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CDK Inhibitors (cdk + inhibitor)
Selected Abstracts2-(Benzylsulfanyl)-6-chloro-9-isopropylpurine, a Valuable Intermediate in the Synthesis of Diaminopurine Cyclin Dependent Kinase InhibitorsEUROPEAN JOURNAL OF ORGANIC CHEMISTRY, Issue 5 2005David Taddei Abstract The synthetic potential of a novel precursor of 2,6-diaminopurine CDK inhibitors, 2-(benzylsulfanyl)-6-chloro-9-isopropylpurine, is described. The Traube purine synthesis was chosen to prepare the required 2-(benzylsulfanyl)hypoxanthine intermediate. Attempts to prepare its purin-6-yl methanesulfonic ester analogue failed. Conversion to the 6-chloropurine derivative enabled the introduction of arylamines in the presence of catalytic amounts of acid. Further chemical variety was introduced on the purine through a regioselective Mitsunobu N -9 alkylation. Oxidative cleavage of the 2-(benzylsulfanyl) leaving group with an aliphatic amine was implemented as previously reported. Purvalanol A, a potent CDK inhibitor, was synthesised using this methodology. The template and intermediates were fully characterised by modern spectroscopic techniques and single-crystal X-ray diffraction. (© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2005) [source] p27/Kip1 mediates retinoic acid-induced suppression of ovarian carcinoma cell growthJOURNAL OF CELLULAR PHYSIOLOGY, Issue 2 2004Scott Vuocolo We have investigated the mechanisms by which all-trans retinoic acid (ATRA) causes growth inhibition of ovarian carcinoma cells. As a model, we have studied the CAOV3 cell line, which is sensitive to ATRA, and the SKOV3 cell line, which is resistant. We have found that treatment of CAOV3 cells with ATRA causes a 5,10 fold increase in the protein level of the cyclin dependent kinase inhibitor p27/Kip1. p27/Kip1 protein upregulation is important in ovarian carcinoma as primary tumors are frequently found lacking this protein. The increase in p27/Kip1 is detected by day 3 of ATRA treatment of CAOV3 cells, and is maximal by day 5. Messenger RNA levels of p27/Kip1 do not change in CAOV3 cells following ATRA treatment, however, we have shown that p27/Kip1 mRNA is more stable in ATRA treated CAOV3 cells. Conversely, the ATRA resistant cell line SKOV3 fails to show p27/Kip1 accumulation. Interestingly, the SCF component protein SKP2 appears to be decreased in CAOV3 cells treated with ATRA. We have also shown that the ATRA dependent increase in p27/kip1 protein in CAOV3 cells leads to a decrease in the kinase activity of cyclin dependent kinase 4 (CDK4) following ATRA treatment. Finally, we found that CAOV3 cells stably transfected with a p27/kip1antisense construct, which express lower levels of p27/kip1 following ATRA treatment, and have a higher CDK4 kinase activity are less sensitive to ATRA induced growth suppression. Taken together our data suggest ATRA-induced growth inhibition in CAOV3 ovarian carcinoma cells involves modulation of the CDK inhibitor p27/kip1. J. Cell. Physiol. 199: 237,243, 2004© 2004 Wiley-Liss, Inc. [source] Characterization of benign and malignant prostate epithelial Hoechst 33342 side populationsTHE PROSTATE, Issue 13 2007Mick D. Brown Abstract Background The prostate epithelial stem cell has been proposed as the primary origin of neoplastic change in prostate cancer. However, the isolation and characterization of unexpanded prostate epithelial stem cells have proven problematic. Methods A prostate epithelial side population (SP) has been isolated utilizing a modified Hoechst 33342 dye efflux assay from both benign and malignant prostate tissue. CD45,ve, integrin ,2+ve Hoechst 33342 SP and NSP cells were isolated by FACS, immunophenotyped and functionally characterized in 3D culture. Results FACS analysis revealed a verapamil sensitive SP accounting for 0.93,±,0.12% and 0.57,±,0.11% of the total epithelial population from both benign and malignant prostates. The benign SP phenotype revealed a heterogeneous cell population consisting predominantly of small basal cells containing minimal cytoplasm. Conversely, the malignant SP was of undetermined acinar origin and with a complete loss of expression of the CDK2 inhibitor p21WAF1/Cip1. In vitro androgen-enhanced 3D culture of the benign and malignant SP cells led to the production of spheroids which had acinus like morphology and expressed primitive and basal cell markers. Incorporation of the CD133 marker isolated a further SP sub-fraction accounting for 0.037,±,0.01% of epithelial cells. Conclusions Our observations are consistent with the Hoechst 33342 dye efflux assay isolating a stem cell enriched population which can be further sub-fractionated by CD133 selection. Moreover, the loss of the CDK inhibitor in malignancy is consistent with the hypothesis that neoplastic change originates in the stem cell compartment. Prostate 67: 1384,1396, 2007. © 2007 Wiley-Liss, Inc. [source] Off-Target Decoding of a Multitarget Kinase Inhibitor by Chemical ProteomicsCHEMBIOCHEM, Issue 7 2009Enrico Missner Abstract Unbiased: Chemical proteomics was used to profile compound interactions in an unbiased fashion. We present here the application of different compound-immobilization routes for decoding nonprotein kinase off-targets of the multitarget kinase inhibitor C1, which interacts with distinct compound moieties. Since the approval of the first selective tyrosine kinase inhibitor, imatinib, various drugs have been developed to target protein kinases. However, due to a high degree of structural conservation of the ATP binding site, off-target effects have been reported for several drugs. Here, we report on off-target decoding for a multitarget protein kinase inhibitor by chemical proteomics, by focusing on interactions with nonprotein kinases. We tested two different routes for the immobilization of the inhibitor on a carrier matrix, and thus identified off-targets that interact with distinct compound moieties. Besides several of the kinases known to bind to the compound, the pyridoxal kinase (PDXK), which has been described to interact with the CDK inhibitor (R)-roscovitine, was captured. The PDXK,inhibitor interaction was shown to occur at the substrate binding site rather than at the ATP binding site. In addition, carbonic anhydrase 2 (CA2) binding was demonstrated, and the determination of the IC50 revealed an enzyme inhibition in the submicromolar range. The data demonstrate that different compound immobilization routes for chemical proteomics approaches are a valuable method to improve the knowledge about the off-target profile of a compound. [source] Retinoic acid induces CDK inhibitors and growth arrest specific (Gas) genes in neural crest cellsDEVELOPMENT GROWTH & DIFFERENTIATION, Issue 3 2005Linping Wang Retinoic acid (RA), the active metabolite of vitamin A, regulates cellular growth and differentiation during embryonic development. In excess, this vitamin is also highly teratogenic to animals and humans. The neural crest is particularly sensitive to RA, and high levels adversely affect migration, proliferation and cell death. We investigated potential gene targets of RA associated with neural crest proliferation by determining RA-mediated changes in gene expression over time, using microarrays. Statistical analysis of the top ranked RA-regulated genes identified modest changes in multiple genes previously associated with cell cycle control and proliferation including the cyclin-dependent kinase inhibitors Cdkn1a (p21), Cdkn2b (p15INK4b), and Gas3/PMP22. The expression of p21 and p15INK4b contribute to decreased proliferation by blocking cell cycle progression at G1-S. This checkpoint is pivotal to decisions regulating proliferation, apoptosis, or differentiation. We have also confirmed the overexpression of Gas3/PMP22 in RA-treated neural crests, which is associated with cytoskeletal changes and increased apoptosis. Our results suggest that increases in multiple components of diverse regulatory pathways have an overall cumulative effect on cellular decisions. This heterogeneity contributes to the pleiotropic effects of RA, specifically those affecting proliferation and cell death. [source] 2-(Benzylsulfanyl)-6-chloro-9-isopropylpurine, a Valuable Intermediate in the Synthesis of Diaminopurine Cyclin Dependent Kinase InhibitorsEUROPEAN JOURNAL OF ORGANIC CHEMISTRY, Issue 5 2005David Taddei Abstract The synthetic potential of a novel precursor of 2,6-diaminopurine CDK inhibitors, 2-(benzylsulfanyl)-6-chloro-9-isopropylpurine, is described. The Traube purine synthesis was chosen to prepare the required 2-(benzylsulfanyl)hypoxanthine intermediate. Attempts to prepare its purin-6-yl methanesulfonic ester analogue failed. Conversion to the 6-chloropurine derivative enabled the introduction of arylamines in the presence of catalytic amounts of acid. Further chemical variety was introduced on the purine through a regioselective Mitsunobu N -9 alkylation. Oxidative cleavage of the 2-(benzylsulfanyl) leaving group with an aliphatic amine was implemented as previously reported. Purvalanol A, a potent CDK inhibitor, was synthesised using this methodology. The template and intermediates were fully characterised by modern spectroscopic techniques and single-crystal X-ray diffraction. (© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2005) [source] INK4 proteins, a family of mammalian CDK inhibitors with novel biological functionsIUBMB LIFE, Issue 7 2007Eduardo T. Cánepa Abstract The cyclin D-Cdk4-6/INK4/Rb/E2F pathway plays a key role in controlling cell growth by integrating multiple mitogenic and antimitogenic stimuli. The members of INK4 family, comprising p16INK4a, p15INK4b, p18INK4c, and p19INK4d, block the progression of the cell cycle by binding to either Cdk4 or Cdk6 and inhibiting the action of cyclin D. These INK4 proteins share a similar structure dominated by several ankyrin repeats. Although they appear to be structurally redundant and equally potent as inhibitors, the INK4 family members are differentially expressed during mouse development. The striking diversity in the pattern of expression of INK4 genes suggested that this family of cell cycle inhibitors might have cell lineage-specific or tissue-specific functions. The INK4 proteins are commonly lost or inactivated by mutations in diverse types of cancer, and they represent established or candidate tumor suppressors. Apart from their capacity to arrest cells in the G1-phase of the cell cycle they have been shown to participate in an increasing number of cellular processes. Given their emerging roles in fundamental physiological as well as pathological processes, it is interesting to explore the diverse roles for the individual INK4 family members in different functions other than cell cycle regulation. Extensive studies, over the past few years, uncover the involvement of INK4 proteins in senescence, apoptosis, DNA repair, and multistep oncogenesis. We will focus the discussion here on these unexpected issues. iubmb Life, 59: 419-426, 2007 [source] Relative roles of TGF-,1 and Wnt in the systemic regulation and aging of satellite cell responsesAGING CELL, Issue 6 2009Morgan E. Carlson Summary Muscle stem (satellite) cells are relatively resistant to cell-autonomous aging. Instead, their endogenous signaling profile and regenerative capacity is strongly influenced by the aged P-Smad3, differentiated niche, and by the aged circulation. With respect to muscle fibers, we previously established that a shift from active Notch to excessive transforming growth factor-beta (TGF-,) induces CDK inhibitors in satellite cells, thereby interfering with productive myogenic responses. In contrast, the systemic inhibitor of muscle repair, elevated in old sera, was suggested to be Wnt. Here, we examined the age-dependent myogenic activity of sera TGF-,1, and its potential cross-talk with systemic Wnt. We found that sera TGF-,1 becomes elevated within aged humans and mice, while systemic Wnt remained undetectable in these species. Wnt also failed to inhibit satellite cell myogenicity, while TGF-,1 suppressed regenerative potential in a biphasic fashion. Intriguingly, young levels of TGF-,1 were inhibitory and young sera suppressed myogenesis if TGF-,1 was activated. Our data suggest that platelet-derived sera TGF-,1 levels, or endocrine TGF-,1 levels, do not explain the age-dependent inhibition of muscle regeneration by this cytokine. In vivo, TGF-, neutralizing antibody, or a soluble decoy, failed to reduce systemic TGF-,1 and rescue myogenesis in old mice. However, muscle regeneration was improved by the systemic delivery of a TGF-, receptor kinase inhibitor, which attenuated TGF-, signaling in skeletal muscle. Summarily, these findings argue against the endocrine path of a TGF-,1-dependent block on muscle regeneration, identify physiological modalities of age-imposed changes in TGF-,1, and introduce new therapeutic strategies for the broad restoration of aged organ repair. [source] Activation of cyclin-dependent kinases CDC2 and CDK2 in hepatocellular carcinomaLIVER INTERNATIONAL, Issue 3 2002Kay K. W. Li Abstract: Background: The cyclin-dependent kinases (CDKs) CDC2 and CDK2 are key regulators of the cell cycle. The expression of the CDK alone does not necessary reflect their true activities because they are highly regulated by post-translational mechanisms. Human hepatocellular carcinoma (HCC) is one of the most common cancers in the world, but the kinase activities of CDKs in HCC have not been examined. Methods: Here we examined the protein expression and kinase activities associated with CDC2 and CDK2 in HCC and the corresponding non-tumorous liver tissues. Results: CDC2 and CDK2 are activated in HCC in over 70% and 80% of the cases, respectively, but have little correlation with clinical parameters and PCNA expression. Interestingly, PCNA was readily detectable in extracts from non-tumorous liver, but more than 60% of samples contain higher concentration of PCNA in HCC than the corresponding non-tumorous tissues. CDC2 and CDK2 are generally activated in the same HCC samples, but the extent of their activation varied significantly, suggesting that the pathways leading to the activation of CDC2 and CDK2 can be regulated independently. Both positive regulators of CDK activity like cyclins and CDKs, and negative regulators of CDK activity like p21CIP1/WAF1 and Thr14/Tyr15 phosphorylation were up-regulated in HCC. Conclusion: CDC2 and CDK2 are activated in HCC, and this may be due to a complex interplay between the level of the cyclin, CDK, CDK inhibitors, and inhibitory phosphorylation. [source] Inhibition of proteasome-dependent degradation of Wee1 in G2 -arrested Hep3B cells by TGF,1MOLECULAR CARCINOGENESIS, Issue 4 2003Osamu Hashimoto Abstract Transforming growth factor ,1 (TGF,1)-induced G2 arrest was observed when a proliferation inhibitory function of the retinoblastoma protein (Rb) was compromised, but the mechanism underlying the G2 arrest was poorly characterized compared with that of G1 arrest. In the present study, we characterized G2 arrest induced by TGF,1 (1 ng/mL) in the Rb-negative hepatoma cell line (Hep3B) and compared with G1 arrest in the Rb-positive hepatoma cell line (Huh7). Activities of cyclin-dependent kinases (CDK) 2 and cell division cycle (CDC) 2 were markedly decreased at 24 h, the time when cell-cycle arrest became apparent in both cell lines. However, considerable amounts of inactive CDC2-cyclinB1 complexes were present in the nucleus of G2 -arrested Hep3B but were not present in G1 -arrested Huh7. The inhibitory phosphorylation of CDC2 on Tyr-15 was significantly elevated at 12,24 h, and its levels gradually declined during G2 arrest in Hep3B. In particular, augmentation of CDK inhibitors p21cip1 and p27kip1 and Wee1 kinase and diminution of CDC25C phosphatase coincided with induced Tyr-15 phosphorylation and inhibition of CDC2. Wee1 in Hep3B was unstable and was degraded in a proteasome-dependent manner, but it became substantially stabilized within 6 h of TGF,1 treatment. Moreover, a Wee1 inhibitor, PD0166285, abrogated the TGF,1-induced G2 arrest in Hep3B. These findings suggest that TGF,1 induced G2 arrest in Hep3B at least in part through stabilization of Wee1 and subsequent increase in Tyr-15 phosphorylation and inhibition of CDC2. © 2003 Wiley-Liss, Inc. [source] Alsterpaullone, a novel cyclin-dependent kinase inhibitor, induces apoptosis by activation of caspase-9 due to perturbation in mitochondrial membrane potential,MOLECULAR CARCINOGENESIS, Issue 4 2003Tyler Lahusen Abstract The majority of human neoplasms have aberrations in the retinoblastoma pathway due to hyperactivation of cyclin-dependent kinases (CDK). Based on this observation, novel small molecules, such as flavopiridol and UCN-01, are being developed and are currently being tested in the clinic. Efforts to develop CDK modulators led us to the discovery of a novel class of CDK inhibitors, the paullones [Cancer Res 1999;59:2566]. Initial studies demonstrated that paullones inhibit CDKs in vitro, thereby blocking cell-cycle progression. However, the exact mechanism for the antiproliferative effects of paullones was never explored. In this report, we demonstrate for the first time that the most potent paullone, alsterpaullone (Alp), induced apoptosis and promoted loss in clonogenicity in the Jurkat cell line. Alp caused early activation of both caspase-8 and -9, leading to cleavage of caspase-3 and poly(ADP-ribose) polymerase (PARP). Moreover, apoptosis by Alp was not associated with loss in anti-apoptotic proteins such as XIAP or BCL-XL. Pre-incubation with cell-permeable inhibitors z-Asp(OMe)-Glu(OMe)-Val-Asp(Ome)-fluoromethylketone and benzyloxycarbonyl-Val-Ala-Asp (OMe)-fluoromethylketone (ZVAD) blocked Alp-induced apoptosis. Moreover, the general caspase inhibitor ZVAD blocked the cleavage and activation of most caspases tested except caspase-9. Studies of mitochondrial membrane potential also demonstrated that Alp is able to disrupt mitochondrial potential in the presence of ZVAD, suggesting that the activation of caspase-9 by Alp follows mitochondrial perturbation. Pre-incubation of Jurkat cells with ZVAD did not prevent the depletion of cyclin D3, loss of CDK, or cell-cycle arrest by Alp. In summary, these experiments suggest that Alp activates caspase-9 via mitochondrial perturbation. Active caspase-9 cleaves and activates caspase-8 and caspase-3, leading to apoptosis. In the presence of the general caspase inhibitor ZVAD, the cell-cycle effects of Alp are unaltered while apoptosis is blocked, suggesting that the CDK effects of Alp are not sufficient for Alp-induced apoptosis. Additional studies with paullones are warranted to further characterize their preclinical effects and to explore their potential use in the clinical setting. Published 2003 Wiley-Liss, Inc. [source] Inhibition of cyclin-dependent kinases by olomoucine and roscovitine reduces lipopolysaccharide-induced inflammatory responses via down-regulation of nuclear factor ,BCELL PROLIFERATION, Issue 2 2009R-S. Jhou Objectives:, Initiation and maintenance of pro-inflammatory reactions elicited by bacterial lipopolysaccharide and/or cytokines in the macrophage lineage have been reported to play a crucial role in acute and chronic pathogenic effects. Whether pro-inflammatory responses triggered by lipopolysaccharide in growth arrested cells differ from those in proliferating cells remains unanswered. Materials and methods:, Olomoucine and roscovitine are cyclin-dependent kinase (CDK) inhibitors that prevent progression through the cell cycle. After treatment with CDK inhibitors, expression of pro-inflammatory genes was analysed by reverse transcriptase,polymerase chain reaction. Protein levels of inducible nitric oxide synthase (iNOS) and nuclear factor kappaB (NF-,B) were determined by Western blotting. Promoter activity of iNOS was measured by the luciferase activity assay. Results:, In this study we have demonstrated that both olomoucine and roscovitine inhibit cell proliferation and diminish nitric oxide production and cytokine gene expression, in lipopolysaccharide-stimulated murine RAW264.7 macrophages. In addition, olomoucine reduces iNOS promoter activity and alleviates NF-,B transcription activation. After co-transfection with E2F1 interference RNA, suppression of lipopolysaccharide-mediated iNOS promoter activity and NF-,B activation was observed. Furthermore, we demonstrated that olomoucine-induced growth arrested cells reduce expression of the p65 subunit of NF-,B. Conclusions:, The findings of this study suggest that inhibition of cell-cycle progression is capable of reducing pro-inflammatory responses via down-regulation of NF-,B. [source] Short-period hypoxia increases mouse embryonic stem cell proliferation through cooperation of arachidonic acid and PI3K/Akt signalling pathwaysCELL PROLIFERATION, Issue 2 2008S. H. Lee Hypoxia plays important roles in some early stages of mammalian embryonic development and in various physiological functions. This study examined the effect of arachidonic acid on short-period hypoxia-induced regulation of G1 phase cell-cycle progression and inter-relationships among possible signalling molecules in mouse embryonic stem cells. Hypoxia increased the level of hypoxia-inducible factor-1, (HIF-1,) expression and H2O2 generation in a time-dependent manner. In addition, hypoxia increased the levels of cell-cycle regulatory proteins (cyclin D1, cyclin E, cyclin-dependent kinase 2 (CDK2) and CDK4). Maximum increases in the level of these proteins and retinoblastoma phosphorylation were observed after 12,24 h of exposure to hypoxic conditions, and then decreased. Alternatively, the level of the CDK inhibitors, p21Cip1 and p27Kip1 were decreased. These results were consistent with the results of [3H]-thymidine incorporation and cell counting. Hypoxia also increased the level of [3H]-arachidonic acid release and inhibition of cPLA2 reduced hypoxia-induced increase in levels of the cell-cycle regulatory proteins and [3H]-thymidine incorporation. The level of cyclooxygenase-2 (COX-2) was also increased by hypoxia and inhibition of COX-2 decreased the levels of cell-cycle regulatory proteins and [3H]-thymidine incorporation. Indeed, the percentage of cells in S phase, levels of cell cycle regulatory proteins, and [3H]-thymidine incorporation were further increased in hypoxic conditions with arachidonic acid treatment compared to normoxic conditions. Hypoxia-induced Akt and mitogen-activated protein kinase (MAPK) phosphorylation was inhibited by vitamin C (antioxidant, 10,3 M). In addition, hypoxia-induced increase of cell-cycle regulatory protein expression and [3H]-thymidine incorporation were attenuated by LY294002 (PI3K inhibitor, 10,6 M), Akt inhibitor (10,6 M), rapamycin (mTOR inhibitor, 10,9 M), PD98059 (p44/42 inhibitor, 10,5 M), and SB203580 (p38 MAPK inhibitor, 10,6 M). Furthermore, hypoxia-induced increase of [3H]-arachidonic acid release was blocked by PD98059 or SB203580, but not by LY294002 or Akt inhibitor. In conclusion, arachidonic acid up-regulates short time-period hypoxia-induced G1 phase cyclins D1 and E, and CDK 2 and 4, in mouse embryonic stem cells through the cooperation of PI3K/Akt/mTOR, MAPK and cPLA2 -mediated signal pathways. [source] The cell cycle: a review of regulation, deregulation and therapeutic targets in cancerCELL PROLIFERATION, Issue 3 2003Katrien Vermeulen This review will focus on these mechanisms, i.e. regulation of cyclin-dependent kinases (CDK) by cyclins, CDK inhibitors and phosphorylating events. The quality checkpoints activated after DNA damage are also discussed. The complexity of the regulation of the cell cycle is also reflected in the different alterations leading to aberrant cell proliferation and development of cancer. Consequently, targeting the cell cycle in general and CDK in particular presents unique opportunities for drug discovery. This review provides an overview of deregulation of the cell cycle in cancer. Different families of known CDK inhibitors acting by ATP competition are also discussed. Currently, at least three compounds with CDK inhibitory activity (flavopiridol, UCN-01, roscovitine) have entered clinical trials. [source] TSC-box is essential for the nuclear localization and antiproliferative effect of XTSC-22DEVELOPMENT GROWTH & DIFFERENTIATION, Issue 3 2007Akiko Hashiguchi Transforming growth factor- ,1-stimulated clone 22 (TSC-22) encodes a leucine zipper-containing protein that is highly conserved among various species. Mammalian TSC-22 is a potential tumor suppressor gene. It translocates into nuclei and suppresses cell division upon antiproliferative stimuli. In human colon carcinoma cells, TSC-22 inhibits cell growth by upregulating expression of the p21 gene, a cyclin-dependent kinase (Cdk) inhibitor. We previously showed that the Xenopus laevis homologue of the TSC-22 gene (XTSC-22) is required for cell movement during gastrulation through cell cycle regulation. In this report, we investigated the molecular mechanism of the antiproliferative effect of XTSC-22. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis suggested that XTSC-22 did not affect the expression levels of the p21 family of Cdk inhibitors or other cell cycle regulators. Analysis of deletion mutants of XTSC-22 revealed that nuclear localization of the N-terminal TSC-box is necessary for cell cycle inhibition by XTSC-22. Further experiments suggested that p27Xic1, a key Cdk inhibitor in Xenopus, interacts with XTSC-22. Because p27Xic1 is a cell cycle inhibitor with a nuclear localization signal, it is possible that XTSC-22 suppresses cell division by translocating into the nucleus with p27Xic1, where it may potentiate the intranuclear action of p27Xic1. [source] Cell proliferation and cell cycle control: a mini reviewINTERNATIONAL JOURNAL OF CLINICAL PRACTICE, Issue 12 2004C.H. Golias Summary Tumourigenesis is the result of cell cycle disorganisation, leading to an uncontrolled cellular proliferation. Specific cellular processes-mechanisms that control cell cycle progression and checkpoint traversation through the intermitotic phases are deregulated. Normally, these events are highly conserved due to the existence of conservatory mechanisms and molecules such as cell cycle genes and their products: cyclins, cyclin dependent kinases (Cdks), Cdk inhibitors (CKI) and extra cellular factors (i.e. growth factors). Revolutionary techniques using laser cytometry and commercial software are available to quantify and evaluate cell cycle processes and cellular growth. S-phase fraction measurements, including ploidy values, using histograms and estimation of indices such as the mitotic index and tumour-doubling time indices, provide adequate information to the clinician to evaluate tumour aggressiveness, prognosis and the strategies for radiotherapy and chemotherapy in experimental researches. [source] Osteoclast Differentiation by RANKL Requires NF-,B-Mediated Downregulation of Cyclin-Dependent Kinase 6 (Cdk6),JOURNAL OF BONE AND MINERAL RESEARCH, Issue 7 2004Toru Ogasawara Abstract This study investigated the involvement of cell cycle factors in RANKL-induced osteoclast differentiation. Among the G1 cell cycle factors, Cdk6 was found to be a key molecule in determining the differentiation rate of osteoclasts as a downstream effector of the NF-,B signaling. Introduction: A temporal arrest in the G1 phase of the cell cycle is a prerequisite for cell differentiation, making it possible that cell cycle factors regulate not only the proliferation but also the differentiation of cells. This study investigated cell cycle factors that critically influence differentiation of the murine monocytic RAW264.7 cells to osteoclasts induced by RANKL. Materials and Methods: Growth-arrested RAW cells were stimulated with serum in the presence or absence of soluble RANKL (100 ng/ml). Expressions of the G1 cell cycle factors cyclin D1, D2, D3, E, cyclin-dependent kinase (Cdk) 2, 4, 6, and Cdk inhibitors (p18 and p27) were determined by Western blot analysis. Involvement of NF-,B and c- jun N-terminal kinase (JNK) pathways was examined by overexpressing dominant negative mutants of the I,B kinase 2 (IKKDN) gene and mitogen-activated protein kinase kinase 7 (MKK7DN) gene, respectively, using the adenovirus vectors. To determine the direct effect of Cdk6 on osteoclast differentiation, stable clones of RAW cells transfected with Cdk6 cDNA were established. Osteoclast differentiation was determined by TRACP staining, and cell cycle regulation was determined by BrdU uptake and flow cytometric analysis. Results and Conclusion: Among the cell cycle factors examined, the Cdk6 level was downregulated by RANKL synchronously with the appearance of multinucleated osteoclasts. Inhibition of the NF-,B pathway by IKKDN overexpression, but not that of the JNK pathway by MKK7DN overexpression, caused the decreases in both Cdk6 downregulation and osteoclastogenesis by RANKL. RAW cells overexpressing Cdk6 resist RANKL-induced osteoclastogenesis; however, cell cycle regulation was not affected by the levels of Cdk6 overexpression, suggesting that the inhibitory effect of Cdk6 on osteoclast differentiation was not exerted through cell cycle regulation. These results indicate that Cdk6 is a critical regulator of RANKL-induced osteoclast differentiation and that its NF-,B-mediated downregulation is essential for efficient osteoclast differentiation. [source] Glucose metabolism and proliferation in glia: role of astrocytic gap junctionsJOURNAL OF NEUROCHEMISTRY, Issue 4 2006Arantxa Tabernero Abstract Astrocytes play a well-established role in brain metabolism, being a key element in the capture of energetic compounds from the circulation and in their delivery to active neurons. Their metabolic status is affected in many pathological situations, such as gliomas, which are the most common brain tumors. This proliferative dysfunction is associated with changes in gap junctional communication, a property strongly developed in normal astrocytes studied both in vitro and in vivo. Here, we summarize and discuss the findings that have lead to the identification of a link between gap junctions, glucose uptake, and proliferation. Indeed, the inhibition of gap junctional communication is associated with an increase in glucose uptake due to a rapid change in the localization of both GLUT-1 and type I hexokinase. This effect persists due to the up-regulation of GLUT-1 and type I hexokinase and to the induction of GLUT-3 and type II hexokinase. In addition, cyclins D1 and D3 have been found to act as sensors of the inhibition of gap junctions and have been proposed to play the role of mediators in the mitogenic effect observed. Conversely, in C6 glioma cells, characterized by a low level of intercellular communication, an increase in gap junctional communication reduces glucose uptake by releasing type I and type II hexokinases from the mitochondria and decreases the exacerbated rate of proliferation due to the up-regulation of the Cdk inhibitors p21 and p27. Identification of the molecular actors involved in these pathways should allow the determination of potential therapeutic targets that could lead to the testing of alternative strategies to prevent, or at least slow down, the proliferation of glioma cells. [source] Cyclin-dependent kinase inhibitors for treating cancerMEDICINAL RESEARCH REVIEWS, Issue 6 2001Peter L. Toogood Abstract Cyclin dependent kinases (Cdks) are essential enzymes for the control of cell cycle progression. Inhibitors of cyclin-dependent kinases are anticipated to possess therapeutic utility against a wide variety of proliferative diseases, especially cancer. The field of published small molecule Cdk inhibitors is briefly reviewed here as background to a summary of work on a class of pyrido[2,3- d]pyrimidine Cdk inhibitors. Compounds from this class are described that display potency against cyclin D/Cdk4 up to IC50,=,0.004 ,M. Good to moderate selectivity for cyclin D/Cdk4 is also reported for compounds in this structural class. Structure-activity relationship data are presented for substitution at the C2 and N8 positions and these data are interpreted in the context of a binding model that is based on the Cdk2 crystal structure. A representative cyclin D/Cdk4 inhibitor (compound 56) is demonstrated to selectively inhibit the proliferation of an Rb+ cell line vs. a matched Rb, cell line and to produce a distinct G1 block consistent with cyclin D/Cdk4 inhibition in cells. © 2001 John Wiley & Sons, Inc. Med Res Rev, 21, No. 6, 487,498, 2001 [source] | ||||||||||||||||