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

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

LncRNAH19 has been implicated as having both oncogenic and tumor suppression properties in cancer. LncRNAH19 transcripts also serve as a precursor for miR-675. However, it is unknown whether LncRNAH19 and miR-675 are involved in the migration and invasion of hepatocellular carcinoma (HCC) cells. The purpose of this study was to investigate the effect and mechanism of LncRNAH19 and miR-675 on migration and invasion of HCC cells. The migration and invasion of HCC cells were measured by Transwell migration and invasion assays after transfection of HCC cells with miR-675 inhibitors and LncRNAH19siRNA. The levels of LncRNAH19 and miR-675 were detected by quantitative reverse transcriptase real-time polymerase chain reaction (qRT-PCR), and the protein expression of AKT, GSK-3β and Cdc25A by Western blotting analysis. The expression levels of LncRNAH19 and miR-675 were higher in MHCC-97H cells than in L02, Huh-7 and HepG2 cells. Transwell migration assay revealed that the miR-675 inhibitor and LncRNAH19siRNA could significantly increase the migration of HCC cells (P<0.01) as compared with the control group. Transwell invasion assay demonstrated that the miR-675 inhibitor and LncRNAH19siRNA could significantly increase the invasion of HCC cells (P<0.01) as compared with the control group. Western blotting analysis showed that the expression levels of AKT and Cdc25A were significantly increased (P<0.05), and the expression level of GSK-3β was significantly decreased (P<0.05) after treatment with miR-675 inhibitors and LncRNAH19siRNA as compared with the control group. These findings suggested that inhibition of LncRNAH19 and miR-675 expression can promote migration and invasion of HCC cells via AKT/GSK-3β/Cdc25A signaling pathway.
Blotting, Western ; Carcinoma, Hepatocellular ; genetics ; metabolism ; pathology ; Cell Line ; Cell Line, Tumor ; Cell Movement ; genetics ; Down-Regulation ; Gene Expression Regulation, Neoplastic ; Gene Knockdown Techniques ; Glycogen Synthase Kinase 3 ; metabolism ; Glycogen Synthase Kinase 3 beta ; Hep G2 Cells ; Humans ; Liver Neoplasms ; genetics ; metabolism ; pathology ; MicroRNAs ; genetics ; Neoplasm Invasiveness ; Proto-Oncogene Proteins c-akt ; metabolism ; RNA Interference ; RNA, Long Noncoding ; genetics ; Reverse Transcriptase Polymerase Chain Reaction ; Signal Transduction ; cdc25 Phosphatases ; metabolism

Biomarker identification is crucial for the selection of patients who might benefit from radiotherapy. To explore potential markers for response and prognosis in patients with locally advanced esophageal carcinoma treated with radiotherapy followed by surgery, we evaluated the expression of cell cycle checkpoint-related proteins Chk2, Cdc25C, and Cyclin D1. A total of 56 patients with locally advanced esophageal squamous cell carcinoma were treated with radiotherapy followed by surgery. Pretreatment tumor biopsy specimens were analyzed for Chk2, Cdc25C, and Cyclin D1 expression by immunohistochemistry. High expression of Chk2, Cyclin D1, and Cdc25C was observed in 44 (78.6%), 15 (26.8%), and 27 (48.2%) patients, respectively. The median survival was 16 months (range, 3-154 months), with a 5-year overall survival rate of 19.6%. Overexpression of Chk2 was associated with smoking (P = 0.021), overexpression of Cdc25C was associated with patient age (P = 0.033) and tumor length (P = 0.001), and overexpression of Cdc25C was associated with pathologic complete response (P = 0.038). Univariate analysis demonstrated that overexpression of Cdc25C and pathologic complete response was associated with better survival. In multivariate analysis, Cdc25C was the most significant independent predictor of better survival (P = 0.014) for patients treated with radiotherapy followed by surgery. Overexpression of Cdc25C was significantly associated with pathologic complete response and better survival of patients with locally advanced esophageal cancer treated with radiotherapy followed by surgery. These results suggest that Cdc25C may be a biomarker of treatment response and good prognosis for esophageal carcinoma patients. Thus, immunohistochemical staining of Cdc25C in a pretreatment specimen may be a useful method of identifying optimal treatment for patients with esophageal carcinoma.
Adult ; Aged ; Carcinoma, Squamous Cell ; metabolism ; pathology ; radiotherapy ; surgery ; Checkpoint Kinase 2 ; metabolism ; Combined Modality Therapy ; Cyclin D1 ; metabolism ; Esophageal Neoplasms ; metabolism ; pathology ; radiotherapy ; surgery ; Female ; Follow-Up Studies ; Humans ; Male ; Middle Aged ; Neoplasm Staging ; Particle Accelerators ; Proportional Hazards Models ; Smoking ; Survival Rate ; cdc25 Phosphatases ; metabolism

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

OBJECTIVETo assess the effects of LiCl on prostate cancer growth and to explore the underlying mechanisms.

METHODSEffects of LiCl on cell growth in vitro and in vivo were determined by cell counting and xenografts of prostate cancer cells. Alterations in cell proliferation and the expression of DNA replication-related protein were determined by MTT assay, BrdU incorporation and Western blot.

RESULTSCompared to PBS control group, the number of prostate cancer cells (PC-3) were lower treated with 10 mmol/L LiCl, the number was 1.9×10(5), 4.8×10(5) and the difference was significant (P < 0.05). The inhibition rate of cellular proliferation were 50%, 95% and 98%, respectively, in LiCl group, NaCl and KCl control group, the difference was significant (P < 0.05). The A-Value of BrdU incorporation was 1.5, 1.3 treated with 10 mmol/L, 30 mmol/L LiCl, while the A-value of BrdU incorporation was 4 in PBS control group, the difference was significant (P < 0.05). On the protein level, LiCl downregulates expression of cdc 6, cyclins A and cyclins E, and cdc 25C, and upregulates expression of the CDK inhibitor p21(CIP1). The mean volume and weight of xenograft tumor were 50 mm(3) and 296 mg after LiCl intraperitoneal injection, But PBS control group were 180 mm(3) and 957 mg, the difference was significant (P < 0.05).

CONCLUSIONLiCl disrupts DNA replication and suppresses tumor growth of prostate cancer cells in vitro and in vivo.

Animals ; Antineoplastic Agents ; pharmacology ; Cell Cycle Proteins ; metabolism ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Cyclin A ; metabolism ; Cyclin E ; metabolism ; Cyclin-Dependent Kinase Inhibitor p21 ; metabolism ; DNA Replication ; drug effects ; Humans ; Lithium Chloride ; pharmacology ; Male ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Neoplasm Transplantation ; Nuclear Proteins ; metabolism ; Prostatic Neoplasms ; metabolism ; pathology ; Tumor Burden ; drug effects ; cdc25 Phosphatases ; metabolism

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

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

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

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