No abstract

No abstract

The pathways by which oncogenes, such as MLL-AF9, initiate transformation and leukemia in humans and mice are incompletely defined. In a study of target cells and oncogene dosage, we found that Mll-AF9, when under endogenous regulatory control, efficiently transformed LSK (Lin(-)Sca1(+)c-kit(+)) stem cells, while committed granulocyte-monocyte progenitors (GMPs) were transformation resistant and did not cause leukemia. Mll-AF9 was expressed at higher levels in hematopoietic stem (HSC) than GMP cells. Mll-AF9 gene dosage effects were directly shown in experiments where GMPs were efficiently transformed by the high dosage of Mll-AF9 resulting from retroviral transduction. Mll-AF9 upregulated expression of 192 genes in both LSK and progenitor cells, but to higher levels in LSKs than in committed myeloid progenitors.

MCM-41/ZSM-5 composites were prepared using a dual templating method through a process of two-step crystallization. Mesoporous MCM-41 was first synthesized using the self-assembling of surfactant cetyltrimethylammonium bromide and subsequently the amorphous wall of MCM-41 was recrystallized with a structure-directing agent tetrapropylammonium bromide, which was introduced into the MCM-41 wall through a pretreatment process. A solid to solid-phase transformation mechanism was presented for the recrystallization of MCM-41 framework. Two kinds of stable MCM-41/ZSM-5 composites can be synthesized during the course of recrystallization. Crystallized mesoporous MCM-41 containing only short-range ordered ZSM-5 structure was first synthesized in the early stage of the recrystallization. With the increase of recrystallization time, some discrete micron-sized ZSM-5 crystals were produced and firmly attached to the loose aggregates of crystallized MCM-41, and another kind of MCM-41/ZSM-5 composite containing interconnected mesopore and micropore was therefore obtained. Because of improved acidity and a 2-fold pore system, both MCM-41/ZSM-5 composites are more advantageous than amorphous MCM-41 and a mechanical mixture of MCM-41 and ZSM-5 in acid catalysis.

No abstract

The 2 most frequent human MLL hematopoietic malignancies involve either AF4 or AF9 as fusion partners; each has distinct biology but the role of the fusion partner is not clear. We produced Mll-AF4 knock-in (KI) mice by homologous recombination in embryonic stem cells and compared them with Mll-AF9 KI mice. Young Mll-AF4 mice had lymphoid and myeloid deregulation manifest by increased lymphoid and myeloid cells in hematopoietic organs. In vitro, bone marrow cells from young mice formed unique mixed pro-B lymphoid (B220 ؉ CD19 ؉ CD43 ؉ sIgM ؊ , PAX5 ؉ , TdT ؉ , IgH rearranged)/myeloid (CD11b/Mac1 ؉ , c-fms ؉ , lysozyme ؉ ) colonies when grown in IL-7-and Flt3 ligand-containing media. Mixed lymphoid/myeloid hyperplasia and hematologic malignancies (most frequently B-cell lymphomas) developed in Mll-AF4 mice after prolonged latency; long latency to malignancy indicates that Mll-AF4-induced lymphoid/myeloid deregulation alone is insufficient to produce malignancy. In contrast, young Mll-AF9 mice had predominately myeloid deregulation in vivo and in vitro and developed myeloid malignancies. The early onset of distinct mixed lymphoid/myeloid lineage deregulation in Mll-AF4 mice shows evidence for both "instructive" and "noninstructive" roles for AF4 and AF9 as partners in MLL fusion genes. The molecular basis for "instruction" and secondary cooperating mutations can now be studied in our Mll-AF4 model. IntroductionThe myeloid/mixed lymphoid leukemia gene (MLL) on human chromosome 11 was first described from a cell line derived from a patient with a hematologic malignancy that resulted from a reciprocal translocation involving chromosome 4. 1 MLL was subsequently shown to partner with many other genes to result in hematologic malignancy. 2 The fusion of MLL to AF4 family members, LAF4 and AF5, results in malignancies that are the most common and unique among the MLL fusion gene malignancies in that they are generally lymphoid or lymphoid/myeloid in type but rarely purely myeloid. They are also unique because of the high frequency in infants, extensive spread beyond the hematopoietic compartment, and a poor outcome with treatment. [3][4][5][6][7][8][9][10] In contrast to MLL-AF4, the fusion of MLL with most other partners, including the second most common partner AF9, 8 results in myeloid malignancies.To date, no murine model of MLL-AF4 translocation has been reported and thus neither the premalignant early events nor the eventual malignancies have been defined. In this study, we produced Mll-AF4 knock-in (KI) mice and compare them with Mll-AF9 mice developed previously. 11 The KI models, which have the advantage of having a single copy of the fusion gene in all stem/progenitor cells, permit control of bias introduced by a variable number of gene copies in the various progenitor populations in other models.The mechanisms for the association between the MLL partner gene and type of malignancy have not been elucidated. The MLL partner may be "instructive" in directing the selective expansion and transformation of cells t...

IntroductionLeukemias with MLL gene rearrangements occur most frequently in infants and as secondary malignancies and are associated with poor outcomes. Several studies have demonstrated that both human and murine MLL-rearranged leukemias have high expression of HOXA9 and MEIS1. 1-5 Recently, we found that the expression of 5Ј Hox-a genes and Meis1 in murine hematopoietic cells is proportional to the level of transformation, suggesting that the MLL-fusion gene induced overexpression of these genes is central to the pathogenesis of leukemia. 6 Wong et al have recently shown that retrovirally expressed MLL-fusion genes are incapable of transforming Meis1 Ϫ/Ϫ murine fetal liver cells. 7 In the current study, we report on Meis1 inhibition in murine Mll-AF9 knockin leukemia. The knockin model closely mimics the human disease because each cell contains a single copy of the gene, expressed from the endogenous promoter and the mice develop myeloid leukemia. 8 A cell line, derived from a leukemic Mll-AF9 knockin mouse with high Meis1 expression, was used in the current study. 9 We found that Meis1 is required for the growth and survival of Mll-AF9 leukemia cells in vitro and for growth of leukemia in vivo. Gene profiling data suggested mechanisms by which Meis1 might mediate these growth-and survival-promoting effects in this cell line. Finally, we show that human MLL-fusion gene leukemia cell lines also require MEIS1 for growth. Methods Cell cultureThe 4166 cell line was established from a leukemic Mll-AF9 knockin mouse. The human cell lines were obtained from ATCC (Manassas, VA). 10 Lentivirus shRNAsLentivirus short hairpin RNA (shRNA) clones were obtained from OpenBiosystems (Huntsville, AL); details are provided in the "Lentivirus" section in Document S1 (available on the Blood website; see the Supplemental Materials link at the top of the online article). For ease of description, these clones were designated M23, M24, M25, M26, and M27. The manually designed construct was labeled M1456. Cell-cycle and apoptosis analysis by flow cytometryThe 4166 cells were transduced with Meis1 shRNA at multiplicities of infection (MOI) of 10 to 100, and the cells were harvested at days 2 through 5 after transduction. Nuclei were stained with propidium iodide (PI), and analysis of nuclear DNA content was performed using the CellQuest-Pro software (BD Biosciences, San Jose, CA). Apoptosis was detected using the CaspaTag caspase activity kit (Millipore, Billerica, MA) as described previously. 11 Western blotting and immunohistochemistryWestern blotting was performed using anti-Meis1 antibody (Upstate Biotechnology, Charlottesville, VA) with antiactin (Sigma-Aldrich, St Louis, MO) used as a loading control. Cell-surface marker expression was determined by immunohistochemistry as previously described. 9 Myeloid colony-forming assayCells were cultured in methylcellulose medium under myeloid conditions, and colonies were counted and scored at 7 days as previously described. 9 TransplantationsFor in vivo experiments, 4166 cells were transd...

No abstract