技术资料

BCR-ABL and v-ABL are oncogenic forms of the Abl tyrosine kinase that can cause leukemias in mice and humans. ABL oncogenes trigger multiple signaling pathways whose contribution to transformation varies among cell types. Activation of phosphoinositide 3-kinase (PI3K) is essential for ABL-dependent proliferation and survival in some cell types,and global PI3K inhibitors can enhance the antileukemia effects of the Abl kinase inhibitor imatinib. Although a significant fraction of BCR-ABL-induced human leukemias are of B-cell origin,little is known about PI3K signaling mechanisms in B-lineage cells transformed by ABL oncogenes. Here we show that activation of class I(A) PI3K and downstream inactivation of FOXO transcription factors are essential for survival of murine pro/pre-B cells transformed by v-ABL or BCR-ABL. In addition,analysis of mice lacking individual PI3K genes indicates that products of the Pik3r1 gene contribute to transformation efficiency by BCR-ABL. These findings establish a role for PI3K signaling in B-lineage transformation by ABL oncogenes. View Publication

NUP98-Hox fusion genes are newly identified oncogenes isolated in myeloid leukemias. Intriguingly,only Abd-B Hox genes have been reported as fusion partners,indicating that they may have unique overlapping leukemogenic properties. To address this hypothesis,we engineered novel NUP98 fusions with Hox genes not previously identified as fusion partners: the Abd-B-like gene HOXA10 and two Antennepedia-like genes,HOXB3 and HOXB4. Notably,NUP98-HOXA10 and NUP98-HOXB3 but not NUP98-HOXB4 induced leukemia in a murine transplant model,which is consistent with the reported leukemogenic potential ability of HOXA10 and HOXB3 but not HOXB4. Thus,the ability of Hox genes to induce leukemia as NUP98 fusion partners,although apparently redundant for Abd-B-like activity,is not restricted to this group,but rather is determined by the intrinsic leukemogenic potential of the Hox partner. We also show that the potent leukemogenic activity of Abd-B-like Hox genes is correlated with their strong ability to block hematopoietic differentiation. Conversely,coexpression of the Hox cofactor Meis1 alleviated the requirement of a strong intrinsic Hox-transforming potential to induce leukemia. Our results support a model in which many if not all Hox genes can be leukemogenic and point to striking functional overlap not previously appreciated,presumably reflecting common regulated pathways. View Publication

Human immunodeficiency virus type 1 (HIV-1)-associated dementia (HAD) is correlated with increased monocyte migration to the brain,and the incidence of HAD among otherwise asymptomatic subjects appears to be lower in India than in the United States and Europe (1 to 2% versus 15 to 30%). Because of the genetic differences between HIV-1 strains circulating in these regions,we sought to identify viral determinants associated with this difference. We targeted Tat protein for these studies in view of its association with monocyte chemotactic function. Analyses of Tat sequences representing nine subtypes revealed that at least six amino acid residues are differentially conserved in subtype C Tat (C-Tat). Of these,cysteine (at position 31) was highly (textgreater99%) conserved in non-subtype C viruses and more than 90% of subtype C viruses encoded a serine. We hypothesized a compromised chemotactic function of C-Tat due to the disruption of CC motif and tested it with the wild type C-Tat (CS) and its two isogenic variants (CC and SC) derived by site-directed mutagenesis. We found that the CS natural variant was defective for monocyte chemotactic activity without a loss in the transactivation property. While the CC mutant is functionally competent for both the functions,in contrast,the SC mutant was defective in both. Therefore,the loss of the C-Tat chemotactic property may underlie the reduced incidence of HAD; although not presenting conclusive evidence,this study provides the first evidence for a potential epidemiologic phenomenon associated with biological differences in the subtype C viruses. View Publication

The development of immunodeficient mouse xenograft models has greatly facilitated the investigation of some human hematopoietic malignancies,but application of this approach to the myelodysplastic syndromes (MDSs) has proven difficult. We now show that cells from most MDS patients (including all subtypes) repopulate nonobese diabetic-severe combined immunodeficient (scid)/scid-beta2 microglobulin null (NOD/SCID-beta2m(-/-)) mice at least transiently and produce abnormal differentiation patterns in this model. Normal marrow transplants initially produce predominantly erythroid cells and later predominantly B-lymphoid cells in these mice,whereas most MDS samples produced predominantly granulopoietic cells. In 4 of 4 MDS cases,the regenerated cells showed the same clonal markers (trisomy 8,n = 3; and 5q-,n = 1) as the original sample and,in one instance,regenerated trisomy 8(+) B-lymphoid as well as myeloid cells were identified. Interestingly,the enhanced growth of normal marrow obtained in NOD/SCID-beta2m(-/-) mice engineered to produce human interleukin-3,granulocyte-macrophage colony-stimulating factor,and Steel factor was seen only with 1 of 7 MDS samples. These findings support the concept that human MDS originates in a transplantable multilineage hematopoietic stem cell whose genetic alteration may affect patterns of differentiation and responsiveness to hematopoietic growth factors. They also demonstrate the potential of this new murine xenotransplant model for future investigations of MDS. View Publication

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Pharmacological inhibitors of glycogen synthase kinase-3 (GSK-3) and cyclin-dependent kinases have a promising potential for applications against several neurodegenerative diseases such as Alzheimer's disease. Indirubins,a family of bis-indoles isolated from various natural sources,are potent inhibitors of several kinases,including GSK-3. Using the cocrystal structures of various indirubins with GSK-3beta,CDK2 and CDK5/p25,we have modeled the binding of indirubins within the ATP-binding pocket of these kinases. This modeling approach provided some insight into the molecular basis of indirubins' action and selectivity and allowed us to forecast some improvements of this family of bis-indoles as kinase inhibitors. Predicted molecules,including 6-substituted and 5,6-disubstituted indirubins,were synthesized and evaluated as CDK and GSK-3 inhibitors. Control,kinase-inactive indirubins were obtained by introduction of a methyl substitution on N1. View Publication

Acute myeloid leukemia (AML) is characterized by the block of differentiation,deregulated apoptosis,and an increased self-renewal of hematopoietic precursors. It is unclear whether the self-renewal of leukemic blasts results from the cumulative effects of blocked differentiation and impaired apoptosis or whether there are mechanisms directly increasing self-renewal. The AML-associated translocation products (AATPs) promyelocytic leukemia/retinoic acid receptor alpha (PML/RAR alpha),promyelocytic leukemia zinc finger (PLZF)/RAR alpha (X-RAR alpha),and AML-1/ETO block hematopoietic differentiation. The AATPs activate the Wnt signaling by up-regulating gamma-catenin. Activation of the Wnt signaling augments self-renewal of hematopoietic stem cells (HSCs). Therefore,we investigated how AATPs influence self-renewal of HSCs and evaluated the role of gamma-catenin in the determination of the phenotype of HSCs expressing AATPs. Here we show that the AATPs directly activate the gamma-catenin promoter. The crucial role of gamma-catenin in increasing the self-renewal of HSCs upon expression of AATPs is demonstrated by (i) the abrogation of replating efficiency upon hindrance of gamma-catenin expression through RNA interference,and (ii) the augmentation of replating efficiency of HSCs upon overexpression of gamma-catenin itself. In addition,the inoculation of gamma-catenin-transduced HSCs into irradiated recipient mice establishes the clinical picture of AML. These data provide the first evidence that the aberrant activation of Wnt signaling by the AATP decisively contributes to the pathogenesis of AML. View Publication

Lysophosphatidic acid (LPA),the smallest and structurally simplest phospholipid,is a platelet-derived serum factor that evokes a wide range of biological effects,including stimulation of fibroblast proliferation,platelet aggregation,cellular motility,tumour cell invasiveness and neurite retraction. This review summarizes recent insights into the mode of action of LPA. LPA appears to activate its own G-protein-coupled receptor(s) to initiate both classic and novel signal cascades. Of particular interest is LPA's ability to activate the Ras pathway and to stimulate protein tyrosine phosphorylation in concert with remodelling of the actin cytoskeleton. View Publication

We previously reported a transgenic mouse model expressing herpesvirus thymidine kinase (TK) gene under the control of a 2.3-kilobase fragment of the rat collagen alpha1 type I promoter (Col2.3 Delta TK). This construct confers lineage-specific expression in developing osteoblasts,allowing the conditional ablation of osteoblast lineage after treatment with ganciclovir (GCV). After GCV treatment these mice have profound alterations on bone formation leading to a progressive bone loss. In addition,treated animals also lose bone marrow cellularity. In this report we characterized hematopoietic parameters in GCV-treated Col2.3 Delta TK mice,and we show that after treatment transgenic animals lose lymphoid,erythroid,and myeloid progenitors in the bone marrow,followed by decreases in the number of hematopoietic stem cells (HSCs). Together with the decrease in bone marrow hematopoiesis,active extramedullary hematopoiesis was observed in the spleen and liver,as measured by an increase in peripheral HSCs and active primary in vitro hematopoiesis. After withdrawal of GCV,osteoblasts reappeared in the bone compartment together with a recovery of medullary and decrease in extramedullary hematopoiesis. These observations directly demonstrate the role of osteoblasts in hematopoiesis and provide a model to study the interactions between the mesenchymal and hematopoietic compartments in the marrow. View Publication

The t(12;21)(p13;q22) translocation is the most common chromosomal abnormality yet identified in any pediatric leukemia and gives rise to the TEL-AML1 fusion product. To investigate the effects of TEL-AML1 on hematopoiesis,fetal liver hematopoietic progenitor cells (HPCs) were transduced with retroviral vectors expressing this fusion protein. We show that TEL-AML1 dramatically alters differentiation of HPCs in vitro,preferentially promoting B-lymphocyte development,enhancing self-renewal of B-cell precursors,and leading to the establishment of long-term growth factor-dependent pre-B-cell lines. However,it had no effect on myeloid development in vitro. Further experiments were performed to determine whether TEL-AML1 also demonstrates lineage-specific activity in vivo. TEL-AML1-expressing HPCs displayed a competitive advantage in reconstituting both B-cell and myeloid lineages in vivo but had no effect on reconstitution of the T-cell lineage. Despite promoting these alterations in hematopoiesis,TEL-AML1 did not induce leukemia in transplanted mice. Our study provides a unique insight into the role of TEL-AML1 in leukemia predisposition and a potential model to study the mechanism of leukemogenesis associated with this fusion. View Publication

Combinatorial libraries were screened for molecules that induce mouse myogenic lineage committed cells to dedifferentiate in vitro. A 2,6-disubstituted purine,reversine,was discovered that induces lineage reversal of C2C12 cells to become multipotent progenitor cells which can redifferentiate into osteoblasts and adipocytes. This and other such molecules are likely to provide new insights into the molecular mechanisms that control cellular dedifferentiation and may ultimately be useful to in vivo stem cell biology and therapy. View Publication

Creation of fusion genes by balanced chromosomal translocations is one of the hallmarks of acute myeloid leukemia (AML) and is considered one of the key leukemogenic events in this disease. In t(12;13)(p13;q12) AML,ectopic expression of the homeobox gene CDX2 was detected in addition to expression of the ETV6-CDX2 fusion gene,generated by the chromosomal translocation. Here we show in a murine model of t(12;13)(p13;q12) AML that myeloid leukemogenesis is induced by the ectopic expression of CDX2 and not by the ETV6-CDX2 chimeric gene. Mice transplanted with bone marrow cells retrovirally engineered to express Cdx2 rapidly succumbed to fatal and transplantable AML. The transforming capacity of Cdx2 depended on an intact homeodomain and the N-terminal transactivation domain. Transplantation of bone marrow cells expressing ETV6-CDX2 failed to induce leukemia. Furthermore,coexpression of ETV6-CDX2 and Cdx2 in bone marrow cells did not accelerate the course of disease in transplanted mice compared to Cdx2 alone. These data demonstrate that activation of a protooncogene by a balanced chromosomal translocation can be the pivotal leukemogenic event in AML,characterized by the expression of a leukemia-specific fusion gene. Furthermore,these findings link protooncogene activation to myeloid leukemogenesis,an oncogenic mechanism so far associated mainly with lymphoid leukemias and lymphomas. View Publication