MLL-AF9-mediated immortalization of human hematopoietic cells along different lineages changes during ontogeny.

The MLL-AF9 fusion gene is associated with aggressive leukemias of both the myeloid and lymphoid lineage in infants, whereas in adults, this translocation is mainly associated with acute myeloid leukemia. These observations suggest that differences exist between fetal and adult tissues in terms of the 'cell of origin' from which the leukemia develops. Here we show that depending on extrinsic cues, human neonatal CD34(+) cells are readily immortalized along either the myeloid or lymphoid lineage upon MLL-AF9 expression and give rise to mainly lymphoid leukemia in immunocompromised mice. In contrast, immortalization of adult bone marrow CD34(+) cells is more difficult to achieve and is myeloid-biased, even when MLL-AF9 is expressed in purified hematopoietic stem cells (HSCs). Transcriptome analysis identified enrichment of HSC but not progenitor gene signatures in MLL-AF9-expressing cells. Although not observed in adult cells, neonatal cells expressing MLL-AF9 were enriched for gene signatures associated with poor prognosis, resistance to chemotherapeutic agents and MYC signaling. These results indicate that neonatal cells are inherently more prone to MLL-AF9-mediated immortalization than adult cells and suggest that intrinsic properties of the cell of origin, in addition to extrinsic cues, dictate lineage of the immortalized cell.

Identification by differential display of transcripts regulated during hematopoietic differentiation.

The polymerase chain reaction-based differential display method (DDRT-PCR) was used to identify mRNAs differentially expressed during the maturation of human CD34+ progenitor cells stimulated to differentiate in vitro towards granulomonocytic or erythroid lineages with a mixture of hemopoietins (kit ligand + interleukin 3 + GM-CSF in the absence or presence of erythropoietin, respectively). Three cDNA transcripts (B32, B41, and B56) display differential expression during cytokine-induced maturation of CD34+ cells. These clones have no homology with already-described sequences. Primer extension cofirmed the presence of the corresponding mRNA. The levels of mRNA corresponding to B32 are enhanced in the later phases of the granulomonocytic as well as in the erythroid differentiation of CD34+ cells. The mRNA identified by B41 was induced by a late stage in only granulomonocytic differentiation of CD34+ cells. The mRNA corresponding to B56 was instead present in nonstimulated CD34+ cells, declined in the early stages of differentiation, and reappeared at later stages in cells treated with both combinations of cytokines. Expression of these genes was detected in a number of acute myelogenous leukemias, as well as in some leukemic cell lines. B32 and B41 were downregulated in KG-1 cells induced to differentiate towards the monocytic lineage, whereas the levels of B56 were unchanged. In K562 cells, clones B41 and B56 were downregulated only in the late phases of PMA-induced megakaryocytic differentiation and during erythroid differentiation. B32 was rapidly downregulated when K562 cells were induced to differentiate towards either megakaryocytic or erythroid phenotypes. These transcripts represent novel hematopoietic cDNAs that should prove of value for the study of human blood cells and their disorders.

Differentially expressed mRNAs as a consequence of oxidative stress in intact cells.

Intracellular redox conditions influence the activity of several transcription factors leading to a modulation of the expression of the genes controlled by these factors. We examined the changes in cell transcription patterns after oxidative stress induced by diethylmaleate (DEM). Using the differential display technique we identified several differentially expressed sequence tags, four of which are identical or highly homologous to sequences contained in the human cDNAs encoding vimentin, c-fos, cytochrome oxidase IV and ribosomal protein L4; another one corresponds to a transcript of the mitochondrial genome of unknown function. The remaining five cDNAs are not recorded in any sequence data bank. One of these, named Rox3, lights up two mRNA species of approximately 3400 and 3600 bp, significantly increased after treatment with DEM or with other oxidizing agents. This increase appears precociously after exposure to DEM and it is completely prevented by pretreatment with N-acetylcysteine. The Rox3 fragment was used to screen a cDNA library; one fully sequenced clone showed 100% homology with the putative human guanine nucleotide regulatory protein nep1.

The DNA-binding efficiency of Sp1 is affected by redox changes.

We have previously demonstrated that the DNA-binding efficiency of Sp1 is greatly decreased in nuclear extracts from 30-month-old rat tissues compared to those from young ones, although its gene appears to be normally expressed. As reactive oxygen intermediates are known to accumulate in aged animals, we investigated the effect of oxidation on the Sp1 DNA-binding activity. Electrophoretic mobility shift assays and DNase I footprintings showed that high concentrations of dithiothreitol, added to the aged tissue extracts, fully restore the Sp1 DNA-binding efficiency. However, in young nuclear extracts hydrogen peroxide treatment strongly decreases the Sp1 DNA-binding activity that is restored by the treatment with high dithiothreitol concentrations. To ascertain whether the oxidative stress is directed toward the Sp1 molecule alone, or whether it acts on unknown Sp1 cofactor(s) necessary for DNA binding, we purified Sp1 from young rat liver and demonstrated that when the purified protein is added to aged nuclear extracts, it efficiently binds to its DNA cis-element. Moreover, purified Sp1 treated with hydrogen peroxide lost its ability to bind its cis-element and the DNA-binding efficiency was fully restored after incubation with dithiothreitol.

Immortalization of a cell line showing some characteristics of the oligodendrocyte phenotype.

We have used the polyoma middle T oncogene to immortalize cells from rat embryo encephalon. Immunostaining experiments with monoclonal antibodies demonstrated that the cells of one of the obtained lines, named CEINGE CL3, are stained by anti-vimentin and anti-S100 antibodies, are not stained by anti-neurofilaments (NF) or anti-glial fibrillary acidic-protein (GFAP) antibodies. Only a subset of the CEINGE CL3 cells (20-30%) is stained by an anti-galactocerebroside antibody. Northern blot analysis demonstrated that these cells express low levels of proteolipid protein mRNA, whereas polymerase chain reaction (PCR) amplification failed to evidentiate the presence of both NF and GFAP mRNAs. Either retinoic acid or forskolin treatments or a combination of them are able to induce morphological changes that are accompanied by a complete growth arrest.

Sp1 DNA binding efficiency is highly reduced in nuclear extracts from aged rat tissues.

To explore the role of transcriptional factors in the genesis of the senescent phenotype, nuclear extracts from 4- and 30-month-old rat brains were analyzed for the presence of DNA-binding proteins able to interact with double-stranded oligonucleotides containing recognition sites for sequence-specific DNA-binding factors. Gel shift assays revealed that the DNA-binding efficiency of Sp1 is significantly reduced in aged animals compared to young ones, whereas CTF/NF1 and AP1 from young and old rat nuclear extracts bind their DNA targets with the same efficiency. The quantitative analysis of Sp1 by immunoblotting indicated that equivalent quantities and degrees of heterogeneity of Sp1 protein are present in both nuclear extracts, suggesting that the observed difference is not due to a different expression of this transcriptional factor. DNase I footprinting of the heavy chain ferritin gene promoter, which contains a Sp1 binding site, demonstrated that the nuclear extract from 30-month-old rat brain does not protect the region involved in the regulation of the H ferritin gene by Sp1. This results in a reduction of about 50% of the expression of the H ferritin mRNA in aged rat brains. Furthermore, the Sp1 binding sites present in the SV40 early promoter are not protected in a DNase I footprinting assay where a nuclear extract from 30-month-old rat brain was used as a source of DNA binding proteins. Liver nuclear extracts prepared from young and aged rats demonstrated that a decrease of Sp1 binding efficiency is similarly present in this tissue.