技术资料

PURPOSE: Genz-644282 [8,9-dimethoxy-5-(2-N-methylaminoethyl)-2,3-methylenedioxy-5H-dibenzo[c,h][1,6]naphthyridin-6-one] has emerged as a promising candidate for antitumor agents. This report describes the bone marrow colony-forming unit,granulocyte macrophage (CFU-GM) and tumor cell CFU activity of topoisomerase I (Top1) inhibitors,such as Genz-644282,topotecan,irinotecan/SN-38,and ARC-111,and examines their activity in several human tumor xenograft models. EXPERIMENTAL DESIGN: Colony-forming assays were conducted with mouse and human bone marrow and eight human tumor cell lines. In addition,29 human tumor cell lines representing a range of histology and potential resistance mechanisms were assayed for sensitivity to Genz-644282 in a 72-hour exposure assay. The efficacy of Genz-644282 was compared with standard anticancer drugs (i.e.,irinotecan,docetaxel,and dacarbazine) in human tumor xenografts of colon cancer,renal cell carcinoma,non-small cell lung cancer,and melanoma. RESULTS: Human bone marrow CFU-GM was more sensitive to the Top1 inhibitors than was mouse bone marrow CFU-GM. The ratio of mouse to human IC(90) values was more than 10 for the camptothecins and less than 10 for Genz-644282,which had more potency as a cytotoxic agent toward human tumor cells in culture than the camptothecins in the colony-forming and 72-hour proliferation assays. Genz-644282 has superior or equal antitumor activity in the human tumor xenografts than the standard drug comparators. CONCLUSIONS: On the basis of preclinical activity and safety,Genz-644282 was selected for development and is currently undergoing phase 1 clinical trial. View Publication

Ionising radiation (IR) is a known carcinogen and poses a significant risk to the haematopoietic system for the development of leukaemia in part by induction of genomic instability. Induction of chronic oxidative stress has been assumed to play an important role in mediating the effect of IR on the haematopoietic system. However,there was no direct evidence to support this hypothesis prior to our studies. In our recent studies,we showed that exposure of mice to total body irradiation (TBI) induces persistent oxidative stress selectively in haematopoietic stem cells (HSCs) at least in part via up-regulation of nicotinamide adenine dinucleotide phosphate oxidase (NOX) 4. Now,we found that post-TBI treatment with diphenylene iodonium (DPI),a pan NOX inhibitor,not only significantly reduces TBI-induced increases in reactive oxygen species (ROS) production,oxidative DNA damage and DNA double-strand breaks in HSCs but also dramatically decreases the number of cells with unstable chromosomal aberrations in the clonal progeny of irradiated HSCs. The effects of DPI are comparable to Mn (III) meso-tetrakis (N-ethylpyridinium-2-yl) porphyrin,a superoxide dismutase mimetic and a potent antioxidant. These findings demonstrate that increased production of ROS by NOX in HSCs mediates the induction of haematopoietic genomic instability by IR and that NOX may represent a novel molecular target to inhibit TBI-induced genomic instability. View Publication

Acute myeloid leukemia (AML) is an aggressive malignancy with a relapse rate approaching 50%,despite aggressive chemotherapy. New therapies for AML are targeted at signal transduction pathways known to support blast survival,such as the Stat3 pathway. Aberrant activation of Stat3 has been demonstrated in many different malignancies,including AML,and this finding is frequently associated with more aggressive disease. The objectives of this study were: (1) to characterize Stat3 signaling patterns in AML cells lines and primary pediatric samples; and (2) to test the efficacy and potency of a novel Stat3 inhibitor in inducing apoptosis in AML cells. We found that Stat3 was constitutively activated in 6 of 7 AML cell lines and 6 of 18 primary pediatric AML samples. Moreover,constitutively phosphorylated Stat3 was frequent in samples with normal karyotype but uncommon in samples with t(8;21). Most cell lines and primary samples responded to G-CSF stimulation,although the sensitivity and magnitude of the response varied dramatically. Our novel small-molecule Stat3 inhibitor,C188-9,inhibited G-CSF-induced Stat3 phosphorylation,induced apoptosis in AML cell lines and primary samples,and inhibited AML blast colony formation with potencies in the low micromolar range. Therefore,Stat3 inhibition may be a valuable strategy for targeted therapies for AML. View Publication

We recently demonstrated that lack of type I IFN signaling (IFNAR knockout) in lymphocyte-deficient mice (IFrag(-/-)) results in bone marrow (BM) failure after Pneumocystis lung infection,whereas lymphocyte-deficient mice with intact IFNAR (RAG(-/-)) had normal hematopoiesis. In the current work,we performed studies to define further the mechanisms involved in the induction of BM failure in this system. BM chimera experiments revealed that IFNAR expression was required on BM-derived but not stroma-derived cells to prevent BM failure. Signals elicited after day 7 postinfection appeared critical in determining BM cell fate. We observed caspase-8- and caspase-9-mediated apoptotic cell death,beginning with neutrophils. Death of myeloid precursors was associated with secondary oxidative stress,and decreasing colony-forming activity in BM cell cultures. Treatment with N-acetylcysteine could slow the progression of,but not prevent,BM failure. Type I IFN signaling has previously been shown to expand the neutrophil life span and regulate the expression of some antiapoptotic factors. Quantitative RT-PCR demonstrated reduced mRNA abundance for the antiapoptotic factors BCL-2,IAP2,MCL-1,and others in BM cells from IFrag(-/-) compared with that in BM cells from RAG(-/-) mice at day 7. mRNA and protein for the proapoptotic cytokine TNF-α was increased,whereas mRNA for the growth factors G-CSF and GM-CSF was reduced. In vivo anti-TNF-α treatment improved precursor cell survival and activity in culture. Thus,we propose that lack of type I IFN signaling results in decreased resistance to inflammation-induced proapoptotic stressors and impaired replenishment by precursors after systemic responses to Pneumocystis lung infection. Our finding may have implications in understanding mechanisms underlying regenerative BM depression/failure during complex immune deficiencies such as AIDS. View Publication

We studied leukemic stem cells (LSCs) in a Smad4(-/-) mouse model of acute myelogenous leukemia (AML) induced either by the HOXA9 gene or by the fusion oncogene NUP98-HOXA9. Although Hoxa9-Smad4 complexes accumulate in the cytoplasm of normal hematopoietic stem cells and progenitor cells (HSPCs) transduced with these oncogenes,there is no cytoplasmic stabilization of HOXA9 in Smad4(-/-) HSPCs,and as a consequence increased levels of Hoxa9 is observed in the nucleus leading to increased immortalization in vitro. Loss of Smad4 accelerates the development of leukemia in vivo because of an increase in transformation of HSPCs. Therefore,the cytoplasmic binding of Hoxa9 by Smad4 is a mechanism to protect Hoxa9-induced transformation of normal HSPCs. Because Smad4 is a potent tumor suppressor involved in growth control,we developed a strategy to modify the subcellular distribution of Smad4. We successfully disrupted the interaction between Hoxa9 and Smad4 to activate the TGF-β pathway and apoptosis,leading to a loss of LSCs. Together,these findings reveal a major role for Smad4 in the negative regulation of leukemia initiation and maintenance induced by HOXA9/NUP98-HOXA9 and provide strong evidence that antagonizing Smad4 stabilization by these oncoproteins might be a promising novel therapeutic approach in leukemia. View Publication

A population of CD133(+)Lin(-)CD45(-) very small embryonic/epiblast-like stem cells (VSELs) has been purified by multiparameter sorting from umbilical cord blood (UCB). To speed up isolation of these cells,we employed anti-CD133-conjugated paramagnetic beads followed by staining with Aldefluor to detect aldehyde dehydrogenase (ALDH) activity; we subsequently sorted CD45(-)/GlyA(-)/CD133(+)/ALDH(high) and CD45(-)/GlyA(-)/CD133(+)/ALDH(low) cells,which are enriched for VSELs,and CD45(+)/GlyA /CD133(+)/ALDH(high) and CD45(+)/GlyA(-)/CD133(+)/ALDH(low) cells,which are enriched for hematopoietic stem/progenitor cells (HSPCs). Although freshly isolated CD45(-) VSELs did not grow hematopoietic colonies,the same cells,when activated/expanded over OP9 stromal support,acquired hematopoietic potential and grew colonies composed of CD45(+) hematopoietic cells in methylcellulose cultures. We also observed that CD45(-)/GlyA(-)/CD133(+)/ALDH(high) VSELs grew colonies earlier than CD45(-)/GlyA(-)/CD133(+)/ALDH(low) VSELs,which suggests that the latter cells need more time to acquire hematopoietic commitment. In support of this possibility,real-time polymerase chain reaction analysis confirmed that,whereas freshly isolated CD45(-)/GlyA(-)/CD133(+)/ALDH(high) VSELs express more hematopoietic transcripts (for example,c-myb),CD45(-)/GlyA(-)/CD133(+)/ALDH(low) VSELs exhibit higher levels of pluripotent stem cell markers (for example,Oct-4). More importantly,hematopoietic cells derived from VSELs that were co-cultured over OP9 support were able to establish human lympho-hematopoietic chimerism in lethally irradiated non-obese diabetic/severe combined immunodeficiency mice 4-6 weeks after transplantation. Overall,our data suggest that UCB-VSELs correspond to the most primitive population of HSPCs in UCB. View Publication

Mast cells (MCs) play an important role in the regulation of protective adaptive immune responses against pathogens. However,it is still unclear whether MCs promote such host defense responses via direct effects on T cells or rather by modifying the functions of antigen-presenting cells. To identify the underlying mechanisms of the immunoregulatory capacity of MCs,we investigated the impact of MCs on dendritic cell (DC) maturation and function. We found that murine peritoneal MCs underwent direct crosstalk with immature DCs that induced DC maturation as evidenced by enhanced expression of costimulatory molecules. Furthermore,the MC/DC interaction resulted in the release of the T-cell modulating cytokines IFN-γ,IL-2,IL-6 and TGF-β into coculture supernatants and increased the IL-12p70,IFN-γ,IL-6 and TGF-β secretion of LPS-matured DCs. Such MC-primed" DCs subsequently induced efficient CD4+ T-cell proliferation. Surprisingly� View Publication

4-Hydroxy-5-methoxy-2,3-dihydro-1H-[1,3]benzodioxolo[5,6-c]pyrrolo[1,2-f]-phenanthridium chloride (NK314) is a benzo[c] phenanthridine alkaloid that inhibits topoisomerase IIα,leading to the generation of DNA double-strand breaks (DSBs) and activating the G(2) checkpoint pathway. The purpose of the present studies was to investigate the DNA intercalating properties of NK314,to evaluate the DNA repair mechanisms activated in cells that may lead to resistance to NK314,and to develop mechanism-based combination strategies to maximize the antitumor effect of the compound. A DNA unwinding assay indicated that NK314 intercalates in DNA,a property that likely cooperates with its ability to trap topoisomerase IIα in its cleavage complex form. The consequence of this is the formation of DNA DSBs,as demonstrated by pulsed-field gel electrophoresis and H2AX phosphorylation. Clonogenic assays demonstrated a significant sensitization in NK314-treated cells deficient in DNA-dependent protein kinase (DNA-PK) catalytic subunit,Ku80,ataxia telangiectasia mutated (ATM),BRCA2,or XRCC3 compared with wild-type cells,indicating that both nonhomologous end-joining and homologous recombination DNA repair pathways contribute to cell survival. Furthermore,both the DNA-PK inhibitor 8-(4-dibenzothienyl)-2-(4-morpholinyl)-4H-1-benzopyran-4-one (NU7441) and the ATM inhibitor 2-(4-morpholinyl)-6-(1-thianthrenyl)-4H-pyran-4-one (KU55933) significantly sensitized cells to NK314. We conclude that DNA-PK and ATM contribute to cell survival in response to NK314 and could be potential targets for abrogating resistance and maximizing the antitumor effect of NK314. View Publication

Recent evidence suggests that enhanced aldehyde dehydrogenase (ALDH) activity is a hallmark of cancer stem cells (CSC) measurable by the aldefluor assay. ALDH1A1,one of 19 ALDH isoforms expressed in humans,was generally believed to be responsible for the ALDH activity of CSCs. More recently,experiments with murine hematopoietic stem cells,murine progenitor pancreatic cells,and human breast CSCs indicate that other ALDH isoforms,particularly ALDH1A3,significantly contribute to aldefluor positivity,which may be tissue and cancer specific. Therefore,potential prognostic application involving the use of CSC prevalence in tumor tissue to predict patient outcome requires the identification and quantification of specific ALDH isoforms. Herein we review the suggested roles of ALDH in CSC biology and the immunohistological studies testing the potential application of ALDH isoforms as novel cancer prognostic indicators. View Publication

CREB-binding protein (CREBBP) is important for the cell-autonomous regulation of hematopoiesis,including the stem cell compartment. In the present study,we show that CREBBP plays an equally pivotal role in microenvironment-mediated regulation of hematopoiesis. We found that the BM microenvironment of Crebbp(+/-) mice was unable to properly maintain the immature stem cell and progenitor cell pools. Instead,it stimulates myeloid differentiation,which progresses into a myeloproliferation phenotype. Alterations in the BM microenvironment resulting from haploinsufficiency of Crebbp included a marked decrease in trabecular bone that was predominantly caused by increased osteoclastogenesis. Although CFU-fibroblast (CFU-F) and total osteoblast numbers were decreased,the bone formation rate was similar to that found in wild-type mice. At the molecular level,we found that the known hematopoietic modulators matrix metallopeptidase-9 (MMP9) and kit ligand (KITL) were decreased with heterozygous levels of Crebbp. Lastly,potentially important regulatory proteins,endothelial cell adhesion molecule 1 (ESAM1) and cadherin 5 (CDH5),were increased on Crebbp(+/-) endothelial cells. Our findings reveal that a full dose of Crebbp is essential in the BM microenvironment to maintain proper hematopoiesis and to prevent excessive myeloproliferation. View Publication

Chemotherapy is the most common way to treat malignancies,but myelosuppression,one of its common side-effects,is a formidable problem. The present study described the protective role of dammarane sapogenins (DS),an active fraction from oriental ginseng,on myelosuppression induced by cyclophosphamide (CP) in mice. DS was orally administered at different dosages (37.5,75,and 150 mg/kg) for 10 d after CP administration (200 mg/kg intraperitoneally). The results showed that DS increased the number of white blood cells (WBC) on day 3 and day 7 (P textless 0.05),such that WBC levels were increased by 105.7 ± 29.5% at 75 mg/kg of DS on day 3 (P textless 0.05,compared with the CP group). Similar results were observed in red blood cells and platelets in DS-treated groups. The colony-forming assay demonstrated that the depressed numbers of CFU-GM (colony-forming unit-granulocyte and macrophage),CFU-E (colony-forming unit-erythroid),BFU-E (burst-forming unit-erythroid),CFU-Meg (colony-forming unit-megakaryocyte) and CFU-GEMM (colony-forming unit-granulocyte,-erythrocyte,-monocyte and -megakaryocyte) induced by CP were significantly reversed after DS treatment. Moreover,the ameliorative effect of DS on myelosuppression was also observed in the femur by hematoxylin/eosin staining. In DS-treated groups,ConA-induced splenocyte proliferation was enhanced significantly at all the doses (37.5,75,150 mg/kg) on day 3 at the rate of 50.3 ± 8.0%,77.6 ± 8.5% and 44.5 ± 8.4%,respectively,while lipopolysaccharide-induced proliferation was increased mainly on day 7 (P textless 0.01),with an increased rate of 39.8 ± 5.6%,34.9 ± 6.6% and 38.3 ± 7.3%,respectively. The thymus index was also markedly increased by 70.4% and 36.6% at 75 mg/kg on days 3 and 7,respectively,as compared with the CP group. In summary,DS has a protective function against CP-induced myelosuppression. Its mechanism might be related to stimulating hematopoiesis recovery,as well as enhancing the immunological function. View Publication

The stem-cell pool is considered to be maintained by a balance between symmetric and asymmetric division of stem cells. The cell polarity model proposes that the facultative use of symmetric and asymmetric cell division is orchestrated by a polarity complex consisting of partitioning-defective proteins Par3 and Par6,and atypical protein kinase C (aPKCζ and aPKCλ),which regulates planar symmetry of dividing stem cells with respect to the signaling microenvironment. However,the role of the polarity complex is unexplored in mammalian adult stem-cell functions. Here we report that,in contrast to accepted paradigms,polarization and activity of adult hematopoietic stem cell (HSC) do not depend on either aPKCζ or aPKCλ or both in vivo. Mice,having constitutive and hematopoietic-specific (Vav1-Cre) deletion of aPKCζ and aPKCλ,respectively,have normal hematopoiesis,including normal HSC self-renewal,engraftment,differentiation,and interaction with the bone marrow microenvironment. Furthermore,inducible complete deletion of aPKCλ (Mx1-Cre) in aPKCζ(-/-) HSC does not affect HSC polarization,self-renewal,engraftment,or lineage repopulation. In addition,aPKCζ- and aPKCλ-deficient HSCs elicited a normal pattern of hematopoietic recovery secondary to myeloablative stress. Taken together,the expression of aPKCζ,aPKCλ,or both are dispensable for primitive and adult HSC fate determination in steady-state and stress hematopoiesis,contrary to the hypothesis of a unique,evolutionary conserved aPKCζ/λ-directed cell polarity signaling mechanism in mammalian HSC fate determination. View Publication