The t(8;21)(q22;q22) translocation, present in approximately 5% of adult acute myeloid leukemia (AML) cases, produces the AML1/ETO (AE) fusion protein. Dysregulation of the Pit/Oct/Unc (POU) ...domain-containing transcription factor POU4F1 is a recurring abnormality in t(8;21) AML. In this study, we showed that POU4F1 overexpression is highly correlated with, but not caused by, AE. We observed that AE markedly increases the self-renewal capacity of myeloid progenitors from murine bone marrow or fetal liver and drives the expansion of these cells in liquid culture. POU4F1 is neither necessary nor sufficient for these AE-dependent properties, suggesting that it contributes to leukemia through novel mechanisms. To identify targets of POU4F1, we performed gene expression profiling in primary mouse cells with genetically defined levels of POU4F1 and identified 140 differentially expressed genes. This expression signature was significantly enriched in human t(8;21) AML samples and was sufficient to cluster t(8;21) AML samples in an unsupervised hierarchical analysis. Among the most highly differentially expressed genes, half are known AML1/ETO targets, implying that the unique transcriptional signature of t(8;21) AML is, in part, attributable to POU4F1 and not AML1/ETO itself. These genes provide novel candidates for understanding the biology and developing therapeutic approaches for t(8;21) AML.
Mammary epithelium can functionally regenerate upon transplantation. This renewal capacity has been classically ascribed to the function of a multipotent mammary gland stem cell population, which has ...been hypothesized to be a primary target in the etiology of breast cancer. Several complementary approaches were employed in this study to identify and enrich mammary epithelial cells that retain stem cell characteristics. Using long-term BrdU labeling, a population of label retaining cells (LRCs) that lack expression of differentiation markers has been identified. LRCs isolated from mammary primary cultures were enriched for stem cell antigen-1 (Sca-1) and Hoechst dye-effluxing "side population" properties. Sca-1(pos) cells in the mammary gland were localized to the luminal epithelia by using Sca-1(+/GFP) mice, were progesterone receptor-negative, and did not bind peanut lectin. Finally, the Sca-1(pos) population is enriched for functional stem/progenitor cells, as demonstrated by its increased regenerative potential compared with Sca-1(neg) cells when transplanted into the cleared mammary fat pads of host mice.
Patients who undergo autologous stem cell transplantation (ASCT) for lymphoma have a significant risk of therapy-related acute myeloid leukemia and myelodysplasia (t-AML/MDS). Compared to that seen ...in other indications such as breast cancer, multiple myeloma or germ cell tumors, there is a substantially increased risk for t-AML/MDS following ASCT for lymphoma. This risk has largely been attributed to the extent of pre-transplant chemotherapy and radiation therapy. In many of the larger series to date, it has not been possible to directly implicate autologous transplantation itself as a risk factor for t-AML/MDS. Although pre-transplant therapy is certainly an important factor in the development of t-AML/MDS, specific components of the autologous transplantation procedure itself may also contribute to the risk of t-AML/MDS. Specifically, priming chemotherapy, total body irradiation, and the extensive cellular proliferation which occurs during engraftment may all play a role in the development of t-AML/MDS. Furthermore, there is an increasing body of evidence that certain inherited polymorphisms in genes governing drug metabolism, DNA repair and leukemogenesis may influence susceptibility to t-AML/MDS. In this paper, we review the evidence implicating the above risk factors for t-AML/MDS, present a potential mechanism for t-AML/MDS and propose interventions to reduce the rate of t-AML/MDS in lymphoma patients.
CD8
+ cytotoxic lymphocytes, natural killer cells and lymphokine-activated killer cells depend primarily on the perforin/granzyme system to kill their targets, while CD4
+ T cells utilize Fas and ...other mechanisms to induce cell death. The molecular mechanisms used by these pathways to induce target cell apoptosis may converge on common death substrates.
This study was focused on studying the role of Sca-1 (Ly-6 A/E) in hematopoietic stem/progenitor cell self-renewal, activation, and lineage fate.
Sca-1
−/− bone marrow cells were transplanted into ...wild-type recipient mice and assessed for self-renewal activity and lineage choice. In addition, Sca-1
−/− mice were injected with 5-FU and Lin
− cells were analyzed. Sca-1 was also overexpressed in mouse and human stem/progenitor cells to assess the effect of Sca-1 overexpression on stem/progenitor differentiation and proliferation.
Self-renewal of Sca-1
−/− HSC appeared to be normal, but lineage skewing was observed in B cells, NK cells, and granulocytes/macrophages derived from Sca-1
−/− HSC. There was also a decrease in c-kit expression on activated Sca-1
−/− progenitor cells. Overexpression of mouse Sca-1 decreased the in vitro myeloid activity of both mouse and human progenitors.
These data indicate that Sca-1 plays a role in hematopoietic progenitor/stem cell lineage fate and c-kit expression. In addition, mouse Sca-1 overexpression affects human as well as mouse stem/progenitor cell activity, suggesting the possibility of a functional human Sca-1 homologue.
The natural killer gene complex (NKC) on chromosome 6 contains clusters of genes that encode both activation and inhibitory receptors expressed on mouse natural killer (NK) cells. NKC genes, ...particularly belonging to the Nkrp1 and Ly49 gene families, display haplotype differences between different mouse strains and allelic polymorphisms of individual genes, as previously revealed by conventional analysis in a small number of inbred mouse strains. Herein we used array-based comparative genomic hybridization (aCGH) to efficiently compare the NKC in 21 mouse strains to the reference C57BL/6 strain. By using unsupervised clustering methods, we could sort these variations into the same groups as determined by previous RFLP analyses of Nkrp1 and Ly49 genes. Prospective analyses of aCGH and RFLP data validated these relationships. Moreover, aCGH data predicted monoclonal antibody reactivity with an allospecific determinant on molecules expressed by NK cells. Taken together, these data demonstrate the structural variation in the NKC between mouse strains as well as the usefulness of aCGH in analysis of complex, polymorphic gene clusters.
The t(8;21)(q22;q22) translocation, present in 10-15% of acute myeloid leukemia (AML) cases, generates the AML1/ETO fusion protein. To study the role of AML1/ETO in the pathogenesis of AML, we used ...the Ly6A locus that encodes the well characterized hematopoietic stem cell marker, Sca1, to target expression of AML1/ETO to the hematopoietic stem cell compartment in mice. Whereas germ-line expression of AML1/ETO from the AML1 promoter results in embryonic lethality, heterozygous Sca1 +/AML1-ETO ires EGFP (abbreviated Sca +/AE ) mutant mice are born in Mendelian ratios with no apparent abnormalities in growth or fertility. Hematopoietic cells from Sca +/AE mice have markedly extended survival in vitro and increasing myeloid clonogenic progenitor output over time. Sca +/AE mice develop a spontaneous myeloproliferative disorder with a latency of 6 months and a penetrance of 82% at 14 months. These results reinforce the notion that the phenotype of murine transgenic models of human leukemia is critically dependent on the cellular compartment targeted by the transgene. This model should provide a useful platform to analyze the effect of AML1/ETO on hematopoiesis and its potential cooperation with other mutations in the pathogenesis of leukemia.
Graft-versus-host disease (GvHD) is the major limiting toxicity of allogeneic bone marrow transplantation. T cells are important mediators of GvHD, but the molecular mechanisms that they use to ...induce GvHD are controversial. Three effector pathways have been described for cytotoxic T lymphocytes: one requires perforin and granzymes, the second Fas (APO-1; CD95) and its ligand. Thirdly, secreted molecules (e.g., TNF-alpha, gamma-IFN) can also mediate cytotoxicity. Together, these mechanisms appear to account for virtually all cytotoxicity induced by activated CTL in standard in vitro lytic assays. Using transplants across histocompatibility barriers, we were able to analyze the contributions of these effector molecules to cell-mediated cytotoxicity in vivo in a GvHD model. We found that Fas ligand is an important independent mediator of class II-restricted acute murine GvHD, while perforin/granzyme-dependent mechanisms have only a minor role in that compartment. In contrast, perforin/ granzyme-dependent mechanisms are required for class I-restricted acute murine GvHD, while Fas ligand is not. The perforin/granzyme pathway may therefore represent a novel target for anti-GvHD drug design. In support of this approach, we provide additional data suggesting that specific perforin/granzyme inhibitors should not adversely affect hematopoietic recovery after transplantation.
A complete molecular description of the syndromes of graft-versus-host disease (GVHD) and graft rejection could have a significant impact on clinical bone marrow transplantation. Recent in vitro ...experiments (Heusel et al. Cell 76:977, 1994 and Shresta et al, Proc Natl Acad Sci USA 92:5679, 1995) have shown that the putative mediators of these two syndromes, cytotoxic lymphocytes (CTL) and natural killer (NK) cells, respectively, initiate a program of cell death (apoptosis) in susceptible target tissues in a manner critically dependent on the serine protease Granzyme B (gzm B). In the present study, we have analyzed the phenotype of gzm B-deficient mice using experimental transplant models designed to isolate their CD8 CTL, CD4+ CTL, and NK compartments. We found a significant impairment in class l-dependent GVHD mediated by gzm B -/- CD8+ CTL, whereas class ll-dependent GVHD was not altered using gzm B -/-CD4+ effectors. In a hybrid resistance model, gzm B -/-hosts rejected haplo-identical marrow grafts as efficiently as did their wild-type littermates. This result is surprising in light of a severe defect in the ability of gzm B -/- NK cells to induce apoptosis in susceptible targets in vitro. These in vivo data define a significant role for gzm B in cytotoxicity mediated by CD8+ CTL, but not by CD4+ CTL. Furthermore, these results do not support a model of hybrid resistance in which NK cells play a pivotal role.
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