技术资料

Fanconi anemia (FA) is a rare inherited genomic instability syndrome representing one of the best examples of hematopoietic stem cell deficiency. Although FA might be an excellent candidate for bone marrow (BM) genetic correction ex vivo,knockout animal models are not sufficient to guide preclinical steps,and gene therapy attempts have proven disappointing so far. Contributing to these poor results is a characteristic and dramatic early BM-cells die-off when placed in culture. We show here that human primary FA BM cell survival can be ameliorated by using specific culture conditions that limit oxidative stress. When coupled with retrovirus-mediated transfer of the main complementation group FANCA-cDNA,we could achieve long-term reconstitution of the stem cell compartment both in vitro and in vivo. Gene-corrected BM cultures grew for textgreater120 days,and after cultured cell transplantation into NOD/SCID mice,clonogenic human cells carrying the FANCA transgene could be detected 6 months after transduction. By comparison,untransduced cells died in culture by 15 days. Of necessity for ethical reasons,experiments were conducted on a very limited number of primary BM cells. By using low cytokine regimen and conditions matching regulatory requirements,a contingent of gene-corrected cells slowly emerges with an unmet potential for in vivo engraftment. Future therapeutic applications of stem cells might be expanding from these data. In addition,we provide a model of gene-corrected human primary cell growth that carries the potential to better delineate the combined role of both DNA damage and oxidative stress in the pathogenesis of FA. View Publication

Current therapies for delayed- or nonunion bone fractures are still largely ineffective. Previous studies indicated that the VEGF homolog placental growth factor (PlGF) has a more significant role in disease than in health. Therefore we investigated the role of PlGF in a model of semi-stabilized bone fracture healing. Fracture repair in mice lacking PlGF was impaired and characterized by a massive accumulation of cartilage in the callus,reminiscent of delayed- or nonunion fractures. PlGF was required for the early recruitment of inflammatory cells and the vascularization of the fracture wound. Interestingly,however,PlGF also played a role in the subsequent stages of the repair process. Indeed in vivo and in vitro findings indicated that PlGF induced the proliferation and osteogenic differentiation of mesenchymal progenitors and stimulated cartilage turnover by particular MMPs. Later in the process,PlGF was required for the remodeling of the newly formed bone by stimulating osteoclast differentiation. As PlGF expression was increased throughout the process of bone repair and all the important cell types involved expressed its receptor VEGFR-1,the present data suggest that PlGF is required for mediating and coordinating the key aspects of fracture repair. Therefore PlGF may potentially offer therapeutic advantages for fracture repair. View Publication

The t(16:16) and inv(16) are associated with FAB M4Eo myeloid leukemias and result in fusion of the CBFB gene to the MYH11 gene (encoding smooth muscle myosin heavy chain [SMMHC]). Knockout of CBFbeta causes embryonic lethality due to lack of definitive hematopoiesis. Although knock-in of CBFB-MYH11 is not sufficient to cause disease,expression increases the incidence of leukemia when combined with cooperating events. Although mouse models are valuable tools in the study of leukemogenesis,little is known about the contribution of CBFbeta-SMMHC to human hematopoietic stem and progenitor cell self-renewal. We introduced the CBFbeta-MYH11 cDNA into human CD34+ cells via retroviral transduction. Transduced cells displayed an initial repression of progenitor activity but eventually dominated the culture,resulting in the proliferation of clonal populations for up to 7 months. Long-term cultures displayed a myelomonocytic morphology while retaining multilineage progenitor activity and engraftment in NOD/SCID-B2M-/- mice. Progenitor cells from long-term cultures showed altered expression of genes defining inv(16) identified in microarray studies of human patient samples. This system will be useful in examining the effects of CBFbeta-SMMHC on gene expression in the human preleukemic cell,in characterizing the effect of this oncogene on human stem cell biology,and in defining its contribution to the development of leukemia. View Publication

SALL4,a human homolog to Drosophila spalt,is a novel zinc finger transcriptional factor essential for development. We cloned SALL4 and its isoforms (SALL4A and SALL4B). Through immunohistochemistry and real-time reverse-transcription-polymerase chain reaction (RT-PCR),we demonstrated that SALL4 was constitutively expressed in human primary acute myeloid leukemia (AML,n = 81),and directly tested the leukemogenic potential of constitutive expression of SALL4 in a murine model. SALL4B transgenic mice developed myelodysplastic syndrome (MDS)-like features and subsequently AML that was transplantable. Increased apoptosis associated with dysmyelopoiesis was evident in transgenic mouse marrow and colony-formation (CFU) assays. Both isoforms could bind to beta-catenin and synergistically enhanced the Wnt/beta-catenin signaling pathway. Our data suggest that the constitutive expression of SALL4 causes MDS/AML,most likely through the Wnt/beta-catenin pathway. Our murine model provides a useful platform to study human MDS/AML transformation,as well as the Wnt/beta-catenin pathway's role in the pathogenesis of leukemia stem cells. View Publication

The B-cell receptor (BCR) transmits life and death signals throughout B-cell development,and altered BCR signaling may be required for survival of B-lymphoma cells. We used single-cell signaling profiles to compare follicular lymphoma (FL) B cells and nonmalignant host B cells within individual patient biopsies and identified BCR-mediated signaling events specific to lymphoma B cells. Expression of CD20,Bcl-2,and BCR light chain isotype (kappa or lambda) distinguished FL tumor B-cell and nontumor host B-cell subsets within FL patient biopsies. BCR-mediated signaling via phosphorylation of Btk,Syk,Erk1/2,and p38 occurred more rapidly in tumor B cells from FL samples than in infiltrating nontumor B cells,achieved greater levels of per-cell signaling,and sustained this level of signaling for hours longer than nontumor B cells. The timing and magnitude of BCR-mediated signaling in nontumor B cells within an FL sample instead resembled that observed in mature B cells from the peripheral blood of healthy subjects. BCR signaling pathways that are potentiated specifically in lymphoma cells should provide new targets for therapeutic attention. View Publication

We previously identified a rearrangement of mixed-lineage leukemia (MLL) gene (also known as ALL-1,HRX,and HTRX1),consisting of an in-frame partial tandem duplication (PTD) of exons 5 through 11 in the absence of a partner gene,occurring in approximately 4%-7% of patients with acute myeloid leukemia (AML) and normal cytogenetics,and associated with a poor prognosis. The mechanism by which the MLL PTD contributes to aberrant hematopoiesis and/or leukemia is unknown. To examine this,we generated a mouse knockin model in which exons 5 through 11 of the murine Mll gene were targeted to intron 4 of the endogenous Mll locus. Mll(PTD/WT) mice exhibit an alteration in the boundaries of normal homeobox (Hox) gene expression during embryogenesis,resulting in axial skeletal defects and increased numbers of hematopoietic progenitor cells. Mll(PTD/WT) mice overexpress Hoxa7,Hoxa9,and Hoxa10 in spleen,BM,and blood. An increase in histone H3/H4 acetylation and histone H3 lysine 4 (Lys4) methylation within the Hoxa7 and Hoxa9 promoters provides an epigenetic mechanism by which this overexpression occurs in vivo and an etiologic role for MLL PTD gain of function in the genesis of AML. View Publication

Recognition of tumor-associated Ags (TAAs) on tumor cells by CTLs and the subsequent tumor cell death are assumed to be dependent on TAA protein expression and to correlate directly with the level of peptide displayed in the binding site of the HLA class I molecule. In this study we evaluated whether the levels of Her-2/neu protein expression on human tumor cell lines directly correlate with HLA-A*0201/Her2/neu peptide presentation and CTL recognition. We developed a TCR mimic (TCRm) mAb designated 1B8 that specifically recognizes the HLA-A2.1/Her2/neu peptide (369-377) (Her2(369)-A2) complex. TCRm mAb staining intensity varied for the five human tumor cell lines analyzed,suggesting quantitative differences in levels of the Her2(369)-A2 complex on these cells. Analysis of tumor cell lines pretreated with IFN-gamma and TNF-alpha for Her2/neu protein and HLA-A2 molecule expression did not reveal a direct correlation between the levels of Her2/neu Ag,HLA-A2 molecule,and Her2(369)-A2 complex expression. However,compared with untreated cells,cytokine-treated cell lines showed an increase in Her2(369)-A2 epitope density that directly correlated with enhanced tumor cell death (p = 0.05). Although a trend was observed between tumor cell lysis and the level of the Her2(369)-A2 complex for untreated cells,the association was not significant. These findings suggest that tumor cell susceptibility to CTL-mediated lysis may be predicted based on the level of specific peptide-MHC class I expression rather than on the total level of TAA expression. Further,these studies demonstrate the potential of the TCRm mAb for validation of endogenous HLA-peptide epitopes on tumor cells. View Publication

Recent population studies provide clues that the use of metformin may be associated with reduced incidence and improved prognosis of certain cancers. This drug is widely used in the treatment of type 2 diabetes,where it is often referred to as an insulin sensitizer" because it not only lowers blood glucose but also reduces the hyperinsulinemia associated with insulin resistance. As insulin and insulin-like growth factors stimulate proliferation of many normal and transformed cell types� View Publication

Overcoming imatinib mesylate (IM) resistance and disease persistence in patients with chronic myeloid leukemia (CML) is of considerable importance to the issue of potential cure. Here we asked whether autocrine signaling contributes to survival of BCR/ABL+ cells in the presence of IM and nilotinib (NI; AMN107),a novel,more selective Abl inhibitor. Conditioned media (CM) of IM-resistant LAMA84 cell clones (R-CM) was found to substantially protect IM-naive LAMA cells and primary CML progenitors from IM- or NI-induced cell death. This was due to an increased secretion of the granulocyte-macrophage colony-stimulating factor (GM-CSF),which was identified as the causative factor mediating IM resistance in R-CM. GM-CSF elicited IM and NI drug resistance via a BCR/ABL-independent activation of the janus kinases 2 (JAK-2)/signal transducer and activator of transcription 5 (STAT-5) signaling pathway in GM-CSF receptor alpha receptor (CD116)-expressing cells,including primary CD34+/CD116+ GM progenitors (GMPs). Elevated mRNA and protein levels of GM-CSF were detected in IM-resistant patient samples,suggesting a contribution of GM-CSF secretion for IM and NI resistance in vivo. Importantly,inhibition of JAK-2 with AG490 abrogated GM-CSF-mediated STAT-5 phosphorylation and NI resistance in vitro. Together,adaptive autocrine secretion of GM-CSF mediates BCR/ABL-independent IM and NI resistance via activation of the antiapoptotic JAK-2/STAT-5 pathway. Inhibition of JAK-2 overcomes GM-CSF-induced IM and NI progenitor cell resistance,providing a rationale for the application of JAK-2 inhibitors to eradicate residual disease in CML. View Publication

Multiple myeloma is characterized by increased osteoclast activity that results in bone destruction and lytic lesions. With the prolonged overall patient survival achieved by new treatment modalities,additional drugs are required to inhibit bone destruction. We focused on a novel and more potent structural analog of the nonsteroidal anti-inflammatory drug etodolac,known as SDX-308,and its effects on osteoclastogenesis and multiple myeloma cells. SDX-101 is another structural analog of etodolac that is already used in clinical trials for the treatment of B-cell chronic lymphocytic leukemia (B-CLL). Compared with SDX-101,a 10-fold lower concentration of SDX-308 induced potent (60%-80%) inhibition of osteoclast formation,and a 10- to 100-fold lower concentration inhibited multiple myeloma cell proliferation. Bone resorption was completely inhibited by SDX-308,as determined in dentin-based bone resorption assays. SDX-308 decreased constitutive and RANKL-stimulated NF-kappaB activation and osteoclast formation in an osteoclast cellular model,RAW 264.7. SDX-308 effectively suppressed TNF-alpha-induced IKK-gamma and IkappaB-alpha phosphorylation and degradation and subsequent NF-kappaB activation in human multiple myeloma cells. These results indicate that SDX-308 effectively inhibits multiple myeloma cell proliferation and osteoclast activity,potentially by controlling NF-kappaB activation signaling. We propose that SDX-308 is a promising therapeutic candidate to inhibit multiple myeloma growth and osteoclast activity and that it should receive attention for further study. View Publication