Skin fibroblast-like (FL) cells from patients with Werner’s syndrome (adult progeria) regularly demonstrate frequent pseudodiploidy involving variable structural rearrangements that are clonal: variegated translocation mosaicism (VTM). Ninety-two percent of 1,538 metaphases from 29 independent strains derived from five patients with Werner’s syndrome demonstrated this cytogenetic abnormality. In contrast, only eight (8.4%) of 95 non-Werner’s syndrome FL cell cultures demonstrated VTM: seven with low-grade VTM (approximately 5% of 300 metaphases), and one with VTM affecting 90–100% of metaphases. Unlike the cytogenetic abnormalities observed in the terminal stages of normal FL cell cultures, VTM occurs throughout the entire lifespan of Werner’s syndrome cultures. Ten of the identifiable break points in 1,005 banded metaphases accounted for 27% of all definable rearrangements. Baseline sister chromatid exchanges were not increased. Co-cultivation of Werner’s syndrome and normal strains did not induce VTM in the normal strain. The relationship between VTM and the reduced growth potential of Werner’s syndrome FL cells is not yet understood, nor is the relationship between these in vitro abnormalities and the presumptive single gene defect that causes the progeroid clinical manifestations of Werner’s syndrome.

The term “variegated translocation mosaicism” is used to describe the repeated occurrence, within cultures of human skin fibroblasts, of a multiplicity of chromosomal rearrangements. With respect to the frequencies of such cytogenetically aberrant clones we found that they (1) were not detectable in routine diagnostic skin fibroblast cultures from 29 subjects with a wide variety of indications for biopsy; (2) were not detectable during in vitro aging of diploid strains with four normal individuals; (3) could be detected after rescue from bacterial contamination of a culture from an otherwise normal diploid male; (4) occurred with high frequencies in independent cultures from another apparently normal subject; (5) occurred with high frequencies in multiple biopsies obtained at autopsy from a patient with Werner’s syndrome who died of sepsis; (6) were of pseudodiploid nature; and (7) involved a different spectrum of chromosomes in different individuals. A consistent association with mycoplasma contamination could not be made.

The growth of 20 independently derived skin fibroblastlike (FL) cell strains from three individuals with Werner syndrome (adult progeria) was compared with the growth of ten FL cell strains from normal individuals. Population growth rates and total replicative life spans of Werner syndrome strains averaged 53% and 27%, respectively, of the growth rates and life spans of non-Werner strains. In the first few passages, four Werner syndrome strains demonstrated population growth rates in the low normal range, but the longest-lived Werner syndrome strain had only 75% of the total replicative potential of the shortest-lived normal strain. Exponential growth rates, cloning efficiencies, and saturation densities of Werner strains were also reduced, whereas cell attachment was normal. Viable cells (identified by dye exclusion) were maintained in post-replicative Werner syndrome and control cultures for periods of at least 10 months; there was no evidence of accelerated post-replicative senescence of cell death of Werner syndrome FL cells. Cocultivation of Werner syndrome and normal strains did not influence population growth rates of either strain. Two proliferating hybrid clones were obtained from fusions of normal and Werner syndrome FL cell strains and these hybrids displayed the reduced growth potential typical of Werner syndrome FL cells. These studies confirm that low growth rate and sharply reduced replicative life span are characteristic of cultured skin FL cells from patients with Werner syndrome, and they suggest that these characteristics are not affected by complementation with non-Werner FL cells.

Skin fibroblast-like (FL) cells from patients with Werner’s syndrome (adult progeria) demonstrate multiple, stable, structural chromosome rearrangements (variegated translocation mosaicism) which can be used to identify cytogenetically marked clones of cells within a mass culture. We have cytogenetically followed eight FL cell strains (from two patients) throughout their entire in vitro replicative lifespans, and we show a correlation between the expansion and attenuation of individual clones and the growth of the mass cultures. One strain, which was aged several times, demonstrated a generally reproducible pattern of clonal succession but, surprisingly, also demonstrated, in two parallel derivative cultures, the late emergence of two relatively rapidly growing clones that had not been observed in the parental culture. These observations suggest that clonal succession and clonal attenuation occur in mass cultures, as had been predicted on the basis of dilute-plating cloning experiments. Our results may have implications for models of in vitro cellular senescence. In addition, there are interesting parallels with the tissue hyperplasia associated with in vivo aging, and this observation is compatible with the suggestion that skin FL cells in vitro provide a model for hyperplasia in vivo.

No abstract

Summary. In this paper we describe two types of i(Xq), in three patients. A classification is proposed for at least seven different types ofhuman i(Xq)s or X longarm duplications described by banding in the literature. Type 1 reported here and also in the literature may be the most common. It consists of a single visible centromere, metacentric, length similar to number 3, G-banding interpreted as i(X)(qter->cen--*qter), one C-band like a normal X. Type 2 reported here may not have a counterpart in the literature; it exhibits a single visible centromere, submetacentric, length similar to number 3, extra G-and C-bands in region ql.The classification summarized in this paper implies that different breakpoints are involved in the production of human X long-arm isochromosomes or duplications. Some include duplications of short arm. Morphological differences in i(Xq)s will complicate their use for studying the effect of X chromosome structure on phenotype, unless differences are defined clearly. It seems important to resolve the question of whether these reported abnormal X chromosomes involve rearrangements between the same or two X chromosomes.We also report X chromosome defects in three generations of a family; both the mother and maternal grandmother of one 45,Xi(Xq)/45,X patient are themselves mosaics for 45,X/46,XX/46,X,r(X). This family suggests that familial predisposition to X chromosome abnormality includes isochromosome formation, as well as ring formation and mosaicism.

No abstract

Two sister strains of skin fibroblast-like (FL) cells from a patient with Werner's syndrome (adult progeria) were grown in regular tissue culture medium or medium supplemented with the radical-scavenging enzymes superoxide dismutase and catalase. Differences in proliferative potential were shown to be due to strain variation rather than to treatment with the enzymes. Two independent strains were also grown in ambient (18%) or reduced (1%) concentrations of oxygen. All cultures (untreated, treated with enzymes, treated with reduced oxygen exposure) displayed the limited in vitro growth potential and cytogenetic abnormality characteristic of Werner's syndrome FL cells; thus the underlying defect in Werner's syndrome does not appear to be related to defective protection against or repair of damage by free radicals.