Human telomerase, a cellular reverse transcriptase (hTERT), is a nuclear ribonucleoprotein enzyme complex that catalyzes the synthesis and extension of telomeric DNA. This enzyme is specifically activated in most malignant tumors but is usually inactive in normal somatic cells, suggesting that telomerase plays an important role in cellular immortalization and tumorigenesis. Terminal maturation of tumor cells has been associated with the repression of telomerase activity. Using maturation-sensitive and -resistant NB4 cell lines, we analyzed the pattern of telomerase expression during the therapeutic treatment of acute promyelocytic leukemia (APL) by retinoids. Two pathways leading to the down-regulation of hTERT and telomerase activity were identified. The first pathway results in a rapid down-regulation of telomerase that is associated with retinoic acid receptor (RAR)-dependent maturation of NB4 cells. Furthermore, during NB4 cell maturation, obtained independently of RAR by retinoic X receptor (RXR)-specific agonists (rexinoids), no change in telomerase activity was observed, suggesting that hTERT regulation requires a specific signaling and occurs autonomously. A second pathway of hTERT regulation, identified in the RAR-responsive, maturation-resistant NB4-R1 cell line, results in a down-regulation of telomerase that develops slowly during two weeks of all-trans retinoic acid (ATRA) treatment. This pathway leads to telomere shortening, growth arrest, and cell death, all events that are overcome by ectopic expression of hTERT. These findings demonstrate a clear and full dissociation between the process of tumor cell maturation and the regulation of hTERT mRNA expression and telomerase activity by retinoids. We propose telomerase expression as an efficient and selective target of retinoids in the therapy of tumors.H uman telomerase, a ribonucleoprotein enzyme, extends chromosome ends with (TTAGGG)n telomeric sequences, and thus plays a key role in maintaining telomere length and in cellular replicative lifespan (1-3). Several observations indicate that telomeres, DNA-protein structures located at the ends of eukaryotic chromosomes, are important in the immortalization process (4). In most human normal cells, telomeric DNA is progressively lost with each round of cell division (5). Thus, telomeres shorten to a critical length and signal the onset of senescence (6). In contrast, telomere length is stable in immortalized cells, including tumor cells. This stabilization of telomere length seems to be achieved through the induction of a telomerase activity (7). The human telomerase is composed of template RNA components (hTR; ref. 8) and two proteins, telomerase-associated protein-1 (TP-1; ref. 9) and telomerase reverse transcriptase (hTERT), which is thought to be the enzyme's catalytic subunit (10)(11)(12)(13)(14). The level of expression of hTERT is the rate-limiting component of this complex: most normal human somatic cells do not have detectable telomerase activity and lack expression of hTERT, whereas most imm...

CXCR4, the stromal cell-derived factor-1 receptor, plays an important role in the migration of hematopoietic progenitor/stem cells. The surface and cytoplasmic expression of CXCR4 on human hematopoietic CD34 + cells was investigated. We show that its surface expression is low, whereas a large part of CXCR4 protein is sequestered intracellularly. Using confocal microscopy, we demonstrated that CXCR4 is colocalized with EEA-1, Rab5, Rab4, and Rab11, which are localized in early and recycling endosomes. No significant colocalization of CXCR4 with lysosomal markers CD63 and Lamp-1 was detected.Using antibody feeding experiments, we report a role for CXCR4 constitutive endocytosis in subcellular localization in stably transduced UT7-CXCR4-GFP and CD34 + cells. Agonist-independent endocytosis of CXCR4 occurs through clathrin-coated vesicles. These data implicate a constitutive endocytosis in the regulation of CXCR4 membrane expression and suggest that constitutive endocytosis may be involved in the regulation of trafficking the human hematopoietic progenitor/stem cells to and in the bone marrow microenvironment.

Previous observations suggested that functional antagonism between FLI-1 and EKLF might be involved in the commitment toward erythrocytic or megakaryocytic differentiation. We show here, using inducible shRNA expression, that EKLF knockdown in mouse erythroleukemia (MEL) cells decreases erythrocytic and increases megakaryocytic as well as Fli-1 gene expression. Chromatin immunoprecipitation analyses revealed that the increase in megakaryocytic gene expression is associated with a marked increase in RNA pol II and FLI-1 occupancy at their promoters, albeit FLI-1 protein levels are only minimally affected. Similarly, we show that human CD34 ؉ progenitors infected with shRNA lentivirus allowing EKLF knockdown generate an increased number of differentiated megakaryocytic cells associated with increased levels of megakaryocytic and Fli-1 gene transcripts. Single-cell progeny analysis of a cell population enriched in bipotent progenitors revealed that EKLF knockdown increases the number of megakaryocytic at the expense of erythrocytic colonies. Taken together, these data indicate that EKLF restricts megakaryocytic differentiation to the benefit of erythrocytic differentiation and suggest that this might be at least partially mediated by the inhibition of FLI-1 recruitment to megakaryocytic and Fli-1 gene promoters. (Blood. 2008; 112:576-584) IntroductionErythrocytic and megakaryocytic lineages derive from a common bipotent progenitor (megakaryocyte/erythroid progenitor, MEP) able to generate only erythrocytic or megakaryocytic progenitors. 1,2 A common bipotent precursor, precursor for erythroid and megakaryocytic cells (PEM), has also been characterized in the spleen of anemic mice. 3,4 Despite this very close proximity, the molecular mechanisms controlling commitment toward either one of these 2 lineages remain poorly understood.Two main models have been proposed to explain the commitment of multipotent hematopoietic progenitors. 5,6 According to the "instructive model," lineage commitment is dictated by specific extracellular signals such as cytokines. Although erythropoietin and thrombopoietin enhance the proliferation, survival, and differentiation of already committed erythrocytic and megakaryocytic progenitors expressing erythropoietin-or thrombopoietin-specific receptors, it is now well established that they have no instructive role in the commitment. 7,8 Available data are more compatible with a "stochastic model," suggesting that commitment is dictated by the spontaneous formation of specific and mutually exclusive combinations of transcription factors. 5,6 At least 10 different transcription factors involved in erythrocytic and/or megakaryocytic differentiation have been identified. [9][10][11][12] Most of them, such as GATA-1, NF-E2, TAL-1, LMO2, FOG1, and GFI-1B, are involved in the regulation of both erythrocytic and megakaryocytic differentiation. However, there is no indication that differential expression of either one of these 6 factors might be involved in the commitment decision. Several transgenic...