OBJECTIVETo investigate the content and activity of M-phase promoting factor (MPF) in pleomorphic adenoma, mucoepidermoid carcinoma, buccal carcinoma and normal tissue, in order to evaluate the role of MPF in the development of tumor and the relationship between MPF and malignant degree.

METHODSThe content and activity of MPF were assessed by immunobloting and Gollicano method.

RESULTSThe cdc2 and cyclinB (two subunits of MPF) were found both in normal and tumor tissues, and their content in tumor was higher than normal tissues. Buccal carcinoma was 64% higher than normal tissues. The activity of MPF in carcinoma was higher than normal tissue and had positive relation with the malignant extent.

CONCLUSIONSThe content and activity of MPF in tumor are higher than normal tissue. PKC can activate MPF. These results show PKC may promote tumor proliferation by activating MPF and also, the activity of MPF has some relation with malignant extent.

CDC2 Protein Kinase ; analysis ; Cyclin B ; analysis ; Humans ; Immunoblotting ; Maturation-Promoting Factor ; analysis ; Mouth ; chemistry ; Mouth Neoplasms ; chemistry ; Protein Kinase C ; physiology

OBJECTIVE: Previously, we identified that transketolase (Tkt), an important enzyme in the pentose phosphate pathway, is highly expressed at 2 hours of spontaneous maturation in oocytes. Therefore, this study was performed to determine the function of Tkt in meiotic cell cycle regulation, especially at the point of germinal vesicle breakdown (GVBD). METHODS: We evaluated the loss-of-function of Tkt by microinjecting Tkt double-stranded RNAs (dsRNAs) into germinal vesicle-stage oocytes, and the oocytes were cultured in vitro to evaluate phenotypic changes during oocyte maturation. In addition to maturation rates, meiotic spindle and chromosome rearrangements, and changes in expression of other enzymes in the pentose phosphate pathway were determined after Tkt RNA interference (RNAi). RESULTS: Despite the complete and specific knockdown of Tkt expression, GVBD occurred and meiosis was arrested at the metaphase I (MI) stage. The arrested oocytes exhibited spindle loss, chromosomal aggregation, and declined maturation promoting factor and mitogen-activated protein kinase activities. The modified expression of two enzymes in the pentose phosphate pathway, Prps1 and Rbks, after Tkt RNAi and decreased maturation rates were amended when ribose-5-phosphate was supplemented in the culture medium, suggesting that the Tkt and pentose phosphate pathway are important for the maturation process. CONCLUSION: We concluded that Tkt and its associated pentose phosphate pathway play an important role in the MI-MII transition of the oocytes' meiotic cell cycle, but not in the process of GVBD.
Cell Cycle ; Maturation-Promoting Factor ; Meiosis ; Metaphase ; Oocytes ; Pentose Phosphate Pathway ; Protein Kinases ; Ribosemonophosphates ; RNA Interference ; RNA, Double-Stranded ; Transketolase

OBJECTIVE: In the previous study, we compiled the differentially expressed genes during early folliculogenesis.1 Objective of the present study was to identify downstream target genes of transcription factors (TFs) using bioinformatics for selecting the target TFs among the gene lists for further functional analysis. MATERIALS AND METHODS: By using bioinformatics tools, constituent domains were identified from database searches using Gene Ontology, MGI, and Entrez Gene. Downstream target proteins/genes of each TF were identified from database searches using TF database (TRANSFAC(R) 6.0) and eukaryotic promoter database (EPD). RESULTS: DNA binding and trans-activation domains of all TFs listed previously were identified, and the list of downstream target proteins/genes was obtained from searche of TF database and promoter database. Based on the known function of identified downstream genes and the domains, 3 (HNF4, PPARg, and TBX2) out of 26 TFs were selected for further functional analysis. The genes of wee1-like protein kinase and p21WAF1 (cdk inhibitor) were identified as potential downstream target genes of HNF4 and TBX2, respectively. PPARg, through protein-protein interaction with other protein partners, acts as a transcription regulator of genes of EGFR, p21WAF1, cycD1, p53, and VEGF. Among the selected 3 TFs, further study is in progress for HNF4 and TBX2, since wee1-like protein kinase and cdk inhibitor may involved in regulating maturation promoting factor (MPF) activity during early folliculogenesis. CONCLUSIONS: Approach used in the present study, in silico analysis of downstream target genes, was useful for analyzing list of TFs obtained from high-throughput cDNA microarray study. To verify its binding and functions of the selected TFs in early folliculogenesis, EMSA and further relevant characterizations are under investigation.
Computational Biology ; Computer Simulation* ; DNA ; Gene Ontology ; Maturation-Promoting Factor ; Oligonucleotide Array Sequence Analysis ; Protein Kinases ; Transcription Factors* ; Vascular Endothelial Growth Factor A

It is important to study the mechanism of oocyte maturation because oocyte maturation is essential for the female procreation. The present study was designed to observe the effects of protooncogenes c-erbB(2) and c-myb on oocyte maturation and the upstream and downstream relationship with mitogen-activated protein kinase (MAPK) and maturation promoting factor (MPF). The investigation was designed as follows: (1) In order to explore the effects of protooncogenes on oocyte maturation, the dose- and time-dependent effects of c-erbB(2) antisense oligodeoxynucleotide (ASODN) and c-myb ASODN on oocyte maturation were examined, and the effects of oocyte microinjection with recombinant c-erbB(2) and c-myb proteins on oocyte maturation were investigated; (2) In order to study the upstream and downstream relationship among protooncogenes of c-erbB(2), c-myb and protein kinases of MAPK and MPF in regulating oocyte maturation, mouse oocytes were cultured in the medium treated with c-erbB(2) ASODN, c-myb ASODN, PD98059 (the MAPK inhibitor) or roscovitine (the MPF inhibitor) for 8 h, respectively, and the expressions of c-erbB(2) mRNA, c-myb mRNA, MAPK and MPF were examined. The results showed that both c-erbB(2) ASODN and c-myb ASODN inhibited the rate of germinal vesicle breakdown (GVBD) and the first polar (PB1) extrusion of denuded oocytes (DOs) in a dose- and time-dependent way, and delayed their maturation time significantly. When recombinant c-erbB(2) and c-myb proteins were microinjected into cytoplasm of germinal vesicle stage oocyte, we found that the GVBD rate increased by 23.1% (P<0.05) and 32.2% (P<0.05), respectively, for 6-hour culture, and the PB1 extrusion rate increased by 17.3% (P<0.05) and 23.5% (P<0.05), respectively, for 12-hour culture. RT-PCR showed that the mRNA expressions of c-erbB(2) and c-myb were detected in oocytes; c c-erbB(2) ASODN inhibited c-erbB(2) mRNA and c-myb mRNA expressions; c-myb ASODN inhibited c-myb mRNA expression but had no effect on c-erbB(2) mRNA expression. Nonsense tat ODN had no effects on the expressions of c-erbB(2) mRNA and c-myb mRNA. Neither PD98059 nor roscovitine changed the expressions of c-erbB(2) mRNA and c-myb mRNA though both of them inhibited recombinant c-erbB(2) and c-myb proteins-induced oocyte maturation. Furthermore, MAPK phosphorylation and cyclin B1 synthesis in oocytes were inhibited remarkably when oocytes were treated with c-erbB(2) ASODN, c-myb ASODN, PD98059 and roscovitine. Nonsense tat ODN had no effects on MAPK phosphorylation and cyclinB1 content. The results suggest that protooncogenes c-erbB(2) and c-myb play an important role in oocyte maturation; the effects of c-erbB(2) and c-myb depend upon the action of MAPK and MPF, and their activation is the event that occurs downstream of c-erbB(2) and c-myb in the maturation signal pathway.
Animals ; Female ; Maturation-Promoting Factor ; metabolism ; Mice ; Microinjections ; Mitogen-Activated Protein Kinases ; metabolism ; Oocytes ; physiology ; Oogenesis ; Proto-Oncogene Proteins c-myb ; metabolism ; Receptor, ErbB-2 ; metabolism ; Signal Transduction

AIMTo explore the effect of Cdc25B overexpression on the development of mouse two-cell embryos.

METHODSThe pBSK-Cdc25B was in vitro transcribed into 5'-capped mRNA for microinjection by using mMESSAGE mMACHINE kit. The Cdc25B mRNA was microinjected into mouse embryos at two-cell stage in order to observe the embryonic development and cleavage rate. Using protein kinase activity assay and Western blot to detect the MPF activity as well as the phosphorylation status of Cdc2-Tyr15 in Cdc25B overexpression group respectively.

RESULTSThe mouse embryos with Cdc25B overexpression developed to the four-cell stage 48 h after the hCG injection with the percentage of cleavage over 40% compared with the embryos in control groups which still remained at the two-cell stage. Moreover, MPF activity increased significantly after Cdc25B mRNA injection. The phosphorylation status of Cdc2-Tyr15 was coincident with MPF activity.

CONCLUSIONThe results indicate that Cdc25B overexpression in early mouse two-cell embryos reverses two-cell block and promotes their development into four-cell stage by activating MPF.

Animals ; Cell Cycle ; Cell Division ; Embryo, Mammalian ; cytology ; Embryonic Development ; physiology ; Female ; Male ; Maturation-Promoting Factor ; metabolism ; Mice ; Microinjections ; Mitosis ; RNA, Messenger ; metabolism ; Zygote ; growth & development ; metabolism ; cdc25 Phosphatases ; physiology

AIMThe mechanisms of cytokines in regulating oocyte maturation is still little known. The present study attempt to investigate whether the protooncogene of c-erbB2, c-myb are involved in introducing of cytokines to regulate oocyte maturation.

METHODSThis research used mouse GV stage oocyte culture model in vitro and RT-PCR, Western blotting method to explore the effect of EGF, TNFalpha, ET-1 and NO on oocyte maturation; to analyze the c-erbB2 mRNA and c-myb mRNA expression and the phosphorylation of MAPK and cyclinB1 expression in oocytes affected by above cytokines.

RESULTSEGF(10 microg/L) stimulated meiosis of oocytes significantly, the level of c-erbB2 mRNA, c-myb mRNA were increased, and promoted the phosphorylation of MAPK and cyclinB1 expression; TNFalpha (1 microg/L) and ET-1 ((10(-1) mol/L) had the results to EGF. Low dose of SNP (10(-5)mol/L) had no effect on oocyte maturation, but could significantly reverse the suppression of dbcAMP on oocyte maturation.

CONCLUSIONc-erbB2 and c-myb were involved in introducing of cytokines to regulate oocyte maturation, might be the middle link in connection of the cytokines with MAPK and MPF in regulation oocyte maturation.

Animals ; Cells, Cultured ; Cytokines ; physiology ; Epidermal Growth Factor ; physiology ; Female ; Intercellular Signaling Peptides and Proteins ; physiology ; Maturation-Promoting Factor ; genetics ; metabolism ; Mice ; Mitogen-Activated Protein Kinases ; metabolism ; Oocytes ; cytology ; growth & development ; physiology ; Oogenesis ; drug effects ; physiology ; Proto-Oncogene Proteins c-myb ; genetics ; metabolism ; RNA, Messenger ; genetics ; metabolism ; Receptor, ErbB-2 ; genetics ; metabolism ; Tumor Necrosis Factor-alpha ; physiology

OBJECTIVE: To investigate the effect of CDK1 interference regulation of PLK1, Aurora B and TRF1 on the proliferation of leukemia cells. METHODS: The human myelogenous leukemia cell line HL-60 was selected as the research object, and the effect of TRF1 expression and its changes on cell proliferation and cycle was investigated by regulating intracellular CDK1 expression. The objects were divided into 5 groups, including control group, shRNA-NC group, CDK1-shRNA group, pcDNA group and pcDNA-CDK1 group. RT-PCR was used to detect the CDK1 expression of cells in each group; colony formation was used to detect the proliferation of the cells. Western blot was used to detect the expression of CDK1, PLK1, Aurora B, TRF1, and cyclin p53, p27, cyclinA. RESULTS: The phosphorylation level of PLK1, Aurora B and the expression of TRF1 in the CDK1-shRNA group were significantly down-regulated as compared with those in the control group (P<0.05). Compared with the control group, the cells in CDK1-shRNA group showed lower clone formation rate, the increasing of cycle-associated proteins p53 and p27 and the decreasing of cyclinA expression (P<0.05). It was shown that interfered CDK1 expression could inhibit the proliferation of HL-60 cells and prolong the time that they enter mitosis, thereby extending the cell cycle. Compared with the control group, the overexpressed CDK1 in the pcDNA-CDK1 group made the phosphorylation level of PLK1, Aurora B, and TRF1 expression increase significantly (P<0.05), also the colony formation rate (P<0.05). The cycle-related proteins p53 and p27 was down-regulated, while cyclinA expression was up-regulate significantly (P<0.05). The results indicted that overexpressed CDK1 could stimulate adverse reactions, thereby promoting the proliferation of HL-60 cells and shortening the cell cycle. CONCLUSION Knocking out CDK1 can inhibit the phosphorylation of PLK1 and Aurora B and negatively regulate TRF1, thereby inhibiting the proliferation of leukemia cells.
CDC2 Protein Kinase ; Cell Cycle Proteins/genetics* ; Cell Proliferation ; Humans ; Leukemia ; Mitosis ; Phosphorylation ; Proto-Oncogene Proteins/genetics*

PURPOSE: The cyclin-dependent kinase 1 (Cdk1) and cyclin B complex performs important roles in the transition from the G2 to M phase in the cell cycle through removal of inhibitory phosphates on Cdk1, and Cdc25B, which is a dual-specific phosphatase, mediates these dephosphorylation events. However, measuring Cdc25B activity by existing methods is hampered by inadequate nonspecific substrates and the need to use a radiolabeled isotope. The present study aimed to develop an improved method with which to properly measure Cdc25B activity using a novel nonradioisotopic assay and Cdc25B overexpression cell lines. MATERIALS AND METHODS: A nonradioisotopic Cdk1 kinase assay, based on Western blotting for retinoblastoma protein and histone H1, was used to analyze Cdc25B activity. Also, stable Cdc25B2 and Cdc25B3 overexpression HeLa cell lines were constructed using the tetracycline-regulated expression system and were applied as a tool for screening for inhibitors of Cdc25B. RESULTS: The present study developed and optimized a nonradioisotopic assay method to properly measure Cdc25B activity. Furthermore, we constructed stable Cdc25B2 and Cdc25B3 overexpression HeLa cell lines for the establishment of a strong assay system with which to evaluate the specificity of Cdc25B inhibitors under conditions similar to the intracellular environment. These methods were confirmed as useful tools for measuring Cdc25B activity. CONCLUSION: The nonradioisotopic Cdk1 kinase assay and Cdc25B overexpression cell lines developed in this study can be conveniently used as tools for screening inhibitors of Cdc25B phosphatase as anticancer drugs.
Blotting, Western ; CDC2 Protein Kinase ; cdc25 Phosphatases ; Cell Cycle ; Cell Division ; Cell Line* ; Cyclin B ; HeLa Cells ; Histones ; Humans ; Mass Screening* ; Methods ; Phosphates ; Retinoblastoma Protein ; Sensitivity and Specificity

WEE1 is an important factor for histone transcription, chromosome condensation and regulation of cell cycle progression. WEE1 kinase can phosphorylate Cdc2 and down-regulate Cdc2 kinase activity. It can regulate G2 to M phase transition and cell mitosis. It plays a key role in chromosome condensation delay and histone synthesis, suggesting the important functions of WEE1 in the occurrence and development in cancer. At present, a multiple WEE1 inhibitors have been discovered. A great progress has been made in combination of WEE1 inhibitors with DNA damage treatment (chemotherapy or radiotherapy), which makes WEE1 an important target in cancer treatment.
CDC2 Protein Kinase ; metabolism ; Cell Cycle ; Cell Cycle Proteins ; metabolism ; DNA Damage ; Histones ; metabolism ; Humans ; Neoplasms ; metabolism ; Nuclear Proteins ; metabolism ; Phosphorylation ; Protein-Tyrosine Kinases ; metabolism

OBJECTIVE: To investigate the expression of eIF3P170, cdc2, cyclinB1 and cyclinD1 in developing cardiac myocytes, and the correlation between eIF3P170 with cdc2, cyclin D1, and cyclin B1 in mice. METHODS: Mouse cardiac myocytes were obtained at different time points. RT-PCR was employed to detect the expression of eIF3P170, cdc2, cyclin D1 and cyclin B1 mRNA. RESULTS: Expressions of eIF3P170, cdc2, cyclinD1 and cyclinB1 mRNA were higher in the embryonic Day 13, 15, 18 and postnatal Day 1, 2, 3, 5. Expressions at postnatal Day 5 reached the highest (all P values<0.05 vs other time points), and then the expressions of these genes gradually decreased to the weakest at postnatal Day 30 (all P values<0.05 vs other time points). The mRNA expression of eIF3P170 was positively correlated with cdc2, cyclin D1 and cyclin B1 mRNA expression respectively. CONCLUSION The mRNA expressions of eIF3 P170, cdc2, cyclin D1 and cyclin B1 in the embryo and the early life after birth are high. They reach the maximum at postnatal Day 5, then gradually decreased.
Animals ; CDC2 Protein Kinase ; metabolism ; Cell Cycle ; Cyclin B1 ; metabolism ; Cyclin D1 ; metabolism ; Eukaryotic Initiation Factor-3 ; metabolism ; Mice ; Myocytes, Cardiac ; cytology ; metabolism ; RNA, Messenger