Radiation sensitivity of Down's syndrome fibroblasts might be due to overexpressed Cu/Zn-superoxide dismutase (EC 1.15.1.1).

Trisomy 21 (Down's syndrome, DS) is the most frequent chromosomal aberration. Triplication of a small region of chromosome 21, the fragment 21q22 is sufficient to cause the DS phenotype including immunodeficiency, premature aging, neurodegenerations, mental retardation and an increased risk of leukemia. Chromosomal aberrations caused by X-ray irradiation were observed in DS lymphocytes and DS fibroblasts, but the correlation to cell death or repair deficiency was not clear. We approached this problem and report here on a profound X-ray repair deficiency of DS cells. With a colorimetric viability assay we observed an UV sensitivity of DS fibroblasts at doses beyond 14 Jm-2 but no significant X-ray sensitivity. By the nucleoid sedimentation technique, a deficient restoration of nucleoids in DS cells after X-ray irradiation was demonstrated. The same features apply for cells, which contain an overexpressed Cu/Zn-superoxide dismutase (SOD-1) gene. Radiation sensitivity of DS cells and SOD-1 overexpressing cells resemble those of ataxia telangiectasia (AT) fibroblasts. Additionally, DS and AT cells exert lack of inhibition of DNA synthesis after X-ray irradiation.

Werner syndrome: characterization of mutations in the WRN gene in an affected family.

Affected and unaffected members of a Caucasian family with Werner syndrome were analyzed for mutations in the recently described Werner syndrome (WRN) gene and for their relevance to phenotypic expression of chromosomal instability and x-ray hypersensitivity. Two distinct molecular alterations were documented in the family. Analysis of the genomic DNA revealed a single-base exchange from A to T at an intron-exon boundary in the otherwise strongly conserved 5' donor splice site. Consequently, exon 30 is spliced together with the intron. The ensuing structure could be confirmed by the presence and calculated size of the resulting RNA fragments. The patients, all compound heterozygotes, had a 1-bp deletion in the first third of the coding sequence in the other allele. The genotypes of the family members for these mutations were determined and consequences for the cellular phenotype of the otherwise unaffected heterozygotes are documented.

Werner syndrome: studies in an affected family reveal a cellular phenotype of unaffected siblings.

Werner syndrome is an inherited disease with symptoms of presenescence. The primary defect site either on the protein or at the DNA level is not known, nor is it possible to identify a heterozygous phenotype. On the basis of cellular peculiarities expressed in the homozygotes-lifespan reduction of cells in culture, length of population doubling time and chromosomal instability-we searched for a 'Werner-like' phenotype in otherwise phenotypically unaffected siblings. We established primary fibroblasts from eight members of a Tyrolean family, two of whom had been diagnosed as typical Werner syndrome, as well as from unrelated healthy young and old volunteers. Determination of the lifespan of each strain and studies on population doubling time and chromosomal instability revealed similar cellular characteristics in all family members, albeit to a lesser extent with the siblings than with the homozygotes when compared to age-matched controls. These features, also apparent in cultivated fibroblasts from old but healthy controls, appear to be indicative of Werner syndrome when expressed in young or middle aged persons. The possible identification of otherwise clinically healthy gene carriers of Werner syndrome is of utmost importance for genetic counselling and medical surveillance for this disorder.

Correlation between senescence and DNA repair in cells from young and old individuals and in premature aging syndromes.

Cellular aging appears to be related to and perhaps caused by diminished DNA repair. To elucidate direct correlations between DNA repair capacity and senescence various parameters of cellular aging and DNA repair were studied simultaneously. Of special interest are features of DNA repair and senescence in cultured diploid fibroblasts derived either from healthy young or elderly probands as well as from patients suffering from premature senescence syndromes (Werner syndrome, Cockayne syndrome, ataxia telangiectasia and Down syndrome). Here we demonstrate the striking parallelism between reduced maximal lifespan, elevated levels of spontaneous chromosomal breaks, higher incidence of formation of micronuclei, a significant prolongation of cell cycle duration and a diminished reactivation of in vitro injured plasmid after transfection in cells from old individuals and from patients with premature senescence syndromes, suggesting a causal relationship between senescence and DNA damage.

Synergistic and antagonistic interactions of transcription factors in the regulation of milk protein gene expression. Mechanisms of cross-talk between signalling pathways.

The stage and tissue specific expression of milk protein genes in the mammary gland is controlled by modular response regions with multiple binding sites for distinct classes of transcription factors, which either co-operate or are antagonistic. In addition, the activity of some of these factors is individually control-led by diverse extracellular signals. A well studied paradigm for a synergistic co-operation is the activation of beta-casein gene transcription by prolactin and glucocorticoids mediated by the signal transducer and activator of transcription STAT5 and the glucocorticoid receptor (GR). As an example for an antagonistic interaction we can demonstrate inhibition of prolactin signalling by TNF-alpha, which is mediated by NF-kappa B. In both cases, the interactions occur at several levels: For GR and STAT5, the synergy is discussed to be promoted by protein-protein interactions. Furthermore, we can demonstrate a co-operation between GR and STAT5 in DNA binding by a mechanism, which is dependent on the integrity of the DNA binding domain of the GR and on the existence of half-palindromic GR binding sites in the hormone response region. Indirect effects of glucocorticoids by modulation of the expression of secondary genes are also important. They might account for the observed enhancement of prolactin induced tyrosine phosphorylation of STAT5 by glucocorticoids. For NF-kappa B and STAT5, one component of the antagonism is the inhibition of STAT5 tyrosine phosphorylation by activation of NF-kappa B. Another potential mechanism is the inhibition of DNA binding of STAT5 due to overlapping binding sites for STAT5 and NF-kappa B in the beta-casein gene promoter. Thus, synergistic and antagonistic interactions between GR, NF-kappa B, and STAT5 involve (a) cross-talk mechanisms influencing the activation of STAT5 and (b) promoter-dependent interactions modulating the DNA binding activity of the transcription factors.

Improvement of the DNA repair capacity of human fibroblasts by autolysates of Lactobacillus gasseri.

Autolysates of Lactobacillus gasseri were tested for their ability to improve repair capacity in cultured human fibroblasts. In the course of this research on molecular mechanisms of hereditary DNA repair deficiencies and presenility syndromes, a significant and reproducible effect on repair capacity in these cells by an autolysate of the gram-positive bacterium Lactobacillus gasseri was found.

Chromosomal instability in a woman with infertility and two unaffected brothers: a new familial chromosomal breakage syndrome?

Repeated chromosomal analysis of peripheral blood lymphocytes and skin fibroblasts from a woman referred for amenorrhoea, streak gonads, hyperthyroidism, adiposity and elevated alpha-fetoprotein levels but no other manifestations of known chromosomal breakage syndromes demonstrated an increased spontaneous chromosomal breakage rate (ISCBR). Chromatid and chromosomal breaks were more numerous than sporadic rearrangements and dicentric chromosomes. Exposure of the cells to mitomycin C, diepoxybutane, X-rays or UV irradiation induced an increase in chromosomal and chromatid abnormalities over that in controls. A micronucleus assay demonstrated an increase in the incidence of formation of micronuclei and the population doubling time of the fibroblasts of the proposita was delayed. Chromosomal analysis was performed on lymphocytes of the parents and of five sibs of the proposita. Two brothers had chromosomal abnormalities identical to those of the patient and elevated alpha-fetoprotein levels, however, without any clinical abnormalities. The parents were affected by only a moderate ISCBR whereas two brothers and one sister were chromosomally normal. The clinical, chromosomal and biochemical findings in this family represent a novel chromosomal instability syndrome.