技术资料

Nuclear accumulation of beta-catenin is a key event for the development of colorectal cancer. Little is known,however,about the mechanisms underlying translocation of beta-catenin from the cytoplasm or the membrane to the nucleus. The present study examined whether signal transducers and activators of transcription 3 (STAT3) activation is involved in the nuclear accumulation of beta-catenin in colorectal cancer cells. Of the 90 primary colorectal cancer tissues,40 (44.4%) were positive for nuclear staining of p-STAT3 and 63 (70.0%) were positive for nuclear staining of beta-catenin. The nuclear staining of both p-STAT3 and beta-catenin were observed predominantly in the periphery of the cancer tissues. Importantly,of the 40 tumors with p-STAT3 nuclear staining,37 (92.5%) were also positive for nuclear beta-catenin staining and there was a significant correlation between p-STAT3 and beta-catenin nuclear staining (P textless 0.01). Coexpression of nuclear p-STAT3 and beta-catenin was associated with lower patient survival (P textless 0.01). In an in vitro study using a human colon cancer cell line,SW480,inhibition of STAT3 by dominant negative STAT3 or the Janus kinase inhibitor,AG490,induced translocation of beta-catenin from the nucleus to the cytoplasm or membrane. Luciferase assays revealed that STAT3 inhibition resulted in significant suppression of beta-catenin/T-cell factor transcription in association with significant inhibition of cell proliferation (P textless 0.05). These findings suggest that in colorectal cancer,STAT3 activation is involved in the nuclear accumulation of beta-catenin,resulting in poor patient survival. View Publication

Phosphatase and tensin homologue deleted on chromosome 10 (PTEN) and Akt are important regulators of the phosphatidylinositol 3-kinase (PI3K) pathway and thus are important to the regulation of a wide spectrum of tumor-related biological processes. Akt regulates several critical cellular functions,including cell cycle progression; cell migration,invasion,and survival; and angiogenesis. Decreased expression of PTEN and overexpression of the Akt proto-oncogene,which is located downstream of PI3K,have been shown in a variety of cancers,including glioblastoma. Novel small-molecule inhibitors of receptors and signaling pathways,including inhibitors of the PI3K pathway,have shown antitumor activity,but inhibitors of Akt have not been examined. In this study,we tested our hypothesis that the pharmacologic inhibition of Akt has an antiproliferative effect on gliomas. We showed that two newly developed Akt inhibitors,KP-372-1 and KP-372-2 (herein called KP-1 and KP-2),effectively inhibited the PI3K/Akt signaling cascade. KP-1 and KP-2 blocked both the basal and epidermal growth factor-induced phosphorylation of Akt Ser473 at 125 and 250 nmol/L,which,in turn,reduced the activation of intracellular downstream targets of Akt,including GSK-3beta and p70s6k. Furthermore,the treatment of U87 and U251 glioma cells with 125 to 250 nmol/L KP-1 and KP2 for 48 hours inhibited cell growth by approximately 50%. This decrease in cell growth stemmed from the induction of apoptosis. Collectively,these results provide a strong rationale for the pharmacologic targeting of Akt for the treatment of gliomas. View Publication

The pathogenesis of malarial anemia is multifactorial,and the mechanisms responsible for its high mortality are poorly understood. Studies indicate that host mediators produced during malaria infection may suppress erythroid progenitor development (Miller,K.L.,J.C. Schooley,K.L. Smith,B. Kullgren,L.J. Mahlmann,and P.H. Silverman. 1989. Exp. Hematol. 17:379-385; Yap,G.S.,and M.M. Stevenson. 1991. Ann. NY Acad. Sci. 628:279-281). We describe an intrinsic role for macrophage migration inhibitory factor (MIF) in the development of the anemic complications and bone marrow suppression that are associated with malaria infection. At concentrations found in the circulation of malaria-infected patients,MIF suppressed erythropoietin-dependent erythroid colony formation. MIF synergized with tumor necrosis factor and gamma interferon,which are known antagonists of hematopoiesis,even when these cytokines were present in subinhibitory concentrations. MIF inhibited erythroid differentiation and hemoglobin production,and it antagonized the pattern of mitogen-activated protein kinase phosphorylation that normally occurs during erythroid progenitor differentiation. Infection of MIF knockout mice with Plasmodium chabaudi resulted in less severe anemia,improved erythroid progenitor development,and increased survival compared with wild-type controls. We also found that human mononuclear cells carrying highly expressed MIF alleles produced more MIF when stimulated with the malarial product hemozoin compared with cells carrying low expression MIF alleles. These data suggest that polymorphisms at the MIF locus may influence the levels of MIF produced in the innate response to malaria infection and the likelihood of anemic complications. View Publication

Chronic phase-to-blast crisis transition in chronic myelogenous leukemia (CML) is associated with differentiation arrest and down-regulation of C/EBPalpha,a transcription factor essential for granulocyte differentiation. Patients with CML in blast crisis (CML-BC) became rapidly resistant to therapy with the breakpoint cluster region-Abelson murine leukemia (BCR/ABL) kinase inhibitor imatinib (STI571) because of mutations in the kinase domain that interfere with drug binding. We show here that the restoration of C/EBPalpha activity in STI571-sensitive or -resistant 32D-BCR/ABL cells induced granulocyte differentiation,inhibited proliferation in vitro and in mice,and suppressed leukemogenesis. Moreover,activation of C/EBPalpha eradicated leukemia in 4 of 10 and in 6 of 7 mice injected with STI571-sensitive or -resistant 32D-BCR/ABL cells,respectively. Differentiation induction and proliferation inhibition were required for optimal suppression of leukemogenesis,as indicated by the effects of p42 C/EBPalpha,which were more potent than those of K298E C/EBPalpha,a mutant defective in DNA binding and transcription activation that failed to induce granulocyte differentiation. Activation of C/EBPalpha in blast cells from 4 patients with CML-BC,including one resistant to STI571 and BMS-354825 and carrying the T315I Abl kinase domain mutation,also induced granulocyte differentiation. Thus,these data indicate that C/EBPalpha has potent antileukemia effects even in cells resistant to ATP-binding competitive tyrosine kinase inhibitors,and they portend the development of anti-leukemia therapies that rely on C/EBPalpha activation. View Publication

Mechanisms of lethality of the three-substituted indolinone and putatively selective cyclin-dependent kinase (CDK)2 inhibitor 3-[1-(3H-imidazol-4-yl)-meth-(Z)-ylidene]-5-methoxy-1,3-dihydro-indol-2-one (SU9516) were examined in human leukemia cells. Exposure of U937 and other leukemia cells to SU9516 concentrations textgreater or =5 microM rapidly (i.e.,within 4 h) induced cytochrome c release,Bax mitochondrial translocation,and apoptosis in association with pronounced down-regulation of the antiapoptotic protein Mcl-1. These effects were associated with inhibition of phosphorylation of the carboxyl-terminal domain (CTD) of RNA polymerase (Pol) II on serine 2 but not serine 5. Reverse transcription-polymerase chain reaction analysis revealed pronounced down-regulation of Mcl-1 mRNA levels in SU9516-treated cells. Similar results were obtained in Jurkat and HL-60 leukemia cells. Furthermore,cotreatment with the proteasome inhibitor N-benzoyloxycarbonyl (Z)-Leu-Leu-leucinal (MG132) blocked SU9516-mediated Mcl-1 down-regulation,implicating proteasomal degradation in diminished expression of this protein. Ectopic expression of Mcl-1 largely blocked SU9516-induced cytochrome c release,Bax translocation,and apoptosis,whereas knockdown of Mcl-1 by small interfering RNA potentiated SU9516 lethality,confirming the functional contribution of Mcl-1 down-regulation to SU9516-induced cell death. It is noteworthy that SU9516 treatment resulted in a marked increase in reactive oxygen species production,which was diminished,along with cell death,by the free radical scavenger N-acetylcysteine (NAC). We were surprised to find that NAC blocked SU9516-mediated inhibition of RNA Pol II CTD phosphorylation on serine 2,reductions in Mcl-1 mRNA levels,and Mcl-1 down-regulation. Together,these findings suggest that SU9516 kills leukemic cells through inhibition of RNA Pol II CTD phosphorylation in association with oxidative damage and down-regulation of Mcl-1 at the transcriptional level,culminating in mitochondrial injury and cell death. View Publication

RATIONALE: Adults with cystic fibrosis (CF) are at increased risk of developing osteoporosis. During infective exacerbations,increased production of proinflammatory cytokines and markers of bone resorption have been reported. OBJECTIVE: The aim of this study is to investigate the growth and proliferation of potential osteoclast precursor cells before,during,and after intravenous antibiotic treatment of infective exacerbations in patients with CF. METHODS: Hematopoietic precursor cell growth was examined using colony formation assays using Methocult culture medium. Circulating potential osteoclast precursors were identified using four-color flow cytometry by CD14,CD33,CD34,and CD45 expression. RESULTS: At the start of an infective exacerbation increases in hematopoietic precursor colony formation (15.42 colonies/10(5) cells plated,p = 0.025),proliferation (28.5%,p textless 0.001),and the numbers of circulating potential osteoclast precursors (6.5%,p textless 0.001) were seen in comparison with baseline levels. These increases declined after treatment with intravenous antibiotics to a level close to baseline. CONCLUSIONS: The results demonstrate an increase in the production of potential osteoclast precursors in the peripheral blood during CF infective exacerbations. This may result in increased bone resorption and contribute to bone loss in patients with CF. View Publication

Cell replacement therapy is a promising approach for the treatment of cardiac diseases. It is,however,challenged by a limited supply of appropriate cells. Therefore,we have investigated whether functional cardiomyocytes can be efficiently generated from human embryonic stem cells (hESCs). In this study,we developed an efficient protocol for the generation of functional cardiomyocytes from hESCs by combining hanging drop culture and 5-azacytidine,a well-known demethylating agent,and then evaluated the expression of cardiac-specific markers. hESCs were cultured both in the medium without or with 0.1,1,or 10 microM of 5-azacytidine under a hanging drop culture. The expression of several cardiac-specific markers was determined by real-time PCR,RT-PCR,immunofluorescence,and confocal microscopy. To verify the structural and functional properties of hESC-derived cardiomyocytes,we performed electron microscopy and electrophysiological recording. The efficiency of beating cell generation was significantly improved in the hanging drop culture compared with that in suspension culture. Treatment of hESCs with 0.1 microM of 5-azacytidine for 1-3 days significantly increased the number of beating cells and simultaneously enhanced the expression of cardiac-specific markers. Transmission electron microscopy and electrophysiological recording showed that hESC-derived cardiomyocytes acquired structural and functional properties of cardiomyocytes. In conclusion,these results suggest that differentiation of hESCs into cardiomyocytes can be enhanced by the combination of hanging drop culture and 5-azacytidine treatment. Also the methylation status of genes related to cardiomyocyte development may play an important role in the differentiation of hESCs into cardiomyocytes. View Publication

BACKGROUND & AIMS: Bone marrow (BM) cells may transdifferentiate into or fuse with organ parenchymal cells. BM therapy shows promise in murine models of cirrhosis,and clinical trials of bone marrow stem cell therapy for organ healing are underway. However,the BM may contribute to scar-forming myofibroblasts in various organs including the liver. We have studied this axis of regeneration and scarring in murine models of cirrhosis,including an assessment of the temporal and functional contribution of the BM-derived myofibroblasts. METHODS: Female mice were lethally irradiated and received male BM transplants. Carbon tetrachloride or thioacetamide was used to induce cirrhosis. BM-derived cells were tracked through in situ hybridization for the Y chromosome. BM transplants from 2 strains of transgenic mice were used to detect intrahepatic collagen production. RESULTS: In the cirrhotic liver,the contribution of BM to parenchymal regeneration was minor (0.6%); by contrast,the BM contributed significantly to hepatic stellate cell (68%) and myofibroblast (70%) populations. These BM-derived cells were found to be active for collagen type 1 transcription in 2 independent assays and could influence the fibrotic response to organ injury. These BM-derived myofibroblasts did not occur through cell fusion between BM-derived cells and indigenous hepatic cells but,instead,originated largely from the BM's mesenchymal stem cells. CONCLUSIONS: The BM contributes functionally and significantly to liver fibrosis and is a potential therapeutic target in liver fibrosis. Clinical trials of BM cell therapy for liver regeneration should be vigilant for the possibility of enhanced organ fibrosis. View Publication

To search for new indicators of self-renewing hematopoietic stem cells (HSCs),highly purified populations were isolated from adult mouse marrow,micromanipulated into a specially designed microscopic array,and cultured for 4 days in 300 ng/ml Steel factor,20 ng/ml IL-11,and 1 ng/ml flt3-ligand. During this period,each cell and its progeny were imaged at 3-min intervals by using digital time-lapse photography. Individual clones were then harvested and assayed for HSCs in mice by using a 4-month multilineage repopulation endpoint (textgreater1% contribution to lymphoid and myeloid lineages). In a first experiment,6 of 14 initial cells (43%) and 17 of 61 clones (28%) had HSC activity,demonstrating that HSC self-renewal divisions had occurred in vitro. Characteristics associated with HSC activity included longer cell-cycle times and the absence of uropodia on a majority of cells within the clone during the final 12 h of culture. Combining these criteria maximized the distinction of clones with HSC activity from those without and identified a subset of 27 of the 61 clones. These 27 clones included all 17 clones that had HSC activity; a detection efficiency of 63% (2.26 times more frequently than in the original group). The utility of these characteristics for discriminating HSC-containing clones was confirmed in two independent experiments where all HSC-containing clones were identified at a similar 2- to 3-fold-greater efficiency. These studies illustrate the potential of this monitoring system to detect new features of proliferating HSCs that are predictive of self-renewal divisions. View Publication

Supplementation of mesenchymal stem cells (MSCs) during hematopoietic stem cell (HSC) transplantation alleviates complications such as graft-versus-host disease,leading to a speedy recovery of hematopoiesis. To meet this clinical demand,a fast MSC expansion method is required. In the present study,we examined the feasibility of using a rotary bioreactor system to expand MSCs from isolated bone marrow mononuclear cells. The cells were cultured in a rotary bioreactor with Myelocult medium containing a combination of supplementary factors,including stem cell factor and interleukin-3 and -6. After 8 days of culture,total cell numbers,Stro-1(+)CD44(+)CD34(-) MSCs,and CD34(+)CD44(+)Stro-1(-) HSCs were increased 9-,29-,and 8-fold,respectively. Colony-forming efficiency-fibroblast per day of the bioreactor-treated cells was 1.44-fold higher than that of the cells without bioreactor treatment. The bioreactor-expanded MSCs showed expression of primitive MSC markers endoglin (SH2) and vimentin,whereas markers associated with lineage differentiation,including osteocalcin (osteogenesis),type II collagen (chondrogenesis),and C/EBP-alpha (CCAAT/enhancer-binding protein-alpha) (adipogenesis),were not detected. Upon induction,the bioreactor-expanded MSCs were able to differentiate into osteoblasts,chondrocytes,and adipocytes. We conclude that the rotary bioreactor with the modified Myelocult medium reported in this study may be used to rapidly expand MSCs. View Publication

Human immunodeficiency virus type 1 (HIV-1) R5 isolates that predominantly use CCR5 as a coreceptor are frequently described as macrophage tropic. Here,we compare macrophage tropism conferred by HIV-1 R5 envelopes that were derived directly by PCR from patient tissue. This approach avoids potentially selective culture protocols used in virus isolation. Envelopes were amplified (i) from blood and semen of adult patients and (ii) from plasma of pediatric patients. The phenotypes of these envelopes were compared to those conferred by an extended panel of envelopes derived from brain and lymph node that we reported previously. Our results show that R5 envelopes vary by up to 1,000-fold in their capacity to confer infection of primary macrophages. Highly macrophage-tropic envelopes were predominate in brain but were infrequent in semen,blood,and lymph node samples. We also confirmed that the presence of N283 in the C2 CD4 binding site of gp120 is associated with HIV-1 envelopes from the brain but absent from macrophage-tropic envelopes amplified from blood and semen. Finally,we compared infection of macrophages,CD4(+) T cells,and peripheral blood mononuclear cells (PBMCs) conferred by macrophage-tropic and non-macrophage-tropic envelopes in the context of full-length replication competent viral clones. Non-macrophage-tropic envelopes conferred low-level infection of macrophages yet infected CD4(+) T cells and PBMCs as efficiently as highly macrophage-tropic brain envelopes. The lack of macrophage tropism for the majority of the envelopes amplified from lymph node,blood,and semen is striking and contrasts with the current consensus that R5 primary isolates are generally macrophage tropic. The extensive variation in R5 tropism reported here is likely to have an important impact on pathogenesis and on the capacity of HIV-1 to transmit. View Publication

Purine nucleoside phosphorylase (PNP) deficiency in humans results in T lymphocytopenia. Forodesine,a potent inhibitor of PNP,was designed based on the transition-state structure stabilized by the enzyme. Previous studies established that forodesine in the presence of deoxyguanosine (dGuo) inhibits the proliferation of T lymphocytes. A phase 1 clinical trial of forodesine in T-cell malignancies demonstrated significant antileukemic activity with an increase in intracellular dGuo triphosphate (dGTP). High accumulation of dGTP in T cells may be dependent on the levels of deoxynucleoside kinases. Because B-cell chronic lymphocytic leukemia (B-CLL) cells have high activity of deoxycytidine kinase (dCK),we hypothesized that these lymphocytes would respond to forodesine. This postulate was tested in primary lymphocytes during in vitro investigations. Lymphocytes from 12 patients with CLL were incubated with forodesine and dGuo. These CLL cells showed a wide variation in the accumulation of intracellular dGTP without any effect on other deoxynucleotides. This was associated with DNA damage-induced p53 stabilization,phosphorylation of p53 at Ser15,and activation of p21. The dGTP accumulation was related to induction of apoptosis measured by caspase activation,changes in mitochondrial membrane potential,and PARP cleavage. Based on these data,a phase 2 clinical trial of forodesine has been initiated for CLL patients. View Publication