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.
However, IGFBP-3 protein accumulated to higher levels in conditioned medium of old cells than in medium of WS and young cells, in that order, under the same conditions.
Five selected mRNAs studied in greater detail [alpha 1(I) procollagen, fibronectin, insulinlike growth factor-binding protein-3, WS3-10, and WS9-14] showed higher mRNA levels in both WS and late-passage normal HDF than in early-passage normal HDF at various intervals following serum depletion/repletion and after subculture and growth from sparse to high-density confluent arrest.
Five selected mRNAs studied in greater detail [alpha 1(I) procollagen, fibronectin, insulinlike growth factor-binding protein-3, WS3-10, and WS9-14] showed higher mRNA levels in both WS and late-passage normal HDF than in early-passage normal HDF at various intervals following serum depletion/repletion and after subculture and growth from sparse to high-density confluent arrest.
In Werner syndrome cultures expression of metalloproteinase and TIMP-1 mRNAs was similar to the level of expression observed in late-passage cell cultures.
Cellular insulin-like growth factor binding protein-3 (IGFBP-3) mRNA and IGFBP-3 levels in conditioned medium were consistently higher in cultures of late passage normal (old) fibroblasts and prematurely senescent fibroblasts derived from Werner syndrome (WS) during quiescence induced by serum depletion and during the renewed growth ensuing after serum repletion, compared to cultures of early passage normal (young) fibroblasts.
Here we present the results of a search for a region that exhibits linkage disequilibrium with the disorder, under the assumption that identification of such a region may provide an alternative method of narrowing down the location of WRN, the gene responsible for WS.
The augmentation in PAI-1 output of fibroblasts as a direct function of chronological age and during in vitro senescence suggests that PAI-1 may play an important role in the reduced capacity for wound healing and the increasing tendency to thrombogenesis and atherogenesis seen during biological aging and in particular in persons with Werner syndrome.
Several independent lines of evidence did not support that hypothesis: (1) activity gels showed normal enzyme activity and electrophoretic mobility; (2) nucleotide sequence analysis of the entire coding region failed to reveal mutations (although indicated mistakes in the published sequence); (3) single-strand conformation polymorphism (SSCP) and heteroduplex analyses failed to reveal evidence of mutations in the promoter region; (4) a newly discerned polymorphism failed to reveal evidence of homozygosity by descent in a consanguineous patient; and 5) fluorescence in situ hybridization (FISH) analysis placed the DNA polymerase beta gene centromeric to D8S135 at 8p11.2 and thus beyond the region of peak LOD scores for WS.
These mutations change the structure of DNA polymerase beta and thus the capacity of the DNA repair system would be impaired, which may account for the high mutation rate observed in WS.
These results suggest that the patients with Werner's syndrome express normal insulin receptors and that the primary genetic lesion for insulin resistance is not in the insulin receptor gene.
To determine whether IL-1 expression may be promoted by in vivo aging, we analyzed the expression of IL-1 and of inducible mRNAs in HDFs from two normal individuals 55 and 92 years old and in HDFs from a patient with premature aging caused by Werner's syndrome.
By reverse transcription polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA), we detected expression of IL-1 alpha and beta mRNA and protein in early passage HDFs from both normal individuals and the Werner's syndrome patient.