N-substituted benzamides inhibit NFkappaB activation and induce apoptosis by separate mechanisms.

Benzamides have been in clinical use for many years in treatment against various disorders. A recent application is that as a sensitizer for radio- or chemotherapies. We have here analysed the mechanism of action of N-substituted benzamides using an in vitro system. We found that while procainamide was biologically inert in our system, the addition of a chloride in the 3' position of the benzamide ring created a compound (declopramide) that induced rapid apoptosis. Furthermore, declopramide also inhibited NFkappaB activation by inhibition of IkappaBbeta breakdown. An acetylated variant of declopramide, N-acetyl declopramide, showed no effect with regard to rapid apoptosis induction but was a potent inhibitor of NFkappaB activation. In fact, the addition of an acetyl group to procainamide in the 4' position was sufficient to convert this biologically inactive substance to a potent inhibitor of NFkappaB activation. These findings suggest two potential mechanisms, induction of early apoptosis and inhibition of NFkappaB mediated salvage from apoptosis, for the biological effect of N-substituted benzamides as radio- and chemo-sensitizers. In addition it suggests that N-substituted benzamides are potential candidates for the development of anti-inflammatory compounds using NFkappaB as a drug target.

Transcriptional regulation in B cell differentiation.

B cell development is a multistage differentiation process that ultimately generates antibody-secreting plasma cells. This model developmental pathway is governed by a choreographed pattern of expression from genes encoding stage and lineage-specific proteins. This is achieved by the interaction of distinct transcription factors with regulatory elements, placing these proteins in a central position in B cell ontogeny. The importance of distinct transcription factors is also supported by the notion that B cell development is disrupted in mice bearing homozygous null alleles of certain factors involved in the control of B-cell-specific genes. In this review we compare and contrast the transcriptional regulation of well-studied B lymphoid restricted genes, on a gene by gene basis, focusing on the functional structure of transcription control regions and interacting transcription factors.

Spi-C, a novel Ets protein that is temporally regulated during B lymphocyte development.

A novel Ets protein was isolated by yeast one-hybrid screening of a cDNA library made from lipopolysaccharide-stimulated mouse splenic B cells, using the SP6 kappa promoter kappaY element as a bait. The novel Ets protein was most closely related to PU.1 and Spi-B within the DNA binding Ets domain and was therefore named Spi-C. However, Spi-C may represent a novel subgroup within the Ets protein family, as it differed significantly from Spi-B and PU.1 within helix 1 of the Ets domain. Spi-C was encoded by a single-copy gene that was mapped to chromosome 10, region C. Spi-C interacted with DNA similarly to PU.1 as judged by methylation interference, band-shift and site selection analysis, and activated transcription of a kappaY element reporter gene upon co-transfection of HeLa cells. Spi-C RNA was expressed in mature B lymphocytes and at lower levels in macrophages. Furthermore, pre-B cell and plasma cell lines were Spi-C-negative, suggesting that Spi-C might be a regulatory molecule during a specific phase of B lymphoid development.

Differentiation-specific, octamer-dependent costimulation of kappa transcription.

By mutational analysis of the octamer-TATA box intervening region in the mouse SP6 kappa promoter, we have mapped two octamer-dependent, costimulatory regions, A and B. The A region was active in late B cells only, while the B region was active throughout B cell differentiation. The B region was TATA proximal and contained a heptamer and an E box of the E2A type that is common in Vkappa promoters. Mutation of the heptamer element did not decrease transcriptional stimulation from this region, but mutations in, or immediately 5' of, the E box core sequence did. A protein binding to this region could be detected in nuclear extracts. The complex could only partially be competed with a muE5 binding site and could not be supershifted with Abs raised to E2A gene products, indicating that it may represent a novel E-box binding complex. The A region was located proximal to the octamer and contained a CCCT element that is conserved both with regard to position and sequence in human VkappaII promoters. By mutational analysis, the transcriptional stimulatory activity was mapped to the CCCT element that also is part of an early B cell factor (EBF) binding site. In late B cells, a novel protein (FA), which did not bind to the EBF binding site in the mb1 promoter, interacted with the A region. This protein was found to be expressed at lower levels in early B cells as well as in HeLa cells. Thus, the octamer-flanking sequence contains positive control elements that may act independently but that differ in the stage of B cell differentiation at which they are active. One of these factors is an example of an ubiquitously expressed transcription factor that participate in differentiation-specific transcriptional activation.

Conserved sequence elements in K promoters from mice and humans: implications for transcriptional regulation and repertoire expression.

Promoter region sequences of human and mouse Igk-V genes were aligned and found to be conserved for about 200-300 base pairs (bp) within subgroups/families. No promoter similarity was found between IGKV promoters from different human subgroups. Related mouse Igk-V gene families were conserved in the promoter region but no similarity was evident when promoters from unrelated Igk-V gene families were compared. Most of the human IGKV promoter subgroups were shown to have mouse counterparts with a similarity region that extended about 150 bp upstream of the translational start codon. All promoters contained an octamer sequence element. The consensus octamer/decamer sequence was favored but only seven residues within the octamer element were strictly conserved. Furthermore, there was also sequence conservation immediately 3' of the octamer where either an A or a G residue was conserved. In addition, other DNA elements were also conserved both within the Igk-V subgroups/families and between mouse and human promoters from related subgroups/families. In several of the subgroups/families an E box of the E2A type was conserved 5' of the octamer and a CCCT element was conserved within the IGKV subgroup II and its related mouse Igk-V families. We conclude from this study that conservation of additional sequence elements besides the octamer is a common feature in Igk-V promoters but that distinct elements are conserved only within a given subgroup/family. Thus, the conservation appears to have operated at the level of function rather than at the level of recognition sequence for defined transcription factors.

Oct proteins are qualitative rather than quantitative regulators of kappa transcription.

The 3' flanking sequence of kappa promoter octamers was found to contain either a conserved A or G residue which increased the affinity of the octamer core motif for Octl and Oct2A. By transient transfections it was shown that decreasing the affinity of an octamer for Oct binding was crippling the transcription unit when the octamer was used in a minimal promoter, while it had only marginal effects when it was analysed in the context of an intact kappa promoter. As the octamer in a kappa promoter was replaced by a TAATGARAT motif with equal affinity for Oct protein binding the latter could still participate in synergistic transcriptional stimulation. Thus, the synergistic interactions involved in kappa promoter transcriptional stimulation are dependent on the presence of Oct proteins but not on the octamer DNA motif per se. Since the transcriptional coactivator OCA-B cannot interact with Oct protein bound to the TAATGARAT motif, the role of OCA-B in these interactions seems to be limited.