Systematic growth studies, cocultivation, and cell hybridization studies of Werner syndrome cultured skin fibroblasts

Abstract

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.

The gene responsible for Werner syndrome may be a cell division "counting" gene.

“…In mammalian cells, the duration of the S phase is almost always 8-10 hr. If all the cells in these young cultures were growing, this would imply a generation (intermitotic) time of between 1 and 4 days, which is consistent with reported data (22,23). Therefore, at the beginning of the cultures most of the cells in the population were reproductively active.…”

“…The kinetic difference between the normal cultures and the cultures from the Werner syndrome patients was not due to a difference in the cell-plating efficiency (22).…”

Mitogen-mediated protein phosphorylation in Werner's syndrome fibroblasts.

“…Thus, phosphorylation of several phosphotyrosine- (15). We have shown here that WF-1 fibroblasts derived from a Japanese patient exhibit a prolonged doubling time and reduced life span as has been seen in other Werner's fibroblasts (14). Furthermore, our analysis revealed significant difference in protein phosphorylation patterns of normal and Werner's fibroblasts.…”

“…Fibroblasts from patients with Werner's syndrome usually show a prolonged doubling time and reduced life span (14). Studies including cocultivation and cell fusion of normal and Werner's fibroblasts showed no evidence for complementation between these cells (14).…”

Functional Link Between Myc and the Werner Gene in Tumorigenesis

“…1 There is much debate on whether organismal aging and cellular senescence may share common pathways. 8,9 In support of this connection, WS derived cells have a diminished lifespan and exhibit signs of accelerated senescence in culture, 10 while immortalized cells exhibit elevated levels of WRN relative to mortal cells. 11 This observation prompted us to investigate a possible role for WRN in the replication potential of cells following oncogenic stimulation.…”