Inhibition of cellular transition from G1-resting to G1-prereplicative phase by aminonucleoside or puromycin

Summary Human embryonic lung fibroblasts (IMR-90 and WI-38) were arrested in the G₁ phase of the cell cycle by serum deprivation and high population density. Within 1 hr after the addition of medium containing fresh serum, these cells showed an increase in rRNA synthesis. The inclusion of 100 μg per ml aminonucleoside of puromycin (AMS) in the fresh medium eliminated the serum stimulation of rRNA synthesis and prevented the cells from making the G₁-resting phase to G₁-prereplicative phase transition. AMS also prevented the synthesis of HnRNA normally found within 10 hr after serum stimulation. Serum-stimulated RNA synthesis in starved, SV-40 transformed fibroblasts (WI-38-VA-13 cells) was inhibited, but not completely prevented, by AMS indicating that transformed cells may produce specific RNA's that are not AMS-sensitive and that may be responsible for the failure of transformed cells to be arrested in G₁. This work was supported by PHS Research Grant CA19750-02 from the National Cancer Institute. These results were reported previously in a preliminary form (7).     

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cells exposed to fresh medium plus 10% serum, or in WI-38 confluent monolayers in which the conditioned medium has been replaced by fresh medium with 0.3% serum (a change that does not cause stimulation of cellular proliferation in WI-38 cells).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.     

Late G1 amino acid restriction point in human dermal fibroblasts

Human dermal fibroblasts arrested in G0 by maintenance in medium supplemented with 0.1% serum were not restimulated to divide when fresh medium containing 10% dialyzed serum but lacking group B amino acids (cystine, isoleucine, lysine, phenylalanine and tyrosine) was added. Unlike rodent cells, the addition of fresh serum-supplemented medium lacking only isoleucine did not cause a growth arrest. The amino acid sensitive growth arrest in human fibroblasts was dependent both on presynchronization in G0 as well as a prestarvation for amino acids prior to stimulation with high serum. When cells were restimulated in the absence of amino acids, they arrested predominantly in G1, although a small percentage of cells entered early S phase. When medium containing a complete complement of amino acids was then added, cells initiated DNA synthesis following a minimum lag of 2-3 hr. Growth arrested cells initiated DNA synthesis even when complete unsupplemented medium was added, although the addition of high concentrations of insulin or 10% serum increased the rate of entry.     

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.     

Analysis of the growth factor requirements for stimulation of WI-38 cells after extended periods of density-dependent growth arrest

When cultures of WI-38 human diploid fibroblasts reach high cell densities, they cease to proliferate and enter a viable state of quiescence. WI-38 cells can remain in this quiescent state for long periods of time; however, the longer the cells remain growth arrested, the more time they require to leave G0, progress through G1, and enter S after stimulation with fresh serum. The experiments presented here compare the response of long-term quiescent WI-38 cells (stimulated 26 days after plating) and short-term quiescent WI-38 cells (stimulated 12 days after plating) to treatment with a variety of individual purified growth factors instead of whole serum. Our results show that the qualitative and quantitative growth factor requirements necessary to stimulate G1 progression and entry into S were the same for both short- and long-term quiescent WI-38 cells, in that the same defined medium (supplemented with epidermal growth factor [EGF], recombinant human insulin-like growth factor 1 [IGF-1], and dexamethasone [DEX]) stimulated both populations of cells to proliferate with the same kinetics and to the same extent as serum. However, the long-term quiescent WI-38 cells were found to exhibit a difference in the time during which either serum or these individual growth factors were required to be present during the prereplicative period. We believe that this difference may be the cause of the prolongation of the prereplicative phase after stimulation of long-term density-arrested WI-38 cells.     

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.     

T98G: an anchorage-independent human tumor cell line that exhibits stationary phase G1 arrest in vitro.

T98 and T98G are two related cell lines that were derived from a human glioblastoma multiforma tumor. T98G has almost twice as many chromosomes as T98, suggesting that it is a polyploid variant of T98. Three aspects of control of cellular proliferation were studied in T98 and T98G cells in comparison to WI-38 normal human diploid cells. WI-38 cells have the following properties: (1) they can undergo only a limited number of population doublings in vitro; (2) they cannot proliferate without anchorage; and (3) they become arrested in G1 phase under stationary phase conditions. T98 cells differ from normal cells in all three of these properties, as do many other transformed cell lines. However, the derivative of T98, namely T98G, expresses an unique combination of normal and transformed aspects of the control of cellular proliferation. T98G cells are like normal cells in that they become arrested in G1 phase under stationary phase conditions, yet they also exhibit the transformed characteristics of anchorage independence and immortality. Thus, T98G cells demonstrate that transformation to immortality and anchorage independence can exist without concomitant loss of the normal mechanism for G1 arrest in response to stationary phase conditions. This result supports the hypothesis that each of these three aspects of control of cellular proliferation can be altered independently. Partially transformed cell lines, such as T98G, should be useful for sorting out the biochemical changes associated with transformation in each of these aspects.     

Changes in template activity and structure of nuclei from WI-38 cells in the prereplicative phase.

Quiescent confluent monolayers of WI-38 fibroblasts were stimulated to proliferate by either adding 10% fetal calf serum or by trypsinization and replating at lower density. The length of the prereplicative phase was 12 hr after serum stimulation and 18 hr after trypsinization and replating at lower density. Nuclei were isolated from WI-38 cells at different time intervals after either type of stimulation and their template activity, circular dichroism spectra, and ability to bind ethidium bromide were investigated. All these parameters were similarly increased after either type of stimulation. However, these changes, like the onset of DNA synthesis, were delayed 6 hr in cells trypsinized and replated at lower density. While there were no detectable changes in nuclear protein content after serum stimulation, at least 40% of nuclear protein, mostly nonhistone chromosomal proteins, were lost after trypsinization. The amount of nuclear proteins returned to prestimulation levels only 6-8 hr after replating. These data seem to suggest that nonhistone chromosomal proteins lost by trypsinization are essential for the entrance of WI-38 cells into the "prereplicative phase".     

Synthesis of DNA-binding proteins during the cell cycle of WI-38 cells

Synthesis of DNA-binding proteins was investigated in WI-38 human diploid fibroblast cultures after stimulation with serum containing medium. Density-inhibited confluent monolayers of young (phase II) and aging (phase III) WI-38 cells can be stimulated to synthesize DNA by replacing the medium with fresh medium containing 10% fetal calf serum. Of the phase II cells, 35–50% showed a partially synchronized burst of DNA-synthesizing activity between 15 and 24 h whereas only 4–6% of phase III cells showed DNA-synthesizing activity at 20 h, and that cell fraction was increasing even at 38 h. This suggests either an extremely prolonged G 1 in stimulated phase III cells, or a heterogeneity of the population (e.g., a mixed population of pre- and postmitotic cells) for phase III cells. At various times after the change of medium, DNA-binding protein synthesis was examined in these stimulated cultures. Protein of mol. wt 20 000–25 000 D accumulated rapidly during early G 1 and declined thereafter, whereas larger protein (40 000 and 68 000 D) accumulated during the late G 1 or G 1-S transition period indicating that accumulation of these proteins is associated with the onset of DNA synthesis in the serum-stimulated cells. In cultures where the DNA synthesis has been reduced or inhibited by an excess of thymidine, hydroxyurea or dibutyryl cAMP, the accumulation of the larger proteins (40 000 and 68 000 D) was neglible as compared with non-stimulated cultures. Hydrocortisone did not exert any effect on the DNA-binding protein synthesis in phase II cells. However, it seems to increase the cell fraction which can respond to the serum factor in phase III cells as evidenced from the pattern of DNA-binding proteins synthesis.     

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.     

An adenovirus early region 1A protein is required for maximal viral DNA replication in growth-arrested human cells.

Two closely related adenovirus early region 1A proteins are expressed in transformed cells. The smaller of these, which is 243 amino acids in length, is required for the transformation of primary rat cells and for the transformation of immortalized rat cells to anchorage-independent growth. This protein is not required for productive infection of exponentially growing HeLa cells but is required for maximal replication in growth (G0)-arrested human lung fibroblasts (WI-38 cells). To determine the function of this protein in viral replication in these G0-arrested cells, we compared viral early mRNA, early protein, and late protein synthesis after infection with wild type or a mutant which does not express the protein. No differences were found. However, viral DNA synthesis by the mutant was delayed and decreased to 20 to 30% that of wild type in these cells. Viral DNA synthesis was much less defective in growing WI-38 cells, and in the transformed human HeLa cell line it occurred at wild-type levels. Furthermore, the mutant which can express only the 243-amino-acid early region 1A protein induced cellular DNA synthesis in G0-arrested rat cells to the same level as wild-type virus. A mutant which can express only the 289-amino-acid early region 1A protein induced less cellular DNA synthesis in G0-arrested rat cells. We propose that the early region 1A 243-amino-acid protein alters the physiology of arrested permissive cells to allow maximal viral DNA replication. In nonpermissive rodent cells, the 243-amino-acid protein drives G0-arrested cells into S phase. This activity is probably important for the immortalization of primary cells.     

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.     

Inducers of DNA synthesis: levels higher in transformed cells than in normal cells

The objective of this study was to determine whether transformed cells have greater DNA synthesis-inducing ability (DSIA) than normal cells when fused with G1 phase cells. HeLa cells synchronized in G1 phase, prelabeled with large latex beads, were fused separately with (a) quiescent human diploid fibroblasts (HDF), (b) HDF partially synchronized in late G1, and random populations of (c) HeLa, (d) WI-38, (e) SV-40 transformed WI-38, (f) CHO, (g) chemically transformed mouse cells (AKR-MCA), and (h) T98G human glioblastoma cells (all prelabeled with small latex beads) using UV-inactivated Sendai virus. The fusion mixture was incubated with [3H] thymidine, sampled at regular intervals, and processed for radioautography. Among the heterodikaryons, the frequency of those with a labeled and an unlabeled nuclei (L/U) were scored as a function of time after fusion. The faster the induction of DNA synthesis in HeLa G1, the steeper the drop in the L/U class and hence the higher DSIA in the S phase cells. The DSIA, which is indicative of the intracellular levels of the inducers of DNA synthesis, was the highest in HeLa and virally transformed WI-38 cells and the lowest in normal human diploid fibroblasts (HDF) while those of chemically and spontaneously transformed cells are intermediate between these two extremes. Higher level of DNA synthesis inducers appears to be one of the pleotropic effects of transformation by DNA tumor viruses. These studies also revealed that initiation of DNA synthesis per se is regulated by the presence of inducers and not by inhibitors.     

Serum-dependent regulation of proliferation of cultured rat fibroblasts in G1 and G2 phases

We reported that: (i) 3Y1tsF121 cells, a temperature-sensitive (ts) mutant of rat 3Y1 fibroblasts, are reversibly arrested either in the G1 or in the G2 phase, at the nonpermissive temperature. (ii) Cells retain the ability to resume proliferation at the permissive temperature after prolonged arrest in the G1 phase (for 5 days), whereas they lose it after prolonged arrest in the G2 phase (over 24 h). (iii) The G1 arrest is overcome at the nonpermissive temperature by the addition of fresh serum (H. Zaitsu and G. Kimura (1984) J. Cell. Physiol. 119, 82; (1985) J. Cell. Physiol. 124, 177). In the present study, the G2 arrest was overcome by exposing the cells to fresh serum, at the nonpermissive temperature. The G2 arrest occurred only at a higher cell density than that of the G1 arrest. The efficiency of the overcome was higher in the case of the G2 arrest than in case of the G1 arrest. When cells synchronized at the G1/S border by aphidicolin at the permissive temperature were released from the block, they divided in the absence of serum, at the permissive temperature. Even if they had passed through the previous G2 phase in a very high concentration of fresh serum at the permissive temperature, mitotic cells did not enter the S phase in the absence of serum, even at the permissive temperature. When the cells arrested in the G1 phase (not in G0) due to the ts defect were incubated in the absence of serum at the permissive temperature, only 34% entered the S phase and only 15% divided. These results suggest that (i) the ts defect in 3Y1tsF121 limiting cellular proliferation in both the G1 and the G2 phases is probably due to a single mutational event, and is a serum-requiring event. (ii) Preparation of the serum-requiring event which is required for the G2 traverse is completed in the G1 phase, under ordinary conditions. (iii) However, cells are able to fulfill the serum-requiring event in the G2 phase as well as in the G1 phase when the preparation is below the required level. (iv) The commitment to DNA synthesis is not necessarily a commitment to cell division. (v) Cells are arrested in the G1 phase more safely and more effectively than in the G2 phase, by the serum-related mechanism.     

Time of c-fos and c-myc expression in human diploid fibroblasts stimulated to proliferate after prolonged periods in quiescence

When human diploid fibroblasts such as WI-38 cells become crowded, they enter a viable state of quiescence (G0) in which they can remain for prolonged periods of time. These quiescent cells can be induced to re-enter the cell cycle by addition of fresh serum. However, cells held in G0 for long periods before stimulation require more time to enter DNA synthesis as compared to cells held in a quiescent state for short periods. We have used this model system to determine if a close temporal coupling exists between the time of expression of two proto-oncogenes associated with cell growth, c-fos and c-myc, and the time of entry into DNA synthesis. WI-38 cells were stimulated to enter DNA synthesis by the addition of fresh culture medium and serum at various lengths of time after plating, ranging from 7 to 34 days. At hourly intervals thereafter, cells were harvested and total RNA was isolated. These samples were then analyzed by RNase protection assay to determine the levels of c-fos and c-myc mRNA. Our results show that the time and pattern of c-fos and c-myc mRNA accumulation after stimulation is determined only by the time which the cells are treated with serum even when they exhibit a 19-h delay in the entry into DNA synthesis. In all of our experiments, c-fos could be detected 0.5 h after stimulation and remained detectable for approximately 2 h. Likewise, the peak of c-myc accumulation occurred at about 3 h after serum addition, regardless of how long it took to initiate DNA synthesis. These results suggest that the time of c-fos and c-myc induction clearly is not the only factor which determines the length of the prereplicative period and thus the ultimate time of initiation of DNA synthesis.     

Kinetics of G1 transit following brief starvation for serum factors

Growing fibroblasts such as 3T3 cells are well-known to enter a quiescent state (G0) after many hours of serum deprivation. They emerge from G0 upon readdition of serum and initiate DNA synthesis about 12 h later. In this paper, we analyzed the effects of brief periods of serum deprivation on the ability of cells in G1 to initiate DNA synthesis. Exponentially growing 3T3 fibroblasts were briefly deprived of serum and their progress into S phase was monitored by autoradiography of labeled nuclei. When 10% serum was added back to cultures deprived of serum for a few hours, the progress of G1 cells into S phase was delayed for intervals far in excess of the length of the serum deprivation. Longer serum starvations resulted in longer excess delays. Several transformed 3T3 derivatives were markedly less sensitive to this serum-induced G1 regression following deprivation. When 1 microgram/ml insulin (rather than 10% serum) was added back to the starved cultures, the G1 cells entered S phase immediately. Delay in S phase entry following serum readdition was completely prevented if insulin (and, to a lesser extent, EGF) was present during the starvation, was diminished if a lower serum concentration was used for readdition, and was partially abolished if 10% serum plus insulin was restored to the cultures. The above results, then, suggest that serum deprivation sensitizes the cells to an unidentified serum component which sets the cells back in G1, unless insulin is present to maintain the flow of cells into S.     

INHIBITION OF CELL GROWTH IN THE G1 PHASE BY ADENOSINE 3'',5''-CYCLIC MONOPHOSPHATE

Incorporation of tritiated thymidine into acid-precipitable material was used to measure the rate of DNA synthesis in secondary cultures of human diploid fibroblasts. Confluent cultures of human diploid fibroblasts, which are synchronized in the G₁ phase due to contact inhibition, were released from growth inhibition either by the addition of fresh medium to the cultures or by trypsinization and replating at nonconfluent densities. Either treatment resulted in a synchronous wave of DNA synthesis beginning 10–15 h after treatment and peaking at 20–25 h. In confluent cultures stimulated by fresh medium, either the addition of 0.25 mM N⁶, O²-dibutyryl-adenosine 3',5'-cyclic monophosphate (db-cAMP) to the medium in the interval 4–8 h after stimulation or the replacement of the fresh medium in that same 4 h interval with the depleted medium present on the cells for the 2 day period before stimulation delayed the synchronous onset of DNA synthesis in the cultures by about 4 h. In nonconfluent cultures freshly seeded from trypsinized confluent cultures, this same depleted medium obtained after a 2 day incubation of fresh medium on confluent cultures is shown to support the progress of the cells into S phase; however, the addition of 0.25 mM db-cAMP to the medium 3½ h after replating still partially prevented the initiation of DNA synthesis in the cultures. The results are discussed in terms of the role of serum and cAMP in the control of cell growth in fibroblast cultures.     

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.     

Synthesis of nuclear acidic proteins in density-inhibited fibroblasts stimulated to proliferate

Previous work from this laboratory (Rovera and Baserga, 1971) has shown that, when density-inhibited WI-38 human diploid fibroblasts are stimulated to proliferate by a change of medium, the synthesis of nuclear acidic proteins increases within 30 minutes after stimulation; several hours before DNA synthesis begins to increase. Similar results have now been obtained with density-inhibited 3T6 mouse fibroblasts, also stimulated by a change of medium. Gel electrophoretic analysis of nuclear acidic proteins in both WI-38 human diploid fibroblasts and 3T6 mouse fibroblasts stimulated to proliferate indicates that the increased synthesis of nuclear acidic proteins is limited to certain classes of proteins while other classes are totally unaffected. The increase in nuclear acidic proteins synthesis is inhibited when WI-38 cells or 3T6 cells are stimulated in the presence of 5-azacytidine (10 μg/ml), a treatment which also inhibits the subsequent stimulation of DNA synthesis. These results, confirming and extending similar findings previously reported in other models of stimulated DNA synthesis, lend further support to the hypothesis that nuclear acidic proteins may play a critical role in the control of DNA synthesis and cell division in mammalian cells.