Cyr61 participates in the pathogenesis of acute lymphoblastic leukemia by enhancing cellular survival via the AKT/NF-κB signaling pathway.

Cyr61 (CCN1) is the product of a growth factor-inducible immediate early gene and is involved in cell adhesion, survival, proliferation, and differentiation. Cyr61 is overexpressed in human tumors and is involved in the development of tumors. However, the role that Cyr61 plays in acute lymphoblastic leukemia (ALL) cells remains undetermined. The aim of this study was to identify the role of Cyr61 in regulating ALL cell survival. Here, we found that the level of Cyr61 was increased in the plasma and bone marrow (BM) from ALL patients compared with samples from normal control patients. Furthermore, we observed that Cyr61 could effectively stimulate Jurkat (T ALL cell lines), Nalm-6 (B ALL cell lines), and primary ALL cell survival. Mechanistically, we showed that Cyr61 stimulated ALL cell survival via the AKT/NF-κB signaling pathways and the consequent up-regulation of Bcl-2. Taken together, our study is the first to reveal that Cyr61 is elevated in ALL and promotes cell survival through the AKT/NF-κB pathway by up-regulating Bcl-2. Our findings suggest that Cyr61 plays an important role in the pathogenesis of ALL.

Mature dendritic cells cause Th17/Treg imbalance by secreting TGF-ß1 and IL-6 in the pathogenesis of experimental autoimmune encephalomyelitis.

Multiple sclerosis (MS) is generally acknowledged to be an autoimmune disease, but its etiology remains unknown. The most intensively studied animal model of MS is experimental autoimmune encephalomyelitis (EAE). Dendritic cells (DCs), the professional antigen presenting cells (APCs), have gained increasing attention because they connect innate and adaptive immunity. The aim of this study was to determine the role of mature DCs in the pathogenesis of EAE. It was found that the number of mature DCs in the EAE spleen increased compared to the control group (p < 0.05). And there was an imbalance between Th17 (effector) and Treg (regulatory) in EAE. The data showed that mature DCs can regulate the differentiation of Th17 and Treg in EAE. In addition, there was a significant difference in secretion of TGF-ß1 and IL-6 between mature DCs from mice with EAE and controls. The present data suggest that mature DCs cause an imbalance between Th17 and Treg by secreting TGF-ß1 and IL-6 in the pathogenesis of EAE disease. Thus, targeting DC may be an effective strategy for treating MS.

[Th17/Treg unbalance is involved in the pathogenesis of experimental autoimmune encephalomyelitis].

OBJECTIVE: To investigate the role of Th17/Treg unbalance in the pathogenesis of experimental autoimmune encephalomyelitis (EAE). METHODS: EAE was modeled in mice and the number of regulatory T cells (Tregs) in spleen of EAE mice was detected by flow cytometry. The expressions of Foxp3 and RoR-γt mRNA in the spleen of EAE mice and IL-17 mRNA in the brain of EAE mice were evaluated by real-time quantitative PCR and the levels of IL-6, TGF-ß and IL-17 in the serum of EAE mice were examined by ELISA. RESULTS: Compared with control group, the number of CD4(+)CD25(+) Foxp3(+) Tregs and the expression of Foxp3 mRNA in the spleen of EAE mice dramatically decreased in the early and peak stage of EAE (P<0.05), but increased in chronic stage of EAE (P<0.05); the RoR-γt mRNA expression from mouse spleen at the early stage of EAE was significant raised (P<0.05), but was not significantly different at the peak and chronic stage of EAE from that in control group (P>0.05). The levels of IL-6 and TGF-ß in the serum of EAE group dramatically increased compared with control group (P<0.05). With the development of EAE, the level of IL-6 gradually decreased, and there was no statistical difference in the chronic stage of EAE compared with control group (P>0.05). However, the level of TGF-ß was higher than that in control group in the chronic stage of EAE (P<0.05). Compared with those in control group, the concentration of IL-17A and the expression of IL-17 mRNA dramatically increased in different stages of EAE group, especially in peak stage (P<0.05). CONCLUSION: Th17/Treg unbalance may be involved in the pathogenesis of EAE.

[Expression changes of genes related to calcium pathway in all-trans retinoic acid-induced differentiation of APL cells].

In order to investigate the role of calcium pathway in myeloid differentiation, the expression level of genes related to calcium pathway in all-trans retinoic acid (ATRA)-induced NB4 cell differentiation was detected by cDNA microarray, some of which were further confirmed by quantitative real time RT-PCR. At the same time, the expressions of these genes in NB4-R1 cells treated with ATRA and 8-CPT-cAM P alone or in combination, and in differentiation of primary cells from ATRA-induced newly diagnosed APL patients were detected by real time RT-PCR. The results showed that during differentiation of ATRA-induced NB4 cells, the expressions of genes related to calcium concentration had changed, the expression of downstream effectors in calcium pathway was up-regulated and confirmed by real time RT-PCR assay. The expression of genes related to calcium concentration did not change significantly when NB4-R1 cells were treated by ATRA or 8-CPT-cAMP alone, but expression changes of those genes were similar to the changes in ATRA-induced NB4 cell differentiation when NB4-R1 cells were treated by ATRA combined with 8-CPT-cAMP. In addition, the expression changes of those genes in ATRA-induced primary cells of patients with APL were also similar to changes in ATRA-induced NB4 cell differentiation. It is concluded that calcium pathway may be involved in ATRA-induced differentiation in APL cell.

[Expression level changes of inhibitor of differentiation 1 during ATRA-induced acute promyelocytic leukemia cells differentiation].

OBJECTIVE: To study the role of inhibitor of differentiation 1 (ID1) in ATRA-induced acute promyelocytic leukemia (APL) cells differentiation. METHODS: The expression of ID1 was detected by cDNA microarray, cycloheximide inhibition test, real-time RT-PCR and western blot. RESULTS: The expression of ID1 gene was up-regulated in ATRA-induced NB4 cells and APL cells from two patients and was independent on other proteins synthesis. ID1 expression level reached the peak at 2 h in NB4 cells induced by ATRA, its relative expression level was (359.4 +/- 48.7)-fold greater than control. ID1 expression level reached the peak at 2 h in bone marrow cells from APL patents treated with ATRA, and its level detected 3 times in one of the patient was (311.1 +/- 48.7) fold of control. The expression of ID1 protein was not up-regulated in ATRA resistant NB4-R2 cells after ATRA treatment. CONCLUSION: ID1 may be involved in ATRA-induced granulocytic differentiation as an ATRA-targeted gene.

Coordination of intrinsic, extrinsic, and endoplasmic reticulum-mediated apoptosis by imatinib mesylate combined with arsenic trioxide in chronic myeloid leukemia.

A treatment strategy that combines arsenic trioxide (ATO) with the tyrosine kinase inhibitor imatinib mesylate (STI571, Gleevec) appears to induce markedly more cell apoptosis than imatinib mesylate alone in chronic myeloid leukemia (CML). To understand the mechanisms underlying the synergistic/additive action of these agents, we applied cDNA microarrays, component plane presentation integrated self-organizing map (CPP-SOM), and methods of protein biochemistry to study cell apoptosis induced by imatinib mesylate, ATO, and the combination of the 2 agents in the CML cell line K562. Numerous features with temporospatial relationships were revealed, indicating the coordinated regulation of molecular networks from various aspects of proapoptotic and apoptotic activities in CML. Imatinib mesylate appears to induce mainly the intrinsic pathway of cell apoptosis, whereas ATO induces the endoplasmic reticulum (ER) stress-mediated pathway of cell apoptosis, and the combination of the 2 agents seems to more effectively induce the intrinsic, extrinsic, and ER stress-mediated pathways of cell apoptosis, which results in a more effective and efficient induction of programmed cell death in K562 cells. This finding appears to be supported also by data derived from bone marrow cells of 2 patients with CML, one in chronic phase and the other in blast-crisis phase of the disease.

Systems analysis of transcriptome and proteome in retinoic acid/arsenic trioxide-induced cell differentiation/apoptosis of promyelocytic leukemia.

Understanding the complexity and dynamics of cancer cells in response to effective therapy requires hypothesis-driven, quantitative, and high-throughput measurement of genes and proteins at both spatial and temporal levels. This study was designed to gain insights into molecular networks underlying the clinical synergy between retinoic acid (RA) and arsenic trioxide (ATO) in acute promyelocytic leukemia (APL), which results in a high-quality disease-free survival in most patients after consolidation with conventional chemotherapy. We have applied an approach integrating cDNA microarray, 2D gel electrophoresis with MS, and methods of computational biology to study the effects on APL cell line NB4 treated with RA, ATO, and the combination of the two agents and collected in a time series. Numerous features were revealed that indicated the coordinated regulation of molecular networks from various aspects of granulocytic differentiation and apoptosis at the transcriptome and proteome levels. These features include an array of transcription factors and cofactors, activation of calcium signaling, stimulation of the IFN pathway, activation of the proteasome system, degradation of the PML-RARalpha oncoprotein, restoration of the nuclear body, cell-cycle arrest, and gain of apoptotic potential. Hence, this investigation has provided not only a detailed understanding of the combined therapeutic effects of RA/ATO in APL but also a road map to approach hematopoietic malignancies at the systems level.

NMR structure and regulated expression in APL cell of human SH3BGRL3.

SH3 domain binding glutamic acid-rich protein like 3 (SH3BGRL3) is the new member of thioredoxin (TRX) super family, whose posttranslational modified form was identified as tumor necrosis factor alpha (TNF-alpha) inhibitory protein, TIP-B1. In this paper, we determined its solution structure by multi-dimensional nuclear magnetic resonance spectroscopy. The overall structure of human SH3BGRL3 conformed to a TRX-like fold. To understand its function in vivo, the upregulated expression in acute promyelocytic leukemia cell line NB4 at both mRNA and protein level was elucidated. Immunofluorescence and immunohistochemistry staining with monoclonal antibody against SH3BGRL3 demonstrated that it was a cytoplasmic protein in both NB4 cell and human tissues. These results, as a whole, indicate that SH3BGRL3 may function as a regulator in all-trans retinoic acid-induced pathway.

[Establishment of CPP-SOM integrated cDNA microarray technology].

OBJECTIVE: To get an insight into the molecular mechanisms of diseases development and targeted therapy at the transcriptome level and search for potential therapeutic targets. METHODS: The present researchers established a cDNA microarray platform and applied component plane presentation integrated self-organizing map (CPP-SOM) to the microarray data obtained from a differentiation model, all trans retinoic acid-induced differentiation in NB4 cells. RESULTS: The platform included 12630 unique clones, including 9436 known genes. By CPP-SOM, the researchers were able to not only well classify the regulated genes into functionally distinct categories but also depict transcriptional changes throughout the process of the development of diseases or drug treatment. CONCLUSION: The platform has proven to be steady and reliable, and the CPP-SOM could serve as an important and good tool for analysis of microarray data.

All-trans retinoic acid/As2O3 combination yields a high quality remission and survival in newly diagnosed acute promyelocytic leukemia.

Both all-trans retinoic acid (ATRA) and arsenic trioxide (As(2)O(3)) have proven to be very effective in obtaining high clinical complete remission (CR) rates in acute promyelocytic leukemia (APL), but they had not been used jointly in an integrated treatment protocol for remission induction or maintenance among newly diagnosed APL patients. In this study, 61 newly diagnosed APL subjects were randomized into three treatment groups, namely by ATRA, As(2)O(3), and the combination of the two drugs. CR was determined by hematological analysis, tumor burden was examined with real-time quantitative RT-PCR of the PML-RAR alpha (promyelocytic leukemia-retinoic acid receptor alpha) fusion transcripts, and side effects were evaluated by means of clinical examinations. Mechanisms possibly involved were also investigated with cellular and molecular biology methods. Although CR rates in three groups were all high (> or =90%), the time to achieve CR differed significantly, with that of the combination group being the shortest one. Earlier recovery of platelet count was also found in this group. The disease burden as reflected by fold change of PML-RAR alpha transcripts at CR decreased more significantly in combined therapy as compared with ATRA or As(2)O(3) mono-therapy (P < 0.01). This difference persisted after consolidation (P < 0.05). Importantly, all 20 cases in the combination group remained in CR whereas 7 of 37 cases treated with mono-therapy relapsed (P < 0.05) after a follow-up of 8-30 months (median: 18 months). Synergism of ATRA and As(2)O(3) on apoptosis and degradation of PML-RAR alpha oncoprotein might provide a plausible explanation for superior efficacy of combination therapy in clinic. In conclusion, the ATRA/As(2)O(3) combination for remission/maintenance therapy of APL brings much better results than either of the two drugs used alone in terms of the quality of CR and the status of the disease-free survival.

[Preliminary analysis of gene expression profiles in oral cancer with microarray technique].

OBJECTIVE: To study the difference in gene expression between oral squamous cell carcinoma tissue and their surrounding normal tissue by microarray so as to investigate the preliminary mechanism of pathogenesis of oral cancer. METHODS: The tissues from 5 patients with oral squamous cell carcinoma tissue and their surrounding normal tissue from the same patients were analyzed by cDNA microarray technology(including 4124 genes). Total RNAs were isolated from two tissues, and then were reversely transcribed to cDNAs with the incorporations of fluorescent dUTP,for preparing the hybridization probes. The mixed probes were then hybridized to the cDNA microarray. After high-stringent washing, the cDNA microarray was scanned for the fluorescent signals and showed the differences between the two tissues. Bioinformatical analysis of those genes had been performed. RESULTS: Among the 4124 target genes, there were 37(0.89%)genes whose expression levels differed between the carcinoma and their surrounding normal tissues in all 5 cases. Bioinformatical analysis of those genes suggested that they may be related to the multistep process of carcinogenesis. CONCLUSION: cDNA microarray technique can simultaneously screen the different expressions of genes from 2 different kinds of tissue. Further analysis of the obtained genes will help to understand the molecular mechanism of malignant carcinoma.