Cyclin dependent kinase inhibitors prevent apoptosis of postmitotic mouse motoneurons

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.     

Slowed N-type calcium channel (CaV2.2) deactivation by the cyclin-dependent kinase inhibitor roscovitine

The lack of a calcium channel agonist (e.g., BayK8644) for CaV2 channels has impeded their investigation. Roscovitine, a potent inhibitor of cyclin-dependent kinases 1, 2, and 5, has recently been reported to slow the deactivation of P/Q-type calcium channels (CaV2.1). We show that roscovitine also slows deactivation (EC(50) approximately 53 microM) of N-type calcium channels (CaV2.2) and investigate gating alterations induced by roscovitine. The onset of slowed deactivation was rapid ( approximately 2 s), which contrasts with a slower effect of roscovitine to inhibit N-current (EC(50) approximately 300 microM). Slow deactivation was specific to roscovitine, since it could not be induced by a closely related cyclin-dependent kinase inhibitor, olomoucine (300 microM). Intracellularly applied roscovitine failed to slow deactivation, which implies an extracellular binding site. The roscovitine-induced slow deactivation was accompanied by a slight left shift in the activation-voltage relationship, slower activation at negative potentials, and increased inactivation. Additional data showed that roscovitine preferentially binds to the open channel to slow deactivation. A model where roscovitine reduced a backward rate constant between two open states was able to reproduce the effect of roscovitine on both activation and deactivation.     

Activation of the Rb/E2F1 pathway by the nonproliferative p38 MAPK during Fas (APO1/CD95)-mediated neuronal apoptosis

Aberrant activation of the Rb/E2F1 pathway in cycling cells, in response to mitogenic or nonmitogenic stress signals, leads to apoptosis through hyperphosphorylation of Rb. To test whether in postmitotic neurons the Rb/E2F1 pathway can be activated by the nonmitogenic stress signaling, we examined the role of the p38 stress-activated protein kinase (SAPK) in regulating Rb phosphorylation in response to Fas (CD95/APO1)-mediated apoptosis of cultured cerebellar granule neurons (CGNs). Anti-Fas antibody induced a dramatic and early activation of p38. Activated p38 was correlated with the induction of hyperphosphorylation of both endogenous and exogenous Rb. The p38-selective inhibitor, SB203580, attenuated such an increase in pRb phosphorylation and significantly protected CGNs from Fas-induced apoptosis. The cyclin-dependent kinase-mediated Rb phosphorylation played a lesser role in this neuronal death paradigm, since cyclin-dependent kinase inhibitors, such as olomoucine, roscovitine, and flavopiridol, did not significantly prevent anti-Fas antibody-evoked neuronal apoptosis. Hyperphosphorylation of Rb by p38 SAPK resulted in the release of Rb-bound E2F1. Increased E2F1 modulated neuronal apoptosis, since E2F1-/- CGNs were significantly less susceptible to Fas-mediated apoptosis in comparison with the wild-type CGNs. Taken together, these studies demonstrate that neuronal Rb/E2F1 is modulated by the nonproliferative p38 SAPK in Fas-mediated neuronal apoptosis.     

Apoptosis of metastatic prostate cancer cells by a combination of cyclin-dependent kinase and AKT inhibitors

Effective treatments for advanced prostate cancer are much needed. Toward this goal, we show apoptosis and impaired long-term survival of androgen-independent prostate cancer cells (PC3 and PC3 derivatives) co-treated with the cyclin-dependent kinase (CDK) inhibitor roscovitine and an AKT inhibitor (LY294002 or API-2). Apoptosis of PC3 cells by the drug combination required caspase-9 but not caspase-8 activity and thus is mitochondria-dependent. Roscovitine reduced amounts of the caspase inhibitor XIAP, and API-2 increased amounts of the BH3-only protein Bim. PC3 cells apoptosed when co-treated with API-2 and either cdk9 siRNA, dominant-negative cdk9, or the cdk9 inhibitor DRB; they did not apoptose when co-treated with API-2 and XIAP siRNA. Bax accumulated in mitochondria in response to API-2, whereas release of cytochrome c from mitochondria required both API-2 and roscovitine. We suggest that roscovitine elicits events that activate Bax once it translocates to mitochondria and that inactivation of cdk9 signals these events and the down-regulation of XIAP. Collectively, our data show apoptosis of prostate cancer cells by a drug combination and identify Bax activation as a basis of cooperation.     

Roscovitine, a novel cyclin-dependent kinase inhibitor, characterizes restriction point and G2/M transition in tobacco BY-2 cell suspension

Although the developmental programs of plants and animals differ, key regulatory components of their cell cycle have been conserved. Particular attention has been paid to the role of the complexes between highly conserved cyclin and cyclin-dependent kinases in regulating progression through the cell cycle. The recent demonstration that roscovitine is a potent and selective inhibitor of the animal cyclin-dependent kinases cdc2 (CDK1), CDK2 and CDK5 prompted an investigation into its effects on progression through the plant cell cycle. Roscovitine induced arrests both in late G1 and late G2 phase in BY-2 tobacco cell suspensions. Both blocks were fully reversible when roscovitine was used at concentrations similar to those used in the animal system. Stationary-phase cells subcultured in the presence of roscovitine were arrested at a 2C DNA content. This arrest was more efficient without exogenous addition of plant growth regulator. Roscovitine induced a block in G1 earlier than that induced by aphidicolin. S-phase synchronized cells treated with roscovitine were arrested at a 4C DNA content at the G2/ M transition. The expression analysis of a mitotic cyclin (NTCYC1) indicated that the roscovitine-induced G2 block probably occurs in late G2. Finally, cells in metaphase were insensitive to roscovitine. The purified CDK/cyclin kinase activities of late G1 and early M arrested cells were inhibited in vitro by roscovitine. The implications of these experimental observations for the requirement for CDK activity during progression through the plant cell cycle are discussed.     

Roscovitine sensitizes breast cancer cells to TRAIL-induced apoptosis through a pleiotropic mechanism

The tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL/APO2L) is a member of the TNF gene superfamily that induces apoptosis upon engagement of cognate death receptors. While TRAIL is relatively non-toxic to normal cells, it selectively induces apoptosis in many transformed cells. Nevertheless, breast tumor cells are particularly resistant to the effects of TRAIL. Here we report that, in combination with the cyclin-dependent kinase inhibitor roscovitine, exposure to TRAIL induced marked apoptosis in the majority of TRAIL-resistant breast cancer cell lines examined. Roscovitine facilitated TRAIL death-inducing signaling complex formation and the activation of caspase-8. The cFLIP(L) and cFLIP(S) FLICE-inhibitory proteins were significantly down-regulated following exposure to roscovitine and, indeed, the knockdown of cFLIP isoforms by siRNA sensitized breast tumor cells to TRAIL-induced apoptosis. In addition, we demonstrate that roscovitine strongly suppressed Mcl-1 expression and up-regulated E2F1 protein levels in breast tumor cells. Significantly, the silencing of Mcl-1 by siRNA sensitized breast tumor cells to TRAIL-induced apoptosis. Furthermore, the knockdown of E2F1 protein by siRNA reduced the sensitizing effect of roscovitine in TRAIL-induced apoptosis. In summary, our results reveal a pleitropic mechanism for the pro-apoptotic influence of roscovitine, highlighting its potential as an antitumor agent in breast cancer in combination with TRAIL.     

Cytokinin N-glucosylation inhibitors suppress deactivation of exogenous cytokinins in radish, but their effect on active endogenous cytokinins is counteracted by other regulatory mechanisms

The prolongation of the effect of exogenous cytokinins by inhibition of their inactivation was studied in Raphanus sativus L. cv. Rampouch. As in radish the main way of cytokinin inactivation is their N- glucosylation, inhibitors of this pathway, papaverine, theophylline and olomoucine, as well as two olomoucine analogues, bohemine and roscovitine, were tested. The latter ones, which function as potent inhibitors of cyclin-dependent kinases, have also been found to effectively inhibit cytokinin N- glucosylation. Incubation (24 h) of de-rooted radish seedlings with inhibitors resulted in c . 50% decrease of the conversion of model cytokinins, [³H]dihydrozeatin and [³H]N⁶-benzyladenine, to the corresponding 7 N- glucosides. Simultaneously the level of the non-metabolized cytokinin bases was elevated. The activity of cytokinin oxidase/dehydrogenase (EC 1.5.99.12) was suppressed in a dose dependent manner. The concentration of physiologically active endogenous cytokinins was not increased significantly by inhibitor application. The inhibition of N- glucosylation was in the case of olomoucine, bohemine and roscovitine accompanied by the accumulation of physiologically nonactive cis -zeatin derivatives. The impact of inhibitors on the endogenous cytokinin pool seems to be balanced by the mechanisms involved in the maintenance of cytokinin homeostasis.     

The cyclin-dependent kinase inhibitor roscovitine inhibits kinase activity, cell proliferation, multicellular development, and Cdk5 nuclear translocation in Dictyostelium discoideum

Roscovitine, a cyclin-dependent kinase (Cdk) inhibitor, inhibited kinase activity and the axenic growth of Dictyostelium discoideum at micromolar concentrations. Growth was almost fully rescued in 50 μM and ≈ 50% rescued in 100 μM roscovitine-treated cultures by the over-expression of Cdk5-GFP. This supports the importance of Cdk5 function during cell proliferation in Dictyostelium and indicates that Cdk5 is a primary target of the drug. Roscovitine did not affect the expression of Cdk5 protein during axenic growth but did inhibit its nuclear translocation. This novel result suggests that the effects of roscovitine could be due in part to altering Cdk5 translocation in other systems as well. Kinase activity was inhibited by roscovitine in assays using AX3 whole cell lysates, but not in assays using lysates from Cdk5-GFP over-expressing cells. At higher concentrations, roscovitine impaired slug and fruiting body formation. Fruiting bodies that did form were small and produced relatively fewer spores many of which were round. However, roscovitine did not affect stalk cell differentiation. Together with previous findings, these data reveal that roscovitine inhibits Cdk5 during growth and as yet undefined Cdks during mid-late development.     

Polyamine depletion enhances the roscovitine-induced apoptosis through the activation of mitochondria in HCT116 colon carcinoma cells

Small molecule inhibitors of cyclin-dependent kinases (CDKs) show high therapeutic potential in various cancer types which are characterized by the accumulation of transformed cells due to impaired apoptotic machinery. Roscovitine, a CDK inhibitor showed to be a potent apoptotic inducer in several cancer cells. Polyamines, putrescine, spermidine and spermine, are biogenic amines involved in many cellular processes, including apoptosis. In this study, we explored the potential role of polyamines in roscovitine-induced apoptosis in HCT116 colon cancer cells. Roscovitine induced apoptosis by activating mitochondrial pathway caspases and modulating the expression of Bcl-2 family members. Depletion of polyamines by treatment with difluoromethylornithine (DFMO) increased roscovitine-induced apoptosis. Transient silencing of ornithine decarboxylase, polyamine biosynthesis enzyme and special target of DFMO also increased roscovitine-induced apoptosis in HCT116 cells. Interestingly, additional putrescine treatment was found pro-apoptotic due to the presence of non-functional ornithine decarboxylase (ODC). Finally, roscovitine altered polyamine catabolic pathway and led to decrease in putrescine and spermidine levels. Therefore, the metabolic regulation of polyamines may dictate the power of roscovitine induced apoptotic responses in HCT116 colon cancer cells.     

Neurotrophic Factors Prevent Ceramide-Induced Apoptosis Downstream of c-Jun N-Terminal Kinase Activation in PC12 Cells

Abstract: Neurotrophic factors prevent apoptosis of PC12 cells in serum-free medium. The present study determines whether neurotrophic factors can prevent ceramide-induced apoptosis in PC12 cells and investigates the role that c-Jun N-terminal kinase (JNK) activation may play in this system. Ceramide-induced apoptosis was inhibited by nerve growth factor, basic fibroblast growth factor, pituitary adenylyl cyclase-activating peptide, 4-(8-chlorophenylthio)cyclic AMP, and the caspase inhibitor benzyloxycarbonyl-Val-Ala- dl -Asp fluoromethyl ketone (zVAD-FMK). It was surprising that inhibition of extracellular signal-regulated kinase and/or phosphatidylinositol 3-kinase did not markedly block the protective effects exerted by neurotrophic factors against ceramide-induced apoptosis, suggesting that neurotrophic factors can promote survival independently of these signaling pathways. Treatment of PC12 cells with ceramide resulted in a time-dependent increase in JNK activity. However, neither neurotrophic factors nor zVAD-FMK attenuated ceramide-stimulated JNK activation. Further experiments indicated that ceramide-induced apoptosis in PC12 cells requires new protein synthesis, and that nerve growth factor and zVAD-FMK can prevent apoptosis after JNK activity has been detected. These results indicate that ceramide-induced JNK activation is an early event and may be required for the expression of essential components of the apoptotic machinery. It is anticipated that neurotrophic factors inhibit ceramide-induced apoptosis by affecting signaling events downstream of JNK activation.     

Cdk5 Modulation of mitogen-activated protein kinase signaling regulates neuronal survival

Cdk5, a cyclin-dependent kinase, is critical for neuronal development, neuronal migration, cortical lamination, and survival. Its survival role is based, in part, on "cross-talk" interactions with apoptotic and survival signaling pathways. Previously, we showed that Cdk5 phosphorylation of mitogen-activated protein kinase kinase (MEK)1 inhibits transient activation induced by nerve growth factor (NGF) in PC12 cells. To further explore the nature of this inhibition, we studied the kinetics of NGF activation of extracellular signal-regulated kinase (Erk)1/2 in cortical neurons with or without roscovitine, an inhibitor of Cdk5. NGF alone induced an Erk1/2-transient activation that peaked in 15 min and declined rapidly to baseline. Roscovitine, alone or with NGF, reached peak Erk1/2 activation in 30 min that was sustained for 48 h. Moreover, the sustained Erk1/2 activation induced apoptosis in cortical neurons. Significantly, pharmacological application of the MEK1 inhibitor PD98095 to roscovitine-treated cortical neurons prevented apoptosis. These results were also confirmed by knocking down Cdk5 activity in cortical neurons with Cdk5 small interference RNA. Apoptosis was correlated with a significant shift of phosphorylated tau and neurofilaments from axons to neuronal cell bodies. These results suggest that survival of cortical neurons is also dependent on tight Cdk5 modulation of the mitogen-activated protein kinase signaling pathway.     

Protein synthesis and mRNA storage in cattle oocytes maintained under meiotic block by roscovitine inhibition of MPF activity

Roscovitine, a specific inhibitor of MPF kinase activity, has been shown to block efficiently and reversibly the meiotic resumption of oocytes from different species, including cattle. In view to verify that oocytes maintain germinal vesicle like molecular activities under roscovitine treatment, we compared in the present study the M-phase Promoting Factor (MPF) and Mitogen Activated Protein (MAP) kinase activities; protein synthesis and phosphorylation patterns in oocytes and cumulus cells; and CDK1 and Cyclin B messengers storage under control culture and under roscovitine inhibition. We observed that roscovitine induced a full and reversible inhibition of MPF kinase activity and of the activating phosphorylation of both ERK1/2 MAPK. During in vivo maturation, there was a highly significant increase in the relative mRNA level of both cyclin B1 and CDK1 whereas during in vitro culture, the relative amount of CDK1 messenger was reduced. These messengers may be used as markers for the optimization of in vitro maturation treatment. Roscovitine reversibly prevented this drop in relative quantities of CDK1 messenger. Oocytes cultured in the presence of roscovitine maintained a GV like profile of protein synthesis except that two proteins of 48 and 64 kDa specific of matured oocytes also appeared under roscovitine treatment. However, roscovitine did not prevent most of the modifications of protein phosphorylation pattern observed during maturation. In conclusion, results of this study revealed that the use of roscovitine did not prevent all the events related to maturation of bovine oocytes.     

Requirement of p38 stress-activated MAP kinase for cell death in the developing retina depends on the stage of cell differentiation

The p38 members of the mitogen-activated protein kinase (MAPK) superfamily are activated by both environmental stress and endogenous signals, and may have either permissive or inhibitory roles upon both cell proliferation and cell death in the retina. We have previously shown that anisomycin, a protein synthesis inhibitor, and 2-aminopurine, a specific inhibitor of the double stranded-RNA dependent protein kinase, block apoptosis of ganglion cells induced by axotomy, and induce apoptosis of cells in the neuroblastic layer in developing rat retina. Using a specific inhibitor, we found that p38-stress activated MAP kinase is required for the death of post-mitotic cells induced by anisomycin, but not for the death of proliferating cells induced by 2-aminopurine, nor of axon-damaged retinal ganglion cells. We also show that p38 activation occurs either upstream of or parallel to the requirement for cyclic AMP to block apoptosis of post-mitotic cells, since the cyclic AMP-producing agent forskolin did not prevent p38 phosphorylation induced by anisomycin. Finally, the lack of immunostaining for phospho-p38 in apoptotic profiles suggests that p38 activation does not kill retinal cells directly, but more likely through the mediation of neighboring cells.     

Key role for cyclin-dependent kinases in the first and second meiotic divisions of rat spermatocytes

In all systems examined so far, the G2/M phase transition is controlled by the M-phase promoting factor (MPF), a complex of cdc2 (CDK1) and cyclin B1. Histone H1 kinase activity and MPF components are present in pachytene spermatocytes (PS). However, it has not been demonstrated yet that direct inhibition of MPF activity prevents the G2/M transition in these cells. When roscovitine, a potent inhibitor of CDK1, CDK2, and CDK5 activities, was added to cocultures of PS with Sertoli cells, the number of both secondary spermatocytes and round spermatids formed were lower than in control cultures, despite similar cell viability. This effect of roscovitine was reversible, did not involve the Sertoli cells, and was dependent on the concentration of the inhibitor. Roscovitine did not modify the amount of MPF in these germ cells but inhibited the CDK1- or CDK2-associated histone H1 kinase activity of PS. Hence a functional relationship between cyclin-dependent kinase activity and the spontaneous processing of the first meiotic division and, for the first time, of the second meiotic division of male germ cells is shown.     

Angiostatin-induced inhibition of endothelial cell proliferation/apoptosis is associated with the down-regulation of cell cycle regulatory protein cdk5

Endothelial cells (ECs) are quiescent in normal blood vessels, but undergo rapid bursts of proliferation after vascular injury, hypoxia or induced by powerful angiogenic cytokines like fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF). Deregulated proliferation of ECs facilitates angiogenic processes and promotes tumor growth. In dividing cells, cell cycle-associated protein kinases, which are referred as cyclin-dependent kinases (cdks), regulate proliferation, differentiation, senescence, and apoptosis. Cyclin-dependent kinase-5 (cdk5) is expressed in neuronal cells and plays an important role in neurite outgrowth, of neuronal migration and neurogenesis, its functions in non-neuronal cells are unclear. Here, we show for the first time that the cdk5 is expressed at high levels in proliferating bovine aortic endothelial (BAE) cells, by contrast insignificant low levels of cdk5 expression in quiescent BAE cells. In addition, bFGF up-regulates cdk5 expression in a dose-dependent fashion. Interestingly, temporal expression data suggests that cdk5 expression is very low between 24-48 h, but high level of cdk5 expression was detected during 60-72 h. This later time corresponds to the time of completion of one cell cycle (doubling of cell population) of BAE cell culture. Angiostatin (AS), a powerful inhibitor of angiogenesis inhibits ECs proliferation in dose-dependent manner with concomitant down-regulation of cdk5 expression. The role of cdk5 in ECs, proliferation and apoptosis was confirmed by selective inhibition of cdk5 expression by the purine derivative roscovitine, which inhibits bFGF-stimulated BAE cells proliferation and induces apoptosis in dose-specific manner. By contrast, the roscovitine analog olomoucine, which is a specific inhibitor of cdk4, but not of cdk5 failed to affect ECs proliferation and apoptosis. These data suggest for the first time that neuron specific protein cdk5 may have significant role in the regulation of ECs proliferation, apoptosis, and angiogenesis and extends beyond its role in neurogenesis.     

Ethanol Induces Apoptosis in Cerebellar Granule Neurons by Inhibiting Insulin-Like Growth Factor 1 Signaling

Abstract: The ability of ethanol to interfere with insulin-like growth factor 1 (IGF-1)-mediated cell survival was examined in primary cultured cerebellar granule neurons. Cells underwent apoptosis when switched from medium containing 25 m M K⁺ to one containing 5 m M K⁺. IGF-1 protected granule neurons from apoptosis in medium containing 5 m M K⁺. Ethanol inhibited IGF-1-mediated neuronal survival but did not inhibit IGF-1 receptor binding or the neurotrophic action of elevated K⁺, and failed to potentiate cell death in the presence of 5 m M K⁺. Inhibition of neuronal survival by ethanol was not reversed by increasing the concentration of IGF-1. Significant inhibition by ethanol (15–20%) was observed at 1 m M and was half-maximal at 45 m M . The inhibition of IGF-1 protection by ethanol corresponded to a marked reduction in the phosphorylation of insulin receptor substrate 1, the binding of phosphatidylinositol 3-kinase (PI 3-kinase), and a block of IGF-1-stimulated PI 3-kinase activity. The neurotrophic response of IGF-1 was also inhibited by the PI 3-kinase inhibitor LY294002, the protein kinase C inhibitor chelerythrine chloride, and the protein kinase A inhibitor KT5720, but unaffected by the mitogen-activated protein kinase kinase inhibitor PD 98059. These data demonstrate that ethanol promotes cell death in cerebellar granule neurons by inhibiting the antiapoptotic action of IGF-1.     

Caspase-3在roscovitine诱发PC12细胞凋亡中发挥重要作用

我们已证实周期蛋白激酶（cyclin-dependent kinases）cdk2、cdc2和cdk5抑制剂roscovitine诱导PC12细胞凋亡。本实验应用caspase-3免疫细胞化学与hoechst 33342荧光化学双标、MTT比色法细胞活性测定和Western blot方法,研究了caspase-3在roscovitine所致PC12细胞凋亡中的作用。结果显示,roscovitine（50μmol／L）处理PC12细胞12h,细胞核染色质凝缩及核碎片形成,同时胞浆中出现caspase-3阳性标志,caspase-3阳性细胞占细胞总数的42％。非特异性caspases抑制剂Z-VAD-FMK（50μmol／L）和caspase-3特异性抑制剂Z-DEVD-FMK（100μmol／L）可部分降低roscovitine所致的细胞死亡,使细胞存活率分别由29．03％（roscovitine）增至58．06％（Z-VAD-FMK＋roscovitine）和45．16％（Z-DEVD-FMK＋roscovitine）：用单克隆non-erythroid α-spectrin抗体检测roscovitine处理组细胞匀浆提取液,表明caspase-3裂解的特异性spectfin 120kDa蛋白产物较对照组显著增加。提示细胞凋亡成分caspases参与roscovitine所敛的细胞凋亡,其中caspase-3发挥重要作用。