Effect of nuclear transcription factor RelB on the proteasome inhibitor-sensitivity of chronic lymphocytic leukemia cells / 中华血液学杂志

<p><b>OBJECTIVE</b>To investigate the effect of nuclear transcription factor RelB on the proteasome inhibitor-sensitivity of chronic lymphocytic leukemia (CLL) cells.</p><p><b>METHODS</b>The mRNA expression of RelB in CD5⁺ CD19⁺ CLL cells from BM was analyzed by reverse transcription PCR (RT-PCR). The RelB activity was examined by electromobility shift assay (EMSA) and an ELISA-based NF-κB family transcription factor activity assay. CLL cells were classified into RelB+ and RelB- groups according to RelB activity. The frequencies of cell death of CLL cells cultured with human bone marrow stromal cells (hBMSCs) after treatment with PS-341, MG-132 or fludarabine were determined by PI staining.</p><p><b>RESULTS</b>RelB mRNA expression and RelB activity could be detected in CLL cells at variable levels. Fludarabine (10 μmol/L), MG-132 (1 μmol/L) and PS-341 (1 μmol/L) could induce cell death of RelB+ and RelB- CLL cells co-cultured with hBMSCs in a time dependent manner. There was no significant difference in the fludarabine sensitivity between RelB+ and RelB- CLL cells, and the frequencies of cell death of RelB+ and RelB- CLL cells were (61.11 ± 6.91)% and (67.57 ± 9.45)%, respectively, when treated with fludarabine for 72 h. RelB+ CLL cells were more sensitive to MG-132 than RelB- CLL cells for 72 h, and the frequencies of cell death were (66.22 ± 3.39)% and (51.07 ± 5.93)%, respectively. RelB+ CLL cells were more sensitive to PS-341 than RelB- CLL cells for 24 and 48 h treatment, and the frequencies of cell death of RelB+ and RelB- CLL cells were (75.50 ± 4.66)% and (66.32 ± 10.20)% for 24 h, (92.11 ± 3.14)% and (85.84 ± 5.81)% for 48 h treatment, respectively.</p><p><b>CONCLUSION</b>The alternative NF-κB activity was detected in bone marrow derived CLL cells. Enhancement of RelB activity may increase CLL cells' sensitivity to proteasome inhibitor bortezomib and MG-132. However, the sensitivity of CLL cells to fludarabine had no relationship to RelB activity.</p>

Comparison and analysis between CLL-hBMSC and N-hBMSC / 中国实验血液学杂志

This study was purpose to compare and analyze the chronic lymphocytic leukemia human bone marrow stromal cells (CLL-hBMSC) and normal hBMSC (N-hBMSC) so as to provide theoretical evidence for establishment of CLL-hBMSC interaction model to imitate CLL microenvironment. Mononuclear cells (MNC) were isolated from bone marrow of CLL patients and healthy donors and then were cultured, hBMSC were established by expanding for at least five passages. The mRNA expression of adhesion molecules, such as vascular cell adhesion molecule 1 (VCAM-1) and intercellular adhesion molecule 1 (ICAM-1), was analyzed by real-time PCR. The mRNA and protein expression of lymphotoxin beta receptor (LTβR) were determined by real-time PCR and Western blot, respectively. The individual NF-κB members at protein level of CLL-hBMSC and N-hBMSC were examined by Western blot. The effect of LTα1β2 on individual NF-κB family members at protein level in CLL-hBMSC and N-hBMSC was also examined by Western blot. The death of CLL cells was determined by flow cytometry with PI staining when cultured with or without CLL-hBMSC and N-hBMSC at different time points. The results showed that the hBMSC could be established successfully from bone marrow of CLL patients, which were similar to N-hBMSC. Adhesion molecules, such as VCAM-1 and ICAM-1, were found to be expressed at similar mRNA levels in CLL-hBMSC and N-hBMSC. LTβR expressions at mRNA and protein levels were comparable between CLL-hBMSC and N-hBMSC. The protein expression of the individual NF-κB family members could be detected in CLL-hBMSC and N-hBMSC with similar expression levels. LTα1β2 stimulation activated both the classical ( RelA/p50 ) and alternative ( RelB/p52 ) NF-κB complexes in CLL-hBMSC and N-hBMSC. The capacities of CLL-hBMSC and N-hBMSC to protect CLL cell survival were similar. It is concluded that there is no statistical difference between bone marrow from healthy donors and CLL patients in the efficiency of generating of hBMSC. LTβR-NF-κB signaling molecules are expressed and activated on hBMSC with a similar pattern.

Function of alternative NF-κB activity in B-cell chronic lymphocytic leukemia cells / 中华血液学杂志

<p><b>OBJECTIVE</b>To investigate the function of alternative NF-κB activity in B-cell chronic lymphocytic leukemia cells (B-CLL).</p><p><b>METHODS</b>The mRNA expression of individual NF-κB subunits in CD5⁺CD19⁺ cells (CLL B-cells) from bone marrow (BM) of 56 patients with B-CLL was analyzed by quantitative RT-PCR. An ELISA-based NF-κB family transcription factor activity assay was performed to quantify the κB DNA-binding activity in nuclear extracts from CLL B-cells. Cell death of CLL-B cells was determined by PI staining, RelA and RelB expression at protein level of CLL B-cells by Western blot analyses.</p><p><b>RESULTS</b>The expression levels of RelA, p50, RelB and p52 mRNA in CLL B-cells were all higher than that of normal B cells with statistical significance (P<0.05). RelA was activated in almost all the patients detected while RelB activity was induced in part of samples. The average RelA activity in CLL B-cells was increased compared to that in normal B cells while the average RelB activity was similar to that of normal B cells. When cultured in vitro for 24, 48 and 72 hours, the frequencies of cell death of CLL B-cells from RelA⁺/RelB⁻ group were(35.54±4.43)%,(50.92±8.44)%, and(49.24±8.16)%, respectively; that of the RelA⁺/RelB⁺ group were (20.65±2.37)%, (18.17±1.36)%, and (26.55±4.08)%, respectively. When the cells from RelA+/RelB⁻ group were co-cultured with bone marrow stromal cells (hBMSCs), the frequencies of cell death of CLL B-cells were decreased compared to that of the cells cultured alone, while the frequencies of cell death of RelA⁺/RelB⁻ CLL B-cells were higher than that of CLL B-cells from RelA⁺/RelB⁺ group when co-cultured with hBMSCs. RelA and RelB expression in CLL-B cells from the RelA⁺/RelB⁻ group was induced after co-cultured with hBMSCs for 48 h. RelB was reduced in the cytoplasm and increased in the nucleus in CLL-B cells from the RelA+/RelB+ group.</p><p><b>CONCLUSION</b>The alternative NF-κB was indeed activated and presented heterogeneous in CLL B-cells from BM. Activation of RelB combined with RelA activity could provide the survival advantage to CLL B-cells from BM. Co-culture with hBMSCs could protect CLL-B cells through the induction of RelA and Rel B expressions.</p>

Effect of RelB on HIV-1 Vpr-mediated transcription activation and cell G2/M arrest / 病毒学报

Vpr, an auxiliary protein of HIV-1(Human immunodeficiency virus type 1), exerts important functions to promote viral replication and AIDS progression. In this study, we performed a yeast two-hybrid screening assay using human cDNA library to further investigate the molecular mechanism of various functions of Vpr RelB, a key protein in NF-kappaB signaling pathway, was identified as a Vpr interaction protein by co-immunoprecipitation. Further investigations indicated that RelB not only promoted the Vpr-mediated activation of NF-kappaB reporter gene, but also enhanced the transactivation of HIV LTR. Moreover, the results showed that RelB promoted Vpr-induced cell cycle G2/M arrest. Collectively, these results indicated that RelB might interact with Vpr and regulate its transcriptional activation and cell cycle arrest.

NF-κB subunits regulate maspin expression in prostate cancer cells in vitro / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To explore how NF-κB family members regulate maspin expression in prostate cancer cells.</p><p><b>METHODS</b>The expression of NF-κB subunits and maspin was detected by Western blot analysis in prostate cancer DU145, PC-3, and LNCaP cell lines. RNA interference was performed to analyze whether RelB- or RelA-deletion affectes cell death as well as the expression of NF-κB subunits and maspin. The impact of RelB-silencing in DU145 cells was investigated by flow cytometry. The regulation of RelB on maspin expression in the prostate cancer PC-3 cells was also examined via stable transfection of RelB expression plasmid.</p><p><b>RESULTS</b>RelA, p50, RelB, and p52 were constitutively expressed in androgen-independent prostate cancer DU145 and PC-3 cells, while RelB had the highest expression in DU145 cells. Low expression of maspin was detected in LNCaP and DU145 cells, but elevated expression in PC-3 cells. RelB-silencing in DU145 cells by siRNA interference upregulated the endogenous expression of maspin and induced cell apoptosis (13.3±4.2)%. Overexpression of RelB in PC-3 cells inhibited the endogenous expression of maspin. RelA-silecing had no significant influence on the endogenous expression of maspin.</p><p><b>CONCLUSIONS</b>The classical and alternative NF-κB activitions are sustained in androgen-independent prostate cancer cell lines. The expressions of RelB and maspin are inversely correlated in these cancer cells. The expression of RelB negatively regulates the endogenous expression of maspin, then interferes the cell survival. RelA is not involved in the regulation of maspin expression.</p>

RNA interference of RelB enhances the radiosensitivity of prostate cancer cell line RM-1 in mice / 中华男科学杂志

<p><b>OBJECTIVE</b>To investigate the effect of RNA interference of the RelB gene on the radiosensitivity of the mouse prostate cancer cell line RM-1 and its mechanism.</p><p><b>METHODS</b>We constructed RelB siRNA-expressing lentiviral vectors targeting the RelB gene with the molecular biological technique, and determined the expressions of RelB mRNA and protein on radiation after transfection with siRelB mediated by liposome using RT-PCR and Western blot, respectively. We also detected the apoptosis of RM-1 cells by FCM assay and their radiosensitivity by clonogenic assay.</p><p><b>RESULTS</b>The expressions of RelB mRNA and protein were significantly lower in the RM-1 cells than in the control and negative interference groups after transfection with RelB siRNA (P < 0.05), while the apoptosis of RM-1 cells remarkably higher in the siRelB-RM-1 than in the control group after radiation treatment (P < 0.05). The activity of MnSOD was markedly decreased (P < 0.05), and the radiosensitization rate of the RM-1 cells in the RelB-RM-1 group was 5.13 after radiation treatment.</p><p><b>CONCLUSION</b>RNA interference of the RelB gene could enhance the radiosensitivity of the mouse prostate cancer cell line RM-1, which might be associated with its inhibition of Mn-SOD expression and induction of cell apoptosis.</p>

Relationship between surface molecules and RelB gene expression in DC2.4 cell line / 南方医科大学学报

<p><b>OBJECTIVE</b>To explore the relationship between the expression of the nuclear factor-kappaB transcription factor RelB gene and the surface molecules in DC2.4 cell line.</p><p><b>METHODS</b>DC2.4 cell line was cultured in complete RPMI 1640 medium, whose morphology was observed with optical microscope and the intracellular structures with transmission electron microscope. Flow cytometry was performed to analyze the surface markers of the cells, including MHC-II, CD86 and CD40, and RelB mRNA expression was detected by RT-PCR.</p><p><b>RESULTS</b>Under optical microscope, the cells appeared irregular in shape with obvious dendritic cell processes, and electron microscopy revealed homogenous fat drops and phagocytic vesicles in the cytoplasm. Flow cytometry demonstrated low expression levels of MHC-II and CD40, but high level of CD86 molecules. Low-level expression of RelB mRNA was detected by RT-PCR, resembling its expression level in bone marrow-derived DC with immature phenotype.</p><p><b>CONCLUSION</b>DC2.4 is a mouse bone marrow dendritic cell line with strong phagocytic capacity, and the low expression of both RelB gene and surface CD40 molecules suggests an immature dendritic cell line.</p>

Identification of effective siRNA sequence for RelB silencing in murine dendritic cells with siRNA cassette / 南方医科大学学报

<p><b>OBJECTIVE</b>To construct small interfering RNA (siRNA) expression cassette targeting murine RelB gene and identify the most effective siRNA sequence against RelB gene in murine bone marrow-derived dendritic cells (DCs).</p><p><b>METHODS</b>Three expression cassettes namely R1/siRNA, R2/siRNA and R3/siRNA targeting the sites 1027, 302 and 1121 of RelB gene, respectively, were constructed by PCR approach and transfected into cultured murine myeloid DCs by catione liposome Advant-Gene. After incubation for 24 hours in a incubator containing 5% CO(2) at 37 degrees C, the DCs were stimulated by lipopolysaccharide (LPS), and RelB gene expression in DCs were then detected by RT-PCR and immunofluorescence.</p><p><b>RESULTS</b>RT-PCR and immunofluorescence assay showed that the expression of RelB gene in DCs transfected with R2/siRNA could not be upregulated by LPS stimulation, but transfection with R1/siRNA or R3/siRNA failed to produce such effect.</p><p><b>CONCLUSION</b>R2/siRNA is an effective sequence for RelB silencing, and can be a useful means to construct new tolerogenic DC, RNAi RelB DC, for clinical immunotolerance induction.</p>