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  • American Society of Hematology  (3)
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  • American Society of Hematology  (3)
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  • 1
    In: Blood, American Society of Hematology, Vol. 108, No. 11 ( 2006-11-01), p. 2212-2212
    Abstract: In T-cell acute lymphoblastic leukaemia (T-ALL) alternative t(5;14)(q35;q32.2) forms effect leukemic dysregulation of either TLX3 or NKX2-5 homeobox genes at 5q35 by juxtaposition with 3′-BCL11B at 14q32.2. Putative regulatory sequences underlying ectopic homeobox gene activation in t(5;14), and their mode of action have remained poorly understood mainly because breakpoints at 14q32.2 are widely scattered over the ~1 Mbp genomic desert region. We pooled cytogenetic data from t(5;14) cell lines together with published clinical data to refine the BCL11B downstream breakpoint cluster region (bcr). Ectopic homeobox gene dysregulation was investigated by DNA-i(nhibitory-treatments) with 26-mer double-stranded DNA oligo(nucleotide)s directed against putative enhancers using NKX2-5 expression as endpoints. Enhancer targets were provisionally identified from orphan T-cell DNase-I hypersensive sites (DNaseI-HS) located between 3′-BCL11B and VRK1. NKX2-5 downregulation in t(5;14) PEER cells was almost entirely restricted to DNAi targeting enhancers within the distal bcr and was dose- and sequence-dependent. Interestingly, enhancers near 3′-BCL11B regulated that gene only. These data imply that enhancer-promoter distances and/or locations are important for long-range gene regulation. Chromatin immunoprecipation assays showed that the four most effectual NKX2-5 ectopic enhancers were hyperacetylated. These enhancers clustered ~1 Mbp downstream of BCL11B, within a region displaying multiple regulatory stigmata, including a TCRA-enhancer motif, and abyssal sequence-conservation (“5-Way Regulatory Potential”). Paradoxically, although TLX3/NKX2-5 promoter/exon-1 regions were hypo-acetylated, their expression decreased after TSA treatment, implying extrinsic regulation by factor(s) subject to acetylation-control. PU.1 is known to get transcriptionally repressed by TSA and potentially binds TLX3/NKX2-5 upstream promoter regions. Knockdown of PU.1 effected downregulation of both homeobox genes. Moreover, genomic analysis showed preferential enrichment near validated ectopic enhancers of binding sites for the PU.1-cofactor HMGA1, knockdown of which also inhibited NKX2-5 in PEER cells. Analysis of nuclear matrix attachment (NMA) in PEER cells showed enhanced attachment near to the most effectual enhancer cluster which was alleviated by TSA-treatment. Interestingly, the juxtapositional genomic regions of “active” ins(14;5) rearrangements driving NKX2-5 expression exhibited tight NMA, forming structures reminiscent of “active chromatin hubs”. These findings lead us to propose that HMGA1 and PU.1 co-regulate ectopic homeobox gene expression in t(5;14) T-ALL by interactions mediated at the nuclear matrix, possibly mediated by SATB1 binding. Our data document homeobox gene dysregulation by a novel regulatory region at 3′-BCL11B responsive to HDAC-inhibition and highlight a novel class of potential therapeutic target amid “junk” DNA.
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
    RVK:
    RVK:
    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2006
    detail.hit.zdb_id: 1468538-3
    detail.hit.zdb_id: 80069-7
    Location Call Number Limitation Availability
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  • 2
    In: Blood, American Society of Hematology, Vol. 106, No. 11 ( 2005-11-16), p. 845-845
    Abstract: The zinc-finger transcription factor BCL11B (14q32) is a key translocation target in pediatric T-cell acute lymphoblastic leukemia (T-ALL) where it ectopically activates NK-family homeobox genes, chiefly TLX3, or NKX2-5, via the recurrent t(5;14)(q35;q32). However, the mechanism underlying leukemic activation by BCL11B remains obscure. Breakpoints at 14q32 are dispersed over about 1.2 Mbp downstream of BCL11B amid a non-coding genomic “desert”. Detailed cytogenetic analysis of T-ALL cell lines, including one carrying a complex double-insertion enabled the putative breakpoint target zone to be narrowed to ~300 kbp covering the central and distal parts of the 3′-BCL11B region. Interestingly, Crawford et al. (PNAS101: 992–7, 2004) have identified a series of DNase-I hypersensitive sites (DHS) in T-cells amid this genomic desert including a unique cluster near the most distal breakpoint to serve as candidate BCL11B transcriptional enhancers. Accordingly, we used the TRANSFAC database to identify multiple transcription factor (TF) binding site sequences nearby these DHS. To investigate the role of DHS in activating homeobox transcription in T-ALL, we designed double-stranded 26-mer oligos matching each of 9 DHS which were used to transfect a T-ALL cell line (PEER) in which NKX2-5 is inserted about 1 Mbp downstream of BCL11B and thereby activated. Such oligos may inhibit TF binding by directly blocking access to DHS or act as decoys to sequester TF. We found that oligos targeting DHS in the central and distal regions of 3′-BCL11B, close to the insertion junctions in PEER cells, were effective in down-regulating NKX2-5 transcription in a dose dependent manner. In contrast, oligos targeting DHS near 3′-BCL11B were able to down-regulate BCL11B but not NKX2-5. Treatments with neither mutated DHS oligos, nor other control oligos caused inhibition. Transcription of ESTs present at 3′-BCL11B was unaffected by DHS-oligo transfection indicating specific targeting of BCL11B or NKX2-5 by their respective DHS. Analysis of t(5;14) T-ALL cell lines by Halo-FISH, showed the selective homing of 3′-BCL11B chromatin to the nuclear matrix when juxtaposed with NKX2-5, whereas 3′-BCL11B chromatin present on non-participant chromosomes was extruded to surrounding halos inimical to transcription. Taken together, our data show the presence of multiple DHS covering the ~1.2 Mbp breakpoint dispersal region at 3′-BCL11B in T-ALL cells which appear to serve as enhancers of BCL11B or nearby translocated NK-family homeobox genes. These multiple enhancers may be required for the precise timing of BCL11B transcription which is critical to thymocyte development - a question which will be addressed by planned future studies. As well as revealing novel aspects underlying the transcriptional control of BCL11B in T-cells, our data highlight a potential therapeutic target in leukemia within “junk DNA”.
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
    RVK:
    RVK:
    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2005
    detail.hit.zdb_id: 1468538-3
    detail.hit.zdb_id: 80069-7
    Location Call Number Limitation Availability
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  • 3
    Online Resource
    Online Resource
    American Society of Hematology ; 2005
    In:  Blood Vol. 106, No. 11 ( 2005-11-16), p. 4340-4340
    In: Blood, American Society of Hematology, Vol. 106, No. 11 ( 2005-11-16), p. 4340-4340
    Abstract: The & lt;Guide to Leukemia-Lymphoma Cell Lines & gt; summarizes the salient characteristics of 560 cell lines: precursor B (85), mature B (155), plasma cell leukemia/myeloma (66), immature T (55), mature T (21), natural killer (10), Hodgkin lymphoma (10), anaplastic large cell lymphoma (16), myelocytic (62), monocytic (33) and erythrocytic-megakaryocytic cell lines (45). Along with other improvements, the advent of continuous human leukemia-lymphoma (LL including myeloma) cell lines as a rich resource of abundant, accessible and manipulable living cells has contributed significantly to a better understanding of the pathophysiology of hematopoietic tumors. The first LL cell lines, Burkitt lymphoma-derived lines, were established in 1963. Since then more than 1500 cell lines have been described, some 500 of them in detail. The major advantages of continuous cell lines is the unlimited supply and worldwide availability of identical cell material and the infinite viable storability in liquid nitrogen. LL cell lines are characterized generally by monoclonal origin and differentiation arrest, sustained proliferation in vitro with preservation of most cellular features, and specific genetic alterations characteristic of their tumor of origin. Recent transcriptional profiling studies have confirmed that LL cell lines stably retain the aberrant profiles typical of their tumors of origin. The most practical classification of LL cell lines assigns them to one of the physiologically occurring cell lineages, based on their immunophenotype, genotype and functional features. Truly malignant cell lines should be discerned from Epstein-Barr virus-immortalized normal cells. The efficiency of cell line establishment is rather low and the deliberate establishment of new LL cell lines remains by and large an unpredictable process. Difficulties in establishing continuous cell lines may be caused by the inappropriate selection of nutrients and growth factors. Clearly, a generally suitable microenvironment for hematopoietic cells, either malignant or normal, cannot yet be defined. The characterization and publication of new LL cell lines should provide important and informative core data, attesting to their scientific significance. Large percentages of LL cell lines are contaminated with mycoplasma (about 20%) or are cross-contaminated with other cell lines (14% in our DNA fingerprinting analysis on 622 samples covering 560 LL cell lines). Solutions to these problems are sensitive detection, effective elimination and rigorous prevention of mycoplasma infection and proper, regular authentication of cell lines. The underlying cause appears to be negligent cell culture practice. The willingness of investigators to make their LL cell lines available to others is all too often limited. There is a need in the scientific community for clean and authenticated high quality LL cell lines to which every scientist has access. These are offered by public cell line banks like the DSMZ which holds 169 LL cell lines which all have undergone a strict quality, identity and characterization program (immunoprofile, karyotype, DNA fingerprint, virus/mycoplasma check). An example of the practical utility of LL cell lines are the recent advances in studies of classical and molecular cytogenetics which in large part were made possible by cell lines. We propose a list of the most useful, robust and publicly available reference cell lines which may be used for a variety of experimental purposes. The & lt;Guide to Leukemia-Lymphoma Cell Lines & gt; is available on CD; inquire at & lt;hdr@dsmz.de & gt;.
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
    RVK:
    RVK:
    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2005
    detail.hit.zdb_id: 1468538-3
    detail.hit.zdb_id: 80069-7
    Location Call Number Limitation Availability
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