PURPOSE: To analyze the effect of the growth control on human breast cancer cells with genistein treatment and to investigate the mechanism of genistein-induced G2/M arrest in T47D and MDA-MB231 breast carcinoma cells by Cdc25C expression. METHODS: We analysed the proliferartion of the two cell lines by using MTT proliferation assay, flow cytometric analysis, real-time quantitative RT-PCR and western blotting and investigated the effect of genistein on cell survival, cellular toxicity, cell cycle progression-related genes and their mRNA and protein alterations. RESULTS: The DNA flow cytometric analysis of both cell lines treated with genistein showed a dose-dependent growth inhibition and accumulation in the G2/M phase of cell cycle. The expression of p21 mRNA and protein increased in both cell lines following genistein treatment but p27 expression was unchanged. Furthermore, decreased Cdc25C expression with decreased polo-like kinase (PLK) 1 expression and increased PLK3 expression were observed after genistein treatment. The decreased level of Cdc25C in the nucleus was associated with decreased phosphorylation of Cdc25C by PLK1. The expression of PLK3 was increased with a dose-dependent and a time-dependent manner and was associated with decreased Cdc25C expression. Check point kinase (CHK) 1 and CHK2 revealed different expression patterns each other. The CHK1 expression was independent of the presence of genestein. CHK2 expression increased in MDA-MB231 cells associated with decreased Cdc25C expression but not in T47D. CONCLUSION: These results suggest that genistein induces a G2/M arrest in human breast cancer cells, the mechanism of which is due, in part, to decreased in Cdc25C phosphatase by a regulatory effect of PLK1, PLK3, and CHK2 as well as increased expression of the cyclin dependent kinase inhibitor p21(WAF1/CIP1).
Blotting, Western ; Breast ; Breast Neoplasms ; cdc25 Phosphatases ; Cell Cycle ; Cell Line ; Cell Survival ; Cyclins ; DNA ; Genistein ; Humans ; Phosphorylation ; Phosphotransferases ; RNA, Messenger

Fifteen 3-(1', 2'-di-O-cyclohexylidendioxyethyl)-5-aryl-3a, 6a-dihydro-4, 6-dioxo-pyrrolino[3', 4'-d] isoxazoline derivatives (3a-3o) were synthesized by 1, 3-dipolar cycloaddition reaction of N-arylmaleimides and the nitrile oxide in situ generated from 2, 3-O-cyclohexylidene-D-glycerohydroximoyl chloride, in the presence of triethylamine. The structures of the target compounds 3a-3o were characterized by 1H NMR, IR and elemental analysis. The preliminary bioassay on the compounds showed that some compounds possess in vitro anticancer activity and the leukocyte common antigen activity to a different extent. The compounds 3e, 3h, 3j and 31 showed Cdc25A phosphatase inhibitory activity of 60.6%, 58.6%, 51.4% and 98.4% respectively at the test concentration of 20 microg x mL(-1), and among them 31 had inhibition rate of 86.97% even at the concentration as low as 5 microg x mL(-1), indicating worthy to be future studied. The compounds 3e, 31 and 3n showed an inhibitory activity of 57.7%, 74.4% and 77.3% on CD45 protein tyrosine phosphatase A, respectively, at the test concentration of 20 micromol x mL(-1). The structure-activity relationship of 3-(1', 2'-di-O-cyclohexylidendioxyethyl)-5-aryl-3a, 6a- dihydro-4, 6-dioxo-pyrrolino[3', 4'-d]isoxazoline derivatives was also discussed.
Antineoplastic Agents ; chemical synthesis ; chemistry ; pharmacology ; Isoxazoles ; chemical synthesis ; chemistry ; pharmacology ; Leukocyte Common Antigens ; antagonists & inhibitors ; Pyrroles ; chemical synthesis ; chemistry ; pharmacology ; Structure-Activity Relationship ; cdc25 Phosphatases ; antagonists & inhibitors

OBJECTIVE: This study was undertaken to quantitatively detect Cdc25A, Cdc25B and Cdc25C in cervical carcinoma and determine the relationship between the expression of mRNA and protein of cell division cycle (Cdc)25 phosphatase and various clinicopathologic prognostic factors of cervical carcinoma. METHODS: 39 patients diagnosed with cervical carcinoma between February 2000 to March 2005 and 10 patients with benign gynecologic disease were enrolled in this study. A reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blot analysis were used to analyze the expression of Cdc25 phosphatase mRNA and protein in fresh invasive cervical cancer tissue and normal cervix tissue. RESULTS: The mRNA expressions of Cdc25A, Cdc25B and Cdc25C in the cancer tissues were significantly greater than in the control (p=0.02, 0.01, 0.02), respectively. A Western blot analysis yielded same results (p=0.01, 0.02, 0.01). There were also significant relationships between the age and the Cdc25B mRNA expression (p=0.03), between the cell type and the Cdc25C mRNA expression (p=0.04). However, other clinicopathologic prognostic factors including stage, subtype, SCC Ag level, DNA flow cytometry, lymph node metastasis, lymphovascular space invasion and HPV positivity were not statistically significant. CONCLUSION: Our results show that Cdc25A, Cdc25B and Cdc25C expression levels were significantly greater in cervical cancer patient group than in those of control group. Thus Cdc25 phosphatase might play an important role in carcinogenesis of cervical carcinoma. Further studies based on the correlation between Cdc25 phosphatase and survival rate would be need to support Cdc25 phosphatase as a prognostic factor of cervical carcinoma.
Blotting, Western ; Carcinogenesis ; cdc25 Phosphatases ; Cell Cycle ; Cervix Uteri ; DNA ; Female ; Flow Cytometry ; Genital Diseases, Female ; Humans ; Lymph Nodes ; Neoplasm Metastasis ; RNA, Messenger ; Survival Rate ; Uterine Cervical Neoplasms

OBJECTIVETo study the expression of Galectin-3 and CDC25B mRNA in gastric carcinoma and their correlation with clinical-pathological features and the survival time.

METHODSTissue microarray (TMA) technique and in situ hybridization were used to detect the expression of Galectin-3 and CDC25B mRNA in 220 gastric carcinoma specimens and 31 normal gastric mucosa samples.

RESULTSIn situ hybridization results revealed that from the 220 cases, the positive expression rate of Galectin-3 and CDC25B mRNA were 58.6% and 54.1%, respectively. There was significant relationship between the Galectin-3 mRNA expression and tumor diameter, advanced TNM stage, invasion depth, vessel invasion, lymph node and distant metastasis. There was significant relationship between CDC25B mRNA expression and tumor diameter, advanced TNM stage, vessel invasion, lymph node and distant metastasis. In addition, there was apositive relationship of Galectin-3 and CDC25B mRNA expression. Finally, the mean survival time in cases with Galectin-3 and CDC25B mRNA positive expression was significantly shorter than those without Galectin-3 and CDC25B expression.

CONCLUSIONThe expression of Galectin-3 and CDC25B mRNA appears to act as a promoting factor in the onset and development of gastric cancer. It can be used as a marker of prognosis of gastric carcinoma in clinical practice.

Female ; Galectin 3 ; genetics ; metabolism ; Gene Expression ; genetics ; Gene Expression Regulation, Neoplastic ; Humans ; Male ; Prognosis ; RNA, Messenger ; metabolism ; Stomach Neoplasms ; genetics ; metabolism ; pathology ; cdc25 Phosphatases ; genetics ; metabolism

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

OBJECTIVE: To investigate the expression of CDC25A in non- small cell lung cancer (NSCLC) tissues and explore its correlation with the clinicpathological features of the patients and the expressions of let-7a1 and let-7c. METHODS: We collected surgical specimens of pathologically confirmed NSCLC tissues and paired adjacent lung tissues from 44 patients and tissues of benign lung lesions from 9 patients. The expressions of CDC25A protein and mRNA in the tissues were detected by immunohistochemistry and fluorescence quantitative RT-PCR, respectively; the expressions of let-7a1 and let-7c mRNA were detected using tail-adding fluorescence quantitative RT-PCR. RESULTS: The positivity rate of CDC25A protein expression was significantly higher in NSCLC tissues than in the adjacent tissues and benign pulmonary lesions ( CONCLUSIONS The expression level of CDC25A is significantly increased in NSCLC with a negative correlation with Let-7c expression, which identifies CDC25A as a possible downstream target gene of Let-7c.
Carcinoma, Non-Small-Cell Lung/genetics* ; Gene Expression Regulation, Neoplastic ; Humans ; Lung ; Lung Neoplasms/genetics* ; Lymphatic Metastasis ; MicroRNAs ; RNA, Messenger/genetics* ; cdc25 Phosphatases

To further test whether protein kinase A (PKA) can affect the mitotic cell cycle, one-cell stage mouse embryos at S phase (22 h after hCG injection) were incubated in M16 medium containing various concentrations of H-89, a PKA inhibitor. With increasing concentrations of H-89 (0-50 μmol/L), the G(2) phase of eggs was decreased and the cleavage rate was accelerated. A concentration of 40 μmol/L H-89 led to all of the mouse eggs entering the M phase of mitosis. Furthermore, to study the role of PKA in regulating the phosphorylation status of S149 and S321 sites of cell division cycle 25B (CDC25B) on one-cell stage fertilized mouse eggs, pBSK-CDC25B-WT, pBSK-CDC25B-S149A, pBSK-CDC25B-S321A and pBSK-CDC25B-S149A/S321A were transcribed into mRNAs in vitro, then mRNAs were microinjected into S phase of mouse fertilized eggs and cultured in M16 medium pretreated with H-89. Then, the cleavage of fertilized eggs, maturation promoting factor (MPF) activity and phosphorylation status of CDC2-Tyr15 were observed. In the presence of 40 μmol/L H-89, the cleavage rate of fertilized eggs in CDC25B-S/A-mRNAs and CDC25B-WT-mRNA injected groups was significantly higher than that in the control groups, and the peak of MPF activity appeared in the CDC25B-S/A-mRNAs and CDC25B-WT-mRNA injected groups earlier than that in the control groups. CDC2-Tyr15 phosphorylation state was consistent with MPF activity. In conclusion, the present study suggests that PKA regulates the early development of mouse embryos by phosphorylation of S149 and S321 of CDC25B, which plays an important role in the regulation of G(2)/M transition in the mitotic cell cycle of fertilized mouse eggs.
Animals ; Cyclic AMP-Dependent Protein Kinases ; genetics ; physiology ; Embryonic Development ; physiology ; Female ; Male ; Mice ; Microinjections ; Mitosis ; drug effects ; Phosphorylation ; Serine ; genetics ; metabolism ; Zygote ; cytology ; growth & development ; cdc25 Phosphatases ; genetics ; metabolism

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

OBJECTIVETo explore the molecular elements responsible for the enhanced apoptotic susceptibility in U937 cells mediated by silencing atactic telangiectasis mutation (ATM) gene.

METHODSTwo U937 cell mutants, U937-ASPI3K (ATM gene negative) and U937-pZEOSV2 (+) (ATM gene positive) were used as a cell model system. Apoptosis was examined by measuring free nucleosome concentrations in U937 cells. Western blotting was employed to measure nuclear protein abundances of cdc25A, cdc25B, cdc25C, total p34cdc2, p34cdc2 (Thr161) or p34cdc2 (Thr14, Tyr15). RT-PCR was used to estimate cdc25 transcript levels.

RESULTSU937-ASPI3K exhibited an enhanced apoptotic susceptibility to lower dosage of irradiation, which could not be blocked by protein synthesis inhibitor. Protein serine-theronine phosphatase inhibitor or cyclin-dependent kinase (CDK) inhibitors could abolish the enhancement. Upon irradiation, p34cdc2 in U937-pZEOSV2 (+) was in an inactive state owing to phosphorylation of Thr 14 and Tyr15, which was associated with a dramatic decrease of nuclear cdc25A, cdc25B and cdc25C poteins. In contrast, p34cdc2 in U937-ASPI3K was in an active state owing to the low phosphorylation of Thr14 and Tyr15, which was associated with constant nuclear cdc25A, cdc25B and cdc25C protein abundance before and after irradiation. The responsive decrease of nuclear cdc25 proteins occurred at the post-transcription level.

CONCLUSIONSilencing ATM gene blocks the irradiation induced responsive decrease of nuclear cdc25 proteins, resulting in an abnormal activation p34cdc2 is the critical molecular mechanism for the enhanced apoptotic responses.

Apoptosis ; drug effects ; genetics ; radiation effects ; Ataxia Telangiectasia Mutated Proteins ; Blotting, Western ; CDC2 Protein Kinase ; genetics ; metabolism ; Cell Cycle Proteins ; genetics ; metabolism ; Cyclin-Dependent Kinases ; genetics ; metabolism ; Cycloheximide ; pharmacology ; DNA-Binding Proteins ; Enzyme Inhibitors ; pharmacology ; Gene Silencing ; drug effects ; radiation effects ; Humans ; Nuclear Proteins ; genetics ; metabolism ; Oxazoles ; pharmacology ; Phosphoprotein Phosphatases ; antagonists & inhibitors ; Protein Synthesis Inhibitors ; pharmacology ; Protein-Serine-Threonine Kinases ; genetics ; Reverse Transcriptase Polymerase Chain Reaction ; Tumor Suppressor Proteins ; U937 Cells ; cdc25 Phosphatases ; genetics ; metabolism

BACKGROUNDExpression of polo-like kinase 1 (Plk1) is elevated in lung cancer and has been proposed as having prognostic value and related to resistance to chemotherapy and radiation. In addition, Plk1 has several functions in mitotic progression. In this study, the authors investigated the effect of Plk1 depletion on cell cycle progression and proliferation in A549 cells, a lung cancer cell line.

METHODSA recombinant plasmid containing antisense RNA targeting Plk1 (pcDNA3-Plk1) was transfected into A549 cells. Reverse transcription-polymerase chain reaction and Western blot were used to examine Plk1 gene expression. Cell proliferation was evaluated by direct cell counting and bromodeoxyuridine (BrdU) labelling. Cell cycle and apoptosis were examined by flow cytometry. Expression of alpha-tubulin was detected by immunofluorescence, and the inhibition rate (IR) by chemotherapeutic agents was determined by 3-(4,5-dimethylthiazol-2-yl) 2,5-diphenyl-tetrazolium bromide assay.

RESULTSAfter transfection into A549 cells, pcDNA3-Plk1 reduced Plk1 mRNA by 46.75% for 24 hours and by 61.84% for 48 hours. Plk1 protein was significantly decreased simultaneously (P < 0.05). Abnormal morphological changes of cells and growth inhibition were observed in pcDNA3-Plk1 transfected groups. The BrdU labelling index was 25.59% 48 hours after transfection, which was significantly lower than that of the control groups (P < 0.05). Forty-eight hours after transfection, there showed absence of microtubule polymerization and spindle abnormalities in staining for alpha-tubulin. A549 cells showed a strong G2/M arrest and apoptosis 72 hours post transfection. IR of vinorelbine in pcDNA3-Plk1 transfected groups was significantly higher than that of the other groups (P < 0.05, respectively).

CONCLUSIONSPlk1 depletion interferes with spindle formation, induces cell cycle arrest and apoptosis, and consequently inhibits cell proliferation in A549 cells. Moreover, it sensitizes lung cancer cells to chemotherapy.

Apoptosis ; drug effects ; Bromodeoxyuridine ; metabolism ; Cell Cycle ; drug effects ; Cell Cycle Proteins ; metabolism ; Cell Division ; drug effects ; Cell Line, Tumor ; Humans ; Lung Neoplasms ; drug therapy ; pathology ; Protein Kinases ; genetics ; physiology ; Protein-Serine-Threonine Kinases ; Proto-Oncogene Proteins ; RNA, Antisense ; pharmacology ; Transfection ; Vinblastine ; analogs & derivatives ; pharmacology ; cdc25 Phosphatases ; metabolism