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The LAZ3/BCL6 gene encoding a Zinc-finger nuclear protein is altered in Non-Hodgkin's Lymphomas (NHLs) by translocations, mutations and/or deletions clustered in its 5' non coding region, in a 3.3 Kbp EcoRI fragment which thus defines the Major Translocation Cluster (MTC). In the present study, we describe at the molecular level the deletions found in the MTC of two (NHL) cases using, (i) DNA obtained from a patient (GUI) with a monosomy 3 and three microdeletions of 101, 22, 25 bp in its unique untranslocated 3q27 allele; (ii) a cell line derived from a patient (VAL) carrying a t(3;4) (q27;p11) translocation and a 2.4 Kbp deletion in the untranslocated allele. As the MTC is recurrently subject to alterations, we have cloned and sequenced the murine equivalent of the human MTC and promoter region in an attempt to identify sequences well conserved in mammals that may be thus important for the LAZ3/BCL6 gene regulation. We show that the human and mouse 5' upstream regions of the LAZ3/BCL6 gene although mainly intronic share a particularly high homology of 79% on the overall sequence. Strikingly, the small sequences which are deleted in patient (GUI) are highly conserved (81%, 100% and 92% respectively). Furthermore, they may play a role in the pathogenesis since proteins prepared from B cell lines and HeLa nuclear extracts bind to these sequences in gel retardation assays. Although a large part of this region is intronic, the high conservation of its sequence and the frequency of alterations in NHLs suggest that they are likely to be significant for the regulation of the LAZ3/BCL6 gene.

In B-cell malignancies, the t(11;14)(q13;q32) at the 11q13 BCL1 locus is characterized by a scattering of breakpoint sites along a 100 kb genomic region, between the BCL1 major translocation cluster (MTC) and the PRAD1 (also termed cyclin D1 or CCND1) gene. Recently, the 11q13 breakpoint region was extended on both sides, centromeric to the MTC and telomeric to PRAD1. We report here the molecular cloning of a new t(11;14) breakpoint site, 20 kb centromeric to the MTC, from a patient with prolymphocytic leukemia. We subcloned a non-repetitive DNA fragment near the breakpoint and mapped this new 11q13 probe (pHO11c) relative to already identified breakpoint sites, using long- and short-range physical mapping within the BCL1 locus. Rearrangements in the BCL1 locus are associated with deregulation of the PRAD1 gene, which is often overexpressed, particularly in mantle-cell malignancies. The detectable but weak PRAD1 expression in the case we present suggests that this breakpoint centromeric to the MTC still lies inside the BCL1 locus boundaries. We think that attention should be focused on this region centromeric to the BCL1-MTC, where the investigation of previously unidentified translocations may increase understanding of the PRAD1 gene deregulation in t(11;14) associated pathologies.

Objective. Cultured synovial fibroblast-like cells from 3 patients with rheumatoid arthritis (RA) and 3 patients with osteoarthritis (OA) were evaluated for their potential to secrete cysteine proteinases spontaneously and after stimulation by tumor necrosis factor α (TNFα) or interleukin-1 (IL-1). Methods. Culture media and cell lysates were analyzed before and after high performance liquid chromatography (HPLC) using the enzymatic substrate, Z-Phe-Arg-AMC, and by immunoblotting with anti–cathepsin B antiserum. Immunolocalization of cathepsin B was studied on cell monolayers. Results. Latent cysteine proteinase activity was found to be secreted spontaneously by cultured synovial fibroblast-like cells. This activity was increased after treatment with either TNFα or IL-1. Stimulated protease activity was eluted by HPLC at a peak coincident with that of purified cathepsin B. By immunoblot, cell supernatants contained a 43-kd form of cathepsin B, while cell lysates contained a 30-kd form, consistent, respectively, with cathepsin B before and after cleavage of its propeptide. An intracellular increase in cathepsin B after treatment with TNFα was also seen with immunohistochemical studies. Conclusion. TNFα (in the 6 cases studied) and IL-1 (in 4 cases) stimulated the secretion of a latent cysteine proteinase activity from synovial fibroblast-like cells, which appears to represent primarily cathepsin B.

A hybrid molecule, which combines an anilinoacridine chromophore related to the antitumour drug amsacrine (m-AMSA) and a bispyrrole moiety analogous to the antiviral agent netropsin, has been examined for its ability to bind chromatin and to modulate the activity of topoisomerase II. The results show that the presence of histones does not alter the bimodal DNA binding process. Intercalation of the acridine and groove binding of the netropsin part of the drug are both observed with chromatin preparations. Moreover, the hybrid has a clear topoisomerase II-DNA cleavable complex-inducing activity close to that of m-AMSA. The role of the two parts of the hybrid ligand is discussed in relation to ternary complex formation. Two cell lines (L1210 leukemia and MCF7 mammary carcinoma) were compared in their sensitivity to the tested ligand. The drug, which appears to be an efficient growth inhibitor of leukemic cells in vitro, reveals moderate activity against P388 leukemia in vivo. The biological activity of the hybrid may derive from a mechanism that involves DNA binding and topoisomerase II inhibition. This study demonstrates that agents which intercalate and bind to the minor groove of DNA simultaneously represent a new class of drugs interfering with topoisomerase II and provide opportunities for the development of new antitumour agents.