Conformational changes in chromatin from density inhibited WI-38 fibroblasts stimulated to proliferate.
Quiescent confluent monolayers of WI-38 human diploid fibroblasts can be stimulated to proliferate by replacing the old medium with fresh medium plus 10% serum. Circular dichroism spectra of chromatin from stimulated cells between 2 and 10 hrs after stimulation show an increase in positive ellipticity maxima and a blue shift in the 250-300 nm region. These changes are reversed when the stimulated cells enter DNA synthesis (which, in the present conditions, begins to increase at 12-15 hrs and reaches a peak at 20 hrs). The circular dichroism changes occurring 3 hrs after stimulation have been studied in greater detail. They consist in a 35% (average) increase in positive ellipticity and a blue shift in the 250-300 nm region. Changes in the gamma less than 244 nm region are less consistent. The differences between chromatins of stimulated and unstimulated cells are abolished when both chromatins are washed with 0.25 M NaC1. This procedure removes 10-12% of chromosomal proteins, which chromatograph with non-histone proteins. DNA, RNA and histones could not be detected in the 0.25 M NaC1 extract. In gel electrophoretic profiles of radioactively labelled chromosomal proteins from stimulated and unstimulated WI-38 cells there were no detectable differences between histones. The non-histone proteins of stimulated cells showed one radioactive peak which was increased above the level of non-histone proteins from control cells. These results show that structural changes occur in the chromatin of WI-38 cells stimulated to proliferate several hrs before the onset of DNA synthesis. The fact that differences in the chromatins can be abolished by washing with 0.25 M NaC1 could give a clue as to the mechanisms responsible for these structural changes.
D000818:Animals; D002478:Cells, Cultured; D002843:Chromatin; D002875:Chromosomes; D005347:Fibroblasts; D006657:Histones / Q000032:analysis; D006801:Humans; D066298:In Vitro Techniques; D008938:Mitosis; D011487:Protein Conformation; D011506:Proteins / Q000032:analysis
D002843:Chromatin; D006657:Histones; D011506:Proteins