Ribosome Biosynthesis Is not Necessary for Initiation of DNA Replication

Abstract

Synthesis of mature 28-S ribosomal RNA and 60-S ribosomal subunits is inhibited in baby hamster kidney (BHK) cell line ts 422E at non-permissive temperature (39 "C). This leads to a 66 decrease of total ribosomes per cell, a marked imbalance between the large and small ribosomal subunits in the cytoplasm and a decrease of cells per dish after prolonged culture at 39 "C. However, inhibition of ribosome synthesis does not affect progression of cells through the GI period of the cell division cycle, the length of the pre-replicative period, and the rate of entry of cells into S phase. In contrast to culture at non-permissive temperature, culture of BHK ts 422E cells in the presence of 0.04 pg/ml actinomycin D at 33 "C inhibits markedly the entry into S period. It is concluded that low doses of actinomycin D exert their inhibitory effect on cell growth by preventing maturation and transport of mRNA rather than by interfering with ribosome synthesis. Microfluorometric analysis revealed only slight differences in the distribution of BHK ts 422E cells in GI, S and GZ phases of the cycle either when cultured at 33°C or at 39°C. When too few ribosomes per cell are produced in BHK ts 422E cells at 39 "C, cells do not seem to be arrested reversibly at a specific point of the cell cycle but rather to die at random.The nucleolus has been considered to play a prominent role in cell proliferation. There are many reports which demonstrate that the nucleolus is markedly activated in cells stimulated to proliferate. An enhanced synthesis of rRNA in stimulated cells has been shown by an increased incorporation of radioactive precursors into nucleolar [l-51 or cytoplasmic rRNA [6-101 as well as by an increased activity of nucleolar RNA polymerase A [5, 11 -131. On the other hand, selective inhibition of nucleolar RNA synthesis by low doses of actinomycin or by lucanthone at the beginning of the GI period caused a delay of the entry of cells into the S phase [14-171. These findings were interpreted in terms of the dependence of DNA replication upon the contuined supply of ribosomal particles. Since, however, in all these studies only the biosynthesis of pre-rRNA was concerned and neither the processing of rRNA nor the assembly of mature ribosomal subunits has been studied, the question of whether growth stimulation of mammalian cells is necessarily correlated with an increased biosynthesis of ribosomes cannot yet be answered. The purpose of this work was to investigate Ahhreviations. BHK, baby hamster kidney; pre-rRNA, precursor to ribosomal RNA.to what extent, if at all, ribosome formation is a prerequisite for cells to traverse GI and enter S phase. We have used a temperature-sensitive mutant of BHK cells, ts 422E3, that is defective in the processing of 28-S rRNA at the non-permissive temperature [ 18, 191. The results suggest that the initiation of DNA replication is not related to an accumulation of cytoplasmic ribosomes during the GI period. EXPERIMENTAL PROCEDURESCell Culture

Evidence of p53-Dependent Cross-Talk between Ribosome Biogenesis and the Cell Cycle: Effects of Nucleolar Protein Bop1 on G1/S Transition

“…In addition, previous studies with mammalian cells indicate that interference with ribosome biogenesis by other means can also inhibit cell proliferation. For example, the ts422E temperature-sensitive mutant of the Syrian hamster cell line BHK21 that is unable to produce mature 28S rRNA and 60S ribosome subunits was rapidly growth arrested at the nonpermissive temperature (38,61); the nature of this growth defect, however, has not been clearly defined (20,41). Antisense-mediated inhibition of the nucleolar protein p120, a human homolog of the yeast Nop2p that is implicated in biosynthesis of the large ribosome subunit, arrested human lymphocytes in G 1 (17).…”

Calcium, cyclic AMP and protein kinase C ? partners in mitogenesis

“…However, this reactivation and expansion of the protein-synthesizing machinery is actually not needed for G O cells to wake up and transit G r Thus, reactivating a temperaturesensitive avian sarcoma virus's oncogenie/mitogenis pp60 ..... tyrosine • PK product or a temperature-sensitive Kirsten sarcoma virus's oncogenic/ mitogenic 21kDa K-RAS Gc~(or N,)-like protein causes their serum-deprived G O NRK rat kidney cell hosts to transit G 1, replicate DNA and ultimately divide without stimulating RNA or protein synthesis [277][278][279]. Moreover, mutant BHK hamster cells that cannot process the 45S ribosomal RNA precursor and make ribosomes at certain non-permissive temperatures can be stimulated by serum to transit G~ phase and replicate DNA at these same restrictive temperatures [280,281]. However, it is the stimulation of only one or a few master CDC genes (e.g., c-rnyc?)…”

The role of the G1 period in the life cycle of eukaryotic cells

“…The absence of a strong correlation between cell size and the time of entry into S phase is in conformity with the published reports. A number of investigators have previously shown that initiation of DNA synthesis can occur in mammalian cells without a concomitant accumulation of RNA (Grummt et al, 1979;Pochron et al, 1980;Mora et al, 1980) or protein (Ronning et al, 1981) or increase in cell size (Zetterberg and Engstrom, 1981;Zetterberg et al, 1982). From our data it is clear that slowing down the chromosome replication cycle does result in a shortening of GI period but only to a limited extent.…”