）和周期素依赖性激酶4（CDK4）表达的影响。方法：SD乳鼠皮质星形胶质细胞原代培养,并纯化鉴定;予不同浓度（2和4μmol·L^-1）的蛋白酶体抑制剂（lactacystin）对第二代星形胶质细胞进行短期（12h）急性干预处理,应用免疫荧光及Westernblot检测星形胶质细胞cyclinD1和CDK4表达的水平。结果：纯化传代的皮质星形胶质细胞经胶质纤维酸性蛋白（GFAP）免疫荧光鉴定,其阳性率可达99％;lactacystin2和4μmol·L^-1可诱导星形胶质细胞cyclinDl和CDK4表达的下降,与对照组相比差异有显著统计学意义（P〈0．01）。结论：一定程度蛋白酶体活性抑制可诱导培养的星形胶质细胞cyclinD1和CDK4表达的减少,从而影响胶质细胞细胞周期,促进胶质细胞分化。提示蛋白酶体功能障碍后可能通过影响胶质细胞细胞周期来参与阿尔茨海默病的病理改变。     

Kainic acid-induced apoptosis in cerebellar granule neurons: an attempt at cell cycle re-entry

This study was undertaken to investigate whether kainic acid (KA) may regulate the expression of several proteins which plays an important role in cell-cycle progression in cerebellar granule neurons (CGNs). KA induced decrease in MTT values in a concentration dependent way. Flow cytometric analysis showed that KA was able to induce 30% apoptosis in CGNs. Apoptotic nuclear condensation were detected 24 h of exposure to KA (200 microM). An associated marked increase in DNA synthesis, measured by BrdU incorporation, was observed. Western blot analysis showed that KA induced an increase in the expression of Cdk2, cyclin E and E2F-1. It is proposed that, in post-mitotic cells like CGNs, re-entry cell cycle could be responsible for the apoptotic effect of KA.