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.     

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.     

Apoptosis of Central and Peripheral Neurons Can Be Prevented with Cyclin-Dependent Kinase/Mitogen-Activated Protein Kinase Inhibitors

Abstract: Previous studies have indicated that certain members of the cyclin-dependent kinase/mitogen-activated protein kinase superfamily are involved in apoptosis of neuronal cells. Here, we have examined programmed cell death induced by withdrawal of neurotrophic support from CNS (rat retinal) and PNS (chick sympathetic, sensory, and ciliary) neurons. All four neuron types were equally rescued by the purine analogues olomoucine and roscovitine. Olomoucine inhibits multiple cyclin-dependent and mitogen-activated protein kinases with similar potency. Roscovitine is a more selective cyclin-dependent kinase inhibitor; but, so is butyrolactone I, which did not prevent retinal ganglion cell death. The specific p38^MAPK inhibitor SB-203580 did not prevent apoptosis in retinal ganglion cells. Death of these cells in the absence of neurotrophic factors was accompanied by morphological changes indicative of apoptosis, including nuclear condensation and fragmentation. Treatment with olomoucine or roscovitine not only prevented these apoptotic changes in retinal ganglion cells but also blocked neurite outgrowth. The survival-promoting activity of olomoucine correlated with its in vitro IC₅₀ for c-Jun N-terminal kinase-1 and its potency to repress c- jun induction in live PC12 cells. Roscovitine was more potent in rescuing neurons than in inhibiting Jun kinase. Thus, the antiapoptotic action of roscovitine might be due to inhibition of additional kinases.     

Cables links Cdk5 and c-Abl and facilitates Cdk5 tyrosine phosphorylation, kinase upregulation, and neurite outgrowth

Cyclin-dependent kinase 5 (Cdk5) is a small serine/threonine kinase that plays a pivotal role during development of the CNS. Cables, a novel protein, interacts with Cdk5 in brain lysates. Cables also binds to and is a substrate of the c-Abl tyrosine kinase. Active c-Abl kinase leads to Cdk5 tyrosine phosphorylation, and this phosphorylation is enhanced by Cables. Phosphorylation of Cdk5 by c-Abl occurs on tyrosine 15 (Y15), which is stimulatory for p35/Cdk5 kinase activity. Expression of antisense Cables in primary cortical neurons inhibited neurite outgrowth. Furthermore, expression of active Abl resulted in lengthening of neurites. The data provide evidence for a Cables-mediated interplay between the Cdk5 and c-Abl signaling pathways in the developing nervous system.     

Involvement of Calpain/p35-p25/Cdk5/NMDAR Signaling Pathway in Glutamate-Induced Neurotoxicity in Cultured Rat Retinal Neurons

We investigated possible involvement of a calpain/p35-p25/cyclin-dependent kinase 5 (Cdk5) signaling pathway in modifying NMDA receptors (NMDARs) in glutamate-induced injury of cultured rat retinal neurons. Glutamate treatment decreased cell viability and induced cell apoptosis, which was accompanied by an increase in Cdk5 and p-Cdk5(T15) protein levels. The Cdk5 inhibitor roscovitine rescued the cell viability and inhibited the cell apoptosis. In addition, the protein levels of both calpain 2 and calpain-specific alpha-spectrin breakdown products (SBDPs), which are both Ca(2+)-dependent, were elevated in glutamate-induced cell injury. The protein levels of Cdk5, p-Cdk5(T15), calpain 2 and SBDPs tended to decline with glutamate treatments of more than 9 h. Furthermore, the elevation of SBDPs was attenuated by either D-APV, a NMDAR antagonist, or CNQX, a non-NMDAR antagonist, but was hardly changed by the inhibitors of intracellular calcium stores dantrolene and xestospongin. Moreover, the Cdk5 co-activator p35 was significantly up-regulated, whereas its cleaved product p25 expression showed a transient increase. Glutamate treatment for less than 9 h also considerably enhanced the ratio of the Cdk5-phosphorylated NMDAR subunit NR2A at Ser1232 site (p-NR2A(S1232)) and NR2A (p-NR2A(S1232)/NR2A), and caused a translocation of p-NR2A(S1232) from the cytosol to the plasma membrane. The enhanced p-NR2A(S1232) was inhibited by roscovitine, but augmented by over-expression of Cdk5. Calcium imaging experiments further showed that intracellular Ca(2+) concentrations ([Ca(2+)](i)) of retinal cells were steadily increased following glutamate treatments of 2 h, 6 h and 9 h. All these results suggest that the activation of the calpain/p35-p25/Cdk5 signaling pathway may contribute to glutamate neurotoxicity in the retina by up-regulating p-NR2A(S1232) expression.     

Aspirin provides cyclin-dependent kinase 5-dependent protection against subsequent hypoxia/reoxygenation damage in culture

Aspirin [acetylsalicylic acid (ASA)] is an anti-inflammatory drug that protects against cellular injury by inhibiting cyclooxygenases (COX), inducible nitric oxide synthase (iNOS) and p44/42 mitogen-activated protein kinase (p44/42 MAPK), or by preventing translocation of nuclear factor kappaB (NF-kappaB). We studied the effect of ASA pre-treatment on neuronal survival after hypoxia/reoxygenation damage in rat spinal cord (SC) cultures. In this injury model, COX, iNOS and NF-kappaB played no role in the early neuronal death. A 20-h treatment with 3 mm ASA prior to hypoxia/reoxygenation blocked the hypoxia/reoxygenation-induced lactate dehydrogenase (LDH) release from neurons. This neuroprotection was associated with increased phosphorylation of neurofilaments, which are substrates of p44/42 MAPK and cyclin-dependent kinase 5 (Cdk5). PD90859, a p44/42 MAPK inhibitor, had no effect on ASA-induced tolerance, but olomoucine and roscovitine, Cdk5 inhibitors, reduced ASA neuroprotection. Hypoxia/reoxygenation alone reduced both the protein amount and activity of Cdk5, and this reduction was inhibited by pre-treatment with ASA. Moreover, the protein amount of a neuronal Cdk5 activator, p35, recovered after reoxygenation only in ASA-treated samples. The prevention of the loss in Cdk5 activity during reoxygenation was crucial for ASA-induced protection, because co-administration of Cdk5 inhibitors at the onset ofreoxygenation abolished the protection. In conclusion, pre-treatment with ASA induces tolerance against hypoxia/reoxygenation damage in spinal cord cultures by restoring Cdk5 and p35 protein expression.     

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.     

Cadherins and synaptic plasticity: activity-dependent cyclin-dependent kinase 5 regulation of synaptic beta-catenin-cadherin interactions

Cyclin-dependent kinase 5 (Cdk5)/p35 kinase activity is known to decrease the affinity of beta-catenin for cadherin in developing cortical neurons. Our recent work demonstrated that depolarization causes an increased affinity between beta-catenin and cadherin. Here, we examine whether Cdk5/p35 regulates beta-catenin-cadherin affinity in response to neural activity. In hippocampal neurons depolarization caused a significant decrease in Cdk5 kinase activity, without changing the protein levels of either Cdk5 or p35, suggesting that the proteasome pathway is not involved. Decreasing Cdk5 kinase activity with the inhibitor roscovitine increased the amount of beta-catenin that was co-immunoprecipitated with cadherin. Inhibiting Cdk5 activity also resulted in a redistribution of EGFP-beta-catenin from the dendritic shaft to the spines, where cadherins are highly concentrated. The redistribution of beta-catenin induced by roscovitine is similar to that induced by depolarization. Interestingly, the redistribution induced by the Cdk5 inhibitor was completely blocked by either a tyrosine phosphatase inhibitor, orthovanadate or by point mutations of beta-catenin Tyr-654 to Glu or Phe. Immunoprecipitation studies further revealed that roscovitine increases the affinity of the wild-type, but not mutated, EGFP-beta-catenin for cadherin. These results suggest that Cdk5 activity regulates the affinity of beta-catenin for cadherin by changing the phosphorylation level of beta-catenin Tyr-654.     

Bestatin inhibits cell growth, cell division, and spore cell differentiation in Dictyostelium discoideum

Bestatin methyl ester (BME) is an inhibitor of Zn(2+)-binding aminopeptidases that inhibits cell proliferation and induces apoptosis in normal and cancer cells. We have used Dictyostelium as a model organism to study the effects of BME. Only two Zn(2+)-binding aminopeptidases have been identified in Dictyostelium to date, puromycin-sensitive aminopeptidase A and B (PsaA and PsaB). PSA from other organisms is known to regulate cell division and differentiation. Here we show that PsaA is differentially expressed throughout growth and development of Dictyostelium, and its expression is regulated by developmental morphogens. We present evidence that BME specifically interacts with PsaA and inhibits its aminopeptidase activity. Treatment of cells with BME inhibited the rate of cell growth and the frequency of cell division in growing cells and inhibited spore cell differentiation during late development. Overexpression of PsaA-GFP (where GFP is green fluorescent protein) also inhibited spore cell differentiation but did not affect growth. Using chimeras, we have identified that nuclear versus cytoplasmic localization of PsaA affects the choice between stalk or spore cell differentiation pathway. Cells that overexpressed PsaA-GFP (primarily nuclear) differentiated into stalk cells, while cells that overexpressed PsaAΔNLS2-GFP (cytoplasmic) differentiated into spores. In conclusion, we have identified that BME inhibits cell growth, division, and differentiation in Dictyostelium likely through inhibition of PsaA.     

GTP-dependent secretion from neutrophils is regulated by Cdk5

We have previously shown evidence for the existence of a calcium-independent, GTP-regulated mechanism of secretion from neutrophils, but this secretory mechanism remains to be fully elucidated. Cyclin-dependent kinase 5 (Cdk5), the various substrates of which include Munc18 and synapsin 1, has been implicated in neuronal secretion. Although the Cdk5 activator, p35, and Cdk5-p35 activity are primarily associated with neurons, we report here that p35 also exists in neutrophils and that an active Cdk5-p35 complex is present in these cells. Cdk5-p35 activity in human neutrophils is mostly localized in secretory granules, which show an increase in Cdk5-p35 level and activity upon GTP stimulation. The potent Cdk5 inhibitor, roscovitine, completely blocks GTP-stimulated granule Cdk5 activity, which accompanies lactoferrin secretion from neutrophil-specific granules. Roscovitine also inhibits GTP-induced lactoferrin secretion and surface localization of the secretion markers, CD63 and CD66b, to a certain extent. Furthermore, neutrophils from wild-type mice treated with roscovitine and neutrophils from p35(-/-) mice exhibit comparable surface expression levels of both CD63 and CD66b upon GTP stimulation. Although our data suggest that other molecules control GTP-induced secretion from neutrophils, it is clear that Cdk5-p35 is required to elicit the maximum GTP-induced secretory response. Our observation that multiple proteins in neutrophil granules serve as specific substrates of Cdk5 further supports the premise that the kinase is a key component of the GTP-regulated secretory apparatus in neutrophils.     

Roscovitine in combination with calcium ionophore induces oocyte activation through reduction of M-phase promoting factor activity in mice

Summary The aim of the present study was to determine oocyte activation and change in M-phase promoting factor (MPF) activity induced by treatment with calcium ionophore and roscovitine in comparison with those induced by treatment with roscovitine alone and treatment with calcium ionophore and puromycin in mice. Freshly ovulated oocytes obtained from 6-8-week-old mice were divided into five groups (no activation treatment; 5 μM calcium ionophore A23187; 50 μM roscovitine; 5 μM calcium ionophore and 10 μg/ml puromycin; and 5 μM calcium ionophore and 50 μM roscovitine) and were incubated for 6 h. Oocyte activation, assessed by morphological changes, and changes in MPF activity in the five groups at 0, 2, 4 and 6 h of incubation were examined. Activated oocytes were defined as oocytes with at least one pronucleus. Oocytes treated with roscovitine alone were not activated during the 6-h incubation period. All of the oocytes in the calcium ionophore with puromycin group and in the calcium ionophore with roscovitine group were activated. The percentage activity of MPF in oocytes treated with roscovitine alone was decreased after 2 h and increased after 4 h of incubation. The percentage activity of MPF in oocytes treated with calcium ionophore and roscovitine was significantly decreased with suppression of MPF activity being maintained for 6 h, and this change was similar to that in oocytes treated with calcium ionophore and puromycin. Roscovitine with calcium ionophore is effective for induction of oocyte activation through suppression of MPF activity in mice.     

Regulation of N-cadherin-mediated adhesion by the p35-Cdk5 kinase

BACKGROUND: The p35-Cdk5 kinase has been implicated in a variety of functions in the central nervous system (CNS), including axon outgrowth, axon guidance, fasciculation, and neuronal migration during cortical development. In p35(-/-) mice, embryonic cortical neurons are unable to migrate past their predecessors, leading to an inversion of cortical layers in the adult cortex. RESULTS: In order to identify molecules important for p35-Cdk5-dependent function in the cortex, we screened for p35-interacting proteins using the two-hybrid system. In this study, we report the identification of a novel interaction between p35 and the versatile cell adhesion signaling molecule beta-catenin. The p35 and beta-catenin proteins interacted in vitro and colocalized in transfected COS cells. In addition, the p35-Cdk5 kinase was associated with a beta-catenin-N-cadherin complex in the cortex. In N-cadherin-mediated aggregation assays, inhibition of Cdk5 kinase activity using the Cdk5 inhibitor roscovitine led to the formation of larger aggregates of embryonic cortical neurons. This finding was recapitulated in p35(-/-) cortical neurons, which aggregated to a greater degree than wild-type neurons. In addition, introduction of active p35-Cdk5 kinase into COS cells led to a decreased beta-catenin-N-cadherin interaction and loss of cell adhesion. CONCLUSIONS: The association between p35-Cdk5 and an N-cadherin adhesion complex in cortical neurons and the modulation of N-cadherin-mediated aggregation by p35-Cdk5 suggests that the p35-Cdk5 kinase is involved in the regulation of N-cadherin-mediated adhesion in cortical neurons.     

Abl deregulates Cdk5 kinase activity and subcellular localization in Drosophila neurodegeneration

Although Abl functions in mature neurons, work to date has not addressed Abl's role on Cdk5 in neurodegeneration. We found that beta-amyloid (Abeta42) initiated Abl kinase activity and that blockade of Abl kinase rescued both Drosophila and mammalian neuronal cells from cell death. We also found activated Abl kinase to be necessary for the binding, activation, and translocalization of Cdk5 in Drosophila neuronal cells. Conversion of p35 into p25 was not observed in Abeta42-triggered Drosophila neurodegeneration, suggesting that Cdk5 activation and protein translocalization can be p25-independent. Our genetic studies also showed that abl mutations repressed Abeta42-induced Cdk5 activity and neurodegeneration in Drosophila eyes. Although Abeta42 induced conversion of p35 to p25 in mammalian cells, it did not sufficiently induce Cdk5 activation when c-Abl kinase activity was suppressed. Therefore, we propose that Abl and p35/p25 cooperate in promoting Cdk5-pY15, which deregulates Cdk5 activity and subcellular localization in Abeta42-triggered neurodegeneration.     

Cyclin-dependent kinase 2 (Cdk2) is required for centrosome duplication in mammalian cells

Centrosome duplication is indispensable for the formation of the bipolar mitotic spindle. Surprisingly, even if DNA replication or mitosis is inhibited, centrosome duplication can still occur [1] [2] [3] [4] [5]. Thus, it remains unknown how centrosome duplication is coordinated with the cell cycle. Here, we show that centrosome duplication requires cyclin-dependent kinase 2 (Cdk2) in mammalian cells. We have found that in Chinese hamster ovary (CHO) cells, whereas centrosome duplication is not inhibited by hydroxyurea (HU) treatment, which arrests the cells in S phase, it is inhibited by mimosine treatment, which arrests the cells in late G1 phase. Cdk2 activity was higher in HU-treated cells than in mimosine-treated cells. Remarkably, inhibition of the Cdk2 activity in HU-treated cells with butyrolactone I or roscovitine [6], or by expression of the Cdk inhibitor p21(Waf1/Cip1), blocked the continued centrosome duplication. Moreover, overexpression of Cdk2 reversed the inhibition of centrosome duplication by mimosine treatment. These results indicate a requirement of Cdk2 activity for centrosome duplication and therefore suggest an underlying mechanism for the coordination of centrosome duplication with the cell cycle.     

Inhibition of Cdk6 expression through p38 MAP kinase is involved in differentiation of mouse prechondrocyte ATDC5

Because a temporal arrest in the G1-phase of the cell cycle is a prerequisite for cell differentiation, this study investigated the involvement of cell cycle factors in the differentiation of cultured mouse prechondrocyte cell line ATDC5. Among the G1 cell cycle factors examined, both protein and mRNA levels of cyclin-dependent kinase (Cdk6) were downregulated during the culture in a differentiation medium. The protein degradation of Cdk6 was not involved in this downregulation because proteasome inhibitors did not reverse the protein level. When inhibitors of p38 MAPK, ERK-1/2, and PI3K/Akt were added to the culture, only a p38 MAPK inhibitor SB203580 blocked the decrease in the Cdk6 protein level by the differentiation medium, indicating that the Cdk6 inhibition was mediated by p38 MAPK pathway. In fact, p38 MAPK was confirmed to be phosphorylated during differentiation of ATDC5 cells. Enforced expression of Cdk6 in ATDC5 cells blocked the chondrocyte differentiation and inhibited Sox5 and Sox6 expressions. However, the Cdk6 overexpression did not affect the proliferation or the cell cycle progression, suggesting that the inhibitory effect of Cdk6 on the differentiation was exerted by a mechanism largely independent of its cell cycle regulation. These results indicate that Cdk6 may be a regulator of chondrocyte differentiation and that its p38-mediated downregulation is involved in the efficient differentiation.     

Effects of cell cycle dependent kinases inhibitor on nuclear and cytoplasmic maturation of porcine oocytes

The aims of this study were to assess the effectiveness of roscovitine, a potent inhibitor of cell cyclin kinases, to prevent meiotic resumption in porcine oocytes, and to test the subsequent fertilisability and developmental competence of these oocytes. Roscovitine blocked porcine oocytes at the GV stage during 22-44 hr of culture. This effect was dose-dependent, and a concentration of 25 microM was sufficient to prevent meiotic resumption in 92+/-5% of the oocytes after 22 hr in the presence of EGF and FSH. Cumulus expansion was also inhibited under these conditions. The histone H1 kinase activity in oocytes was inhibited in a dose-dependent way, and maintained at a basal level with 25 microM of roscovitine. Synthesis of proteins of 29, 47 and 79 kDa, normally synthesized during maturation, was inhibited too. All these effects were fully reversible. However, the kinetics of maturation were accelerated after roscovitine removal, and the acceleration was more pronounced after 44 hr of inhibition than after 22 hr. Fertilization of oocytes blocked for 22 hr before a 44 hr maturation was decreased compared to control, but was not different from that of oocytes matured for 66 hr. The developmental competence was decreased for the oocytes cultured for 66 hr, including or not an inhibition period, but it was less reduced for oocytes maintained under inhibition for 22 hr. Roscovitine may thus protect oocytes against the aging mechanisms responsible for developmental competence loss, but not against loss of fertilisability. In conclusion, roscovitine provides a useful tool to study the morphological and biochemical basis of porcine oocyte terminal differentiation.     

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.     

Modulating zucchini cotyledon plate meristem activity by interactions between the cycline-dependent kinase inhibitor roscovitine and cytokinins

The inhibitors of cytokinin N-glucosylation are known to influence the growth of some plant objects including cotyledons. The use of the plate meristem of zucchini cotyledon as an experimental system allowed us to study for the first time the way in which the changes in the cell division are integrated in this growth reaction. Roscovitine, a potent inhibitor of cytokinin N-glucosylation and cycline-dependent kinases, did not show to have an effect on the meristem activity when applied in 100 μM to cultivated zucchini cotyledons, and acted as an inhibitor in concentrations higher than 400 μM. A 200 μM roscovitine stimulated both palisade cell division and growth. In different seed batches, 400 μM roscovitine acted as a stimulator or an inhibitor. A much stronger stimulating effect on growth and cell division was observed after application of benzyladenine (BA, 10 μM). In contrast to BA, roscovitine provoked a formation of principally flat lamina. In combined treatments, it lowered the stimulating effect of BA; 400 μM roscovitine combined with BA severely suppressed the growth and division activity. This cellular behavior and changes in cotyledon growth could be due to the roscovitine-provoked changes in endogenous cytokinin levels via the inhibition of cytokinin N-glucosylation. Roscovitine-caused stimulation of cell growth and division is stronger in the marginal meristem than that registered in central regions of the cotyledon blade. In this region it also changed the pattern of cell division and lowered the adhesion between the clusters, which enhanced the appearance of local ruptures of the cotyledon edges. The first palisade layer of the plate meristem of cultured zucchini cotyledons, the natural mono-layer of proliferating palisade cells, may be used for screening the inhibitors of cycline-dependent kinases and cytokinin N-glucosylation with regard to their effects on cell division and growth.