Template activity of chromatin during stimulation of cellular proliferation in human diploid fibroblasts

1. Contact-inhibited confluent monolayers of WI-38 human diploid fibroblasts can be stimulated to divide by replacing the medium with fresh medium containing 30% foetal calf serum. 2. Of the cells 40–75% are stimulated to divide with a peak DNA synthesis between 15 and 21h and a peak mitotic index between 28 and 30h after stimulation. 3. In the first 12h before the initiation of DNA synthesis there is a biphasic increase in the incorporation of [³H]uridine into RNA of whole cells. 4. This is paralleled by a similar biphasic stimulation of chromatin template activity measured in vitro in a system in which purified cell chromatin is incubated with an exogenous RNA polymerase isolated from Escherichia coli. 5. The changes in chromatin template activity are believed to represent activation of the genome, with more sites available for RNA synthesis, and to account almost entirely for the changes in RNA synthesis occurring in the whole cell.     

Effect of vitamin A on epithelial morphogenesis in vitro

Quiescent confluent monolayers of WI-38 human diploid fibroblasts and of 3T6 mouse fibroblasts were stimulated to proliferate by nutritional changes. WI-38 cells had a stringent requirement for serum factor(s) but 3T6 did not require serum in order to proliferate again. In both cell lines there was an early increase in the synthesis of non-histone chromosomal proteins shortly after stimulation of cellular proliferation and this increase was linearly correlated to the number of cells entering the S phase several hours later. Only WI-38 diploid fibroblasts, however, showed an early increase in chromatin template activity 1 h after stimulation of cellular proliferation, while chromatin template activity in 3T6 cells remained unchanged. It is suggested that the activation of gene function represents a critical step for the passage of WI-38 cells in the G0 resting phase to the G1 phase of the cell cycle. It is also suggested that 3T6 cells are unable to enter or stay in a G0 phase but can be arrested predominantly in the G1 phase by nutritional deficit, probably amino acid starvation.     

Changes in enzymic activity during cultivation of human cells in vitro

The composition of chromatin, its template activity and the activity of certain chromatin-associated enzymes, including DNA polymerase (DP) and soluble RNase, DNase, DP and seryl tRNA synthetase, were examined in early and late passage of WI-38 cells and of WI-38VA13 cells.No significant changes in soluble RNase, DNase, seryl tRNA synthetase or soluble and chromatin-associated DP were found with increasing passage of WI-38 cells. The activity of seryl tRNA synthetase and DP in WI38VA13 cells was, however, significantly higher than WI-38 cells in all passages. A decline in RNA synthesizing activity of chromatin, an increase in the proportion of RNA and histone in chromatin, as well as an increase in the activities of ‘chromatin-associated enzymes’ (RNase, DNase, protease, nucleoside triphosphatase, DPN pyrophosphorylase) were noted in WI-38 cells with increasing passages. Although RNA synthesizing activity of chromatin from WI38VA13 cells was lower than that from WI-38 cells, the former also were much lower in ‘chromatin-associated enzymes’. An increase of chromatin-associated enzymes responsible for RNA, DNA and protein degradation in WI-38 cells in successive passages, and a much lower activity of these enzymes in WI-38VA13 cells (which have an indefinite doubling potential in vitro) suggests that an elevation in the activity of these enzymes, which would seriously interfere with the chromatin function, could result in ‘aging’ of WI-38 cells.     

Effect of cyclic AMP on chromatin-bound protein kinases in WI-38 fibroblasts stimulated to proliferate.

When resting confluent monolayers of WI-38 fibroblasts are stimulated to proliferate by serum, DNA synthesis begins to increase between 15-18 h after stimulation. Chromatin-bound protein kinase activity increases in stimulated cells within 1 h after the nutritional change, concomitant with an increase in the template activity of nuclear chromatin. Addition of dibutyryl 3' : 5'-cyclic adenosine monophosphate (dibutyryl cyclic) AMP to the stimulating medium inhibits the entrance of cells into S phase, but only if dibutyryl cyclic AMP (5-10(-4) M) is added before the onset of DNA synthesis. The increases in chromatin template activity and in the chromatin-bound kinase activity are not inhibited by dibutyryl cyclic AMP in the early hours after stimulation, but are completely inhibited after the 5th hour from the nutritional change. This seems to indicate that in stimulated WI-38 cells, dibutyryl cyclic AMP exerts its inhibitory action somewhere between 5 and 12 h after stimulation. A number of protein kinase activities were extracted from chromatin with 0.3 M NaCl and partially resolved on a phosphocellulose column. Two distinct peaks of protein kinase activity appeared to be markedly increased in WI-38 cells 6 h after serum stimulation. Both peaks of increased activity were inhibited by dibutyryl cyclic AMP in vivo. Adenosine, sodium butyrate and adenosine 5'-monophosphate (AMP) do not inhibit the increase in DNA synthesis nor the increase in protein kinase activity. The results suggest that stimulation of cell proliferation in confluent monolayers of WI-38 cells causes an increase (or the new appearance) of certain chromatin-bound protein kinases, and that this increase is inhibited by cyclic AMP in vivo.     

Changes in the G0 state of WI-38 fibroblasts at different times after confluence

Some events in the prereplicative phase of WI-38 human diploid fibroblasts stimulated to proliferate are found to be a function of the length of time the cells have been quiescent. At 5 days after plating, when the cells first become confluent, the prereplicative phase upon stimulation by a nutritional change is relatively short, DNA synthesis begins at 8 h after stimulation, and there is no increase in chromatin template activity. At 9 days after plating the prereplicative phase of stimulated cells is lengthened to 14 h and there is an increase in chromatin template activity within 1 h of stimulation. Finally, in 18-day cells, the prereplicative phase is lengthened even further to 20 h, and there is a lag after stimulation before the increase in chromatin template activity. It is proposed that confluent WI-38 cells initially arrest in G 1, subsequently pass into G 0, and continue to go deeper into G 0 as they remain quiescent.     

Circular dichroism and ethidium bromide binding studies of chromatin from WI-38 fibroblasts stimulated to proliferate.

Confluent monolayers of WI-38 diploid fibroblasts can be stimulated to proliferate by fresh serum. In the first 3 h after stimulation (that is, several hours before DNA replication) the chromatin of stimulated cells show structrual changes which include: (1) an increase in maximum positive ellipticity and a blue shift in the 250-300 nm region of circular dichroism spectra; and (2) an increase,in isolated chromatin, of the number of binding sites for the intercalating dye, ethidium bromide.The differences between chromtin of stimulated and chromatin of unstimulated cells are abolised when bother chromatins are treated with 0.25 M NaCL.     

Changes in membrane transport function in G0 and G1 cells

Confluent quiescent monolayers of aneuploid and euploid cells in culture can be stimulated to proliferate by appropriate nutritional changes. In confluent monolayers of WI-38 human diploid fibroblasts the uptake of cycloleucine is increased three hours after these cells are stimulated to proliferate by a change of medium plus 10% serum. No changes in the uptake of cycloleucine are observed in logarithmically-growing WI-38 cells exposed to fresh medium plus 10% serum, or in WI-38 confluent monolayers in which the conditioned medium has been replaced by fresh medium with 0.3% serum (a change that does not cause stimulation of cellular proliferation in WI-38 cells). In 3T6 cells in the stationary phase stimulated to proliferate by nutritional changes, there is a prompt increase in the uptake of cycloleucine, within one hour after stimulation of cell proliferation. Similar results were obtained with stationary 2RA cells which are SV-40 transformed WI-38 fibroblasts. In addition, chromatin template activity which is known to increase in the early stages after stimulation of confluent WI-38 cells, was unchanged in confluent 3T6 or 2RA cells stimulated to proliferate. These results show that at least two of the very early biochemical events occurring in response to stimulation of cell proliferation are different in WI-38 diploid cells and in aneuploid 2RA or 3T6 cells. It is proposed that WI-38 cells in the stationary phase are arrested in the G₀ phase of the cell cycle, while 2RA and 3T6 cells are arrested in the G₁ phase.     

Effects of prolonged quiescence on neuclei and chromatin of WI-38 fibroblasts.

DNA synthesis and cell division are markedly reduced in confluent mono-layers of WI-38 diploid fibroblasts, but resume again if the depleted medium is replaced by fresh medium containing 10% fetal calf serum. If the cells are kept quiescent for prolonged periods of time after confluence (1 or 2 weeks), the fraction of cells that can be stimulated to proliferate by fresh serum decreases and the length of the prereplicative phase increases. The template activity of isolated nuclei decreases with increasing time of quiescence, and parallel changes occur in chromatin as evidenced by circular dichroism spectra and capacity to bind the intercalating dye, ethidium bromide. When WI-38 cells are stimulated to proliferate after prolonged quiescence, the increase in template activity of nuclei is delayed by several hours in comparison to cells stimulated after short periods of quiescence. Two distinct steps, both requiring serum, can be identified in the prereplicative phase of cells stimulated to proliferative after prolonged quiescence. We interpret the results as indicating that, during prolonged quiescence, WI-38 fibroblasts go into a deeper GO state from which they can be rescued only after prolonged stimulation. In this respect, prolonged quiescence may bear some resemblance to the process of aging.     

Control of RNA synthesis by chromatin proteins.

The effect of chromatin proteins on template activity has been studied. Using both E. coli RNA polymerase and calf thymmus polymerase B we have measured the number of initiation sites on chromatin and various histone-DNA complexes. Chromatin can be reconstituted with histone proteins alone and this complex is still a restricted template for RNA synthesis. The removal of histone f1 causes a large increase in the template activity. Chromatin is then treated with Micrococcal nuclease and the DNA fragments protected from nuclease attack ("covered DNA") are isolated. Alternatively, the chromatin is titrated with poly-D-lysine, and by successive treatment with Pronase and nuclease, the DNA regions accessible to polylysine are isolated ("open DNA"). Both fractions were tested for template activity. It was found that RNA polymerase initiation sites are distributed equally in open and covered region DNA.     

In situ studies on the effect of acid extraction on the DNA template activity of mature avian erythrocytes.

Exogenous E. coli RNA polymerase was used to determine the in situ DNA template activity of ethanol/acetone fixed avian erythrocytes. No RNA polymerase-catalyzed incorporation of 3H-UTP was detected in mature avian erythrocytes while simultaneously fixed avian lymphocytes did exhibit incorporation of 3H-UTP. Nuclei of mature erythrocytes which were subjected to treatments known to remove histones showed dramatic increases in RNA polymerase-catalyzed incorporation of 3H-UTP. The chromatin of treated cells was presumed to be more accessible to RNA polymerase as determined by the increase in RNA polymerase-catalyzed incorporation of 3H-UTP. Incubation of acid-treated nuclei in poly-L-lysine prior to incubation with RNA polymerase failed to inhibit the incorporation of 3H-UTP. Possible mechanisms for the inactivation of avian erythrocyte nuclei are discussed.     

大鼠衰老过程中肝细胞核及染色质的体外转录活性

用同位素掺入法研究不同年龄大鼠的肝细胞核及染色质体外转录活性,所得结果表明:(1)老年大鼠肝细胞核的转录起始能力较断乳鼠及青年鼠分别下降68％及56％。(2)大鼠肝细胞核内与染色质结合的RNA聚合酶所致的转录活性随增龄呈近似线性下降,而不与染色质结合的RNA聚合酶所致的转录活性随增龄则无变化。(3)老年大鼠肝染色质体外转录活性较断乳鼠及青年鼠分别下降52％及35％。这些结果提示。老年大鼠肝染色质功能的改变可能是转录活性改变的主要原因。     

Translational control of protein synthesis in stimulated WI-38 fibroblasts.

A cell-free protein synthesis system employing ribosomes from WI-38 human diploid fibroblasts was developed and its optimum MgC12 and KC1 levels and pH value found. The rate at which ribosomes are able to incorporate radioactive leucine into proteins ([14C]leucine incorporation/10 min/100 mug rRNA) and the number of growing peptide chains [3H]puromycinpeptides formed/100 mug rRNA) was determined. When confluent monolayers of WI-38 cells were stimulated to proliferate by serum, a transient increase in the rate of peptide elongation by ribosomes was observed at 60 min after stimulation. This increase was not affected by the presence of actinomycin D (10 mug/ml) in the stimulating medium. A change in the relative amount of certain ribosome-associated proteins accompanied the increased elongation rate of peptide growth. The alteration in associated proteins could not be accounted for by an increased synthesis of protein. Finally, the early activation of ribosomes in stimulated WI-38 cells appears to result from the removal of an inhibitor(s) of ribosome function.     

Chromatin template activity during aging in WI38 cells

A reduction in the chromatin template activity of WI38 cells of different ages has been demonstrated using (1) the endogenous RNA polymerase of the host cell and (2) exogenously supplied E. coli RNA polymerase. The consistency between these two methods suggests that the reduction in chromatin template activity of these cells is age-associated and may represent changes in gene activity during in vitro aging.     

Isolation and characterization of nonhistone chromosomal protein C-14 which stimulates RNA synthesis.

The nonhistone chromatin protein, C-14, was extracted from chromatin of Novikoff hepatoma ascites cells and isolated in high purity as shown by its migration as a single dense spot on two-dimensional polyacrylamide gels. Its mobility on sodium dodecyl sulfate gels is consistent with a molecular weight of approximately 70 000. The amino acid composition shows that protein C-14 has an acidic:basic amino acid ratio of 1.8. Its amino terminal amino acid is lysine. Protein C-14 stimulated the incorporation of [3H]UMP into RNA by approximately 30% when added to naked DNA and homologous RNA polymerase I. A 30% stimulation of [3H]UMP incorporation into RNA was also found when protein C-14 was added to an E. coli RNA polymerase system containing either E. coli or Novikoff hepatoma DNA.     

Release of template restriction in chromatin by nuclear 4.5s RNA.

The stimulatory mechanism of RNA synthesis of calf-thymus chromatin by nuclear 4.5 S RNA from the homologous tissue was investigated by using exogenously added Escherichia coli RNA polymerase. The RNA synthesis was initiated at low concentration of salt, and then the chain elongation was achieved at high concentration of ammonium sulfate in the presence of polyvinyl sulfate. Under these conditions the number of binding sites of RNA polymerase on chromatin which were capable of initiating RNA chain was increased by the addition of the 4.5 S RNA. This stimulation was presumed to result from the release of template restriction in chromatin. The polyvinyl salt minimized ribonuclease activity without changing the RNA polymerase activity bound to the template. Neither rearrangement nor release of chromatin proteins affected the amount or size of RNA produced. Preliminary analysis suggested that the molecular species of RNA produced upon the addition of the 4.5 S RNA from various tissues seemed to be heterologous.