技术资料

PURPOSE: To develop a new oncolytic herpes simplex virus (oHSV) for glioblastoma (GBM) therapy that will be effective in glioblastoma stem cells (GSC),an important and untargeted component of GBM. One approach to enhance oHSV efficacy is by combination with other therapeutic modalities. EXPERIMENTAL DESIGN: MG18L,containing a U(S)3 deletion and an inactivating LacZ insertion in U(L)39,was constructed for the treatment of brain tumors. Safety was evaluated after intracerebral injection in HSV-susceptible mice. The efficacy of MG18L in human GSCs and glioma cell lines in vitro was compared with other oHSVs,alone or in combination with phosphoinositide-3-kinase (PI3K)/Akt inhibitors (LY294002,triciribine,GDC-0941,and BEZ235). Cytotoxic interactions between MG18L and PI3K/Akt inhibitors were determined using Chou-Talalay analysis. In vivo efficacy studies were conducted using a clinically relevant mouse model of GSC-derived GBM. RESULTS: MG18L was severely neuroattenuated in mice,replicated well in GSCs,and had anti-GBM activity in vivo. PI3K/Akt inhibitors displayed significant but variable antiproliferative activities in GSCs,whereas their combination with MG18L synergized in killing GSCs and glioma cell lines,but not human astrocytes,through enhanced induction of apoptosis. Importantly,synergy was independent of inhibitor sensitivity. In vivo,the combination of MG18L and LY294002 significantly prolonged survival of mice,as compared with either agent alone,achieving 50% long-term survival in GBM-bearing mice. CONCLUSIONS: This study establishes a novel therapeutic strategy: oHSV manipulation of critical oncogenic pathways to sensitize cancer cells to molecularly targeted drugs. MG18L is a promising agent for the treatment of GBM,being especially effective when combined with PI3K/Akt pathway-targeted agents. View Publication

Retinoic acid (RA) is used to treat leukemia and other cancers through its ability to promote cancer cell differentiation. Strategies to enhance the anticancer effects of RA could deepen and broaden its beneficial therapeutic applications. In this study,we describe a receptor cross-talk system that addresses this issue. RA effects are mediated by RAR/RXR receptors that we show are modified by interactions with the aryl hydrocarbon receptor (AhR),a protein functioning both as a transcription factor and a ligand-dependent adaptor in an ubiquitin ligase complex. RAR/RXR and AhR pathways cross-talk at the levels of ligand-receptor and also receptor-promoter interactions. Here,we assessed the role of AhR during RA-induced differentiation and a hypothesized convergence at Oct4,a transcription factor believed to maintain stem cell characteristics. RA upregulated AhR and downregulated Oct4 during differentiation of HL-60 promyelocytic leukemia cells. AhR overexpression in stable transfectants downregulated Oct4 and also decreased ALDH1 activity,another stem cell-associated factor,enhancing RA-induced differentiation as indicated by cell differentiation markers associated with early (CD38 and CD11b) and late (neutrophilic respiratory burst) responses. AhR overexpression also increased levels of activated Raf1,which is known to help propel RA-induced differentiation. RNA interference-mediated knockdown of Oct4 enhanced RA-induced differentiation and G(0) cell-cycle arrest relative to parental cells. Consistent with the hypothesized importance of Oct4 downregulation for differentiation,parental cells rendered resistant to RA by biweekly high RA exposure displayed elevated Oct4 levels that failed to be downregulated. Together,our results suggested that therapeutic effects of RA-induced leukemia differentiation depend on AhR and its ability to downregulate the stem cell factor Oct4. 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

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

BACKGROUND: Ewing's sarcoma cell lines may represent a good in vitro model for the understanding of tumor biology in this heterogeneous group of diseases. In the present study,we report the establishment and characterization of a primary Ewing's sarcoma cell line (LDS-Falck 01). MATERIALS AND METHODS: LDS-Falck 01 was generated from a malignant pleural effusion of a patient with metastatic peripheral primitive neuroectodermal tumor arising from the chest wall. Extensive characterization of the cells was accomplished using immunocytochemical,RT-PCR and cytogenetic studies. RESULTS: In vitro LDS-Falck 01 cells had both anchorage-dependent and -independent growth patterns. Immunocytochemical studies showed that cells were PAS-,vimentin-,CD99- and NSE-positive,EGFR- and CD117-negative. Cytogenetic analysis revealed a complex hyperdiploid karyotype with multiple chromosomal aberrations including an unbalanced translocation t(11;22)(q24;q12). The EWS/FLI1 chimeric transcript type 1 was detected. CONCLUSION: This cell line may represent a valid tool for investigating the biomolecular characteristics of this group of neoplasms and their sensitivity to therapeutic agents. View Publication

BACKGROUND: Cancer stem cells are a chemotherapy-resistant population capable of self-renewal and of regenerating the bulk tumor,thereby causing relapse and patient death. Ewing's sarcoma,the second most common form of bone tumor in adolescents and young adults,follows a clinical pattern consistent with the Cancer Stem Cell model - remission is easily achieved,even for patients with metastatic disease,but relapse remains frequent and is usually fatal. METHODOLOGY/PRINCIPAL FINDINGS: We have isolated a subpopulation of Ewing's sarcoma cells,from both human cell lines and human xenografts grown in immune deficient mice,which express high aldehyde dehydrogenase (ALDH(high)) activity and are enriched for clonogenicity,sphere-formation,and tumor initiation. The ALDH(high) cells are resistant to chemotherapy in vitro,but this can be overcome by the ATP binding cassette transport protein inhibitor,verapamil. Importantly,these cells are not resistant to YK-4-279,a small molecule inhibitor of EWS-FLI1 that is selectively toxic to Ewing's sarcoma cells both in vitro and in vivo. CONCLUSIONS/SIGNIFICANCE: Ewing's sarcoma contains an ALDH(high) stem-like population of chemotherapy-resistant cells that retain sensitivity to EWS-FLI1 inhibition. Inhibiting the EWS-FLI1 oncoprotein may prove to be an effective means of improving patient outcomes by targeting Ewing's sarcoma stem cells that survive standard chemotherapy. View Publication

Constant deregulation of Id1 and Id3 has been implicated in a wide range of carcinomas. However,underlying molecular evidence for the joint role of Id1 and Id3 in the tumorigenicity of small cell lung cancer (SCLC) is sparse. Investigating the biological significance of elevated expression in SCLC cells,we found that Id1 and Id3 co-suppression resulted in significant reduction of proliferation rate,invasiveness and anchorage-independent growth. Suppressing both Id1 and Id3 expression also greatly reduced the average size of tumors produced by transfectant cells when inoculated subcutaneously into nude mice. Further investigation revealed that suppressed expression of Id1 and Id3 was accompanied by decreased angiogenesis and increased apoptosis. Therefore,the SCLC tumorigenicity suppression effect of double knockdown of Id1 and Id3 may be regulated through pathways of apoptosis and angiogenesis. View Publication

Human T-cell lymphotropic virus type 1 (HTLV-1) is the etiologic agent of adult T-cell leukemia,an aggressive CD4(+) malignancy. Although HTLV-2 is highly homologous to HTLV-1,infection with HTLV-2 has not been associated with lymphoproliferative disorders. Lentivirus-mediated transduction of CD34(+) cells with HTLV-1 Tax (Tax1) induced G(0)/G(1) cell cycle arrest and resulted in the concomitant suppression of multilineage hematopoiesis in vitro. Tax1 induced transcriptional upregulation of the cdk inhibitors p21(cip1/waf1) (p21) and p27(kip1) (p27),and marked suppression of hematopoiesis in immature (CD34(+)/CD38(-)) hematopoietic progenitor cells in comparison to CD34(+)/CD38(+) cells. HTLV-1 infection of CD34(+) cells also induced p21 and p27 expression. Tax1 also protected CD34(+) cells from serum withdrawal-mediated apoptosis. In contrast,HTLV-2 Tax (Tax2) did not detectably alter p21 or p27 gene expression,failed to induce cell cycle arrest,failed to suppress hematopoiesis in CD34(+) cells,and did not protect cells from programmed cell death. A Tax2/Tax1 chimera encoding the C-terminal 53 amino acids of Tax1 fused to Tax2 (Tax(221)) displayed a phenotype in CD34(+) cells similar to that of Tax1,suggesting that unique domains encoded within the C terminus of Tax1 may account for the phenotypes displayed in human hematopoietic progenitor cells. These remarkable differences in the activities of Tax1 and Tax2 in CD34(+) hematopoietic progenitor cells may underlie the sharp differences observed in the pathogenesis resulting from infection with HTLV-1 and HTLV-2. View Publication

The B-cell receptor (BCR) transmits life and death signals throughout B-cell development,and altered BCR signaling may be required for survival of B-lymphoma cells. We used single-cell signaling profiles to compare follicular lymphoma (FL) B cells and nonmalignant host B cells within individual patient biopsies and identified BCR-mediated signaling events specific to lymphoma B cells. Expression of CD20,Bcl-2,and BCR light chain isotype (kappa or lambda) distinguished FL tumor B-cell and nontumor host B-cell subsets within FL patient biopsies. BCR-mediated signaling via phosphorylation of Btk,Syk,Erk1/2,and p38 occurred more rapidly in tumor B cells from FL samples than in infiltrating nontumor B cells,achieved greater levels of per-cell signaling,and sustained this level of signaling for hours longer than nontumor B cells. The timing and magnitude of BCR-mediated signaling in nontumor B cells within an FL sample instead resembled that observed in mature B cells from the peripheral blood of healthy subjects. BCR signaling pathways that are potentiated specifically in lymphoma cells should provide new targets for therapeutic attention. View Publication

Recognition of tumor-associated Ags (TAAs) on tumor cells by CTLs and the subsequent tumor cell death are assumed to be dependent on TAA protein expression and to correlate directly with the level of peptide displayed in the binding site of the HLA class I molecule. In this study we evaluated whether the levels of Her-2/neu protein expression on human tumor cell lines directly correlate with HLA-A*0201/Her2/neu peptide presentation and CTL recognition. We developed a TCR mimic (TCRm) mAb designated 1B8 that specifically recognizes the HLA-A2.1/Her2/neu peptide (369-377) (Her2(369)-A2) complex. TCRm mAb staining intensity varied for the five human tumor cell lines analyzed,suggesting quantitative differences in levels of the Her2(369)-A2 complex on these cells. Analysis of tumor cell lines pretreated with IFN-gamma and TNF-alpha for Her2/neu protein and HLA-A2 molecule expression did not reveal a direct correlation between the levels of Her2/neu Ag,HLA-A2 molecule,and Her2(369)-A2 complex expression. However,compared with untreated cells,cytokine-treated cell lines showed an increase in Her2(369)-A2 epitope density that directly correlated with enhanced tumor cell death (p = 0.05). Although a trend was observed between tumor cell lysis and the level of the Her2(369)-A2 complex for untreated cells,the association was not significant. These findings suggest that tumor cell susceptibility to CTL-mediated lysis may be predicted based on the level of specific peptide-MHC class I expression rather than on the total level of TAA expression. Further,these studies demonstrate the potential of the TCRm mAb for validation of endogenous HLA-peptide epitopes on tumor cells. View Publication

BACKGROUND: Cancer stem cells are presumed to have virtually unlimited proliferative and self-renewal abilities and to be highly resistant to chemotherapy,a feature that is associated with overexpression of ATP-binding cassette transporters. We investigated whether prolonged continuous selection of cells for drug resistance enriches cultures for cancer stem-like cells. METHODS: Cancer stem cells were defined as CD44+/CD24�?� cells that could self-renew (ie,generate cells with the tumorigenic CD44+/CD24�?� phenotype),differentiate,invade,and form tumors in vivo. We used doxorubicin-selected MCF-7/ADR cells,weakly tumorigenic parental MCF-7 cells,and MCF-7/MDR,an MCF-7 subline with forced expression of ABCB1 protein. Cells were examined for cell surface markers and side-population fractions by microarray and flow cytometry,with in vitro invasion assays,and for ability to form mammospheres. Xenograft tumors were generated in mice to examine tumorigenicity (n = 52). The mRNA expression of multidrug resistance genes was examined in putative cancer stem cells and pathway analysis of statistically significantly differentially expressed genes was performed. All statistical tests were two-sided. RESULTS: Pathway analysis showed that MCF-7/ADR cells express mRNAs from ABCB1 and other genes also found in breast cancer stem cells (eg,CD44,TGFB1,and SNAI1). MCF-7/ADR cells were highly invasive,formed mammospheres,and were tumorigenic in mice. In contrast to parental MCF-7 cells,more than 30% of MCF-7/ADR cells had a CD44+/CD24�?� phenotype,could self-renew,and differentiate (ie,produce CD44+/CD24�?� and CD44+/CD24+ cells) and overexpressed various multidrug resistance-linked genes (including ABCB1,CCNE1,and MMP9). MCF-7/ADR cells were statistically significantly more invasive in Matrigel than parental MCF-7 cells (MCF-7 cells = 0.82 cell per field and MCF-7/ADR = 7.51 cells per field,difference = 6.69 cells per field,95% confidence interval = 4.82 to 8.55 cells per field,P textless .001). No enrichment in the CD44+/CD24�?� or CD133+ population was detected in MCF-7/MDR. CONCLUSION: The cell population with cancer stem cell characteristics increased after prolonged continuous selection for doxorubicin resistance. View Publication