技术资料

The human ATP-binding cassette superfamily G (White) member 2 (ABCG2) gene and its murine homologue breast cancer resistance protein 1 (Bcrp1) are recently described ATP-binding cassette transporters associated with drug resistance in tumor cell lines,including the MCF-7 cell line,selected for its resistance to mitoxantrone (MCF-7/MitoR). Infection of MCF-7 cells with the retroviral vector containing ABCG2 cDNA (G1-ABCG2) resulted in cells (MCF-7/ABCG2) that were resistant to mitoxantrone at levels similar to those observed in MCF-7/MitoR cells. Previous studies have shown that pluripotent hematopoietic stem cells overexpress the multidrug-resistant transport (MDR1) gene and efflux rhodamine,a substrate for the MDR1 transporter. Other studies have identified a primitive hematopoietic stem cell population,or side population (SP) cells,which are identified by their efflux of the fluorescent dye,Hoechst 33342. In an attempt to identify the transport genes responsible for this phenotype,we examined the uptake of Hoechst 33342 into MCF-7,MCF-7/MitoR,and MCF-7 cells infected with a retroviral vector expressing the ABCG2 gene (MCF-7/ABCG2). MCF-7/MitoR cells as well as MCF-7/ABCG2 cells demonstrated lower levels of Hoechst 33342 uptake compared with the parental MCF-7 cells. We also examined the level of the mouse Bcrp1 RNA in SP cells and non-SP cells isolated from mouse hematopoietic cells. Mouse SP cells expressed relatively high levels of Bcrp1 mRNA relative to non-SP cells. These results suggest that Hoechst 33342 is a substrate for the ABCG2 transporter and that ABCG2/Bcrp1 expression may serve as a marker for hematopoietic stem cells in hematopoietic cells. View Publication

Sir2p is an NAD(+)-dependent histone deacetylase required for chromatin-dependent silencing in yeast. In a cell-based screen for inhibitors of Sir2p,we identified a compound,splitomicin,that creates a conditional phenocopy of a sir2 deletion mutant in Saccharomyces cerevisiae. Cells grown in the presence of the drug have silencing defects at telomeres,silent mating-type loci,and the ribosomal DNA. In addition,whole genome microarray experiments show that splitomicin selectively inhibits Sir2p. In vitro,splitomicin inhibits NAD(+)-dependent histone deacetylase activity (HDA) of the Sir2 protein. Mutations in SIR2 that confer resistance to the drug map to the likely acetylated histone tail binding domain of the protein. By using splitomicin as a chemical genetic probe,we demonstrate that continuous HDA of Sir2p is required for maintaining a silenced state in nondividing cells. View Publication

Two distinct dendritic cell (DC) subpopulations have been evidenced in mice on the basis of their differential CD8alpha expression and their localization in lymphoid organs. Several reports suggest that CD8alpha(+) and CD8alpha(-) DC subsets could be functionally different. In this study,using a panel of MHC class I- and/or class II-restricted peptides,we analyzed CD4(+) and CD8(+) T cell responses obtained after i.v. injection of freshly purified peptide-pulsed DC subsets. First,we showed that both DC subsets efficiently induce specific CTL responses and Th1 cytokine production in the absence of CD4(+) T cell priming. Second,we showed that in vivo activation of CD4(+) T cells by CD8alpha(+) or CD8alpha(-) DC,injected i.v.,leads to a nonpolarized Th response with production of both Th1 and Th2 cytokines. The CD8alpha(-) subset induced a higher production of Th2 cytokines such as IL-4 and IL-10 than the CD8alpha(+) subset. However,IL-5 was produced by CD4(+) T cells activated by both DC subsets. When both CD4(+) and CD8(+) T cells were primed by DC injected i.v.,a similar pattern of cytokines was observed,but,under these conditions,Th1 cytokines were mainly produced by CD8(+) T cells,while Th2 cytokines were produced by CD4(+) T cells. Thus,this study clearly shows that CD4(+) T cell responses do not influence the development of specific CD8(+) T cell cytotoxic responses induced either by CD8alpha(+) or CD8alpha(-) DC subsets. View Publication

AG-490 is a member of the tyrphostin family of tyrosine kinase inhibitors. While AG-490 has been considered to be a Janus kinase (JAK)2-specific inhibitor,these conclusions were primarily drawn from acute lymphoblastic leukemia cells that lack readily detectable levels of JAK3. In the present study,evidence is provided that clearly demonstrates AG-490 potently suppresses IL-2-induced T cell proliferation,a non-JAK2-dependent signal,in a dose-dependent manner in T cell lines D10 and CTLL-2. AG-490 blocked JAK3 activation and phosphorylation of its downstream counterpart substrates,STATs. Inhibition of JAK3 by AG-490 also compromised the Shc/Ras/Raf/mitogen-activated protein kinase (MAPK) signaling pathways as measured by phosphorylation of Shc and extracellular signal-related kinase 1 and 2 (ERK1/2). AG-490 effectively inhibited tyrosine phosphorylation and DNA binding activities of several transcription factors including STAT1,-3,-5a,and -5b and activating protein-1 (AP-1) as judged by Western blot analysis and electrophoretic mobility shift assay. These data suggest that AG-490 is a potent inhibitor of the JAK3/STAT,JAK3/AP-1,and JAK3/MAPK pathways and their cellular consequences. Taken together,these findings support the notion that AG-490 possesses previously unrecognized clinical potential as an immunotherapeutic drug due to its inhibitory effects on T cell-derived signaling pathways. View Publication

Pluripotent ES cells can be used to generate a wide variety of cell populations in vitro in a manner resembling embryonic development. Recent advances in controlling ES cell differentiation,combined with the power of genetic and biochemical manipulation,are providing insights into cell biology and the determination of cell fate. View Publication

Recent studies have indicated that the development of cyclin-dependent kinase (cdk)2 inhibitors that deregulate E2F are a plausible pharmacological strategy for novel antineoplastic agents. We show here that 3-[1-(3H-Imidazol-4-yl)-meth-(Z)-ylidene]-5-methoxy-1,3-dihydro-indol-2-one (SU9516),a novel 3-substituted indolinone compound,binds to and selectively inhibits the activity of cdk2. This inhibition results in a time-dependent decrease (4-64%) in the phosphorylation of the retinoblastoma protein pRb,an increase in caspase-3 activation (5-84%),and alterations in cell cycle resulting in either a G(0)-G(1) or a G(2)-M block. We also report here cell line differences in the cdk-dependent phosphorylation of pRb. These findings demonstrate that SU9516 is a selective cdk2 inhibitor and support the theory that compounds that inhibit cdk2 are viable resources in the development of new antineoplastic agents. View Publication

Valproic acid is widely used to treat epilepsy and bipolar disorder and is also a potent teratogen,but its mechanisms of action in any of these settings are unknown. We report that valproic acid activates Wntdependent gene expression,similar to lithium,the mainstay of therapy for bipolar disorder. Valproic acid,however,acts through a distinct pathway that involves direct inhibition of histone deacetylase (IC(50) for HDAC1 = 0.4 mm). At therapeutic levels,valproic acid mimics the histone deacetylase inhibitor trichostatin A,causing hyperacetylation of histones in cultured cells. Valproic acid,like trichostatin A,also activates transcription from diverse exogenous and endogenous promoters. Furthermore,valproic acid and trichostatin A have remarkably similar teratogenic effects in vertebrate embryos,while non-teratogenic analogues of valproic acid do not inhibit histone deacetylase and do not activate transcription. Based on these observations,we propose that inhibition of histone deacetylase provides a mechanism for valproic acid-induced birth defects and could also explain the efficacy of valproic acid in the treatment of bipolar disorder. View Publication

Although the source of embryonic stem (ES) cells presents ethical concerns,their use may lead to many clinical benefits if differentiated cell types can be derived from them and used to assemble functional organs. In pancreas,insulin is produced and secreted by specialized structures,islets of Langerhans. Diabetes,which affects 16 million people in the United States,results from abnormal function of pancreatic islets. We have generated cells expressing insulin and other pancreatic endocrine hormones from mouse ES cells. The cells self-assemble to form three-dimensional clusters similar in topology to normal pancreatic islets where pancreatic cell types are in close association with neurons. Glucose triggers insulin release from these cell clusters by mechanisms similar to those employed in vivo. When injected into diabetic mice,the insulin-producing cells undergo rapid vascularization and maintain a clustered,islet-like organization. View Publication

Calmodulin-binding sites on target proteins show considerable variation in primary sequence; hence compounds that block the access of calmodulin to these binding sites may be more selective than compounds that inactivate calmodulin. Suramin and its analogue NF307 inhibit the interaction of calmodulin with the ryanodine receptor. We have investigated whether inhibition of calmodulin binding to target proteins is a general property of these compounds. Suramin inhibited binding of [(125)I]calmodulin to porcine brain membranes and to sarcoplasmic reticulum from skeletal muscle (IC(50)=4.9+/-1.2 microM and 19.9+/-1.8 microM,respectively) and blocked the cross-linking of [(125)I]calmodulin to some,but not all,target proteins in brain membranes by [(125)I]calmodulin. Four calmodulin-binding proteins were purified [ryanodine receptor-1 (RyR1) from rabbit skeletal muscle,neuronal NO synthase (nNOS) from Sf9 cells,G-protein betagamma dimers (Gbetagamma) from porcine brain and a glutathione S-transferase-fusion protein comprising the C-terminal calmodulin-binding domain of the metabotropic glutamate receptor 7A (GST-CmGluR7A) from bacterial lysates]. Three of the proteins employed (Gbetagamma,GST-CmGluR7A and RyR1) display a comparable affinity for calmodulin (in the range of 50-70 nM). Nevertheless,suramin and NF307 only blocked the binding of Gbetagamma and RyR1 to calmodulin-Sepharose. In contrast,the association of GST-CmGluR7A and nNOS was not impaired,whereas excess calmodulin uniformly displaced all proteins from the matrix. Thus suramin and NF307 are prototypes of a new class of calmodulin antagonists that do not interact directly with calmodulin but with calmodulin-recognition sites. In addition,these compounds discriminate among calmodulin-binding motifs. View Publication

High expression of the Breast Cancer Resistance Protein (BCRP) gene has been shown to be involved in resistance to chemotherapeutic drugs. Knowledge of the localization of BCRP protein in normal tissues may help unravel the normal function of this protein. Therefore,we characterized the tissue distribution and cellular localization of BCRP in frozen sections of normal human tissues. For this purpose,we used the recently described monoclonal antibody BXP-34 and another independently developed monoclonal antibody directed against BCRP,BXP-21. Both monoclonal antibodies show specific BCRP plasma membrane staining on cytospins obtained from topotecan- or mitoxantrone-selected cell lines,as well as from BCRP-transfected cell lines. Immunoprecipitation experiments using either BXP-21 or BXP-34 yielded a clear M(r) 72,000 BCRP band from BCRP-overexpressing tumor cells. In the topotecan-selected T8 and mitoxantrone-selected MX3 tumor cell lines,BCRP turned out to be differentially glycosylated. In contrast to BXP-34,BXP-21 is able to detect the M(r) 72,000 BCRP protein on immunoblots and is reactive with BCRP in formalin-fixed,paraffin-embedded tissues. Using BXP-21 and BXP-34,prominent staining of BCRP was observed in placental syncytiotrophoblasts,in the epithelium of the small intestine and colon,in the liver canalicular membrane,and in ducts and lobules of the breast. Furthermore,BCRP was present in veinous and capillary endothelium,but not in arterial endothelium in all of the tissues investigated. In the tissues studied,the mRNA levels of BCRP were assessed using reverse transcription-PCR,and these corresponded with the levels of BCRP protein estimated from immunohistochemical staining. The presence of BCRP at the placental syncytiotrophoblasts is consistent with the hypothesis of a protective role of BCRP for the fetus. The apical localization in the epithelium of the small intestine and colon indicates a possible role of BCRP in the regulation of the uptake of p.o. administered BCRP substrates by back-transport of substrate drugs entering from the gut lumen. Therefore,it may be useful to attempt to modulate the uptake of p.o. delivered BCRP substrates,e.g.,topotecan or irinotecan,by using a BCRP inhibitor. Clinical trials testing this hypothesis have been initiated in our institute. View Publication

A careful prognostic evaluation of patients referred for high-dose therapy (HDT) is warranted to identify those who maximally benefit from HDT as well as those who clearly fail current HDT and are candidates for more innovative treatments. In a series of 110 patients with myeloma who received HDT as first-line therapy,times to event (disease progression and death) were studied through proportional hazard models,in relation to different prognostic factors,including a chromosome 13 fluorescence in situ hybridization (FISH) analysis using a D13S319 probe. Delta13 was detected in 42 patients (38%). Follow-up time among surviving patients and survival time were 48 +/- 3 and 51 +/- 7 months,respectively (median +/- SE). In the univariate analysis,Delta13 was the most powerful adverse prognostic factor for all times to event,especially for the survival time (P textless.0001) and was followed by beta2-microglobulin (beta2m) levels 2.5 mg/L or higher (P =.0001). The comparison of survival prognostic models including beta2m 2.5 mg/L or greater and another factor favored the Delta13/beta2m combination. In 22 patients (20%) with no unfavorable factor,the median survival time was not reached at 111 months. In contrast,among 55 patients (50%) with one unfavorable factor and 33 patients (30%) with 2 unfavorable factors,median survival times were 47.3 +/- 4.6 months and 25.3 +/- 3.2 months,respectively (P textless.0001). We conclude that delta13,adequately detected by FISH analysis,is a very strong factor related to poor survival,especially when associated with a beta2m level of 2.5 mg/L or higher. Routine FISH Delta13 assessment is strongly recommended for patients considered for HDT. View Publication

The effect of resiquimod (R-848),an immune-response modifier that is similar to imiquimod,on recurrent herpes simplex virus (HSV) was evaluated using the guinea pig model of genital herpes. Guinea pigs were intravaginally infected with HSV-2 and then were randomized on day 14 to receive nothing or 0.1 mL/kg per dose of subcutaneous resiquimod,given either daily,every other day,or weekly from days 15-35. During a 3-week course of therapy,recurrences in all 3 treated groups were reduced by textgreater80%,compared with the control group. After therapy,recurrences remained significantly (Ptextless.05) decreased in all 3 groups for the next 3 weeks. The group treated weekly developed the fewest recurrences. Significant increases in interleukin-2 levels,produced by incubation of mononuclear cells with HSV-2 antigens,but not in circulating antibody also were detected in the treated groups. Resiquimod treatment may offer significant advantages to present antiviral therapies for the control of recurrent genital herpes. View Publication