Glioma stem/progenitor cells (GSPCs) are considered to be responsible for the initiation, propagation, and recurrence of gliomas. The factors determining their differentiation remain poorly defined. Accumulating evidences indicate that alterations in autophagy may influence cell fate during mammalian development and differentiation. Here, we investigated the role of autophagy in GSPC differentiation. SU-2 cells were treated with rapamycin, 3-methyladenine (3-MA) plus rapamycin, E64d plus rapamycin, or untreated as control. SU-2 cell xenografts in nude mice were treated with rapamycin or 3-MA plus rapamycin, or untreated as control. Western blotting and immunocytochemistry showed up-regulation of microtubule-associated protein light chain-3 (LC3)-II in rapamycin-treated cells. The neurosphere formation rate and the number of cells in each neurosphere were significantly lower in the rapamycin treatment group than in other groups. Real-time PCR and immunocytochemistry showed down-regulation of stem/progenitor cell markers and up-regulation of differentiation markers in rapamycin-treated cells. Transmission electron microscopy revealed autophagy activation in rapamycin-treated tumor cells in mice. Immunohistochemistry revealed decreased Nestin-positive cells and increased GFAP-positive cells in rapamycin-treated tumor sections. These results indicate that rapamycin induces differentiation of GSPCs by activating autophagy.
Adenine ; analogs & derivatives ; pharmacology ; Animals ; Antibiotics, Antineoplastic ; pharmacology ; Autophagy ; drug effects ; Brain Neoplasms ; metabolism ; pathology ; Cell Differentiation ; drug effects ; Cell Line, Tumor ; Female ; Glial Fibrillary Acidic Protein ; genetics ; metabolism ; Glioma ; metabolism ; pathology ; Humans ; Leucine ; analogs & derivatives ; pharmacology ; Male ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Microtubule-Associated Proteins ; metabolism ; Neoplastic Stem Cells ; pathology ; RNA, Messenger ; metabolism ; Sirolimus ; pharmacology ; Xenograft Model Antitumor Assays

OBJECTIVETo explore the influence of inhibition of hypoxia-inducible factor-1 alpha (HIF-1 alpha) by RNA interference (RNAi) on tumorigenesis of human myeloma cell line (HMCL) RPMI8226 cells in nude mice.

METHODRNAi vector of HIF-1 alpha was constructed with commercial shRNA expression vector pSilencer 2. 1-U6 hygro. RT-PCR and western blot were used to detect HIF-1 alpha mRNA and protein expression respectively. Vascular endothelial growth factor (VEGF) secretion and cell cycle changes were detected by ELISA and flow cytometry respectively. Expression of target gene of HIF-1 alpha, VEGF and Glut-1 were tested under hypoxia condition. Tumorigenesis was observed after transfected cells were injected subcutaneously in nude mice.

RESULTSAfter interference, expression of HIF-1 alpha decreased significantly at both mRNA and protein level. Under normoxia condition, VEGF concentrations in HIF-la inhibited cells (RPMI8226-il and RPMI8226-i2) and non-inhibited cells (RPMI8226-c and RPMI8226) showed no differences. While under hypoxia condition, VEGF concentration in the above four cells was (506.0 +/- 53.2), (494.7 +/- 63.1), (984.4 +/- 61.9) and (938.2 +/- 62.2) pg/ml, respectively, being significantly lower in RPMI8226-il and RPMI8226-i2 cells than in RPMI8226-c and RPMI8226 cells (P <0.05). HIF-1 alpha interference was found to suppress the cells shift from S-phase to G1 induced by hypoxia. VEGF and Glut-1 expressions were markedly attenuated (P <0.05). The growth rate of HIF-1 alpha inhibition tumors in subcutaneous xenograft model decreased drastically.

CONCLUSIONSRNAi inhibits HIF-1 alpha expression. Reduced tumor growth by HIF-1 alpha inhibition may partly through inhibiton of angiogenesis and glycolysis metabolism.

Animals ; Cell Cycle ; Cell Line, Tumor ; Genetic Vectors ; Glucose Transporter Type 1 ; metabolism ; Humans ; Hypoxia-Inducible Factor 1, alpha Subunit ; genetics ; metabolism ; Mice ; Mice, Nude ; Multiple Myeloma ; metabolism ; pathology ; RNA Interference ; Vascular Endothelial Growth Factor A ; metabolism ; Xenograft Model Antitumor Assays

The aim of this research was to understand the influence of rhG-CSF on the sphingosine kinase (SphK) activity of monocytes. The peripheral blood monocytes were collected from 6 peripheral blood progenitor cell donors on the fifth day of mobilization with rhG-CSF and from 5 blood donors' buffy coats. The mRNA expressions of monocyte G-CSF receptor and SphK were tested with RT-PCR. The changes of SphK activity of monocytes were assayed after being treated with rhG-CSF. The results showed that the two kinds monocytes collected from both blood donors and peripheral blood progenitor cell donors mobilized with rhG-CSF expressed mRNA of G-CSF receptor and SphK. The SphK activity of monocytes collected from blood donors was not changed significantly after being treated with rhG-CSF (P > 0.05). The SphK activity of monocytes collected from peripheral blood progenitor cell donors transiently increased by (39.6 - 87.2)% after being treated by means of rhG-CSF (P < 0.05) without obviously dose-dependent effect. It is concluded that the SphK activity of monocytes collected from peripheral blood progenitor cell donors can be activated by rhG-CSF.
Granulocyte Colony-Stimulating Factor ; pharmacology ; Hematopoietic Stem Cell Mobilization ; Humans ; Monocytes ; cytology ; enzymology ; Phosphotransferases (Alcohol Group Acceptor) ; drug effects ; metabolism ; Receptors, Granulocyte Colony-Stimulating Factor ; biosynthesis ; genetics ; Recombinant Proteins

The study was to understand the impact on the proliferation and cytotoxicity of donor's T cells during mobilization with rhG-CSF. The peripheral blood mononuclear cells (PBMNC) were collected from 15 donors before mobilization and on fifth day of mobilization with rhG-CSF. After the PBMNC were activated with 500 ng/ml of CD3 monoclonal antibody and 500 microg/ml of rhIL-2 for 96 hours, the activated T cells were collected for testing proliferation, cytotoxicity, Fas expression, perforin and Fas ligand (FasL) mRNA expression, the IFN-gamma concentration in the culture medium of the activated T cells was determined by radioimmunoassay. The results showed that the proliferation activity of T lymphocytes and the cytotoxicity of T cells activated with CD3 monoclonal antibody and rhIL-2 were reduced markedly after mobilization with rhG-CSF (P < 0.05). The Fas molecule expression in the activated T cells was very high both before and after mobilization with rhG-CSF (P > 0.10). The activated T cells expressed perforin mRNA and didn't express FasL mRNA both before and after mobilization with rhG-CSF. The concentration of IFN-gamma in the culture medium of the activated T cells decreased significantly after mobilization with rhG-CSF (P < 0.01). It is concluded that activity of proliferation and cytotoxicity of donor's T cells is impaired after mobilization with rhG-CSF.
Adolescent ; Adult ; Cell Proliferation ; drug effects ; Cells, Cultured ; Fas Ligand Protein ; biosynthesis ; genetics ; Female ; Granulocyte-Macrophage Colony-Stimulating Factor ; administration & dosage ; pharmacology ; Hematopoietic Stem Cell Mobilization ; methods ; Humans ; Male ; Middle Aged ; RNA, Messenger ; biosynthesis ; genetics ; Recombinant Proteins ; T-Lymphocytes ; cytology ; drug effects ; T-Lymphocytes, Cytotoxic ; drug effects ; immunology ; fas Receptor ; biosynthesis ; genetics

Paf1 complex was identified in yeast and characterized to function in transcription and its related events. We identified the Drosophila homological components of paf1, CDC73 and RTF1 of paf1 complex. The genes encoding Drosophila paf1, CDC73 and RTF1 were cloned and expressed. With the purified recombinant proteins of truncated components of paf1 complex, antibodies against the Drosophila paf1, CDC73 and RTF1 were generated. These antibodies have been shown to be able to detect the endogenous paf1 subunits as well as their human counterparts in the HeLa extract. On Drosophila polytene chromosomes, these antibodies have been demonstrated to locate the paf1 complex at actively transcribing sites, which co-localized with phosphorylated RNA polymerase II, indicating that paf1 complex in Drosophila is involved in transcription or the events coupling with transcription.
Animals ; Antibodies ; chemistry ; Drosophila Proteins ; immunology ; Drosophila melanogaster

OBJECTIVETo explore the impact of mobilization with recombinant human granulocyte colony stimulated factor (rhG-CSF) on the migration and adhesive function and their related signal mechanism mediated by the CXCR4 and lymphocyte function antigen-1 (LFA-1) molecules on the surfaces of CD4(+) T cells.

METHODSBefore and at day 5 on rhG-CSF mobilization, the expression rates of CXCR4 and LFA-1 (CD11a) on CD4(+) T cells in the peripheral blood were detected by tricolor fluorescence labeling, and the migration and adhesive activities of CD4(+) T cells to stroma cell-derived factor 1 alpha (SDF-1 alpha) and intercellular adhesion molecule-1 (ICAM-1) were also tested.

RESULTSThe expression of CXCR4 on CD4(+) T lymphocytes was (84.58 +/- 20.31)% before mobilization and (81.23 +/- 22.46)% at day 5 on mobilization. The expression of LFA-1 on CD4(+) T lymphocytes before and at day 5 on mobilization was 100%. There was no significant difference in the expression CXCR-4 and LFA-1 on CD4(+) T lymphocytes whether mobilization (P > 0.05). SDF-1 alpha induced 4 hours' CD4(+) T cells migration didn't change markedly before and after mobilization \[(28.5 +/- 10.3)% vs (31.2 +/- 8.9)%\] (P > 0.05). The adhesive activity of CD4(+) T cells to ICAM-1 was decreased from (85.59 +/- 14.21)% to (61.45 +/- 15.07)% after mobilization (P < 0.05).

CONCLUSIONSThe expression of CXCR4 and LFA-1 on CD4(+) T lymphocytes didn't change markedly during rhG-CSF mobilization, but the adhesive activity of CD4(+) T cells to ICAM-1 was frustrated after that.

CD4-Positive T-Lymphocytes ; drug effects ; immunology ; metabolism ; physiology ; Cell Adhesion ; Cell Movement ; Cells, Cultured ; Chemokine CXCL12 ; physiology ; Granulocyte Colony-Stimulating Factor ; pharmacology ; Humans ; Intercellular Adhesion Molecule-1 ; physiology ; Lymphocyte Function-Associated Antigen-1 ; metabolism ; Receptors, CXCR4 ; metabolism ; Recombinant Proteins

Chronic myelogenous leukemia (CML) is a clonal myeloproliferative disease of transformed hematopoietic progenitor cells. It is now clear that the chimeric bcr/abl P210(bcr/abl) fusion protein, which is generated by the reciprocal translocation t (9; 22), inhibits apoptosis and increase proliferation. P210(bcr/abl) plays a central role in the pathophysiology of CML. The purpose of this study was to construct a cell line model that bcr/abl expression can be regulated by Tet-off inducing-expression-system. The full-length b3a2 bcr/abl cDNA was subcloned into the pTRE2hyg expression vector to construct the pT2-P210 plasmid. 293 cells were firstly transfected with Tet-off plasmid and the clone that the Tet-off system can work effectively after transfected with pTRE2hyg-LUC was selected by luciferase activity assay. The pT2-P210 plasmid was then transfected into the selected clone and cells were then selected for hygromycin B and G418 resistance. The results showed that individual subclones expressing bcr/abl after withdrawing doxycycline were 293pT2-P210 cell line. In conclusion, selected 293pT2-P210 cells are cells that bcr/abl expression can be regulated by Tet-off inducing-expression-system. They are suitable thoroughly to study the function of bcr/abl fusion gene and its signal regulation mechanism.
Base Sequence ; Cell Line, Transformed ; cytology ; physiology ; Chromosomes, Human, Pair 22 ; genetics ; Chromosomes, Human, Pair 9 ; genetics ; Fusion Proteins, bcr-abl ; biosynthesis ; genetics ; Gene Expression Regulation, Neoplastic ; Genes, abl ; genetics ; Humans ; Leukemia, Myelogenous, Chronic, BCR-ABL Positive ; genetics ; metabolism ; pathology ; Models, Genetic ; Molecular Sequence Data ; Proto-Oncogene Proteins c-bcr ; genetics ; Transfection ; Translocation, Genetic ; Tumor Cells, Cultured

Sphingosine 1-phosphate (S1P), which can be impacted by different growth factors through sphingosine kinase (SphK), is a bioactive lipid produced by metabolism of sphingolipid with various biological responses. Recombinant human granulocyte-colony-stimulating factor (rhG-CSF) have been used widely in peripheral blood stem/progenitor cell mobilization. This study was aimed to investigate the effects of rhG-CSF on S1P concentration in plasma of donors. The peripheral blood mononuclear cells and blood plasma were collected from 8 peripheral blood progenitor cell donors before mobilization and on the fifth day of mobilization with rhG-CSF. The SphK mRNA expression of blood mononuclear cells were detected by RT-PCR. The changes of S1P concentration were assayed with SphK enzyme catalyzed reaction. The results showed that both kinds blood mononuclear cells collected before and after rhG-CSF mobilization expressed SphK mRNA. The S1P concentration is low in donor's plasma both before and after mobilization with rhG-CSF, and there was no markedly change of S1P concentration in plasma before and after mobilization (P > 0.05). In conclusion, mobilization with rhG-CSF does not impact S1P concentration in donors' plasma.
Blood Donors ; Granulocyte Colony-Stimulating Factor ; therapeutic use ; Hematopoietic Stem Cell Mobilization ; Humans ; Lysophospholipids ; blood ; RNA, Messenger ; blood ; Recombinant Proteins ; Sphingosine ; analogs & derivatives ; blood

OBJECTIVETo analyze the efficacy and quality of life and safety for paclitaxel and carboplatin (TC) and TC combined with endostar in the treatment of advanced non-small cell lung cancer (NSCLC).

METHODSThis is a prospective, multicenter, randomized, double-blind, placebo-controlled clinical study. A total of 126 cases of untreated advanced NSCLC were enrolled in this study. There were 63 patients in the TC control arm and TC combined endostar arm, respectively. All enrolled patients were continuously followed-up for disease progression and death.

RESULTSThe objective response rate (ORR) of TC combined with endostar arm was 39.3%, and that of TC control arm was 23.0%, P = 0.078. The progression-free survival rates for TC combined with endostar arm and TC control arm were 78.3% and 58.8%, respectively, in 24 weeks (P = 0.017). The hazard ratio for the risk of disease progression was 0.35 (95%CI 0.13 to 0.90, P = 0.030). The median time to progression (TTP) of the TC combined with endostar arm was 7.1 months and TC arm 6.3 months (P > 0.05). The follow-up results showed that the median survival time (mOS) of the TC + Endostar arm was 17.6 months; (95%CI 13.4 to 21.7 months), and the TC + placebo arm 15.8 months (95%CI 9.4 to 22.9 months) (P > 0.05). The quality of life scores (LCSS patient scale) after treatment of the TC combined with endostar arm was improved, and that of the TC group was improved after completion of two cycles and three cycles of treatment. The quality of life scores compared with baseline after the completion of one cycle treatment was significantly improved for both the TC combined with endostar arm (P = 0.028 and), and TC arm (P = 0.036). It Indicated that TC combined with endostar treatment improved the patient's quality of life in the early treatment. The difference of adverse and serious adverse event rates between the two groups was not significant (P > 0.05).

CONCLUSIONSCompared with TC alone treatmrnt, TC combined with endostar treatment can reduce the risk of disease progression at early time (24 weeks), increase the ORR, and can be used as first-line treatment for advanced NSCLC. The TC combined with endostar treatment has good safety and tolerability, improves the quality of life, and not increases serious adverse effects and toxicity for patients with advanced NSCLC.

Antineoplastic Agents ; adverse effects ; therapeutic use ; Antineoplastic Combined Chemotherapy Protocols ; adverse effects ; therapeutic use ; Carboplatin ; administration & dosage ; Carcinoma, Non-Small-Cell Lung ; drug therapy ; pathology ; Disease Progression ; Disease-Free Survival ; Double-Blind Method ; Endostatins ; adverse effects ; therapeutic use ; Follow-Up Studies ; Humans ; Leukopenia ; chemically induced ; Lung Neoplasms ; drug therapy ; pathology ; Nausea ; chemically induced ; Neoplasm Staging ; Paclitaxel ; administration & dosage ; Prospective Studies ; Quality of Life ; Remission Induction

This study was aimed to investigate the effect of AML1-ETO fusion protein on the anti-apoptotic gene BCL-2 in leukemic cells and to explore its role in leukemogenesis. The apoptotic levels of U937-WT, U937-Mock and U937-A/E1-4 cells were examined by flow cytometry. And cleaved caspase-3 protein expression was detected by Western blot. BCL-2 gene expression both in AML1-ETO-expressing cells or U937 nonexpressing cells and in leukemia cells of AML patients with or without t(8;21) was assessed by quantitative PCR. The chromatin immunoprecipitation (ChIP)-based PCR was used to investigate the direct interaction between the AML1-ETO and BCL-2 promoter in AML1-ETO positive leukemia cell line. The results indicated that in U937-A/E cells but not in U937-WT or U937-Mock cells, apoptotic cells statistically significantly increased, and AML1-ETO expression also significantly enhanced activation of caspase-3. AML1-ETO-expressing cell subclones displayed significantly low levels of BCL-2 mRNA in comparison with the non-transfected U937. In primary bone marrow cells of acute myeloid leukemia containing AML1-ETO, levels of BCL-2 mRNA were markedly lower as compared with other acute myeloid leukemias lacking this translocation. The enriched regions in transfected cells were located within BCL-2 promoter. It is concluded that BCL-2 is the direct target gene of AML1-ETO. AML1-ETO can down-regulate the expression of BCL-2.
Core Binding Factor Alpha 2 Subunit ; genetics ; metabolism ; Gene Expression Regulation, Leukemic ; Humans ; Leukemia, Myeloid, Acute ; genetics ; metabolism ; Oncogene Proteins, Fusion ; genetics ; metabolism ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; RUNX1 Translocation Partner 1 Protein ; U937 Cells