Primary cultures of mouse embryonic fibroblasts were effected and cells passaged as described previously. Such cultures underwent 10-12 population doublings before the cessation of growth. Cells were maintained in a nondividing state by decreasing the serum concentration of the medium to 0.1%. Nuclei were isolated, and RNA polymerase was assayed. The total RNA synthesizing capacity of nuclei isolated from mid passage (4th to 7th generation) fibroblasts was found to be equal to, or slightly higher than that of early passage cell nuclei with the endogenous template. However, this fell in late passage cells to a value 50% of that of young cell nuclei. Such an effect is consistent with cells aging both in vitro and in vivo. In young cells, a highly active free RNA polymerase activity exists, which increases linearly with the concentration of poly(dA DT) added. The concentration of this exogenous template used here was rate limiting. RNA polymerase II comprises about 80% of this activity, as opposed to only 40-50% of the bound enzyme as judged by sensitivity to α amanitin. Sarcosyl, an anionic detergent known to stimulate RNA polymerase II activity by histone removal, stimulated the polymerase activity 2-3 fold. However, in populations at the end of their lifespans in vitro, this effect was greatly decreased. To determine to what extent these changes result from, rather than cause, the cessation of mitosis during senescence in vitro, second generation fibroblasts were held in the G0 phase by serum restriction and their characteristics compared with those of early and late passage cells. Young quiescent cells did, indeed, show changes in their RNA polymerase activities equivalent to those found in late passage fibroblasts. Free polymerase disappeared from the nuclei relatively rapidly, within 18 hr of serum removal. At this time, the endogenous enzyme was still susceptible to stimulation by sarcosyl. However, more prolonged periods in the absence of mitosis resulted in an impaired stimulatory effect. The role of free RNA polymerase in the nuclei of eukaryotic cells is unknown. The decreased ability of sarcosyl to stimulate the bound enzyme may be related to aging. As sarcosyl acts by histone removal it is possible that histones are more tightly bound to the DNA of old cells; the cells on the G0 phase cells suggest that this process is a consequence of the nonmitotic state. During each cell cycle histones are removed from and reapplied to the DNA. It is known that histones are only synthesized during the S phase. Hence in a nondividing cell, there is a long term association between nonturning over protein and nonreplicating DNA. Such an event may favor the formation of secondary alterations in the chromatin, such as cross links. This would, in part, explain why cells held in the G0 phase have an impaired response to mitogenic stimulation.
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