Mechanism of R-loop in myelodysplastic syndromes and the treatment progress / 白血病·淋巴瘤

Almost 50% myelodysplastic syndromes (MDS) patients have different splicing factor mutations, including SF3B1, SRSF2, U2AF1. Different splicing factor mutations cause the various mechanisms of slicing abnormality and eventually lead to the similar MDS phenotypes, indicating that splicing factor mutations might generate the common pathopoiesia pathway different from slicing abnormality. Recent studies have shown that SF3B1, U2AF1 and SRSF2 mutations could contribute to the accumulation of R-loop, cause DNA damage and repair abnormality, activate ATR-Chk1 pathway and finally promote apoptosis and tumorigenesis. This paper reviews the role of R-loop in the pathogenesis of MDS and the progress of related targeted drugs.

Study on the aging of leukemia K562 cells induced by ginsenoside Rg1 / 医学研究生学报

Objective　At present, the main studies of ginsenoside Rg1 are almost on the field of solid tumors and acute leukemias, and few on chronic leukemias. We aims to figure out the role of ATR-Chk1 pathway on cell aging in ginsenoside Rg1-treated leukemia K562 cells.　Methods　K562 cells were treated with ginsenoside Rg1 at different concentrations and divided into a control group (with 50 μL PBS culture solution) and 5 μmol/L ginsenoside group, 10 μmol/L ginsenoside group, 20 μmol/L ginsenoside group, 40 μmol/L ginsenoside group, 80 μmol/L ginsenoside group. CCK-8 assay，colony formation assay and flow cytometry for cell cycle detection were used to determine the effect of ginsenoside Rg1 on the aging of K562 cells. SA-β-Gal staining and Wright’s staining were used to observe the morphological changes of K562 cells’ aging. Real-time quantitative PCR and Western blot were used to detect the changes of ATR and Chk1 expression.　Results　The colony formation rate of K562 cells in the 20 μmol/L ginsenoside group was significantly lower than that in the other groups (P<0.05). CCK-8 test results showed that K562 cell proliferation of ginsenoside Rg1 induced groups was higher than that of the control group at 24, 48, and 72 hours (P<0.05). K562 cell proliferation inhibition rate was the highest in 20 μmol/L ginsenoside group for 48 hours treatment (P<0.05). The rate of SA-β-Gal positive cells [(95.833 ± 1.528) %] in 20 μmol/L ginsenoside-treated K562 cells for 48 h was significantly higher than that of the control group [(3.083 ± 0.764) %]. Cells blocked in G0/G1 phase and entered S and G2/M phases were significantly higher and lower than those in the control group, respectively (P<0.05).The ATR and Chk1 mRNA expression levels [(0.0117 ± 0.0038) %, (0.0120 ± 0.0021) %] were significantly higher than that of the control group ([0.0027 ± 0.0006) %, (0.0058 ± 0.0019) %) (P<0.05). ATR and Chk1 relative protein expression levels [(19.370 ± 0.994) %, (43.520 ± 1.236) %] were significantly increased compared with that of the control group [(17.080 ± 1.274) %, (39.100 ± 0.969) %) (P<0.05).　Conclusion　Ginsenoside Rg1 can induce the aging of K562 cells by regulating the ATR-Chk1 pathway, providing a new target for clinical leukemia treatment.

Expression of Chk1 in glioblastoma and its association with tumor biological behavior and prognosis survival / 实用肿瘤学杂志

Objective The aim of this study was to investigate the expression of cell cycle checkpoint kinase 1(Chk1)gene in glioblastoma cells( GBM) and its correlation with GBM cell proliferation,tumorigenic activity and prognosis. Methods The ex-pression of Chk1 in GBM cells was selected and analyzed by TCGA database and brain tumor molecular database( Rembrandt),and the level of Chk1 expression in GBM cells was detected by molecular biology techniques such as Western blot and Real-Time PCR. The expression of Chk1 was silenced by siRNA to investigate its effect on proliferation and colony-forming ability of GBM cells. The prognosis survival of GBM patients accompanying with Chk1 expression was analyzed by immunohistochemical staining and Rembrandt database. Results The results of TCGA database and Rembrandt showed that Chk1 gene was highly expressed in GBM tissues. West-ern blot and Real-Time PCR also showed that Chk1 gene was highly expressed in GBM cells. Lentiviral transfection siRNA-specific silencing of Chk1 significantly inhibited proliferation and colony-forming ability of U87 cells( P<0. 01 and P<0. 05). Prognostic survival analysis showed that GBM patients with low expression of Chk1 gene had a significantly better clinical outcome than those of GBM patients with high expression of Chk1 gene(P<0. 001). Conclusion Chk1 gene is overexpressed in GBM cells,up-regula-tion of Chk1 gene expression can promote the growth and proliferation of GBM cells,and Chk1 gene is associated with poor prognosis in GBM patients.

Protective effects of Panax notoginseng saponins on testicular DNA damage in natural aging rats based on ATR/Chk1/P53 signal pathway / 中草药

Objective: To explore the protective effects of Panax notoginseng saponins (PNS) on testicular DNA damage in natural aging rats based on ATR/Chk1/P53 signal pathway. Methods: SPF SD rats were randomly divided into five groups, with 10 rats in each group: young control group, aging model group, low, medium, and high dose of PNS-treated group. From 18 months, rats of PNS low, medium, and high dose groups were received PNS (10, 30, and 60 mg/kg )by stomach lavaging for 6 times per week for 6 months, respectively. The rats were weighed and euthanized by exsanguination under diethyl ether anesthesia, and testis were immediately removed, weighed, and the index of testis was calculated. The testicular tissue morphology was observed by HE staining, the expression and locations of γ-H2AX and ATR were detected by immunohistochemical analysis, and the relative expression levels of γ-H2AX, Chk1, p-P53, and P21 in testicular tissue were detected by Western blotting. Results: Compared with young control group, the obvious changes of seminiferous tubule were observed in aging model group, accompanied with a reduction in weight and index of testis. However, PNS treatment in some extent improved seminiferous tubule structure, weight, and index of testis. In addition, the protein expression levels of γ-H2AX and the numbers of γ-H2AX-positive cells in testis were significantly upregulated in aging model group relative to young control group. Furthermore, the numbers of ATR-positive cells and the protein expression levels of Chk1, p-P53, and P21 were significantly upregulated in testis of rats in aging model group when compared with young control group. Conversely, PNS significantly restored these changes of aging induced DNA damage response related proteins. Conclusion: PNS attenuates testicular DNA damage of natural aging rats, which may be associated with ATR/Chk1/P53 signaling.

Mechanisms underlying the synergistic damage to human squamous cell carcinoma cell line COLO-16 by everolimus and cisplatin: a preliminary study / 中华皮肤科杂志

Objective To investigate molecular mechanisms underlying the synergistic damage to the human squamous cell carcinoma cell line COLO-16 by everolimus and cisplatin.Methods In the signaling pathway experiment,COLO-16 cells were divided into 4 groups:control group receiving no treatment,50,100 and 200 nmol/L everolimus groups treated with 50,100 and 200 nmol/L everolimus respectively.In the combined experiment,COLO-16 cells were divided into another 4 groups:control group,50 nmol/L everolimus group,25 mol/L cisplatin group,and 50 nmol/L everolimus + 25 mol/L cisplatin group.Western blot analysis was performed to analyze changes in mammalian target of rapamycin (mTOR) pathway,Akt pathway,DNA damage-related pathway and Csk homologous kinase (Chk) pathway.Results After the treatment with everolimus at different concentrations of 50,100 and 200 nmol/L for 12 and 24 hours,the phosphorylation levels of mTOR at ser2448 and ser2481 as well as Rictor at thr1 135 in COLO-16 cells were all decreased compared with the control group.However,there were no significant changes in the phosphorylation levels of downstream signals ULK1 at ser757,p70 S6 at thr389 and PKCα at thr638/64.The treatment with everolimus did not change the total protein level and phosphorylation of Akt.After the treatment with cisplatin for 12 and 24 hours,the phosphorylation levels of Rictor at thr1135 and Chk1 at ser345 were significantly increased,but the treatment with everolimus alone showed no such effects.After the combined treatment with everolimus and cisplatin for 12 and 24 hours,the upregulation of Chk1 and Rictor phosphorylation were significantly inhibited compared with the cisplatin alone group.Conclusions mTOR signaling is sensitive to everolimus in COLO-16 cells,but its targeted pathway is not regulated simultaneously to develop a cascade reaction.Everolimus may increase the cisplatin-induced death of COLO-16 cells by inhibiting the activation of Chk 1,but can not aggravate DNA damage induced by cisplatin.

Impacts of Chk1 and Chk2 gene expressions on sperm concentration and motility / 中华男科学杂志

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.

Mechanisms underlying the synergistic damage to human squamous cell carcinoma cell line COLO-16 by everolimus and cisplatin: a preliminary study / 中华皮肤科杂志

Objective To investigate molecular mechanisms underlying the synergistic damage to the human squamous cell carcinoma cell line COLO-16 by everolimus and cisplatin.Methods In the signaling pathway experiment,COLO-16 cells were divided into 4 groups:control group receiving no treatment,50,100 and 200 nmol/L everolimus groups treated with 50,100 and 200 nmol/L everolimus respectively.In the combined experiment,COLO-16 cells were divided into another 4 groups:control group,50 nmol/L everolimus group,25 mol/L cisplatin group,and 50 nmol/L everolimus + 25 mol/L cisplatin group.Western blot analysis was performed to analyze changes in mammalian target of rapamycin (mTOR) pathway,Akt pathway,DNA damage-related pathway and Csk homologous kinase (Chk) pathway.Results After the treatment with everolimus at different concentrations of 50,100 and 200 nmol/L for 12 and 24 hours,the phosphorylation levels of mTOR at ser2448 and ser2481 as well as Rictor at thr1 135 in COLO-16 cells were all decreased compared with the control group.However,there were no significant changes in the phosphorylation levels of downstream signals ULK1 at ser757,p70 S6 at thr389 and PKCα at thr638/64.The treatment with everolimus did not change the total protein level and phosphorylation of Akt.After the treatment with cisplatin for 12 and 24 hours,the phosphorylation levels of Rictor at thr1135 and Chk1 at ser345 were significantly increased,but the treatment with everolimus alone showed no such effects.After the combined treatment with everolimus and cisplatin for 12 and 24 hours,the upregulation of Chk1 and Rictor phosphorylation were significantly inhibited compared with the cisplatin alone group.Conclusions mTOR signaling is sensitive to everolimus in COLO-16 cells,but its targeted pathway is not regulated simultaneously to develop a cascade reaction.Everolimus may increase the cisplatin-induced death of COLO-16 cells by inhibiting the activation of Chk 1,but can not aggravate DNA damage induced by cisplatin.

Overexpression of Chk1/2 gene affects G2/M arrest in MGC803 cells induced by diallyl disulfide / 中国药理学通报

Aim To investigate the effects of diallyl di-sulfide( DADS) on G2/M arrest in Chk1/MGC803 and Chk2/MGC803 cells so as to establish stable human gastric cancer MGC803 cells with overexpression of Chk1/2 gene. Methods The colony formation, flow cytometry, RT-PCR and Western blot were used to de-tect the proliferation, cell cycle, and expression of Chk1/2 mRNA and protein, p-Chk1/2, CDC25C and cyclinB1, respectively. Results The colony formation showed that the colony forming efficiency in Chk1/MGC803 and Chk2/MGC803 cells treated by 30 mg· L-1 DADS was lower than in control group and vector group ( P <0. 05 ) . Flow cytometry demonstrated that 41. 3%, 57. 4%, 68. 9% and 42. 9% of G2/M cells in Chk1/MGC803 were increased than in MGC803 and Chk2/MGC803 , respectively after treated by DADS in 12,24, 36 and 48 h(P <0. 05). At the same time, RT-PCR disclosed that expression of Chk1 and Chk2 mRNA had no marked change. Western blot showed that total proteins of Chk1 and Chk2 and p-Chk2 had invisible change, but expression of p-Chk1 was up-reg-ulated, and CDC25C and cyclinB1 were down-regula-ted time-dependently in Chk1/MGC803 cells ( P <0. 05 ) . Conclusion DADS arrests MGC803 cells at G2/M by increasing p-Chk1 expression to cause down-regulation of CDC25C and cyclinB1 simultaneously.

DADS induces G2/M arrest through Chk1/Cdc25C/CyclinB1/CDK1 pathway in human leukemia HL-60 cells / 中国药理学通报

Aim To study the effects of cycle arrest and molecular mechanism in human leukemia HL-60 cells induced by diallyl disulfide ( DADS ) . Methods Cell count, colony formation in soft agar experiments and flow cytometry analysis were employed to observe the DADS-induced cell growth inhibition and the effect of cycle arrest in HL-60 cells. The expressions of Chk1/2 and its downstream element in HL-60 cells were detected by Western blot. Results Cell count revealed that population doubling time increased to 35. 03 h and 71. 82 h, respectively, from 19. 14 h in HL-60 cells treated with 60 and 120 μmol·L-1 DADS ( P0. 05). The expression of Cdc25C, CyclinB1 and CDK1 decreased after treated with 60 μmol·L-1 DADS in a time-dependent manner ( P0. 05 ) . Conclusion DADS can in-hibit the proliferation of HL-60 cells, and induce G2/M arrest through Chk1/Cdc25 C/CyclinB1/CDK1 path-way.

Expression and significance of Plk1, Chk1/2 protein in primary hepatic carcinoma tissue and HepG2 cell / 中国免疫学杂志

Objective:To investigate the expression of Plk1 ( Polo-like kinase 1 ) and Chk1/2 ( Checkpoint kinase 1/2 ) in primary hepatic carcinoma tissue and HepG2 cell. Methods: Using immunohistochemistry chemical method detected expression of Plk1,Chk1/2 protein in 40 cases of primary hepatic carcinoma tissue and 16 cases of non-tumor tissue of liver. Western blot was applied to detect the expression of Plk1 and Chk1/2 protein in HepG2 cells, and gray value was measured by using the quantitative analysis. Results:The positive rate of Plk1,Chk1/2 protein expression in primary hepatic carcinoma was 57. 5%,75. 0% and 22. 5%respectively,compared with positive rate in the liver of non-tumor tissue were 0%,25. 0% and 56. 3%. The expression of Plk1 and Chk1 protein in primary hepatic carcinoma tissue is higher than that in non-tumor tissue of liver,and the difference was statistically sig-nificant( PChk1>Chk2. Conclusion: Plk1,Chk1 protein in primary hepatic carcinoma was up-regulated,while Chk2 protein was down-regulated in these tissues. The expression degree was Plk1> Chk1>Chk2. There were relatively selective expression in primary hepatic carcinoma tissue of Plk1,Chk1 protein,then Plk1 and Chk1 might be ideal targets for therapy of primary hepatic carcinoma.

Chk1 gene scijencing potentiates human hepatoma Huh7 cells to curcumin-induced apoptosis / 中国癌症杂志

Background and purpose: Checkpoint kinase 1 and 2 have been proposed to be potential therapeutic targets to sensitize cancers to radio- or chemo-therapeutics. However, little is known about whether Chk1/2 is also a suitable target for sensitizing cancers to curcumin. In the present study, we investigated effects of Chk1/2 siRNA on curcumin-induced apopotosis in hepatoma cell line Huh7 and evaluated the effectiveness of Chkl/2as a therapeutic target to potentiate human hepatoma to curcumin. Methods: Effect of curcumin on the cell cycle checkpoint-associated proteins was detected by Westem blot. The knockdown efficacy of Chk1/2 siRNA was measured by RT-PCR and Westem blot. Effect of Chk1/2 siRNA on curcumin-induced apoptosis in Huh7 cells was evaluated by DAPI staining. Effect of Chk1/2 siRNA on cell cycle distribution in curcumin-treated Huh7 cells was analyzed by flow cytometry. Results: Curcumin significantly inhibited phosphorylation of cell cycle checkpoint-associtaed proteins Chk1(S317), Cdc25C(S216) and Cdk1(Y15). Chk1 siRNA decreased Chk1 mRNA and protein by 95% and 92% and Chk2 siRNA decreased Chk2 mRNA and protein by 60% and 55% respectively as compared with negative control siRNA (P<0.01). Inhibition of Chk1, but not Chk2, increased apoptotic rate from (21.3±1.8)% to (29.5±2.6)% (P<0.05). Neither Chk1 nor Cbk2 siRNA had any impact on cell cycle distribution in Huh7 cells induced by curcumin. Conclusion: Chk1 siRNA sensitized Huh7 cells to curcumin-induced apoptosis, suggesting that Chk1 is a potential therapeutic target to sensitize human hepatoma to curcumin.

Apoptotic Sensitivity to Irradiation Increased after Transfection of chk1 Antisense Chain to HL-60 Cell Line / 华中科技大学学报（医学）（英德文版）

The HL-60 cells were transfected with chk1 antisense and sense chain, and 24 h later subjected to irradiation. Twenty-four h after irradiation, the changes in the chk1 protein expression was assayed by Western blot, and the cell cycles and apoptosis rate detected by FCM. The irradiated apoptosis sensitivity was increased by antisense blocking of chk1 gene in HL-60 cell line with the apoptosis rate being 26.31%, significantly higher than that by the sense blocking (10.34 %,0.025＜P＜0.05). In HL-60 cells transfected with chk1 antisense chain, the G2/M phase arrest was attenuated and the cells in G2/M phase were accounted for 38.42 %, significantly lower than those of the cells transfected with chk1 sense chain (54.64 %, 0. 005＜P＜0.01). It was concluded that antisense blocking of chk1 gene could increase the apoptosis sensitivity to irradiation.

Chk1 gene scilencing potentiates human hepatoma Huh7 cells to curcumin-induced apoptosis / 中国癌症杂志

Background and purpose:Checkpoint kinase 1 and 2 have been proposed to be potential therapeutic targets to sensitize cancers to radioor chemo-therapeutics. However, little is known about whether Chk1/2 is also a suitable target for sensitizing cancers to curcumin. In the present study, we investigated effects of Chk1/2 siRNA on curcumin-induced apopotosis in hepatoma cell line Huh7 and evaluated the effectiveness of Chk1/2 as a therapeutic target to potentiate human hepatoma to curcumin. Methods:Effect of curcumin on the cell cycle checkpoint-associated proteins was detected by Western blot. The knockdown efficacy of Chk1/2 siRNA was measured by RT-PCR and Western blot. Effect of Chk1/2 siRNA on curcumin-induced apoptosis in Huh7 cells was evaluated by DAPI staining. Effect of Chk1/2 siRNA on cell cycle distribution in curcumin-treated Huh7 cells was analyzed by flow cytometry. Results:Curcumin significantly inhibited phosphorylation of cell cycle checkpoint-associtaed proteins Chk1(S317), Cdc25C(S216) and Cdk1(Y15). Chk1 siRNA decreased Chk1 mRNA and protein by 95% and 92% and Chk2 siRNA decreased Chk2 mRNA and protein by 60% and 55% respectively as compared with negative control siRNA (P