The tumor necrosis factor (TNF) superfamily of cytokines includes both soluble and membrane-bound proteins that regulate immune responses. A member of the human TNF family, BLyS (B lymphocyte stimulator), was identified that induced B cell proliferation and immunoglobulin secretion. BLyS expression on human monocytes could be up-regulated by interferon-gamma. Soluble BLyS functioned as a potent B cell growth factor in costimulation assays. Administration of soluble recombinant BLyS to mice disrupted splenic B and T cell zones and resulted in elevated serum immunoglobulin concentrations. The B cell tropism of BLyS is consistent with its receptor expression on B-lineage cells. The biological profile of BLyS suggests it is involved in monocyte-driven B cell activation.

Some cases of hereditary nonpolyposis colorectal cancer (HNPCC) are due to alterations in a mutS-related mismatch repair gene. A search of a large database of expressed sequence tags derived from random complementary DNA clones revealed three additional human mismatch repair genes, all related to the bacterial mutL gene. One of these genes (hMLH1) resides on chromosome 3p21, within 1 centimorgan of markers previously linked to cancer susceptibility in HNPCC kindreds. Mutations of hMLH1 that would disrupt the gene product were identified in such kindreds, demonstrating that this gene is responsible for the disease. These results suggest that defects in any of several mismatch repair genes can cause HNPCC.

Hereditary nonpolyposis colorectal cancer (HNPCC) is one of man's commonest hereditary diseases. Several studies have implicated a defect in DNA mismatch repair in the pathogenesis of this disease. In particular, hMSH2 and hMLH1 homologues of the bacterial DNA mismatch repair genes mutS and mutL, respectively, were shown to be mutated in a subset of HNPCC cases. Here we report the nucleotide sequence, chromosome localization and mutational analysis of hPMS1 and hPMS2, two additional homologues of the prokaryotic mutL gene. Both hPMS1 and hPMS2 were found to be mutated in the germline of HNPCC patients. This doubles the number of genes implicated in HNPCC and may help explain the relatively high incidence of this disease.

NF-~B is an inducible transcription factor comprised of a 50-kD (pS0) and a 65-kD (p65) subunit. Induction of NF-KB activity, which is a critical event in many signal transduction pathways, involves release from a cytoplasmic inhibitory protein, IKB, followed by translocation of the active transcription factor complex into the nucleus. Earlier studies suggested that IKB targets the p65 subunit of NF-KB. However, we demonstrate by in vitro and in vivo methods that the recently cloned IKB/MAD-3 interacts with both the p50 and p65 subunits of NF-KB, as well as c-Rel. Furthermore, an alternatively spliced, dimerization-deficient transforming variant of p65 (p65A) interacts extremely weakly with IKB/MAD-3, suggesting that dimerization is important for interaction. We demonstrate that the conserved nuclear localization sequences (NLSs) of NF-KB and c-Rel are the targets for IKB/MAD-3 interaction. Indirect immunofluorescence experiments demonstrate that IKB/MAD-3 expression retains both p65 and p50 in the cytoplasm. Furthermore, and most important, a p65 that contains an SV40 large T antigen NLS in addition to its own NLS is no longer retained in the cytoplasm in the presence of IKB/MAD-3. We propose that IKB/MAD-3 masks the NLSs of NF-KB and c-Rel and that this constitutes the mechanism for cytoplasmic retention of these proteins.

Analysis of the p50 and p65 subunits of the NF-KB transcription factor complex has revealed that both proteins can interact with related DNA sequences through either homo-or heterodimer formation. In addition, the product of the proto-oncogene c-rel can bind to similar DNA motifs by itself or as a heterodimer with p50 or p65. However, these studies have used a limited number of known KB DNA motifs, and the question of the optimal DNA sequences preferred by each homodimer has not been addressed. Using purified recombinant p50, p65, and c-Rel proteins, optimal DNA-binding motifs were selected from a pool of random oligonucleotides. Alignment of the selected sequences allowed us to predict a consensus sequence for binding of the individual homodimeric Rel-related proteins, and DNA-protein binding analysis of the selected DNA sequences revealed sequence specificity of the proteins. Contrary to previous assumptions, we observed that p65 homodimers can interact with a subset of DNA sequences not recognized by p50 homodimers. Differential binding affinities were also obtained with p50-and c-Rel-selected sequences. Using either a p50-or p65-selected KB motif, which displayed differential binding with respect to the other protein, little to no binding was observed with the heterodimeric NF-KB complex. Similarly, in transfection experiments in which the selective KB binding sites were used to drive the expression of a chloramphenicol acetyltransferase reporter construct, the p65-and p50-selected motifs were activated only in the presence of p65 and p50/65 (a chimeric protein with the p50 DNA binding domain and p65 activation domain) expression vectors, respectively, and neither demonstrated a significant response to stimuli that induce NF-KB activity. These findings demonstrate that interaction of both subunits of the heterodimeric NF-KB complex with DNA is required for DNA binding and transcriptional activation and suggest that transcriptional activation mediated by the individual rel-related proteins will differ dramatically, depending on the specific KB motifs present.