New benzo(b)thiophenesulphonamide 1,1-dioxide derivatives induce a reactive oxygen species-mediated process of apoptosis in tumour cells.

In this work, we describe the process of cell death induced by a series of new benzo(b)thiophenesulphonamide 1,1-dioxide derivatives (BTS) that have been selected as candidate antineoplastic drugs. Human leukaemic CCRF-CEM cells incubated with BTS undergo a typical apoptotic process that includes cell shrinkage, phosphatidylserine translocation to the cell surface, mitochondrial dysfunction, caspase activation, chromatin condensation and internucleosomal DNA degradation. Mitochondrial alterations included dissipation of the mitochondrial membrane potential, oxidation of the phospholipid cardiolipin, release of cytochrome c and uncoupling of the mitochondrial respiratory chain, leading to a decrease of the intracellular ATP pool. Activation of caspase-8, -9 and -3 takes place during BTS-induced apoptosis. Either the addition of the specific caspase-8 inhibitor Z-IETD-fmk, or the overexpression of the antiapoptotic protein Bcl-2 significantly prevented BTS-induced apoptosis, suggesting the involvement of both caspase-8-regulated and mitochondria-dependent signalling pathways in this process. BTS induce a significant increase in the production and accumulation of intracellular reactive oxygen species (ROS) that can be observed within minutes after drug addition. Moreover, cytochrome c release, caspase-3 activation and cell death can be completely abrogated by a previous incubation with the antioxidant N-acetyl-cysteine. These results suggest that ROS are essential mediators in BTS-induced apoptosis.

cAMP activates transcription of the human glucocorticoid receptor gene promoter.

Glucocorticoids and cAMP regulate, either in a synergistic or additive fashion, the transcription of multiple genes, although some antagonistic effects of dexamethasone on cAMP-activated transcription have been described. The increased glucocorticoid receptor (GR) mediated response of some cell types, as a result of augmented cAMP, has been considered to be mainly due to an increased stability of GR mRNA, although other plausible explanations should not be ruled out. We studied the possibility that GR transcription itself could be affected by cAMP levels. HeLa cells were transfected with human GR (hGR) promoter constructs and their transcriptional activity determined after inducing a cAMP increase with forskolin. We found that forskolin almost doubled the transcriptional activity of the promoter construct spanning -2995 to +38 of the hGR, whereas no significant variations were observed with shorter chimeras containing sequences downstream -979. Shift mobility showed binding of CREB in vitro to a putative cAMP responsive element located at -1000, suggesting that hGR may be upregulated by cAMP at the transcriptional level, thus adding a new mechanism ascribable to this second messenger, which in conjunction with the cAMP-induced GR mRNA increased stability, would lead to a more precise control of the amount of GR protein within the cell.

Familial glucocorticoid resistance caused by a splice site deletion in the human glucocorticoid receptor gene.

The clinical syndrome of generalized, compensated glucocorticoid resistance is characterized by increased cortisol secretion without clinical evidence of hyper- or hypocortisolism, and manifestations of androgen and/or mineralocorticoid excess. This condition results from partial failure of the glucocorticoid receptor (GR) to modulate transcription of its target genes. We studied the molecular mechanisms of this syndrome in a Dutch kindred, whose affected members had hypercortisolism and approximately half of normal GRs, and whose proband was a young woman with manifestations of hyperandrogenism. Using the polymerase chain reaction to amplify and sequence each of the nine exons of the GR gene alpha, along with their 5'- and 3'-flanking regions, we identified a 4-base deletion at the 3'-boundary of exon 6 in one GR allele (delta 4), which removed a donor splice site in all three affected members studied. In contrast, the sequence of exon 6 in the two unaffected siblings was normal. A single nucleotide substitution causing an amino acid substitution in the amino terminal domain of the GR (asparagine to serine, codon 363) was also discovered in exon 2 of the other allele (G1220) in the proband, in one of her affected brothers and in her unaffected sister. The functional importance of this mutation was tested in a cotransfection study using the recombinant expression vector pRShGR-Ser363 and the glucocorticoid responsive vector mouse mammary tumor virus-chloramphenicol transferase. This amino acid substitution did not alter the function of the glucocorticoid receptor. Using reverse transcription-polymerase chain reaction we could only identify messenger RNA transcripts of the G1220-allele but not of the delta 4-allele in the affected members of this family who were heterozygous for the G1220 mutation. This deletion in the glucocorticoid receptor gene was, thus, associated with the expression of only one allele and a decrease of GR protein by 50% in affected members of this glucocorticoid resistant family. The mutation identified in exon 2 did not segregate with the disease and appears to be of no functional significance. The presence of the null allele was apparently compensated for by increased cortisol production at the expense of concurrent hyperandrogenism.

A systematic search for a bipolar predisposing locus on chromosome 5.

Chromosome 5 markers spanning the pter to the qter were used to examine linkage to bipolar illness in 14 pedigrees. Twenty-four loci were examined in 237 individuals, of whom 69 were either bipolars or schizoaffectives. Marker genotypes were determined for each individual and lod scores were calculated under a dominant disease model with a maximum penetrance of 85%, a disease gene frequency of 0.015, a variable age of onset, and a phenocopy rate of 0.001. Under the assumption that bipolar illness is genetically homogeneous, the total lod scores from all pedigrees with each marker were uniformly lower than -2.0, suggesting the absence of linkage to disease at any of these loci. Multipoint analysis allowed exclusion of intervals between markers. When lod scores were calculated allowing for heterogeneity, no subset of linked families was found. These results indicate that in our pedigree series almost the entire mapped region of chromosome 5 can be excluded for linkage to bipolar illness.

The genomic structure of the human glucocorticoid receptor.

We have determined the structure of the human glucocorticoid receptor (hGR) gene after the isolation and characterization of cosmid clones mapping to discrete regions of the cDNA. The gene contains a total of 10 exons and has a minimum size of 80 kilobases. Exon 1 consists solely of 5'-untranslated sequence, and exon 2 encodes the amino-terminal portion of the receptor. The two putative zinc fingers are separately encoded by two exons, and a total of five exons combine to form the cortisol-binding domain. By restriction mapping and sequence analysis of cosmids located on the 3'-end of the gene, we have established that the two receptor isoforms, hGR alpha and hGR beta, originate from the same gene by alternative splicing. Each hGR isoform is encoded by nine exons, of which the first eight are identical, whereas the ninth exons are heterologous. Multiple GC boxes and no obvious TATA or CAAT elements have been found in the 5'-flanking region. S1 nuclease analysis yielded one major band, and the transcription start site is localized to the *C residue within TAC*CCTC. Alignment of sequences around the splice junctions of hGR with those of other members of the steroid receptor superfamily revealed three different splice positions within the DNA-binding domain. This comparison also permitted the prediction of the positions of the splice sites and the sizes of the putative exons in the human mineralocorticoid receptor.