Effect of dimethyl fumarate on rats with chronic pancreatitis.

OBJECTIVE: To discuss the effect of dimethyl fumarate (DMF) on rats with l-arginine induced chronic pancreatitis (CP). METHODS: Male Wistar rats were given DMF treatment (25 mg/kg) by oral lavage method; then Wistar rats were given the intraperitoneal injection of l-arginine for 5 times (250 mg/100 kg, twice per time, each interval of 1 h) for building of CP model. Rats were divided into control group, CP group, DMF group and CP + DMF group. Rats in CP + DMF group were given the oral intragastric administration of DMF (25 mg/kg), while rats in control group and CP group were given the equal volume of normal saline. The weight of rats was evaluated and the intraperitoneal glucose tolerance test was performed (IPGTT, 2 g/kg). The islet of rats was isolated and then flow cytometry was employed to evaluate the quality and activity of islets. Meanwhile, the histology of non-endocrine tissues and levels of myeloperoxidase (MPO) and malondialdehyde (MDA) were detected. RESULTS: Compared with control group, the weight of rats in CP group was significantly reduced at week 2, 4 and 6; the blood glucose significantly increased, AUC increased, the histopathological scores of pancreatic atrophy, acinar injury, edema and cellular infiltration increased, levels of MDA and MPO increased, the islet equivalent and islet activity decreased at 0, 30, 60, 120 and 180 min. Compared with CP group, the weight of rats in CP + DMF group significantly increased at week 2, 4 and 6; the blood glucose significantly decreased, AUC decreased, the histopathological scores of pancreatic atrophy, acinar injury, edema and cellular infiltration decreased, levels of MDA and MPO decreased, the islet equivalent and islet activity increased at 0, 30, 60, 120 and 180 min. CONCLUSIONS: DMF treatment can improve CP induced by l-arginine and islet function in rats.

[Effects of acute myeloid leukemia cell supernatant on the proliferation and apoptosis of CD4+ and CD8+ T cell subsets].

To study the effects of supernatant derived from acute myeloid leukemia (AML) cell lines on proliferation and apoptosis of CD4(+) and CD8(+) T cell subsets and to investigate the mechanism by which AML escapes from immune recognition, lymphocytes were labeled with CFSE and were stimulated with anti-CD3 and anti-CD28 in presence or absence of supernatants from three AML cell lines (HL-60, NB4, U937). After culture, cell suspensions were labeled with 7AAD and CD4 PE (or CD8 PE). Cells were then detected by flow cytometry and their proliferation and apoptosis were analyzed. The results showed that supernatants from two of three cell lines (HL-60 and NB4) inhibited the proliferation of CD4(+) and CD8(+) T cells, and the degree of inhibition showed a dose-dependent way. Similarly, the apoptosis of stimulated CD4(+) T cells was inhibited, but stimulated CD8(+) T cells remained unaffected by supernatant from HL-60 and NB4. In contrary, the apoptosis of proliferative CD8(+) T cells were increased significantly by HL-60 and NB4 supernatant. It is concluded that soluble factors derived from AML cell lines inhibit the proliferation of CD4(+) and CD8(+) T cells and induce the apoptosis of proliferative CD8(+) T cells, that may be one of the mechanisms by which the immunity was suppressed.

[Correlation of immunophenotype to cytogenetics and clinical features of adult acute myeloid leukemia].

BACKGROUND & OBJECTIVE: New WHO classification has been rapidly used in diagnosis of leukemia. Based on coexpression and correlation of lineage-associated antigens, multiparameter high-resolution flow cytometry has been developed to precisely identify lineage characteristics of leukemia. Some immunophenotypes correlate with cytogenetic abnormality and prognosis. This study was to analyze immunophenotype of naive acute myeloid leukemia (AML), and explore its correlations to cytogenetics, clinical features, and FAB subtype of AML. METHODS: Multiparameter high-resolution flow cytometry with a panel of 25 different monoclonal antibodies was used to analyze the surface and cytoplasmic antigens expressions of 96 adults with AML; G-binding technique was used to analyze karyotype of 73 of the 96 patients. RESULTS: In these AML patients, some antigens were correlated with FAB subtypes:expression of CD2 was enhanced in AML-M3; HLA-DR, CD34, and CD56 were absent in AML-M3; expression of CD19 was increased in AML-M2; expressions of CD14 and CD56 were enhanced in AML-M5; MPO was absent in AML-M0. Karyotype abnormality was detected in 40(54.8%) patients. CD22, CD56, and TdT expressions were correlated with karyotype abnormality. t(8; 21) was only detected in 10 AML-M2 patients with high expressions of CD15, CD19, CD34, and CD56; no lymphoid lineage antigens were detected in 7 AML-M3 patients with t (15; 17). Expressions of CD4 and TdT were positively correlated with patient's age; expressions of CD7 and CD14 were positively correlated with high white blood cell count; expressions of CD4, CD14, and CD56 were positively correlated with high platelet count. CONCLUSIONS: The abnormal antigen expression of AML is tightly linked with karyotype abnormality. Detection of immunophenotype may help to diagnose and classify AML.

[Effect of curcumin on STAT5 signaling pathway in primary CML cells].

To study the effect of curcumin on signaling pathway of signal transducers and activators of transcription (STAT5) in primary newly-diagnosed chronic myelocytic leukemia (CML) cells, and to explore the clinical significance of curcumin in the treatment of primary CML cells, the cells were randomly divided into 3 groups: normal control group, CML cells group, and curcumin group; the cellular proliferation was assayed by MTT test; the expression of cellular STAT5 mRNA in CML cells was detected by RT-PCR; the activation of STAT5 in CML cell was detected by electrophoretic mobility shift assay (EMSA). The results showed that the cellular proliferation of curcumin group (OD value 0.640 +/- 0.073) was decreased, as compared with that of the CML cells group (OD value 0.856 +/- 0.083, P <0.01). The expression levels of STAT5 mRNA in CML cells group (integral ratio of OD 1.782 +/- 0.156) were significantly greater than that in the normal control group (integral ratio of OD 0.289 +/- 0.025, P <0.01). The expression of STAT5 mRNA in curcumin group (integral ratio of OD 1.398 +/- 0.126) was significantly decreased as compared with that in the CML cells group (P <0.01). The activation of STAT5 was significantly increased in CML cells group (gray value 5323.375 +/- 515.640) as compared with that in the normal control group (gray value 2943.000 +/- 273.377, P <0.01). The activation of STAT5 of curcumin group (gray value 4331.750 +/- 398.035) was significantly decreased as compared with that of CML cells group (P <0.01). It is concluded that the cellular proliferation and the expression of STAT5 mRNA are increased in the primary CML cells. The activation of STAT5 in primary CML cells is markedly enhanced. STAT5 signaling pathway may be involved in the proliferation of primary CML cells. Curcumin can inhibit the cellular proliferation and the expression of STAT5 mRNA, and down-regulate the activation of STAT5 in primary CML cells. Curcumin may be used in treatment of leukemia.

[Effect of trichostatin A on histone acetylation level and apoptosis in HL-60 cells].

In order to explore the underlying mechanism of high effects and low toxicity of trichostatin A (TSA), the effect of TSA on growth inhibition, histone acetylation level and apoptosis in HL-60 cells and normal human peripheral blood mononuclear cells (NPBMNC) were examined using MTT method, immunocytochemistry technology, and Annexin-V-FITC/PI double staining flow cytometry. The results showed that TSA inhibited growth of HL-60 in time- and dose-dependent manners, and the IC(50) of 36 hours was 100 ng/ml. The apoptosis induction effect of TSA in HL-60 cells was also time- and dose-dependent. Besides, the dose of TSA showing significant apoptotic cytotoxicity in HL-60 cells did not demonstrate apparent apoptosis induction in NPBMNC within definite dose and time range. The histone acetylation level in HL-60 cells and NPBMNC both showed remarkable increase (P < 0.05) after incubated with 100 ng/ml TSA for 4 hours without statistical difference between them is detected (P > 0.05). It is concluded that TSA shows effects of definite and significant growth inhibition and apoptosis induction on HL-60 cells in time- and dose-dependent manners. TSA is able to selectively induce apoptosis in HL-60 cells with low toxicity in NPBMNC at the same time. The mechanism of this selectivity can not be ascribed to the differential regulation of histone acetylation level between HL-60 cells and NPBMNC.

[Effect of curcumin on STAT5 signaling molecule in K562 cells].

OBJECTIVE: To investigate the effect of curcumin on STAT 5 signaling molecule in K562 cells and its molecular mechanism of antileukemia. METHODS: Cell proliferation was studied by tetrazolium dye assay. The expressions of STAT5 mRNA and protein were assayed by in situ hybridization, and Western blotting respectively. RESULTS: Curcumin could inhibit K562 cell proliferation in a time- and dose-dependent manner. The percentage of STAT5-positive cells was 19% in curcunin group, significantly less than 31% of that in K562 cell group (P < 0.01). The A value of the expression level of STAT5 protein in curcumin group was 15 266 +/- 769, significantly less than 25 781 +/- 1240 of that in K562 cell group (P < 0.01). CONCLUSION: The expressions of STAT5 mRNA and protein in K562 cells were inhibited by curcumin and curcumin could inhibit K562 cell proliferation.

Interaction with general transcription factor IIF (TFIIF) is required for the suppression of activated transcription by RPB5-mediating protein (RMP).

RMP was reported to regulate transcription via competing with HBx to bind the general transcription factor IIB (TFIIB) and interacting with RPB5 subunit of RNA polymerase II as a corepressor of transcription regulator. However, our present research uncovered that RMP also regulates the transcription through interaction with the general transcription factors IIF (TFIIF), which assemble in the preinitiation complex and function in both transcription initiation and elongation. With in vitro pull-down assay and Far-Western analysis, we demonstrated that RMP could bind with bacterially expressed recombinant RAP30 and RAP74 of TFIIF subunits. In the immunoprecipitation assay in COS1 cells cotransfected with FLAG-tagged RMP or its mutants, GST-fused RAP30 and RAP74 were co-immunoprecipitated with RMP in approximately equal molar ratio, which suggests that RAP30 and RAP74 interact with RMP as a TFIIF complex. Interestingly both RAP30 and RAP74 interact with the same domain (D5) of the C-terminal RMP of 118-amino-acid residuals which overlaps with its TFIIB-binding domain. Internal deletion of D5 region of RMP abolished its binding ability with both subunits of TFIIF, while D5 domain alone was sufficient to interact with TFIIF subunits. The result of luciferase assay showed that overexpression of RMP, but not the mutant RMP lacking D5 region, suppressed the transcription activated by Gal-VP16, suggesting that interaction with TFIIF is required for RMP to suppress the activated transcription. The interaction between RMP and TFIIF may be an additional passway for RMP to regulate the transcription, or alternatively TFIIF may cooperate with RPB5 and TFIIB for the corepressor function of RMP.