Insulin-like growth factor 1 is a direct HOXA9 target important for hematopoietic transformation.

HOX homeobox proteins are key oncogenic drivers in hematopoietic malignancies. Here we demonstrate that HOXA1, HOXA6 and predominantly HOXA9 are able to induce the production of insulin-like growth factor 1 (Igf1). In chromatin immunoprecipitations, HOXA9 bound directly to the putative promoter and a DNase-hypersensitive region in the first intron of the Igf1 gene. Transcription rates of the Igf1 gene paralleled HOXA9 activity. Primary cells transformed by HOXA9 expressed functional Igf1 receptors and activated the protein kinase Akt in response to Igf1 stimulation, suggesting the existence of an autocrine signaling loop. Genomic deletion of the Igf1 gene by Cre-mediated recombination increased sensitivity toward apoptosis after serum starvation. In addition, the leukemogenic potential of Igf1-negative, HOXA9-transformed cells was impaired, leading to a significant delay in disease development on transplantation into recipient animals.

Printed disposable colorimetric array for metal ion discrimination.

One of the main limiting factors in optical sensing arrays is the reproducibility in the preparation, typically by spin coating and drop casting techniques, which produce membranes that are not fully homogeneous. In this paper, we increase the discriminatory power of colorimetric arrays by increasing the reproducibility in the preparation by inkjet printing and measuring the color from the image of the array acquired by a digital camera, using the H coordinate of the HSV color space as the analytical parameter, which produces robust and precise measurements. A disposable 31 mm × 19 mm nylon membrane with 35 sensing areas with 7 commercial chromogenic reagents makes it possible to identify 13 metal ions and to determine mixtures with up to 5 ions using a two-stage neural network approach with higher accuracy than with previous approaches.

Efficacy of cyclin-dependent-kinase 9 inhibitors in a murine model of mixed-lineage leukemia.

Mixed-lineage leukemia fusion proteins activate their target genes predominantly by stimulating transcriptional elongation. A core component necessary for this activity is cyclin-dependent kinase 9. Here we explored the effectiveness of small molecules targeting this enzyme as potential therapeutics. A screen of seven compounds with anti-CDK9 activity applied to a panel of leukemia cell lines identified flavopiridol and the experimental inhibitor PC585 as superior in efficacy with inhibitory concentrations in the submicromolar range. Both substances induced rapid dephosphorylation of the RNA polymerase II C-terminal domain, accompanied by downregulation of CDK9-dependent transcripts for MYC and HOXA9. Global gene expression analysis indicated the induction of a general stress response program, culminating in widespread apoptosis. Importantly, colony-forming activity in leukemia lines and primary patient samples could be completely inhibited under conditions that did not affect native precursors from bone marrow. In vivo application in a mouse transplant model significantly delayed disease with PC585 showing also oral activity. These results suggest CDK9 inhibition as novel treatment option for mixed-lineage leukemia.

Feasibility of the use of disposable optical tongue based on neural networks for heavy metal identification and determination.

This study presents the development and characterization of a disposable optical tongue for the simultaneous identification and determination of the heavy metals Zn(II), Cu(II) and Ni(II). The immobilization of two chromogenic reagents, 1-(2-pyridylazo)-2-naphthol and Zincon, and their arrangement forms an array of membranes that work by complexation through a co-extraction equilibrium, producing distinct changes in color in the presence of heavy metals. The color is measured from the image of the tongue acquired by a scanner working in transmission mode using the H parameter (hue) of the HSV color space, which affords robust and precise measurements. The use of artificial neural networks (ANNs) in a two-stage approach based on color parameters, the H feature of the array, makes it possible to identify and determine the analytes. In the first stage, the metals present above a threshold of 10(-7) M are identified with 96% success, regardless of the number of metals present, using the H feature of the two membranes. The second stage reuses the H features in combination with the results of the classification procedure to estimate the concentration of each analyte in the solution with acceptable error. Statistical tests were applied to validate the model over real data, showing a high correlation between the reference and predicted heavy metal ion concentration.

A surface fit approach with a disposable optical tongue for alkaline ion analysis.

A disposable optical tongue for the alkaline ions Na(I) and K(I) is described. The two-sensor layout prepared on a transparent support consists of non-specific polymeric membranes working by ionophore-chromoionophore chemistry. The non-specific behavior of the membranes was controlled by means of the crown ether-type ionophore present. The imaging of the tongue, after reaction for 3 min with the unknown solution, by means of a conventional flatbed scanner working by transmission mode, makes it possible to calculate the H (hue) value of the hue, saturation, value (HSV) color space used as a robust and precise analytical parameter. The modelling of the response of the two-sensor tongue as a sigmoidal surface is used to characterize the behavior of the tongue and as a basis to infer the concentration values. To compute the concentration of two analytes from the two hue values obtained using the optical tongue, a surface fit approach was used. The tongue works over a wide dynamic range (1.0×10(-4)-0.1 M both in Na(I) and K(I)). The sensing membranes show good intramembrane (1.4% RSD) and intermembrane precision (0.71% RSD) and lifetime (around 45 days in darkness). The procedure was used to analyze Na(I) and K(I) in different types of natural waters (tap and mineral), validating the results against a reference procedure.

Full-range optical pH sensor based on imaging techniques.

A new colour-based disposable sensor array for a full pH range (0-14) is described. The pH sensing elements are a set of different pH indicators immobilized in plasticized polymeric membranes working by ion-exchange or co-extraction. The colour changes of the 11 elements of the optical array are obtained from a commercial scanner using the hue or H component of the hue, saturation, value (HSV) colour space, which provides a robust and precise parameter, as the analytical parameter. Three different approaches for pH prediction from the hue H of the array of sensing elements previously equilibrated with an unknown solution were studied: Linear model, Sigmoid competition model and Sigmoid surface model providing mean square errors (MSE) of 0.1115, 0.0751 and 0.2663, respectively, in the full-range studied (0-14). The performance of the optical disposable sensor was tested for pH measurement, validating the results against a potentiometric reference procedure. The proposed method is quick, inexpensive, selective and sensitive and produces results similar to other more complex optical approaches for broad pH sensing.

Alterations of the CxxC domain preclude oncogenic activation of mixed-lineage leukemia 2.

The mixed-lineage leukemia (MLL) family of histone methyltransferases has become notorious for the participation of the founding member, MLL, in fusion proteins that cause acute leukemia. Despite structural conservation, no other MLL homolog has so far been found in a similar arrangement. Here, we show that fusion proteins based on Mll2, the closest relative of MLL, are incapable of transforming hematopoietic cells. Elaborate swap experiments identified the small CxxC zinc-binding region of Mll2 and an adjacent 'post-CxxC' stretch of basic amino acids as the essential determinants for the observed difference. Gel shift experiments indicated that the combined CxxC and post-CxxC domains of MLL and Mll2 possess almost indistinguishable DNA-binding properties in vitro. Within the cellular environment, however, these motifs guided MLL and Mll2 to a largely nonoverlapping target gene repertoire, as evidenced by nuclear localization, reporter assays, and measurements of homeobox gene levels in primary cells expressing MLL and Mll2 fusion proteins. Therefore, the CxxC domain appears to be a promising target for therapies aimed at MLL fusion proteins without affecting the general function of other MLL family members.

The oncoprotein MLL-ENL disturbs hematopoietic lineage determination and transforms a biphenotypic lymphoid/myeloid cell.

Mixed-lineage leukemia (MLL) fusion proteins are associated with a unique class of leukemia that is characterized by the simultaneous expression of lymphoid-specific as well as myeloid-specific genes. Here we report the first experimental model of MLL. Murine bone marrow cells were retrovirally transduced to express the MLL-eleven nineteen leukemia (MLL-ENL) fusion protein. When cultivated in flt-3 ligand, stem cell factor and interleukin-7 (IL-7) in a stroma-free culture system MLL-ENL-transduced as well as control cells showed a wave of B-lymphopoiesis. Whereas the controls exhausted their proliferative capacity in a CD19+/B220+ state, a continuously proliferating CD19-/B220+ cell population emerged in the MLL-ENL-transduced cultures. Despite the lymphoid surface marker, these cells were of monocytoid morphology. The immortalized cells contained unrearranged retrovirus, expressed MLL-ENL mRNA and were able to grow in syngenic recipients. From the diseased animals an MLL-ENL positive, B220+/CD19- cell type could be reisolated and cultivated in vitro. In analogy to human MLL, MLL-ENL-transformed cells not only coexpressed lymphocyte-specific (rag1, rag2, pax5, Tdt) and monocyte-specific genes (lysozyme, c-fms), but also showed rearrangements of the genomic immunoglobulin locus. This model shows that MLL-ENL influences events of early lineage determination and it will enable the investigation of the underlying molecular processes.

Transcriptional activation is a key function encoded by MLL fusion partners.

Chromosomal translocations that fuse the mixed lineage leukemia gene (MLL) to a variety of unrelated partner genes are frequent in pediatric leukemias. The novel combination of genetic material leads to the production of active oncoproteins that depend on the contributions of both constituents. In a search for a common function amongst the diverse group of MLL fusion partners we constructed artificial fusions joining MLL with generic transactivator and repressor domains (acidic blob, GAL4 transactivator domain, Herpes simplex VP16 activation domain, KRAB repressor domain). Of all constructs tested, only MLL-VP16 was able to transform primary bone marrow cells and to induce a block of early myeloid differentiation like an authentic MLL fusion. Interestingly, the transformation capability of the artificial MLL fusions was correlated with the transcriptional potential of the resulting chimeric protein but it was not related to the strength of the isolated transactivation domain that was joined to MLL. These results prove for the first time that a general biological function - transactivation - might be the common denominator of many MLL fusion partners.

MLL-ENL causes a reversible and myc-dependent block of myelomonocytic cell differentiation.

The translocation t(11;19) is a recurrent feature of a subgroup of acute leukemias occurring in infants. This event fuses the genes MLL and ENL and creates the leukemogenic oncoprotein MLL-ENL. We studied the effect of retroviral MLL-ENL expression in primary mouse hematopoietic cells and show here that MLL-ENL requires the oncoprotein Myc to establish a reversible differentiation arrest of a myelomonocytic precursor population. MLL-ENL-transduced cells proliferated as immature myeloid cells in the presence of interleukin 3. The addition of granulocyte colony-stimulating factor reversed the maturation block set by MLL-ENL and induced the development of mature granulocytes and macrophages accompanied by growth arrest. Gene expression analysis indicated a down-regulation of the proto-oncogene c-myc and of several c-myc target genes during granulocyte colony-stimulating factor-mediated differentiation. The role of c-myc in the MLL-ENL transformation pathway was tested by modulating the effective Myc protein concentrations in MLL-ENL transduced cells. Cotransduction of dominant-negative Myc neutralized the MLL-ENL effect and precluded transformation. In contrast, constitutive expression of Myc cooperated with MLL-ENL and caused the transformation of a cell population with an irreversible maturation arrest.

The ENL moiety of the childhood leukemia-associated MLL-ENL oncoprotein recruits human Polycomb 3.

The translocation t(11;19) is frequently found in acute leukemia in infants. This event truncates the proto-oncogene MLL and fuses the 5' end of MLL in frame with the ENL gene. ENL contributes a crucial protein-protein interaction domain to the resulting oncoprotein MLL-ENL. Here we show by yeast two-hybrid assays, GST-pull-down experiments and in a far western blot analysis that this domain is necessary and sufficient to recruit a novel member of the human Polycomb protein family (hPc3). hPc3 RNA was detected throughout the human hematopoietic system. Similar to other Polycomb proteins hPc3 acts as a transcriptional repressor. The ENL-hPc3 interaction was verified by mutual co-precipitation of the proteins from cell extracts. ENL and hPc3 tagged with fluorescent proteins co-localized in living cells in a nuclear dot pattern. An internal region of hPc3 was responsible for binding to ENL. Finally, hPc3 binds to the C-terminus of AF9, another common MLL fusion partner. The recruitment of a repressive function by ENL opens up a new insight into a possible mechanism of leukemogenesis by the fusion protein MLL-ENL.

ENL, the MLL fusion partner in t(11;19), binds to the c-Abl interactor protein 1 (ABI1) that is fused to MLL in t(10;11)+.

Translocations of the chromosomal locus 11q23 that disrupt the MLL gene (alternatively ALL-1 or HRX) are frequently found in children's leukemias. These events fuse the MLL amino terminus in frame with a variety of unrelated proteins. Up to date, 16 different fusion partners have been characterized and more are likely to exist. No general unifying property could yet be detected amongst these proteins. We show here that the frequent MLL fusion partner ENL at 19p13.1 interacts with the human homologue of the mouse Abl-Interactor 1 (ABI1) protein. ABI1 in turn, is fused to MLL in the t(10;11)(p11.2;q23) translocation. ABI1 was identified as an ENL binding protein by a yeast two-hybrid screen. The interaction of ENL and ABI1 could be verified in vitro by far-Western blot assays and GST-pulldown studies as well as in vivo by co-immunoprecipitation experiments. A structure-function analysis identified an internal region of ENL and a composite motif of ABI1 including an SH3 domain as mutual binding partners. These data introduce novel aspects that might contribute to the understanding of the process of leukemogenesis by MLL fusion proteins.

The leukemogenic fusion of MLL with ENL creates a novel transcriptional transactivator.

Translocations affecting the chromosomal locus 11q23 are hallmarks of infant leukemias. These events disrupt the MLL gene (also ALL-1 or HRX) and fuse the MLL amino terminus in frame with a variety of unrelated proteins. The ENL gene on 19p13.1 is a recurrent fusion partner of MLL. Whereas potential functions have been suggested for isolated domains of either MLL or ENL no experimental data exist for the biological properties of the complete chimeric MLL-ENL protein. We show here that the fusion of MLL with ENL creates a novel molecule that is a potent general transcriptional transactivator in transient reporter gene assays. MLL-ENL strongly transactivated several unrelated promoters including the promoter of Hoxa7 a potential target gene for the unaltered MLL protein. This transactivation capability was cell type specific and it was critically dependent on the contributions of the methyltransferase-homology (MT) region of MLL in combination with the C-terminus of ENL. Squelching experiments and gel retardation studies identified the ENL C-terminus as a binding partner for an unknown factor and the MLL MT region as a unique general DNA binding motif. The potential implications of these findings for the leukemogenesis by MLL-ENL are discussed.

RNA-dependent RNA polymerase activity associated with the yeast viral p91/20S RNA ribonucleoprotein complex.

20S RNA is a noninfectious viral single-stranded RNA found in most laboratory strains of the yeast Saccharomyces cerevisiae. 20S RNA encodes a protein of 91 kDa (p91) that contains the common motifs found among RNA-dependent RNA polymerases from RNA viruses. p91 and 20S RNA are noncovalently associated in vivo, forming a ribonucleoprotein complex. We detected an RNA polymerase activity in p91/20S RNA complexes isolated by high-speed centrifugation. The activity was not inhibited by actinomycin D nor alpha-amanitin. The majority of the in vitro products was 20S RNA and the rest was the complementary strands of 20S RNA. Because the extracts were prepared from cells accumulating 20S RNA over its complementary strands, these in vitro products reflect the corresponding activities in vivo. When the p91/20S RNA complexes were subjected to sucrose gradient centrifugation, the polymerase activity cosedimented with the complexes. Furthermore, an RNA polymerase activity was detected in the complex by an antibody-linked polymerase assay using anti-p91 antiserum, suggesting that p91 is present in the active RNA polymerase machinery. These results together indicate that p91 is the RNA-dependent RNA polymerase or a subunit thereof responsible for 20S RNA replication.

Yeast viral 20 S RNA is associated with its cognate RNA-dependent RNA polymerase.

Most Saccharomyces cerevisiae strains carry in their cytoplasm 20 S RNA, a linear single-stranded RNA molecule of 2.5 kilobases in size. 20 S RNA copy number is greatly induced in stress conditions such as starvation, with up to 100,000 copies per cell. 20 S RNA has coding capacity for a protein of 91 kDa (p91) with sequences diagnostic of RNA-dependent RNA polymerases of (+) strand and double-stranded RNA viruses. We detected p91 in 20 S RNA-carrying strains with specific antisera. The amount of p91 in growing cells is higher than that of stationary cells and similar to the one in 20 S RNA-induced cells. Although 20 S RNA is not encapsidated into viral particles, p91 non-covalently forms a ribonucleoprotein complex with 20 S RNA. This suggests a role of p91 in the RNA to RNA synthesis processes required for 20 S RNA replication. Although the strain analyzed also harbors 23 S RNA, a closely related single-stranded RNA, 23 S RNA is not associated with p91 but with its putative RNA polymerase, p104. Similarly, 20 S RNA is not associated with p104 but with p91. These results suggest that 20 S RNA and 23 S RNA replicate independently using their respective cognate RNA polymerases.