BACKGROUND: The ability of transfected synthetic small interfering (si) RNAs to suppress the expression of specific transcripts has proved a useful technique to probe gene function in mammalian cells. However, high production costs limit this technology's utility for many laboratories and experimental situations. Recently, several DNA-based plasmid vectors have been developed that direct transcription of small hairpin RNAs, which are processed into functional siRNAs by cellular enzymes. Although these vectors provide certain advantages over chemically synthesized siRNAs, numerous disadvantages remain including merely transient siRNA expression and low and variable transfection efficiency. RESULTS: To overcome several limitations of plasmid-based siRNA, a retroviral siRNA delivery system was developed based on commerically available vectors. As a pilot study, a vector was designed to target the human Nuclear Dbf2-Related (NDR) kinase. Cells infected with the anti-NDR siRNA virus dramatically downregulate NDR expression, whereas control viruses have no effect on total NDR levels. To confirm and extend these findings, an additional virus was constructed to target a second gene, transcriptional coactivator p75. CONCLUSION: The experiments presented here demonstrate that retroviruses are efficient vectors for delivery of siRNA into mammalian cells. Retrovirus-delivered siRNA provides significant advancement over previously available methods by providing efficient, uniform delivery and immediate selection of stable "knock-down" cells. This development should provide a method to rapidly assess gene function in established cell lines, primary cells, or animals. (+info)
Rituximab reduces the number of peripheral blood B-cells in vitro mainly by effector cell-mediated mechanisms.
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BACKGROUND AND OBJECTIVES: The humanized CD20 mono- clonal antibody, rituximab, has significant anti-tumor activity in patients with B-cell non-Hodgkin's lymphoma and induces depletion of B-cells in vivo. It was the objective of this study to define the contribution of the different mechanisms of action of rituximab on primary normal and malignant B-cells. DESIGN AND METHODS: Primary human B-lymphocytes and effector cell fractions were isolated from peripheral blood of normal donors using an immunomagnetic separation technique. Blood samples from 20 patients with chronic lymphocytic leukemia (CLL) were studied and the B-lymphoblastoid Daudi cell line was used as a control. B-cells were cultured in the presence or absence of rituximab adding a secondary hyper-crosslinking antibody, serum as source of complement or different effector cell fractions. The cells were analyzed by immunofluorescence staining and flow cytometry. RESULTS: In contrast to the B-lymphoblastoid Daudi cell line, the number of highly purified normal peripheral blood CD19+ cells was only minimally affected by rituximab in the presence of autologous serum. A significant reduction in the number of B-cells was observed when mononuclear cells from peripheral blood were added back. To identify the cell type which mediates this effect, CD3+ T-cells, CD56+ cells, and CD14+ monocytes were added to selected CD22+ B-cells. A marked B-cell decrease was only observed in the presence of CD56+ and CD14+ cells in an effector to target ratio of 10:1. The experiments with mononuclear cells from patients with CLL showed a B-cell reduction by rituximab, which was significantly enhanced following addition of granulocyte-macrophage colony-stimulating factor (GM-CSF). INTERPRETATION AND CONCLUSIONS: These data support the important role of cell-mediated mechanisms in the B-cell-depleting action of rituximab and suggest that pre-treatment with GM-CSF could improve the response to rituximab in patients with CLL. (+info)
Reactivation of a silenced H19 gene in human rhabdomyosarcoma by demethylation of DNA but not by histone hyperacetylation.
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BACKGROUND: The active copy of the imprinted gene H19 is turned off by inappropriate methylation in several pediatric tumors including Wilms' Tumour and embryonal rhabdomyosarcoma. H19 controls in cis the linked Insulin-like Growth Factor 2 (IGF2) gene, encoding an important growth factor. Recent work has suggested that methylation of a gene may lead to deacetylation of its associated histones and that silenced genes can be reactivated by increasing histone acetylation levels. RESULTS: Treatment of a rhabdomyosarcoma cell line which has a silent, methylated H19 gene with histone deacetylase (HDAC) inhibitors under conditions which gave maximal hyperacetylation of histone 4, both globally and at the H19 gene itself could not reactivate H19 or affect the active Insulin-like Growth Factor 2 (IGF2) gene, but caused clear up-regulation of the Tissue-type Plasminogen Activator (TPA) gene, a non-imprinted gene known to respond to changes in histone acetylation. In contrast, mild treatment of the cells with the methylation inhibitor 5-AzaC-2'-deoxycytidine (AzaC) on its own was able to reactivate H19. Combining AzaC treatment with HDAC inhibitors gave a reduced rather than enhanced reactivation. These findings were confirmed in mouse primary liver and kidney explants which maintain normal imprinting, where we also found that the silent Igf2 gene could not be reactivated by HDAC inhibitors. CONCLUSION: These results suggest that DNA methylation rather than histone acetylation is the primary determinant of silencing of H19 in rhabdomyosarcoma. (+info)
Mapping common deleted regions on 5p15 in cervical carcinoma and their occurrence in precancerous lesions.
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BACKGROUND: Previous studies have shown that the short arm of chromosome 5 (5p) exhibit frequent genetic changes in invasive cervical carcinoma (CC), and that these changes arise early during the carcinogenesis, in precancerous lesions. These data therefore suggest that loss of candidate tumor suppressor genes located on 5p is associated with the development of CC. However, the precise location of 5p deletions is not known. RESULTS: We performed a detailed deletion mapping of 5p in 60 cases of invasive CC. We found that 60% of the tumors exhibit a 5p loss of heterozygosity (LOH). The patterns of LOH allowed us to identify two minimal regions of deletions, one at 5p15.3 spanning a 5.5 cM genetic distance and a second site of 7 cM at 5p15.2-15.3. In addition, we also identified 5p deletions in 16% lesions of high-grade cervical intraepithelial neoplasia (CIN). 5p LOH was found in 63% of HPV 16 positive tumors, while only 33% tumors with other HPV-types had 5p LOH. The differences in frequency of 5p LOH between tumors harboring HPV16 in combination with other HPV types and tumors harboring HPV16 DNA alone were significantly higher, suggesting a synergistic effect of high-risk types in causing genomic instability. CONCLUSION: These findings implicate the presence of tumor suppressor gene(s) on 5p relevant to CC tumorigenesis. (+info)
The mRNA expression of serotonin 2C subtype receptors uncoupled with inositol hydrolysis in NG108-15 cells.
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Cell culture systems seem to be useful for clarifying the cellular physiological mechanisms of serotonin 2C subtype receptors (5-HT2CR) and related drug action mechanisms. However, there are still few reports about cells that contain intrinsic 5-HT2CR. This report demonstrates by using RT/PCR that 5-HT2CR mRNA exists in splicing variant forms in NGI08-15 cells. The PCR results using a pair of primers that recognized sequences near the third intracellular loop site showed two neighboring bands at about 500 bp upon electrophoresis in acrylamide gels. The sequence analysis demonstrated that one band was the rat 5-HT2CR sequence and the other one was that of the mouse. Serotonin, however, did not enhance the inositol phosphates formation in NG108-15 cells. It has been reported that post-translational modifications of RNA, splicing and editing, occur at the site of the second intracellular loop domain in 5-HT2CR mRNA. Accordingly, a pair of primers that recognized this site were designed. The molecular size of the PCR product was shorter than that expected based on the sequence of the native 5-HT2CR. The fragment lacked the 95 nucleotides of native 5-HT2CR mRNA. This seems to be the reason why serotonin did not enhance inositol phosphates formation in NG108-15 cells. (+info)
Glycerol as a chemical chaperone enhances radiation-induced apoptosis in anaplastic thyroid carcinoma cells.
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INTRODUCTION: Anaplastic thyroid carcinoma, which is one of the most aggressive, malignant tumors in humans, results in an extremely poor prognosis despite chemotherapy and radiotherapy. The present study was designed to evaluate therapeutic effects of radiation by glycerol on p53-mutant anaplastic thyroid carcinoma cells (8305c cells). To examine the effectiveness of glycerol in radiation induced lethality for anaplastic thyroid carcinoma 8305c cells, we performed colony formation assay and apoptosis analysis. RESULTS: Apoptosis was analyzed with Hoechst 33342 staining and DNA ladder formation assay. 8305c cells became radiosensitive when glycerol was added to culture medium before X-ray irradiation. Apoptosis was induced by X-rays in the presence of glycerol. However, there was little apoptosis induced by X-ray irradiation or glycerol alone. The binding activity of whole cell extracts to bax promoter region was induced by X-rays in the presence of glycerol but not by X-rays alone. CONCLUSION: These findings suggest that glycerol is effective against radiotherapy of p53-mutant thyroid carcinomas. (+info)
Plasma PGE-2 levels and altered cytokine profiles in adherent peripheral blood mononuclear cells in non-small cell lung cancer (NSCLC).
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INTRODUCTION: PGE-2 is constitutively produced by many non-small cell lung cancers (NSCLC) and its immunosuppressive effects have been linked to altered immune responses in lung cancer. We asked whether elevated levels of plasma PGE-2 correlated with monocyte IL10 production in the NSCLC environment. Looking for correlation in NSCLC patient blood we assayed plasma from NSCLC patients for PGE2 and IL10; we further evaluated production of IL10 by adherent mononuclear cells from a subset of these patients looking for an altered cytokine profile. RESULTS: Our initial in vitro experiments show that monocyte IL10 induction correlates with tumor cell PGE-2 production, confirming similar reports in the literature. Data show plasma PGE-2 levels in 38 NSCLC patients are elevated compared to normal controls. Plasma IL10 levels were not significantly elevated; however, adherent monocytes derived from NSCLC patient blood did produce significantly more IL10 in 24 hr primary culture than those from normal controls (p < 0.01). The association of elevated plasma PGE-2 and monocyte derived IL-10 was not significant. CONCLUSIONS: Elevated plasma PGE-2 and monocyte IL10 production are associated with NSCLC. The biological significance to elevated PGE-2 levels in NSCLC are unclear. Further investigation of each as a nonspecific marker for NSCLC tumor is warranted. (+info)
Activation of the Syk tyrosine kinase is insufficient for downstream signal transduction in B lymphocytes.
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BACKGROUND: Immature B lymphocytes and certain B cell lymphomas undergo apoptotic cell death following activation of the B cell antigen receptor (BCR) signal transduction pathway. Several biochemical changes occur in response to BCR engagement, including activation of the Syk tyrosine kinase. Although Syk activation appears to be necessary for some downstream biochemical and cellular responses, the signaling events that precede Syk activation remain ill defined. In addition, the requirements for complete activation of the Syk-dependent signaling step remain to be elucidated. RESULTS: A mutant form of Syk carrying a combination of a K395A substitution in the kinase domain and substitutions of three phenylalanines (3F) for the three C-terminal tyrosines was expressed in a murine B cell lymphoma cell line, BCL1.3B3 to interfere with normal Syk regulation as a means to examine the Syk activation step in BCR signaling. Introduction of this kinase-inactive mutant led to the constitutive activation of the endogenous wildtype Syk enzyme in the absence of receptor engagement through a 'dominant-positive' effect. Under these conditions, Syk kinase activation occurred in the absence of phosphorylation on Syk tyrosine residues. Although Syk appears to be required for BCR-induced apoptosis in several systems, no increase in spontaneous cell death was observed in these cells. Surprisingly, although the endogenous Syk kinase was enzymatically active, no enhancement in the phosphorylation of cytoplasmic proteins, including phospholipase Cgamma2 (PLCgamma2), a direct Syk target, was observed. CONCLUSION: These data indicate that activation of Syk kinase enzymatic activity is insufficient for Syk-dependent signal transduction. This observation suggests that other events are required for efficient signaling. We speculate that localization of the active enzyme to a receptor complex specifically assembled for signal transduction may be the missing event. (+info)