To establish a basis for deep investigation of the role of microRNA (miRNA) in the regulation of hematopoiesis, differential expression profiles of miRNA between human cord blood CD34(+)CD38(-) and CD34(+)CD38(+) cells were analyzed. Mononuclear cells from cord blood (CB) of healthy donors were separated by Ficoll-Hypaque density gradients. CD34(+)CD38(-) and CD34(+)CD38(+) cells were sorted by using FACS Vantage SE. Their mRNA were then extracted and hybridized to miRNA microarray chip. The resulting data were analyzed with GeneSpring and informatics technique. The results showed that eleven miRNAs were found to be downregulated and 73 miRNAs to be upregulated by at least two-fold in the CD34(+)CD38(+) cells of CB, compared with the CD34(+)CD38(-) cells, which maintained CD34(+)CD38(-) cells' self-renewal and multiple lineage potential, that were defined as "stemness" miRNAs. 12 of the 84 genes (14.29%) were common to 33 hematopoietic-expressed miRNAs expressed by CD34(+) cells from both peripheral blood and bone marrow in Georgantas's study, which included 10 upregulated miRNAs (hsa-miR-23b, -26b, -92, -107, -130a, -181a, -197, -213, -222, -223) and 2 downregulated ones (hsa-miR-16a, -155). Some "stemness" miRNAs undergo CD34 antigen-like expression pattern during development and commmitted differeniation of hematopoietic stem cell/progenitors. Hematopoiesis-associated miRNA clusters and putative target genes could be found with informatics technique. It is concluded that the hematopoietic "stemness" miRNAs play important roles in normal hematopoiesis: miRNA expression profiles of hematopoietic stem cell/progenitors --> their gene expression profiles --> their self-renewal and lineage-commmitted differeniation.
ADP-ribosyl Cyclase 1 ; immunology ; Antigens, CD34 ; immunology ; Fetal Blood ; immunology ; metabolism ; Gene Expression Profiling ; Hematopoietic Stem Cells ; cytology ; immunology ; physiology ; Humans ; MicroRNAs ; genetics ; metabolism

ADP-ribosyl cyclase (ADPR-cyclase) produces a Ca2+-mobilizing second messenger, cyclic ADP- ribose (cADPR), from beta-NAD+. A prototype of mammalian ADPR-cyclases is a lymphocyte antigen CD38. Accumulating evidence indicates that ADPR-cyclases other than CD38 are expressed in various cells and organs. In this study, we discovered a small molecule inhibitor of kidney ADPR-cyclase. This compound inhibited kidney ADPR-cyclase activity but not CD38, spleen, heart or brain ADPR-cyclase activity in vitro. Characterization of the compound in a cell-based system revealed that an extracellular calcium-sensing receptor (CaSR)- mediated cADPR production and a later long-lasting increase in intracellular Ca2+ concentration ([Ca2+]i) in mouse mesangial cells were inhibited by the pre-treatment with this compound. In contrast, the compound did not block CD3/TCR-induced cADPR production and the increase of [Ca2+]i in Jurkat T cells, which express CD38 exclusively. The long-lasting Ca2+ signal generated by both receptors was inhibited by pre-treatment with an antagonistic cADPR derivative, 8-Br-cADPR, indicating that the Ca2+ signal is mediated by the ADPR-cyclse metabolite, cADPR. Moreover, among structurally similar compounds tested, the compound inhibited most potently the cADPR production and Ca2+ signal induced by CaSR. These findings provide evidence for existence of a distinct ADPR-cyclase in the kidney and basis for the development of tissue specific inhibitors.
Receptors, Calcium-Sensing/metabolism ; Rats, Sprague-Dawley ; Rats ; Mice ; Kidney/*enzymology ; Humans ; Enzyme Inhibitors/chemistry/*pharmacology ; Cyclic ADP-Ribose/*metabolism ; Cell Line ; *Calcium Signaling ; Azo Compounds/chemistry/*pharmacology ; Animals ; ADP-ribosyl Cyclase/*antagonists & inhibitors/*metabolism

Leukemia seems to depend on a small population of "leukemia stem cells (LSCs)" for its growth and metastasis. However, the precise surviving mechanisms of LSCs remain obscure. Cellular senescence is an important obstacle for production and surviving of tumor cells. In this study we investigated the activated state of a pathway, in which reactive oxygen species (ROS) induces cellular senescence through DNA damage and phophorylation of p38 MAPK (p38), in myeloid leukemic CD34(+)CD38(-) cells. Bone marrow samples were obtained from patients with acute myeloid leukemia (AML, n=11) and chronic myeloid leukemia (CML, n=9). CD34(+)CD38(-) cells were isolated from mononuclear cells from these bone marrow samples, and K562 and KG1a cells (two kinds of myeloid leukemia cell lines) by mini-magnetic activated cell sorting. Hematopoietic stem cells (HSCs) from human cord blood served as controls. Intracellular ROS level was detected by flow cytometry. DNA damage defined as the γH2AX level was measured by immunofluorescence staining. Real-time RT-PCR was used to detect the expression of p21, a senescence-associated gene. Western blotting and immunofluorescence staining were employed to determine the p38 expression and activation. The proliferation and apoptosis of CD34(+)CD38(-) cells were detected by MTT assay and flow cytometry. Our results showed that ROS and DNA damage were substantially accumulated and p38 was less phosphorated in myeloid leukemic CD34(+)CD38(-) cells as compared with HSCs and H(2)O(2)-induced senescent HSCs. Furthermore, over-phosphorylation of p38 by anisomycin, a selective activator of p38, induced both the senescence-like growth arrest and apoptosis of CD34(+)CD38(-) cells from K562 and KG1a cell lines. These findings suggested that, although excessive accumulation of oxidative DNA damage was present in LSCs, the relatively decreased phosphorylation of p38 might help leukemic cells escape senescence and apoptosis.
ADP-ribosyl Cyclase 1 ; metabolism ; Antigens, CD34 ; metabolism ; Cellular Senescence ; Female ; Humans ; Leukemia, Myeloid, Acute ; enzymology ; pathology ; Male ; Neoplastic Stem Cells ; metabolism ; pathology ; Oxidative Stress ; Phosphorylation ; Reactive Oxygen Species ; metabolism ; Tumor Cells, Cultured ; p38 Mitogen-Activated Protein Kinases ; metabolism

ADP-ribosyl Cyclase 1 ; metabolism ; Aged ; Epstein-Barr Virus Infections ; Herpesvirus 4, Human ; isolation & purification ; Humans ; Interferon Regulatory Factors ; metabolism ; Ki-67 Antigen ; metabolism ; Male ; Nasal Cavity ; Nose Neoplasms ; metabolism ; pathology ; surgery ; virology ; Plasmacytoma ; metabolism ; pathology ; surgery ; virology

OBJECTIVETo investigate the expression of Notch1 on the membrane of bone marrow CD38(+)CD138(+) plasma cells in the patients with multiple myeloma (MM), and explore the importance of Notch signaling pathway in the formation and progression of MM.

METHODSThirty three MM patients and 15 healthy controls were enrolled in this study. The expression of Notch1 on the membrane of bone marrow CD38(+)CD138(+) and CD38(+)CD138(-) plasma cells were analyzed by flow cytometry. The clinical data of MM patients were also analyzed.

RESULTSThe ratio of Notch1 on the membrane of CD38(+)CD138(+) plasma cells of MM patients was (60.21 ± 25.06)% which was significantly higher than those of CD38(+)CD138(-) plasma cells of MM patients (39.84 ± 18.94)% (P = 0.000) and controls (38.34 ± 19.39)% (P = 0.004). There was no statistical difference between the two latter groups (P > 0.05). The expression of Notch1 on CD38(+)CD138(+)plasma cells from 24 newly diagnosed MM patients was correlated to the level of malignant plasma cells in there bone marrow (r = 0.914, P = 0.000), serum level of lactate dehydrogenase (LDH) (r = 0.754, P = 0.007), and β(2)-MG(r = 0.716, P = 0.013). The ratio of Notch1 on the membrane of CD38(+)CD138(+) plasma cells of MM patients who had renal dysfunction was correlated to their abnormal serum creatinine levels. The expression of Notch1 on CD38(+)CD138(+) plasma cells from 17 MM patients who received VD (bortezamib and dexamethasone) chemotherapy was correlated to the ratio of plasma cell reduction after the first VD chemotherapy (r = 0.842, P = 0.000).

CONCLUSIONThe expression of Notch1 on the membrane of CD38(+)CD138(+) plasma cells of MM patients was significantly higher than those of CD38(+)CD138(-) plasma cells of MM patients and controls. Notch1 overexpressed plasma cells were sensitive to the early VD therapy, and correlated to the progression and long term outcome of MM.

ADP-ribosyl Cyclase 1 ; immunology ; Adult ; Aged ; Aged, 80 and over ; Bone Marrow ; metabolism ; Case-Control Studies ; Cell Count ; Female ; Humans ; Male ; Middle Aged ; Multiple Myeloma ; immunology ; metabolism ; Plasma Cells ; immunology ; metabolism ; Prognosis ; Receptor, Notch1 ; metabolism ; Syndecan-1 ; immunology

OBJECTIVETo investigate CLLU1 expression and its relationship with clinical stage, expression of CD38 and ZAP-70, and chromosome abnormalities in patients with chronic lymphoid leukemia (CLL).

METHODSFifty CLL patients were studied. Semiquantitative RT-PCR was performed to detect CLLU1 expression levels; three-color flow cytometry to detect the ZAP-70 and CD38 expression, interphase fluorescence in situ hybridization (FISH) to detect chromosomal aberrations.

RESULTSThe expression of CLLU1 mRNA was detected in 26 of the 50 cases (52%), of them 7 cases (26.92%) in Binet A and 19 (73. 08%) in Binet B + C. The expression levels of CLLU1 were significantly higher in Binet stage B + C than in Binet stage A (P < 0. 01). Among 24 CD38+ CLL cases, 17 (70. 83%) expressed high CLLU1 mRNA, whereas in 26 CD38- CLL patients only 9 (34.62%) were CLLU1 positive. The expression of CLLU1 in CD38+ CLL was significantly higher than that in CD38- CLL (P < 0.05). However, no significant difference of CLLU1 expression was found between ZAP-70+ and ZAP-70- patients (P > 0. 05), and between chromosomal aberrations (P > 0. 05).

CONCLUSIONSCLLU1 expression was significantly associated with clinical stage and CD38 expression, and might be an important prognostic factor in CLL patients.

ADP-ribosyl Cyclase 1 ; metabolism ; Adult ; Aged ; Female ; Humans ; Leukemia, Lymphocytic, Chronic, B-Cell ; genetics ; metabolism ; pathology ; Male ; Middle Aged ; Neoplasm Proteins ; metabolism ; Neoplasm Staging ; Prognosis ; ZAP-70 Protein-Tyrosine Kinase ; metabolism

This study was aimed to explore the prognostic significance of telomere length in patients with chronic lymphocytic leukemia (CLL) and to analyze relation of telomere length with Binet stage, IgVH mutation status, CD38, ZAP-70 expression as well as other clinical features. 35 CLL patients who contained 80% or more tumor cells in the peripheral blood or bone marrow samples were selected as objects studied, while 13 healthy donors were served as normal controls. The telomere relative length was detected by using a real-time fluorescent quantitative polymerase chain reaction method (qPCR); the expression of CD38 and ZAP-70 protein were detected by flow cytometry, the IgVH mutation was detected by multiplex PCR. The results showed that the mean telomere relative length in CLL patients and normal controls were 0.384 and 0.443 respectively, but the difference between them was not significant (p > 0.05). The telomere length was significantly correlated with Binet stages and IgVH mutation status. Patients in Binet stage B and C showed significantly shorter telomeres than those in Binet stage A (p = 0.001). Mean telomere relative lengths in patients without IgVH mutation were shorter than those in patients with IgVH mutation (p = 0.015). No relation of telomere length with sex, age, ZAP-70 protein and CD38 were found (p > 0.05). It is concluded that telomere length may have a prognostic significance for CLL patients. Combining telomere length and IgVH mutation status may achieve a better prognostic subclassification for CLL patients.
ADP-ribosyl Cyclase 1 ; metabolism ; Adult ; Aged ; Aged, 80 and over ; Case-Control Studies ; Female ; Humans ; Leukemia, Lymphocytic, Chronic, B-Cell ; genetics ; metabolism ; Male ; Middle Aged ; Mutation ; Prognosis ; Telomere ; chemistry ; genetics ; metabolism ; ZAP-70 Protein-Tyrosine Kinase ; metabolism

This study was aimed to analyze the clinical and laboratorial characteristics of patients with chronic lymphocytic leukemia (CLL), as well as their relationship with outcomes of patients. The clinical and laboratorial data of 40 CLL patients admitted from 2004 to 2010 in our hospital were analyzed retrospectively. The results indicated that the most of CLL attacked the elderly male patients with median age 66 (from 42 to 80). Flow cytometric analysis showed that 25 cases were positive for typical immunophenotype of CLL. On the other hand, all the patients clearly expressed CD19 and CD5, 7 cases (17.5%) and 14 cases (35%) were positive for the expression of CD38 and Zap70 respectively. 8 cases harbored a mutated immunoglobulin heavy-chain (VH) gene, among them 4 cases belong to VH3 family. Interphase FISH analysis showed that P53 deletion, RB1 deletion, trisomy 12 and normal chromosome were detected in 6, 3, 1, and 5 cases, respectively. The median PFS in 31 patients received treatment of fludarabine based chemotherapy was 48 months (95%CI: 39 - 57 months), among them 27 cases (87.1%) achieved CR + PR. While PFS was 14 months (95%CI: 10 - 18 months, P < 0.001) in 9 patients received other treatment regimen, out of them only 3 cases (33.3%) achieved CR + PR. Patients with normal level of serum β2-microglobulin at diagnosis showed significantly higher overall survival (78%, 95%CI: 69% - 87%) in 36 months than those with abnormal level of serum β2-microglobulin (47%, 95%CI: 35% - 59%, P = 0.004). Significant difference in the rate of CR + PR was noted in the Zap70 positive group (50%) and in negative group (88.5%, P = 0.006). All of 8 patients with IgVH mutation displayed CR after treatment, while 4 cases (66.7%) archived CR among 6 patients without IgVH mutation. It is concluded that CLL is characterized by high heterogeneity in both clinical features and molecular markers, which are associated with prediction of outcomes for patients. The treatment with fludarabine-based chemotherapy results in a major benefit and long survival for patients with CLL.
ADP-ribosyl Cyclase 1 ; metabolism ; Adult ; Aged ; Aged, 80 and over ; Female ; Flow Cytometry ; Humans ; Immunoglobulin Variable Region ; genetics ; Leukemia, Lymphocytic, Chronic, B-Cell ; genetics ; metabolism ; Male ; Middle Aged ; Mutation ; Retrospective Studies ; ZAP-70 Protein-Tyrosine Kinase ; metabolism

OBJECTIVETo investigate lipoprotein lipase (LPL) and serum thymidine kinase (TK) levels in chronic lymphocytic leukemia (CLL) and their correlations with other prognostic factors.

METHODSLPL expression level in peripheral blood samples of 58 CLL patients was detected by semi-quantitative reverse transcription PCR (RT-PCR). Serum TK1 level in 39 CLL patients was detected by enhanced chemiluminescence (ECL) and TK monoclonal antibody (Anti-TK mAb). IgVH mutation status was detected by multiplex PCR and sequencing of purified PCR products. The expression of ZAP-70 protein and CD38 were determined by flow cytometry . Panel probes and fluorescence in situ hybridization (FISH) were used to detect cytogenetic aberrations.

RESULTSThe median LPL expression level in CLL was 0.26 (0 -6.29), while undetectable in normal controls. LPL expression level was significantly correlated with IgVH mutation status, Binet stages, CD38 and cytogenetic aberrations. Patients with unmutated IgVH genes had higher LPL than those with IgVH mutations (P = 0.010). Patients in Binet stage B and C had higher LPL than those in stage A (P = 0.011). LPL level was higher in patients with CD38 > or = 30% (P = 0.001). Higher LPL level was found in patients with unfavorable cytogenetic aberrations (deletion in 17p13 or 11q22) than those with favorable cytogenetics (deletion in 13q as the sole abnormality) (P = 0.002). LPL level was not significantly correlated with sex, age, and ZAP-70 protein (P > 0.05). The level of TK1 was higher in CLL patients than that in normal control (P < 0.05). Patients with higher level of absolute lymphocyte count (ALC), lactate dehydrogenase (LDH), unmutated IgVH genes and ZAP-70 had higher levels of TK1 than those with lower level of ALC, LDH, mutated IgVH genes and ZAP-70 (P = 0.018, P = 0.018, P = 0.030 and P = 0.038, respectively). TK1 level had no correlation with sex, age, Binet stages, CD38, and cytogenetic aberrations (P > 0.05).

CONCLUSIONSLPL expression and serum TK1 levels significantly correlate with other CLL prognostic factors and could be predictive markers for IgVH mutation status. LPL and serum TK1 might be applied to the assessment of prognosis in CLL patients.

ADP-ribosyl Cyclase 1 ; metabolism ; Adult ; Aged ; Aged, 80 and over ; Female ; Humans ; Immunoglobulin Heavy Chains ; genetics ; Leukemia, Lymphocytic, Chronic, B-Cell ; enzymology ; metabolism ; Lipoprotein Lipase ; blood ; Male ; Middle Aged ; Mutation ; Thymidine Kinase ; blood ; ZAP-70 Protein-Tyrosine Kinase ; metabolism

The aim of this study was to investigate bag3 gene expression in chronic lymphocytic leukemia (CLL)patients and its association with clinical prognosis. A total of 46 blood samples from untreated CLL patients were collected, SYBR Green-based real-time PCR was used to detect the bag3 mRNA expression, and its association with prognostic index was analyzed by statistical software. The results showed that the median values of bag3 level detected by real-time PCR in 46 CLL patients and normal controls were 0.021 (0.0007 - 1.124) and 0.0025 (0.0005 - 0.014) respectively, the former was significantly higher than the latter. The bag3 level in drug-resistant group was obviously higher as compared with the drug-responsive group. No association was found between bag3 expression and patient clinical baseline information (gender and age) as well as established prognostic factors (lymphocyte count, disease stage, IgVH mutation status, cytogenetics analysis and CD38, ZAP 70 expression). It is concluded that the bag3 expression in CLL patients is markedly higher than that in normal controls, while the high bag3 level in CML patients is probably related with drug resistance, but is not related with clinically established prognostic factors.
ADP-ribosyl Cyclase 1 ; metabolism ; Adaptor Proteins, Signal Transducing ; genetics ; metabolism ; Adult ; Aged ; Aged, 80 and over ; Apoptosis Regulatory Proteins ; Female ; Gene Expression ; Humans ; Leukemia, Lymphocytic, Chronic, B-Cell ; diagnosis ; genetics ; Male ; Middle Aged ; Prognosis ; ZAP-70 Protein-Tyrosine Kinase ; metabolism