技术资料

Dual EGFR/erbB2 inhibition is an attractive therapeutic strategy for epithelial tumors,as ligand-induced erbB2/EGFR heterodimerization triggers potent proliferative and survival signals. Here we show that a small molecule,GW572016,potently inhibits both EGFR and erbB2 tyrosine kinases leading to growth arrest and/or apoptosis in EGFR and erbB2-dependent tumor cell lines. GW572016 markedly reduced tyrosine phosphorylation of EGFR and erbB2,and inhibited activation of Erk1/2 and AKT,downstream effectors of proliferation and cell survival,respectively. Complete inhibition of activated AKT in erbB2 overexpressing cells correlated with a 23-fold increase in apoptosis compared with vehicle controls. EGF,often elevated in cancer patients,did not reverse the inhibitory effects of GW572016. These observations were reproduced in vivo,where GW572016 treatment inhibited activation of EGFR,erbB2,Erk1/2 and AKT in human tumor xenografts. Erk1/2 and AKT represent potential biomarkers to assess the clinical activity of GW572016. Inhibition of activated AKT in EGFR or erbB2-dependent tumors by GW572016 may lead to tumor regressions when used as a monotherapy,or may enhance the anti-tumor activity of chemotherapeutics,since constitutive activation of AKT has been linked to chemo-resistance. View Publication

Transfer of therapeutic genes to human hematopoietic stem cells (HSCs) using complex vectors at clinically relevant efficiencies remains a major challenge. Recently we described a stable retroviral vector that sustains long-term expression of green fluorescent protein (GFP) and a human beta-globin gene in the erythroid progeny of transduced murine HSCs. We now report the efficient transduction of primitive human CD34(+) fetal liver or cord blood cells with this vector and expression of the beta-globin transgene in the erythroid progeny of these human cells for at least 2 months. After growth factor prestimulation and then a 2- to 3-day exposure to the virus,35% to 55% GFP(+) progeny were seen in assays of transduced colony-forming cells,primitive erythroid precursors that generate large numbers of glycophorin A(+) cells in 3-week suspension cultures,and 6-week long-term culture-initiating cells. In immunodeficient mice injected with unselected infected cells,5% to 15% of the human cells regenerated in the marrow (including the erythroid cells) were GFP(+) 3 and 6 weeks after transplantation. Importantly,the numbers of GFP(+) human lymphoid and either granulopoietic or erythroid cells in individual mice 6 weeks after transplantation were significantly correlated,indicative of the initial transduction of human multipotent cells with in vivo repopulating activity. Expression of the transduced beta-globin gene in human cells obtained directly from the mice or after their differentiation into erythroid cells in vitro was demonstrated by reverse transcriptase-polymerase chain reaction using specific primers. These experiments represent a significant step toward the realization of a gene therapy approach for human beta-globin gene disorders. View Publication

BACKGROUND: At therapeutic concentrations,the antineoplastic agent taxol selectively perturbs mitotic spindle microtubules. Taxol has recently been shown to induce apoptosis,similar to the mechanism of cell death induced by other antineoplastic agents. However,taxol has shown efficacy against drug-refractory cancers,raising the possibility that this pharmacological agent may trigger an alternative apoptotic pathway. MATERIALS AND METHODS: The kinetics and IC50 of mitotic (M) block,aberrant mitosis,and cytotoxicity following taxol treatment were analyzed in human cell lines as well as normal mouse embryo fibroblasts (MEFs) and MEFs derived from p53-null mice. Apoptosis was followed by DNA gel electrophoresis and by in situ DNA end-labeling (TUNEL). RESULTS: Taxol induced two forms of cell cycle arrest: either directly in early M at prophase or,for those cells progressing through aberrant mitosis,arrest in G1 as multimininucleated cells. TUNEL labeling revealed that DNA nicking occurred within 30 min of the arrest in prophase. In contrast,G1-arrested,multimininucleated cells became TUNEL positive only after several days. In the subset of cells that became blocked directly in prophase,both wt p53-expressing and p53-null MEFs responded similarly to taxol,showing rapid onset of DNA nicking and apoptosis. However,p53-null MEFs progressing through aberrant mitosis failed to arrest in the subsequent G1 phase or to become TUNEL positive,and remained viable. CONCLUSIONS: Taxol induces two forms of cell cycle arrest,which in turn induce two independent apoptotic pathways. Arrest in prophase induces rapid onset of a p53-independent pathway,whereas G1-block and the resulting slow (3-5 days) apoptotic pathway are p53 dependent. View Publication

Taxol stabilizes microtubules,prevents tubulin depolymerization,and promotes tubulin bundling and is one of the most effective drugs for the treatment of metastatic breast and ovarian cancer. Although its interaction with tubulin has been well characterized,the mechanism by which taxol induces growth arrest and cytotoxicity is not well understood. Herein,we show that taxol induced dose- and time-dependent accumulation of the cyclin inhibitor p21WAF1 in both p53 wild-type and p53-null cells,although the degree of induction was greater in cells expressing wild-type p53. In MCF7 cells,wild-type p53 protein was also induced after taxol treatment,and this induction was mediated primarily by increased protein stability. Taxol induced both p21WAF1 and wild-type p53 optimally in MCF7 cells after 20-24-h exposure with an EC50(3) of 5 nM. In p53-null PC3M cells,p21WAF1 was similarly induced after 24-h exposure to taxol. Coincident with these biochemical effects,taxol altered the electrophoretic mobility of c-raf-1 and stimulated mitogen activated protein kinase. Previous depletion of c-raf-1 inhibited both the p21WAF1- and p53-inducing properties of taxol,as well as the activation of MAP kinase. These data suggest that induction of p21WAF1 by taxol requires c-raf-1 activity,but that it is not strictly dependent on wild-type p53. Furthermore,the ability of taxol to both induce wild-type p53 in MCF7 cells and activate MAP kinase is also dependent on c-raf-1 expression. View Publication

Cancer initiation and progression have been attributed to newly discovered subpopulations of self-renewing,highly tumorigenic,drug-resistant tumor cells termed cancer stem cells. Recently,we and others reported a new phenotypic plasticity wherein highly tumorigenic,drug-resistant cell populations could arise not only from pre-existing cancer stem-like populations but also from cancer cells lacking these properties. In the current study,we hypothesized that this newfound phenotypic plasticity may be mediated by PI3K/Akt and Wnt/β-catenin signaling,pathways previously implicated in carcinogenesis,pluripotency and drug resistance. Using GFP expression,Hoechst dye exclusion and fluorescence activated cell sorting (FACS) of cancer cell lines,we identified and tracked cancer stem-like side populations (SP) of cancer cells characterized by high tumorigenicity and drug resistance. We found that pharmacological inhibition or genetic depletion of PI3K and AKT markedly reduced the spontaneous conversion of nonside population (NSP) cells into cancer stem-like SP cells,whereas PI3K/Akt activation conversely enhanced NSP to SP conversion. PI3K/AKT signaling was mediated through downstream phosphorylation of GSK3β,which led to activation and accumulation of β-catenin. Accordingly,pharmacological or genetic perturbation of GSK3β or β-catenin dramatically impacted conversion of NSP to SP. Further downstream,β-catenin's effects on NSP-SP equilibrium were dependent upon its interaction with CBP,a KAT3 family coactivator. These studies provide a mechanistic model wherein PI3K/Akt/β-catenin/CBP signaling mediates phenotypic plasticity in and out of a drug-resistant,highly tumorigenic state. Therefore,targeting this pathway has unique potential for overcoming the therapy resistance and disease progression attributed to the cancer stem-like phenotype. View Publication

The aim of the present study was to determine the effect of AZD8055 on proliferation,apoptosis and glycolysis in the human cervical cancer cell line HeLa and to investigate the underlying mechanism(s) of action. HeLa human cervical cancer cells were treated with 10 nM AZD8055 for 24,48 or 72 h. MTT was used to determine cell proliferation. Annexin V/propidium iodide staining was used to determine cell apoptosis analyzed by fluorescence-activated cell sorting (FACS). Glycolytic activity was determined by measuring the activity of the key enzyme lactate dehydrogenase (LDH) and lactate production. RNA and protein expression were examined by qRT-PCR and western blotting,respectively. Treatment with AZD8055 inhibited proliferation and glycolysis,and induced apoptosis in HeLa cells in a time-dependent manner. During the prolonged treatment with AZD8055,the phosphorylation of mammalian target of rapamycin (mTOR) C1 substrates p70S6K and phosphorylation of the mTORC2 substrate Akt were deregulated,suggesting that the activity of mTOR was downregulated. Furthermore,our study showed that the expression of miR-143 was upregulated in a time-dependent manner in HeLa cells treated with AZD8055. In summary,the present study reveals a novel antitumor mechanism of AZD8055 in HeLa human cervical cancer cells. View Publication