Alkaloids ; pharmacology ; Cisplatin ; pharmacology ; Fluorouracil ; pharmacology ; Hep G2 Cells ; Humans ; Quinolizines ; pharmacology ; Tumor Necrosis Factor Ligand Superfamily Member 13 ; metabolism

The study was purposed to detect BAFF/APRIL gene expression changes in bone marrow mononuclear cells (BMMNC) and myeloma cell line U266 after interference with glucocorticoid and bortezomib. After separation of BMMNC from 7 patients with multiple myeloma, BAFF/APRIL mRNA expression in BMMNC and U266 cell line was detected by real-time PCR after treated with dexamethasone 100, 200 µg/ml, methylprednisolone 100, 200 µg/ml, bortezomib 0.1 µg/ml alone and dexamethasone or methylprednisolone combined with bortezomib respectively for 48 hours. The results showed that U266 cells and BMMNC of untreated MM patients highly expressed BAFF/APRIL genes. When dexamethasone, methylprednisolone or bortezomib was added to U266 cells or BMMNC alone, BAFF/APRIL gene expression decreased as compared with the blank control (p < 0.01). The inhibiting effect of bortezomib to BAFF/APRIL expression was obviously strong(p < 0.05). When dexamethasone or methylprednisolone combined with bortezomib, the BAFF/APRIL gene expression further decreased compared with dexamethasone or methylprednisolone alone (p < 0.01). As compared with the group of methylprednisolone combined with bortezomib, BAFF/APRIL gene expression decreased in dexamethasone combined with bortezomib with a statistically significant difference (p < 0.05). It is concluded that the expression of BAFF/APRIL gene is down-regulated after bing treated with glucocorticoids and bortezomib, which suggests that besides the glucocorticoid receptor and proteasomes targets, BAFF/APRIL and their receptor sites may be new targets of glucocorticoids and bortezomib.
B-Cell Activating Factor ; genetics ; metabolism ; Boronic Acids ; pharmacology ; Bortezomib ; Cell Line, Tumor ; Gene Expression Regulation, Neoplastic ; drug effects ; Glucocorticoids ; pharmacology ; Humans ; Multiple Myeloma ; metabolism ; Pyrazines ; pharmacology ; RNA, Messenger ; genetics ; Tumor Necrosis Factor Ligand Superfamily Member 13 ; genetics ; metabolism

OBJECTIVETo investigate the effects of lentivirus-mediated RNA interference (RNAi) targeting a proliferation-inducing ligand (APRIL) on the chemosensitivity to 5-FU of colorectal cancer cell line LoVo.

METHODSThe lentiviral vector siRNA-APRIL was constructed and verified by PCR and DNA sequencing. LoVo cells were transfected with siRNA-APRIL plasmid, non-targeting siRNA plasmid, or empty plasmid. Forty-eight hours after the transfection, the cells were examined for APRIL expression using Western blot. Seventy-two hours after treatment with 10 µg/ml 5-FU, flow cytometry was used to detect the cell apoptosis and cell cycle changes. The cell growth inhibition rate following 5-FU exposure was detected by MTT assay.

RESULTSPCR analysis and DNA sequencing demonstrated that the RNAi sequence targeting APRIL gene was successfully inserted into the lentiviral vector. siRNA-APRIL transfection resulted in obviously reduced expression of APRIL in LoVo cells. After 5-FU exposure, the apoptosis rate of siRNA-APRIL-transfected cells were increased to (21.12∓3.35)%, significantly higher than that in cells transfected with the non-targeting plasmid or the empty plasmid [(13.06∓1.92)% and (12.28∓1.79)%, respectively, P<0.01]; the cell number in G0/G1 phase increased while that in G2/M phase decreased in siRNA-APRIL-transfected cells. The growth inhibition rate in siRNA-APRIL group was (59.67∓5.03)%, significantly higher than that in the other two groups [(42.33∓4.16)% and (39.67∓4.73)%, respectively, P<0.01].

CONCLUSIONLentivirus-mediated RNAi targeting APRIL can effectively suppress the expression of APRIL in LoVo cells and enhance the chemosensitivity of the cells to 5-FU.

Apoptosis ; drug effects ; Cell Cycle ; drug effects ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Colorectal Neoplasms ; drug therapy ; metabolism ; pathology ; Drug Resistance, Neoplasm ; Fluorouracil ; pharmacology ; Humans ; Lentivirus ; genetics ; RNA Interference ; Tumor Necrosis Factor Ligand Superfamily Member 13 ; genetics ; metabolism

OBJECTIVETo investigate the roles of B-lymphocyte stimulator/ a proliferation-inducing ligand (BLyS/April) in immunological pathogenesis of idiopathic thrombocytopenic purpura (ITP).

METHODSThirty ITP children and 30 age-matched healthy children were studied. Reverse-transcription PCR (RT-PCR) and real-time PCR were used to evaluate the mRNA levels of BLyS/ April, receptors for BLyS/April (BR3, BCMA and TACI) and cytokines in ITP patients. Flow cytometry was performed to measure relative mean fluorescence intensity (relative MFI) for platelet-associated immunoglobulin G (PAIgG).

RESULTS(1) The transcription levels of BLyS/April in monocytes/macrophage [(8.30 +/- 2.31) x 10(-1) and (7.51 +/- 1.93) x 10(-3), respectively] were significantly up-regulated in acute ITP compared with that in healthy controls [(3.95 +/- 1.04) x 10(-1) and (3.08 +/- 0.82) x 10(-3), respectively] (P < 0.0.1). (2) Expression levels of the BLyS/April receptors BR3, BCMA and TACI mRNA were remarkably raised during acute phase of ITP (P < 0.01). (3) The mRNA levels of cytokines, including IL-4, IL-5, IL-6, IL-10 and IL-15, were significantly higher in acute phase ITP than in healthy controls (P < 0.01). (4) The mRNA levels of IL-10 and IFN-alpha were significantly elevated in acute phase of ITP. (5) Relative MFI of acute phase ITP patients (67.4 +/- 28.1) was higher than that in healthy controls (19.5 +/- 8.5) (P < 0.01), and there was a significant positive correlation between relative MFI and BLyS/April as well as their receptors (BR3, BCMA and TACI) (r = 0.56, 0.53, 0.62, 0.70, 0.45, respectively, P < 0.01), relative MFI in ITP patients decreased after treatment.

CONCLUSIONOver-expression of BLyS/April may be one of factors contributed to the immunological dysfunction in ITP.

Acute Disease ; B-Cell Activating Factor ; metabolism ; Case-Control Studies ; Child, Preschool ; Female ; Humans ; Infant ; Macrophages ; immunology ; metabolism ; Male ; Monocytes ; immunology ; metabolism ; Purpura, Thrombocytopenic, Idiopathic ; immunology ; metabolism ; Tumor Necrosis Factor Ligand Superfamily Member 13 ; metabolism

BCMA is one of the transmembrane receptors belonging to BAFF and APRIL. In order to identify the feasibility of sBCMA as decoy receptor and obtain active sBCMA for its structural and functional research, full length of hBCMA was amplified with total RNA from Raji cell line by RT-PCR, and the cDNA encoding the extracelluar soluble domain of hBCMA was inserted into pET43.1a(+) vector. The recombinant vector pET43.1a(+)-sBCMA was transformed into E. coli Origami B(DE3) pLyS which is helpful for disulfide bond construction of expression proteins. After IPTG induction, the recombinant protein was expressed as soluble fusion protein, sBCMA-NusA-His6, and identified by western blotting. Then the target protein was purified by Ni(+)-chelating Sepharose Fast Flow. The binding activity between recombinant sBCMA and BAFF was detected by ELISA. Also, Recombinant sBCMA inhibited proliferation of mouse B cell stimulating by rhsBAFF. It was proved that recombinant sBCMA has good bioactivity and the method to express those proteins rich in disulfide bond is feasible and effectual.
B-Cell Activating Factor ; chemistry ; B-Cell Maturation Antigen ; biosynthesis ; genetics ; Cloning, Molecular ; DNA, Complementary ; genetics ; Disulfides ; chemistry ; Escherichia coli ; genetics ; metabolism ; Humans ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; Solubility ; Tumor Necrosis Factor Ligand Superfamily Member 13 ; chemistry

This study was aimed to prepare the polyclonal antibody against the soluble proliferation-inducing ligand (sAPRIL) antigen and to investigate its effects in suppressing sAPRIL mediated lymphocyte proliferation. Mutated recombinant sAPRIL protein, which lacks biological activity but maintains immunogenicity, was used as antigen to immunize humanized SCID mice. Sera were obtained at 6 weeks after immunization. Indirect ELISA and Western blot were used to detect the antibody titer and specificity. The inhibition of polyclonal antibodies on Raji and Jurkat cell proliferation stimulated by sAPRIL was assessed by the MTT assay. The results showed that the mutant of sAPRIL could induce the production of polyclonal antibodies against human sAPRIL. Western blot and indirect ELISA analyses indicated that the anti-serum had higher specificity with a titer of 1:640. Functional analysis revealed that these polyclonal antibodies significantly inhibited the proliferation of Raji and Jurkat cell stimulated by sAPRIL (p < 0.05). It is concluded the polyclonal antibody against human sAPRIL is successfully prepared, which can inhibit the proliferation of Raji and Jurkat cells stimulated by sAPRIL in vitro.
Animals ; Antibodies ; genetics ; immunology ; pharmacology ; Antibody Specificity ; immunology ; Cell Proliferation ; drug effects ; Cloning, Molecular ; Humans ; Immune Sera ; analysis ; immunology ; Jurkat Cells ; Mice ; Tumor Necrosis Factor Ligand Superfamily Member 13 ; genetics ; immunology

This study was aimed to quantitatively detect the levels of april mRNA expression in leukemia patients so as to provide theoretical basis for the target therapy directing at april in leukemia. Real time fluorescent quantitative PCR was used to detect the relative expression level of april mRNA in newly diagnosed leukemia patients and to analyze the changes of its expression level in various type of leukemia. The results showed that the april mRNA expression level in acute leukemia (AL) patients was significantly higher than that in normal controls, there was statistical difference between them (p < 0.05); april mRNA expression level in acute myeloid leukemia (AML) patients was significantly higher than that in normal controls (p < 0.05) and positively correlated with white blood cell count ≥ 20.0 × 10(9)/L (p < 0.05), but not related with extramedullary infiltration and the expression of CD34. Except for acute promyelocytic leukemia (APL), april mRNA expression level was negatively correlated with sensitivity of patients to chemotherapy. april mRNA expression levels in acute lymphoid leukemia (ALL) and chronic myeloid leukemia (CML) patients were not higher than that in normal controls, there was no statistical difference between them (p > 0.05). It is concluded that april gene overexpression exits in AML patients. APRIL protein produced by AML cells probably plays an important role in abnormal proliferation and drug-resistance of AML cells.
Adolescent ; Adult ; Aged ; Aged, 80 and over ; Case-Control Studies ; Female ; Humans ; Leukemia ; genetics ; therapy ; Male ; Middle Aged ; RNA, Messenger ; genetics ; Tumor Necrosis Factor Ligand Superfamily Member 13 ; genetics ; Young Adult

OBJECTIVETo explore the relationship between a proliferation-inducing ligand (APRIL) expression in primary tumor foci of breast cancer and the patients' prognosis.

METHODSParaffin sections of surgical specimens were retrospectively collected from 130 stage I-III breast cancer patients who received surgery between January 2000 and December 2002 in our hospital. Immunohistochemistry was used to assess APRIL expression intensity in the tumor cells and density of interstitial APRIL-positive cells, and their association was analyzed with the density of interstitial CD4⁺ and CD8⁺ cells and with the histopathologic features, overall survival (OS), and disease-free survival (DFS) of the patients.

RESULTSAPRIL positive staining was found in the cytoplasm of the tumor cells, interstitial cells, and the extracellular matrix. APRIL intensity in the tumor cells was positively correlated with the density of interstitial APRIL-positive cells (P=0.009) and Ki67 (P=0.003). The density of interstitial APRIL-positive cells was positively correlated with the density of interstitial CD4⁺ cells (P<0.001) and CD8⁺ cells (P<0.001). In hormone receptor negative patients (ER- and PR-), multivariate COX regression identified the density of interstitial APRIL-positive cells as a positive prognostic factor for DFS (HR=0.313, 95% CI=0.107-0.920, P=0.035). CONCLUSIONSl APRIL is widely expressed in the interstitial immune cells in breast cancer. APRIL staining intensity in the tumor cells is positively correlated with tumor proliferation, indicating that the immune cells might promote tumor proliferation by secreting APRIL. A greater density of interstitial APRIL-positive cells is associated with a good prognosis in hormone receptor-negative patients.

Breast Neoplasms ; diagnosis ; metabolism ; CD4-Positive T-Lymphocytes ; CD8-Positive T-Lymphocytes ; Cell Transformation, Neoplastic ; Cytoplasm ; Disease-Free Survival ; Female ; Humans ; Immunohistochemistry ; Prognosis ; Retrospective Studies ; Tumor Necrosis Factor Ligand Superfamily Member 13 ; metabolism

B lymphocyte stimulator (BLyS), a tumor neurosis factor ligand superfamily, is an important factor of B cell survival and activation. However, BLyS also regulates T cell activation and survival, playing key roles in T cell-mediated autoimmune disorders. In the paper, we introduced the mechanisms of BLyS and a proliferation-inducing ligand (APRIL) regulating T cell responses and their roles in rheumatoid arthritis (RA).
Animals ; Antibodies, Monoclonal, Humanized ; therapeutic use ; Arthritis, Rheumatoid ; drug therapy ; metabolism ; pathology ; B-Cell Activating Factor ; metabolism ; B-Cell Activation Factor Receptor ; metabolism ; B-Cell Maturation Antigen ; metabolism ; B-Lymphocytes ; metabolism ; pathology ; Humans ; Lymphocyte Activation ; Recombinant Fusion Proteins ; therapeutic use ; T-Lymphocytes ; metabolism ; pathology ; Transmembrane Activator and CAML Interactor Protein ; metabolism ; Tumor Necrosis Factor Ligand Superfamily Member 13 ; metabolism

OBJECTIVETo study the effect of pGCsi-H1-APRIL on the growth of human colorectal cancer cells in transplated tumor in nude mice and to improve the effect of APRIL on proliferation and apoptosis of colorectal cancer (CRC).

METHODSHuman CRC model was established in nude mice, and the nude mice were treated with APRIL siRNA twice per week for 2 weeks. APRIL mRNA expression was surveyed by PCR and APRIL protein expression was detected by immunohistochemistry. The expression of PCNA protein was detected by ELISA. The expression of bcl-2 and bcl-xl was assessed by immunohistochemical staining, and TUNEL staining was used to detect apoptosis.

RESULTSThe expression of APRIL mRNA in the APRIL siRNA group was (0.13 ± 0.05) × 10(-3), significantly lower than that in the vector group (0.95 ± 0.04) × 10(-3) and the PBS group (0.96 ± 0.05) × 10(-3). The expression of APRIL protein in the APRIL siRNA group was (87.5 ± 5.0)% lower than that in the vector and PBS groups (P < 0.05). APRIL siRNA significantly suppressed the growth of SW480 tumor: the IR (inhibitory rate) of APRIL siRNA group was (60.7 ± 1.5)% (P < 0.05). The expression of PCNA in APRIL siRNA group was (176.8 ± 18.1) ng/ml, was (56.5 ± 2.0)% lower than that of PBS group (328.4 ± 22.8) ng/ml. Furthermore, the expressions of anti-apoptosis proteins bcl-2 and bcl-xl of APRIL siRNA group were (82.6 ± 4.5)% and (79.2 ± 3.5)% lower than those of the PBS group. The apoptotic rate of the APRIL siRNA group was 40.1% ± 2.5%, significantly higher than that in the vector group (2.5 ± 0.1)% and PBS group (2.5 ± 0.2)% (P < 0.05).

CONCLUSIONAPRIL siRNA may significantly suppress the growth and promote apoptosis in transplanted tumor of human colorectal cancer in nude mice. APRIL may become a candidate gene of gene therapy of human colorectal cancer.

Animals ; Apoptosis ; Cell Line, Tumor ; Cell Proliferation ; Colorectal Neoplasms ; genetics ; metabolism ; pathology ; Female ; Humans ; Ligands ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Neoplasm Transplantation ; Proliferating Cell Nuclear Antigen ; metabolism ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; RNA, Messenger ; metabolism ; RNA, Small Interfering ; genetics ; Random Allocation ; Tumor Necrosis Factor Ligand Superfamily Member 13 ; biosynthesis ; genetics ; bcl-X Protein ; metabolism