Micro RNAs (miRNA) regulate gene expression by hybridization and recruitment of multi-protein complexes to complementary mRNA target sequences. miRNA function can transiently be antagonized by antagomirs—chemically modified oligonucleotides complementary to individual miRNAs. Here, we describe the induction of stable loss-of-function phenotypes for specific miRNAs by lentivirus-mediated antagomir expression. Lentivirally expressed antagomirs are transcribed from a H1-promoter located within the lentiviral 3′LTR and were directed against miRNAs encoded on the polycistronic miR17-92 transcript. Functional silencing of miR-18a, miR-19b and miR-20a by the corresponding antagomirs specifically relieves miRNA-mediated reporter gene repression. Inhibition of miRNA function correlates to reduction of ‘miRNA’ amplification by miRNA-specific quantitative RT-PCR. Furthermore, protein expression of E2F-1, a known miR-20 target, is enhanced by lentivirally expressed anti-miR-20 antagomirs in a dose-dependent manner, whereas over-expression of miR-20a reduces E2F-1 levels. Finally, combined over-expression of specific miRNAs and antagomirs reveals individual and complementary functions of miR-18a and miR-20a and demonstrates specific miRNA impact on cell proliferation in a cell culture model.

The results of this study demonstrate that the detection of tyrosinase mRNA in cells in the peripheral blood by RT-PCR may be a useful prognostic marker for predicting tumor progression and poor clinical outcome in patients with malignant melanoma.

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MicroRNAs (miRNAs) are small, noncoding RNAs that regulate gene expression by sequence-specific targeting of multiple mRNAs. Although lineage-, maturation-, and disease-specific miRNA expression has been described, miRNAdependent phenotypes and miRNAregulated signaling in hematopoietic cells are largely unknown. Combining functional genomics, biochemical analysis, and unbiased and hypothesis-driven miRNA target prediction, we show that lentivirally over-expressed miR-125b blocks G-CSF-induced granulocytic differentiation and enables G-CSF-dependent proliferation of murine 32D cells. In primary lineage-negative cells, miR-125b over-expression enhances colonyformation in vitro and promotes myelopoiesis in mouse bone marrow chimeras. We identified Stat3 and confirmed Bak1 as miR-125b target genes with approximately 30% and 50% reduction in protein expression, respectively. However, genespecific RNAi reveals that this reduction, alone and in combination, is not sufficient to block G-CSF-dependent differentiation. STAT3 protein expression, DNAbinding, and transcriptional activity but not induction of tyrosine-phosphorylation and nuclear translocation are reduced upon enforced miR-125b expression, indicating miR-125b-mediated reduction of one or more STAT3 cofactors. Indeed, we identified c-Jun and Jund as potential miR-125b targets and demonstrated reduced protein expression in 32D/ miR-125b cells. Interestingly, gene-specific silencing of JUND but not c-JUN partially mimics the miR-125b over-expression phenotype. These data demonstrate coordinated regulation of several signaling pathways by miR-125b linked to distinct phenotypes in myeloid cells. IntroductionMicroRNAs (miRNAs) represent an emerging class of noncoding single-stranded RNAs of approximately 22 nucleotides 1,2 that play an important role in posttranscriptional regulation of gene expression. miRNAs are processed from primary primiRNA transcripts to pre-miRNAs and mature miRNAs in a multistep process. Mature miRNAs are incorporated into and subsequently recruit a multi-protein effector complex RISC (RNA-induced silencing complex) to complementary miRNAbinding sites located preferentially within the 3ЈUTR of target mRNAs. Sequence-specific binding of RISC results in reduced mRNA translation and/or degradation through RNA interference (RNAi). 3 The interaction between a miRNA and its target mRNAs usually requires complementarity only within so-called seed sequence (miRNA nucleotides 2-8). Hence, a single miRNA has the potential to regulate hundreds of proteins 4-6 but resulting target protein repression is typically relatively mild. 5,6 Thereby, the ratio of regulatory RNAs to target mRNAs may modulate the silencing activity with a negative correlation between target abundance and target down-regulation. 7 To identify miRNA target genes, several prediction programs based on the hybridization energy of complementary miRNA/mRNAs sequences have been described. [8][9][10][11][12] However, the use of these programs is error-prone and identification of miRNA tar...

Recently, we have documented a hematopoietic NKL-code mapping physiological expression patterns of NKL homeobox genes in early hematopoiesis and in lymphopoiesis, which spotlights genes deregulated in lymphoid malignancies. Here, we enlarge this map to include normal NKL homeobox gene expressions in myelopoiesis by analyzing public expression profiling data and primary samples from developing and mature myeloid cells. We thus uncovered differential activities of six NKL homeobox genes, namely DLX2, HHEX, HLX, HMX1, NKX3-1 and VENTX. We further examined public expression profiling data of 251 acute myeloid leukemia (AML) and 183 myelodysplastic syndrome (MDS) patients, thereby identifying 24 deregulated genes. These results revealed frequent deregulation of NKL homeobox genes in myeloid malignancies. For detailed analysis we focused on NKL homeobox gene NANOG, which acts as a stem cell factor and is correspondingly expressed alone in hematopoietic progenitor cells. We detected aberrant expression of NANOG in a small subset of AML patients and in AML cell line NOMO-1, which served as a model. Karyotyping and genomic profiling discounted rearrangements of the NANOG locus at 12p13. But gene expression analyses of AML patients and AML cell lines after knockdown and overexpression of NANOG revealed regulators and target genes. Accordingly, NKL homeobox genes HHEX, DLX5 and DLX6, stem cell factors STAT3 and TET2, and the NOTCH-pathway were located upstream of NANOG while NKL homeobox genes HLX and VENTX, transcription factors KLF4 and MYB, and anti-apoptosis-factor MIR17HG represented target genes. In conclusion, we have extended the NKL-code to the myeloid lineage and thus identified several NKL homeobox genes deregulated in AML and MDS. These data indicate a common oncogenic role of NKL homeobox genes in both lymphoid and myeloid malignancies. For misexpressed NANOG we identified an aberrant regulatory network, which contributes to the understanding of the oncogenic activity of NKL homeobox genes.

The use of NDV as biological adjuvant in vaccines against human cancer is still actual in several clinical treatment protocols. In this study, we have investigated in vitro-effects of Newcastle disease virus (NDV) strain 73-T on isolated mononuclear blood cells and cultured tumor cells. Cellular cytotoxicity of PBMC freshly isolated from healthy donors against tumor cells was enhanced significantly (p < 0.01) after coincubation of NDV with effector cells. NDV failed to enhance cytotoxicity of effector cells when PBMC were stimulated three days with 500 IU recombinant interleukin-2 (rIL-2) per ml prior to coincubation with the virus. No significant enhancement of cellular lysis was seen when only target cells were coincubated with NDV. As shown by depletion of various lymphocyte subsets, NK cells were the predominant mediator of lysis. Enhancement of cytotoxicity correlated with the induction of interferon-alpha (IFN-alpha) in PBMC by NDV. NDV also induced high amounts of tumor necrosis factor-alpha (TNF-alpha) in PBMC. Induction of interferon-gamma (IFN-gamma) was weak. A direct cytopathic effect (CPE) of NDV on different target cells was detected by colorimetric measurement of metabolic cell activity. The human tumor cell lines A-498, A-704, Caki-1, Caki-2, and K-562 and the fibroblast line MRC-5 showed progressive cellular destruction 48 h after infection with NDV, whereas PBMC and Daudi cells remained unaffected during the observation period. The nontransformed monkey kidney cell line CV-1 and the transformed monkey kidney cell line COS-1 were both lysed by NDV with marginal difference in time course of CPE. Our results indicate a reasonable potential of pleiotropic modifications of the immune response against tumors by NDV.

Summary Interleukin 10 (IL-10) is an immunosuppressive factor and has been detected in tumour cell cultures of renal cell carcinoma and of malignant melanoma. IL-10 has been described as a cytokine of the Th2 response; it is able to suppress antigen-presenting cells (APCs) and may lead to down-regulation of HLA class I and II molecules on dendritic cells and to anergy of T-lymphocytes. We evaluated pretreatment serum levels of soluble IL-10 and various clinical parameters to determine their prognostic value in 80 advanced renal cell carcinoma patients seen at our institution between May 1990 and April 1996. For statistical evaluation we used both univariate and multivariate Cox proportional hazards models. An elevated pretreatment serum level of IL-10 was a statistically independent predictor of unfavourable outcome (P < 0.0028), in addition to the well-known clinical and biochemical risk factors. These data support risk stratification for future therapeutic trials and identify a predictor which needs to be validated in prospective studies and may potentially influence decision making in palliative management of patients with metastatic renal cell carcinoma. These data also suggest a potential role of IL-10 in the development of advanced renal cell carcinoma and in the future design of therapeutic strategies.

Signal transducers and activators of transcription (STATs) are latent cytoplasmic transcription factors linking extracellular signals to target gene transcription. Hematopoietic cells express two highly conserved STAT5-isoforms (STAT5A/STAT5B), and STAT5 is directly activated by JAK2 downstream of several cytokine receptors and the oncogenic BCR-ABL tyrosine kinase. Using an IL-3-dependent cell line with inducible BCR-ABL-expression we compared STAT5-activation by IL-3 and BCR-ABL in a STAT5-isoform specific manner. RNAi targeting of STAT5B strongly inhibits BCR-ABL-dependent cell proliferation, and STAT5B but not STAT5A is essential for BCL-XL-expression in the presence of BCR-ABL. Although BCR-ABL induces STAT5-tyrosine phosphorylation independent of JAK2-kinase activity, BCR-ABL is less efficient in inducing active STAT5A:STAT5B-heterodimerization than IL-3, leaving constitutive STAT5A and STAT5B-homodimerization unaffected. In comparison to IL-3, nuclear accumulation of a STAT5A-eGFP fusion protein is reduced by BCR-ABL, and BCR-ABL tyrosine kinase activity induces STAT5A-eGFP translocation to the cell membrane and co-localization with the IL-3 receptor. Furthermore, BCR-ABL-dependent phosphorylation of Y682 in STAT5A was detected by mass-spectrometry. Finally, RNAi targeting STAT5B but not STAT5A sensitizes human BCR-ABL-positive cell lines to imatinib-treatment. These data demonstrate differences between IL-3 and BCR-ABL-mediated STAT5-activation and isoform-specific effects, indicating therapeutic options for isoform-specific STAT5-inhibition in BCR-ABL-positive leukemia.