Experimental Study on the Mechanism of Mangiferin Inhibiting Malignant Biological Characteristics of Multiple Myeloma and Exerting Anticancer Effect / 中国实验血液学杂志

OBJECTIVE@#To investigate the effect of pure Chinese herbal extract Mangiferin on the malignant biological behaviors of multiple myeloma (MM) cells, and to analyze the molecular mechanism of the anti-myeloma effect of Mangiferin, so as to provide experimental basis for MM replacement therapy.@*METHODS@#U266 and RPMI8226 of human MM cell lines were intervened with different concentrations of Mangiferin. Cell proliferation was detected by CCK-8 method. Annexin V/PI double staining flow cytometry was used to detect cell apoptosis. Western blot was used to detect the expression of apoptosis and related signaling pathway proteins, and real-time quantitative polymerase chain reaction (qRT-PCR) was used to detect the expression of matrix metalloproteinase (MMP) and CXC chemokine receptor (CXCR) family.@*RESULTS@#Mangiferin could inhibit the proliferation activity of U266 and RPMI8226 cells and induce cells apoptosis. After Mangiferin intervened in U266, RPMI8226 cells for 48 h, the expression of Bcl-2 family pro-apoptotic protein Bax was up-regulated, while the expression of survivin and Bcl-xL proteins was down-regulated and caspase-3 was hydrolyzed and activated to promote cell apoptosis, besides, the expression of Bcl-2 protein in U266 cells was also significantly down-regulated to induce apoptosis (P<0.05). After Mangiferin intervenes in MM cells, it can not only increase the expression level of tumor suppressor p53, but also induce programmed cell death of MM cells by inhibiting the expression of anti-apoptotic molecules and down-regulating the phosphorylation levels of AKT and NF-κB. In addition, after the intervention of Mangiferin, the expressions of CXCR4, MMP2 and MMP9 in U266 cells were down-regulated (P<0.05), while there is no effect on the expressions of CXCR2, CXCR7 and MMP13 (P>0.05). However, the expressions of CXCR4, MMP9, and MMP13 in RPMI8226 cells were down-regulated (P<0.01), the expression of MMP2 was weakly affected, and the expression of CXCR2 and CXCR7 was basically not affected (P>0.05).@*CONCLUSION@#Mangiferin can inhibit the proliferation and induce apoptosis of MM cells, and its mechanism may be related to inhibiting the activation of NF-κB signaling pathway, affecting the expression of Bcl-2 family proteins, and inhibiting the expression of core members of MMP and CXCR family.

Clinical Features and Outcomes of the Patients with B-Cell Chronic Lymphoproliferative Disease in the New Drug Era / 中国实验血液学杂志

OBJECTIVE@#To analyze the clinical characteristics of the patients with B-cell chronic lymphoproliferative disease（B-CLPD） in the new drug era and the effect of new drug treatment on efficacy and survival.@*METHODS@#The clinical and laboratory data of 200 cases B-CLPD patients diagnosed between April 2015 and August 2021 were analyzed retrospectively. The clinical efficacy and survival of the patients under different treatments including Bruton tyrosine kinase（BTK） inhibitors， rituximab， and chemotherapy alone were analyzed. The prognostic factors affecting the survival of patients were analyzed by univarite analysis and multivariate analysis.@*RESULTS@#There were 119 male（59.5％） and 81 female（40.5%） in 200 cases B-CLPD patients， the sex ratio(male/female) was 1.5∶1 with median age of 61（30- 91） years old. The distribution of subtypes were as fallows: 51 cases (25.5%) of chronic lymphocytic leukemia/small lymphocytic lymphoma（CLL/SLL）， 64（32.0%） cases of follicular lymphoma（FL）， 40（20.0%） cases mantle cell lymphoma（MCL）， 30（15.0%） cases of marginal zone lymphoma（MZL）， 10（5%） cases of lymphoplasmacytic lymphoma/waldenstrom macroglobulinemia（LPL/WM）， 5（2.5%） cases of B cell chronic lymphoproliferative disorders unclassified（B-CLPD-U） . The main clinical manifestation of 102 patients was lymph node enlargement, 32 cases were complicated with B symptoms. Among CLL/SLL patients， there were 12（23.5%） cases in Binet A and 39（76.5%） cases in Binet B/C. There were 29 patients（20.9%） in Ann Arbor or Lugano stage I-II and 110 cases（79.1%） in stage III-IV of other subtypes. The complete remission（CR） rate was 43.1%（25/58）， 40.2%（39/97）， 7.1%（1/14）， and overaIl response rate（ORR） was 87.9%（51/58）， 62.9%（61/97）， 28.6%（4/14） in the groups of BTK inhibitors， rituximab-based therapy， and chemotherapy alone. The 3-year OS rate and PFS rate in all patients was 79.2% and 72.4% respectively. The 3-year OS rate of patient with MZL, CLL/SLL, FL,WM was 94.7%, 87.7%, 86.8% and 83.3% respectively, while the 3-year OS rate of MCL was only 40.6%, which was significantly lower than other subtypes. The median OS of patients treated with BTK inhibitors and rituximab-based therapy was 20.5 and 18.5 months respectively， and the 3-year OS rate was 97.4% and 90.7%. However， the median PFS of patients receiving chemotherapy alone was 4 months， and the 1-year OS rate was 52.7%， which was statistically significant compared with the other two groups（P<0.05）. Univarite analysis showed that anemia， elevated lactate dehydrogenase， elevated β2-microglobulin， and splenomegaly were the poor prognostic factors for OS（P<0.05）， elevated lactate dehydrogenase was also poor prognostic factors for PFS（P<0.05）. Multifactor analysis showed that anemia and elevated lactate dehydrogenase were the independent poor prognostic factors for survival（P<0.05）.@*CONCLUSION@#The clinical features of B-CLPD was various， anemia and elevated lactate dehydrogenase are the prognostic factors for poor survival. BTK inhibitors and new immunotherapy can improve the survival and prognosis of patients in the new drug era.

The Effects and Regulatory Mechanism of Targeting CXC Chemokine Receptor 1/2 Combined with Ara-C on the Malignant Biological Behaviors of U937 Cells of Acute Myeloid Leukemia / 中国实验血液学杂志

OBJECTIVE@#To investigate and analyze the effect of CXC chemokine receptor 1/2 (CXCR1/2) targeting inhibitor Reparixin combined with cytarabine (Ara-C) on the malignant biological behaviors of acute myeloid leukemia cells and its effect on the expression of the CXCR family, while exploring the accompanying molecular mechanism, providing scientific basis and reference for new molecular markers and targeted therapy for AML.@*METHODS@#Acute myeloid leukemia U937 cells were treated with different concentrations of Reparixin, Ara-C alone or in combination, and the cell morphology was observed under an inverted microscope; Wright-Giemsa staining was used to detect cell morphological changes; CCK-8 method was used to detect cell proliferation; the ability of cell invasion was detected by Transwell chamber method; the ability of colony formation was detected by colony formation assay; cell apoptosis was detected by Hoechst 33258 fluorescent staining and Annexin V/PI double-staining flow cytometry; monodansylcadaverine(MDC) staining was used to detect cell autophagy; the expression of apoptosis, autophagy and related signaling pathway proteins was detected by Western blot and the expression changes of CXCR family were detected by real-time quantitative polymerase chain reaction (qRT-PCR).@*RESULTS@#Reparixin could inhibit the proliferation, invasion, migration and clone formation ability of U937 cells. Compared with the single drug group, when U937 cells were intervened by Reparixin combined with Ara-C, the malignant biological behaviors such as proliferation, invasion and colony formation were significantly decreased, and the levels of apoptosis and autophagy were significantly increased (P<0.01). After Reparixin combined with Ara-C intervenes in U937 cells, it can up-regulate the expression of the pro-apoptotic protein Bax and significantly down-regulate the expression of the anti-apoptotic protein Bcl-2, and also hydrolyze and activate Caspase-3, thereby inducing cell apoptosis. Reparixin combined with Ara-C could up-regulate the expressions of LC3Ⅱ and Beclin-1 proteins in U937 cells, and the ratio of LC3Ⅱ/LC3Ⅰ in cells was significantly up-regulated compared with single drug or control group (P<0.01). MDC result showed that the green granules of vesicles increased significantly, and a large number of broken cells were seen (P<0.01). Reparixin combined with Ara-C can significantly inhibit the phosphorylation level of PI3K, AKT and NF-κB signaling molecule, inhibit the malignant biological behavior of cells by inhibiting the activation of PI3K/AKT/NF-κB pathway, and induce programmed cell death. Ara-C intervention in U937 cells had no effect on the expression of CXCR family (P>0.05). The expression of CXCR1, CXCR2, and CXCR4 mRNA could be down-regulated by Reparixin single-agent intervention in U937 cells (P<0.05), and the expression of CXCR2 was more significantly down-regulated than the control group and other CXCRs (P<0.01). When Reparixin and Ara-C intervened in combination, the down-regulated levels of CXCR1 and CXCR2 were more significant than those in the single-drug group (P<0.01), while the relative expressions of CXCR4 and CXCR7 mRNA had no significant difference compared with the single-drug group (P>0.05).@*CONCLUSION@#Reparixin combined with Ara-C can synergistically inhibit the malignant biological behaviors of U937 cells such as proliferation, invasion, migration and clone formation, and induce autophagy and apoptosis. The mechanism may be related to affecting the proteins expression of Bcl-2 family and down-regulating the proteins expression of CXCR family, while inhibiting the PI3K/AKT/NF-κB signaling pathway.

Effect of miR-203/CREB1 Signaling Regulation Mediated by DNA Methylation on the Proliferation and Apoptosis of Multiple Myeloma Cells / 中国实验血液学杂志

OBJECTIVE@#To investigate the effect of miR-203/CREB1 signaling regulation mediated by DNA methylation on the proliferation, invasion and apoptosis of multiple myeloma (MM) cells.@*METHODS@#The methylation level of miR-203 in the RPMI 8226 cells was detected by bisulfite sequcucing polymerase chain reaction (BSP). The mRNA expression of miR-203 was measured by quantitative real-time polymerase chain reaction. RPMI 8226 cells were treated with DNA methyltransferase inhibitor 5-Aza-2'-deoxycytidine (5-Aza-CdR). The miR-203 mimic in MM cell line RPMI 8226 was transfected to establish overexpressed miR-203 cell. The proliferation, invasion ability and apoptosis of RPMI 8226 cell was detected by CCK-8 assay, Transwell, and flow cytometry, respectively. The targeting relationship between miR-203 and CREB1 was verified by double luciferase report assay. Western blot was used to detect the expression of CREB1 protein.@*RESULTS@#Hypermethylation of miR-203 promoter region and low expression level of miR-203 mRNA were detected in the RPMI 8226 cells, which showed that demethylation could induce the expression of miR-203. The proliferation and invasion ability of RPMI 8226 cells after treated by 5-Aza-CdR were inhibited, and showed statistical significance as compared with blank control group (both P<0.05)，while the apoptosis rate was promoted (P<0.05). The proliferation, invasion ability and apoptosis of overexpressed miR-203 were the same as the demethylation group. Double luciferase report assay confirmed that CREB1 was the direct target of miR-203. The protein level of CREB1 was inhibited by demethylation and showed statistical significance as compared with control group (P<0.05).@*CONCLUSION@#MiR-203 targeting CREB1 mediated by DNA methylation leads to maintain the malignant biological behaviors of MM cells.

A Case-Control Study on Receptor Gene Polymorphism and Risk Suffering from Adult Acute Leukemia in Fujian Area / 中国实验血液学杂志

OBJECTIVE@#To investigate the correlation of receptor gene (P2X7, VDR and SLC19A1) polymorphisms with risk suffering from acute leukemia (AL) in Fujian area.@*METHODS@#Ninety-three cases of newly diagnosed AL as AL group and 90 persons not suffered from hematologic and other tumors as control group were selected and used for comparative analysis of receptor gene polymorphisms and risk suffering from AL between case and control groups. The bone marrow and peripheral blood were collected, from which the DNA was extracted. The PCR-RFLP was used to detect 8 SNP sites (P2X7: rs208294, rs2230911, rs3751143; VDR: rs2228570, rs7975232; SLC194A1: rs1051266, rs1131596, rs3788200) of receptor genes related with the environment response, and the genotypes analysis was used to the correlation of receptor gene polymorphisms with risk suffering from adult AL.@*RESULTS@#The unvariate logistic analysis showed that as compared with control group, P2X7 rs208294 T>C mutation and rs3751143 A>C mutation in codominant model, dominant model and over-dominant model were higher in case group, moreover the differences were statistically significant (PA mutation could increase the risk suffering from AL (PC mutation is one of protective factors against adult acute leukemia.

Oral Mucosal Diffuse Large B-cell Lymphoma Caused by Long-term Oral Methotrexate:Report of One Case / 中国医学科学院学报

A case of primary oral mucosal diffuse large B-cell lymphoma(DLBCL)due to long-term use of methotrexate(MTX)for the treatment of rheumatoid arthritis(RA)was admitted to the Department of Hematology,Fujian Medical University Union Hospital.We analyzed and discussed the clinical features,diagnosis and treatment,and prognosis of specific malignant lymphoma induced by MTX in this RA patient.Our purpose is to improve the awareness and knowledge of other iatrogenic immunodeficiency-associated lymphoproliferative disorders of clinicians and pathologists.This study provides a new reference for the clinical diagnosis and treatment of MTX-associated DLBCL.

The Effect of MicroRNA-143 on the Proliferation and Apoptosis of Leukemia Cell Line U-937 / 中国实验血液学杂志

OBJECTIVE@#To investigate the expression of microRNA-143 (miR-143) in patients with acute myeloid leukemia (AML), and the effect of miR-143 over-expression on the proliferation and apoptosis of AML cells. And to verify the targeting relationship between miR-143 and long non-coding RNA MALAT-1. So as to explore the possible regulatory mechanism of miR-143 in the pathogenesis of AML.@*METHODS@#The expression of miR-143 in bone marrow cells of AML patients was detected by RT-qPCR. After miR-143 was over-expressed in U-937 cell lines, the proliferation of U-937 cell lines was detected by CCK-8, clone formation assay, and flow cytometry. In addition, cell apoptosis was detected by Hoechst 33258 fluorescence staining and flow cytometry. At the same time, bioinformatics, RT-qPCR and dual luciferase reporter gene assay were used to predicted and verified the targeting relationship between miR-143 and MALAT-1.@*RESULTS@#Expression of miR-143 in AML patients was significantly lower than those in normal controls. Over-expressed miR-143 could inhibit the proliferation of U-937 cells and promote the apoptosis of the cell. The miR-143 binding site was located on the MALAT-1 RNA sequence, and MALAT-1 was down-regulated in U-937 cells after over-expressed miR-143. However, the expression of miR-143 showed no significantly changed after MALAT-1 silencing.@*CONCLUSION@#Expression of miR-143 in AML patients is lower than that in normal controls. Over-expression of miR-143 can inhibit the proliferation of U-937 cells and promote its apoptosis. And its mechanism may be related to its targe regulation of MALAT-1.

Research Advances on Abnormal Secretion and Function Abnormality of Exosomes Derived from Lymphoma Cells Caused by Latent EB Virus--Review / 中国实验血液学杂志

Abstract　　The Latent infection cansed by Epstein-Barr virus (EBV) closely relates with the occurrence and development of several kinds of lymphoma. Exosome (EXO) is functional bilayer membrane structural corpuscles which are secreted by cells contain proteins, lipids and nucleic acids. In recent years, researches showed that EXO play an important role in the occurrence and development of tumors. Therefore, the resenrches which compare the differences in quantity and contents of EXO secreted by cells between EBV negative lymphoma and EBV positive lymphoma and the clarify the influence of EXO on biological behaviors of lymphoma cells and immune cells have the important, significance for understanding the mechanisms related with effect of latent EBV on the occurrence and development of lymphoma by exosome pathway. This review focuses on research progress about the effect of latent EBV on amounts, contents and functions of EXO secreted by lymphoma cells.

Clinical features and prognosis of children with mature B-cell non-Hodgkin's lymphoma: an analysis of 28 cases / 中国当代儿科杂志

<p><b>OBJECTIVE</b>To study the clinical features and treatment outcome of children with mature B-cell non-Hodgkin's lymphoma (B-NHL).</p><p><b>METHODS</b>A total of 28 previously untreated children with mature B-NHL were enrolled and given the chemotherapy regimen of CCCG-B-NHL-2010. Among them, 20 were given rituximab in addition to chemotherapy. The children were followed up for 31 months (ranged 4-70 months). A retrospective analysis was performed for the clinical features of these children. The Kaplan-Meier method was used for survival analysis. A univariate analysis was performed to investigate the prognostic factors.</p><p><b>RESULTS</b>Among the 28 children, 17 (61%) had Burkitt lymphoma, 8 (29%) had diffuse large B-cell lymphoma (DLBCL), and 3 (11%) had unclassifiable B-cell lymphoma. As for the initial symptom, 13 (46%) had cervical mass, 10 (36%) had maxillofacial mass, 9 (32%) had hepatosplenomegaly, 5 (18%) had abdominal mass, and 5 (18%) had exophthalmos. Of all children, 14 had a lactate dehydrogenase (LDH) level of <500 IU/L, 3 had a level of 500-1 000 IU/L, and 11 had a level of ≥ 1 000 IU/L. After two courses of chemotherapy, 21 children achieved complete remission and 7 achieved partial remission. At the end of follow-up, 24 achieved continuous complete remission and 4 experienced recurrence. The 2-year event-free survival rate was (85.7± 6.6)%. The children with bone marrow infiltration suggested by bone marrow biopsy, serum LDH ≥500 IU/L, and bone marrow tumor cells >25% had a low 2-year cumulative survival rate.</p><p><b>CONCLUSIONS</b>The CCCG-B-NHL 2010 chemotherapy regimen combined with rituximab has a satisfactory effect in the treatment of children with B-NHL. Bone marrow infiltration on bone marrow biopsy is associated with poor prognosis.</p>

Effects of Triptolide on MMP-9 Expression and Inducing Apoptosis of Multiple Myeloma Cells by Inhibiting SMYD3 / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To investigate the effect of triptolide(TPL) on proliferation and apoptosis of RPMI8226 cells and its mechanism.</p><p><b>METHODS</b>MTT assay was used to measure the proliferation of RPMI8226 cells after treatment with different concentration (10, 20, 40, 80 and 160 nmol/L) of TPL for different incubation time (24 h, 48 h and 72 h). The cell apoptosis was detected by flow cytometry, the mRNA expressions of SMYD3 and MMP-9 were measured by quantitative real-time PCR, the protein level of H3K4me2 and H3K4me3 in RPMI8226 cells was assayed by Western blot.</p><p><b>RESULTS</b>TPL inhibited RPMI8226 cell proliferation, and the inhibitory rate of cell proliferation increased significantly in a dose- and time-dependent manner(P<0.05), the RPMI8226 cell apoptosis was induced by treatment with 40, 80 and 160 nmol/L TPL (P<0.05), the qRT-PCR showed that treatment of RPMI8226 cells with TPL down-regulated the mRNA expression of SMYD3 in a dose-dependent manner(P<0.05). Compared with the blank group, the mRNA expression level of MMP-9 in RPMI8226 cells transfected by siRNA-SMYD3 was significantly depressed. Western blot showed that the protein levels of H3K4me2 and H3K4me3 were decreased in a dose-dependent manner after TPL treatment(P<0.05). Compared with the blank group and siRNA negative group, the protein level of H3K4me2 and H3K4me3 in RPMI8226 cells transfected by siRNA-SMYD3 also were significantly depressed(P<0.05).</p><p><b>CONCLUSION</b>TPL can significantly inhibit the proliferation of RPMI8226 cells and induce their apoptosis, which may be related to the inhibition of SMYD3 expression by TPL- down-regulating the H3K4 methylation and the activating the MMP-9 transcription.</p>

Application of Bisulfite Sequencing PCR in Detecting the Abnormal Methylation of Suppressor Gene of Wnt Signaling Pathway in Acute Promyelocytic Leukemia / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To detect the abnormal methylation of the CPG island in the suppressor gene promoter region of the Wnt signaling pathway in cell strain NB4 of the acute promyelocytic leukemia by using the bisulfite sequcucing PCR(BSP), to screan the hyper-methylated suppressor gene of the Wnt signaling pathway and to evaluatc the potency of BSP in the methylation study.</p><p><b>METHODS</b>The strain NB4 cells of the acute promyelocytic leukemia patients were used as the object, the mononuclear cells from 20 normal persons were used as the controls. The DNA was extracted and processed by bisulfite, the target sequences were amplified with PCR, then the abnormal methylation of the suppressor genes of the Wnt signaling pathway in the NB4 cells was analyzed by BSP, and the advantage and disadvantage of BSP were evaluated by comparison with the Methylation specific PCR and Pyrosequencing.</p><p><b>RESULTS</b>The methylated rate of suppressor genes of the Wnt signaling pathways in the NB4 cells detected by BSP was as follows: the gene WIF1 95.26%, the gene DKK3 86%, the gene SFRP1 81.67%, the gene SFRP2 95.71%, the gene SFRP4 85%, and the gene SFRP5 95%; while the methylations in the control group were respectively as follows: the gene WIF-1 1.5%, the gene DKK3 4.2%, the gene SFRP1 0%, the gene SFRP2 0.9%, the gene SFRP4 2.5%, and the gene SFRP5 1.75%. A more significant methylation happened in the suppressor genes promoter of the Wnt signaling pathway in the NB4 cells as compared with the control group.</p><p><b>CONCLUSION</b>Many hypermethylated suppressor genes are found in the Wnt signaling pathway of the acute promyelocytic leukemia NB4 cells, which may be served as one of the early diagnosis index and therapeutic target of the acute promyelocytic leukemia.</p>

MicroRNA-193b expression in newly diagnosed leukemia patients and its significance / 中国实验血液学杂志

This study was aimed to quantitatively detect the levels of microRNA-193b (miR-193b) in leukemia patients and explore its significance. Real time fluorescent quantitative PCR was used to detect the relative expression level of miR-193b. The expression changes of miR-193b in various types of leukemia were analyzed. Then the relationship among miR-193b expression, parts of laboratory index and the response to chemotherapy was analyzed as well. The results showed that miR-193b expression level in acute promyelocytic leukemia (APL) and chronic myeloid leukemia (CML) patients was not lower than that in normal group (P > 0.05). Except for APL, miR-193b expression level in acute myeloid leukemia (AML) patients was lower than that in normal group (P < 0.05). In AML (except for APL) patients, there was no correlation between white blood cell count (P > 0.05), the expression of CD34 (P > 0.05) and miR-193b expression level, but there was negative correlation between chemotherapy response and miR-193b expression level (P < 0.05). It is concluded that miR-193b expression level may be correlated with susceptibility of cells to chemotherapy in AML (except for APL) patients. miR-193b maybe become a new target in AML (except for APL) therapy.

Expression of Hippo signaling pathway core element MST1 in acute leukemia patients and its significance / 中国实验血液学杂志

This study was aimed to investigate the expression of Hippo signaling pathway core element MST1 gene in acute leukemia (AL) patients, and explore its relation with AL pathogenesis and prognosis. 50 newly diagnosed patients with AL, 33 normal people, 23 patients with AL in complete remission, 12 refractory or relapsed patients were tested for the expression of MST1 gene by real-time quantitative PCR, Western blot was used to further validate the level of MST1 protein expression. And combined with clinical data, prognostic factors of the patients were analyzed. The results indicated that compared with the normal people, the expression level of MST1 in newly diagnosed patients with AL significantly decreased (P < 0.05), but significantly increased in AL patients with complete remission (CR), the difference of expression was statistically significant before CR and after CR (P < 0.05). Compared with refractory or relapsed patients, the expression level of MST1 gene in newly diagnosed patients was not significantly different (P > 0.05). Besides, the expression level of MST1 between the patients with CR and the normal people was not significantly different (P > 0.05). It is concluded that the low expression of MST1 may be related with the pathogenesis and prognosis of AL.

Differential expression of miR-143 in bone marrow cells between acute leukemia patients and normal people and its significance / 中国实验血液学杂志

MicroRNA abnormality is closely related to the development of acute leukemia. This study was aimed to investigate the differential expression of miR-143 in bone marrow cells between patients with acute leukemia and normal people. Bone marrow cells from 50 AL patients and 20 normal people were collected respectively and the total RNA was isolated routinely. The quantitative real-time PCR (Q-PCR) method was used to detect the expression levels of miR-143 in these specimens. The results showed that compared with the normal people, the expression of miR-143 in AL patients was significantly decreased (p < 0.05), which significantly increased after complete remission; besides, the expression of miR-143 was negatively correlated with the expression of DNMT3A mRNA, a known target gene of miR-143. It is concluded that the expression of miR-143 in bone marrow cells of AL patients is down-regulated which may be related with the development of acute leukemia.

Methylation status of IEX-1 gene promotor CpG island in malignant hematopoietic cell lines / 中国实验血液学杂志

This study was to purposed to explore the methylation status changes of IEX-1 gene promoter CpG island and its relevance with occurrence of hematologic malignancies. The methylation status of IEX-1 gene promoter CpG island in 9 NB4, HL-60 U937, Raji, CA46, Jurkat, K562, CEM and Molt4 hematologic malignant cell lines was detected by using methylation-specific PCR, the methylation status of IEX-1 gene promoter CpG island in normal peripheral blood mononuclear cells treated by M. sssI enzyme and the methylation status of IEX-1 gene promoter CpG island in normal peripheral blood mononuclear cells untreated were used as positive and negative controls respectively. The results showed that the hypermethylation of IEX-1 gene promoter CpG islands was detected in NB4, Molt4 and Raji cell lines, as well as in normal peripheral blood mononuclear cells treated by M. sssl enzyme; the partial methylation status was found in CA46, CEM, U937, K562, HL-60 and Jurkat cell lines; the unmethylation status was observed in untreated normal peripheral blood mononuclear cells. It is concluded that the changes of methylation status of gene IEX-1 promoter CpG island correlates with hematologic malignancies to a certain extent.

Effect of triptolide on reversing hypermethylation of apc gene in Jurkat cells and its possible mechanisms / 中国实验血液学杂志

This study was aimed to investigate the effect of traditional Chinese medicine, Triptolide (TPL) on reversing hypermethylation of antioncogene (apc gene) in acute lymphoblastic leukemia cell line Jurkat in vitro and to explore its mechanisms. The effects of TPL on cell growth, proliferation and cell cycle were detected by growth curve, MTT assay, colony formation test and flow cytometry, respectively. The effect of TPL on apc gene methylation of Jurkat cells was analyzed by nested methylation specific PCR; the expressions of apc gene, dnnt3a, dnmt3b mRNA were measured by RT-PCR; the protein expression of apc gene was detected by Western blot. The results showed that as compared with untreated control cells, the TPL of different concentrations could significantly inhibit growth and proliferation of Jurkat cells in dose-and time-dependent manners with IC₅₀ 19.7 ng/ml at 48 hours. All cytosines in CpG dinucleotides in untreated Jurkat cells had no changed, while all cytosines in Jurkat cells treated with TPL had been converted to thymidine suggesting the methylation of apc gene in Jurkat cells. The TPL could reverse hypermethylation of apc gene and induced the mRNA and protein expression of apc gene in dose-dependent manner. It is concluded that the small dose of TPL can obviously suppress the proliferation of Jurkat cells, activate and up-regulate the expression of apc gene through demethylation of apc gene resulting from DNMT and/or direct action, thereby inhibit the proliferation rate of Jurkat cells.

Effect of epigallocatechin-3-galate on human acute monocytic leukemia cell line U937 and its relevant mechanism / 中国实验血液学杂志

The purpose of this study was to explore the effect of epigallocatechin-3-galate (EGCG) on acute monocytic leukemia cell line U937 and its relevant mechanism. The viability of U937 cells were assayed by SRB method. The cell cycle of U937 cells was analyzed by flow cytometry. The mRNA and protein expression of p16 gene were detected by RT-PCR and Western blot, respectively. Methylation level of U937 cells was analyzed by n-MSP. The mRNA expression of DNA methyltransferase 1 (DNMT1), DNMT3A and DNMT3B genes were analyzed by RT-PCR. The results showed that EGCG could inhibit the growth of U937 cells significantly in dose-and time-dependent manners (r=0.71), and induce the G0/G1 arrest of U937 cells in dose-dependent manner. EGCG could up-regulate the mRNA and protein expression of P16 gene in U937 cells in dose-dependent manner. EGCG could down-regulate the methylation level of p16 gene in U937 cells in dose-dependent manner. EGCG could down-regulate the mRNA expression of DNMT3A, DNMT3B genes, while did not influence the mRNA expression of DNMT1 gene. It is concluded that EGCG can up-regulate the mRNA and protein expression of p16 gene by demethylation or/and by inhibiting DNMT3A and DNMT3B genes, leading, in turn, to G0/G1 arrest and growth inhibition of U937 cells.

Arsenic trioxide reverses hypermethylation of p16 and activates its transcription in malignant lymphoma cell line CA46 / 中国实验血液学杂志

This study was aimed to investigate the reversing effect of arsenic trioxide (As2O3) on methylation status and the regulatory effect on transcription of malignant lymphoma cell line CA46 p16 gene as well as their possibe mechanisms. The hypermethylated malignant lymphoma cell line CA46 was used as a subject of experiment for studying relation of gene methylation with expression. The effect of As2O3 on the proliferation and viability of CA46 was detected by SRB method, the change of p16 methylation status after exposure to As2O3 was determined by nMSP, the expressions of p16, DNMT1, DNMT3A, DNMT3B mRNA were assayed by RT-PCR, the influence of As2O3 on CA46 cell cycle was analyzed by flow cytometry using analytical method for DNA ploidy. The results showed that the methylation level of p16 gene was obviously reduced after treatment with As2O3 for 72 hours and the hypermethylation of p16 gene was successfully reversed; the expression of p16 gene in untreated (control) group was low while it was enhanced in treated groups; the gray scale ratios of p16 gene to beta-actin in groups treated with As2O3 of concentration 0.5, 1.0 and 2.0 micromol/L were 0.33+/-0.10, 0.57+/-0.11 and 0.67+/-0.09 respectively, exhibiting a significant difference in comparison with 0.73+/-0.13 of positive control (p<0.01); as compared with the untreated group, the expression of DNMT3A and DNMT3B in treated groups was obviously down-regulated in a concentration-dependent manner, while expression of DNMT1 was nearly unchanged; as compared with control, all the 3 different concentrations of As2O3 could inhibit the proliferation of CA46 cells and increase the cell number in G0/G1 phase. It is concluded that the As2O3 may up-regulate the expression of p16 gene, recover the activity of p16 gene, thereby promote the regulatory function on cell cycle resul-ting in arrest of cells in G0/G1 phase and inhibit growth of tumor cells through depressing the expression of DNMT3A and DNMT3B and/or directly reversing the methylation status of p16 gene.

Inhibitory effect of VPA on multiple myeloma U266 cell proliferation and regulation of histone acetylation / 中国实验血液学杂志

This study was aimed to investigate the effects of sodium valproate (VPA) on the proliferation and regulation of histone acetylation of multiple myeloma cell line U266. U266 cells were treated with VPA. Cell proliferation was determined by CCK-8 assay, and cell cycle were analyzed by flow cytometry (FCM). The expression level of HDAC1 mRNA was detected by RT-PCR, and the protein levels of HDAC1 and histone H3, H4 acetylation was detected by Western blot. The results showed that the VPA inhibited the proliferation of U266 cells in concentration-and time-dependent manners.After exposure to different concentrations of VPA for 48 hours, the proportion of G(0)/G(1) cells increased, while the proportion of S phase cells decreased. The cell cycle was arrested obviously in G(0)/G(1) phase (p < 0.05). The expression of HDAC1 mRNA was inhibited, and the protein level of HDAC1 was down-regulated, while the histone H3/H4 acetylation was up-regulated in U266 cells. It is concluded that the VPA can inhibit cell proliferation of U266 and induce G(0)/G(1) phase arrest. The increase of histone H3/H4 acetylation resulting from inhibiting HDAC1 by VPA might be considered as a possible mechanism.

Mechanism of As(2)O(3) on hdpr1 gene demethylation in Jurkat cell line / 中国实验血液学杂志

This study was purposed to investigate the effect of As(2)O(3) on the demethylation of anti-oncogene-hdpr1 gene of acute lymphoblastic leukemia cell line Jurkat in vitro and its mechanism. The inhibitory effect of As(2)O(3) on the proliferation of Jurkat cells was assayed by CCK-8; the change of Jurkat cell cycle was detected by flow cytometry before and after using As(2)O(3); the effect of As(2)O(3) on the methylation model of hdpr1 gene was analyzed by methylation-specific PCR, and the effect of As(2)O(3) on the expression of hdpr1 mRNA was analyzed by semiquantitative RT-PCR. The results showed that the proliferation rate of Jurkat cells was decreased significantly after being treated with As(2)O(3), and in dose-and time-dependent manner; As(2)O(3) blocked Jurkat cell cycle in G(0)/G(1) phase in dose-dependent manner. As(2)O(3) could reverse hypermethylation of hdpr1 gene and induce its mRNA reexpression, and down-regulate the dnmt1, dnmt3a, dnmt3b mRNA expression level also in dose-dependent manner. It is concluded that the As(2)O(3) suppresses the proliferation of Jurkat cells and blocks cell cycle is G(0)/G(1), its possible mechanism may be down-regulating mRNA expression level of dnmt1, dnmt3a and dnmt3b, induce demethylation of hdpr1 gene from abnormal hypermethylation status and activates its reexpression.