OBJECTIVETo investigate the expression of the Chk1 gene in human sperm and its clinical significance.

METHODSWe collected 80 semen samples and divided them into 4 groups of equal number: normal, oligospermia, asthenozoospermia and oligoasthenozoospermia. The Chk1 expression and its relative level were detected by Western blot and RT-PCR, sperm DNA damage and gradient changes assessed by DNA ladder analysis, and sperm apoptosis determined by Annexin V/PI double staining in each group.

RESULTSThe Chk1 gene was expressed in all the four groups, but with significant differences (P < 0.01); the relative levels of CHK1 protein were similar to those of Chk1 mRNA in the normal, oligospermia, asthenozoospermia and oligoasthenozoospermia groups, which were 1.00 +/- 0.22, 0.76 +/- 0.10, 0.45 +/- 0.08 and 0.37 +/- 0.07, respectively. DNA ladder analysis showed a marked DNA ladder in the asthenozoospermia and oligoasthenozoospermia groups. Sperm apoptosis was markedly increased in the oligospermia, asthenozoospermia, oligoasthenozoospermia and 100% graded sperm groups ([ 8.3 +/- 0.60]%, [11.6 +/- 0.92]%, [12.5 +/- 1.43]% and [17.0 +/- 1.98]%), as compared with the normal group ([7.6 +/- 0.34]%) (P < 0.05).

CONCLUSIONChk1 is expressed in human sperm, but differently in different semen quality groups. And its expression is correlated with sperm DNA damage and apoptosis; its reduction may lead to declined sperm repair and increased sperm apoptosis and thus affect semen quality.

Apoptosis ; Checkpoint Kinase 1 ; DNA Damage ; Humans ; Male ; Protein Kinases ; genetics ; Semen Analysis ; Spermatozoa ; metabolism

Cell Cycle Checkpoints/genetics* ; Checkpoint Kinase 1/metabolism* ; Heterozygote ; Humans ; Mitosis/genetics* ; Mutation ; Zygote/metabolism*

OBJECTIVETo explore the increasing effect of blocking Chk1 and /or Chk2 gene by Chk1 or Chk2-specific antisense oligodeoxynucleotides (AsODN) on apoptosis in HeLa cell line after irradiation and its mechanism of action.

METHODSAsynchronized HeLa cells were exposed to (60)Co-irradiation at different dosage to activate G(2)/M checkpoint arrest. The cell cycle profiles were observed in HeLa cells after irradiation at a range of various doses and different time points by flow cytometry. In the experimental groups, Chk1/2 sODN and AsODN alone or in combination were transfected into HeLa cells, and the cells were exposed to (60)Co-irradiation at 24 h after transfection. The changes of Chk1/2 protein expression were assayed by Western blot and confocal laser scanning microscopy (Confocal), and the cell cycles, apoptosis rates and cell cycle specific apoptosis were detected by annexin V-PI labeling and flow cytometry.

RESULTSApoptotic response was significantly increased in the Hela cells after G(2)/M arrest and was inversed to activation of G(2)/M checkpoint. Either Chk1 or Chk2-specific AsODN consistently enhanced DNA damage-induced apoptosis by 90% approximately 120%, compared to corresponding sODN control (P < 0.05). Unexpectedly, combined use of Chk1- and Chk2-specific AsODN did not produce synergistic effect as compared to treatment with Chk1- or Chk2-specific AsODN alone (P > 0.05). While irradiated HeLa cells underwent apoptosis preferentially in G(1)-phase, apoptosis occurred in either of G(1)-, S- or G(2)/M -phase in the presence of Chk1 and/or Chk2 AsODN.

CONCLUSIONThe radioresistance is mainly induced by activating the cell cycle checkpoint signal transduction pathway after irradiation, and abrogating of the key effector Chk1 and Chk2 may increase the apoptotic sensitivity to irradiation due to changes of the pattern of cell cycle specific apoptosis.

Apoptosis ; radiation effects ; Cell Cycle ; radiation effects ; Checkpoint Kinase 1 ; Checkpoint Kinase 2 ; Cobalt Radioisotopes ; Down-Regulation ; Gene Expression Regulation, Neoplastic ; HeLa Cells ; Humans ; Oligodeoxyribonucleotides, Antisense ; genetics ; Protein Kinases ; genetics ; metabolism ; Protein-Serine-Threonine Kinases ; genetics ; metabolism ; Transfection

OBJECTIVETo block signal transduction of cell cycle checkpoints by antisense blocking of chk1/2 gene to increase the radiation sensitivity of HL-60 cell line.

METHODTo transfect the HL-60 cell with chk1/2 antisense and sense chain alone and in combination, expose the cells to irradiation at 24 h after the transfection, the chk1 protein change was assayed by Western blot and the cell cycles and annexin V apoptosis rates by FCM.

RESULTSThe irradiated apoptosis sensitivity was increased by antisense blocking of chk1 gene in HL-60 cell line, the apoptotic rate was 26.31% being significantly higher than that of the sense blocking (10.34%) (P < 0.05), Furthermore, the G(2)/M phase blocking phenomenon decreased and a synergic effect was observed in antisense blocking both the chk1 and chk2 genes.

CONCLUSIONAntisense blocking of chk1/chk2 could increase the apoptotic sensitivity to irradiation.

Apoptosis ; genetics ; radiation effects ; Cell Cycle ; radiation effects ; Checkpoint Kinase 1 ; Checkpoint Kinase 2 ; HL-60 Cells ; Humans ; Oligonucleotides, Antisense ; genetics ; Protein Kinases ; genetics ; Protein-Serine-Threonine Kinases ; genetics ; Radiation Tolerance ; Transfection

In order to investigate the change of cell-cycle of K562 cells induced by cisplatin (DDP) and role of antisense oligonucleotide targeting Chk1/2 on apoptosis of K562 cell induced by DDP, the change of cell-cycle was observed by means of flow cytometry after different intervals in which the K562 cell were treated by DDP. Chk1/2 protein expression was investigated by Western blot and confocal microscopy in best condition of transfection of antisense oligonucleotide targeting Chk1/2 by lipofection. Apoptosis of K562 induced by DDP was investigated by flow cytometry after transfection of antisense oligonucleotide targeting Chk1/2. The results showed that K562 cells were arrested at S phase at 10 micromol/L of DDP. Transfection with antisense oligonucleotide targeting Chk1/2 could inhibit expression of Chk1/2 at different levels. The frequency of apoptosis induced by DDP was increased when transfected with antisense oligonucleotide targeting Chk1 and/or Chk2. The effect of antisense oligonucleotide targeting Chk1 and Chk2 synchronously exceeded that of antisense oligonucleotide targeting either Chk1 or Chk2 alone. In conclusion, Chk1 and Chk2 may be regarded as targets of therapy for leukemia.
Antineoplastic Agents ; pharmacology ; Apoptosis ; drug effects ; Cell Cycle ; drug effects ; Checkpoint Kinase 1 ; Checkpoint Kinase 2 ; Cisplatin ; pharmacology ; Humans ; K562 Cells ; Oligonucleotides, Antisense ; pharmacology ; Protein Kinases ; physiology ; Protein-Serine-Threonine Kinases ; physiology

Objective: To study the correlation of the gene expressions of Chk1 and Chk2 with sperm concentration and motility. METHODS: According to sperm concentration and motility (percentage of progressively motile sperm), we divided 80 semen samples into four groups of equal number: normal control, oligozoospermia (OS), asthenospermia (AS), and oligoasthenozoospermia (OAS). We detected the sperm DNA fragmentation index (DFI) and viability and determined the expressions of Chk1 and Chk2 in the sperm by RT-PCR and Western blot. RESULTS: Statistically significant differences were not found in sperm DFI among the control, OS, AS, and OAS groups (21.24±6.93, 19.67±7.64, 21.52±6.92, and 19.28±11.55, P>0.05), but observed in sperm concentration, progressive motility, and viability between the DFI >30% and DFI ≤30% groups (P<0.01). Compared with the normal control, sperm viability was remarkably decreased in the OS, AS, and OAS groups (［83.48±9.87］% vs ［63.86±9.16］%, ［50.45±16.99］%, and ［39.21±15.74］%, P<0.05). RT-PCR showed remarkable differences among the control, OS, AS, and OAS groups in the relative expression level of Chk1 mRNA (0.73±0.22, 0.62±0.14, 1.03±0.39, and 0.92±0.071, P<0.01), which was correlated positively with sperm concentration (b = 80.661, P<0.01) but negatively with sperm motility (b = －19.275, P < 0.01), as well as in that of Chk2 mRNA (0.66±0.30, 0.27±0.09, 0.59±0.19, and 0.42 ± 0.11, P<0.01), which was correlated negatively with sperm concentration (b = －90.809, P<0.01) but positively with sperm motility (b = 27.507, P <0.01). The relative expression levels of the Chk1 protein were significantly different among the four groups (0.63±0.05, 0.42±0.03, 1.13±0.08, and 0.87±0.07, P<0.01), which was correlated positively with sperm concentration (b = 55.74, P<0.01) but negatively with sperm motility (b =－22.649, P<0.01), and so were those of the Chk2 protein (1.23±0.36, 0.37±0.16, 0.87±0.08, and 0.68±0.12, P<0.01), which was correlated negatively with sperm concentration (b =－53.001, P<0.01) but positively with sperm motility (b = 16.676, P < 0.01). CONCLUSIONS Chk1 and Chk2 are significantly expressed in human sperm. In case of sperm DNA damage, up-regulated Chk1 expression may enhance sperm apoptosis and lead to asthenospermia, while increased Chk2 expression may inhibit spermatogenesis and result in oligospermia.
Apoptosis ; Asthenozoospermia ; genetics ; Checkpoint Kinase 1 ; genetics ; metabolism ; Checkpoint Kinase 2 ; genetics ; metabolism ; DNA Damage ; DNA Fragmentation ; Gene Expression ; Humans ; Male ; Oligospermia ; genetics ; Semen Analysis ; Sperm Count ; Sperm Motility ; genetics ; Spermatozoa ; physiology

CHEK1 gene is known to play an important role in tumor progression by cell cycle control. However, the association between CHEK1 and the prognosis of esophageal squamous cell carcinoma (ESCC) is unclear. In this study, we explored the association between genetic variants in CHEK1 gene and prognosis of ESCC patients treated with radical resection. A total of 131 thoracic ESCC patients who underwent radical resection were included in this retrospective study and genotyped using the MassArray method. According to the univariate Cox hazard analysis, the GT/TT genotype of CHEK1 rs555752 was shown to be strongly related to a decreased overall survival (OS) (HR=2.560, 95% CI: 1.415-4.631, P=0.002) and disease-free survival (DFS) (HR=2.160, 95% CI: 1.258-3.710, P=0.005). Furthermore, according to the multivariate Cox hazard analysis and multiple testing, patients with the GT/TT genotype of CHEK1 rs555752 had a notably decreased OS (HR=2.735, 95% CI: 1.468-5.096, P=0.002, Pc=0.006) and DFS (HR=2.282, 95% CI: 1.292-4.023, P=0.004, Pc=0.012). In conclusion, genetic variants of the CHEK1 gene are significantly related to OS and DFS of ESCC patients, and may therefore be predictors of the prognosis of thoracic ESCC after surgery.
Adult ; Aged ; Carcinoma, Squamous Cell ; genetics ; pathology ; surgery ; Checkpoint Kinase 1 ; genetics ; Disease-Free Survival ; Esophageal Neoplasms ; genetics ; pathology ; surgery ; Female ; Humans ; Male ; Middle Aged ; Polymorphism, Single Nucleotide

OBJECTIVETo explore the regulatory effect of the GAGA element-related protein (GRP) on the Drosophila GAGA-dependent promoter activity in Jurkat cells.

METHODSDrosophila GAGA (dGAGA) factor (dGAF), GRP, and either the chloramphenicol acetyltransferase (CAT) reporter plasmid driven by the GAGA element-containing promoter of ftz gene or its mutant GAGA control were selectively co-transfected into Jurkat cells. Promoter activity analyses were performed by analyzing the RNA expression of the CAT in a real-time-RT PCR system, with pRC-CMV-betaGal co-transfected with the CAT reporter as a transfection efficiency control. Electrophoretic mobility shift assays (EMSA) were carried out to examine the binding profile of Jurkat nuclear extracts with a biotin labeled probe-containing dGAGA.

RESULTSIn Jurkat cells, GRP, either singly or combined with dGAF, elevated the activity of wild type dGAGA-containing ftz promoter dose-dependently in certain range. However, when the level of GRP was excessively high, it reduced or even fully inhibited the promoter activity of the ftz gene. On the contrary, either GRP or dGAF could not activate the ftz promoter with mutant GAGA. EMSA profile showed a specific band composed of the GAGA element and its binding proteins from Jurkat cells.

CONCLUSIONSHuman GAGA element-binding proteins exist in Jurkat cells. Its may either directly regulate the gene via GAGA elements or mediate the biphasic regulation of relevant gene in a GRP dose-dependent way.

Checkpoint Kinase 1 ; DNA-Binding Proteins ; genetics ; Drosophila Proteins ; genetics ; Gene Expression Regulation ; Humans ; Jurkat Cells ; Plasmids ; genetics ; Promoter Regions, Genetic ; Transcription Factors ; genetics ; Transfection

OBJECTIVE: To screen genes associated with poor prognosis of hepatocellular carcinoma (HCC) and to explore the clinical significance of these genes. METHODS: The proper expression profile data of HCC was obtained from the Gene Expression Omnibus (GEO) database, and the differentially expressed genes (DEGs) were identified by differential expression analysis. The DAVID and String database were used for function enrichment analysis and to construct the protein-protein interaction (PPI) network respectively. The Cancer Genome Atlas (TCGA) database and the Cox Proportional Hazard Model were used for prognosis analysis of the DEGs. RESULTS: A eligible human HCC data set (GSE84402) met the requirements. A total of 1141 differentially expressed genes were identified, including 720 up-regulated and 421 down-regulated genes. The results of function enrichment analysis and PPI network performed that CDK1、CDC6、CCNA2、CHEK1、CENPE 、PIK3R1、RACGAP1、BIRC5、KIF11 and CYP2B6 were prognosis key genes. And the prognosis analysis showed that the expressions of CDC6、PIK3R1、KIF11 and RACGAP1 were increased, and the expression of CENPE was decreased, which was closely related to prognosis of HCC. CONCLUSION CDC6、CENPE、PIK3R1、KIF11 and RACGAP1 may be closely related to poor prognosis of HCC, and can be used as molecular biomarkers for future research of HCC prognosis.
Carcinoma, Hepatocellular ; diagnosis ; genetics ; Checkpoint Kinase 1 ; Computational Biology ; Down-Regulation ; Gene Expression Profiling ; Genes, Neoplasm ; Humans ; Liver Neoplasms ; diagnosis ; genetics ; Prognosis ; Up-Regulation

OBJECTIVETo observe the effect of RNA interference on CHK1 and CHK2 expression and change of G2/M phase arrest in esophageal carcinoma cells after irradiation.

METHODSFour sequences short hairhip RNA (shRNA) of each CHK1 and CHK2 genes were constructed and connected with vector of pENTR/U6 plasmid, respectively, and then transfected into Eca109 cells with lipofectamine 2000 reagent. Protein and mRNA expression of CHK1 and CHK2 genes were detected with Western blotting and RT-PCR, respectively. Cell cycling was measured by flow cytometry after 5 Gy irradiation. Cell survival rate after 5 Gy irradiation was evaluated by clonegenetic assay.

RESULTSFour shRNA vector each of CHK1 and CHK2 genes were successfully constructed and transfected into Ecal09 cells, respectively. Protein expression of CHK1 and CHK2 were obviously decreased. Their mRNA expressions were also decreased after transfected with shRNA of CHK1 and CHK2. Arrest of G2/M stage in Eca109 cells were obviously decreased only in cells transfected with CHK1 shRNA but not with CHK2 shRNA at 12 h after 5 Gy irradiation. In first progeny Eca109 cells transfected with CHK1 and CHK2 shRNA, expression of CHK1 and CHK2 protein was also decreased. The level of phosphorylated CHK2-T68 expression was decreased at 1 h after 5 Gy irradiation, and at 72 h only transfected with CHK2 shRNA but not with CHK1 shRNA. Phosphorylation level of CHK1-S345 was not increased after transfected with CHK1 or CHK2 shRNA, but arrest of G2/M stage still remained at 12 h after 5 Gy irradiation and at 72 h accordingly. The cell survival rate was decreased in Eca109 cells transfected with CHK1 or CHK2 shRNA after 5 Gy irradiation.

CONCLUSIONAfter transfected with shRNA of CHK1 or CHK2, their expressions of mRNA and protein in Ecal09 cells are markedly inhibited and this inhibition effect can be observed in their first progeny cells and at least hold for 3 days. Arrest of G2/M phase can be reduced after irradiation when teansfected with shRNA of CHK1 and the radiosensitivity of Ec109 cells can be increased.

Blotting, Western ; Cell Division ; genetics ; physiology ; radiation effects ; Cell Line, Tumor ; Cell Survival ; genetics ; physiology ; radiation effects ; Checkpoint Kinase 1 ; Checkpoint Kinase 2 ; Esophageal Neoplasms ; genetics ; pathology ; physiopathology ; G2 Phase ; genetics ; physiology ; radiation effects ; Gamma Rays ; Genetic Vectors ; Humans ; Protein Kinases ; genetics ; metabolism ; Protein-Serine-Threonine Kinases ; genetics ; metabolism ; RNA Interference ; RNA, Small Interfering ; genetics ; Reverse Transcriptase Polymerase Chain Reaction ; Transfection