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

Expression of CDC25A in non-small cell lung cancer and its relationship with let-7 gene / 南方医科大学学报

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.

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.

Development of a Novel Nonradioisotopic Assay and Cdc25B Overexpression Cell Lines for Use in Screening for Cdc25B Inhibitors

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.

Downregulation of LncRNAH19 and MiR-675 promotes migration and invasion of human hepatocellular carcinoma cells through AKT/GSK-3β/Cdc25A signaling pathway / 华中科技大学学报（医学）（英德文版）

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.

Overexpression of Cdc25C predicts response to radiotherapy and survival in esophageal squamous cell carcinoma patients treated with radiotherapy followed by surgery / 癌症

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.

PKA-regulated phosphorylation status of S149 and S321 sites of CDC25B inhibits mitosis of fertilized mouse eggs / 生理学报

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.

LiCl suppresses tumor growth and inhibits DNA replication in prostate cancer / 中华病理学杂志

<p><b>OBJECTIVE</b>To assess the effects of LiCl on prostate cancer growth and to explore the underlying mechanisms.</p><p><b>METHODS</b>Effects 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.</p><p><b>RESULTS</b>Compared 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).</p><p><b>CONCLUSION</b>LiCl disrupts DNA replication and suppresses tumor growth of prostate cancer cells in vitro and in vivo.</p>

Inhibitory activity of dioxy-pyrrolino 3', 4'-disoxazoline derivatives containing (1', 2'-O-cyclohexylidendioxyethyl) against Cdc25A and CD45 / 药学学报

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.

Molecular Mechanism of the G2/M Arrest in Breast Cancer Cell Lines (T47D and MDA-MB231) Induced by Genistein / 한국유방암학회지

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).

Study on the expression and significance of Galectin-3 and CDC25B mRNA in human gastric carcinoma / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To study the expression of Galectin-3 and CDC25B mRNA in gastric carcinoma and their correlation with clinical-pathological features and the survival time.</p><p><b>METHODS</b>Tissue 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.</p><p><b>RESULTS</b>In 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.</p><p><b>CONCLUSION</b>The 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.</p>

Cdc25B overexpression reverses two-cell block in mouse embryos / 中国应用生理学杂志

<p><b>AIM</b>To explore the effect of Cdc25B overexpression on the development of mouse two-cell embryos.</p><p><b>METHODS</b>The 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.</p><p><b>RESULTS</b>The 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.</p><p><b>CONCLUSION</b>The 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.</p>

Induction of G2 /M phase arrest and apoptosis of MCF-7 cells by novel benzofuran lignan via suppressing cell cycle proteins / 药学学报

In the present study, a newly synthesized benzofuran lignan 4-formyl-2-(4-hydroxy-3methoxyphenyl)-5-(2-methoxycarbonyethyl)-7-methoxy-benzo [b] furan (ERJT-12) was tested for its antiproliferative activity on human tumor cells. The related mechanisms were also investigated. In vitro growth inhibitory effects of ERJT-12 on various cancer cell lines were determined by MTT assay. Cell cycle distribution and apoptosis were detected by flow cytometry. The integrity of DNA was assessed by agarose gel electrophoresis. Activation of Caspase-3/7 and Caspase-6 was measured by colorimetric assay. The expressions of cell cycle proteins cell divide cycle 25c (Cdc25c), cyclin dependent kinase 1 (CDK1), CyclinB1 and apoptosis-related proteins Bax and Bcl-2 were detected by Western blotting. MTT assay showed that ERJT-12 inhibited the proliferation of several cancer cell lines including multidrug resistant cells. MCF-7 cells were markedly arrested at gap2/mitosis (G2/M) phase after treatment with ERJT-12 and progressed into apoptosis. The increased activities of Caspase-3/7 and Caspase-6 in MCF-7 cells were observed. The expression of CyclinB1 was down-regulated. The activities of Cdc25c and CDK1 protein were suppressed and Bcl-2 protein was phosphorylated. ERJT-12 displays potent antiproliferative activity towards cancer cells through suppressing cell cycle proteins, arresting cell cycle at G2/M phase and inducing apoptosis. It might be a novel candidate for cancer therapy.

Expression of Cdc25A, Cdc25B and Cdc25C in cervical carcinoma / 대한산부인과학회지

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.

Screening the active constituents of Chinese medicinal herbs as potent inhibitors of Cdc25 tyrosine phosphatase, an activator of the mitosis-inducing p34cdc2 kinase / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

<p><b>OBJECTIVE</b>To screen and evaluate the active constituents of Chinese medicinal herbs as potent inhibitors of Cdc25 phosphatase.</p><p><b>METHODS</b>The affinity chromatography purified glutashione-S-transferase/Cdc25A phosphatase fusion protein and Cdc2/cyclin B from the extracts of starfish M phase oocytes are used as the cell cycle-specific targets for screening the antimitotic constituents. We tested 9 extracts isolated from the Chinese medicinal herbs and vegetables including the agents currently used in cancer treatment by measuring the inhibition of Cdc25A phosphatase and Cdc2 kinase activity. The antitumor activity of the extracts was also evaluated by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay and flow cytometry.</p><p><b>RESULTS</b>Cdc25A inhibitory activity and antitumor activity are detected in the extracts isolated from three Chinese medicinal herbs Agrimona pilosa; Herba solani lyrati; Galla chinesis.</p><p><b>CONCLUSION</b>We found three extracts isolated from Chinese medicinal herbs have potential inhibitory activity of Cdc25 phosphatase using a highly specific mechanism-based screen assay for antimitotic drug discovery.</p>

Investigation on the molecular mechanisms of anti-hepatocarcinoma herbs of traditional Chinese medicine by cell cycle microarray / 中国中药杂志

<p><b>OBJECTIVE</b>To design DNA microarray and investigate the molecular anti-tumor mechanism of herbs of traditional Chinese medicine.</p><p><b>METHOD</b>cDNA microarrays consisting of 56 probes representing 24 human cell cycle genes were constructed, Four anti-hepatocarcinoma herbs including Radix Linderae, Hebra Artemisiae Annuae, Radix Amebiae, Radix Astragli, were chosen. Effects of herbs on SMMC-7721 cell cycle were observed by flow cytometry assay. Effects of herbs on cell cycle gene expression in SMMC-7721 cells were analyzed by comparing hybridization of Dig-Labeled cDNAs from herb-treated cells and cDNAs from untreated cells.</p><p><b>RESULT</b>Expressions of cell cycle geneswere changed in different degrees after herbs treated. Some genes were down-regulated and some genes were up-regulated. The changes in gene expression agreed with the results of flow cytometry assay.</p><p><b>CONCLUSION</b>The results suggest that these herbs may have effects on cell cycle and DNA damage checkpoint genes which may be the mechanism of the herbs, and DNA microarray can be used to investigate the biological function of extracts of traditional Chinese medicine.</p>

Dysregulation of the TGF-beta postreceptor signaling pathway in cell lines derived from primary or metastatic ovarian cancer / 华中科技大学学报（医学）（英德文版）

Transforming growth factor-beta (TGF-beta) may cause cell cycle arrest, terminal differentiation, or apoptosis in most normal epithelial cells, whereas most malignant cell lines are resistant to TGF-beta. Mechanisms of resistance to TGF-beta caused by modulation of cell cycle regulators and/or inactivation of components of the TGF-beta signaling transduction pathway such as C-myc and Smad4 are not well understood. To investigate the potential association between loss of sensitivity to TGF-beta and expression status of transforming growth factor receptor II (TbetaR II) , Smad4, CDC25A and C-myc in 14 cell lines derived from ovarian cancer, the expression levels of these genes were detected by semi-quantitative RT-PCR. Normal ovarian surface tissues were used as controls. The expression of TbetaR II was detectable in all of 14 cell lines. The expression of Smad4 was decreased in 10 cell lines and 9 cell lines overexpressed CDC25A, as compared to normal controls. CDC25A gene was overexpressed with 88% (8/9) in tumorigenic cell lines as determined by xenografts in nude mice, and only in 20% (1/5) of non-tumorigenic cell lines (P<0.05). C-myc was not overexpressed in any of these cell lines. The loss of sensitivity to TGF-beta of cell lines derived from ovarian cancers may be related to a decreased expression of Smad4, which mediates TGF-beta induced growth inhibition, and/or an overexpression of CDC25A. This overexpression of CDC25A correlates with increased tumorigenicity of ovarian cancer cell lines. The loss of sensitivity to TGF-beta is not associated with a lack of TbetaR II.

The initiation of G2/M checkpoint by diallyl disulfide requires the activation of p38 MAP kinase in HL-60 cells / 中华血液学杂志

<p><b>OBJECTIVE</b>To explore the molecular mechanisms of G(2)/M checkpoint initiated by diallyl disulfide (DADS) in HL-60 cells.</p><p><b>METHODS</b>Cell viability was determined by MTT assay. Cell cycle was assayed by flow cytometry. The expression of phospho-p38, Cdc25B and Cdc2, and p38 mRNA were measured by Western blotting and RT-PCR, respectively.</p><p><b>RESULTS</b>After treatment with DADS at 5 - 160 micro mol/L for 0 - 72 h, the growth of HL-60 cells were suppressed in a concentration-dependent manner and the inhibitory effect of DADS (20 micro mol/L) was similar to that of ATRA (10 nmol/L) (P > 0.05). Incubation of HL-60 cells with DADS (20 micro mol/L) for 12 h could activate G(2)/M checkpoint and increase the expression of phospho-p38 MAPK, followed by the expression of phospho-Cdc25B and phospho-Cdc2 (P < 0.05). SB202190, a specific inhibitor of p38 MAPK, markedly blocked the phosphorylation of p38 MAPK, Cdc25B and Cdc2 (P < 0.05).</p><p><b>CONCLUSION</b>DADS could induce the G(2)/M arrest in HL-60 cells which may be involved in the activation of p38 MAP kinase.</p>

Effect of antisense RNA targeting polo-like kinase 1 on cell cycle and proliferation in A549 cells / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Expression 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.</p><p><b>METHODS</b>A 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.</p><p><b>RESULTS</b>After 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).</p><p><b>CONCLUSIONS</b>Plk1 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.</p>

The relationship between ATM gene silence inducing apoptosis susceptibility and abnormal CDK activity / 中华血液学杂志

<p><b>OBJECTIVE</b>To explore the molecular elements responsible for the enhanced apoptotic susceptibility in U937 cells mediated by silencing atactic telangiectasis mutation (ATM) gene.</p><p><b>METHODS</b>Two 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.</p><p><b>RESULTS</b>U937-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.</p><p><b>CONCLUSION</b>Silencing 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.</p>