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.     

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.     

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.     

Rapid induction of competence formation is PDGF-isoform specific.

Platelet-derived growth factor (PDGF) stimulates the expression of a number of genes associated with entry of quiescent Balb/c-3T3 fibroblasts into the cell cycle. We determined that two of these genes, c-myc and c-fos, are induced equivalently in medium supplemented with platelet-poor plasma (PPP) and either PDGF-BB or PDGF-AA. The rate at which fibroblasts entered S phase was also similar in PDGF-BB- and AA-treated cells as was the expression of the late G1 gene, thymidine kinase (TK). However, PDGF-AA must be present for a period of 16 h to stimulate the proliferation of 90% of the cells, whereas PDGF-BB was required for only 4 h. Exposure of cells to PDGF-AA for 4 h, a time during which maximum expression of c-fos and c-myc occurred, only induced 20% of the cells in a quiescent population to enter the cell cycle. Therefore, PDGF-AA-mediated expression of the immediate early genes c-fos and c-myc may be necessary but is not sufficient to rapidly stimulate density-arrested Balb/c-3T3 fibroblasts into the competent state. Thus, these data suggest that PDGF-AA and PDGF-BB initiate traverse of the cell cycle by distinct mechanisms.     

Variations in c-fos mRNA expression during serum induction and the synchronous cell cycle

Induction of c-fos mRNA levels associated with the stimulation of growth by fetal bovine serum following quiescence was examined in three cell types following brief (24 h) serum starvation. Starved NIH-3T3 and HeLa S3 cells experienced c-fos mRNA induction 20-30 min after addition of serum. In contrast, Swiss-3T3 cells expressed c-fos constitutively following serum starvation. The pattern of oncogene expression coincided with the level of quiescence of each cell line prior to induction. Serum inductions of c-fos expression was dependent upon the response of each cell line to serum starvation, c-fos expression was also examined in HeLa S3 cells that had been separated into sequential cell cycle phases by centrifugal elutriation, c-fos expression peaked during the earliest part of the synchronous G1 phase. The amount of c-fos mRNA measured was approximately twice that found during other cell cycle phases. This suggests that, in addition to its role during the transition from quiescence, the c-fos gene product may play a regulatory role during the earliest part of G1 phase of the continuous cell cycle.     

Role of c-myc and other genes in interleukin 2 regulated CT6 T lymphocytes and their malignant variants

The cloned murine cytotoxic T cell line CT6 solely requires interleukin 2 (IL 2) for viability and cell cycle progression. Treatment of G arrested cultures of CT6 cells with recombinant IL 2 induces the rapid sequential expression of the nuclear proto-oncogenes c-fos, c-myc, and c-myb but does not affect the expression of several cytosolic or membrane-associated proto-oncogenes. A comparison of early genes induced by growth factor treatment of quiescent NIH/3T3 fibroblasts and CT6 cells demonstrated that only c-fos and c-myc induction is shared in the two different lineages. Factor-independent lines derived from CT6 cells show no mitogenic response to IL 2, yet binding of IL 2 with its receptor in the cells was capable of inducing the expression of c-fos and c-myc. In factor-independent cell lines, c-myc was uniformly expressed at high constitutive levels, suggesting that c-myc abrogates growth factor requirements of these cells. The levels of c-myc expression in the factor-independent lines was not due to an autocrine production of IL 2 but may be a consequence of constitutively activated IL 2 receptors.     

Mechanism of induction of cellular DNA synthesis by the adenovirus E1A 12S cDNA product

The mechanism of induction of DNA synthesis in quiescent rat 3Y1 cells by the adenovirus E1A gene was investigated using the 3Y1 derivative cell lines g12-21, gn12RB1, and gn12RB2. The g12-21 cells express the E1A 12S cDNA and the latter two cells express both the E1A 12S cDNA and the human retinoblastoma susceptibility (Rb) gene at different levels in response to dexamethasone (dex). The cDNA sequences of E1A-inducible cell cycle-dependent genes, clone 3 and clone 16, were isolated by differential screening of a cDNA library constructed from dex-treated g12-21 cells. The quiescent 3Y1 cells induced c-fos and c-myc expression within 2 h after serum stimulation and expressed clone 16 and clone 3 transiently at around 8 h before the onset of DNA synthesis (10 h). In contrast, the quiescent g12-21 cells treated with dex expressed a high level of E1A at 6 to 8 h after treatment and expressed clone 16 and clone 3 at around 8 h without stimulation of c-fos and c-myc expression, suggesting that E1A bypasses the cell cycle early in G1. The half-maximal rate of DNA synthesis was reached in a much shorter time in dex-treated g12-21 cells (12 h) than in serum-treated 3Y1 cells (18 h), suggesting that E1A also bypasses the cell cycle at the G1/S boundary. The gn12RB1 and gn12RB2 cells were unable to induce DNA synthesis in response to dex presumably due to lower levels of E1A expression, although gn12RB2 but not gn12RB1 cells could express clone 16 and clone 3. These results suggest that the level of E1A required for bypass at the G1/S boundary is higher than that required early in G1.     

Effects of epidermal growth factor and platelet-derived growth factor on c-fos and c-myc mRNA levels in normal human fibroblasts

The mRNA levels of two proto-oncogenes, c-fos and c-myc, were determined in human foreskin fibroblasts exposed to epidermal growth factor (EGF) or platelet-derived growth factor (PDGF) in a serum-free, defined medium (MCDB 104). Untreated, quiescent cells were found to have low or undetectable levels of c-fos and c-myc mRNA. Within 10 min after the addition of EGF or PDGF the c-fos mRNA level increased, reached a peak at 30 min, and then declined to the control level after 60 min. The level of c-myc mRNA increased somewhat later and peaked after 8 h in cultures treated with either of the growth factors. The c-myc mRNA level remained elevated throughout the 24 h of investigation. The concentrations of EGF and PDGF required for a maximal effect on c-fos or c-myc expression were found to be similar to those that give maximal effect on cell proliferation. Both c-fos and c-myc mRNA expression were super-induced by the addition of cycloheximide. The addition of neutralizing PDGF antibodies to cultures that had received PDGF 4 h earlier inhibited the subsequent increase in the c-myc mRNA level, indicating that the effect of PDGF on c-myc expression is not caused by a "hit and run," mechanism. Density-inhibited cells responded to EGF and PDGF by an increase in c-fos and c-myc mRNA levels in the absence of any mitogenic response. The present results conform to the view that the c-fos and c-myc proto-oncogenes may be important (or necessary) but not sufficient for the initiation of DNA synthesis. Moreover, the finding that both EGF and PDGF increase c-fos and c-myc expression supports our previous suggestion that these two growth factors may in part act via a common intracellular pathway in the prereplicative phase of human fibroblasts.     

细胞松驰素B促微丝解聚对DNA合成的作用

利用微丝（ｍｉｃｒｏｆｉｌａｍｅｎｔ,ＭＦ）解聚药物细胞松驰素Ｂ（ｃｙｔｏｃｈａｌａｓｉｎＢ,ＣＢ）处理Ｇ_０期小鼠Ｃ_３Ｈ_（１０）Ｔ_（１／２）成纤维细胞,对Ｇ_０至Ｓ期ＤＮＡ合成,胸腺嘧啶核苷激酶（ｔｈｙｍｉｄｉｎｅｋｉｎａｓｅ,ＴＫ）活性、ＴＫ基因表达、钙调素（ｃａｌｍｏｄｕｌｉｎ,ＣａＭ）水平和一些细胞周期早期基因的表达进行了观察,Ｇ_０期细胞经３ｍｇ／ＬＣＢ处理２ｈ,促ＭＦ解聚增强了血清对Ｓ期细胞ＴＫ活性、ＴＫ基因表达和ＤＮＡ合成的刺激作用,并促进细胞提前进入Ｓ期．血清刺激Ｇ_０期细胞进入晚Ｇ_１期和Ｓ期时,ＣａＭ水平明显升高,而ＣＢ预处理则使ＣａＭ含量进一步增加,特别是ＣＢ处理促使Ｓ期ＣａＭ增加向核内转移．ＣＢ处理明显增强血清对ｃ－ｊｕｎ、ｃ－ｆｏｓ和ｃ－ｍｙｃ基因表达的刺激作用,而ＰＫＣ抑制剂Ｈ_７则抑制ＣＢ处理对这些基因转录的刺激作用,说明ＣＢ使Ｇ_０期细胞ＭＦ解聚刺激ｃ－ｊｕｎ、ｃ－ｆｏｓ和ｃ－ｍｙｃ的转录活性与ＰＫＣ的作用有关．结果表明Ｇ_０至Ｓ期早期ＭＦ的重组可促进细胞进入Ｓ期,增强ＤＮＡ合成．     

Dimethyl sulfoxide inhibits the expression of early growth-response genes and arrests fibroblasts at quiescence

We have previously shown that dimethyl sulfoxide (DMSO) treatment of mouse embryo fibroblasts (MEF) at the early hours of mitogenic stimuli resulted in the inhibition of DNA and protein synthesis; delayed treatment of serum-stimulated cells with DMSO had little effect on the synthesis of these macromolecules. Here, we demonstrate the specific inhibition of expression of early growth response genes by DMSO in serum-stimulated MEF. The expression of interleukin 6, and of oncogenes c-myc and c-fos were inhibited when the cells were treated with 2% DMSO from the beginning of serum-stimulated growth but not after 3 h of mitogenic stimuli. Although the actin gene is an early serum-response gene, its expression was not affected by DMSO. The synthesis of another serum-induced protein, the plasminogen activator inhibitor-1 was blocked during concurrent and delayed (after 3 h of stimulation) treatment of serum-stimulated fibroblasts with DMSO. The expression of glyceraldehyde-3-phosphate dehydrogenase gene was not affected by DMSO. These results indicate that the expression of non-growth-related genes are either not affected or affected nonspecifically both at early and late stages of serum-induced growth of mouse embryo fibroblasts. The serum-induced expression of c-fos gene was abolished by DMSO treatment of MEF while the phorbol 12-myristate 13-acetate-induced expression of fos gene was not, indicating that the PMA signaling pathway was refractory to DMSO. Treatment of cells with medium containing 2% DMSO for 24-48 h prevents them from progression into cell cycle by preventing the expression of genes involved in G0-G1 transition of quiescent cells.     

Heparin suppresses specific second messenger pathways for protooncogene expression in rat vascular smooth muscle cells.

We investigated the molecular mechanisms underlying the ability of heparin to inhibit vascular smooth muscle cell (VSMC) growth. Previous experiments have shown that heparin inhibits induction of c-fos and c-myc protooncogene mRNA in rat VSMC stimulated by phorbol 12-myristate 13-acetate (PMA) but not when stimulated by epidermal growth factor (EGF) (Pukac, L. A., Castellot, J. J., Wright, T. C., Caleb, B. L., and Karnovsky, M. J. (1990) Cell Regul. 1, 435-443). The present experiments show that these mitogens activate distinct second messenger pathways in VSMC, because PMA but not EGF induction of c-fos and c-myc mRNA was suppressed in protein kinase C (PKC) down-regulated VSMC; this suggests that EGF does not act through a PKC-dependent pathway for induction of these genes. Heparin inhibited serum stimulation of c-fos mRNA in control VSMC, but heparin did not inhibit the smaller but significant serum stimulation of c-fos mRNA in PKC down-regulated VSMC, indicating that heparin may selectively inhibit PKC-dependent, but not PKC-independent, stimulation of gene expression. To further determine if heparin inhibits non-PKC pathways, VSMC were treated with dibutyryl cAMP, 3-isobutyl-1-methyl-xanthine, and Ca2+ ionophore A23187; stimulation of c-fos mRNA by this treatment was not inhibited by heparin. DNA synthesis and cell proliferation were inhibited in rat VSMC exposed briefly to heparin during the G0/G1 phase of the cell cycle. These experiments indicate heparin can act early in the cell cycle and suggest PKC-dependent but not PKC-independent signaling pathways for gene expression are selectively sensitive to heparin inhibition.     

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.