Mistletoe lectin activates caspase-8/FLICE independently of death receptor signaling and enhances anticancer drug-induced apoptosis.
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Mistletoe lectin I (ML-I) is a major active component in plant extracts of Viscum album that is increasingly used in adjuvant cancer therapy. ML-I exerts potent immunomodulating and cytotoxic effects, although its mechanism of action is largely unknown. We show that treatment of leukemic T- and B-cell lines with ML-I induced apoptosis, which required the prior activation of proteases of the caspase family. The involvement of caspases is demonstrated because (a) a peptide caspase inhibitor almost completely prevented ML-I-induced cell death and (b) proteolytic activation of caspase-8, caspase-9, and caspase-3 was observed. Because caspase-8 has been implicated as a regulator of apoptosis mediated by death receptors, we further investigated a potential receptor involvement in ML-I-induced effects. Cell death triggered by ML-I was neither attenuated in cell clones resistant to CD95 nor in cells that were rendered refractory to other death receptors by overexpressing a dominant-negative FADD mutant. In contrast, ML-I triggered a receptor-independent mitochondria-controlled apoptotic pathway because it rapidly induced the release of cytochrome c into the cytosol. Because ML-I was also observed to enhance the cytotoxic effect of chemotherapeutic drugs, these data may provide a molecular basis for clinical trials using MLs in anticancer therapy. (+info)
Quantitative analysis of CD79b, CD5 and CD19 in mature B-cell lymphoproliferative disorders.
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BACKGROUND AND OBJECTIVE: Distinction between B-cell chronic leukemias can be difficult due to overlap in cell morphology and immunologic features. We investigated, by quantitative flow cytometry, the expression of CD79b, CD5 and CD19 in cells from a variety of B-cell disorders to see whether this analysis adds further information useful to the diagnosis and characterization of these diseases. DESIGN AND METHODS: Peripheral blood cells from 6 normal individuals were used as reference controls. The diseases of the 63 patients investigated comprised: 29 chronic lymphocytic leukemia (CLL), six of them with atypical morphology, 6 B-cell prolymphocytic leukemia (PLL), 12 splenic lymphoma with villous lymphocytes (SLVL) and 16 mantle-cell (Mc) lymphoma in leukemic phase. The study was carried out by triple immunostaining with directly conjugated monoclonal antibodies (MoAb) against CD79b, CD5 and CD19 and quantitative estimation of the antigens per cell assessed with standard microbeads (Quantum Simply Cellular). RESULTS: Compared to normal B-cells, the number of CD19 molecules was significantly lower in cells from all of the B-cell disorders except PLL. The intensity of CD5 in leukemic B-cells was significantly higher in CLL cells, including atypical cases, and Mc lymphoma than in normal B-cells, whilst PLL and SLVL had values similar to those of normal B-lymphocytes. CD79b was expressed at lower levels in all types of leukemic cells compared to normal B-lymphocytes but differences were statistically significant in CLL, Mc lymphoma and SLVL. The number of CD79b molecules per cell was significantly lower in typical CLL than in the remaining B-cell diseases whilst the comparison of CD5 and CD19 intensity between CLL and non-CLL samples failed to show any statistically significant difference. INTERPRETATION AND CONCLUSIONS: Distinct antigen density patterns for the various conditions emerged from this analysis: Typical CLL was characterized by moderate CD5 and weak or negative CD79b expression. Mc lymphoma showed an homogeneous pattern, characterized by similar expression of CD5 than CLL but significantly stronger expression of CD79b whilst PLL and SLVL had weak CD5 and moderate CD79b expression. Atypical CLL had an intermediate pattern of CD79b antigen expression ranging from weak to moderate with bright CD5. Unlike CD5 and CD79b, CD19 did not discriminate the various B-cell disorders but only between normal and leukemic cells. (+info)
A DNA damage repair mechanism is involved in the origin of chromosomal translocations t(4;11) in primary leukemic cells.
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Some chromosomal translocations involved in the origin of leukemias and lymphomas are due to malfunctions of the recombinatorial machinery of immunoglobulin and T-cell receptor-genes. This mechanism has also been proposed for translocations t(4;11)(q21;q23), which are regularly associated with acute pro-B cell leukemias in early childhood. Here, reciprocal chromosomal breakpoints in primary biopsy material of fourteen t(4;11)-leukemia patients were analysed. In all cases, duplications, deletions and inversions of less than a few hundred nucleotides indicative of malfunctioning DNA repair mechanisms were observed. We concluded that these translocation events were initiated by several DNA strand breaks on both participating chromosomes and subsequent DNA repair by 'error-prone-repair' mechanisms, but not by the action of recombinases of the immune system. (+info)
SLUG, a ces-1-related zinc finger transcription factor gene with antiapoptotic activity, is a downstream target of the E2A-HLF oncoprotein.
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The E2A-HLF fusion gene transforms human pro-B lymphocytes by interfering with an early step in apoptotic signaling. In a search for E2A-HLF-responsive genes, we identified a zinc finger transcription factor, SLUG, whose product belongs to the Snail family of developmental regulatory proteins. Importantly, SLUG bears close homology to the CES-1 protein of C. elegans, which acts downstream of CES-2 in a neuron-specific cell death pathway. Consistent with the postulated role of CES-1 as an antiapoptotic transcription factor, SLUG was nearly as active as Bcl-2 or Bcl-xL in promoting the survival of IL-3-dependent murine pro-B cells deprived of the cytokine. We conclude that SLUG is an evolutionarily conserved transcriptional repressor whose activation by E2A-HLF promotes the aberrant survival and eventual malignant transformation of mammalian pro-B cells otherwise slated for apoptotic death. (+info)
Molecular diagnostics on microfabricated electrophoretic devices: from slab gel- to capillary- to microchip-based assays for T- and B-cell lymphoproliferative disorders.
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BACKGROUND: Current methods for molecular-based diagnosis of disease rely heavily on modern molecular biology techniques for interrogating the genome for aberrant DNA sequences. These techniques typically include amplification of the target DNA sequences followed by separation of the amplified fragments by slab gel electrophoresis. As a result of the labor-intensive, time-consuming nature of slab gel electrophoresis, alternative electrophoretic formats have been developed in the form of capillary electrophoresis and, more recently, multichannel microchip electrophoresis. METHODS: Capillary electrophoresis was explored as an alternative to slab gel electrophoresis for the analysis of PCR-amplified products indicative of T- and B-cell malignancies as a means of defining the elements for silica microchip-based diagnosis. Capillary-based separations were replicated on electrophoretic microchips. RESULTS: The microchip-based electrophoretic separation effectively resolved PCR-amplified fragments from the variable region of the T-cell receptor-gamma gene (150-250 bp range) and the immunoglobulin heavy chain gene (80-140 bp range), yielding diagnostically relevant information regarding the presence of clonal DNA populations. Although hydroxyethylcellulose provided adequate separation power, the need for a coated microchannel for effective resolution necessitated additional preparative steps. In addition, preliminary data are shown indicating that polyvinylpyrrolidone may provide an adequate matrix without the need for microchannel coating. CONCLUSIONS: Separation of B- and T-cell gene rearrangement PCR products on microchips provides diagnostic information in dramatically reduced time (160 s vs 2.5 h) with no loss of diagnostic capacity when compared with current methodologies. As illustrated, this technology and methodology holds great potential for extrapolation to the abundance of similar molecular biology-based techniques. (+info)
Functional characterization of TEL/AML1 fusion protein in the regulation of human CR1 gene promoter.
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The TEL/AML1 fusion gene occurs in childhood B-cell acute lymphoblastic leukemia (ALL) as a result of the translocation of human chromosome 12;21. Using reporter gene assays, we have functionally characterized TEL, AML1 and TEL/AML1 fusion proteins in the regulation of the human CR1 gene. Analysis of transcription activities showed that AML1 increased the CR1 promoter activity and that TEL repressed the basal activity of the promoter. Increased activities of the CR1 promoter by AML1 protein were reduced by the TEL protein in a concentration-dependent manner. When TEL/AML1 and AML1 proteins are present in cells at the same time, the TEL/AML1 protein inhibits the transactivation activities of AML1 protein on the human CR1 promoter even though TEL/AML1 retains the transactivation domain of AML1. A mutation analysis of the human CR1 promoter revealed that the binding sites for TEL and AML1 are necessary for the action of TEL and TEL/AML1, respectively. Thus, production of the TEL/AML1 protein by translocation of human chromosome 12;21 may contribute to leukemogenesis by the specific inhibition of AML1-dependent activation of myeloid promoters. (+info)
Activation of Hex and mEg5 by retroviral insertion may contribute to mouse B-cell leukemia.
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AKXD recombinant inbred mice develop a variety of leukemias and lymphomas due to retrovirally mediated insertional activation of cellular proto-oncogenes. We describe a new retroviral insertion site that is the most frequent genetic alteration in AKXD B-cell leukemias. Multiple genes flank the site of viral insertion, but the expression of just two, Hex and mEg5, is significantly upregulated. Hex is a divergent homeobox gene that is transiently expressed in many hematopoietic lineages, suggesting an involvement in cellular differentiation. mEg5 is a member of the bim-C subfamily of kinesin related proteins that are necessary for spindle formation and stabilization during mitosis. Our data provide the first genetic evidence for the activation of these genes in leukemia, and suggest that unscheduled expression of Hex and mEg5 contributes to the development of B-cell leukemia. In addition, this work highlights the use of genomic approaches for the study of position effect mutations. (+info)
Distribution and pattern of BCL-6 mutations throughout the spectrum of B-cell neoplasia.
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BCL-6 mutations are accumulated during B-cell transit through the germinal center (GC) and provide a histogenetic marker for B-cell tumors. On the basis of a comprehensive analysis of 308 B-cell neoplasms, we (1) expand the spectrum of tumors associated with BCL-6 mutations; (2) corroborate the notion that mutations cluster with GC and post-GC B-cell neoplasms; and (3) identify heterogeneous mutation frequency among B-lineage diffuse large cell lymphoma (B-DLCL) subsets. Mutations are virtually absent in acute lymphoblastic leukemia (P <.001) and mantle cell lymphoma (P <.05), whereas they occur frequently in GC or post-GC neoplasms, including lymphoplasmacytoid lymphoma, follicular lymphoma, MALT lymphomas, B-DLCL and Burkitt lymphoma. Among B-DLCL, mutations occur frequently in systemic nodal B-DLCL, primary extranodal B-DLCL, CD5(+) B-DLCL, CD30(+) B-DLCL, and primary splenic B-DLCL, suggesting a similar histogenesis of these B-DLCL subsets. Conversely, mutations are rare in primary mediastinal B-DLCL with sclerosis (10.0%; P <.01), supporting a distinct histogenesis for this lymphoma. Longitudinal follow-up of B-DLCL transformed from follicular lymphoma shows that they BCL-6 mutations may accumulate during histologic progression. Mutations also occur in some B-cell chronic lymphocytic leukemias, small lymphocytic lymphomas, and hairy cell leukemias, consistent with the hypothesis that a fraction of these lymphoproliferations are related to GC-like cells. Finally, the molecular pattern of 193 mutational events reinforces the hypothesis that mutations of BCL-6 and immunoglobulin genes are caused by similar mechanisms. (Blood. 2000;95:651-659) (+info)