植物细胞周期调控因子及其功能的研究进展

细胞周期调控因子能通过影响细胞周期对植物细胞的生长、分裂和分化产生作用,进而调节植物的生长发育。本文综述了近几年来植物细胞周期调控因子中细胞周期蛋白（cyclin,CYC）、周期蛋白依赖激酶（cyclin-dependent kinase,CDK）等的作用机理及研究进展,阐述了各调控因子在植物生长发育过程中的作用。     

周期蛋白和周期蛋白依赖性激酶及相关激酶抑制剂在细胞周期进程中的调控机制研究进展

在细胞发育过程中,细胞周期起着至关重要的作用。细胞周期进程主要受细胞周期蛋白依赖性激酶(cyclin dependent kinase, CDK)、周期蛋白和内源性CDK抑制剂(cyclin-dependent kinase inhibitors,CKI)调控。其中,CDK是主要的细胞周期调节因子,可与周期蛋白结合形成周期蛋白-CDK复合物,从而使数百种底物磷酸化,调控分裂间期和有丝分裂进程。各类细胞周期蛋白的活性异常,可引起不受控制的癌细胞增殖,导致癌症的发生与发展。因此,了解CDK的活性变化情况、周期蛋白-CDK的组装以及CKI的作用,将有助于了解细胞周期进程中潜在的调控过程,为癌症与疾病的治疗和CKI治疗药物的研发提供基础。本文关注了CDK激活和灭活的关键事件,并总结了周期蛋白-CDK在特定时期及位置的调控过程,以及相关CKI治疗药物在癌症及疾病中的研究进展,最后简单阐述了细胞周期进程研究面临的问题和存在的挑战,以期为后续细胞周期进程的深入研究提供参考和思路。     

Cyclin D1, cdk4, and Bim are involved in thrombin-induced apoptosis in cultured cortical neurons

Thrombin, a multifunctional serine protease, is neurotoxic in vitro and in vivo. Thrombin has been shown to be increased in Alzheimer's disease (AD) and other neuropathological conditions and could be a mediator of pathological neuronal cell death in the brain. The mechanisms of thrombin-induced neuronal cell death are incompletely understood. The objective of this study is to explore mechanisms that contribute to thrombin-induced neuronal apoptosis focusing on the role of cell cycle regulators and the pro-apoptotic protein Bim (Bcl-2-interacting mediator of cell death) in this process. Our data show that thrombin treatment of primary cerebral cortical cultures results in dose-dependent apoptotic cell death. Exposure of neuronal cultures to thrombin leads to induction of cell cycle proteins cyclin D1 and cyclin E, at both mRNA and protein levels. In addition, thrombin treatment causes the appearance of cyclin-dependent kinase 4 (cdk4) and expression of the pro-apoptotic protein Bim. Inhibition of cdk4 prevents both induction of Bim expression and thrombin-induced neuronal apoptosis. These data demonstrate that thrombin-induced apoptosis proceeds via cell cycle activation involving cdk4 resulting in induction of Bim. Thus, cell cycle proteins could be therapeutic targets in diseases such as AD where thrombin has been implicated.     

The Mck1 GSK-3 kinase inhibits the activity of Clb2-Cdk1 post-nuclear division

The glycogen synthase kinase-3 homolog, Mck1, has been implicated in many cellular functions, from sporulation to calcium stress response in budding yeast. Here, we report a novel function for Mck1 in the inhibition of Clb2-Cdk1 activity post nuclear division. Clb2-Cdk1, the major mitotic cyclin-Cdk complex in yeast, accumulates before anaphase and must be inhibited in telophase for cells to exit mitosis and enter into the next cell cycle. We show that the mck1Δ mutant is highly sensitive to increased Clb2-Cdk1 activity caused either by overexpression of Clb2 or the Cdk1-activating phosphatase Mih1. Deletion of the Cdk1 inhibitory kinase, SWE1, in combination with a mck1Δ mutant results in a synthetic growth defect, suggesting that Mck1 and Swe1 function in parallel pathways to inhibit Clb2-Cdk1. We find that mck1Δ strains have a delay in mitotic exit as well as elevated levels of Clb2-Cdk1 activity post-nuclear division. Using a co-immunoprecipitation assay, we identify a physical interaction between Mck1 and both Clb2 and Mih1. Finally, we demonstrate that phosphorylation of purified Clb2 by Cdk1 is inhibited by catalytically active Mck1 but not catalytically inactive Mck1 in vitro. We propose that Mck1 inhibits the activity of Clb2-Cdk1 via interaction with Clb2. The mammalian glycogen synthase kinase-3 homolog has been implicated in cyclin inhibition, suggesting a conserved cell cycle function for both yeast and mammalian glycogen synthase kinases.     

The Mck1 GSK-3 kinase inhibits the activity of Clb2-Cdk1 post-nuclear division

The glycogen synthase kinase-3 homolog, Mck1, has been implicated in many cellular functions, from sporulation to calcium stress response in budding yeast. Here, we report a novel function for Mck1 in the inhibition of Clb2-Cdk1 activity post nuclear division. Clb2-Cdk1, the major mitotic cyclin-Cdk complex in yeast, accumulates before anaphase and must be inhibited in telophase for cells to exit mitosis and enter into the next cell cycle. We show that the mck1Δ mutant is highly sensitive to increased Clb2-Cdk1 activity caused either by overexpression of Clb2 or the Cdk1-activating phosphatase Mih1. Deletion of the Cdk1 inhibitory kinase, SWE1, in combination with a mck1Δ mutant results in a synthetic growth defect, suggesting that Mck1 and Swe1 function in parallel pathways to inhibit Clb2-Cdk1. We find that mck1Δ strains have a delay in mitotic exit as well as elevated levels of Clb2-Cdk1 activity post-nuclear division. Using a co-immunoprecipitation assay, we identify a physical interaction between Mck1 and both Clb2 and Mih1. Finally, we demonstrate that phosphorylation of purified Clb2 by Cdk1 is inhibited by catalytically active Mck1 but not catalytically inactive Mck1 in vitro. We propose that Mck1 inhibits the activity of Clb2-Cdk1 via interaction with Clb2. The mammalian glycogen synthase kinase-3 homolog has been implicated in cyclin inhibition, suggesting a conserved cell cycle function for both yeast and mammalian glycogen synthase kinases.     

Essential embryonic roles of the CKI-1 cyclin-dependent kinase inhibitor in cell-cycle exit and morphogenesis in C elegans

Following a phase of rapid proliferation, cells in developing embryos must decide when to cease division and then whether to survive and differentiate or instead undergo programmed death. In screens for genes that regulate embryonic patterning of the endoderm in Caenorhabditis elegans, we identified overlapping chromosomal deletions that define a gene required for these decisions. These deletions result in embryonic hyperplasia in multiple somatic tissues, excessive numbers of cell corpses, and profound defects in morphogenesis and differentiation. However, cell-cycle arrest of the germline is unaffected. Cell lineage analysis of these mutants revealed that cells that normally stop dividing earlier than their close relatives instead undergo an extra round of division. These deletions define a genomic region that includes cki-1 and cki-2, adjacent genes encoding members of the Cip/Kip family of cyclin-dependent kinase inhibitors. cki-1 alone can rescue the cell proliferation, programmed cell death, and differentiation and morphogenesis defects observed in these mutants. In contrast, cki-2 is not capable of significantly rescuing these phenotypes. RNA interference of cki-1 leads to embryonic lethality with phenotypes similar to, or more severe than, the deletion mutants. cki-1 and -2 gene reporters show distinct expression patterns; while both are expressed at around the time that embryonic cells exit the cell cycle, cki-2 also shows marked expression starting early in embryogenesis, when rapid cell division occurs. Our findings demonstrate that cki-1 activity plays an essential role in embryonic cell cycle arrest, differentiation and morphogenesis, and suggest that it may be required to suppress programmed cell death or engulfment of cell corpses.     

Cell cycle function of a rice B2-type cyclin interacting with a B-type cyclin-dependent kinase

Cyclin-dependent kinases (CDKs) are involved in the control of cell cycle progression. Plant A-type CDKs are functional homologs of yeast Cdc2/Cdc28 and are expressed throughout the cell cycle. In contrast, B-type CDK (CDKB) is a family of mitotic CDKs expressed during the S/M phase, and its precise function remains unknown. Here, we identified two B2-type cyclins, CycB2;1 and CycB2;2, as a specific partner of rice CDKB2;1. The CDKB2;1-CycB2 complexes produced in insect cells showed a significant level of kinase activity in vitro, suggesting that CycB2 binds to and activates CDKB2. We then expressed green fluorescent protein (GFP)-fused CDKB2;1 and CycB2;2 in tobacco BY2 cells to investigate their subcellular localization during mitosis. Surprisingly, the fluorescence signal of CDKB2;1-GFP was tightly associated with chromosome alignment as well as with spindle structure during the metaphase. During the telophase, the signal was localized to the spindle midzone and the separating sister chromosomes, and then to the phragmoplast. On the other hand, the CycB2;2-GFP fluorescence signal was detected in nuclei during the interphase and prophase, moved to the metaphase chromosomes, and then disappeared completely after the cells passed through the metaphase. Co-localization of CDKB2;1-GFP and CycB2;2-GFP on chromosomes aligned at the center of the metaphase cells suggests that the CDKB2-CycB2 complex may function in retaining chromosomes at the metaphase plate. Overexpression of CycB2;2 in rice plants resulted in acceleration of root growth without any increase in cell size, indicating that CycB2;2 promoted cell division probably through association with CDKB2 in the root meristem.     

Inhibition of cell division but not nuclear division by 1-O- octadecyl-2-O-methyl-Sn-glycero-3-phosphocholine

1-O-Octadecyl-2-O-methyl-glycero-3-phosphocholine (ET-18-OCH(3)) selectively inhibits the growth of cancer cells. Here we show that in some cell types ET-18-OCH(3)and liposome-associated ET-18-OCH(3)inhibit cell division without concurrent inhibition of nuclear division, leading to multinucleate cell formation, and cell death through apoptosis. Cell cycle analysis revealed that ET-18-OCH(3)-treated U-937 cells continued to move through the cell cycle, but many cells were not able to divide and instead accumulated as tetraploid cells or octaploid cells in the G0/G1 phase of the cell cycle. Inhibition of cytokinesis has been shown to be paralleled by activation of U-937 cells, including upregulation of some cell-surface markers, acquisition of phagocytic activity, and secretion of tumor necrosis factor (TNF)-alpha (Pushkareva et al., 2000). Furthermore, treatment of cells with ET-18-OCH(3)results in the accumulation of apoptotic cells in time- and dose-dependent manner. It is possible that inhibition of cytokinesis may be related to cytoskeletal effects.     

Endoreduplication and development: rule without dividing?

Endoreduplication, a strategy to amplify nuclear DNA without cell division, is very common but poorly understood in plants. Recent findings in Drosophila provide a first picture of the molecular mechanism, which appears to be conserved between plants and animals. In Arabidopsis, the study of trichomes, leaf epidermis and hypocotyl cells sheds new light on the developmental regulation of this process, and its relation to cell expansion.     

Cell cycle regulation in mouse heart during embryonic and postnatal stages

The regulation of cardiomyocyte proliferation is important for heart development and function. Proliferation levels of mouse cardiomyocytes are high during early embryogenesis and start to decrease at midgestation. Many cardiomyocytes undergo mitosis without cytokinesis, resulting in binucleated cardiomyocytes during early postnatal stages, following which the cell cycle arrests irreversibly. It remains unknown how the proliferation pattern is regulated, and how the irreversible cell cycle arrest occurs. To clarify the mechanisms, fundamental information about cell cycle regulators in cardiomyocytes and cell cycle patterns during embryonic and postnatal stages is necessary. Here, we show that the expression, complex formation, and activity of main cyclins and cyclin‐dependent kinases (CDKs) changed in a synchronous manner during embryonic and postnatal stages. These levels decreased from midgestation to birth, and then showed one wave in which the peak was around postnatal day 5. Detailed analysis of the complexes suggested that CDK activities were inhibited before the protein levels decreased. Analysis of DNA content distribution patterns in mono‐ and binucleated cardiomyocytes after birth revealed changes in cell cycle distribution patterns and the transition from mono‐ to binucleated cells. These analyses indicated that the wave of cell cycle regulator expression or activities during postnatal stages mainly produced binucleated cells from mononucleated cells. The data obtained should provide a basis for the analysis of cell cycle regulation in cardiomyocytes during embryonic and postnatal stages.     

Garlic Compound, Diallyl Disulfide Induces Cell Cycle Arrest in Prostate Cancer Cell Line PC-3

Prostate cancer is the most predominant cancer in men and related death rate increases every year. Till date, there is no effective therapy for androgen independent prostate cancer. Previous studies reported that aged garlic extract suppresses cancer growth. In the present study, diallyl disulfide [DADS], oil soluble organosulfur compound of garlic, was studied for its antiproliferative and induction of cell cycle arrest on prostate cancer cells in vitro. The suppression of cell growth was assessed by MTT assay. Induction of cell cycle arrest was assessed and confirmed by propidium iodide staining in flowcytometric analysis and western blotting analysis of major cell cycle regulator proteins. The results showed that DADS inhibited the growth of prostate cancer cells in a dose dependent manner, compared to the control. At 25 μM and 40 μM concentrations, DADS induced cell cycle arrest at G2/M transition in PC-3 cells. Western blotting analysis of cyclin A, B₁ and cyclin dependent kinase 1 [CDK1] revealed that DADS inhibited the cell cycle by downregulating CDK1 expression. It is concluded that DADS, inhibits proliferation of prostate cancer cells through cell cycle arrest. Dose dependent effect of DADS on PC-3 cell line was observed in the present study.     

Isolation of acdc28 mutation that abrogates the dependence of S phase on completion of M phase of the budding yeast cell cycle

We have isolated a mutation in the budding yeastSaccharomyces cerevisisae CDC28 gene that allowscdc13 cells, carrying damaged DNA, to continue with the cell division cycle. Whilecdc13 mutant cells are arrested as largebudded cells at the nonpermissive temperature 37‡C, thecdc13 cdc28 double mutant culture showed cells with one or more buds, most of which showed apical growth. The additional buds emerged without the intervening steps of nuclear division and cell separation. We suggest that thecdc28 mutation abrogates a checkpoint function and allows cells with damaged or incompletely replicated DNA an entry to another round of cell cycle and bypasses the mitotic phase of the cell cycle.     

The effect of cadmium on cell cycle control in suspension culture cells of soybean

Heavy metals inhibit plant growth. This proces may be directly or indirectly connected with mechanisms regulating cell division. We analyzed the effect of Cd²⁺ on cell cycle progression in partially synchronized soybean (Glycine max) cell suspension culture and followed the expression of cell cycle genes (cyclin B1 and cyclin-dependent kinase A - CDK-A). We have checked the hypothesis that Cd²⁺-induced impairment of cell division is connected with DNA damage. The [³H]-thymidine incorporation in cell cultures synchronized either with hydroxyurea (HU) or phosphate starvation have shown, that Cd²⁺ strongly affects the S phase of soybean cell cycle, by causing the earlier entry of cells into S phase and by decreasing the rate of DNA synthesis. RT-PCR analysis indicated that Cd²⁺ decreases the level of cyclin B1 mRNA and has no effect on CDK-A mRNA. The result of comet assay indicated the damaging effect of Cd²⁺ on DNA of soybean cells. We suggest that Cd²⁺ affects plant cell cycle at two major checkpoints: the G1/S — by damaging of DNA, and G2/M - by decreasing the level of cyclin B1 mRNA     

Immunodetection of four mitotic cyclins and the Cdc2a protein kinase in the maize root: Their distribution in cell development and dedifferentiation

Summary Cyclin proteins and cyclin-dependent kinases play a key role in the regulation of cell division. We have therefore studied the relationship of the level of four mitotic cyclin proteins and the Cdc2a kinase protein to cell division in maize root tissue with respect to cessation of division as cells leave the primary meristem region, resumption of division in formation of lateral-root primordia, and induced division following wounding. All four mitotic cyclins and Cdc2a were most abundant in dividing cells. The only examined cell cycle protein which was restricted to dividing tissue was cyclin ZmCycB1;2 (previously ZmIb) and may thus be a limiting factor for cell division. All other cyclin proteins, i.e., ZmCycB1;1 (previously ZmIa), ZmCycA1;1 (previously ZmII), and ZmCycB2;1 (previously ZmIII), and the Cdc2a kinase declined shortly after cells had ceased division. The distance from the root tip at which cells ceased division was tissue-specific and reflected the distance at which decrease of cell cycle proteins was detected. Whereas cyclin ZmCycB1;2 rapidly declined to a hardly detectable level in either nucleus or cytoplasm, in the nuclei of nondividing cells there was persistence of Cdc2a and of cyclins ZmCycB1;1, ZmCycCA1;1, and ZmCycB2;1, indicating that there are plant cyclins which are tightly linked to cell division and others that persist, especially in the nuclei, in nondividing cells. The transition from division to differentiation may thus partly be triggered and enforced by the decrease of the cell cycle proteins and especially the decline of cyclins in the cytoplasm. In the resumption of cell division, both in lateral-root formation and in wound response, high nuclear and low cytoplasmic accumulation of cyclin ZmCycB2;1 was the first visible sign of cell dedifferentiation, implying a role for cyclin ZmCycB2;1 in the G₀–G₁ phase transition. Next, cytoplasmic accumulation of cyclin ZmCycA1;1, followed by a rearrangement of cortical microtubules, was observed and since both the cyclins ZmCycA1;1 and ZmCycB2;1 were found at places of high tubulin concentration, they may function in the microtubule rearrangement for cell division. When the nuclei of dedifferentiating cells had visibly enlarged, all cyclins and Cdc2a accumulated there, possibly contributing to DNA replication and preparation for mitosis. Later, presumably during G₂ phase, cytoplasmic accumulation was observed for Cdc2a at low levels, as observed in G₂ phase cells of the primary meristem, and for cyclins ZmCycB1;1 and ZmCycB1;2 accumulation was observed above the levels found in undisturbed meristems, suggesting special contributions to late dedifferentiation processes in both wound-induced and lateral meristems.Abbreviations CDK cyclin-dependent kinase - LRP lateral-root primordium - Mt microtubule - FITC fluorescein isothiocyanate - TRITC tetramethylrhodamine isothiocyanate Dedicated to Professor Walter Gustav Url on the occasion of his 70th birthday     

CDK inhibitors,p21 and p27, participate in cell cycle exit of mammalian cardiomyocytes

Mammalian cardiomyocytes actively proliferate during embryonic stages, following which cardiomyocytes exit their cell cycle after birth. The irreversible cell cycle exit inhibits cardiac regeneration by the proliferation of pre-existing cardiomyocytes. Exactly how the cell cycle exit occurs remains largely unknown. Previously, we showed that cyclin E- and cyclin A-CDK activities are inhibited before the CDKs levels decrease in postnatal stages. This result suggests that factors such as CDK inhibitors (CKIs) inhibit CDK activities, and contribute to the cell cycle exit. In the present study, we focused on a Cip/Kip family, which can inhibit cyclin E- and cyclin A-CDK activities. Expression of p21^Cip1 and p27^Kip1 but not p57^Kip2 showed a peak around postnatal day 5, when cyclin E- and cyclin A-CDK activities start to decrease. p21^Cip1 and p27^Kip1 bound to cyclin E, cyclin A and CDK2 at postnatal stages. Cell cycle distribution patterns of postnatal cardiomyocytes in p21^Cip1 and p27^Kip1 knockout mice showed failure in the cell cycle exit at G1-phase, and endoreplication. These results indicate that p21^Cip1 and p27^Kip play important roles in the cell cycle exit of postnatal cardiomyocytes.     

CDKIs p18INK4c and p57Kip2 are involved in quiescence of CML leukemic stem cells after treatment with TKI

Chronic Myeloid Leukemia (CML) is sustained by a small population of cells with stem cell characteristics known as Leukemic Stem Cells that are positive to BCR-ABL fusion protein, involved with several abnormalities in cell proliferation, expansion, apoptosis and cell cycle regulation. Current treatment options for CML involve the use of Tirosine Kinase Inhibitor (Imatinib, Nilotinib and Dasatinib), that efficiently reduce proliferation proliferative cells but do not kill non proliferating CML primitive cells that remain and contributes to the persistence of the disease.

In order to understand the role of Cyclin Dependent Kinase Inhibitors in CML LSC permanence after TKI treatment, in this study we analyzed cell cycle status, the levels of several CDKIs and the subcellular localization of such molecules in different CML cell lines, as well as primary CD34⁺CD38^?lin^? LSC and HSC.

Our results demonstrate that cellular location of p18^INK4c and p57^Kip2 seems to be implicated in the antiproliferative activity of Imatinib and Dasatinib in CML cells and also suggest that the permanence of quiescent stem cells after TKI treatment could be associated with a decrease in p18^INK4c and p57^Kip2 nuclear location. The differences in p18^INK4cand p57^Kip2activities in CML and normal stem cells suggest a different cell cycle regulation and provide a platform that could be considered in the development of new therapeutic options to eliminate LSC.     

Meiotic weakness: the first division

Cell division is probably the most dramatic event in the life of a cell : the entire genetic material has to be equally distributed into the two daughter cells. Segregation errors have severe consequences and lead to either cell death or the generation of aneuploid cells and may cause the formation of tumors or tumor promoting mutations in somatic cells. In meiosis, they provoke the generation of aneuploid embryos and/or spontaneous abortions. Trisomies in humans, such as trisomy 21, are due to the missegregation of one chromosome in the first meiotic division in the oocyte. This review deals with the molecular mechanisms regulating the two meiotic divisions required for the generation of female haploid germ cells. Here we focus mainly on spindle assembly, and cell cycle regulation especially during the first meiotic division in mouse oocytes (excellent reviews have been written on the peculiar aspects of cell cycle regulation in meiosis II, such as the CSF arrest).     

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.     

Polyamine depletion partially reduces the radiation-induced cell death via cell cycle delay mediated by thioredoxin

In previous studies, polyamine depletion by DFMO (α-difluoromethylornithine)-treatment reduced H₂O₂-induced apoptotic cell death by reduction of ferric ion uptake. In the present study, we analyzed the reduction of radiation-induced cell death by polyamine depletion. Exposure of HT29 cells to radiation induced severe cell death, but when cells were pretreated with DFMO, a specific inhibitor of polyamine biosynthesis, radiation-induced cell death was reduced to 50–60% of control. Cell cycle analysis showed that, in these cells, the time to reach the G₂/M phase arrest was delayed for 20–24 h compared to the control cells, at which stage the fate of cells exposed to ionizing radiation is determined. DFMO-treated cells also showed a low level of thioredoxin, which is a high-level determinant of the cellular fate. To investigate the relationship between the G₂/M phase arrest and the reduction of thioredoxin caused by polyamine depletion, we also analyzed thioredoxin-antisensed (asTRX) HT29 cells as for DFMO-treated cells. In asTRX-transfected cells, the γ-irradiation-induced G₂/M phase arrest was also significantly delayed and radiation-induced cell death was profoundly reduced, as in the DFMO-treated cells. Both sets of cells showed a decrease of cyclin D1 and an increment of HSP25, which are involved in radiation-induced cell cycle progress. Overall, these results suggest that polyamines are essential for normal cell death of HT29 cells triggered by γ-radiation and that this is partially mediated by the regulation of thioredoxin expression.