Regulation of human ornithine decarboxylase expression following prolonged quiescence: Role for the c-Myc/Max protein complex

WI-38 cells can remain quiescent for long periods of time and still be induced to reenter the cell cycle by the addition of fresh serum. However, the longer these cells remain growth arrested, the more time they require to enter S phase. This prolongation of the prereplicative phase has been localized to a point early in G₁, after the induction of “immediate early” G₁ genes such as c-fos and c-jun but before maximal expression of “early” G₁ genes such as ornithine decarboxylase (ODC). Understanding the molecular basis for ODC mRNA induction can therefore provide information about the molecular events which regulate the progression of cells out of long-term quiescence into G₁ and subsequently into DNA synthesis. Studies utilizing electrophoretic mobility shift assays (EMSA) of nuclear extracts from short- and long-term quiescent WI-38 cells identified a region of the human ODC promoter at ?491 bp to ?474 bp which exhibited a protein binding pattern that correlated with the temporal pattern of ODC mRNA expression. The presence of a CACGTG element within this fragment, studies with antibodies against c-Myc and Max, the use of purified recombinant c-Myc protein in the mobility shift assay, and antisense studies suggest that these proteins can specifically bind this portion of the human ODC promoter in a manner consistent with growth-associated modulation of the expression of ODC and other early G₁ genes following prolonged quiescence. These studies suggest a role for the c-Myc/Max protein complex in regulating events involved in the progression of cells out of long-term quiescence into G₁ and subsequently into S. © 1995 Wiley-Liss, Inc.     

WI-38 cell long-term quiescence model system: A valuable tool to study molecular events that regulate growth

A number of cell culture model systems have been used to study the regulation of cell cycle progression at the molecular level. In this paper we describe the WI-38 cell long-term quiescence model system. By modulating the length of time that WI-38 cells are density arrested, it is possible to proportionately alter the length of the prereplicative or G-1 phase which the cell traverses after growth factor stimulation in preparation for entry into DNA synthesis. Through studies aimed at understanding the cause and molecular nature of the prolongation of the prereplicative phase, we have determined that gene expression plays an important role in establishing growth factor “competence” and that once the cell becomes “competent” there is a defined order to the molecular events that follow during the remainder of G-1. More specifically, we have determined that the prolongation represents a delay in the ability of long term quiescent cells to become fully “competent” to respond to growth factors which regulate progression through G-1 into S. This prolongation appears to occur as a result of changes during long term quiescence in the ability of immediate early G-1 specific genes (such as c-myc) to activate the expression of early G-1 specific genes (such as ornithine decarboxylase). While ODC is the first and thus far only growth associated gene identified as a target of c-myc (and the Myc/Max protein complex), it is likely that further studies in this model system will reveal other early G-1 growth regulatory genes. We anticipate that future follow-up studies in this model system will provide additional valuable information abuot the function of growth-regulatory genes in controlling growth factor responsiveness and cell cycle progression.     

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.     

Colchicine activates cell cycle-dependent genes in growth-arrested rat 3Y1 cells

When growth-arrested 3Y1 cells (Fischer rat fibroblasts) were exposed to 3 X 10(-5) M colchicine, they entered S phase after a 12-h lag period which is the same as that in serum-stimulated cells. The expression of genes such as c-fos, c-myc, JE, KC, ornithine decarboxylase, and histone H3, analyzed by Northern blotting, increased in a cell-cycle dependent manner after colchicine treatment. The increased level of mRNAs was much smaller in colchicine-stimulated cells than in serum-stimulated cells, corresponding to the lower frequency of the former cells entering S phase. The course of the prereplicative phase seems to be similar in terms of the expression of cell cycle-dependent genes in cells stimulated with colchicine and in those stimulated with serum.     

Comparative effects of butyrate and N6, 2'-O-dibutyryladenosine-3':5'-cyclic monophosphate on growth, differentiation and gene expression in U937 human monoblastoid cells.

Administration of 1mM sodium butyrate or N6,2'-O-dibutyryladenosine 3':5'-cyclic monophosphate (dbcAMP) inhibits the growth activity of U937 human monoblastoid cells by blocking them at the G1 or at the G1 + G2 phases of the cell cycle, respectively. Both agents induce the differentiation of U937 cells, as proved by the increased expression of the maturation-associated CD11b antigen and by the increased capacity to reduce nitroblue tetrazolium. RNA blot assays indicate that butyrate and dbcAMP decrease the expression of ornithine decarboxylase and c-myc genes, and stimulate the expression of the vimentin gene. However, while dbcAMP induces c-fos mRNA accumulation, butyrate did not affect the expression of this proto-oncogene.     

Selective induction of c-jun and jun-B but not c-fos or c-myc during mitogenesis in SV40-transformed cells at the predifferentiation growth arrest state.

Insulin has recently been reported to function as a complete mitogen for SV40 large T antigen-transformed 3T3 T-cells, designated CSV3-1, but not for nontransformed 3T3 T-cells (H. Wang and R. E. Scott, J. Cell. Physiol., 147: 102-110, 1991). It is now reported that sodium orthovanadate mimics this effect of insulin. For example, when exposed to 1-5 microM vanadate, most predifferentiation growth-arrested CSV3-1 cells undergo DNA synthesis within 24 h, but neither vanadate nor insulin induces mitogenesis in nontransformed 3T3 T-cells. To investigate the possible mechanisms by which mitogenesis is induced in CSV3-1 cells, the effects of insulin and vanadate on the expression of growth-related genes were examined. Whereas insulin and vanadate had no effect on the expression of c-fos, c-myc, c-jun, jun-B, or ornithine decarboxylase activity in nontransformed 3T3 T-cells, insulin and vanadate showed different effects on the expression of these genes in CSV3-1 cells. Insulin induced a rapid and transient accumulation of c-fos mRNA followed by induction of c-myc, c-jun, jun-B, and ornithine decarboxylase. In contrast, vanadate induced the expression of c-jun, jun-B, and ornithine decarboxylase without inducing c-fos and c-myc. These observations suggest that SV40 large T antigen may play an important role in insulin- and vanadate-induced mitogenesis and that insulin and vanadate may mediate their mitogenic effects by different signal transduction pathways.     

Expression of growth-regulated genes in tsJT60 cells, a temperature-sensitive mutant of the cell cycle

We have investigated the expression of growth-regulated genes in tsJT60 cells, a temperature-sensitive (ts) mutant of Fischer rat cells, which, on the basis of its kinetic behavior, can be classified as a G0 mutant. It grows normally at 34 degrees C and also at 39.5 degrees C if shifted to the higher temperature during exponential growth. However, if the cell population is first made quiescent by serum deprivation, subsequent stimulation by serum induces the cells to enter S phase at 34 degrees C but not at 39.5 degrees C. A panel of growth-regulated genes was used that included three protooncogenes (c-fos, c-myc, and p53), several genes that are induced in G0 cells stimulated by growth factors (beta-actin, 2A9, 2F1, vimentin, JE-3, KC-1, and ornithine decarboxylase), and an S-phase gene (histone H3). The expression of these growth-regulated genes was studied in both tsJT60 cells and its parental cell line, rat 3Y1 cells. All the genes tested, except histone H3, are similarly induced when quiescent tsJT60 cells are stimulated by serum at either permissive or restrictive temperatures. These results raise intriguing questions on the nature of quiescence and the relationship between G0 and G1 in cells in culture.     

Cell cycle dependent gene expression in quiescent stimulated and asynchronously cycling arterial smooth muscle cells in culture.

The expression of a set of cell cycle dependent (CCD) genes (c-fos, c-myc, ornithine decarboxylase (ODC), and thymidine kinase (TK)) was comparatively studied in cultured arterial smooth muscle cells (SMC) during exit from quiescence and exponential proliferation. These genes, which were not expressed in quiescent SMC, were chronologically induced after serum stimulation. c-fos mRNA were rapidly and transiently expressed very early in the G1 phase; c-myc and ODC peaked a few hours after serum stimulation and then remained at an intermediary level throughout the first cell cycle; TK mRNA and activity then appeared at the G1/S boundary and peak in G2/M phases. Except for c-fos, the other genes were also expressed in asynchronously cycling SMC (ACSMC); their expression was studied in elutriated subpopulations representative of cell cycle progression. c-fos mRNA were undetectable in any sorted subpopulations, even in the pure early G1 population. Despite a slight increase as the cell cycle advanced, c-myc and ODC genes were expressed throughout the ACSMC cell cycle. A faint TK activity was found in G1 subpopulations and increased in populations enriched in other phases; in contrast, TK mRNA remained highly expressed in all elutriated subpopulations. This study demonstrates significant modulations in CCD gene expression between quiescent stimulated and asynchronously cycling SMC in culture. This suggests that the events occurring during the emergence of SMC from quiescence are probably different from those in the G1 phase of ACSMC.     

Tyrosine protein kinase expression in long-term quiescent WI-38 cells following growth factor simulation

We have used the WI-38 cell long-term quiescent model system to study the regulation of cell cycle progression at the molecular level. By modulating the length of time that WI-38 cells are density arrested, it is possible to proportionately alter the length of the prereplicative or G-1 phase which the cell traverses after growth factor stimulation in preparation for entry into DNA synthesis. Stimulation of long-and short-term density arrested WI-38 cells with different growth factors or higher concentrations of individual growth factors does not alter the time required by long-term cells to enter S after stimulation. However, the time during the prereplicative period for which these growth factors are needed is different. Long-term quiescent WI-38 cells require EGF to traverse the G-0/G-1 border but do not need and apparently cannot respond to IGF-1 during the first 10 h after EGF stimulation, the length of the prolongation of the prereplicative phase. This suggests that EGF stimulation of long-term quiescent WI-38 cells initiates a series of molecular events which make these cells “competent” to respond to the “progression” growth factor, IGF-1. In light of the well-established role of protein tyrosine kinases in signal transduction, we set out to identify, clone, and analyze the expression of receptor and non-receptor tyrosine kinases which potentially could play a role during the prolongation of the prereplicative phase in making the long-term quiescent WI-38 cells competent to respond to IGF-1. We obtained 49 clones representing 11 different receptor and non-receptor type protein tyrosine kinases. Analysis of expression of these clones revealed a variety of different patterns of expression. However, the most striking pattern was exhibited by IGF-1 receptor. Our results suggest that induction of IGF-1 receptor mRNA by EGF may be an important event in the establishment of competence by EGF in long-term density arrested WI-38 cells. © 1995 Wiley-Liss, Inc.     

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.     

Regulation of ornithine decarboxylase and other cell cycle-dependent genes during senescence of IMR-90 human diploid fibroblasts

Aging of IMR-90 human diploid fibroblasts in vitro is accompanied by significant changes of polyamine metabolism, most notably, a 5-fold decrease of serum-induced activity of ornithine decarboxylase, the key enzyme in the biosynthesis of polyamines (Chen, K. Y., Chang, Z. F., and Liu, A. Y.-C. (1986) J. Cell. Physiol. 129, 142-146). In this paper, we employed Northern blot hybridization and affinity radiolabeling techniques to investigate the molecular basis of this age-associated change of ornithine decarboxylase activity. Since the induction of ornithine decarboxylase by serum is a mid-G1 event, we also examined expressions of other cell cycle-dependent genes that are induced before and after the mid-G1 phase to determine if their expressions may also be age-dependent. Our results demonstrated a 3-fold decrease of the amount of active ornithine decarboxylase molecules that can be labeled by alpha-difluoromethyl[3H]ornithine in senescent IMR-90 cells (population doubling level (PDL) = 52) as compared to young cells (PDL = 22). However, the levels and kinetics of induction of ornithine decarboxylase mRNA in both young and senescent IMR-90 cells were found to be identical throughout a 24-h time period after serum stimulation. The time course and the magnitude of the expression of c-myc, an early G1 gene, were quite similar in young and senescent IMR-90 cells and appeared to be PDL-independent. In contrast, the expression of thymidine kinase, a late G1/S gene, was significantly reduced in senescent IMR-90 cells. Levels of thymidine kinase mRNA and thymidine kinase activity in senescent IMR-90 cells were 6- and 8-fold less than those in young cells, respectively. Based on these data, we proposed that impairment of cell cycling in senescent IMR-90 cells may occur at the late G1/S phase and that decreases of ornithine decarboxylase activity and putrescine accumulation during cell senescence may contribute to this impairment.     

TGF-beta inhibits growth factor-induced DNA synthesis in hamster fibroblasts without affecting the early mitogenic events

Transforming growth factor beta (TGF-beta) was found to inhibit (IC50 = 0.1 ng/ml) alpha-thrombin or FGF-induced mitogenicity in G0-arrested Chinese hamster lung fibroblasts. Growth factor-stimulated cells became rapidly insensitive to TGF-beta addition during their progression through G0/G1 suggesting that an early step of the mitogenic response was the target of TGF-beta action. Surprisingly, none of the well characterized early mitogenic events commonly triggered by growth factors was found to be affected by TGF-beta addition. These responses included: phosphoinositide breakdown, activation of protein kinase C as determined by EGF receptor down-modulation, subsequent rises in pHi, c-fos, and c-myc mRNA levels, ribosomal protein S6 phosphorylation, the increase in RNA and protein synthesis, induction of ornithine decarboxylase. Only the induction of thymidine kinase, a marker of entry in the S phase, was found to be repressed by TGF-beta, with maximal inhibition when TGF-beta was added early in G1. These results indicate that the inhibitory action of TGF-beta does not affect the growth factors signalling pathways but touches an early event different from those so far analyzed.     

Heparin prevents vascular smooth muscle cell progression through the G1 phase of the cell cycle

To gain insight into the mechanism of the antiproliferative effects of heparin on vascular smooth muscle cells (SMC), the influence of this glycosaminoglycan on cell cycle progression and the expression of the c-fos, c-myc, and c-myb proto-oncogenes and two other growth-regulated genes was examined. SMC, synchronized by a serum-deprivation protocol, enter S phase 12-16 h after serum stimulation. Pretreatment with heparin for 48 h blocked the induction of histone H3 RNA, an S phase-expressed product, and prevented cell replication. Thus, heparin prevents entry of cells into S phase. Conversely, heparin had essentially no effect on changes in expression of the c-fos and c-myc proto-oncogenes during the G0 to G1 transition. Normal increases in c-fos and c-myc RNA were observed 30 min and 2 h following serum addition, respectively. However, the increase in expression of the mRNA of the c-myb proto-oncogene and the mitochondrial ATP/ADP carrier protein, 2F1, which begins to occur 8 h following serum addition to SMC, was completely inhibited by heparin. Two-dimensional polyacrylamide gel electrophoresis of the products of a rabbit reticulocyte cell-free translation of RNA isolated at various times confirmed this temporal assessment of the effects of heparin. These results suggest that heparin does not inhibit cell proliferation by blocking the G0 to G1 transition. Rather, heparin may affect a critical event in the mid-G1 phase of the cell cycle which is necessary for subsequent DNA synthesis.     

Cell cycle dependent growth factor regulation of gene expression

The expression of the proto-oncogenes c-fos and c-myc is a rapid response of G0-arrested fibroblasts to serum and peptide growth factors; however, the role of the c-fos and c-myc gene products in subsequent cell cycle transit is not understood. We examined the expression of c-fos and c-myc mRNA in Balb/c 3T3 murine fibroblasts in response to platelet-derived growth factor (PDGF) and platelet-poor plasma, using arrest points associated with density dependent growth inhibition or metabolic inhibition to synchronize cells in S phase of the cell cycle. The expression of c-fos and c-myc mRNA in Balb/c 3T3 cells was differentially regulated with respect to growth factor dependence and cell cycle dependence. c-fos expression was induced in the presence of PDGF and was unaffected by plasma. The induction of c-fos expression in response to PDGF was cell cycle independent, occurring in cells transiting S phase and G2 as well as in G0 arrest. In contrast, c-myc expression was both growth factor and cell cycle dependent. In G0 arrested cells, c-myc expression was PDGF-dependent and plasma-independent, and PDGF was required for maintenance of elevated c-myc levels during G1 transit. In cells transiting S phase, c-myc mRNA was induced in response to PDGF, but was also plasma-dependent in S phase cells that had been "primed" by exposure to PDGF during S phase.