No abstract

The AF10 gene encodes a putative transcription factor containing an N-terminal LAP/PHD zinc finger motif, a functional nuclear localization signal, an AT-hook domain, and a leucine zipper toward the C-terminus. AF10 is involved in 2 distinct chromosomal translocations associated with hematologic malignancy. The chimeric fusion proteins MLL/AF10 and CALM/AF10, resulting from the t(10; 11)(p12;q23) and the t(10;11)(p12;q14), respectively, consistently retain the leucine zipper motif of AF10. This part of the C-terminal region was used as bait in a yeast 2 hybrid screening of a testis complementary DNA library. The leucine zipper interacted with GAS41, a protein previously identified as the product of an amplified gene in a glioblastoma. GAS41 shows significant homology to the Saccharomyces cerevisiae protein ANC1 and to the human MLL fusion partners AF9 and ENL. The interaction was confirmed in vivo. Furthermore, the study showed by coimmunoprecipitation that GAS41 in IntroductionThe disruption of the human homologue of the Drosophila Trithorax (trx) gene, MLL on 11q23 1-4 by chromosomal translocations is a frequent event in human acute leukemia. These translocations, leading to the juxtaposition of genetic elements and formation of MLL fusion genes, occur in approximately 5% to 10% of human acute leukemias, but with a higher frequency in infant leukemias 5 and secondary leukemias. 6 Although the full biological function of MLL is uncertain, it is known to act as a positive regulator of HOX gene expression in development. 7,8 Currently, 20 different translocations affecting the MLL gene have been molecularly cloned and the partner genes identified. 9 Significantly, all such translocations result in in-frame fusions at the messenger RNA level, and, therefore, the rearrangements result in the production of chimeric proteins in which the N-terminus of MLL is consistently fused to the C-terminus encoded by the partner gene. [10][11][12] Because such a diverse group of proteins can be fused to MLL, the role of the fusion proteins in leukemogenesis remains obscure. 13 Although most of the fusion partners are structurally and functionally unrelated to each other, 14 a number are involved in transcriptional regulation. For example, ENL, AF9, and AF4 activate transcription from synthetic reporter genes in vivo. [15][16][17][18][19][20] The AF10 gene is one of the few MLL partner genes to be independently rearranged with a third gene in leukemia, the CALM gene in the t(10;11)(p12;q14) translocation. 21 AF10 complementary DNA (cDNA) encodes a 1084-aa protein, a member of a family of proteins including MLL, all carrying a conserved LAP/PHD finger domain. 22,23 There is also a putative AT-hook motif, 24 a bipartite nuclear localization signal, a leucine zipper domain, and a glutamine-rich region at the C-terminus. The latter is not present in all isoforms, as a result of alternative splicing. 25,26 Although different breakpoints have been described for AF10, the resultant fusion protein in both products consistently loses the...

To investigate the fate of human megakaryocytes, CD34+ hematopoietic progenitor cells were purified from the peripheral blood or bone marrow of healthy donors and seeded in serum-free chemically defined suspension cultures. In the presence of thrombopoietin (TPO; 100 ng/mL), CD34-derived cells showed an eightfold numerical expansion and a progressive maturation along the megakaryocytic lineage. Megakaryocyte maturation was characterized ultrastructurally by the presence of a demarcation membrane system and phenotypically by a high surface expression of αIIbβ3 integrin. The number of mature megakaryocytes peaked at days 12 to 15 of culture. On the other hand, the number of platelets released in the culture supernatant by CD34-derived megakaryocytes peaked at days 18 to 21, when a high percentage of megakaryocytes showed the characteristic features of apoptosis, as evaluated by electron microscopy, terminal deoxynucleotidyl transferase (TdT)-mediated d-UTP-biotin nick end-labeling technique (TUNEL) and uptake of propidium iodide. In other experiments, primary αIIbβ3+ megakaryocytic cells were directly purified from the bone marrow aspirates of normal donors and seeded in serum-free suspension cultures. In the absence of cytokines, αIIbβ3+ megakaryocytes progressively underwent apoptotic cell death. The addition of TPO but not interleukin-3 or erythropoietin showed some protection of αIIbβ3+ cells from apoptosis at early culture times (days 2 to 4), but it did not show any significant effect at later time points. These findings suggest that the terminal phase of the megakaryocyte life span is characterized by the onset of apoptosis, which can be modulated only to a certain extent by TPO.