技术资料

The majority of BRCA1-associated breast cancers are basal cell-like,which is associated with a poor outcome. Using a spontaneous mouse mammary tumor model,we show that platinum compounds,which generate DNA breaks during the repair process,are more effective than doxorubicin in Brca1/p53-mutated tumors. At 0.5 mg/kg of daily cisplatin treatment,80% primary tumors (n = 8) show complete pathologic response. At greater dosages,100% show complete response (n = 19). However,after 2 to 3 months of complete remission following platinum treatment,tumors relapse and become refractory to successive rounds of treatment. Approximately 3.8% to 8.0% (mean,5.9%) of tumor cells express the normal mammary stem cell markers,CD29(hi)24(med),and these cells are tumorigenic,whereas CD29(med)24(-/lo) and CD29(med)24(hi) cells have diminished tumorigenicity or are nontumorigenic,respectively. In partially platinum-responsive primary transplants,6.6% to 11.0% (mean,8.8%) tumor cells are CD29(hi)24(med); these populations significantly increase to 16.5% to 29.2% (mean,22.8%; P textless 0.05) in platinum-refractory secondary tumor transplants. Further,refractory tumor cells have greater colony-forming ability than the primary transplant-derived cells in the presence of cisplatin. Expression of a normal stem cell marker,Nanog,is decreased in the CD29(hi)24(med) populations in the secondary transplants. Top2A expression is also down-regulated in secondary drug-resistant tumor populations and,in one case,was accompanied by genomic deletion of Top2A. These studies identify distinct cancer cell populations for therapeutic targeting in breast cancer and implicate clonal evolution and expansion of cancer stem-like cells as a potential cause of chemoresistance. View Publication

Aberrant expression and/or activity of members of the Src family of nonreceptor protein tyrosine kinases (SFK) are commonly observed in progressive stages of human tumors. In prostate cancer,two SFKs (Src and Lyn) have been specifically implicated in tumor growth and progression. However,there are no data in preclinical models demonstrating potential efficacy of Src inhibitors against prostate cancer growth and/or metastasis. In this study,we used the small molecule SFK/Abl kinase inhibitor dasatinib,currently in clinical trials for solid tumors,to examine in vitro and in vivo effects of inhibiting SFKs in prostate tumor cells. In vitro,dasatinib inhibits both Src and Lyn activity,resulting in decreased cellular proliferation,migration,and invasion. In orthotopic nude mouse models,dasatinib treatment effectively inhibits expression of activated SFKs,resulting in inhibition of both tumor growth and development of lymph node metastases in both androgen-sensitive and androgen-resistant tumors. In primary tumors,SFK inhibition leads to decreased cellular proliferation (determined by immunohistochemistry for proliferating cell nuclear antigen). In vitro,small interfering RNA (siRNA)-mediated inhibition of Lyn affects cellular proliferation; siRNA inhibition of Src affects primarily cellular migration. Therefore,we conclude that SFKs are promising therapeutic targets for treatment of human prostate cancer and that Src and Lyn activities affect different cellular functions required for prostate tumor growth and progression. View Publication

Evi-1 is a zinc-finger transcriptional factor whose inappropriate expression leads to leukemic transformation in mice and humans. Recently,it has been shown that Evi-1 regulates proliferation of hematopoietic stem/progenitor cells at embryonic stage via GATA-2 up-regulation; however,detailed mechanisms underlying Evi-1-mediated early hematopoiesis are not fully understood. We therefore evaluated hematopoietic potential of Evi-1 mutants using a cultivation system of murine para-aortic splanchnopleural (P-Sp) regions,and found that both the first zinc finger domain and the acidic domain were required for Evi-1-mediated hematopoiesis. The hematopoietic potential of Evi-1 mutants was likely to be related to its ability to up-regulate GATA-2 expression. We also showed that the decreased colony forming capacity of Evi-1-deficient P-Sp cells was successfully recovered by inhibition of TGF-b signaling,using ALK5 inhibitor or retroviral transfer of dominant-negative-type Smad3. Our findings suggest that Evi-1 promotes hematopoietic stem/progenitor expansion at the embryonic stage through up-regulation of GATA-2 and repression of TGF-beta signaling. View Publication

T-bet and STAT4 play critical roles in helper T cell differentiation,especially for Th1 cells. However,it is still unknown about the relative importance and redundancy of T-bet and STAT4 for Th1 differentiation. It is also unknown about their independent role of T-bet and STAT4 in the regulation of allergic airway inflammation. In this study,we addressed these issues by comparing T-bet-deficient (T-bet(-/-)) mice,STAT4(-/-) mice,and T-bet- and STAT4-double-deficient (T-bet(-/-)STAT4(-/-)) mice on the same genetic background. Th1 differentiation was severely decreased in T-bet(-/-) mice and STAT4(-/-) mice as compared with that in wild-type mice,but Th1 differentiation was still observed in T-bet(-/-) mice and STAT4(-/-) mice. However,Th1 cells were hardly detected in T-bet(-/-)STAT4(-/-) mice. In contrast,the maintenance of Th17 cells was enhanced in T-bet(-/-) mice but was reduced in STAT4(-/-) mice and T-bet(-/-)STAT4(-/-) mice. In vivo,Ag-induced eosinophil and neutrophil recruitment into the airways was enhanced in T-bet(-/-) mice but was attenuated in STAT4(-/-) mice and T-bet(-/-)STAT4(-/-) mice. Ag-induced IL-17 production in the airways was also diminished in STAT4(-/-) mice and T-bet(-/-)STAT4(-/-) mice. These results indicate that STAT4 not only plays an indispensable role in T-bet-independent Th1 differentiation but also is involved in the maintenance of Th17 cells and the enhancement of allergic airway inflammation. View Publication

BRAF-activating mutations have been reported in several types of cancer,including melanoma ( approximately 70% of cases),thyroid (30-70%),ovarian (15-30%),and colorectal cancer (5-20%). Mutant BRAF has constitutive kinase activity and causes hyperactivation of the mitogen-activated protein kinase pathway. BRAF silencing induces regression of melanoma xenografts,indicating the essential role of BRAF for cell survival. We set up an inducible short hairpin RNA system to compare the role of oncogenic BRAF in thyroid carcinoma versus melanoma cells. Although BRAF knockdown led to apoptosis in the melanoma cell line A375,the anaplastic thyroid carcinoma cell ARO underwent growth arrest upon silencing,with little or no cell death. Reexpression of the thyroid differentiation marker,sodium iodide symporter,was induced after long-term silencing. The different outcome of BRAF down-regulation in the two cell lines was associated with an opposite regulation of p21(CIP1/WAF1) expression levels in response to the block of the BRAF mitogenic signal. These results were confirmed using a specific BRAF small-molecule inhibitor,PLX4032. Restoration of p21(CIP1/WAF1) expression rescued melanoma cells from death. Altogether,our data indicate that oncogenic BRAF inhibition can have a different effect on cell fate depending on the cellular type. Furthermore,we suggest that a BRAF-independent mechanism of cell survival exists in anaplastic thyroid cancer cells. View Publication

Vitamin D has been known to be important for skeletal development and growth but the mechanism whereby it affects these processes is not well understood. We report now that the hormonal metabolite of vitamin D3,namely 1,25-dihydroxyvitamin D3,stimulates chondrogenesis in cultures of stage 24 chick embryo limb bud mesenchymal cells,as evidenced by morphologic changes as well as by increased transcription of collagen type II and core protein genes. This effect appears to be specific to 1,25(OH)2D3 since 24,25(OH)2D3 or D3 does not influence chondrogenesis in this system,and is probably mediated via the specific 1,25(OH)2D3 receptor protein which is undetectable in untreated cells but appears following exposure to the hormone. View Publication

Embryonic stem cells (ESCs),which have characteristics such as self-renewal,indefinite proliferation,and pluripotency,are thought to hold great promise for regenerative medicine. ESCs are generally cultured on mouse embryonic fibroblast (MEF) or MEF conditioned medium (MEF-CM). However,for therapeutic applications,it is preferable for ESCs to be cultured under chemically defined conditions. Here,we report synthetic compounds that allow expansion of undifferentiated mouse ESCs in the absence of MEF,Leukemia Inhibitory Factor (LIF),and Fetal Bovine Serum (FBS). ESCs cultured for more than 30 d in a serum-free medium supplemented with indole derivertives retained their characteristic morphology and expressed markers such as SSEA-1,OCT3/4,Rex-1,Sox2,and Nanog. They consistently differentiated into many types of cells,including neurons,muscle cells,and hepatocytes. These results indicate that our compounds provide a more efficient and safer large-scale culture system for pluripotent ESCs,and hence might contribute to the use of ESCs in therapeutic applications. View Publication

MDS is characterized by ineffective hematopoiesis that leads to peripheral cytopenias. Development of effective treatments has been impeded by limited insight into pathogenic pathways governing dysplastic growth of hematopoietic progenitors. We demonstrate that smad2,a downstream mediator of transforming growth factor-beta (TGF-beta) receptor I kinase (TBRI) activation,is constitutively activated in MDS bone marrow (BM) precursors and is overexpressed in gene expression profiles of MDS CD34(+) cells,providing direct evidence of overactivation of TGF-beta pathway in this disease. Suppression of the TGF-beta signaling by lentiviral shRNA-mediated down-regulation of TBRI leads to in vitro enhancement of hematopoiesis in MDS progenitors. Pharmacologic inhibition of TBRI (alk5) kinase by a small molecule inhibitor,SD-208,inhibits smad2 activation in hematopoietic progenitors,suppresses TGF-beta-mediated gene activation in BM stromal cells,and reverses TGF-beta-mediated cell-cycle arrest in BM CD34(+) cells. Furthermore,SD-208 treatment alleviates anemia and stimulates hematopoiesis in vivo in a novel murine model of bone marrow failure generated by constitutive hepatic expression of TGF-beta1. Moreover,in vitro pharmacologic inhibition of TBRI kinase leads to enhancement of hematopoiesis in varied morphologic MDS subtypes. These data directly implicate TGF-beta signaling in the pathobiology of ineffective hematopoiesis and identify TBRI as a potential therapeutic target in low-risk MDS. View Publication

The regulated production of neurons in the hippocampus throughout life underpins important brain functions such as learning and memory. Surprisingly,however,studies have so far failed to identify a resident hippocampal stem cell capable of providing the renewable source of these neurons. Here,we report that depolarizing levels of KCl produce a threefold increase in the number of neurospheres generated from the adult mouse hippocampus. Most interestingly,however,depolarizing levels of KCl led to the emergence of a small subpopulation of precursors (approximately eight per hippocampus) with the capacity to generate very large neurospheres (textgreater 250 microm in diameter). Many of these contained cells that displayed the cardinal properties of stem cells: multipotentiality and self-renewal. In contrast,the same conditions led to the opposite effect in the other main neurogenic region of the brain,the subventricular zone,in which neurosphere numbers decreased by approximately 40% in response to depolarizing levels of KCl. Most importantly,we also show that the latent hippocampal progenitor population can be activated in vivo in response to prolonged neural activity found in status epilepticus. This work provides the first direct evidence of a latent precursor and stem cell population in the adult hippocampus,which is able to be activated by neural activity. Because the latent population is also demonstrated to reside in the aged animal,defining the precise mechanisms that underlie its activation may provide a means to combat the cognitive deficits associated with a decline in neurogenesis. View Publication

The demonstration that the small synthetic molecule reversine [2-(4-morpholinoanilino)-N6-cyclohexyladenine] promotes the dedifferentiation of committed cells into multipotent progenitor-type cells has raised hopes on the exploitation of this small chemical tool for the generation of stem cells. Here,we show that reversine causes a failure in cytokinesis and induces polyploidization. These effects of reversine are due to the inhibition of Aurora A and B,two related kinases that are implicated in several aspects of mitosis and that are frequently amplified and overexpressed in human tumors. Reversine inhibits the phosphorylation of histone H3,a direct downstream target of Aurora kinases. Similarly to the Aurora kinase inhibitor VX-680,which has recently entered phase II clinical trials for cancer treatment,reversine inhibited colony formation of leukemic cells from patients with acute myeloid leukemia but was significantly less toxic than VX-680 on cells from healthy donors. The crystal structure of the reversine-Aurora B kinase complex shows that reversine is a novel class of ATP-competitive Aurora kinase inhibitors. Thus,although our studies raise serious doubts on the application of reversine in regenerative medicine,they support the paradigm that reversine might be a useful agent in cancer chemotherapy. View Publication

Recent studies have shown that mesenchymal stem cells (MSC) with the potential for cell-mediated therapies and tissue engineering applications can be isolated from extracted dental tissues. Here,we investigated the collection,processing,and cryobiological characteristics of MSC from human teeth processed under current good tissue practices (cGTP). Viable dental pulp-derived MSC (DPSC) cultures were isolated from 31 of 40 teeth examined. Of eight DPSC cultures examined more thoroughly,all expressed appropriate cell surface markers and underwent osteogenic,adipogenic,and chondrogenic differentiation in appropriate differentiation medium,thus meeting criteria to be called MSC. Viable DPSC were obtained up to 120 h postextraction. Efficient recovery of DPSC from cryopreserved intact teeth and second-passage DPSC cultures was achieved. These studies indicate that DPSC isolation is feasible for at least 5 days after tooth extraction,and imply that processing immediately after extraction may not be required for successful banking of DPSC. Further,the recovery of viable DPSC after cryopreservation of intact teeth suggests that minimal processing may be needed for the banking of samples with no immediate plans for expansion and use. These initial studies will facilitate the development of future cGTP protocols for the clinical banking of MSC. View Publication

OBJECTIVE: We used genetically engineered mouse hematopoietic progenitor cells (HPCs) to investigate the therapeutic effects of human apoAI on atherosclerosis in apoE(-/-) mice. METHODS AND RESULTS: Lentiviral constructs expressing either human apoAI (LV-apoAI) or green fluorescent protein (LV-GFP) cDNA under a macrophage specific promoter (CD68) were generated and used for ex vivo transduction of mouse HPCs and macrophages. The transduction efficiency was textgreater25% for HPCs and textgreater70% for macrophages. ApoAI was found in the macrophage culture media,mostly associated with the HDL fraction. Interestingly,a significant increase in mRNA and protein levels for ATP binding cassette A1 (ABCA1) and ABCG1 were found in apoAI-expressing macrophages after acLDL loading. Expression of apoAI significantly increased cholesterol efflux in wild-type and apoE(-/-) macrophages. HPCs transduced with LV-apoAI ex vivo and then transplanted into apoE(-/-) mice caused a 50% reduction in atherosclerotic lesion area compared to GFP controls,without influencing plasma HDL-C levels. CONCLUSIONS: Lentiviral transduction of apoAI into HPCs reduces atherosclerosis in apoE(-/-) mice. Expression of apoAI in macrophages improves cholesterol trafficking in wild-type apoE-producing macrophages and causes upregulation of ABCA1 and ABCG1. These novel observations set the stage for a cell therapy approach to atherosclerosis regression,exploiting the cooperation between apoE and apoAI to maximize cholesterol exit from the plaque. View Publication