技术资料

The chimeric transcription factor E2a-Hlf is an oncoprotein associated with a subset of acute lymphoblastic leukemias of early B-lineage derivation. We employed a retroviral transduction-transplantation approach to evaluate the oncogenic effects of E2a-Hlf on murine B-cell progenitors harvested from adult bone marrow. Expression of E2a-Hlf induced short-lived clusters of primary hematopoietic cells but no long-term growth on preformed bone marrow stromal cell layers comprised of the AC6.21 cell line. Coexpression with Bcl-2,however,resulted in the sustained self-renewal of early preB-I cells that required stromal and interleukin-7 (IL-7) support for growth in vitro. Immortalized cells were unable to induce leukemias after transplantation into nonirradiated syngeneic hosts,unlike the leukemic properties and cytokine independence of preB-I cells transformed by p190(Bcr-Abl) under identical in vitro conditions. However,bone marrow cells expressing E2a-Hlf in combination with Bcl-2,but not E2a-Hlf alone,induced leukemias in irradiated recipients with long latencies,demonstrating both a requirement for suppression of apoptosis and the need for further secondary mutations in leukemia pathogenesis. Coexpression of IL-7 substituted for Bcl-2 to induce the in vitro growth of pre-B cells expressing E2a-Hlf,but leukemic conversion required additional abrogation of undefined stromal requirements and was associated with alterations in the Arf/Mdm2/p53 pathway. Thus,E2a-Hlf enhances the self-renewal of bone marrow B-cell progenitors without inciting a p53 tumor surveillance response or abrogating stromal and cytokine requirements for growth,which are nevertheless abrogated during progression to a leukemogenic phenotype. View Publication

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

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BACKGROUND: Compelling evidence indicates that paclitaxel kills cancer cells through the induction of apoptosis. Paclitaxel binds microtubules and causes kinetic suppression (stabilization) of microtubule dynamics. The consequent arrest of the cell cycle at mitotic phase has been considered to be the cause of paclitaxel-induced cytotoxicity. However,the biochemical events,downstream from paclitaxel's binding to microtubules,that lead to apoptosis are not well understood. METHODS: The authors examined recent scientific literature about the mechanisms by which paclitaxel exerts cytotoxicity. RESULTS: In addition to an arrest of the cell cycle at the mitotic phase in paclitaxel-treated cells,recent discoveries of activation of signaling molecules by paclitaxel and paclitaxel-induced transcriptional activation of various genes indicate that paclitaxel initiates apoptosis through multiple mechanisms. The checkpoint of mitotic spindle assembly,aberrant activation of cyclin-dependent kinases,and the c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK) are shown to be involved in paclitaxel-induced apoptosis. Consistent with observations that microtubules of different status (e.g.,cytoskeletal microtubules vs. mitotic spindles) have different sensitivity to paclitaxel,the concentration of paclitaxel appears to be the major determinant of its apoptogenic mechanisms. CONCLUSIONS: Advances in research of the cell cycle and apoptosis have extended our understanding of the mechanisms of paclitaxel-induced cell death. Further elucidation of resistance and enhancement of paclitaxel-induced apoptosis should expedite the development of better paclitaxel-based regimens for cancer therapy. View Publication

Current treatment options for advanced and hormone refractory prostate cancer are limited and responses to commonly used androgen pathway inhibitors are often unsatisfactory. Our recent results indicated that sodium ionophore monensin is one of the most potent and cancer-specific inhibitors in a systematic sensitivity testing of most known drugs and drug-like molecules in a panel of prostate cancer cell models. Because monensin has been extensively used in veterinary applications to build muscle mass in cattle,the link to prostate cancer and androgen signaling was particularly interesting. Here,we showed that monensin effects at nanomolar concentrations are linked to induction of apoptosis and potent reduction of androgen receptor mRNA and protein in prostate cancer cells. Monensin also elevated intracellular oxidative stress in prostate cancer cells as evidenced by increased generation of intracellular reactive oxygen species and by induction of a transcriptional profile characteristic of an oxidative stress response. Importantly,the antiproliferative effects of monensin were potentiated by combinatorial treatment with the antiandrogens and antagonized by antioxidant vitamin C. Taken together,our results suggest monensin as a potential well-tolerated,in vivo compatible drug with strong proapoptotic effects in prostate cancer cells,and synergistic effects with antiandrogens. Moreover,our data suggest a general strategy by which the effects of antiandrogens could be enhanced by combinatorial administration with agents that increase oxidative stress in prostate cancer cells. View Publication

Normal stem cells and cancer stem cells (CSCs) share similar properties,in that both have the capacity to self-renew and differentiate into multiple cell types. In both the normal stem cell and cancer stem cell fields,there has been a great need for a universal marker that can effectively identify and isolate these rare populations of cells in order to characterize them and use this information for research and therapeutic purposes. Currently,it would appear that certain isoenzymes of the aldehyde dehydrogenase (ALDH) superfamily may be able to fulfill this role as a marker for both normal and cancer stem cells. ALDH has been identified as an important enzyme in the protection of normal hematopoietic stem cells,and is now also widely used as a marker to identify and isolate various types of normal stem cells and CSCs. In addition,emerging evidence suggests that ALDH1 is not only a marker for stem cells,but may also play important functional roles related to self-protection,differentiation,and expansion. This comprehensive review discusses the role that ALDH plays in normal stem cells and CSCs,with focus on ALDH1 and ALDH3A1. Discrepancies in the functional themes between cell types and future perspectives for therapeutic applications will also be discussed. View Publication

To further clarify the transformation from monoclonal gammopathy of undetermined significance (MGUS) to plasma cell myeloma (PCM),we compared interphase fluorescence in situ hybridization (FISH) patterns in 381 MGUS and 301 PCM patients. According to the World Health Organization and the International Myeloma Working Group,a threshold of 10% of bone marrow plasma cells separated MGUS from PCM. After magnetic activated cell sorting for CD138(+) cells,FISH succeeded in 272 of 301 (90.4%) PCM,but in only 302 of 381 (79.3%) MGUS cases (P textless 0.001). Cytogenetic alterations were more frequent in PCM (237 of 272; 87.1%) than MGUS (169 of 302; 56.0%; P = 0.0002). PCM showed a median of two cytogenetic alterations (range,0-9) and MGUS one (range,0-6). Considering only cases with a yield of plasma cells allowing five or more FISH probes,del(13)(q14) was found in 99 of 251 (39.3%) PCM but in only 59 of 267 (22.1%) MGUS (P = 0.0001),del(17p) in 15 PCM (6.0%) and in 6 MGUS (2.2%) patients (P = 0.029). A t(4;14)/IGH-FGFR3 was detected in 28 PCM (11.1%) and 5 MGUS (1.9%; P textless 0.001). The t(11;14)/IGH-CCND1 and the t(14;16)/IGH-MAF showed no significant differences. Cytomorphology detected higher numbers of plasma cells than multiparameter flow cytometry (median ratio 4.25). This study underlines the genetic heterogeneity of MGUS similar to PCM. Genetic analysis might contribute to more diversified monitoring strategies for MGUS patients. View Publication

The p53 tumor suppressor limits proliferation in response to cellular stress through several mechanisms. Here,we test whether the recently described ability of p53 to limit stem cell self-renewal suppresses tumorigenesis in acute myeloid leukemia (AML),an aggressive cancer in which p53 mutations are associated with drug resistance and adverse outcome. Our approach combined mosaic mouse models,Cre-lox technology,and in vivo RNAi to disable p53 and simultaneously activate endogenous Kras(G12D)-a common AML lesion that promotes proliferation but not self-renewal. We show that p53 inactivation strongly cooperates with oncogenic Kras(G12D) to induce aggressive AML,while both lesions on their own induce T-cell malignancies with long latency. This synergy is based on a pivotal role of p53 in limiting aberrant self-renewal of myeloid progenitor cells,such that loss of p53 counters the deleterious effects of oncogenic Kras on these cells and enables them to self-renew indefinitely. Consequently,myeloid progenitor cells expressing oncogenic Kras and lacking p53 become leukemia-initiating cells,resembling cancer stem cells capable of maintaining AML in vivo. Our results establish an efficient new strategy for interrogating oncogene cooperation,and provide strong evidence that the ability of p53 to limit aberrant self-renewal contributes to its tumor suppressor activity. View Publication

Chimeric oncoproteins resulting from fusion of MLL to a wide variety of partnering proteins cause biologically distinctive and clinically aggressive acute leukemias. However,the mechanism of MLL-mediated leukemic transformation is not fully understood. Dot1,the only known histone H3 lysine 79 (H3K79) methyltransferase,has been shown to interact with multiple MLL fusion partners including AF9,ENL,AF10,and AF17. In this study,we utilize a conditional Dot1l deletion model to investigate the role of Dot1 in hematopoietic progenitor cell immortalization by MLL fusion proteins. Western blot and mass spectrometry show that Dot1-deficient cells are depleted of the global H3K79 methylation mark. We find that loss of Dot1 activity attenuates cell viability and colony formation potential of cells immortalized by MLL oncoproteins but not by the leukemic oncoprotein E2a-Pbx1. Although this effect is most pronounced for MLL-AF9,we find that Dot1 contributes to the viability of cells immortalized by other MLL oncoproteins that are not known to directly recruit Dot1. Cells immortalized by MLL fusions also show increased apoptosis,suggesting the involvement of Dot1 in survival pathways. In summary,our data point to a pivotal requirement for Dot1 in MLL fusion protein-mediated leukemogenesis and implicate Dot1 as a potential therapeutic target. View Publication

BACKGROUND: We showed there are specific ALDH1 autoantibodies in ovarian autoimmune disease and ovarian cancer,suggesting a role for ALDH1 in ovarian pathology. However,there is little information on the ovarian expression of ALDH1. Therefore,we compared ALDH1 expression in normal ovary and benign and malignant ovarian tumors to determine if ALDH1 expression is altered in ovarian cancer. Since there is also recent interest in ALDH1 as a cancer stem cell (CSC) marker,we assessed co-expression of ALDH1 with CSC markers in order to determine if ALDH1 is a potential CSC marker in ovarian cancer. METHODS: mRNA and protein expression were compared in normal human ovary and serous ovarian tumors using quantitative Reverse-Transcriptase PCR,Western blot (WB) and semi-quantitative immunohistochemistry (IHC). ALDH1 enzyme activity was confirmed in primary ovarian cells by flow cytometry (FC) using ALDEFLUOR assay. RESULTS: ALDH1 mRNA expression was significantly reduced (p textless 0.01; n = 5) in malignant tumors compared to normal ovaries and benign tumors. The proportion of ALDH1+ cells was significantly lower in malignant tumors (17.1 ± 7.61%; n = 5) compared to normal ovaries (37.4 ± 5.4%; p textless 0.01; n = 5) and benign tumors (31.03 ± 6.68%; p textless 0.05; n = 5). ALDH1+ cells occurred in the stroma and surface epithelium in normal ovary and benign tumors,although surface epithelial expression varied more in benign tumors. Localization of ALDH1 was heterogeneous in malignant tumor cells and little ALDH1 expression occurred in poorly differentiated malignant tumors. In benign tumors the distribution of ALDH1 had features of both normal ovary and malignant tumors. ALDH1 protein expression assessed by IHC,WB and FC was positively correlated (p textless 0.01). ALDH1 did not appear to be co-expressed with the CSC markers CD44,CD117 and CD133 by IHC. CONCLUSIONS: Total ALDH1 expression is significantly reduced in malignant ovarian tumors while it is relatively unchanged in benign tumors compared to normal ovary. Thus,ALDH1 expression in the ovary does not appear to be similar to breast,lung or colon cancer suggesting possible functional differences in these cancers. SIGNIFICANCE: These observations suggest that reduced ALDH1 expression is associated with malignant transformation in ovarian cancer and provides a basis for further study of the mechanism of ALDH1 in this process. View Publication