IntroductionNatural killer (NK) cells are cytotoxic and cytokine-producing lymphocytes, involved in the immune defense against viral infections and tumors. 1 Their homeostasis is regulated by cytokines and membrane associate receptors able to inhibit or activate cellular programs. 2 The inhibitory receptors are well characterized and described extensively in several reviews. [3][4][5] Triggering of NK cells depends largely on NK receptor member D of the lectinlike receptor family (NKG2D) and natural cytotoxicity receptors (NCRs): NKp46, NKp44, NKp30. 6,7 NCRs are involved in the recognition of several tumor cell lines, although their ligands remain elusive. 6 NKG2D recognizes the MHC (major histocompatibility complex) class I chain-related (MIC) protein A (MICA) and B (MICB); both are nonclassic class I molecules. The UL16-binding proteins (ULBP1-3 or RAET1 proteins; ULBP1-3 in this paper) are the second group of NKG2D ligands in humans. MICs are expressed during virus infection or cell transformation; ULBP expression in fresh tumor cells is essentially unknown; only long-term cultured in vitro cell lines have been looked at so far. [8][9][10] Cytotoxic T lymphocytes (CTLs) and interferons (IFNs) have a key role in tumor progression and tumor "immune-editing process." 11 MHC class I molecule loss is a frequent event in tumor progression and could prevent CTL recognition. However, theoretically, NK cells could recognize MHC class I-defective tumors, according with the "missing self hypothesis." 12 So far, only in mouse models NK cells were demonstrated to destroy in vivo lymphoma and melanoma tumors with reduced MHC class I expression and/or with high levels of activating target structures. [13][14][15] Even though almost 30 years ago human NK cells were discovered for their in vitro antitumor cytotoxicity, we still have little information concerning the regulation of their antitumor activity in vivo or ex vivo. 16,17 Therefore, several questions remain to be addressed to understand the antineoplastic potential of human NK lymphocytes:1. HLA class I molecules are reported to be down-regulated during solid tumor progression. 18 19,20 Other hematopoietic-derived cells can stimulate NK lymphocytes as described for dendritic cells (DCs). 21 The B-cell membrane-associated proteins CD40 and CD1 regulate natural killer cell cytotoxicity. [22][23][24][25] Furthermore, NK lymphocytes are specifically activated after bone marrow graft but not by other tissue transplantations. 26 They localize in lymph nodes and spleen, mainly in B-cell follicles and in the marginal zone. 27 Blood, spleen, and bone marrow are the anatomic districts where the highest number and activity of NK cells are present. 1 Taking together these considerations, hematologic malignancies (B-cell-derived tumors in particular) could be considered an appealing system to investigate the potential role of NK cells in the control of tumor progression.Multiple myeloma (MM) is a plasma cell-derived tumor. It is characterized by accumulation of plasma cells in th...

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.

We have previously demonstrated that the DNA-binding efficiency of Spl 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 Spl 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 Spl DNA-binding efficiency. However, in young nuclear extracts hydrogen peroxide treatment strongly decreases the Spl DNA-binding activity that is restored by the treatment with high dithiothreitol concentrations. To ascertain whether the oxidative stress is directed toward the Spl molecule alone, or whether it acts on unknown Spl cofactor(s) necessary for DNA binding, we purified Spl from young rat liver and demonstrated that when the purified protein is added to aged nuclear extracts, it efficiently binds to its DNA ciselement. Moreover, purified Spl treated with hydrogen peroxide lost its ability to bind its ciselement and the DNA-binding efficiency was fully restored after incubation with dithiothreitol.The aging process includes many modifications of gene expression [l-31. An altered transcription rate in aged organisms has been reported for several inducible genes, e.g. those encoding T-kininogen [4] and the heat-shock protein hsp70 [5, 61, and for many genes involved in the functioning of the immune system such as the genes of interleukin-2 and its receptor [7, 81, y-interferon [9] and interleukin-3 [lo]. This phenomenon could be a consequence of age-related changes in the expression andor of the activity of one or more factors regulating the transcriptional machinery. In senescent human fibroblasts, c-jios gene transcription is blocked by a dominant transcriptional repressor that prevents the appearance of the transient increase of c-jiis mRNA levels induced by serum, phorbol esters or CAMP [ll]. Recently, we demonstrated that the transcriptional factor Spl, present in nuclear extracts from aged rat tissues, binds its cognate DNA sequence with a decreased efficiency compared to that of Spl present in nuclear extracts from young animals [12]. In addition, the gene encoding the heavy chain of ferritin, which is Spl regulated, is transcribed at lower levels in aged tissues than in young tissues [12]. A quantitative analysis of the amount of Spl by immunoblotting demonstrated that both the quantities and the degree of heterogeneity of the Spl protein are nearly identical in the two nuclear extracts, suggesting that the observed differences in binding efficiency is not due to a different amount of this transcriptional factor [ 121. We thus concluded that the decreased DNA-binding efficiency could be due to a post-translational alteration of Spl in aged tissues.Protein oxidation in the aged tissues has been extensively investigated [13]. It is caused by the effect of such free...

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Zinc finger protein 521 (EHZF/ZNF521) is a multi-functional transcription co-factor containing 30 zinc fingers and an amino-terminal motif that binds to the nucleosome remodelling and histone deacetylase (NuRD) complex. ZNF521 is believed to be a relevant player in the regulation of the homeostasis of the hematopoietic stem/progenitor cell compartment, however the underlying molecular mechanisms are still largely unknown. Here, we show that this protein plays an important role in the control of B-cell development by inhibiting the activity of early B-cell factor-1 (EBF1), a master factor in B-lineage specification. In particular, our data demonstrate that: (1) ZNF521 binds to EBF1 via its carboxyl-terminal portion and this interaction is required for EBF1 inhibition; (2) NuRD complex recruitment by ZNF521 is not essential for the inhibition of transactivation of EBF1-dependent promoters; (3) ZNF521 represses EBF1 target genes in a human B-lymphoid molecular context; and (4) RNAi-mediated silencing of ZNF521/Zfp521 in primary human and murine hematopoietic progenitors strongly enhances the generation of B-lymphocytes in vitro. Taken together, our data indicate that ZNF521 can antagonize B-cell development and lend support to the notion that it may contribute to conserve the multipotency of primitive lympho-myeloid progenitors by preventing or delaying their EBF1-driven commitment toward the B-cell lineage.