<b>OBJECTIVEb>To investigate the function of alternative NF-&kappa;B activity in B-cell chronic lymphocytic leukemia cells (B-CLL).

<b>METHODSb>The mRNA expression of individual NF-&kappa;B 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-&kappa;B family transcription factor activity assay was performed to quantify the &kappa;B 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.

<b>RESULTSb>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.

<b>CONCLUSIONb>The alternative NF-&kappa;B 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.

Case-Control Studies ; Cell Nucleus ; metabolism ; Humans ; Leukemia, Lymphocytic, Chronic, B-Cell ; metabolism ; NF-kappa B p50 Subunit ; metabolism ; NF-kappa B p52 Subunit ; metabolism ; Transcription Factor RelA ; metabolism ; Transcription Factor RelB ; metabolism ; Tumor Cells, Cultured

<b>OBJECTIVEb>To explore how NF-&kappa;B family members regulate maspin expression in prostate cancer cells.

<b>METHODSb>The expression of NF-&kappa;B 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-&kappa;B 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.

<b>RESULTSb>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.

<b>CONCLUSIONSb>The classical and alternative NF-&kappa;B 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.

Apoptosis ; Cell Line, Tumor ; Gene Silencing ; Humans ; Male ; NF-kappa B ; genetics ; metabolism ; NF-kappa B p50 Subunit ; genetics ; metabolism ; NF-kappa B p52 Subunit ; genetics ; metabolism ; Prostatic Neoplasms ; genetics ; metabolism ; pathology ; RNA, Small Interfering ; genetics ; Serpins ; metabolism ; Transcription Factor RelA ; genetics ; metabolism ; Transcription Factor RelB ; genetics ; metabolism ; Transfection

Nuclear factor &kappa;B (NF-&kappa;B), including RelA, RelB, c-Rel, NF-&kappa;B1, and NF-&kappa;B2, plays a crucial role in immune response, inflammatory reaction, tumorigenesis, and development of peripheral lymphoid organs and lymphocytes. There are two NF-&kappa;B activation pathways: canonical pathway (classical pathway) and noncanonical pathway (alternative pathway). Previous studies focused on the effects of the canonical NF-&kappa;B pathway (mainly p50-RelA) in hematological malignancies. Recently, the noncanonical NF-&kappa;B pathway (mainly p52-RelB) is gradually taken importance in pathogenesis of hematological malignancies. Understanding the relations of the noncanonical pathway with hematological malignancies would provide a new therapeutic approach for these diseases. This review focuses on the noncanonical NF-&kappa;B signaling transduction pathway and its relation to hematologic malignancies.
Hematologic Neoplasms ; metabolism ; Humans ; NF-kappa B ; metabolism ; NF-kappa B p50 Subunit ; metabolism ; NF-kappa B p52 Subunit ; metabolism ; Signal Transduction

Humans ; Lymphoma, Large B-Cell, Diffuse ; diagnosis ; metabolism ; NF-kappa B p50 Subunit ; metabolism ; Transcription Factor RelA ; metabolism

<b>OBJECTIVEb>To investigate the effects of burn sera on the nuclear translocation of endothelial NF-kappaB heterodimers p50/p65 and on the degradation of inhibiting kappaB (IkappaBalpha), in order to explore the role of burn sera on activation of the endothelium.

<b>METHODSb>Cultured human umbilical vein endothelial cells (HUVECs) (ECV-304 strain) were employed as the target cells. The cells were stimulated by sera from healthy volunteers and from burn patients and burn sera together with PDTC (pyrrolidine dithiocarbarnate). The normal cultured cells were taken as the control. The nuclear translocation of endothelial p50/p65 at 30, 60, 120 and 480 mins after the stimulation was observed with laser confocal microscopy, and the endothelial IkappaBalpha protein degradation at 30, 60, 90 and 120 mins after the stimulation was determined by Western blotting.

<b>RESULTSb>When compared to that in control group, the nuclear translocation of p50/p65 took place 30 mins after the endothelial cells were stimulated by burn sera, and it reached the summit at 30 - 60 mins, but recovered to pre-stimulation state at 2hrs. In addition, IkBalpha degradation occurred 30 mins after the cells were stimulated by burn sera (P < 0.01) and peaking at 45 - 60 mins after the stimulation and recovered at 2hrs after the stimulation. The nuclear translocation of endothelial p50/p65 and IkBalpha degradation at 30 and 60 mins after the stimulation by burn sera could be effectively inhibited by PDTC.

<b>CONCLUSIONb>Burn sera might induce the nuclear translocation of endothelial NF-kappaB p50/p65 and IkappaBalpha degradation and activate NF-kappaB, which ultimately lead to the secretion of cytokines from the endothelium.

Active Transport, Cell Nucleus ; Adolescent ; Adult ; Burns ; blood ; Female ; Humans ; I-kappa B Proteins ; analysis ; Male ; Middle Aged ; NF-KappaB Inhibitor alpha ; NF-kappa B ; metabolism ; NF-kappa B p50 Subunit ; Transcription Factor RelA

<b>OBJECTIVEb>To investigate whether hepatitis B virus (HBV) can induce the expression of the host-encoded cytokine interleukin-32 (IL-32) and its effects on host signaling mechanisms related to HBV pathogenesis.

<b>METHODSb>A eukaryotic expression vector harboring an enhanced green fluorescent protein was constructed with HBV genomic sequences (pIRES2-HBV-EGFP) and transfected into HepG2 cells. In addition, the nuclear factor-kappa B (NF-kB) subunits, p50 and p65, were transfected respectively into HepG2 cells. In both cases, 48 hrs after transfection, IL-32 expression was determined at the mRNA and protein levels using real-time PCR and ELISA and western blot, respectively. The HepG2 cells transfected with pIRES2-HBV-EGFP were also treated with the NF-kB inhibitor SN50 at various concentrations, and the effects on IL-32 protein expression 48 hrs later were evaluated by western blot. Significance of between-group differences was assessed by the Student's t-test.

<b>RESULTSb>Transfection with pIRES2-HBV-EGFP led to significantly higher IL-23 expression than transfection with empty vector (mRNA: 2.8-fold higher and protein: 4.5-fold higher; both P less than 0.05). Transfection of p50 and p65 proteins led to significantly higher IL-32 expression (both P less than 0.05), and NF-kB activation was found to be required for HBV-induced IL-32 expression.

<b>CONCLUSIONb>IL-32 expression is induced by HBV in HepG2 cells. This host-encoded cytokine, and its downstream activation of NF-kB, may be involved in the pathogenesis of HBV, especially in the subsequent liver inflammation that accompanies HBV infection.

Genetic Vectors ; Hep G2 Cells ; metabolism ; Hepatitis B virus ; Host-Pathogen Interactions ; Humans ; Interleukins ; metabolism ; NF-kappa B p50 Subunit ; metabolism ; Transcription Factor RelA ; metabolism ; Transfection

<b>OBJECTIVEb>To investigate the effects of burn serum on nuclear translocation of monocytic NF-kappaB heterodimers p50/p65 and the degradation of inhibiting kappaB (IkappaBalpha), so as to further explore the role of burn serum on the activation of monocytes.

<b>METHODSb>Peripheral blood monocytes (PBMCs) isolated from healthy volunteers were employed as the target cells. The cells were stimulated by the serum from healthy volunteers and burn patients, and by burn serum together with pyrrolidine dithiocarbamate (PDTC). Sera from normal healthy volunteers were taken as control. The nuclear translocation of monocytic p50 and p65 at 30th, 60th, 120th and 480th post stimulation minutes (PSM) was observed with laser confocal microscopy. The degradation of monocytic IkappaBalpha protein at 30th, 60th, 90th and 120th PSM was determined by Western blot.

<b>RESULTSb>Compared to that in control group, the nuclear translocation of monocytic p50 and p65 took place 30 min after the PBMCs were stimulated by burn serum, peaking at 30 to 60 min, but it gradually recovered to pre-stimulation state at 2 hrs with decreased intra-nuclear collection. Meanwhile, the IkappaBalpha degradation occurred within 30 min after PBMCs being stimulated by burn serum, and it peaked at 60 mins. However, IkappaBalpha gradually reappeared in the cytoplasm after 2 hrs of stimulation. PDTC (an antioxidants) could effectively inhibit monocytic IkappaBalpha degradation and nuclear translocation of NF-kappaB induced by burn serum.

<b>CONCLUSIONb>Burn serum could induce nuclear translocation of p50 and p65 components of NF-kappaB in monocytes into the nucleus and degradation of IkappaBalpha, leading ultimately to the secretion of cytokines from the PBMCs.

Active Transport, Cell Nucleus ; Burns ; blood ; Cells, Cultured ; Female ; Humans ; I-kappa B Proteins ; metabolism ; Male ; Monocytes ; metabolism ; NF-KappaB Inhibitor alpha ; NF-kappa B p50 Subunit ; metabolism ; Transcription Factor RelA ; metabolism

In this study, immunohistochemistry and Western blot were used to determine whether the expression of NF-kappaB in the hippocampus of prenatally stressed offspring rats is gender-dependent. The results were as follows: In the female offspring rats, the expressions of p65 in the hippocampal dentate gyrus in mid-term stress (MS) and late-term stress (LS) groups were significantly less than that in the control group (P<0.01). There was a significant difference between MS and LS groups (P<0.01). The expressions of p50 in all regions of hippocampus in MS and LS groups were significantly more than that in the control group (P<0.01). A significant difference was also present between MS and LS groups (P<0.01). In the male offspring rats, the expressions of p65 in the hippocampal dentate gyrus in MS and LS groups were evidently more than that in the control group (P<0.01). There was a significant difference between MS and LS groups (P<0.01). The expressions of p50 in all regions of hippocampus in MS and LS groups were significantly less than that in the control group (P<0.05, P<0.01). There was also a significant difference in p65 expression between MS and LS groups (P<0.01). In addition, in the control group the expressions of p65 in the hippocampal dentate gyrus of female offspring rats were significantly more than that of male ones (P<0.01). However, in LS group the expressions of p65 in the hippocampal dentate gyrus of female offspring rats were significantly less than that of male ones (P<0.01). Moreover, there was no significant difference in p65 expression between female and male offspring rats in MS group. In the control group the gender difference in the expression of p50 was only observed in hippocampal CA1 (P<0.01). The expressions of p50 in all regions of hippocampus of female offspring rats were significantly more than that of male ones in LS group (P<0.01). There was no significant difference in p50 expression between female and male offspring rats in MS group. The results of Western blot were similar to those of immunohistochemical study. These results indicate that prenatal stress in different gestational periods significantly affects the expressions of p65 and p50 in hippocampus, and this effect is gender-dependent. This may be one of the mechanisms underlying the gender difference in the ability of learning and memory of the prenatally stressed offspring rats.
Animals ; Female ; Hippocampus ; metabolism ; Male ; NF-kappa B p50 Subunit ; metabolism ; Pregnancy ; Prenatal Exposure Delayed Effects ; Rats ; Sex Factors ; Stress, Physiological ; Transcription Factor RelA ; metabolism

OBJECTIVE: To detect the expression and correlation of cyclooxygenase-2 (COX-2) and nuclear factor-kappa B (NF-kappaB) in nasal polyps co-cultured with clarithromycin, and to investigate their roles in CRS pathogenesis. METHOD: Nasal polyps from 11 patients with chronic rhinosinusitis (CRS) were cultured for 24 hours with different doses of clarithromycin (0, 10(-6), 10(-5), 10(-4) mol/L). Western blot and real time fluorescence quantitative PCR were performed to detect the expressions of COX-2 and NF-kappaB subunits. RESULT: The expression levels of COX-2, NF-kappaBp50 and NF-kappaBp65 were most high in control groups (0 mol/L clarithromycin). The expressions of COX-2, NF-kappaBp50 and NF-kappaBp65 were dose-dependently attenuated as the concentrations of clarithromycin increased. Significantly positive correlation between RNA expressions of COX-2 and NF-kappaB subunits in each CRS group was confirmed by Pearson correlation treatment (P<0.05). CONCLUSION The increased expression of COX-2 is involved in the inflammation in CRS. It indicate that clarithromycin may play an anti-inflammatory effect on CRS by decreasing the synthesis of COX-2 through blocking NF-kappaB pathway.
Adult ; Clarithromycin ; pharmacology ; Cyclooxygenase 2 ; metabolism ; Female ; Humans ; In Vitro Techniques ; Male ; Middle Aged ; NF-kappa B p50 Subunit ; metabolism ; Nasal Polyps ; metabolism ; Transcription Factor RelA ; metabolism

The involvement of NF-kappaB binding activity is known to be important in the mechanism of acute liver injury and in the induction of cyclooxygenase (COX-2). This study was performed to evaluate NF-kappaB binding activity and the expression of COX-2 in chronic liver injury induced by carbon tetrachloride (betaCCI(4)). Liver tissues from Sprague - Dawley rats were collected at 1, 3, 5, and 7th week after intraperitoneal injection of 0.1 mL of betaCCI(4)/100 g body weight twice a week. Reactive oxy-gen species (ROS) were measured in the postmitochondrial fraction by dichlorofluorescein formation with a fluorescent probe. An electrophoretic mobility shift assay was performed for NF-kappaB binding activity. Western blot was performed to measure the level of COX-1, COX-2, p65, p50, and I B proteins. ROS and NF-kappaB activity increased during the CCl4-induced chronic liver injury. The expression of nuclear p65 protein and p50 protein increased compared with that of the control, while the cytoplasmic I B protein decreased as the inflammation persisted. The expression of COX-2 in betaCCI(4)-treated rat liver increased compared with that of the control. It could be suggested that ROS produced by betaCCI(4) treatment increased NF-kappaB binding activity and thereby COX-2 expression, and these might be implicated in the progress of chronic liver damage.
Animals ; Biological Transport ; Carbon Tetrachloride/administration & dosage/*adverse effects ; Carbon Tetrachloride Poisoning/*metabolism/pathology ; Cell Nucleus/metabolism ; Cyclooxygenase 1 ; Cyclooxygenase 2 ; Cytoplasm/metabolism ; I-kappa B Proteins/biosynthesis ; Isoenzymes/*biosynthesis ; Liver/drug effects/*injuries/pathology ; Membrane Proteins ; NF-kappa B/antagonists & inhibitors/*metabolism ; NF-kappa B p50 Subunit ; Prostaglandin-Endoperoxide Synthases/*biosynthesis ; Protein Binding ; Rats ; Rats, Sprague-Dawley ; Reactive Oxygen Species ; Transcription Factor RelA