ABSTRACT
This study was aimed to compare the differential expressions of calcineurin (PP2B, PP3) in the mouse Pre-B cell lines (S9) and the tumor cell lines (S4C2) derived from pre-B lymphocytes, and to clarify its possible mechanism involving in the leukemia cell apoptosis. The quantitative real-time PCR was used to detect the differential expressions of H2AX-associated phosphakinase ATM, ATR, DNA-PKs, JNK1, P38 and the γ-H2AX-related phosphatase PP1, PP2A, calcineurin, PP4, PP6, PP5 between S9 and S4C2 cell lines. CCK-8 assay and flow cytometry were used to detect the effect of imatinib (IM) and cyclosporine A (CsA) on cytotoxicity and apoptosis of 2 cell lines. The Western blot was used to detect the effects of 2 drugs on apoptosis of S9 and S4C2 cell lines. The results showed that the expression level of calcineurin gene in the leukemia cell S4C2 was about 3.5 times of that in S9 cells, while the expression of other genes in these 2 kinds of cells was not significantly different. The apoptosis and toxicity of IM and CsA on S4C2 cells was significantly stronger than that on S9 cells. The expression level of calcineurin in S4C2 cells was higher than that in S9 cells.When CsA inhibited the calcineurin activity, the expression of DNA damage marker γ-H2AX in S9 cells was significantly lower than that in S4C2 cells,while the expression level of γ-H2AX between the two cell lines was no significantly different after treatment with imatinib, the expression level of γ-H2AX in S9 cells was lower than that in S4C2 cells when the two drugs were combined. It is concluded that the calcineurin plays a role of anti-apoptosis in B leukemic cells, cyclosporine A can promote the leukemia cell apoptosis.
Subject(s)
Apoptosis , Calcineurin/metabolism , Leukemia/metabolism , Precursor Cells, B-Lymphoid/metabolism , Animals , Cell Line, Tumor , Cyclosporine , DNA Damage , Flow Cytometry , Mice , Real-Time Polymerase Chain ReactionABSTRACT
This study was purposed to investigate the therapeutic effects of early transfusion of immunized donor lymphocytes after haploidentical transplantation by means of mouse model of nonmyeloablative haploidentical bone marrow transplantation. CB6F1 female mouse was served as recipient and C57BL/6 male mouse was served as donor. Each CB6F1 female mouse was subjected to intravenous transfusion with 1×10(6) erythroleukemia (EL9611) cells at day 4 before transplantation, followed with intraperitoneal injection of Ara-C (0.015 g) respectively at day 2 and day 1, then conditioned for BMT with TBI (450 cGy) at day 1 before transplantation. After conditioning (day 0), each of recipients was transplanted with 6×10(7) mixture of bone marrow and spleen cells from the C57BL/6 mice, and was infused with 6 × 10(7) immunized donor lymphocytes at day 15 after transplantation. All treated animals were evaluated for survival, development of leukemia and aGVHD. The donor CD3(+) cell chimerism and sex determining region Y gene (SRY)in recipients were monitored periodically after transplantation. The results showed tht all mice with only inoculation of 10(6) EL9611 cells survived for 15 ± 1 days (n = 4); all mice of other groups obtained the varying degrees of implantation. SRY could be detected at day 30 and 60 after transplantation. The chimerism of donor CD3(+) cells in mixed bone marrow transplantation (MT) group at day 14, 30 and 60 respectively reached 17.95% ± 12.03%, 37.34% ± 2.78% and 47.06% ± 6.1%. In donor lymphocyte infusion (DLI) group it reached 69.78% ± 12.62%, 75% ± 15.97%, 83.41% ± 16.07% at day 30, 45 and 60 after transplantation. The mice of MT and DLI group survived for 66.66 ± 1.47 days and 78.2 ± 7.82 days. It is concluded that the high tumor burden before transplantation can affect donor cell engraftment and prognosis.Early post-transplanted infusion of immunized lymphocytes from donor can help to improve the therapeutic efficacy and survival.
Subject(s)
Bone Marrow Transplantation/methods , Leukemia, Erythroblastic, Acute/therapy , Lymphocyte Transfusion , Animals , Female , Haplotypes , Male , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Tissue Donors , Transplantation Conditioning/methods , Transplantation, HomologousABSTRACT
OBJECTIVE: To detect the expression profile of NK ligands in acute leukemia cell lines and investigate the differential expression pattern between acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML). METHODS: Using quantitative real-time PCR, 23 NK ligands (MICA, MICB, ULBP-1, ULBP-2, ULBP-3, ULBP-4, HLA-E, HLA-G, CD48, NBTA, HLA-F, LLT-1, PVR, Nectin2, CD72, CD80, ICAM-1, LFA-3, CRACC, Fas, DR4, DR5, TNFR1) were detected in 6 acute leukemia cell lines, including 3 ALL cell lines (CEM, Jurkat T, Reh) and 3 AML cell lines (HL-60, KG-1a, NB4), respectively. Independent-samples t test analysis was performed to determine statistical significance. RESULTS: Using ß-actin as reference gene, the relative expression results showed that the expression of 4 NK ligands between ALL and AML is significantly different. Specifically, the level of ULBP-2 is higher in ALL (CEM: 1, Jurkat T: 0.617, Reh: 0.246) than that in AML (HL-60: 0.000, KG-1a: 0.003, NB4: 0.000)(P = 0.047). However, the expressions of CD48, PVR(PVR-1, PVR-2) and DR4 is higher in AML (HL-60: 13.987, 4.403, 10.334, 8.711; KG-1a: 5.387, 2.900, 7.315, 4.512; NB4: 7.763, 3.248, 7.049, 6.127) than that in ALL (CEM: 1, 1, 1, 1; Jurkat T: 2.035, 1.553, 3.888, 0.449; Reh: 1.559, 0.000, 0.000, 1.304) (P = 0.044, 0.014, 0.014, 0.011). And there're no significant differences between the rest 19 NK ligands. CONCLUSIONS: ULBP-2, CD48, PVR and DR4 might play an important role in the distinct mechanisms in leukemogenesis between ALL and AML and could be potential targets for diagnosis and treatment.
Subject(s)
Leukemia/metabolism , Membrane Proteins/metabolism , Real-Time Polymerase Chain Reaction , Acute Disease , Antigens, CD/genetics , Antigens, CD/metabolism , CD48 Antigen , Cell Line, Tumor , GPI-Linked Proteins/genetics , GPI-Linked Proteins/metabolism , HL-60 Cells , Humans , Intercellular Signaling Peptides and Proteins/genetics , Intercellular Signaling Peptides and Proteins/metabolism , Leukemia/genetics , Leukemia, Myeloid, Acute/genetics , Leukemia, Myeloid, Acute/metabolism , Ligands , Membrane Proteins/genetics , Precursor Cell Lymphoblastic Leukemia-Lymphoma/genetics , Precursor Cell Lymphoblastic Leukemia-Lymphoma/metabolism , Receptors, TNF-Related Apoptosis-Inducing Ligand/genetics , Receptors, TNF-Related Apoptosis-Inducing Ligand/metabolism , Receptors, Virus/genetics , Receptors, Virus/metabolismABSTRACT
AIM: To express DnaJ-homologous chaperon peripherin-binding protein(PBP) gene in E.coli and prepare the rabbit antibody against PBP. METHODS: The PBP cDNA was amplified from the human fetal brain tissue by RT-PCR. After confirmed by DNA sequencing, the PBP-cDNA was cloned into expression vector pET28a and then the PBP gene was expressed in E.coli under the IPTG induction. The expressed protein was purified through Ni-NTA affinity chromatography column. The rabbit antibody against PBP was prepared by immunizing two New Zealand white rabbits using the purified PBP as immunogen. The titer and specificity of the antisera were determined by Western blot. RESULTS: The 720 bp PBP gene was amplified, cloned, and expressed in E.coli. The expressed product existed in the bacterial inclusion body and the supernatant of the bacteria lysate. The purified PBP reached electrophoretic purity. The rabbit antibody against PBP was prepared and its titer was about 1:1,600. Western blot analysis showed that the antibody could bind to the expressed PBP protein specifically. CONCLUSION: The PBP protein was expressed in E.coli and rabbit antibody against PBP was prepared successfully, which lays the foundation for further study on the structure and biological function of PBP.