Haematologica 2004;89: supplement no. 6 - Supplements ...

20Oral Communications: Molecular HematologyCO-03GENE EXPRESSION PROFILING OF HUMAN HEMATOPOIETICSTEM CELLS AND TERMINALLY DIFFERENTIATED MYELOIDCELLSZini R, Salati S, Siena M, Bianchi E, Tenedini E,Tagliafico E, Fogli M, § Lemoli RM, § Ferrari SE,Manfredini RDip. di Scienze Biomediche, Sez. di Chimica Biologica,Università di Modena e Reggio Emilia; § Istitutodi Ematologia e Oncologia Medica “L. & A. Seragnoli”,Università di Bologna, ItalyThe comparison of the gene expression profiles ofhemopoietic stem cells (HSC) and terminally differentiatedmyeloid cells can be remarkable for the molecularreconstruction of myeloid differentiation programsand for the identification of new lineage specificmarkers. In this work we studied the geneexpression profile of CD34 + HSC and normal terminallydifferentiated myeloid cells (monocytes, neutrophiland eosinophil) using Affymetrix HG-U95Av2GeneChip array technology. The global gene expressionanalysis showed a significantly higher complexityof mRNAs in CD34 + HSC population comparedwith terminally differentiated myeloid cells.The functional analysis of the differentially expressedgenes, performed using Gene Ontology (GO) MiningTool software, revealed a general metabolic activationin CD34 + cells (activation of DNA replication,transcription and RNA processing, ribosome and proteinsynthesis), while the majority of the preferentiallyexpressed genes in mature leucocytes werefound belonging to the defense immunity GO category.According to these preliminary observations,we found a preferential expression of G1/S cyclinsand CDKs in CD34 + cells, whereas CDK Inhibitors andgenes involved in immune response, such as inflammatorycytokines and chemokines receptors, cytotoxicgranules proteins, oxidative burst enzymes,MHC class II molecules and components of INFgammapathway are up-regulated in differentiatedmyeloid cells. Moreover, we found up-regulated inCD34 + cells the expression of self-renewal and lineagecommitment-related transcription factors (TFs),whereas leucocyte samples showed a preferentialexpression of TFs involved in maintenance of the terminallydifferentiated phenotypes. This work providesa strong molecular support to essential propertiesof the HSC and of terminally differentiatedmyeloid cells; moreover, in vitro functional assayswill allow us to identify the correlations betweenchanges in gene expression occurring in the commitmentphase and the activation of the myeloiddifferentiation program.CO-04EXPRESSION OF TRANSCRIPTIONAL COREPRESSOR NCOR ORITS RECEPTOR INTERACTION DOMAIN AFFECTS LIGANDBINDING TO WILD TYPE RETINOIC ACID RECEPTOR α ANDPML/RARBrambilla D,* # Fiorini R,* # Racanicchi S, §Maccherani C, § Grignani F, § Nervi C* #*Dipartimento di Istologia ed Embriologia Medica,Università “La Sapienza” Rome; # Parco ScientificoBiomedico di Rome San Raffaele, Rome; § Istituto diMedicina Interna e Scienze Oncologiche, Universitàdi Perugia, ItalyAcute promyelocytic leukemia (APL) is associatedwith reciprocal chromosomal translocations involvingthe RARα locus with either PML or more rarelyPLZF. Such fusion proteins inhibit physisologicalretinoid signalling via the RAR/RXR pathway and thisaction is linked to their oncogenic activity which isachieved through aberrant recruitment of nuclearcorepressor molecules such as NCoR or SMRT andhistone deacetylases. A unique feature of PMLRARαexpressing APL is its sensitivity to retinoic acid (RA)therapy, which induces remission by promoting cellulardifferentiation. To investigate the molecularmechanisms of leukemogenesis by PMLRARα and ofacquired RA resistance we addressed the biologicalrole of the interaction of transcriptional regulatorswith nuclear receptors (NR). To this end we expressedthe transcriptional co-repressor NCoR or its interactiondomains (IDC and IDN) into COS-1 cells (in cotransfectionwith RARα and PML/RARα), U937(expressing RARα) and NB-4 (expressing PMLRARα).In these cells we analyzed: i) the molecular interactionsof the above mentioned molecules withradioactive RA ([H3]-RA) through HPLC; ii) theeffects on RA target promoters and iii) differentiationstatus. An IDC with three aminoacids mutatedto alanine in the receptor interaction domain (IDCmut10)or an antisense IDN (IDNAS) were also usedas controls. The results obtained showed that theover-expression of NCoR or of IDC, its domain thatinteracts with the nuclear receptors, stronglyincreases the RA-binding to RARα and PML/RARα.Moreover, IDC increased PMLRARα binding toretinoic acid also when stably transfected into U937induced to express PMLRARα (U937-PR9). In contrast,the over-expression of IDN (another NCoRdomain that interacts with nuclear receptors), IDCmut10,IDNAS and of transcriptional co-activatorsTIF2 and NSD1 did not significantly modifiy thecapacity of RARα and PML/RARα to bind RA. NCoR,IDC and IDN, modified the conformation of the RAreceptors, as shown by tryptic digestion patterns ofRARand PML/RARα. In vitro binding assays with GSThaematologica vol. 89[suppl. n. 6]:september 2004