52 research outputs found

    A functional assay for microRNA target identification and validation

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    MicroRNAs (miRNA) are a class of small RNA molecules that regulate numerous critical cellular processes and bind to partially complementary sequences resulting in down-regulation of their target genes. Due to the incomplete homology of the miRNA to its target site identification of miRNA target genes is difficult and currently based on computational algorithms predicting large numbers of potential targets for a given miRNA. To enable the identification of biologically relevant miRNA targets, we describe a novel functional assay based on a 3ā€²-UTR-enriched library and a positive/negative selection strategy. As proof of principle we have used mir-130a and its validated target MAFB to test this strategy. Identification of MAFB and five additional targets and their subsequent confirmation as mir-130a targets by western blot analysis and knockdown experiments validates this strategy for the functional identification of miRNA targets

    IL-2 and Antisense TGF-B1 Gene Therapy of the H238 Tumor and Analysis of TGF-B Receptors

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    Transforming growth factor-beta (TGF-p) belongs to a family of immunosuppressive cytokines, capable of regulating macrophages, T, B and NK cells. We have detected the TGF-p transcript being synthesized by H238 cells. This is herpes simplex-Type2 transformed murine fibrosarcoma cell line. The amount of TGF-p formed/106 cells was \u3e2000pg/ml. Previous attempts to control H238 tumorigenicity had sub-optimal efficacies. In addition, studies had shown that the FI238 cell line was not responsive to TGF-p. This project was an attempt to develop an improved modality to reduce H238 tumorigenicity and investigate potential abnormalities in expression of TGF-p receptors in the H238 cell line. We created expression plasmids encoding transgenes for murine IL-2 and antisense TGF-p and stably transfected H238 cells to create populations/clones of H238 cells expressing these transgenes. In vitro, expression analysis using ELISA and RT-PCR showed that H238 cells are capable of expressing functional IL-2 and, in addition, antisense transfected H238 cells showed a significant decrease in TGF-p production. The ultimate test, was to evaluate the tumorigenicity of these stable transfected cells in an immunocompetent BALB/c host, in comparison to wild type H238 cells. Mice injected with wild type and control H238 cells, showed a 80 - 100% tumor incidence. Mice injected with either antisense TGF-p or murine IL-2 transfected H238 cells showed almost no tumor incidence (0-20%). Mitogen stimulation of splenocytes from tumor bearing and non tumor bearing animals showed significant differences. Splenocytes from non tumor bearing animals displayed a higher stimulation index than from tumor bearing animals, probably alluding to lower amounts of tumor derived systemic TGF-p. The non-responsiveness of H238 cells to TGF-p was also confirmed. Our results corroborated Uhm, at al (1993), whereby the authors reported similar results. To evaluate this further, we hypothesized that a defect within the TGF-p receptors may be the cause of this inert response. Total H238 RNA was exctracted and mRNA for TGF-p receptors was amplified using primers specific to the Type 1 and Type 2 TGF-p receptors, these were sequenced to confirm their identity. Our sequence analysis concluded that the TGF-p receptors on H238 cells were essentially normal with no critical mutations which would validate either splicing or truncated products. Protein expression was confirmed using Western blot analysis. Whole cell lysate was also resolved into membrane and cytosolic fractions, and these probed with for the type 1 or the type2 receptor. In comparision, to positive cell lysate controls for these receptors, H238 cell lysates showed a comparitively less amount of receptor expression. Receptor expression was further quantitated using a laser scanning cytometer. Using this procedure, H238 cells showed approximately 50% less expression of the type 2 TGF-p receptor than the type 1 TGF-p receptor. Cytochemical staining for the receptors showed little membrane puncate staining, however, distinct staining was observed in the cytosolic regions of the cells. The lack of pucntate staining may be explained by either the relatively very few numbers of TGF-p receptors expressed/cell or there may be defects in the glycosylation and receptor trafficking. In addtion, the different ratios of the type 1 and type 2 receptor expression may also account for the inertness of H238 cells to TGF-p. This has been shown to be present in other tumor models and may explain the non responsiveness of FI238 cells to TGF-p

    Serum profiling reveals distinctive proteomic markers in the serum from chronic myeloid leukaemia patients

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    We have shown that proteomic profiling of sera from chronic myeloid leukaemia patients at presentation identifies five serum markers whose expression profile is significantly different from that seen in normal controls, non-malignant neutrophilia and in CML patients in HR following treatment with Gleevec. Of particular interest is the persistence of three of the five biomarkers following successful induction of HR, suggesting that those changes are disease specific. We have demonstrated that serum profiling can robustly identify disease groups and disease state in patients with CML compared with non-malignant and normal controls. In addition our data suggest that serum profiling of other haematological malignancies may provide new biomarkers for disease state and stag

    FLT3 and JAK2 mutations in acute myeloid leukemia promote interchromosomal homologous recombination and the potential for copy neutral loss of heterozygosity

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    Acquired copy neutralLOH (CN-LOH) is a frequent occurrence in myeloid malignancies and is often associated with resistance to standard therapeutic modalities and poor survival. Here, we show that constitutive signaling driven by mutated FLT3 and JAK2 confers interchromosomal homologous recombination (iHR), a precedent for CN-LOH. Using a targeted recombination assay, we determined significant iHR activity in internal tandem duplication FLT3 (FLT3-ITD) and JAK2V617F-mutated cells. Sister chromatid exchanges, a surrogate measure of iHR, was significantly elevated in primary FLT3-ITD normal karyotype acute myeloid leukemia (NK-AML) compared with wild-type FLT3 NK-AML. HR was harmonized to S phase of the cell cycle to repair broken chromatids and prevent iHR. Increased HR activity in G0 arrested primary FLT3-ITD NK-AML in contrast to wild-type FLT3 NK-AML. Cells expressing mutated FLT3-ITD demonstrated a relative increase in mutation frequency as detected by thymidine kinase (TK) gene mutation assay. Moreover, resistance was associated with CN-LOH at the TK locus. Treatment of FLT3-ITD- and JAK2V617F-mutant cells with the antioxidant N-acetylcysteine diminished reactive oxygen species (ROS), restoring iHR and HR levels. Our findings show that mutated FLT3-ITD and JAK2 augment ROS production and HR, shifting the cellular milieu toward illegitimate recombination events such as iHR and CN-LOH. Therapeutic reduction of ROS may thus prevent leukemic progression and relapse in myeloid malignancies.</p

    Leukemia associated antigens: their dual role as biomarkers and immunotherapeutic targets for acute myeloid leukemia

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    Leukemia associated antigens (LAAs) are being increasingly identified by methods such as cytotoxic T-lymphocyte (CTL) cloning, serological analysis of recombinant cDNA expression libraries (SEREX) and mass spectrometry (MS). In additional, large scale screening techniques such as microarray, single nucleotide polymorphisms (SNPs), serial analysis of gene expression (SAGE) and 2-dimensional gel electrophoresis (2-DE) have expanded our understanding of the role that tumor antigens play in the biological processes which are perturbed in acute myeloid leukemia (AML). It has become increasingly apparent that these antigens play a dual role, not only as targets for immunotherapy, but also as biomarkers of disease state, stage, response to treatment and survival. We need biomarkers to enable the identification of the patients who are most likely to benefit from specific treatments (conventional and/or novel) and to help clinicians and scientists improve clinical end points and treatment design. Here we describe the LAAs identified in AML, to date, which have already been shown to play a dual role as biomarkers of AML diseas
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