Mitotic waves in the early embryogenesis of Drosophila: Bistability traded for speed.

Early embryogenesis of most metazoans is characterized by rapid and synchronous cleavage divisions. Chemical waves of Cdk1 activity were previously shown to spread across Drosophila embryos, and the underlying molecular processes were dissected. Here, we present the theory of the physical mechanisms that control Cdk1 waves in Drosophila The in vivo dynamics of Cdk1 are captured by a transiently bistable reaction-diffusion model, where time-dependent reaction terms account for the growing level of cyclins and Cdk1 activation across the cell cycle. We identify two distinct regimes. The first one is observed in mutants of the mitotic switch. There, waves are triggered by the classical mechanism of a stable state invading a metastable one. Conversely, waves in wild type reflect a transient phase that preserves the Cdk1 spatial gradients while the overall level of Cdk1 activity is swept upward by the time-dependent reaction terms. This unique mechanism generates a wave-like spreading that differs from bistable waves for its dependence on dynamic parameters and its faster speed. Namely, the speed of "sweep" waves strikingly decreases as the strength of the reaction terms increases and scales as the powers 3/4, -1/2, and 7/12 of Cdk1 molecular diffusivity, noise amplitude, and rate of increase of Cdk1 activity in the cell-cycle S phase, respectively. Theoretical predictions are supported by numerical simulations and experiments that couple quantitative measurements of Cdk1 activity and genetic perturbations of the accumulation rate of cyclins. Finally, our analysis bears upon the inhibition required to suppress Cdk1 waves at the cell-cycle pause for the maternal-to-zygotic transition.

Identification of a protein signature for predicting overall survival of hepatocellular carcinoma: a study based on data mining.

BACKGROUND: Hepatocellular carcinoma (HCC), is the fifth most common cancer in the world and the second most common cause of cancer-related deaths. Over 500,000 new HCC cases are diagnosed each year. Combining advanced genomic analysis with proteomic characterization not only has great potential in the discovery of useful biomarkers but also drives the development of new diagnostic methods. METHODS: This study obtained proteomic data from Clinical Proteomic Tumor Analysis Consortium (CPTAC) and validated in The Cancer Proteome Atlas (TCPA) and TCGA dataset to identify HCC biomarkers and the dysfunctional of proteogenomics. RESULTS: The CPTAC database contained data for 159 patients diagnosed with Hepatitis-B related HCC and 422 differentially expressed proteins (112 upregulated and 310 downregulated proteins). Restricting our analysis to the intersection in survival-related proteins between CPTAC and TCPA database revealed four coverage survival-related proteins including PCNA, MSH6, CDK1, and ASNS. CONCLUSION: This study established a novel protein signature for HCC prognosis prediction using data retrieved from online databases. However, the signatures need to be verified using independent cohorts and functional experiments.

Quantitative reconstitution of mitotic CDK1 activation in somatic cell extracts.

The regulation of mitotic entry in somatic cells differs from embryonic cells, yet it is only for embryonic cells that we have a quantitative understanding of this process. To gain a similar insight into somatic cells, we developed a human cell extract system that recapitulates CDK1 activation and nuclear envelope breakdown in response to mitotic cyclins. As cyclin B concentrations increase, CDK1 activates in a three-stage nonlinear response, creating an ordering of substrate phosphorylations. This response is established by dual regulatory feedback loops involving WEE1/MYT1, which impose a cyclin B threshold, and CDC25, which allows CDK1 to escape the WEE1/MYT1 inhibition. This system also exhibits a complex response to cyclin A. Cyclin A promotes WEE1 phosphorylation to weaken the negative feedback loop and primes mitotic entry through cyclin B. This observation explains the requirement of both cyclin A and cyclin B to initiate mitosis in somatic cells.

The clinical significance of CDK1 expression in oral squamous cell carcinoma.

OBJECTIVES: To evaluate the clinical significance of cyclin-dependent kinase 1 (CDK1) in 77 oral squamous cell carcinomas (OSCC) using immunohistochemical methods. STUDY DESIGN: Immunohistochemical expression of CDK1 was compared with various clinicopathological features in 77 OSCC and 60 controlled epithelia adjacent to the tumours. In addition, correlation of CDK1 expression and prognostic and the 5-year accumulative survival rate of OSCC were investigated. RESULTS: The CDK1 protein was expressed in 52 cases of 77 tumor tissues (67.5%), compared with 21 cases of 60 controlled (35.0%). The expression of CDK1 was significantly correlated with the histological grade of OSCC (P<0.05). The CDK1 protein was over-expressed in recurrent tumors or in those with lymph node metastasis. Statistical analysis showed a significant reduction in the 5-year accumulative survival rate in CDK1 positive cases compared with CDK1 negative cases (P<0.05). Namely, the CDK1 positive patients had poor prognosis. CONCLUSIONS: The expression of CDK1 might serve as malignant degree and prognostic markers for the survival of OSCC.

5,7,3'-Trihydroxy-3,4'-dimethoxyflavone inhibits the tubulin polymerization and activates the sphingomyelin pathway.

Flavonoids are polyphenolic compounds which display a vast array of biological activities and are among the most promising anti-cancer agents. The derivative of quercetin, 5,7,3'-trihydroxy-3,4'-dimethoxyflavone (THDF), is a natural flavonoid that inhibits cell proliferation and induces apoptosis in human leukemia cells. Here we show that THDF induces cell-cycle arrest in the M phase and inhibits tubulin polymerization. This was associated with the accumulation of cyclin B1 and p21(Cip1) , changes in the phosphorylation status of cyclin B1, Cdk1, Cdc25C, and MPM-2, and activation of the acidic sphingomyelinase (ASMase). Moreover, desipramine attenuated THDF-mediated cell death, indicating a crucial role of ASMase in the mechanism of cell death. In vivo studies on the athymic nude mouse xenograft model also confirmed that THDF inhibits growth of human leukemia cells and suggest that this compound may have therapeutic value.

Effects of puberty and gonadotropins on the molecular events controlling meiotic resumption of mouse oocytes.

Although studies suggest that the low competence of oocytes from prepubertal animals is due to their insufficient cytoplasmic maturation and that FSH improves oocyte maturation possibly by retarding meiotic progression and allowing more time for cytoplasmic maturation, the mechanisms by which puberty and gonadotropins regulate meiotic progression require additional detailed studies. For the first time, we observed that while meiotic progression was significantly slower, the maturation-promoting factor (MPF) activity of oocytes was significantly higher in prepubertal than in adult mice. To resolve this contradiction, we specified the molecules regulating the MPF activity and their localization during oocyte maturation in prepubertal and adult mice primed with or without gonadotropins. Our tests using corresponding enzyme regulators suggested that while activities of protein kinase A were unaffected, the activity of adenylate cyclase (ADCY) and phosphodiesterase increased while cell division cycle 2 homolog A (CDC2A) decreased significantly after puberty. While most of the adult oocytes had CDC2A protein concentrated in the germinal vesicle (GV) region, the majority of prepubertal oocytes showed no nuclear concentration of CDC2A. Maximally priming mice with equine chorionic gonadotropin brought the above parameters of prepubertal oocytes close to those in adult oocytes. Together, the results suggest that puberty and gonadotropin control oocyte meiotic progression mainly by regulating the ADCY activity and the concentration of the activated MPF toward the GV region.

DHA alters expression of target proteins of cancer therapy in chemotherapy resistant SW620 colon cancer cells.

Diets rich in n-3 polyunsaturated fatty acids (PUFAs) have been associated with a reduced risk of several types of cancer. Recent reports have suggested that these PUFAs enhance the cytotoxic effect of cancer chemoradiotherapy. The effect of docosahexaenoic acid (DHA) on key cell cycle regulators and target proteins of cancer therapy was investigated in the human malign colon cancer cell line SW620. Cell cycle check point proteins such as p21 and stratifin (14-3-3 sigma) increased at mRNA and protein level, whereas cell cycle progression proteins such as cell division cycle 25 homolog and cyclin-dependent kinase 1 decreased after DHA treatment. Protein levels of inhibitors of apoptosis family members associated with chemotherapy resistance and cancer malignancy, survivin and livin, decreased after the same treatment: likewise the expression of NF-kappaB. Levels of the proapoptotic proteins phosphorylated p38 MAPK and growth arrest-inducible and DNA damage-inducible gene 153/C/EBP-homologous protein (CHOP) increased. The results indicate that DHA treatment causes simultaneous cell cycle arrest in both the G1 and G2 phase. In conclusion, DHA affects several target proteins of chemotherapy in a favorable way. This may explain the observed enhanced chemosensitivity in cancer cells supplemented with n-3 PUFAs and encourage further studies investigating the role of n-3 PUFAs as adjuvant to chemotherapy and radiotherapy in vivo.

Mitochondrial DNA depletion induces radioresistance by suppressing G2 checkpoint activation in human pancreatic cancer cells.

We hypothesized that mitochondrial function regulates cell cycle checkpoint activation and radiosensitivity. Human pancreatic tumor cells (MiaPaCa-2, rho(+)) were depleted of mitochondrial DNA (rho degrees ) by culturing cells in the presence of ethidium bromide. Depletion of mitochondrial DNA was verified by PCR amplification of total DNA using primer pairs specific for mitochondrial DNA. Loss of mitochondrial DNA decreased plating efficiency and the percentage of cells in S phase. Exponential cultures were irradiated with 2, 4 and 6 Gy (dose rate: 0.83 Gy/min) of ionizing radiation and harvested for determination of cell viability, growth and cell cycle phase distributions. Rho degrees cells were radioresistant compared to rho(+) cells, with a dose-modifying factor (DMF) of 1.6. Although cell growth was significantly inhibited in irradiated rho(+) cells compared to unirradiated control cells, the inhibition in Rho degrees cells was minimal. In addition, mitochondrial DNA depletion suppressed radiation-induced G(2) checkpoint activation, which was accompanied by increases in both cyclin B1 and CDK1. These results suggest that mitochondrial function may regulate cell cycle checkpoint activation and radiosensitivity in pancreatic cancer cells.

Determination of the specific activity of CDK1 and CDK2 as a novel prognostic indicator for early breast cancer.

BACKGROUND: We recently established a novel assay for specific activity (SA) of cyclin-dependent kinases (CDKs) using small tumor samples (>/=8 mm(3)). The aim of this study was to investigate the prognostic significance of CDK1SA and CDK2SA in human breast cancer. METHODS: CDK1SA and CDK2SA were determined in 284 breast cancer patients and their prognostic significance was investigated. RESULTS: Tumors with high CDK1SA and high CDK2SA showed significantly poorer 5-year relapse-free survival than those with low CDK1SA and low CDK2SA, respectively (66.9% vs 84.2% for CDK1SA; 43.6% vs 83.6% for CDK2SA). Moreover, combined analysis of CDK1SA and CDK2SA enabled the classification of breast tumors into high-risk and low-risk groups, where tumors in the high-risk group were strongly associated with unfavorable prognosis (5-year relapse-free survival 69.4% for the high-risk group and 91.5% for the low-risk group). Multivariate analysis showed that the risk determined by combined analysis of CDK1SA and CDK2SA is a significant (hazard ratio 3.09, P < 0.001) prognostic indicator for relapse, especially in node-negative patients (hazard ratio 6.73, P < 0.001). CONCLUSION: Determination of CDK1SA and CDK2SA may be useful in the prediction of outcomes in breast cancer patients and has potential for use as a routine laboratory test.

Fertilization differently affects the levels of cyclin B1 and M-phase promoting factor activity in maturing and metaphase II mouse oocytes.

Fertilization affects levels of cyclin B1 and M-phase promoting factor (MPF) activity in maturing and metaphase II mouse oocytes in two distinct ways. In metaphase II oocytes, it leads to a Ca(2)(+)-dependent, continuous degradation of cyclin B1 and inactivation of cyclin dependent kinase (CDC2A)-cyclin B1 complex (MPF). In this paper, we show that neither mono- nor polyspermic fertilization of prometaphase I and metaphase I oocytes triggered degradation of cyclin B1. However, polyspermic fertilization of prometaphase I oocytes led to a transient decrease in MPF activity that lasted for 2 h. The inactivation of MPF in polyspermic prometaphase I oocytes did not depend on the fertilization-induced increase in the cytoplasmic concentration of free Ca(2)(+) ions, but was caused, at least in part, by dephosphorylation of CDC2A at threonine 161 (Thr161). We found that polyspermic fertilization did not affect glutathione levels in prometaphase I oocytes, and concluded that the decrease in MPF activity and dephosphorylation of CDC2A at Thr161 in polyspermic prometaphase I oocytes were not caused by a change in the redox status of the cell induced by an introduction of excessive amount of sperm protamines. Instead, we propose that inactivation of MPF activity in polyspermic maturing oocytes is caused by a change in nucleo-cytoplasmic ratio that leads to a 'titration' of kinases and phosphatases responsible for keeping MPF in an active state. This idea is supported by the finding that oocytes fused with thymocytes rather than spermatozoa also showed a transient decrease in MPF activity.

Sulindac sulfide, an aspirin-like compound, inhibits proliferation, causes cell cycle quiescence, and induces apoptosis in HT-29 colon adenocarcinoma cells.

Nonsteroidal antiinflammatory drugs (NSAIDs), have cancer preventive and tumor regressive effects in the human colon. They lower the incidence of and mortality from colorectal cancer and sulindac reduces the number and size of polyps in patients with familial adenomatous polyposis. We studied the effect of sulindac, and its metabolite sulindac sulfide, on the proliferation of HT-29 colon adenocarcinoma cells. Both compounds reduced the proliferation rate of these cells, changed their morphology, and caused them to accumulate in the G0/G1 phase of the cell cycle. These responses were time- and concentration-dependent and reversible. In addition, these compounds reduced the level and activity of several cyclin-dependent kinases (cdks), which regulate cell cycle progression. Sulindac and sulindac sulfide also induced apoptosis in these cells at concentrations that affected their proliferation, morphology, and cell cycle phase distribution. Sulindac sulfide was approximately sixfold more potent than sulindac in inducing these cellular responses. Our results indicate that inhibition of cell cycle progression and induction of apoptotic cell death contribute to the anti-proliferative effects of sulindac and sulindac sulfide in HT-29 cells. These findings may be relevant to the cancer preventive and tumor regressive effects of these compounds in humans.

The 2,6-disubstituted purine reversine induces growth arrest and polyploidy in human cancer cells.

Reversine (RV) is the synthetic purine identified from a protein kinase-based screen of purine mimetics and it has been shown to induce muscle myoblast differentiation into progenitor cells that can be further converted into other cell lineages. Since protein kinases play a pivotal role in cell cycle control, we hypothesize that RV might affect the proliferation of cancer cells. Herein we report that RV inhibited growth of cultured human tumor cells, respectively, PC-3, HeLa, CWR22Rv1, and DU-145 cells, and induced accumulation of polyploidal cells with > or =4N DNA content. However, RV was without effect on growth of normal prostate epithelial cells. RV-treated PC-3 cells showed enlarged nuclei and an estimated 100-fold increase in cell size. Moreover, PC-3 cells treated with RV for 2-4 days were accompanied by a marked increase in the expression of p21(WAF1), a modest elevation in the levels of cyclin D3 and CDK6 and concomitantly, also a substantial reduction in cyclin B and CDK1. These results suggest that RV may induce polyploidy and increase in cell size by up-regulating p21(WAF1) and cyclin D3/CDK6, while simultaneously suppressing the expression of cyclin B and CDK1.

pp39mos is associated with p34cdc2 kinase in c-mosxe-transformed NIH 3T3 cells.

We investigated the possible interactions between pp39mos and p34cdc2 kinase in NIH 3T3 cells transformed by c-mosxe. pp39mos is coprecipitated with p34cdc2 when using either anti-PSTAIR antibody or p13suc1-Sepharose beads. Likewise, p34cdc2 is coprecipitated with pp39mos when using anti-mos antibody. However, pp39mos was not present in histone H1 kinase-active p34cdc2 complexes precipitated with anti-p34cdc2 C-terminal peptide antibody even during metaphase of the cell cycle. The molar ratio of p34 to pp39mos in the p13suc1 complex is approximately 2:1. Consistent with the tight association between pp39mos and tubulin, tubulin was also present in equivalent amounts with pp39mos and p34 in the p13suc1 complex. This pp39mos-p34cdc2-tubulin complex may be important in transformation by the mos oncogene.

Novel CDC34 (UBC3) ubiquitin-conjugating enzyme mutants obtained by charge-to-alanine scanning mutagenesis.

CDC34 (UBC3) encodes a ubiquitin-conjugating (E2) enzyme required for transition from the G1 phase to the S phase of the budding yeast cell cycle. CDC34 consists of a 170-residue catalytic N-terminal domain onto which is appended an acidic C-terminal domain. A portable determinant of cell cycle function resides in the C-terminal domain, but determinants for specific function must reside in the N-terminal domain as well. We have explored the utility of "charge-to-alanine" scanning mutagenesis to identify novel N-terminal domain mutants of CDC34 that are enzymatically competent with respect to unfacilitated (E3-independent) ubiquitination but that nevertheless are defective with respect to its cell cycle function. Such mutants may reveal determinants of specific in vivo function, such as those required for interaction with substrates or trans-acting regulators of activity and substrate selectivity. Three of 18 "single-scan" mutants (in which small clusters of charged residues were mutated to alanine) were compromised with respect to in vivo function. One mutant (cdc34-109, 111, 113A) targeted a 12-residue segment of the Cdc34 protein not found in most other E2s and was unable to complement a cdc34 null mutant at low copy numbers but could complement a null mutant when overexpressed from an induced GAL1 promoter. Combining adjacent pairs of single-scan mutants to produce "double-scan" mutants yielded four additional mutants, two of which showed heat and cold sensitivity conditional defects. Most of the mutant proteins expressed in Escheria coli displayed unfacilitated (E3-independent) ubiquitin-conjugating activity, but two mutants differed from wild-type and other mutant Cdc34 proteins in the extent of multiubiquitination they catalyzed during an autoubiquitination reation-conjugating enzyme function and have identified additional mutant alleles of CDC34 that will be valuable in further genetic and biochemical studies of Cdc34-dependent ubiquitination.

[Knockdown of DNA-PKcs inhibits cell cycle and its mechanism of drug-resistant Bel7402/5-Fu hepatocellular carcinoma cells].

Objective To study the effect of the knock-down of the DNA-dependent protein kinase catalytic subunit (DNA-PKcs) on the cell cycle of the multidrug-resistant (MDR) Bel7402/5-Fu hepatocellular carcinoma cells and its MDR mechanism. Methods After cationic liposome-mediated siDNA-PKcs oligonucleotide transfection, the drug sensitivity of Bel7402/5-Fu cells to 5-fluorouracil (5-Fu) and adriamycin (ADM) was determined by MTT assay; the cell cycle were detected by flow cytometry; meanwhile, the protein expressions of cell cycle-related proteins P21, cell cycle protein B1 (cyclin B1), cell cycle division protein 2 (CDC2) were tested by Western blotting; the expressions of ataxia telangiectasia mutated (ATM) and p53 at both mRNA and protein levels were detected by real-time PCR and Western blot analysis. Results The MTT results showed siDNA-PKcs increased the chemotherapeutic sensitivity of Bel7402/5-Fu cells to 5-Fu and ADM. The flow cytometric analysis showed siDNA-PKcs decreased the percentage of S-phase cells but increased the percentage of G2/M phase cells. Western blotting showed siDNA-PKcs increased the protein expression of P21 but decreased cyclinB1 and CDC2 proteins. In addition, siDNA-PKcs also increased the expressions of ATM and p53. Conclusion DNA-PKcs silencing increases P21 while decreases cyclin B1 and CDC2 expressions, and finally induces G2/M phase arrest in Bel7402/5-Fu cells, which may be related to ATM-p53 signaling pathway.

Effects of selenium on the proliferation, apoptosis and testosterone production of sheep Leydig cells in vitro.

The objective of this study was to investigate the effects of selenium (Se) on in vitro proliferation, apoptosis and testosterone production of sheep Leydig cells and its underlying mechanism. Leydig cells were collected from 8-month-old sheep and divided into four treatment groups (0, 2.0, 4.0 and 8.0 µmol/L Se). After treatment with Se for 48 h, the MTT and flow cytometric assay were used to detect cell proliferation and apoptosis. Testosterone level in the culture medium was determined by ELISA. The mRNA expression and protein abundance of cell cycle, apoptosis and testosterone synthesis-related genes were detected using real-time PCR and western blot analysis. The results showed that the highest percentage of live and apoptotic cells was obtained in the 2.0 and 8.0 µmol/L group, respectively. In the Se treatment groups, the proliferation rate of Leydig cells and the expression of cell cycle-related genes were decreased with the increasing Se supplementation in the culture medium. The percentage of apoptotic cells was increased with the increasing Se level, which was consistent with the expression of pro-apoptosis genes. The highest GSH-Px activity and lowest ROS content were also observed in the 2.0 µmol/L group. Appropriate Se level (2.0 µmol/L) can significantly increase the expression of p-ERK1/2, StAR and 3ß-HSD, and improve the testosterone synthesis. Compared with the control group, PD0325901 could significantly inhibit the production of testosterone and the protein abundance of p-ERK1/2, StAR and 3ß-HSD. Se treatment can mitigate the inhibition effect of PD0325901 and the testosterone secretion between the 2.0 µmol/L and control group was not significantly different. These results demonstrate that Se can affect the proliferation and apoptosis of Leydig cells by regulating cellular oxidative stress and the expressions of cell cycle and apoptosis-related genes. Se can also enhance the testosterone production of Leydig cells by activating the ERK signaling pathway and the expression of its downstream genes (StAR and 3ß-HSD), which could be closely related to the regulating roles of Se in male fertility and spermatogenesis.

Elevation of cyclin B1, active cdc2, and HuR in cervical neoplasia with human papillomavirus type 18 infection.

Over 30 cervical epitheliotrophic HPV types may lead to altered biological functions that affect the clinical outcome of HPV infection. In order to determine the regulatory mechanism and effect of different HPV subtypes, we performed functional assays on cdc2, cyclinB1 and HuR in human uterine cervical samples. After confirming 22 HPV types among 95 cervical swabs, 10 cervical tissues, and seven established cell lines using a DNA chip, we evaluated the functional activities of G2 molecules assays, that included; western blotting for cyclin B1, cdc2 and phospho-cdc2 (Y15 and T161), immunoprecipitation for cdc2, a nuclear extraction fractional assay, and RT-PCR for cyclin B1. The expression of cyclin B1 was found to be dependent on HPV type, and was particularly overexpressed in high-risk types, whereas cdc2 was ubiquitously expressed irrespective of HPV type. Phospho-cdc2 and cyclin B1, however, were most intense in HPV18 infected cervical samples. Furthermore, the HuR stabilizing factor of the cyclin B1 transcript was upregulated in HPV 18 infected swabs. Moreover, SiHa cell line showed weaker G2 functional activity than the HeLa cell line. This study demonstrates that HPV-18 decreases the fidelity of mitotic checkpoints and increases cdc2-associated histone H1 kinase activity relative to control populations, and further shows that the G2 checkpoint is aberrant by virtue of the stabilization of cyclin B1 mRNA through the upregulation of HuR protein and the functional form of cdc2, especially in cases with HPV 18 infected cervical lesions.

Involvement of Cdc2 in axonal regeneration enhanced by exercise training in rats.

PURPOSE: Physical activity can improve sensorimotor recovery after peripheral nerve injury. We examined the effects of treadmill training (TMT) on axonal regeneration in the injured sciatic nerve of the rat and further investigated cellular and molecular events that underlie enhanced axonal regrowth by training. METHODS: After crush injury of the sciatic nerves, rats were randomly assigned into either TMT or sedentary groups. Three to 14 d after injury, changes in protein levels in the regenerating nerve were analyzed by Western blotting and immunofluorescence staining. Axonal regeneration was assessed by anterograde and retrograde tracing techniques. The animals' functional recovery was determined by the sciatic functional index. RESULTS: We identified enhanced axonal regrowth in the distal stump of the sciatic nerve 7-14 d after injury in the rats with TMT. Cell division cycle 2 (Cdc2) mRNA and protein levels were highly increased in the injured sciatic nerves 3 and 7 d after injury, and decreased to basal levels 14 d later. Daily TMT accelerated distal shift of Cdc2 mRNA and protein induced in the regenerating nerves, and Cdc2 kinase activity was similarly increased in the distal stump by TMT. Cdc2 protein induced by TMT was mainly colocalized with Schwann cell marker S100beta protein, and correlated with axial distribution pattern of bromodeoxyuridine-labeled proliferating cell population in the regenerating nerve. We further demonstrate that axonal regeneration and motor function recovery after injury, both of which were promoted by TMT, were greatly suppressed by in vivo administration of Cdc2 inhibitor roscovitine. CONCLUSION: The present data suggest that Cdc2 kinase activated in the regenerating sciatic nerve may play an important role in TMT-mediated enhancement of axonal regeneration.

Effect of roscovitine on nuclear maturation, MPF and MAP kinase activity and embryo development of prepubertal goat oocytes.

The low number of embryos obtained from IVM-IVF-IVC of prepubertal goat oocytes could be due to an incomplete cytoplasmic maturation. Roscovitine (ROS) inhibits MPF and MAP kinase activity and maintains the oocyte at Germinal Vesicle (GV) stage. The aim of this study was to determine if meiotic activity is arrested in prepubertal goat oocytes cultured with 0, 12.5, 25, 50 and 100 microM of ROS for 24 h. A group of oocytes from adult goats was cultured with 25 microM of ROS to compare the effect of ROS on prepubertal and adult goat oocytes. A sample of oocytes was stained to evaluate the nuclear stage at oocyte collection time and after ROS incubation. IVM-oocytes not exposed to ROS formed the control group. Prepubertal goat IVM-oocytes were inseminated and cultured for 8 days. The percentage of oocytes at GV stage, after exposition to ROS was significantly higher in adult goat oocytes (64.5%) than in prepubertal goat oocytes. No differences were found among 25, 50 and 100 microM ROS concentrations (29, 23 and 26%, oocytes at GV stage, respectively). After 8 days of culture, no differences in total embryos were observed between control oocytes and oocytes treated with 12.5 and 25 microM (45.2, 36.1 and 39.4%, respectively), however the percentage of blastocysts was higher in the control group. Western blot for the MAPK and p34(cdc2) showed that both enzymes were active in prepubertal goat oocytes after 24h of ROS exposition. In conclusion, a low percentage of prepubertal goat oocytes reached GV stage after ROS incubation; possibly because most of them had reinitiated the meiosis inside the follicle. ROS did not affect fertilization or total embryos but ROS showed a negative effect on blastocyst development.

Nuclear signaling induced by ionizing radiation involves colocalization of the activated p56/p53lyn tyrosine kinase with p34cdc2.

The Src-like protein-tyrosine kinase p56/p53lyn associates with cell membranes and transduces signals from activated cell surface receptors. In the present work, cell fractionation and confocal microscopy studies demonstrate expression of Lyn in the nucleus. We also demonstrate that exposure of intact cells to ionizing radiation is associated with selective activation of nuclear Lyn. Similar findings have been obtained following irradiation of purified nuclei. Immunoprecipitation studies of nuclear lysates demonstrate radiation-induced binding of Lyn to p34cdc2. Nuclear colocalization of Lyn with Cdc2 has been confirmed by confocal microscopy. Other studies with glutathione S-transferase-Lyn fusion proteins demonstrate that the binding of Lyn to nuclear Cdc2 is associated with inhibition of Cdc2 activity. These findings suggest that the association of activated Lyn with Cdc2 in the nucleus may contribute to regulation of a DNA damage-dependent premitotic checkpoint.