Timing the spinal cord development with neural progenitor cells losing their proliferative capacity: a theoretical analysis.

In the developing neural tube in chicken and mammals, neural stem cells proliferate and differentiate according to a stereotyped spatiotemporal pattern. Several actors have been identified in the control of this process, from tissue-scale morphogens patterning to intrinsic determinants in neural progenitor cells. In a previous study (Bonnet et al. eLife 7, 2018), we have shown that the CDC25B phosphatase promotes the transition from proliferation to differentiation by stimulating neurogenic divisions, suggesting that it acts as a maturating factor for neural progenitors. In this previous study, we set up a mathematical model linking fixed progenitor modes of division to the dynamics of progenitors and differentiated populations. Here, we extend this model over time to propose a complete dynamical picture of this process. We start from the standard paradigm that progenitors are homogeneous and can perform any type of divisions (proliferative division yielding two progenitors, asymmetric neurogenic divisions yielding one progenitor and one neuron, and terminal symmetric divisions yielding two neurons). We calibrate this model using data published by Saade et al. (Cell Reports 4, 2013) about mode of divisions and population dynamics of progenitors/neurons at different developmental stages. Next, we explore the scenarios in which the progenitor population is actually split into two different pools, one of which is composed of cells that have lost the capacity to perform proliferative divisions. The scenario in which asymmetric neurogenic division would induce such a loss of proliferative capacity appears very relevant.

APC/CCdh1 Enables Removal of Shugoshin-2 from the Arms of Bivalent Chromosomes by Moderating Cyclin-Dependent Kinase Activity.

In mammalian females, germ cells remain arrested as primordial follicles. Resumption of meiosis is heralded by germinal vesicle breakdown, condensation of chromosomes, and their eventual alignment on metaphase plates. At the first meiotic division, anaphase-promoting complex/cyclosome associated with Cdc20 (APC/CCdc20) activates separase and thereby destroys cohesion along chromosome arms. Because cohesion around centromeres is protected by shugoshin-2, sister chromatids remain attached through centromeric/pericentromeric cohesin. We show here that, by promoting proteolysis of cyclins and Cdc25B at the germinal vesicle (GV) stage, APC/C associated with the Cdh1 protein (APC/CCdh1) delays the increase in Cdk1 activity, leading to germinal vesicle breakdown (GVBD). More surprisingly, by moderating the rate at which Cdk1 is activated following GVBD, APC/CCdh1 creates conditions necessary for the removal of shugoshin-2 from chromosome arms by the Aurora B/C kinase, an event crucial for the efficient resolution of chiasmata.

Tributyltin induces G2/M cell cycle arrest via NAD(+)-dependent isocitrate dehydrogenase in human embryonic carcinoma cells.

Organotin compounds, such as tributyltin (TBT), are well-known endocrine-disrupting chemicals (EDCs). We have recently reported that TBT induces growth arrest in the human embryonic carcinoma cell line NT2/D1 at nanomolar levels by inhibiting NAD(+)-dependent isocitrate dehydrogenase (NAD-IDH), which catalyzes the irreversible conversion of isocitrate to α-ketoglutarate. However, the molecular mechanisms by which NAD-IDH mediates TBT toxicity remain unclear. In the present study, we examined whether TBT at nanomolar levels affects cell cycle progression in NT2/D1 cells. Propidium iodide staining revealed that TBT reduced the ratio of cells in the G1 phase and increased the ratio of cells in the G2/M phase. TBT also reduced cell division cycle 25C (cdc25C) and cyclin B1, which are key regulators of G2/M progression. Furthermore, apigenin, an inhibitor of NAD-IDH, mimicked the effects of TBT. The G2/M arrest induced by TBT was abolished by NAD-IDHα knockdown. Treatment with a cell-permeable α-ketoglutarate analogue recovered the effect of TBT, suggesting the involvement of NAD-IDH. Taken together, our data suggest that TBT at nanomolar levels induced G2/M cell cycle arrest via NAD-IDH in NT2/D1 cells. Thus, cell cycle analysis in embryonic cells could be used to assess cytotoxicity associated with nanomolar level exposure of EDCs.

Cell cycle-dependent Cdc25C phosphatase determines cell survival by regulating apoptosis signal-regulating kinase 1.

Cdc25C (cell division cycle 25C) phosphatase triggers entry into mitosis in the cell cycle by dephosphorylating cyclin B-Cdk1. Cdc25C exhibits basal phosphatase activity during interphase and then becomes activated at the G2/M transition after hyperphosphorylation on multiple sites and dissociation from 14-3-3. Although the role of Cdc25C in mitosis has been extensively studied, its function in interphase remains elusive. Here, we show that during interphase Cdc25C suppresses apoptosis signal-regulating kinase 1 (ASK1), a member of mitogen-activated protein (MAP) kinase kinase kinase family that mediates apoptosis. Cdc25C phosphatase dephosphorylates phospho-Thr-838 in the activation loop of ASK1 in vitro and in interphase cells. In addition, knockdown of Cdc25C increases the activity of ASK1 and ASK1 downstream targets in interphase cells, and overexpression of Cdc25C inhibits ASK1-mediated apoptosis, suggesting that Cdc25C binds to and negatively regulates ASK1. Furthermore, we showed that ASK1 kinase activity correlated with Cdc25C activation during mitotic arrest and enhanced ASK1 activity in the presence of activated Cdc25C resulted from the weak association between ASK1 and Cdc25C. In cells synchronized in mitosis following nocodazole treatment, phosphorylation of Thr-838 in the activation loop of ASK1 increased. Compared with hypophosphorylated Cdc25C, which exhibited basal phosphatase activity in interphase, hyperphosphorylated Cdc25C exhibited enhanced phosphatase activity during mitotic arrest, but had significantly reduced affinity to ASK1, suggesting that enhanced ASK1 activity in mitosis was due to reduced binding of hyperphosphorylated Cdc25C to ASK1. These findings suggest that Cdc25C negatively regulates proapoptotic ASK1 in a cell cycle-dependent manner and may play a role in G2/M checkpoint-mediated apoptosis.

miR-195/497 induce postnatal quiescence of skeletal muscle stem cells.

Skeletal muscle stem cells (MuSCs), the major source for skeletal muscle regeneration in vertebrates, are in a state of cell cycle arrest in adult skeletal muscles. Prior evidence suggests that embryonic muscle progenitors proliferate and differentiate to form myofibres and also self-renew, implying that MuSCs, derived from these cells, acquire quiescence later during development. Depletion of Dicer in adult MuSCs promoted their exit from quiescence, suggesting microRNAs are involved in the maintenance of quiescence. Here we identified miR-195 and miR-497 that induce cell cycle arrest by targeting cell cycle genes, Cdc25 and Ccnd. Reduced expression of MyoD in juvenile MuSCs, as a result of overexpressed miR-195/497 or attenuated Cdc25/Ccnd, revealed an intimate link between quiescence and suppression of myogenesis in MuSCs. Transplantation of cultured MuSCs treated with miR-195/497 contributed more efficiently to regenerating muscles of dystrophin-deficient mice, indicating the potential utility of miR-195/497 for stem cell therapies.

Maspin genetically and functionally associates with gastric cancer by regulating cell cycle progression.

Human SERPINB5, commonly known as maspin, has diverse functions as a tumor suppressor. In this study, we discovered that maspin has a novel role in cell cycle control, and common variants were discovered to be associated with gastric cancer. The genotypes of 836 unrelated Korean participants (including 430 with gastric cancer) were examined for 12 tag single-nucleotide polymorphisms (SNPs) and imputed for 178 SNPs in the maspin gene. Susceptibility to diffuse-type gastric cancer was strongly and significantly associated with several SNPs including rs3744941 (C>T) in the promoter (TT versus CC+CT, odds ratio = 0.56 [0.37-0.83], P = 0.0038) and rs8089104 (C>T) in intron 1 (TT+CT versus CC, odds ratio = 1.7 [1.2-2.5], P = 0.0021). No SNPs were associated with susceptibility to intestinal-type gastric cancer. A haplotype of three highly correlated promoter SNPs associated with higher cancer risk showed 40% of the activity of a non-risk-associated haplotype promoter in the diffuse-type gastric cancer cell line MKN45. Maspin downregulation achieved either by a short hairpin RNA targeting maspin or overexpression of the E2F1-DP1 complex in MKN45 cells dramatically accelerated cell cycle progression and caused an increase of active CDC25C levels and a decrease of inactive CDK1 levels. In contrast, maspin upregulation had the opposite effect, substantially retarding cell proliferation. Therefore, our results suggest that a maspin promoter haplotype that reduces maspin gene expression accelerates cell cycle progression and, consequently, is associated with increased susceptibility to diffuse-type gastric cancer. Furthermore, a novel maspin-related pathway is demonstrated to underlie gastric carcinogenesis.

Accelerated elimination of ultraviolet-induced DNA damage through apoptosis in CDC25A-deficient skin.

Cell division cycle 25A (CDC25A) is a dual-specificity phosphatase that removes inhibitory phosphates from cyclin-dependent kinases, allowing cell-cycle progression. Activation of cell-cycle checkpoints following DNA damage results in the degradation of CDC25A, leading to cell-cycle arrest. Ultraviolet (UV) irradiation, which causes most skin cancer, results in both DNA damage and CDC25A degradation. We hypothesized that ablation of CDC25A in the skin would increase cell-cycle arrest following UV irradiation, allowing for improved repair of DNA damage and decreased tumorigenesis. Cdc25a(fl/fl) /Krt14-Cre recombinase mice, with decreased CDC25A in the epithelium of the skin, were generated and exposed to UV. UV-induced DNA damage, in the form of cyclopyrimidine dimers and 8-oxo-deoxyguanosine adducts, was eliminated earlier from CDC25A-deficient epidermis. Surprisingly, loss of CDC25A did not alter epidermal proliferation or cell cycle after UV exposure. However, the UV-induced apoptotic response was prolonged in CDC25A-deficient skin. Double labeling of cleaved caspase-3 and the DNA damage marker γH2A.X revealed many of the apoptotic cells in UV-exposed Cdc25a mutant skin had high levels of DNA damage. Induction of skin tumors by UV irradiation of Cdc25a mutant and control mice on a skin tumor susceptible to v-ras(Ha) Tg.AC mouse background revealed UV-induced papillomas in Cdc25a mutants were significantly smaller than in controls in the first 6 weeks following UV exposure, although there was no difference in tumor multiplicity or incidence. Thus, deletion of Cdc25a increased apoptosis and accelerated the elimination of DNA damage following UV but did not substantially alter cell-cycle regulation or tumorigenesis.

Knockdown of Cdc25B in renal cell carcinoma is associated with decreased malignant features.

Cdc25 phosphatases are important regulators of the cell cycle. Their abnormal expression detected in a number of tumors implies that their dysregulation is involved in malignant transformation. However, the role of Cdc25B in renal cell carcinomas remains unknown. To shed light on influence on renal cell carcinogenesis and subsequent progression, Cdc25B expression was examined by real-time RT-PCR and western blotting in renal cell carcinoma and normal tissues. 65 kDa Cdc25B expression was higher in carcinomas than in the adjacent normal tissues (P<0.05), positive correlations being noted with clinical stage and histopathologic grade (P<0.05). To additionally investigate the role of Cdc25B alteration in the development of renal cell carcinoma, Cdc25B siRNA was used to knockdown the expression of Cdc25B. Down-regulation resulted in slower growth, more G2/M cells, weaker capacity for migration and invasion, and induction of apoptosis in 769-P transfectants. Reduction of 14-3-3 protein expression appeared related to Cdc25B knockdown. These findings suggest an important role of Cdc25B in renal cell carcinoma development and provide a rationale for investigation of Cdc2B-based gene therapy.

Phosphatases driving mitosis: pushing the gas and lifting the brakes.

Entry into and progression through mitosis depends critically on the establishment and maintenance of protein phosphorylation. For this reason, studies on mitotic progression have focused heavily on the activation of MPF (M phase promoting factor), a cyclin-dependent kinase responsible for phosphorylating proteins that execute the dynamic events of mitosis. Recent work, however, has significantly expanded our understanding of mechanisms that allow accumulation of phosphoproteins at M phase, suggesting that mitotic entry relies not only on MPF activation but also on the inhibition of antimitotic phosphatases. It is now clear that there exists a separate, albeit equally important, signaling pathway for the inactivation of protein phosphatases at the G2/M transition. This pathway, which is governed by the kinase Greatwall is essential for both entry into and maintenance of M phase. This chapter will outline the molecular events regulating entry into mitosis, specifically highlighting the role that protein phosphorylation plays in triggering both MPF activation and the inhibition of phosphatase activity that would otherwise prevent accumulation of mitotic phosphoproteins. These intricate regulatory pathways are essential for maintaining normal cell division and preventing inappropriate cell proliferation, a central hallmark of cancer cells.

Regulation of cell cycle components during exposure to anoxia or dehydration stress in the wood frog, Rana sylvatica.

The wood frog (Rana sylvatica) exhibits a well-developed natural anoxia and dehydration tolerance. The degree of stress tolerance depends on numerous biochemical adaptations, including stress-induced hypometabolism that helps to preserve long-term viability by reducing ATP demand. We hypothesized that the mechanisms involved in cell cycle control could act to aid in the establishment of the hypometabolic state required for stress survival. Selected proteins involved in the proliferation of cells were evaluated using immunoblotting in liver and skeletal muscle of wood frogs comparing controls with animals subjected to either 24-hr anoxia exposure under a nitrogen gas atmosphere or dehydration to 40% of total body water lost (all at 5°C). Levels of cyclins (type A, B, D, and E) decreased significantly under both stresses in liver and skeletal muscle. Similar reductions were seen for Cyclin-dependant kinases (Cdk) types 2, 4, and 6 in both liver and skeletal muscle; however, an increase in the relative amount of phosphorylated inactive p-Cdk (Thr14/Tyr15) was observed in liver under both stresses. Levels of positive regulators of Cdk activity (Cdc25 type A and C) were significantly reduced in both tissues under both stresses, whereas negative regulators of Cdk activity (p16(INK4a) and p27(KIP1) ) increased significantly in liver under both anoxia and dehydration stress (but not in muscle). This study provides the first report of differential regulation of cell cycle components in an anoxia and dehydration tolerant vertebrate, the wood frog, suggesting that cell cycle suppression is an active part of stress resistance and life extension in hypometabolic states.

Cdc25A regulates matrix metalloprotease 1 through Foxo1 and mediates metastasis of breast cancer cells.

Cdc25A is a cell cycle-activating phosphatase, and its overexpression in breast cancers has been shown to correlate with poor prognosis. Most recent studies related to Cdc25A and tumor progression have focused on its role in regulating cell cycle progression. However, less is known about how Cdc25A modulates the metastasis of breast cancer cells. In this study, we revealed that Cdc25A enhances Foxo1 stability by dephosphorylating Cdk2, and Foxo1 was shown to directly regulate transcription of the metastatic factor MMP1. Further studies have shown that overexpression of Cdc25A in breast cancer cells enhances metastasis, whereas its downmodulation inhibits metastasis in mouse models, and the effects of Cdc25A on breast cancer cell metastasis are independent of cell proliferation and apoptosis. Furthermore, we have demonstrated that aberrant Cdc25A in breast cancer patient samples directly correlates with the metastatic phenotype. Further insights into this critical role of Cdc25A in the metastasis of breast cancer cells and the trial of an anti-Cdc25A strategy in mouse models may reveal its therapeutic potential in prevention and treatment of breast cancer cell dissemination.

Contributions made by CDC25 phosphatases to proliferation of intestinal epithelial stem and progenitor cells.

The CDC25 protein phosphatases drive cell cycle advancement by activating cyclin-dependent protein kinases (CDKs). Humans and mice encode three family members denoted CDC25A, -B and -C and genes encoding these family members can be disrupted individually with minimal phenotypic consequences in adult mice. However, adult mice globally deleted for all three phosphatases die within one week after Cdc25 disruption. A severe loss of absorptive villi due to a failure of crypt epithelial cells to proliferate was observed in the small intestines of these mice. Because the Cdc25s were globally deleted, the small intestinal phenotype and loss of animal viability could not be solely attributed to an intrinsic defect in the inability of small intestinal stem and progenitor cells to divide. Here, we report the consequences of deleting different combinations of Cdc25s specifically in intestinal epithelial cells. The phenotypes arising in these mice were then compared with those arising in mice globally deleted for the Cdc25s and in mice treated with irinotecan, a chemotherapeutic agent commonly used to treat colorectal cancer. We report that the phenotypes arising in mice globally deleted for the Cdc25s are due to the failure of small intestinal stem and progenitor cells to proliferate and that blocking cell division by inhibiting the cell cycle engine (through Cdc25 loss) versus by inducing DNA damage (via irinotecan) provokes a markedly different response of small intestinal epithelial cells. Finally, we demonstrate that CDC25A and CDC25B but not CDC25C compensate for each other to maintain the proliferative capacity of intestinal epithelial stem and progenitor cells.

Filamin A stimulates cdc25C function and promotes entry into mitosis.

The activity of the dual specificity phosphatase cdc25C is required for mitotic progression though the mechanisms by which cdc25C is activated prior to mitosis in human cells remain unclear. The data presented herein show that the actin binding protein Filamin A forms a complex with cdc25C in vivo and binds preferentially to the mitotic form of cdc25C. Co-expression of Filamin A with cdc25C results in an increase in PCC induced by cdc25C, while knocking down Filamin A expression reduces the levels of PCC induced by cdc25C overexpression. Further, only a Filamin A fragment that forms a complex with both cdc25C and cyclin B1 and retains the dimerization domain can stimulate the ability of cdc25C to induce PCC. These results suggest that Filamin A provides a platform for the assembly of the cyclin B1-cdk1- cdc25C complex resulting in cdk1 activation and mitotic progression.

Involvement of MAPK and PI3K signaling pathway in sterigmatocystin-induced G2 phase arrest in human gastric epithelium cells.

SCOPE: Sterigmatocystin (ST), a mycotoxin commonly found in foodstuff and feedstuff, has been shown to be a carcinogenic mycotoxin in animal models. Many studies showed that the high level of ST contamination in grains might be related to the high incidence of gastric carcinoma in rural areas of China. However, up to now, the potential effects of ST on human gastric epithelium cells remain largely unknown. In this study, we explored the effects of ST on cell-cycle distribution and the regulatory mechanism in immortalized human gastric epithelium cells (GES-1). METHODS AND RESULTS: The effects of ST on the cell cycle distribution of GES-1 cells were determined with flow cytometric (FCM) analysis, Giemsa staining and immunofluorescence staining, while that on the expression of related gene-Cdc25C, Cdc2, CyclinB1 and the complex of CyclinB1-Cdc2 were studied with Western blot, reverse transcription polymerase chain reaction (RT-PCR) and immunoprecipitation assay respectively. We found that ST induced GES-1 cells arrested at G2 phase by regulating the expression of Cdc25C, Cdc2, CyclinB1 and the formation of CyclinB1-Cdc2 complex. Further study suggested JNK, ERK and PI3K/AKT/mTOR pathways to be involved in the process of G2 arrest induced by ST. The specific inhibitors of JNK and ERK reversed the role of ST, whereas that of PI3K/AKT/mTOR reinforced the effect of ST on cell-cycle distribution. CONCLUSION: This study demonstrates that JNK, ERK and PI3K/AKT/mTOR pathways participated in the G2 arrest induced by ST through the deregulation of CyclinB1, Cdc2 and Cdc25C. It may play some roles in the gastric carcinogenesis in ST exposure populations.

Cdc25B is negatively regulated by p53 through Sp1 and NF-Y transcription factors.

Cdc25B phosphatases function as key players in G2/M cell cycle progression by activating the CDK1-cyclinB1 complexes. They also have an essential role in recovery from the G2/M checkpoint activated in response to DNA damage. Overexpression of Cdc25B results in bypass of the G2/M checkpoint and illegitimate entry into mitosis, and also causes replicative stress, leading to genomic instability. Thus, fine-tuning of Cdc25B expression level is critical for correct cell cycle progression and G2 checkpoint recovery. However, the transcriptional regulation of Cdc25B remains largely unknown. Earlier studies have shown that the tumor suppressor p53 overexpression transcriptionally represses Cdc25B; however, the molecular mechanism of this repression has not yet been elucidated, although it was suggested to occur through the induction of p21. Here we show that Cdc25B is downregulated by the basal level of p53 in multiple cell types. This downregulation also occurs in p21-/- cell lines, indicating that p21 is not required for p53-mediated regulation of Cdc25B. Deletion and mutation analyses of the Cdc25B promoter revealed that downregulation by p53 is dependent on the presence of functional Sp1/Sp3 and NF-Y binding sites. Furthermore, chromatin immunoprecipitation analyses show that p53 binds to the Cdc25B promoter and mediates transcriptional attenuation through the Sp1 and NF-Y transcription factors. Our results suggest that the inability to downregulate Cdc25B after loss of p53 might contribute to tumorigenesis.

The regulatory network of cell-cycle progression is fundamentally different in plants versus yeast or metazoans.

Plant growth and proliferation control is coming into a global focus due to recent ecological and economical developments. Plants represent not only the largest food supply for mankind but also may serve as a global source of renewable energies. However, plant breeding has to accomplish a tremendous boost in yield to match the growing demand of a still rapidly increasing human population. Moreover, breeding has to adjust to changing environmental conditions, in particular increased drought. Regulation of cell-cycle control is a major determinant of plant growth and therefore an obvious target for plant breeding. Furthermore, cell-cycle control is also crucial for the DNA damage response, for instance upon irradiation. Thus, an in-depth understanding of plant cell-cycle regulation is of importance beyond a scientific point of view. The mere presence of many conserved core cell-cycle regulators, e.g. CDKs, cyclins, or CDK inhibitors, has formed the idea that the cell cycle in plants is exactly or at least very similarly controlled as in yeast or human cells. Here together with a recent publication we demonstrate that this dogma is not true and show that the control of entry into mitosis is fundamentally different in plants versus yeast or metazoans. Our findings build an important base for the understanding and ultimate modulation of plant growth not only during unperturbed but also under harsh environmental conditions.

Cdc25A-driven proliferation regulates CD62L levels and lymphocyte movement in response to interleukin-7.

OBJECTIVE: Interleukin-7 (IL-7) is a multifunctional cytokine and a promising immunotherapeutic agent. However, because transient T-cell depletion is an immediate outcome of IL-7 administration at supraphysiological doses, we investigated the mechanism by which the IL-7 proliferative signal transduced through Cdc25A, a key activator of cyclin-dependent kinases, could modulate lymphocyte movement. MATERIALS AND METHODS: Employing novel methods of manipulating Cdc25A gene expression, combined with in vitro and in vivo evaluation of IL-7 application, we assessed the expression of activation and homing markers and identified the mechanism by which IL-7 could induce T-cell expansion and alter lymphocyte motility. RESULTS: Constitutively active Cdc25A drove T-cell proliferation independently of IL-7 and resulted in an activated phenotype (CD69(hi), CD44(hi)). Conversely, inhibition of Cdc25A resulted in decreased proliferation, reduced expression of activation markers, and upregulation of the lymph node homing molecule, CD62L, which promoted cell adhesion when engaged by ligand. We found that IL-7 prevented the nuclear translocation of the transcription factor, Foxo1, in a manner dependent on the activity of Cdc25A, resulting in decreased levels of CD62L. In vivo administration of IL-7 decreased lymph node cellularity, while treatment with IL-7, premixed with a neutralizing IL-7 antibody (M25), increased total lymph node cells--with more nuclear Foxo1 detected in cells from mice receiving IL-7 + M25. CONCLUSIONS: These results are consistent with the model that IL-7 drives Cdc25A-mediated T-cell proliferation, which prevents the nuclear translocation of Foxo1, leading to reduced expression of CD62L and the migration of T cells into circulation.

A role for the cell cycle phosphatase Cdc25a in vitamin D-dependent inhibition of adult rat vascular smooth muscle cell proliferation.

We have explored the mechanism(s) underlying 1,25 dihydroxyvitamin D's (1,25(OH)(2)D) suppression of agonist-induced vascular smooth muscle cell (VSMC) proliferation. Quiescent cultured adult rat VSMC were treated with 1,25(OH)(2)D for 48h and endothelin (ET) or angiotensin II (AII) for the final 24h. We show that VSMC responded to 1,25(OH)(2)D or its less hypercalcemic analogue RO 25-6760 with ∼70% inhibition of ET-dependent (3)H-thymidine incorporation. The inhibition was linked to a comparable reduction in ET-stimulated cyclin-dependent kinase 2 (Cdk2) activity and suppression of an ET-induced Cdk2 activator, cell division cycle 25 homolog A (Cdc25A). Both 1,25(OH)(2)D and RO 25-6760 completely inhibited the ET-dependent increase in Cdc25A mRNA and protein levels, phosphatase and promoter activities. 1,25(OH)(2)D also suppressed AII-induced DNA synthesis, Cdk2 activity and Cdc25A gene transcription. Inhibition of Cdc25A gene expression using a siRNA approach resulted in significant inhibition of ET or AII-dependent Cdk2 activity and (3)H-thymidine incorporation. The Cdc25A siRNA-mediated inhibition of ET or AII-induced Cdk2 activity and DNA synthesis was not additive with that produced by 1,25(OH)(2)D treatment. These data demonstrate that 1,25(OH)(2)D inhibits VSMC proliferation through a Cdc25A-dependent mechanism and suggest that this hormone may prove useful in the management of disorders characterized by aberrant proliferation of VSMC in the vascular wall.

Dual mode of regulation of cell division cycle 25 A protein by TRB3.

We have recently demonstrated that TRB3, a novel stress-inducible protein, is an unstable protein regulated by the ubiquitin-proteasome system. The expression level of TRB3 protein is down-regulated by anaphase-promoting complex/cyclosome-cell division cycle division 20 homolog 1 (APC/C(Cdh1)) through its D-box motif. Here we demonstrate that TRB3 regulates the stability of cell division cycle 25 A (Cdc25A), an essential activator of cyclin dependent kinases (CDKs). The expression level of Cdc25A protein is suppressed by over-expression of TRB3, while knockdown of TRB3 enhances the endogenous Cdc25A expression level. On the other hand, Cdc25A degradation induced by DNA damage is significantly rescued by TRB3. When serine residues in the DSG motif, which is the critical sequences for the degradation of Cdc25A induced by DNA damage, is mutated to alanine (Cdc25A(DSG2X)), both stimulatory and protective effects of TRB3 on the Cdc25A degradation is disappeared. TRB3 protein interacts with both wild Cdc25A and mutant Cdc25A(DSG2X). Expression level of the endogenous TRB3 protein is down-regulated in a genotoxic condition. These results suggest TRB3 is a regulator for adjusting the expression level of Cdc25A both in a normal and a genotoxic conditions.

Single amino-acid changes that confer constitutive activation of mTOR are discovered in human cancer.

Mammalian target of rapamycin (mTOR) is a serine/threonine kinase that regulates a variety of cellular functions such as growth, proliferation and autophagy. In a variety of cancer cells, overactivation of mTOR has been reported. In addition, mTOR inhibitors, such as rapamycin and its derivatives, are being evaluated in clinical trials as anticancer drugs. However, no active mutants of mTOR have been identified in human cancer. Here, we report that two different point mutations, S2215Y and R2505P, identified in human cancer genome database confer constitutive activation of mTOR signaling even under nutrient starvation conditions. S2215Y was identified in large intestine adenocarcinoma whereas R2505P was identified in renal cell carcinoma. mTOR complex 1 prepared from cells expressing the mutant mTOR after nutrient starvation still retains the activity to phosphorylate 4E-BP1 in vitro. The cells expressing the mTOR mutant show increased percentage of S-phase cells and exhibit resistance to cell size decrease by amino-acid starvation. The activated mutants are still sensitive to rapamycin. However, they show increased resistance to 1-butanol. Our study points to the idea that mTOR activating mutations can be identified in a wide range of human cancer.