Fosfatase Cdc25B: Interações transientes intramoleculares e complexação com pequenos ligantes / Cdc25B phosphatase: Transient intramolecular interactions and small molecules complexation

Proteínas tirosina-fosfatase (PTPs) possuem papel fundamental na regulação da transdução de sinais e estão envolvidas em diversos processos fundamentais do ciclo celular. As Cdc25 (Cell Division Cycle 25) são fosfatases duais encontradas em todos os organismos eucarióticos e atuam em checkpoints do ciclo celular, permitindo ou inibindo o prosseguimento deste. Este grupo de proteínas pertence à classe de PTPs com atividade baseada em cisteína, apresenta domínio catalítico altamente conservado assim como o motivo catalítico, P-loop. Devido sua função, as Cdc25 são consideradas possíveis alvos terapêuticos para tratamento de câncer e sua interação com pequenas moléculas e inibidores tem sido investigada de forma que análises estruturais e de ligação das Cdc25 com inibidores podem elucidar aspectos importantes do mecanismo de ação destes além de direcionar para o desenho racional de fármacos. Interações cátion-π são interações intra ou intermoleculares não-covalentes que ocorrem entre uma espécie química catiônica, como o grupo guanidino de argininas, e uma das faces de um sistema π rico em elétrons, como dos anéis indólicos de triptofanos. Apesar de pouco discutidas na literatura, quando em comparação às interações não-covalentes mais convencionais, do ponto de vista energético as interações cátion-π são tão importantes na estruturação de proteínas quanto às ligações de hidrogênio ou pontes salinas. De fato estas interações são observadas com frequência em estruturas proteicas resolvidas. O domínio catalítico da Cdc25B possui diversas argininas expostas em sua superfície e um único resíduo de triptofano localizado na região C-terminal flexível, muito próximo do sítio catalítico da proteína. A flexibilidade de proteínas ou de regiões proteicas apresenta importante papel no reconhecimento entre biomoléculas participantes de vias de sinalização e tem sido muito estudada atualmente. Aqui, simulações de dinâmica molecular, experimentos de 1H-15N HSQC RMN, ensaios de cinética de inibição e de ancoragem molecular, evidenciam a existência de contatos cátion-π transientes na superfície de um importante membro da família das Cdc25, a Cdc25B, e de sítios de interação entre inibidores testados e a proteína com destaque a sítios na proximidades do P-loop, região próxima ao C-terminal desordenado, onde se demonstra estabilidade da interação com os pequenos ligantes

Protein tyrosine phosphatase (PTPs) play a fundamental role in the regulation of signal transduction and are involved in several fundamental processes of the cell cycle. Cdc25 (Cell Division Cycle 25) are dual phosphatases found in all eukaryotic organisms and act at checkpoints of the cell cycle, allowing or inhibiting its progression. This group of proteins belongs to the class of PTPs with cysteine-based activity, presenting a highly conserved catalytic domain as well as the catalytic motif, P-loop. Due to their function, Cdc25 are considered possible therapeutic targets for cancer treatment and their interaction with small molecules and inhibitors has been investigated so that structural and binding analyzes of Cdc25 with inhibitors can elucidate important aspects of their mechanism of action besides directing to rational drug design. Cation-π interactions are non-covalent intra- or intermolecular interactions that occur between a cationic chemical species, such as the guanidino group of arginines, and one of the faces of an electron-rich system, such as the indole rings of tryptophans. Although little discussed in the literature, when compared to more conventional non-covalent interactions, from the energetic point of view, cation-π interactions are as important in the structuring of proteins as hydrogen bonds or salt bridges. In fact, these interactions are frequently observed in solved protein structures. The catalytic domain of Cdc25B has several arginines exposed on its surface and a single tryptophan residue located in the flexible C-terminal region, very close to the catalytic site of the protein. The flexibility of proteins or protein regions plays an important role in the recognition between biomolecules participating in signaling pathways and has been extensively studied today. Here, molecular dynamics simulations, 1H-15N HSQC NMR experiments, inhibition kinetics and molecular anchoring assays, evidence the existence of transient cation-π contacts on the surface of an important member of the Cdc25 family, Cdc25B, and of sites of interaction between tested inhibitors and the protein, with emphasis on sites in the vicinity of the P-loop, a region close to the disordered C-terminus, where stability of the interaction with the small ligands is demonstrated

CDC25B Inhibition by Menadione: A Potential New Therapeutical Approach.

Gastric cancer (GC) is the fifth most common type of tumor and the third leading cause of cancer death worldwide. The evolution of gastric carcinogenesis is still poorly understood and, for this reason, preclinical research protocols were established that included the development of gastric cancer cell lines and the establishment of models of gastric carcinogenesis in non-human primates such as Sapajus apella. A comprehensive literature search was performed in relevant databases such as PubMed, ResearchGate, and Google Scholar to identify studies related to the topic. After an in-depth study of these reports, significant data were collected and compiled under appropriate headings. The main result of the studies carried out by the group on GC is the demonstration of the MYC gene overexpression as a common phenomenon in stomach carcinogenesis. Furthermore, we revealed that reducing the expression of the CDC25B gene, regulated by the MYC protein, is a therapeutic strategy against stomach tumors. This review article reveals preclinical evidence that treatment with menadione in experimental models of gastric tumorigenesis, in vivo and in vitro, inhibits the action of the phosphatase CDC25B and, consequently, prevents cell proliferation, invasion, and migration.

Vanadium oxides modify the expression levels of the p21, p53, and Cdc25C proteins in human lymphocytes treated in vitro.

In vitro assays have demonstrated that vanadium compounds interact with biological molecules similar to protein kinases and phosphatases and have also shown that vanadium oxides decrease the proliferation of cells, including human lymphocytes; however, the mechanism, the phase in which the cell cycle is delayed and the proteins involved in this process are unknown. Therefore, we evaluated the effects of vanadium oxides (V2 O3 , V2 O4 and V2 O5 ) in human lymphocyte cultures (concentrations of 2, 4, 8, or 16 µg/ml) on cellular proliferation and the levels of the p53, p21 and Cdc25C proteins. After 24 h of treatment with the different concentrations of vanadium oxides, the cell cycle phases were determined by evaluating the DNA content using flow cytometry, and the levels of the p21, p53 and Cdc25C proteins were assessed by Western blot analysis. The results revealed that the DNA content remained unchanged in every phase of the cell cycle; however, only at high concentrations did protein levels increase. Although, according to previous reports, vanadium oxides induce a delay in proliferation, DNA analysis did not show this occurring in a specific cell cycle phase. Nevertheless, the increases in p53 protein levels may cause this delay.

The prognostic role of FZD6 in esophageal squamous cell carcinoma patients.

BACKGROUND: Esophageal squamous cell carcinoma (ESCC) is a kind of cancer with heterogeneous biological characteristics, which is affected by a complex network of gene interactions. Identification of molecular biomarkers paves the way for individualized therapy based on gene expression profiles, which can overcome the heterogeneity of ESCC. METHODS: In this study, GSE20347, GSE23400 and GSE45670 datasets were retrieved from Gene Expression Omnibus (GEO) database, and the overlapping differentially expressed genes (DEGs) in three datasets were screened. Then the overlapping DEGs function was annotated by Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway-enrichment analysis. The prognostic value of the top five KEGG pathway-related genes were further validated in The Cancer Genome Atlas (TCGA) database. After extensive statistical analysis, four genes (CDC25B, CXCL8, FZD6 and MCM4) were identified as potential prognostic markers. Among the four candidate genes, the prognostic value of FZD6 in ESCC patients has not been evaluated. Therefore, we finally used immunohistochemistry method to evaluate the effect of FZD6 on the prognosis of patients with ESCC. Additionally, we detected the expression level of FZD6 in ESCC cell line and normal esophageal epithelial cell line, and observed the cell viability of ESCC cell line after FZD6 knockdown. RESULTS: The results showed that the overexpression of FZD6 predicted poor overall survival (OS) (P = 0.005) and progression-free survival (PFS) (P = 0.004) in ESCC patients. COX regression analysis showed that N stage (P = 0.026) and FZD6 expression level (P = 0.001) were independent prognostic factors of OS for ESCC patients. Furthermore, compared with normal esophageal epithelial cell line, the up-regulation of FZD6 was detected in ESCC cell line. Knockdown of FZD6 could significantly inhibit the proliferation of ESCC cells (P < 0.001). CONCLUSION: CDC25B, CXCL8, FZD6 and MCM4 were screened as candidate genes for prognosis assessment of patients with ESCC. The prognostic role of FZD6 in ESCC patients was confirmed in current study.

MicroRNA-125a-3p overexpression promotes liver regeneration through targeting proline-rich acidic protein 1.

INTRODUCTION AND OBJECTIVES: Liver regeneration plays a valuable significance for hepatectomies, and is mainly attributed to hepatocyte proliferation. MicroRNA-125a-3p was reported to be highly associated with liver regeneration process. We studied the underlying mechanism of the functional role of miR-125a-3p in liver regeneration. MATERIALS AND METHODS: The miR-125a-3p mimics and inhibitor vector were constructed and transfected into primary human liver HL-7702 cells, the transfected cell viability was detected using cell counting kit-8 (CCK-8). Cell cycle distribution was analyzed by flow cytometry. With Targetscan and OUGene prediction, the potential targets of miR-125 were verified by real-time quantitative PCR (qPCR) and luciferase reporter assays in turn. The overexpression vector of proline-rich acidic protein 1 (PRAP1) was constructed and co-transfected with miR-125a-3p mimics into HL-7702 cells, detecting the changes of proliferative capacity and cell cycle distribution. Western blot and qPCR performed to analyze gene expressions. RESULTS: Overexpressed miR-125a-3p notably increased the hepatocyte viability at 48h, and decreased the number of G1 phase cells (p<0.05). However, miR-125a-3p inhibition suppressed the development of hepatocytes. PRAP1 was the target of miR-125a-3p. After co-transfection with PRAP1 vector, hepatocyte viability was decrease and the G1 phase cell number was increased (p<0.05). More importantly, overexpressed PRAP1 notably decreased the mRNA and protein levels of cyclin D1, cyclin-dependent kinase 2 (CDK2) and cell division cycle 25A (CDC25A). CONCLUSION: The elevated miR-125a-3p positively correlated with hepatocyte viability and cell cycle progression due to the modulation of PRAP1, and miR-125a-3p may contribute to improving liver regeneration.

Overexpression of CDC25C affects the cell cycle of ovarian granulosa cells from adult and young goats

Background: CDC25 is a dual-specificity phosphatase that was first identified in the yeast Schizosaccharomyces pombe as a cell cycle-defective mutant. Although CDC25 is involved in the cell cycle of ovarian granulosa cells, the CDC25 signaling pathway has not been clarified fully. To explore the role of CDC25C in the cell cycle of goat ovarian granulosa cells, a CDC25C-overexpressing vector, pCMV-HA-CDC25C, was constructed and transfected into granulosa cells from adult and young white goats from Jiangsu Nantong. RT-PCR was used to measure CDC25C, CDK1, and WEE1 gene expression levels, and flow cytometry was used to distinguish ovarian granulosa cells in different phases of the cell cycle. Progesterone and estradiol levels in transfected ovarian granulosa cells were also measured. Results: In adult goat follicular granulosa cells transfected with pCMV-HA-CDC25C, CDC25C expression increased significantly, which greatly increased the relative gene expression levels of both CDK1 and WEE1. Additionally, progesterone and estradiol levels were increased in goat follicular granulosa cells overexpressing CDC25C. And the cell cycle results showed that transfection of pCMV-HA-CDC25C leads to a higher proportion of cells in S phase compared to the no vector-transfected groups. Conclusions: The results of this study indicated that the overexpression of CDC25C may increase the gene expression levels of both WEE1 and CDK1 in S phase and accelerate the transition of cells from G1 phase to S phase.

YWHAE silencing induces cell proliferation, invasion and migration through the up-regulation of CDC25B and MYC in gastric cancer cells: new insights about YWHAE role in the tumor development and metastasis process.

We previously observed reduced YWHAE (14-3-3ε) protein expression in a small set of gastric cancer samples. YWHAE may act as a negative regulator of the cyclin CDC25B, which is a transcriptional target of MYC oncogene. The understanding of YWHAE role and its targets is important for the better knowledge of gastric carcinogenesis. Thus, we aimed to evaluate the relationship among YWHAE, CDC25B, and MYC in vitro and in vivo. For this, we analyzed the YWHAE, CDC25B, and MYC expression in YWHA-silenced, CDC25B-silenced, and MYC-silenced gastric cancer cell lines, as well as in gastric cancer and non-neoplastic gastric samples. In gastric cancer cell lines, YWHAE was able to inhibit the cell proliferation, invasion and migration through the reduction of MYC and CDC25B expression. Conversely, MYC induced the cell proliferation, invasion and migration through the induction of CDC25B and the reduction of YWHAE. Most of the tumors presented reduced YWHAE and increased CDC25B expression, which seems to be important for tumor development. Increased MYC expression was a common finding in gastric cancer and has a role in poor prognosis. In the tumor initiation, the opposite role of YWHAE and CDC25B in gastric carcinogenesis seems to be independent of MYC expression. However, the inversely correlation between YWHAE and MYC expression seems to be important for gastric cancer cells invasion and migration. The interaction between YWHAE and MYC and the activation of the pathways related to this interaction play a role in the metastasis process.

Conformational flexibility of the complete catalytic domain of Cdc25B phosphatases.

Cdc25B phosphatases are involved in cell cycle checkpoints and have become a possible target for developing new anticancer drugs. A more rational design of Cdc25B ligands would benefit from detailed knowledge of its tertiary structure. The conformational flexibility of the C-terminal region of the Cdc25B catalytic domain has been debated recently and suggested to play an important structural role. Here, a combination of experimental NMR measurements and molecular dynamics simulations for the complete catalytic domain of the Cdc25B phosphatase is presented. The stability of the C-terminal α-helix is confirmed, but the last 20 residues in the complete catalytic domain are very flexible, partially occlude the active site and may establish transient contacts with the protein core. This flexibility in the C-terminal tail may modulate the molecular recognition of natural substrates and competitive inhibitors by Cdc25B. Proteins 2016; 84:1567-1575. © 2016 Wiley Periodicals, Inc.

A Novel CDC25B Promoter-Based Oncolytic Adenovirus Inhibited Growth of Orthotopic Human Pancreatic Tumors in Different Preclinical Models.

PURPOSE: We decided to construct a novel oncolytic adenovirus whose replication was driven by the CDC25B promoter for its use in preclinical models of pancreatic cancer. EXPERIMENTAL DESIGN: We placed the essential E1A gene under control of the CDC25B promoter. Based on preliminary data, we pseudotyped the adenovirus with a chimeric fiber of serotypes 5/3. We investigated the in vitro lytic effect and the in vivo therapeutic efficacy in combination with gemcitabine on human pancreatic tumor xenografts orthotopically growing in nude mice and in tumors growing in Syrian hamsters. We also assessed biochemical markers of hepatic toxicity and CA19.9 levels. RESULTS: AV25CDC exhibited a strong in vitro lytic effect on pancreatic cancer cells. In vivo administration of AV25CDC combined with gemcitabine in mice harboring subcutaneously growing SW1990 pancreatic tumors almost abrogated tumor growth. Nude mice harboring 15-day-old orthotopic tumors, treated intratumorally or systemically with AV25CDC combined with gemcitabine, exhibited 70% to 80% reduction in tumor size compared with control mice that lasted for at least 60 days. Chemovirotherapy treatment induced a return to normal levels of biochemical parameters of hepatic toxicity; these mice exhibited more than 90% reduction in CA19.9 serum levels compared with control. Chemovirotherapy efficacy was confirmed in mice harboring Mia PaCa-2 tumors and in Syrian hamster harboring HaP-T1 tumors. We observed that viral treatment disrupted tumor architecture and induced an increase in MMP-9 activity that might facilitate gemcitabine penetrability. CONCLUSION: These data demonstrate that AV25CDC is an effective oncolytic agent candidate for pancreatic cancer chemovirotherapy combination.

PVM/MA-shelled selol nanocapsules promote cell cycle arrest in A549 lung adenocarcinoma cells.

BACKGROUND: Selol is an oily mixture of selenitetriacylglycerides that was obtained as a semi-synthetic compound containing selenite. Selol is effective against cancerous cells and less toxic to normal cells compared with inorganic forms of selenite. However, Selol's hydrophobicity hinders its administration in vivo. Therefore, the present study aimed to produce a formulation of Selol nanocapsules (SPN) and to test its effectiveness against pulmonary adenocarcinoma cells (A549). RESULTS: Nanocapsules were produced through an interfacial nanoprecipitation method. The polymer shell was composed of poly(methyl vinyl ether-co-maleic anhydride) (PVM/MA) copolymer. The obtained nanocapsules were monodisperse and stable. Both free Selol (S) and SPN reduced the viability of A549 cells, whereas S induced a greater reduction in non-tumor cell viability than SPN. The suppressor effect of SPN was primarily associated to the G2/M arrest of the cell cycle, as was corroborated by the down-regulations of the CCNB1 and CDC25C genes. Apoptosis and necrosis were induced by Selol in a discrete percentage of A549 cells. SPN also increased the production of reactive oxygen species, leading to oxidative cellular damage and to the overexpression of the GPX1, CYP1A1, BAX and BCL2 genes. CONCLUSIONS: This study presents a stable formulation of PVM/MA-shelled Selol nanocapsules and provides the first demonstration that Selol promotes G2/M arrest in cancerous cells.

Participation of MAPK, PKA and PP2A in the regulation of MPF activity in Bufo arenarum oocytes.

The objectives of the present paper were to study the involvement and possible interactions of both cAMP-PKA and protein phosphatases in Bufo arenarum oocyte maturation and to determine if these pathways are independent or not of the MAP kinase (MAPK) cascade. Our results indicated that the inhibition of PKA by treatment with H-89, an inhibitor of the catalytic subunit of PKA, was capable of inducing GVBD in a dose-dependent manner by a pathway in which Cdc25 phosphatase but not the MAPK cascade is involved. The injection of 50 nl of H-89 10 µM produced GVBD percentages similar to those obtained with treatment with progesterone. In addition, the assays with okadaic acid (OA), a PP2A inhibitor, significantly enhanced the percentage of oocytes that resumed meiosis by a signal transducing pathway in which the activation of the MEK-MAPK pathway is necessary, but in which Cdc25 phosphatase was not involved. Treatment with H-89, was able to overcome the inhibitory effect of PKA on GVBD; however, the inhibition of Cdc25 activity with NaVO3 was able to overcome the induction of GVBD by H-89. Although the connections between PKA and other signalling molecules that regulate oocytes maturation are still unclear, our results suggest that phosphatase Cdc25 may be the direct substrate of PKA. In Xenopus oocytes it was proposed that PP2A, a major Ser/Thr phosphatase present, is a negative regulator of Cdc2 activation. However, in Bufo arenarum oocytes, inhibition of Cdc25 with NaVO3 did not inhibit OA-induced maturation, suggesting that the target of PP2A was not the Cdc25 phosphatase. MAPK activation has been reported to be essential in Xenopus oocytes GVBD. In B. arenarum oocytes we demonstrated that the inhibition of MAPK by PD 98059 prevented the activation of MPF induced by OA, suggesting that the activation of the MAPK cascade produced an inhibition of Myt1 and, in consequence, the activation of MPF without participation of the Cdc25 phosphatase. Our results suggest that in incompetent oocytes of B. arenarum two signal transduction pathways may be involved in the control of MPF activation: (1) the inhibition of phosphatase 2A that through the MEK-MAPK pathway regulates the activity of the Myt1; and (2) the inhibition of AMPc-PKA, which affects the activity of the Cdc25 phosphatase.

Flexibility and inhibitor binding in cdc25 phosphatases.

Cdc25 phosphatases involved in cell cycle checkpoints are now active targets for the development of anti-cancer therapies. Rational drug design would certainly benefit from detailed structural information for Cdc25s. However, only apo- or sulfate-bound crystal structures of the Cdc25 catalytic domain have been described so far. Together with previously available crystalographic data, results from molecular dynamics simulations, bioinformatic analysis, and computer-generated conformational ensembles shown here indicate that the last 30-40 residues in the C-terminus of Cdc25B are partially unfolded or disordered in solution. The effect of C-terminal flexibility upon binding of two potent small molecule inhibitors to Cdc25B is then analyzed by using three structural models with variable levels of flexibility, including an equilibrium distributed ensemble of Cdc25B backbone conformations. The three Cdc25B structural models are used in combination with flexible docking, clustering, and calculation of binding free energies by the linear interaction energy approximation to construct and validate Cdc25B-inhibitor complexes. Two binding sites are identified on top and beside the Cdc25B active site. The diversity of interaction modes found increases with receptor flexibility. Backbone flexibility allows the formation of transient cavities or compact hydrophobic units on the surface of the stable, folded protein core that are unexposed or unavailable for ligand binding in rigid and densely packed crystal structures. The present results may help to speculate on the mechanisms of small molecule complexation to partially unfolded or locally disordered proteins.

The catalytic acid in the dephosphorylation of the Cdk2-pTpY/CycA protein complex by Cdc25B phosphatase.

The development of anticancer therapeutics that target Cdc25 phosphatases is now an active area of research. A complete understanding of the Cdc25 catalytic mechanism would certainly allow a more rational inhibitor design. However, the identity of the catalytic acid used by Cdc25 has been debated and not established unambiguously. Results of molecular dynamics simulations with a calibrated hybrid potential for the first reaction step catalyzed by Cdc25B in complex with its natural substrate, the Cdk2-pTpY/CycA protein complex, are presented here. The calculated reaction free-energy profiles are in very good agreement with experimental measurements and are used to discern between different proposals for the general acid. In addition, the simulations give useful insight on interactions that can be explored for the design of inhibitors specific to Cdc25.

Effect of dehydroleucodine on meiosis reinitiation in Bufo arenarum denuded oocytes.

In amphibian oocytes meiosis, the transition from G2 to M phase is regulated by the maturation promoting factor (MPF), a complex of the cyclin-dependent kinase p34/cdc2 and cyclin B. In immature oocytes there is an inactive complex (pre-MPF), in which cdc2 is phosphorylated on both Thr-161 and Thr-14/Tyr-15 residues. The dephosphorylation of Thr-14/Tyr-15 is necessary for the start of MPF activation and it is induced by the activation of cdc25 phosphatase. Late, to complete the activation, a small amount of active MPF induces an auto-amplification loop of MPF stimulation (MPF amplification). Dehydroleucodine (DhL) is a sesquiterpenic lactone that inhibits mammalian cell proliferation in G2. We asked whether DhL interferes with MPF activation. For this question, the effect of DhL (up to 30 microM) on the resumption of meiosis was evaluated, and visualized by germinal vesicle break down (GVBD), of Bufo arenarum oocytes induced in vitro by either: (i) removing follicle cells; (ii) progesterone stimulation; (iii) VG-content injection; or (iv) injection of mature cytoplasm. The results show that DhL induced GVBD inhibition, in a dose-dependent manner, in spontaneous and progesterone-induced oocyte maturation. Nevertheless, DhL at the doses assayed had no effect on GVBD induced by mature cytoplasm injection, but exerted an inhibitory effect on GVBD induced by GV content. On the basis of these results, we interpreted that DhL does not inhibit MPF amplification and that the target of DhL is any event in the early stages of the cdc25 activation cascade.

Activation of maturation promoting factor in Bufo arenarum oocytes: injection of mature cytoplasm and germinal vesicle contents.

Although progesterone is the established maturation inducer in amphibians, Bufo arenarum oocytes obtained during the reproductive period (spring-summer) resume meiosis with no need of an exogenous hormonal stimulus if deprived of their enveloping follicle cells, a phenomenon called spontaneous maturation. In this species it is possible to obtain oocytes competent and incompetent to undergo spontaneous maturation according to the seasonal period in which animals are captured. Reinitiation of meiosis is regulated by maturation promoting factor (MPF), a complex of the cyclin-dependent kinase p34cdc2 and cyclin B. Although the function and molecule of MPF are common among species, the formation and activation mechanisms of MPF differ according to species. This study was undertaken to evaluate the presence of pre-MPF in Bufo arenarum oocytes incompetent to mature spontaneously and the effect of the injection of mature cytoplasm or germinal vesicle contents on the resumption of meiosis. The results of our treatment of Bufo arenarum immature oocytes incompetent to mature spontaneously with sodium metavanadate (NaVO3) and dexamethasone (DEX) indicates that these oocytes have a pre-MPF, which activates and induces germinal vesicle breakdown (GVBD) by dephosphorylation on Thr-14/Tyr-15 by cdc25 phosphatase and without cyclin B synthesis. The injection of cytoplasm containing active MPF is sufficient to activate an amplification loop that requires the activation of cdc25 and protein kinase C, the decrease in cAMP levels, and is independent of protein synthesis. However, the injection of germinal vesicle content also induces GVBD in the immature receptor oocyte, a process dependent on protein synthesis but not on cdc25 phosphatase or PKC activity.

Effect of insulin on spontaneous and progesterone-induced GVBD on Bufo arenarum denuded oocytes.

Progesterone is considered as the physiological steroid hormone that triggers meiosis reinitiation in amphibian oocytes. Nevertheless, isolated oocytes can be induced to undergo germinal vesicle breakdown (GVBD) in a saline medium by means of treatment with various hormones or inducing agents such as other steroid hormones, insulin or an insulin-like growth factor. It has been demonstrated that Bufo arenarum oocytes obtained during the reproductive period (spring-summer) resume meiosis with no need of an exogenous hormonal stimulus if deprived of their enveloping follicle cells, a phenomenon called spontaneous maturation. This study was undertaken to evaluate the participation of the purine and phosphoinositide pathway in the insulin-induced maturation of oocytes competent and incompetent to mature spontaneously, as well as to determine whether the activation of the maturation promoting factor (MPF) involved the activation of cdc25 phosphatase in Bufo arenarum denuded oocytes. Our results indicate that insulin was able to induce GBVD in oocytes incompetent to mature spontaneously and to enhance spontaneous and progesterone-induced maturation. In addition, high intracellular levels of purines such as cAMP or guanosine can reversibly inhibit the progesterone and insulin-induced maturation process in Bufo arenarum as well as spontaneous maturation. Assays of the inhibition of phosphatidylinositol-4,5-bisphosphate (PIP2) hydrolysis and its turnover by neomycin and lithium chloride respectively exhibited a different response in insulin- or progesterone-treated oocytes, suggesting that phosphoinositide turnover or hydrolysis of PIP2 is involved in progesterone- but not in insulin-induced maturation. In addition, the inhibitory effect of vanadate suggests that an inactive pre-maturation promoting factor (pre-MPF), activated by dephosphorylation of Thr-14 and Tyr-15 on p34cdc2, is present in Bufo arenarum full-grown oocytes; this step would be common to both spontaneous and hormone-induced maturation. The data presented here strongly suggest that insulin initiates at the cell surface a chain of events leading to GVBD. However, our studies point to the existence of certain differences between the steroid and the peptide hormone pathways, although both involve the decrease in intracellular levels of cAMP, the activation of phosphodiesterase (PDE) and the activation of pre-MPF.

Reciprocal Cdc25A and p27 expression in B-cell non-Hodgkin lymphomas.

Cell cycle regulation is often altered in cancer and deregulation of the cell cycle checkpoints is common in human neoplasia. The dual-specificity phosphatase Cdc25A and the cell cycle inhibitor p27 both play an important role in the regulation of the G1-S transition. We evaluated Cdc25A mRNA expression by in situ hybridization and p27 protein expression by immunohistochemistry in 42 histologically indolent B-cell non-Hodgkin lymphoma (NHL and 51 histologically aggressive B-cell NHL. Overexpression of Cdc25A (>50% tumor cells positive) was detected in 5 of 42 cases (12%) of histologically indolent B-cell NHL and in 29 of 51 (57%) of histologically aggressive B-cell NHL (P < 0.001). In contrast, high p27 protein expression (>50% tumor cells positive) was observed in 29 (69%) cases of indolent but in only one case (2%) of aggressive B-cell NHL (P < 0.0001). Thus, overexpression of Cdc25A and concomitant loss of p27 expression are associated with high grade B-cell NHL and may contribute to their aggressive biologic behavior.