ABSTRACT
This study was aimed at exploring the expression pattern of P2X family receptors (P2XR) in peritoneal macrophages and their relationship with the activation states of macrophages in Notch1-induced mouse T-ALL model. After establishment of the leukemia model, F4/80(+) peritoneal macrophages, F4/80(+)CD206(+) M2-like and F4/80(+)CD206(-) M1-like peritoneal macrophages were sorted by flow cytometry based on F4/80 and CD206 surface markers. The expression of P2XR in each cell population was detected by real time RT-PCR. The results showed that macrophages,M1-like and M2-like macrophages moderately expressed P2XR except for P2X5R. The expression of P2XR varied with the development of leukemia. The expression of P2X1R and P2X7R in peritoneal macrophages increased steadily; the expression of P2X2R and P2X3R decreased at late stage of leukemia;the expression of P2X4R slightly decreased at intermediate stage;the expression of P2X6R kept unchanged. At intermediate stage of leukemia, the expression of P2XR in M1-like and M2-like peritoneal macrophages varied. M1-like macrophages expressed higher level of P2X1R than M2-like macrophages, whereas M2-like macrophages expressed higher level of P2X7R than M1-like macrophages, which suggested that the expression of P2XR were related to the activation states. It is concluded that the expression of P2XR in peritoneal macrophages from leukemia mice is related to the progression of leukemia and the activation states of macrophages, which lay a foundation for further studying the role of macrophages in the development of leukemia.
Subject(s)
Animals , Mice , Cytokines , Metabolism , Disease Models, Animal , Macrophages, Peritoneal , Metabolism , Mice, Inbred C57BL , Precursor Cell Lymphoblastic Leukemia-Lymphoma , Metabolism , Receptor, Notch1 , Metabolism , Receptors, Purinergic P2X , Metabolism , Signal TransductionABSTRACT
This study aimed to construct the dual expression vectors of wide type or N187D mutant P2X7 receptor and intracellular domain of Notch1 (ICN1) linked by 2A peptide to coexpress them in leukemia cells so as to lay a foundation for further investigating the role of P2X7 in development of leukemia. Overlap PCR was used to construct the dual expression vectors encoding wide type or N187D mutant type P2X7 receptor and ICN1 linked by the self-cleaving 2A sequence. The results showed that stable expressing cell lines were obtained by retroviral infection followed by cell sorting after DNA sequence analysis. RT-PCR, Western blot, intracellular free calcium concentration analysis were used to verify the functionally successful construction of K562 cell line expressing P2X7 receptor alone or with ICN1. DNA sequence analysis revealed that all construction were right. The infection efficiency of packaged constructed virus ranged from 40% to 70% for K562 cells. Stable infected cell line was obtained by cell sorting. RT-PCR analysis revealed that P2X7 receptor and/or ICN1 could be detected at high level in their stable infected cell lines, respectively. Western blot analysis also showed that P2X7 receptor was highly expressed in cell line infected by virus with P2X7 receptor. Sustained increase in intracellular free calcium concentration ([Ca(2+)]i) could be observed in K562 cells overexpressing either type of P2X7 receptor upon stimulation with BzATP. It is concluded that the wide type or N187D mutant P2X7 receptor and ICN1 are simultaneously and functionally over-express in leukemia cells, which lay a foundation for further studying the role of P2X7 receptor in the development of leukemia.
Subject(s)
Humans , Gene Expression , Genetic Vectors , K562 Cells , RNA, Messenger , Genetics , Receptor, Notch1 , Genetics , Receptors, Purinergic P2X7 , Genetics , Retroviridae , GeneticsABSTRACT
This study was purposed to investigate the expression of ADAR1 isoforms of P110 and P150 during the development of murine leukemia. A Notch1 over-expressing murine T cell acute lymphoblastic leukemia model was used to study the expression of ADAR1. BMMNC were isolated at different stages of disease and CD45.2(+)GFP(+) leukemia cells were sorted by flow cytometry at late stage. The expression of ADAR1 was detected by real time quantitative PCR. The results showed that mouse bone marrow cells from both leukemia and control groups expressed P110 and P150. Difference of P110 and P150 mRNA expression were observed during the development of leukemia. The expression of P110 dramatically increased and was significantly higher than that in control group. However, the expression level of P150 in leukemia group decreased stably and reached one-fourth of that in control group at 14 day. Furthermore, similar expression patterns could be detected in sorted CD45.2(+)GFP(+) leukemia cells. It is concluded that the mRNA expressions of P110 and P150 show diverse patterns in the development of leukemia, suggesting that RNA editing mediated by ADAR1 isoforms may play different roles in leukemia.
Subject(s)
Animals , Mice , Adenosine Deaminase , Genetics , Gene Expression , Precursor T-Cell Lymphoblastic Leukemia-Lymphoma , Genetics , Protein Isoforms , Genetics , RNA Editing , RNA, Messenger , Genetics , RNA-Binding ProteinsABSTRACT
Objective:To investigate the inhibitory effects of melanoma differentiation associated gene-7(mda-7/IL- 24)on lymphoma cell line Namalwa in vitro and in vivo.Methods:Using RT-PCR,the expression of mda-7/IL-24 was examined in 10 malignant hematopoietic cell lines,including Namalwa,Raji,K562,NB4,U937,Ramous,CEM,KG1a, HL60,J6-1,etc.The coding region of mda-7/IL-24 was cloned from LPS-treated human peripheral blood mononuclear cells(PBMC)by RT-PCR,and the eukaryotic expression vector pTarget-IL-24 was constructed.The recombinant vector, after sequenced,was transfected into Namalwa cell line via lipofectamine reagent.The stable expression transfectants were selected by G418.The expression of mda-7/IL-24 mRNA and protein was verified by RT-PCR and Western blotting.MTT assay,colony forming assay,apoptosis detection,and tumorigenesis in nude mice were used to assess the effects of mda- 7/IL-24 on tumor proliferation,growth characteristics,colony forming,apoptosis,and tumorigenesis.Results:Expression of mda-7/IL-24 mRNA was not found in any of the 10 malignant hematopoietic cell lines and the expression of mda-7/IL- 24 mRNA and protein was found in Namalwa cells transfected with recombinant plasmid pTarget-IL-24.Significant de- crease in tumor cell viability was observed in Namalwa cells stably transfected with mda-7/IL-24,compared with control cells transfected with empty plasmid pTarget(P
ABSTRACT
<p><b>OBJECTIVE</b>To clone the entire coding sequence and analyze the function of P2X7 receptor of J6-1 human leukemia cells.</p><p><b>METHODS</b>The entire coding sequence of P2X7 receptor was amplified by RT-PCR and then inserted into pTARGET plasmid to construct an eukaryotic expressing plasmid followed by DNA sequencing. HEK293 cells stably expressing P2X7 receptor were obtained after transfection and screening, and confirmed by RT-PCR and Western blotting. The bleb formation upon agonist stimulation was observed under phase contrast microscope.</p><p><b>RESULTS</b>The entire coding sequence of P2X7 receptor of J6-1 cells was successfully cloned. DNA sequencing analysis revealed a substitution of G559, for A559, causing a substitution of Glu187 for Gln187. The P2X7 receptor derived from J6-1 cells could be functionally expressed in HEK293 cells, in which bleb formation could be detected upon stimulation.</p><p><b>CONCLUSIONS</b>The entire coding sequence of P2X7 receptors was successfully cloned from J6-1 leukemia cells. Other unknown mechanism may contribute to the dysfunction of P2X7 receptor in these cells.</p>
Subject(s)
Humans , Cell Line, Tumor , Cloning, Molecular , DNA, Complementary , Genetics , Gene Expression , Leukemia , Genetics , Metabolism , Receptors, Purinergic P2 , Genetics , Physiology , Receptors, Purinergic P2X7 , Reverse Transcriptase Polymerase Chain Reaction , TransfectionABSTRACT
Regulation of P2X7 receptor expression is of interest because activation of this receptor by extracellular ATP triggers a wide variety of cell functions in leukocytes. However, its expression and modulation in human peripheral blood mononuclear cells (PBMC) and monocytes remain unclear. RT-PCR was used to detect the constitutive level of P2X7 receptor and the levels upon stimulation with bacteria, bacterial product, mitogen and various cytokines in human PBMC and monocytes. P2X7 receptor mRNA was detected in PBMC and monocytes. P2X7 receptor expression in PBMC was up-regulated by interleukin-2, -4, -6 (IL-2, IL-4, IL-6) tumour necrosis factor-alpha (TNF-alpha), lipopolysaccharide (LPS) and heat-inactivated Staphylococcus aureus Cowan strain I (SAC). However, interferon-gamma (IFN-gamma), granulocyte-macrophage colony-stimulating factor (GM-CSF), macrophage colony-stimulating factor (M-CSF) and phytohemagglutinin-M (PHA-M) had little effect on the expression of P2X7 receptor. Furthermore, LPS and M-CSF could up-regulate P2X7 receptor expression in monocytes, while IFN-gamma, TNF-alpha and GM-CSF had weak effects, but pretreatment with these inducers could not further enhance LPS-stimulated P2X7 receptor expression in monocytes. The results obtained demonstrate that inflammatory stimuli drive P2X7 expression, thus supporting the hypothesis that P2X7 receptor may play a role in the inflammatory responses against bacteria infection, which need further verification.