ABSTRACT
<p><b>OBJECTIVE</b>Suppressors of cytokine signaling (SOCS) have been shown to play an important role in regulating cytokines, such as intracellular interferon (IFN) and interleukin (IL), in the Janus kinase-signal transducer and activator of transcription (JAK-STAT) pathway. At present, the association between SOCS and asthma is still under study. The aim of this study is to explore the relationship of SOCS-1 and SOCS-3 mRNA expression in peripheral blood mononuclear cells (PBMCs) with the intracellular IFN-'/IL-4 ratio in CD4+ T cells and specific IgE (sIgE) level in children with asthma.</p><p><b>METHODS</b>BMCs were collected from 44 children with allergic asthma (4-14 years) and 30 healthy children. The intracellular IFN-'/IL-4 ratio in CD4+ T cells was measured by flow cytometry. Total RNAs were extracted from the PBMCs and SOCS-1 and SOCS-3 mRNA expression was measured by SYBR Green I quantitative RT-PCR.</p><p><b>RESULTS</b>Compared with the healthy children, children with allergic asthma showed a lower level of intracellular IFN-' in peripheral blood [(15.7±2.0)% vs (19.1±2.7)%] and IFN-'/IL-4 ratio (3.4±1.5 vs 4.8±2.9) and higher SOCS-1 mRNA expression (-Ct, 11.1±1.9 vs 12.6±2.8). There was a negative relationship between SOCS-1 mRNA expression and the percentage of IFN-'-producing CD4+ T cells in peripheral blood in both asthmatic and healthy children (P<0.05). No correlation was found between SOCS-1 and SOCS-3 expression and sIgE level.</p><p><b>CONCLUSIONS</b>Children with allergic asthma have elevated levels of SOCS-1 mRNA in PBMCs, which is associated with Th2-skewed immune response.</p>
Subject(s)
Adolescent , Child , Child, Preschool , Female , Humans , Male , Asthma , Allergy and Immunology , Cytokines , Genetics , Gene Expression Regulation , Interferon-gamma , Genetics , Interleukin-4 , Genetics , RNA, Messenger , Signal Transduction , Suppressor of Cytokine Signaling 1 Protein , Suppressor of Cytokine Signaling 3 Protein , Suppressor of Cytokine Signaling Proteins , Genetics , Th1 Cells , Allergy and Immunology , Th2 Cells , Allergy and ImmunologyABSTRACT
<p><b>OBJECTIVE</b>To investigate the effect of glucosylceramide synthase (GCS) on P-glycoprotein (P-gp) expression via extracellular signal-regulated kinase (ERK) pathways in leukemia K562/A02 cell line.</p><p><b>METHODS</b>K562/A02 multidrug resistance cells were treated with GCS siRNA and U0126, respectively. Expression of multidrug resistance protein 1 (MDR1) mRNA was analyzed with qRT-PCR. Phosphorylated ERK1/2, total ERK1/2 protein and P-gp in different groups were measured with Western blotting.</p><p><b>RESULTS</b>After treated with U0126, P-ERK1/2 was decreased along with the increased U0126 concentration. P-ERK1/2 and P-gp were apparently down-regulated by U0126 at the concentrations of 20 μmol/L, 40 μmol/L and 60 μmol/L. After being transfected with GCS siRNA, GCS mRNA was inhibited by 70.50% (58.00%-76.00%) in K562/A02 cells. Compared with the negative control, both P-ERK1/2 and P-gp were inhibited significantly after RNAi for 72 hours (P<0.01 and P<0.05, respectively.</p><p><b>CONCLUSION</b>GCS may affect the expression of P-gp by ERK signal transduction pathway in leukemia cells.</p>
Subject(s)
Humans , ATP Binding Cassette Transporter, Subfamily B, Member 1 , Genetics , Metabolism , Cell Line, Tumor , Drug Resistance, Multiple , Drug Resistance, Neoplasm , Glucosyltransferases , Metabolism , K562 Cells , Leukemia , Genetics , Metabolism , MAP Kinase Signaling SystemABSTRACT
<p><b>OBJECTIVE</b>To investigate the correlation of glucosylceramide synthase (GCS) gene and multidrug resistance 1 (MDR1) gene in inducing multidrug resistance in human multidrug-resistant K562/A02 cell line, and search for a novel strategy for reversing multidrug resistance of leukemia cells.</p><p><b>METHODS</b>The expression levels of GCS and MDR1 mRNA in K562 and K562/A02 cells were assayed by RT-PCR. siRNAs targeting the GCS and MDR1 gene were transfected into K562/A02 cells with liposome, respectively. The differential expression of GCS and MDR1 mRNAs, as well as their correlation, were detected by RT-PCR and real time quantitative-PCR(QPCR).</p><p><b>RESULTS</b>The expression level of GCS and MDR1 mRNA was dramatically lower in drug-sensitive K562 cells compared with the K562/A02 cells. The GCS mRNA was inhibited by 73%(59%-82%) and MDR1 mRNA expression was down regulated by 67% (38%-82%) in K562/A02 cells after being transfected with GCS siRNA. The expression level of MDR1 mRNA was inhibited by 81%(63%-91%) and GCS mRNA expression had no apparent change in K562/A02 cells treated with MDR1 small interference RNA(siRNA).</p><p><b>CONCLUSION</b>Positive correlation was detected between the expression of GCS and MDR1 mRNA in K562/A02 cells and MDR1 mRNA expression was down regulated after silencing the GCS gene expression.</p>
Subject(s)
Humans , ATP Binding Cassette Transporter, Subfamily B, Member 1 , Genetics , Cell Line, Tumor , Drug Resistance, Multiple , Genetics , Drug Resistance, Neoplasm , Genetics , Glucosyltransferases , Genetics , RNA, Messenger , Genetics , RNA, Small Interfering , Genetics , Physiology , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
<p><b>OBJECTIVE</b>To investigate the expression of the CDH13 gene and BCR/ABL fusion gene in chronic myeloid leukemia(CML) patients at different stages and explore their relationship.</p><p><b>METHODS</b>RNA was isolated from peripheral blood in 30 healthy adults, 22 primary CML patients and 25 blastic crisis of CML patients. We examined the expression of the CDH13 gene and BCR/ABL fusion gene using EVA Green real-time reverse transcriptase-polymerase chain reaction (RT-PCR).</p><p><b>RESULTS</b>The expression of the BCR/ABL fusion gene in the patients with blastic crisis CML was 4.72 folds as that of the patients with primary CML. The expression of the CDH13 gene in the primary and blastic crisis CML patients was significantly reduced to 36% and 25% of that in healthy controls, respectively. Moreover, negative correlation was found between the expressions of these two genes.</p><p><b>CONCLUSION</b>The study showed that the reduction of the CDH13 expression at different clinical stage of CML may account for the defective cell adhesion in CML, and the expression of the CDH13 gene was probably down-regulated by the BCR/ABL fusion gene.</p>
Subject(s)
Adolescent , Adult , Aged , Female , Humans , Male , Middle Aged , Cadherins , Genetics , Case-Control Studies , Fusion Proteins, bcr-abl , Genetics , Gene Expression Regulation, Neoplastic , Leukemia, Myeloid , Genetics , Pathology , Polymerase Chain ReactionABSTRACT
This study was purposed to explore the expression of glucosylceramide synthase (GCS) in human leukemia cells and its relationship with multidrug resistance. RT-PCR was used to analyze peripheral blood samples from 53 leukemia patients with multidrug resistance/non-resistance, and to detect the expression level of GCS gene in HL-60 cells and HL-60/ADR cells, the expression level was compared with the level of mdr-1. The expressions of GCS protein and P-gp protein in HL-60 cells and HL-60/ADR cells were assayed by Western blot analysis. The results showed that the relative optical density ratio of GCS gene amplified bands in samples of leukemia patients with drug-resistance was significantly higher than that in samples of leukemia patients with drug non-resistance group (P < 0.05), meanwhile the significant enhancement of optical density value of GCS gene amplified bands accompanied by high expression of mdr-1 gene. Their correlation showed positive (P < 0.01, r = 0.6). The GCS mRNA and protein were overexpressed in HL-60/ADR cells, and their expression levels were obviously higher than that in HL-60 cells, meanwhile the expression of mdr-1 mRNA and P-gp also significantly increased in HL-60/ADR cells. It is concluded that the high level of GCS in leukemia patients possibly is associated with multidrug resistance of leukemia cells.