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The mechanism of regulation of fibroblastic cell replication II. Participation of the nucleoli
Author(s) -
Mironescu Stefan,
Ellem K. A. O.,
Love R.
Publication year - 1973
Publication title -
journal of cellular physiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.529
H-Index - 174
eISSN - 1097-4652
pISSN - 0021-9541
DOI - 10.1002/jcp.1040820314
Subject(s) - nucleolus , uridine , mitosis , cell , cell cycle , biology , ploidy , cell growth , period (music) , cell division , embryo , microbiology and biotechnology , nucleus , rna , biochemistry , physics , acoustics , gene
Abstract Cultures of human diploid fibroblasts (HDFs) exhibiting density dependent inhibition of replication (DDIR) resumed their progression through the cell cycle following medium replacement and, after a lag period of two hours, showed a dramatic increase in the incidence of isonucleolinar 4 cells and in the levels of uptake of 3 H‐uridine into the nucleoli. Between five and ten hours after refeeding these nucleolar changes were maximal, leveling off at the highest values, in periods corresponding to late G1 and early S. Concomitantly, a parallel increase in the number of nucleolini per cell occurred. As cells progressed through S and G2 phases the nucleolini decreased in number and reverted to the aniso‐nucleolinar type. The intensity of nucleolar labeling by 3 H‐uridine and its correlate, the frequency of cells with labeled nucleoli, also decreased during these cell cycle stages. Both pre‐ and postreplicative periods of mitotic quiescence were characterized by high levels of anisonucleolinosis (60–80% of the cells) and by very low levels of nucleolar 3 H‐uridine incorporation. The magnitude of these nucleolar changes occurring during G1 stage was found to be strongly dependent on: (1) the length of time of contact between the cells and the fresh medium, at least eight hours of contact being necessary for a maximal response; (2) the amount of serum in the medium, the optimal serum concentration being between 10 and 50%, and (3) the pH of the medium. The nucleolar response was completely abolished at pH values below 7.0. These nucleolar changes were very sensitive to the presence of cycloheximide (10 μg/ml) and actinomycin D (0.003 μg/ml). The behavior of the nucleoli in response to these parameters was similar to the activation response of the cells to initiate DNA synthesis. During the time period of maximal nucleolar (activation) the onset of DNA synthesis as well as the morphological and autoradiographic manifestations of the nucleolar activation were completely inhibited by very low levels of actinomycin D (Ellem and Mironescu, '72), a selective inhibitor of nucleolar RNA synthesis (Perry, '65). This suggested a possible role of nucleolar metabolism, in normal diploid cells, in the initiation of DNA synthesis. Our results, however, seem to indicate that the nucleolar changes are necessary but not sufficient for the subsequent initiation of DNA synthesis, since with graded serum concentrations or medium volumes, smaller levels of a stimulus were needed to produce maximal isonucleolinosis than to effect a maximum replicative response in the cells.