1.Effect of methylation inhibitor in the treatment of leukemia.
Journal of Experimental Hematology 2013;21(2):536-538
More and more studies have found that the occurrence of tumors are directly related to the abnormal expression of oncogene and antioncogene. If the antioncogene is mutated or absent, the function of cells will be weakened and inactivated, the cells will be duplicated repeatedly out of control, then will induce occurrence and metastasis of tumor. For example, SHP-1 tyrosine phosphatase, as an antioncogene, is a key negative regulator in signaling transduction of haematopoietic cells. The decrease and silence of SHP-1 play an important role in tumorigenesis. If the oncogene in leukemia patients lost the effect of negative regulation of antioncogene, the oncogene would be expressed abnormally high, such as the oncogene c-kit (an important member of the class III in the tyrosine kinase receptor family) in many kinds of leukemia cells expresses actively. Studies have shown that the high methylation of promoter region would induce the inactivation of tumor suppressor and active expression of oncogene, therefore, the restoring normal methylation of promoter region will contribute to restoration of normal gene expression, thus achieving the purpose of gene therapy for leukemia. In this article, the methylation, methylation abnormality and leukemia, methylation suppressors and therapy of leukemia are briefly reviewed.
DNA Methylation
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drug effects
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Humans
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Leukemia
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drug therapy
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metabolism
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Protein Tyrosine Phosphatase, Non-Receptor Type 6
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metabolism
2.Inhibitory Effect of Decitabine-inhibeting Methylation of SHP-1 Gene on Proliferation and Apoptosis of MDS Cell Line Skm-1.
Ying HAN ; Jin ZHANG ; Jing JI ; Yan-Bin PANG ; Luo-Ming HUA ; Zong-Hong SHAO
Journal of Experimental Hematology 2019;27(5):1561-1567
OBJECTIVE:
To investigate the phenotype and molecular mechanism of DCA on MDS cell model, and to study the response of chemotherapeutic medicines to MDS cells through multiple dimensions, such as cell proliferation, invasion, migration and apoptosis, thus revealing the molecular mechanism of DCA treatment of MDS and its relationship with SHP-1 gene methylation.
METHODS:
MTT assay was used to determine the survival rate of MDS cells after treated by different concentrations of DCA. The effect of DCA on the invasion and migration of MDS cells was detected by Transwell assay method. Apoplexin V-FITCPI was used to detect apoptosis, the MDS treatment on the mechanism of DCA was investigated by Western blot and Real-time PCR experiment.
RESULTS:
According to the experiment, it was found that tumor proliferation could be inhibited when MDS skm-1 cells was treated by DCA, and the absorbance was lower and the inhibitory effect was more obvious in the 2.0, 5.0 μmol/L DCA group than in the 0.5 μmol/L DCA group and the negative control group. Compared with the control group, the number of MDS skm-1 cells crossing through the transwell upper chamber was significantly decreased after DCA application. After treated with 0.5, 2.0 and 5.0 μmol/L DCA, the apoptosis rate of MDS cells was 4.54%, 9.31% and 16.58% respectively, while the apoptosis rate of the control group was 3.20%, which shows the apoptosis rate increased significantly with the concentration of DCA. After treatment of MDS cell lines with different concentration of DCA, the methylation status of SHP-1 gene was decreased with the increase of drug concentration, the expression of SHP-1 was increased, the expression of STAT3 was decreased and the level of phosphorylation was decreased.
CONCLUSION
By analyzing the phenotypic response of DCA treatment on MDS cells, it was found that interfere with MDS can be performed by inhibiting proliferation, metastasis, and inducing apoptosis in a dose-dependent way. It revealed that the molecular mechanism by DCA treatment can improve the methylation of SHP-1 gene and inhibit the expression of p-STAT3.
Apoptosis
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Cell Line
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Cell Line, Tumor
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Cell Proliferation
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Decitabine
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Humans
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Protein Tyrosine Phosphatase, Non-Receptor Type 6
3.The relationship of SHP1 expression in liver tissues with the activation and proliferation of hepatic stellate cells in vivo during the pathologic processes of hepatic fibrosis in rats.
Li-Sen HAO ; Pan-Pan CHEN ; Li-Min JIN ; Zong-Yuan ZHAN ; Xiao-Shi YANG ; Jing-Xiu JI ; Mei-Yu JIANG ; Yan-Bo MO
Chinese Journal of Applied Physiology 2022;38(1):58-61
4.Expression and kinetic analysis of catalytic domain of protein tyrosine phosphatases SHP-1/SHP-2.
Yi MO ; Wei WANG ; Fangfang LIANG ; Guanyuan FU ; Hesheng JIANG ; Wayne ZHOU
Chinese Journal of Biotechnology 2009;25(2):274-278
In order to express and purify the catalytic domain of SHP-1/SHP-2 (named as D1C and D2C respectively) and determine their kinetics, the constructed D1C and D2C plasmids were transformed into Escherichia coli BL21 and the expression was induced with IPTG. The harvested cells were suspended in extraction buffer. After sonication, the solution was applied to HPLC and the results were confirmed by SDS-PAGE. The purified peptides were further subjected to kinetic specificity study using synthetic phosphotyrosine (pY) as substrate by malachite green method and analyzed by Lineweaver-Burk plot calculation. From this study, we found D1C and D2C were expressed successfully in soluble state in Escherichia coli BL21 and purified efficiently with HPLC system. The molecular weight of D1C was 34.6 kD, and its Michaelis constant (K(m)) was 2.04 mmol catalytic constant (K(cat)) was 44.98 s(-1), specific constant (K(cat)/K(m)) was 22.05 L/(mmol x s); the molecular weigh of D2C was 35.3 kD, and its Michaelis constant (K(m)) was 2.47 mmol, catalytic constant (K(cat)) was 27.45 s(-1), specific constant (K(cat)/K(m)) was L/(mmol x s). The enzyme activity of D1C is stronger than that of D2C.
Catalytic Domain
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Chromatography, High Pressure Liquid
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Escherichia coli
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genetics
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metabolism
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Kinetics
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Plasmids
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Protein Tyrosine Phosphatase, Non-Receptor Type 11
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genetics
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metabolism
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Protein Tyrosine Phosphatase, Non-Receptor Type 6
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genetics
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metabolism
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Recombinant Proteins
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genetics
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metabolism
5.Expression of tyrosine phosphatase containing C-src homology SH-2 in benign prostate hyperplasia.
Journal of Zhejiang University. Medical sciences 2007;36(5):488-492
OBJECTIVETo explore the expression of tyrosine phosphatase containing C-src homology SH-2 (SHP-1 and SHP-2) in benign prostate hyperplasia.
METHODSWith En Vision two-step method, the expression of SHP-1 and SHP-2 was detected in 10 cases of normal prostate tissue, 30 cases of BPH, 20 cases of PIN, 20 cases of high differential Pca and 20 cases of low differential Pca.
RESULTThe expression of SHP-2 in normal group was mainly distributed in the cytoplasm of secretive cells and basal cells, and a little part in the nucleu. In BPH it was distributed equally in the plasm and nucleu. In PIN, high differential Pca and low differential Pca, SHP-2 expressed mainly in nucleu. The average dyeing index of SHP-2 in each group is 0.4, 1.7, 2.1, 2.2 and 2.6. SHP-1 positive expression in normal prostate, BPH, PIN and high differential Pca showed differentiating layer staining in the cytoplasm of secretive cells and basal cells, while not in low differential Pca. The average dyeing index of SHP-1 in each group is 1.8, 1.8, 1.5, 1.2 and 0.4.
CONCLUSIONThere are transformation in signal transduction relation with SHP-1 and SHP-2 in the progress of prostate cell proliferation, differentiation and malignant. The abnormal activation and distribution of SHP-2 might induce prostate reconstruction and hyperplasia, even carcinoma.
Adult ; Aged ; Cell Nucleus ; enzymology ; Cytoplasm ; enzymology ; Humans ; Immunohistochemistry ; Male ; Middle Aged ; Prostatic Hyperplasia ; enzymology ; pathology ; Protein Tyrosine Phosphatase, Non-Receptor Type 11 ; metabolism ; Protein Tyrosine Phosphatase, Non-Receptor Type 6 ; metabolism ; Protein Tyrosine Phosphatases ; metabolism ; SH2 Domain-Containing Protein Tyrosine Phosphatases ; metabolism ; src-Family Kinases ; metabolism
6.Overexpression of SHP-1 Enhances the Sensitivity of K562 Cells to Imatinib.
Ying-Hua LI ; Xue-Dong LIU ; Xiu-Fen GUO ; Xiao LIU ; Jian-Min LUO ; Zhi-Shang LI ; Yong-Xiao ZHANG
Journal of Experimental Hematology 2016;24(1):46-51
OBJECTIVETo explore the effect of overexpression of SH2-containing tyrosine phosphatase 1 (SHP-1) on sensitivity of chronic myelogenous 1eukemia (CML) K562 cell line to imatinib and its related mechamism.
METHODSK562 cells were infected with the lentiviral plasmids containing the specified retroviral vector (pEX-SHP-1-puro-Lv105) or the mock vector (pEX-EGFP-puro-Lv105). The expression of SHP-1 in K562 cells treated with 0.2 µmol/L imatinib (IM) for 72 h was determined by Western blot. After transfection the CCK-8 assay was used to determine the proliferation of the tramfected K562 cells (K562(SHP-1) and K562(EGFP) cells) at 72 h after exposure to different doses of IM, the half inhibitary concentration (IC50) was calculated. The mechanisms of the overexpression effects of SHP-1 and IM on the proliferation in K562 cells was investigated, the BCR-ABL1 activity and the level of tyrosine phosphorylation of CrkL (pCrkL) was measured by flow cytometry; the Western blot was used to detect the expression and activity of these molecules controlling cell growth, including MAPK, AKT, STAT5 and JAK2.
RESULTSAfter exposure of K562 cells to 0.08 µmol/L IM for 72 h, there was no significant change of SHP-1 expression in K562 cells. After exposure to 0.2 µmol/L of IM for 72 h, the inhibitory rate of K562(SHP-1) group was higher than that of K562(EGFP) group (P < 0.05), indicating that overexpression of SHP-1 in K562 cells could enhance the proliferation inhtibition effect of IM on K562 cells. The IC50 of IM in K562(SHP-1) cells was the lower as compared with that of K562(EGFP) cells (P < 0.05) after exposure to different concentrations of IM for 72 h. The slope of K562(SHP-1) cells was the largest ranging 0.02 - 0.16 µmol/L of IM. Overexpression of SHP-1 and IM could inhibit the activity BCR-ABL1, MAPK, AKT, STAT5 and JAK2 signaling pathways in the K562 cell line and displayed a synergistic effect.
CONCLUSIONSHP-1 inhibits BCR-ABL1, MAPK, AKT, STAT5 and JAK2 signaling pathways in K562 cells, the overexpression of SHP-1 can enhance the sensitivity of K562 cells to IM.
Cell Proliferation ; Drug Resistance, Neoplasm ; Genetic Vectors ; Humans ; Imatinib Mesylate ; pharmacology ; K562 Cells ; drug effects ; Phosphorylation ; Protein Tyrosine Phosphatase, Non-Receptor Type 6 ; genetics ; metabolism ; Signal Transduction ; Transfection
7.Inhibitory effect of SHP-1 gene transfer on the proliferation of breast cancer cell line MDA-MB-231.
Ya-lin XIE ; Ji-zhen LIANG ; Fei-ye LIU ; Jun-yi ZHANG ; Jian-ming XIE ; Rong-cheng LUO ; Rong LI
Journal of Southern Medical University 2010;30(5):1024-1027
OBJECTIVETo observe SHP-1 protein expression in breast cancer cell line MDA-MB-231 before and after SHP-1 gene transfer and its effect on the proliferation of MDA-MB-231 cells.
METHODSThe eukaryotic expression vector pEGFP-C3-SHP-1 was constructed and transfected into breast cancer cell line MDA-MB-231 via Lipofectamine 2000, and the positive clones were selected using G418. SHP-1 expression in MDA-MB-231 cells was detected with immunocytochemistry and Western blotting, and the cell growth curve was observed using MTT assay.
RESULTSSHP-1 was highly expressed in transfected MDA-MB-231 cells, whose proliferation was significantly inhibited (P<0.05).
CONCLUSIONSHP-1 gene transfer into MDA-MB-231 cells results in inhibition of the cell proliferation.
Breast Neoplasms ; genetics ; pathology ; Cell Line, Tumor ; Cell Proliferation ; Female ; Gene Transfer Techniques ; Humans ; Protein Tyrosine Phosphatase, Non-Receptor Type 6 ; genetics ; metabolism
8.Mutation analysis of hematopoietic cell phosphatase gene in acute leukemia.
Jian-Min LUO ; Ze-Lin LIU ; Hong-Ling HAO ; Fu-Xu WANG ; Zuo-Ren DONG ; Ohno RYUZO
Journal of Experimental Hematology 2004;12(2):128-132
The hematopoietic cell phosphatase (HCP or SHP-1), the SH2 domain contain protein tyrosine phosphatase, is a crucial negative regulator in the process of hematopoietic cell development, proliferation and receptor-mediated mitogenic signaling pathways, and its mutation is responsible for the over-expansion and inappropriate activation of myelomonocytic population in motheaten mice. The aim of the study was to evaluate the role of the HCP gene in leukemogenesis. Bone marrow and/or peripheral blood from 32 acute myeloid leukemia (AML) patients, 9 acute lymphocytic leukemia (ALL) patients, 8 leukemia cell lines and 50 normal controls were analyzed by reverse transcription-polymerase chain reaction (RT-PCR) based on single strand conformation polymorphism (SSCP) and sequencing. RT-PCR showed that all samples expressed HCP gene, only one missense mutation at codon 225 (AAC to AGC, Asn to Ser) within N-terminal SH2 domain was found in an ALL patient. In addition, four polymorphic base substitutions were detected in codon 69, 85, 86 and 266, respectively. In conclusion, mutation of HCP gene is an infrequent genetic aberration which may only play a role in pathogenesis of a small part of leukemia, however, its significance needs to be further clarified.
Acute Disease
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Cell Line, Tumor
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Humans
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Intracellular Signaling Peptides and Proteins
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Leukemia
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enzymology
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genetics
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Mutation
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Polymorphism, Single-Stranded Conformational
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Protein Tyrosine Phosphatase, Non-Receptor Type 6
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Protein Tyrosine Phosphatases
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genetics
9.Retrovirus-mediated SHP-1 gene expression in human breast cancer MDA-MB-231 cells.
Ji-zhen LIANG ; Rong LI ; Jun-yi ZHANG ; Hang ZHENG ; Rong-cheng LUO
Journal of Southern Medical University 2009;29(5):902-905
OBJECTIVETo construct a retrovirus-mediated expression system carrying human SHP-1 gene to transfer SHP-1 gene in human breast cancer MDA-MB-231 cells.
METHODSThe full-length SHP-1 gene fragment was amplified by RT-PCR from the total RNA extracted from human breast cancer cell line MCF-7 over-expressing SHP-1 protein. The gene fragment was inserted into the vector pLNCX2 to construct the recombinant retroviral plasmid, which was transfected into the packaging cell PT67 via Lipofectamine2000. A cell line stably producing the virus was selected with G418. MDA-MB-231 cells was infected with the virus, and the expression of SHP-1 gene in the positive cell clone was detected with Western blotting.
RESULTSA 1.8 kb cDNA fragment of SHP-1 gene was obtained from MCF-7 cells and successfully inserted into the pLNCX2. A stable cell clone PT67/SHP-1 and virus supernatant were obtained. Expression of SHP-1 protein was detected in the cells infected with the virus.
CONCLUSIONThe recombinant retroviral vector carrying SHP-1 gene has been successfully constructed and MDA- MB-231/SHP-1 cell line expressing SHP-1 has been obtained to allow further functional study of SHP-1 in breast cancer.
Breast Neoplasms ; genetics ; metabolism ; pathology ; Cell Line, Tumor ; Cloning, Molecular ; Genetic Vectors ; genetics ; Humans ; Protein Tyrosine Phosphatase, Non-Receptor Type 6 ; biosynthesis ; genetics ; Recombinant Proteins ; biosynthesis ; genetics ; Retroviridae ; genetics ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; Transfection
10.Experimental study of SHP-1 promoter methylation in myelodysplastic syndromes and its related mechanism.
Yi-Zhuo ZHANG ; Dan-Dan ZHAO ; Wei-Peng ZHAO ; Hai-Feng ZHAO ; Zhi-Gang ZHAO ; Ya-Fei WANG ; Xiao-Xiong WU ; Xiao-Ping HAN ; Wan-Ming DA
Chinese Journal of Hematology 2012;33(2):108-112
OBJECTIVETo explore the role of SHP-1 promoter methylation on the pathogenesis and progression in myelodysplastic syndromes (MDS) and its related mechanism.
METHODS63 MDS patients were divided into low-grade (LG) group and high-grade (HG) group according to IPSS score system. Bone marrow samples were collected. Methylation specific-PCR (MSP) were used to detect the status of SHP-1 promoter methylation in bone marrow (BM) samples from different risk MDS patients and MDS cell line, SKK-1. Western blot was used to detect signal transduction and activator of transcription (STAT3) activation in SKK-1 cell line and MDS patients.
RESULTSNo SHP-1 promoter methylation could be detected in healthy controls BM. Partially methylation was found in SKK-1 cell line. Methylation rate of SHP-1 gene promoter was found in BM of 24.2% of low-grade MDS patients and 63.3% of high-grade MDS patients, the difference between these two groups was statistically significant (P < 0.05); Patients were divided into different groups according to WHO subtype, chromosomal karyotype and blast cells in bone marrow, methylation rates of SHP-1 were significantly higher in RAEB-II, poor karyotype group and samples with 0.11-0.19 blast cells (P < 0.05); The phosphorylation protein of STAT3 was detected in SKK-1 cell line. The expression of phosphorylation STAT3 was significantly higher in HG group than in LG group (66.7% vs 18.2%) (P < 0.05). There was a significant correlation between SHP-1 promoter methylation and STAT3 phosphorylation.
CONCLUSIONAbnormal methylation of SHP-1 gene promoter might have tentative role in the pathogenesis and progression of MDS, which may be involved in STAT3 activation. Detection of SHP-1 promoter methylation may be helpful to evaluate the prognosis of MDS.
Adolescent ; Adult ; Aged ; Case-Control Studies ; Child ; DNA Methylation ; Female ; Humans ; Male ; Middle Aged ; Myelodysplastic Syndromes ; genetics ; metabolism ; Prognosis ; Protein Tyrosine Phosphatase, Non-Receptor Type 6 ; genetics ; metabolism ; STAT3 Transcription Factor ; metabolism ; Young Adult