技术资料

The epithelium of the mammary gland exists in a highly dynamic state,undergoing dramatic morphogenetic changes during puberty,pregnancy,lactation,and regression. The recent identification of stem and progenitor populations in mouse and human mammary tissue has provided evidence that the mammary epithelium is organized in a hierarchical manner. Characterization of these normal epithelial subtypes is an important step toward understanding which cells are predisposed to oncogenesis. This review summarizes progress in the field toward defining constituent cells and key molecular regulators of the mammary epithelial hierarchy. Potential relationships between normal epithelial populations and breast tumor subtypes are discussed,with implications for understanding the cellular etiology underpinning breast tumor heterogeneity. View Publication

To date,studies on T cells in acute myeloid leukemia (AML) have been limited to flow cytometric analysis of whole peripheral blood mononuclear cell (PBMC) specimens or functional work looking at the impact of AML myeloblasts on normal or remission T cells. This lack of information on T cells at the time of presentation with disease is due in part to the difficulty in isolating sufficiently pure T cells from these specimens for further study. Negative immunomagnetic selection has been the method of choice for isolating immune cells for functional studies due to concerns that binding antibodies to the cell surface may induce cellular activation,block ligand-receptor interactions or result in immune clearance. In order specifically to study T cells in presentation AML specimens,we set out to develop a method of isolating highly pure CD4 and CD8 T cells by negative selection from the peripheral blood (PB) of newly diagnosed AML patients. This technique,unlike T cell selection from PB from normal individuals or from patients with chronic lymphocytic leukaemia,was extremely problematic due to properties of the leukaemic myeloblasts. A successful method was eventually optimized requiring the use of a custom antibody cocktail consisting of CD33,CD34,CD123,CD11c and CD36,to deplete myeloblasts. 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

Previous studies have demonstrated leukemic complications in mice after high-copy retroviral gene transfer of the multidrug resistance 1 (MDR1) cDNA,encoding a membrane-located efflux pump expressed in hematopoietic stem cells. In contrast,no such complications or MDR1-associated alterations of hematopoiesis were observed in numerous other studies exploring MDR1 gene transfer into cell lines,mice,dogs,nonhuman primates,and human subjects. Here,we show that leukemias associated with retroviral expression of MDR1 depend on high vector dose,and involve the selection of clones with combinatorial insertional mutagenesis of proto-oncogenes or other signaling genes. Compared with insertion patterns in normal long-term repopulating hematopoietic cells,such hits were overrepresented in leukemic clones,pointing to a causal role. A similar constellation of insertion sites was also observed in a leukemia arising after high-copy retroviral gene transfer of a fluorescent protein. Spectral karyotyping demonstrated additional chromosomal translocations in a subset of cases,indicative of secondary genetic instability. We also show that insertional mutants can be amplified in vitro prior to transplantation. On the basis of these findings,we suggest the use of preclinical dose-escalation studies to define a therapeutic index for retroviral transgene delivery. View Publication

In this study,in an attempt to identify neuroblastoma-associated surface antigens,we generated mAbs against the ACN neuroblastoma cell line. A mAb was selected (5B14) that reacted with all neuroblastoma cell lines analyzed and allowed detection of tumor cell infiltrates in bone marrow aspirates from neuroblastoma patients. In cytofluorimetric analysis,unlike anti-disialoganglioside mAb,5B14 mAb did not display reactivity with normal bone marrow hematopoietic cell precursors,thus representing a highly specific marker for identifying neuroblastoma cells. Molecular analysis revealed that the 5B14 mAb-reactive surface glycoprotein corresponded to the recently identified 4Ig-B7-H3 molecule. Remarkably,mAb-mediated masking of the 4Ig-B7-H3 molecule on cell transfectants or on freshly isolated neuroblastoma cells resulted in enhancement of natural killer-mediated lysis of these target cells. These data suggest that 4Ig-B7-H3 molecules expressed at the tumor cell surface can exert a protective role from natural killer-mediated lysis by interacting with a still undefined inhibitory receptor expressed on natural killer cells. View Publication

Cell enrichment methods that deal with larger volumes of peripheral blood and BM are needed for increased sensitivity of detection,characterization and quantification of isolated tumor cells (ITC). This study was designed to evaluate a new procedure,the RosetteSep-Applied Imaging Rare Event (RARE) detection method,which depletes the majority of the erythrocytes and leucocytes in a peripheral blood (PB) sample,thereby negatively enriching tumor cells if present. This enrichment procedure allows for increased sensitivity,by analyzing a 5-10 fold larger volume of blood,compared with a direct immunocytochemical (ICC) technique,with minimal impact on laboratory workload. Model experiments showed comparable tumor cell recoveries between the two tested methods,both in PB and BM. Clinical samples were evaluated using paired PB and BM samples from 95 carcinoma patients. Analysis of PB results showed that 25.3% had textgreater or = 1 tumor cell detected by the RARE procedure,compared with 5.2% after direct ICC analysis,analyzing a 10-fold larger volume by the RARE procedure. The direct ICC analysis of BM from the same patients revealed 16.8% positive. The ITC detection differed both quantitatively and qualitatively between BM and PB,as samples with high numbers of ITC in BM were still negative in PB. The clinical significance of ITC in blood still needs to be established. However,the easy access of peripheral blood,and the increased sensitivity obtained by increasing the sample volume with the RARE procedure,suggests that the value of peripheral blood analysis should be tested in parallel in studies where ITC detection in BM is performed. View Publication

The aim of this study was to standardize in vitro culture conditions for human acute lymphoblastic leukemia (ALL) cells. The cells were cultured in medium containing 10% fetal calf serum (FCS) and in the four serum-free media X-vivo 10,X-vivo 15,X-vivo 20 and Stem Span. Native ALL blasts could proliferate in all four serum-free media,but the strongest responses were usually observed with Stem Span. Native leukemia blasts were also cultured in the presence of various single cytokines or cytokine combinations. The highest proliferation was usually observed in the presence of Flt3-Ligand (Flt3-L) when single cytokines were examined,and these responses could be further increased especially by combining Flt3-L with interleukin 3 (IL3),IL7 or stem cell factor (SCF). Proliferation could also be increased when ALL blasts were cultured in the presence of two commercially available fibroblast cell lines (Hs27 and HFL1). Based on these results we suggest that in vitro culture conditions for native human ALL blasts can be standardized by using serum-free culture media supplemented with exogenous Flt3-L+IL3+SCF,and the use of accessory cells can also be standardized by using well-characterized fibroblast cell lines. Detectable ALL blast proliferation can then be observed for most patients. Our experimental model can thereby be used for in vitro evaluation of possible antileukemic treatment strategies,and it will then allow comparison of experimental results between different studies. View Publication