CDK4 T172 phosphorylation is central in a CDK7-dependent bidirectional CDK4/CDK2 interplay mediated by p21 phosphorylation at the restriction point

Cell cycle progression, including genome duplication, is orchestrated by cyclin-dependent kinases (CDKs). CDK activation depends on phosphorylation of their T-loop by a CDK-activating kinase (CAK). In animals, the only known CAK for CDK2 and CDK1 is cyclin H-CDK7, which is constitutively active. Therefore, the critical activation step is dephosphorylation of inhibitory sites by Cdc25 phosphatases rather than unrestricted T-loop phosphorylation. Homologous CDK4 and CDK6 bound to cyclins D are master integrators of mitogenic/oncogenic signaling cascades by initiating the inactivation of the central oncosuppressor pRb and cell cycle commitment at the restriction point. Unlike the situation in CDK1 and CDK2 cyclin complexes, and in contrast to the weak but constitutive T177 phosphorylation of CDK6, we have identified the T-loop phosphorylation at T172 as the highly regulated step determining CDK4 activity. Whether both CDK4 and CDK6 phosphorylations are catalyzed by CDK7 remains unclear. To answer this question, we took a chemical-genetics approach by using analogue-sensitive CDK7(as/as) mutant HCT116 cells, in which CDK7 can be specifically inhibited by bulky adenine analogs. Intriguingly, CDK7 inhibition prevented activating phosphorylations of CDK4/6, but for CDK4 this was at least partly dependent on its binding to p21(cip1). In response to CDK7 inhibition, p21-binding to CDK4 increased concomitantly with disappearance of the most abundant phosphorylation of p21, which we localized at S130 and found to be catalyzed by both CDK4 and CDK2. The S130A mutation of p21 prevented the activating CDK4 phosphorylation, and inhibition of CDK4/6 and CDK2 impaired phosphorylations of both p21 and p21-bound CDK4. Therefore, specific CDK7 inhibition revealed the following: a crucial but partly indirect CDK7 involvement in phosphorylation/activation of CDK4 and CDK6; existence of CDK4-activating kinase(s) other than CDK7; and novel CDK7-dependent positive feedbacks mediated by p21 phosphorylation by CDK4 and CDK2 to sustain CDK4 activation, pRb inactivation, and restriction point passage

Intra- and interobserver analysis in the morphological assessment of early stage embryos during an IVF procedure: a multicentre study

Contains fulltext : 96031.pdf (publisher's version ) (Open Access

JNKs function as CDK4-activating kinases by phosphorylating CDK4 and p21

Cyclin D-CDK4/6 are the first cyclin-dependent kinase (CDK) complexes to be activated by mitogenic/oncogenic pathways. They have a central role in the cell multiplication decision and in its deregulation in cancer cells. We identified T172 phosphorylation of CDK4 rather than cyclin D accumulation as the distinctly regulated step determining CDK4 activation. This finding challenges the view that the only identified metazoan CDK-activating kinase, cyclin H-CDK7-Mat1 (CAK), which is constitutively active, is responsible for the activating phosphorylation of all cell cycle CDKs. We previously showed that T172 phosphorylation of CDK4 is conditioned by an adjacent proline (P173), which is not present in CDK6 and CDK1/2. Although CDK7 activity was recently shown to be required for CDK4 activation, we proposed that proline-directed kinases might specifically initiate the activation of CDK4. Here, we report that JNKs, but not ERK1/2 or CAK, can be direct CDK4-activating kinases for cyclin D-CDK4 complexes that are inactivated by p21-mediated stabilization. JNKs and ERK1/2 also phosphorylated p21 at S130 and T57, which might facilitate CDK7-dependent activation of p21-bound CDK4, however, mutation of these sites did not impair the phosphorylation of CDK4 by JNKs. In two selected tumor cells, two different JNK inhibitors inhibited the phosphorylation and activation of cyclin D1-CDK4-p21 but not the activation of cyclin D3-CDK4 that is mainly associated to p27. Specific inhibition by chemical genetics in MEFs confirmed the involvement of JNK2 in cyclin D1-CDK4 activation. Therefore, JNKs could be activating kinases for cyclin D1-CDK4 bound to p21, by independently phosphorylating both CDK4 and p21

Semi-automated morphometric analysis of human embryos can reveal correlations between total embryo volume and clinical pregnancy

what is known already: Morphometric analysis of a large group of embryos has revealed the potential to optimize algorithms for image-analysis systems for the grading of embryos and predicting pregnancy outcomes. study design, size, duration: Oocytes and embryos were obtained from 458 patients who underwent single embryo transfer on Day 3 after IVF/ICSI, between September 2006 and December 2010 at the Leuven University Fertility Center, Belgium. In total, the data set contained 2796 embryos including 458 embryos that were transferred on Day 3. Ongoing pregnancy was defined as the presence of at least one intrauterine gestational sac at 20 weeks. participants/materials, setting, methods: Patients included in this study were younger than 36 years, entering their first (n ¼ 375) or second (n ¼ 83) IVF/ICSI cycle and were only included once. Patients were excluded if the cycle included biopsy for PGD or if donor sperm/donor oocytes were used. Based on the 26 sequential images of the same embryo taken at one time point in different planes, the software calculates the total cytoplasmic volume for each time point, from which any reduction or change in the volume with time can be assessed (which helps interpret the degree of fragmentation) and the size of blastomeres. The diameter of the smallest and largest blastomere and the total volume of each embryo were extracted from the computer-assisted scoring system database and the coefficient of diversity was calculated for Days 1, 2 and 3. A logistic regression analysis was performed to determine the range of embryo volume associated with an increased chance of pregnancy. main results and the role of chance: On Day 3, blastomeres of 8-cell stage embryos were less divergent in size than those of 6-, 7-, 9-cell stage embryos. Although, the coefficients of diversity (ratio of the largest:smallest blastomeres) of implanted embryos tended to be lower than for non-implanted embryos, the difference was only significant for 6-cell stage embryos (P ¼ 0.02). After logistic regression, an association between total embryo volume and pregnancy was observed which had a quadratic nature: both lower and higher volumes were associated with a lower probability of successful pregnancy. A significant association was identified between total embryo volume and pregnancy rate on both Days 2 (P ¼ 0.003) and 3 (P ¼ 0.0003). Diagnostic measures (sensitivity, specificity, positive predictive value, accuracy and c-statistics) of the defined volume range were relatively poor. However, results showed a good negative predictive value [76.86% (95% confidence interval 71.03-82.02) on Day 3]. limitations, reasons for caution: A general disadvantage of studies evaluating the impact of a characteristic on the implantation potential of an embryo is the fact that the best embryo is chosen for transfer. No comparisons can therefore be made with the other embryos. Moreover, the decision process is currently based on a non-automated, standard scoring system, which means that a 'bias' in the selection process is always present. wider implications of the findings: Our results are an important step towards the development of an automated computer-assisted scoring system for the morphological characteristics of human embryos to improve embryo selection for optimizing implantation potential. Total embryo volume appears to be one of the objective characteristics that should be included. study funding/competing interest(s): None. trial registration number: Not applicable

Intra- and inter-observer analysis in the morphological assessment of early-stage embryos

<p>Abstract</p> <p>Background</p> <p>The aim of this study was to determine the intra- and inter-observer variability in the evaluation of embryo quality. Multilevel images of embryos on day 1, day 2 and day 3, were analysed using different morphological parameters.</p> <p>Methods</p> <p>Multilevel images of embryos on day 1, day 2 and day 3, were analysed using a standard scoring system. The kappa coefficient was calculated to measure intra- and inter-observer variability before and after training sessions.</p> <p>Results</p> <p>Good to excellent intra-observer agreement was present for most parameters exceptions being scoring the position of pronuclei and the presence of a cytoplasmic halo on day 1, multinucleation on day 2 and the size of fragments on day 3. Inter-observer agreement was only good to excellent for the number of blastomeres on day 2 and day 3 and the orientation of the cleavage axes on day 2. Training sessions had a positive impact on inter-observer agreement.</p> <p>Conclusion</p> <p>In conclusion, assessment of morphological characteristics of early stage embryos using multilevel images was marked by a high intra-observer and a moderate inter-observer agreement. Training sessions were useful to increase inter-observer agreement.</p

Ecofriendly recycled aggregate concrete and bioreceptivity

Nowadays, it becomes essential to limit environmental impact of building materials and to consider the life cycle of materials used. Recycling of materials from demolition has the dual objective of preserving natural resources and limiting the number of storage sites. The study presented here aims to develop the use of recycled aggregate issued of concrete in total replacement of natural materials (sand and gravel). This work has to be done upstream the studies on bio-corrosion or bio-receptivity of these concrete composed of recycled aggregates. Following an experimental analysis of physical, mechanical and mineralogical properties of recycled aggregates,the influence of these characteristics on choosing formulation parameters of concrete and mortar was studied. It was shown that the use of superplasticizers is necessary to reach satisfactory properties of concrete. The next step of this work will be toanalyse the bio-receptivity of these concrete and compatibilitywith bio-admixture used to decrease bio-receptivity and bio-corrosion; and to develop bio-superplasticizers to replace chemical ones

Bicyclic triterpenoid Iripallidal induces apoptosis and inhibits Akt/mTOR pathway in glioma cells

<p>Abstract</p> <p>Background</p> <p>The highly resistant nature of glioblastoma multiforme (GBM) to chemotherapy prompted us to evaluate the efficacy of bicyclic triterpenoid Iripallidal against GBM in vitro.</p> <p>Methods</p> <p>The effect of Iripallidal on proliferation and apoptosis in glioma cell lines was evaluated by MTS, colony formation and caspase-3 activity. The effect of iripallidal to regulate (i) Akt/mTOR and STAT3 signaling (ii) molecules associated with cell cycle and DNA damage was evaluated by Western blot analysis. The effect of Iripallidal on telomerase activity was also determined.</p> <p>Results</p> <p>Iripallidal (i) induced apoptosis, (ii) inhibited Akt/mTOR and STAT3 signaling, (iii) altered molecules associated with cell cycle and DNA damage, (iv) inhibited telomerase activity and colony forming efficiency of glioma cells. In addition, Iripallidal displayed anti-proliferative activity against non-glioma cancer cell lines of diverse origin.</p> <p>Conclusion</p> <p>The ability of Iripallidal to serve as a dual-inhibitor of Akt/mTOR and STAT3 signaling warrants further investigation into its role as a therapeutic strategy against GBM.</p