Gene expression in quiescent and senescent fibroblasts.
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Antigens, Polyomavirus Transforming↗
Biomedical subjects
J Campisi
Publications and source records attributed to J Campisi.
Ras proteins are essential and selective for the action of insulin-like growth factor 1 late in the G1 phase of the cell cycle in BALB/c murine fibroblasts.
BALB/c 3T3 cells (A31 cells) require the sequential action of growth factors in order to proliferate from a quiescent growth state. Insulin-like growth factor I (IGF-I) is needed late in the G1 phase of the cell cycle, a time at which expression of the c-Ha-ras protooncogene is near maximal. An anti-ras antibody, introduced by microinjection, specifically blocked the ability of IGF-I to stimulate initiation of DNA synthesis. The antibody was specific for IGF-I; it failed to block serum, platelet-derived growth factor, or epidermal growth factor from inducing c-fos mRNA. By contrast, an anti-G alpha-subunit antibody had no effect on IGF-I-stimulated DNA synthesis but inhibited the induction of c-fos mRNA by platelet-derived growth factor or epidermal growth factor. BPA31 cells are tumorigenic A31-derived cells that progress through G1 in the absence of IGF-I. BPA31 cells produced an autocrine IGF-I that was responsible for the loss of late G1 control; the anti-ras antibody arrested the growth of these cells in late G1. The results suggest that ras proteins are essential for an IGF-I-sensitive, G1 control point.
Animals↗
Control of cell proliferation in senescent cells.
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Animals↗
Cellular senescence: a reflection of normal growth control, differentiation, or aging?
Normal cells, with few exceptions, cannot proliferate indefinitely. Cell populations--in vivo and in culture--generally undergo only a limited number of doublings before proliferation invariably and irreversibly ceases. This process has been termed the finite lifespan phenotype or cellular senescence. There is long-standing, albeit indirect, evidence that cellular senescence plays an important role in complex biological processes as diverse as normal growth control, differentiation, development, aging, and tumorigenesis. In recent years, it has been possible to develop a molecular framework for understanding some of the fundamental features of cellular senescence. This framework derives primarily from the physiology, genetics, and molecular biology of cells undergoing senescence in culture. Our understanding of senescence, and the mechanisms that control it, is still in its infancy. Nonetheless, recent data raise some intriguing possibilities regarding potential molecular bases for the links between senescence in culture and normal and abnormal growth control, differentiation, and aging.
Adult↗
Cellular aging and senescence.
Differentiated eukaryotic cells have only a finite capacity for cell division. This limitation is thought to be a cellular manifestation of organismal aging, and a restraint to tumor progression. The molecular basis for cellular senescence is not known, but a molecular framework for understanding this phenomenon has recently been established.
Animals↗
Fundamentals of cell proliferation: control of the cell cycle.
Cell proliferation in higher eukaryotes is controlled by the extracellular environment and the state of differentiation. Many cells exist in a nondividing growth state termed quiescence. Some quiescent cells cannot proliferate and are said to be terminally differentiated. Others can be stimulated to divide in response to environmental signals or when cell replacement is needed. Finally, some cells undergo continual proliferation and differentiation. Growth regulatory factors generally act at specific stages of the cell cycle, most commonly during the first gap phase of the cell cycle. Once cells initiate DNA synthesis, they are generally committed to complete DNA replication. After DNA synthesis, additional signals determine whether cells in the last gap phase proceed through mitosis. In recent years, genes that appear to be critical for progression through the first two gap phases have been identified. Many are proto-oncogenes and therefore can neoplastically transform certain cells when mutated or inappropriately expressed. Growth factors that stimulate proliferation induce the expression of several proto-oncogenes; growth inhibitory factors often suppress proto-oncogene expression. As cells differentiate, the response to extracellular factors changes. In many cases, this may be due to intracellular controls that alter the response of certain proto-oncogenes to external signals.
Animals↗
Posttranslational control of cyclic AMP-dependent protein kinase by phorbol ester in normal but not in chemically transformed 3T3 cells.
Protein kinase C (PKC) and cyclic AMP-dependent protein kinase (PKA) are important for normal cell proliferation. We show that both kinases are down-regulated by a phorbol ester tumor promoter in nontumorigenic murine BALB/c 3T3 fibroblasts (A31 cells), whereas only PKC responded to the phorbol ester in a chemically transformed derivative cell line (DMBA31 cells). In quiescent A31 cells, phorbol dibutyrate (PDBu) caused a 10-fold reduction in PKC activity and a 5-fold reduction in PKA activity. In contrast, PDBu depleted quiescent DMBA31 cells of PKC activity only and had no effect on the PKA activity. In both cell lines, PDBu did not affect the level of PKA regulatory subunits (determined by a cyclic [3H]-AMP binding assay), the levels of mRNA encoding the catalytic and the abundant regulatory subunit (determined by Northern blotting), or the level of the catalytic subunit protein (determined by Western blotting). An in situ gel activity assay confirmed that PDBu reduced the kinase activity of native PKA complexes from A31 cells but not from DMBA31 cells. These results indicate that phorbol esters down-regulate PKA activity by a posttranslational mechanism. They further suggest that the activity of PKC and PKA may be coordinately regulated in nontumorigenic cells and that transformation can disrupt this coordinate regulation.
Animals↗
Repression of c-fos transcription and an altered genetic program in senescent human fibroblasts.
Normal cells in culture invariably undergo senescence, whereby they cease proliferation after a finite number of doublings. Irreversible changes in gene expression occurred in senescent human fetal lung fibroblasts: a non-cell cycle-regulated mRNA was partially repressed; an unusual polyadenylated histone mRNA was expressed; although serum induced c-H-ras, c-myc, and ornithine decarboxylase mRNA normally, ornithine decarboxylase activity was deficient; and serum did not induce mRNA for a replication-dependent histone and for the c-fos proto-oncogene. The loss of c-fos inducibility was the result of a specific, transcriptional block. The results suggest that senescent fibroblasts were unable to proliferate because of, at least in part, selective repression of c-fos; moreover, the multiple changes in gene expression support the view that cellular senescence is a process of terminal differentiation.
Blood↗
Constitutive expression of growth-related mRNAs in proliferating and nonproliferating lung epithelial cells in primary culture: evidence for growth-dependent translational control.
We describe the control of proliferation and growth-related gene expression in primary cultures of epithelial cells derived from rat lung. Type 2 epithelial cells line the gas-exchange surface of the alveoli where they produce and secrete surfactant. When isolated from adult animals, type 2 cells do not proliferate in culture, although they have a limited ability to do so in vivo. We show that type 2 cells isolated from neonatal rats proliferate in culture and that growth can be reversibly arrested by withdrawing serum from the medium. We studied the expression of five genes whose mRNA levels fluctuate with the state of proliferation in several cell systems: the c-myc and c-Ha-ras protooncogenes and the genes encoding actin, ornithine decarboxylase (L-ornithine carboxy-lyase, EC 4.1.1.17), and histone 3.2. All five mRNAs were constitutively expressed at identical levels in proliferating and nonproliferating (serum deprived) neonatal cells and in adult cells. Thus, at the level of mRNA abundance, the expression of these five genes was uncoupled from the growth state of the cells. By contrast, synthesis of the replication-dependent histones and the activity of ornithine decarboxylase were detectable only in proliferating neonatal cells and not in serum-deprived neonatal cells or in adult cells. The results suggest that, in type 2 cells, growth factors might regulate the translation, rather than the mRNA abundance, of at least some growth-related genes and that the ability to respond to this translational control may be developmentally regulated.
Aging↗
Posttranscriptional changes in growth factor-inducible gene regulation caused by antiproliferative interferons.
Growth factors stimulate quiescent fibroblasts to progress through G0/G1, in part by inducing the expression of genes whose products are necessary or permissive for cell proliferation. Interferons, by contrast, inhibit progress through G0/G1 by mechanisms that are poorly understood. We show, in BALB/c murine 3T3 fibroblasts (A31 cells), that alpha/beta-interferon (IFN) had no effect the growth factor-dependent induction of several messenger ribonucleic acids (mRNAs), including those encoding ornithine decarboxylase (odc), fibronectin and the c-fos and c-myc protooncogenes. However, IFN caused an abnormal accumulation of fibronectin and c-myc mRNA on polysomes and markedly increased the stability of c-myc mRNA. Moreover, despite high, induced levels of mRNA, IFN inhibited the serum-stimulated rise in odc enzyme activity and the increased rate of fibronectin protein synthesis. By contrast, IFN had no effect on c-fos protein synthesis, nor did it affect the synthesis of most, but not all, proteins detectable by two-dimensional gel electrophoresis. The data suggest IFN inhibits proliferation by suppressing the expression of a subset of growth factor-inducible genes through a selective, posttranscriptional mechanism.
Animals↗
Growth-factor-inducible gene expression in senescent human fibroblasts.
Human diploid fibroblasts undergo only a finite number of population doublings in culture. At the end of their life span in culture, human fibroblasts enter an irreversible quiescent state, a process termed cellular senescence. Senescent cells fail to proliferate despite an adequate supply of growth factors in the medium and no apparent change in the number or binding properties of cellular growth factor receptors. In proliferating fibroblast cultures, growth factors have been shown to induce the expression of several genes (growth-related genes). In this report, we review some of our findings on the expression of growth-related genes in senescent cells. We find that the mRNAs for some growth-related genes are not induced by growth factors in senescent cells. By contrast, the mRNAs for other such genes actually increase after human fibroblasts have undergone senescence, although these mRNAs are not necessarily translated efficiently. Our results indicate that multiple changes in gene expression occur during cellular senescence and suggest that the failure to proliferate is a consequence of a more complex change in cellular phenotype, akin to the cessation of proliferation that accompanies terminal differentiation.
Cell Survival↗
Heparin suppresses the induction of c-fos and c-myc mRNA in murine fibroblasts by selective inhibition of a protein kinase C-dependent pathway.
Heparin is a complex glycosaminoglycan that inhibits the proliferation of several cell types in culture and in vivo. To begin to define the mechanism(s) by which heparin exerts its antiproliferative effects, we asked whether heparin interferes with the expression of the growth factor-inducible protooncogenes c-fos and c-myc. We show that heparin suppressed the induction of c-fos and c-myc mRNA by serum in murine (BALB/c) 3T3 fibroblasts. Using purified mitogens, we further show that suppression was most marked when protooncogene expression was induced by phorbol 12-myristate 13-acetate, an activator of protein kinase C. By contrast, there was little or no suppression when the cells were stimulated by epidermal growth factor, which, in these cells, utilizes a protein kinase C-independent pathway for the induction of gene expression. Heparin also inhibited the change in cell morphology induced by the phorbol ester but had no effect on the morphological change induced by epidermal growth factor and agents that raise intracellular cAMP. Heparin did not inhibit intracellular protein kinase C activity, phorbol ester-induced down-regulation of protein kinase C, or phosphorylation of the 80-kDa intracellular protein kinase C substrate. These results suggest that heparin inhibits a protein kinase C-dependent pathway for cell proliferation and suppresses the induction of c-fos and c-myc mRNA at a site distal to activation of the kinase.
Animals↗
c-ras-Ha gene expression is regulated by insulin or insulinlike growth factor and by epidermal growth factor in murine fibroblasts.
Although much is known about the structure of ras-encoded proteins, little is known about how expression is regulated. In serum-stimulated murine fibroblasts, c-ras-Ha mRNA levels fluctuated with the growth state but not with the position in the cell cycle. Two types of growth factors regulated c-ras-Ha expression: insulin (IN) or insulinlike growth factor I, each apparently acting through its cognate receptor, and epidermal growth factor (EGF). In quiescent cells, IN or insulinlike growth factor I induced c-ras-Ha mRNA three- to fivefold within 4 h, but thereafter the mRNA declined. By contrast, EGF had little effect in 4 h but induced the mRNA after 4 to 6 h. When quiescent cells were given serum or IN and EGF simultaneously, c-ras-Ha mRNA rose steadily, beginning 1 to 2 h after stimulation, and reached a stable five- to sevenfold elevation in 16 h. Thus, c-ras-Ha gene expression was sequentially regulated by two growth factors, one of which (IN) does not induce expression of other growth-regulated protooncogenes. A transformed derivative cell line that does not require IN for G1 progression has lost early IN-dependent but not late serum-dependent regulation. The results support the possibility that c-ras-Ha and IN action are functionally linked.
Animals↗
Deregulation of the c-myc and N-myc genes in transformed cells.
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Animals↗
Two independent growth factor-generated signals regulate c-fos and c-myc mRNA levels in Swiss 3T3 cells.
Polypeptide growth factors that stimulate cell proliferation bind to cell surface receptors and activate intracellular signal transduction pathways. One major signalling pathway, initiated by phosphatidylinositol (PI) turnover, involves activation of protein kinase C. Some polypeptide growth factors, including mitogens that activate protein kinase C, induce a rapid increase in expression of the proto-oncogenes, c-myc and c-fos. In order to characterize the signal transduction pathways responsible for proto-oncogene activation, we treated Swiss 3T3 cells with the tumor promoter phorbol dibutyrate to generate cells deficient in protein kinase C. These cells were then stimulated with platelet extract, bombesin, or epidermal growth factor (EGF) and the levels of c-myc and c-fos mRNA were determined. Platelet extract or bombesin, which stimulate PI turnover, were substantially weaker inducers of c-myc and c-fos mRNA levels in the protein kinase C-depleted cells, although some variability with platelet extract was noted. EGF, which does not stimulate PI turnover in several cell systems, was by contrast a potent inducer of both proto-oncogenes whether or not the cells were deficient in protein kinase C. Pretreatment of cells with phorbol dibutyrate caused little or no change in the basal levels of c-myc or c-fos mRNA, but led to a small but significant increase in basal levels of ornithine decarboxylase mRNA. These results demonstrate that EGF and growth factors that activate PI turnover induce expression of the c-myc and c-fos proto-oncogenes through different pathways.
Animals↗
Alternative modes of c-myc regulation in growth factor-stimulated and differentiating cells.
We have analysed the regulation of c-myc expression in murine fibroblasts and F9 teratocarcinoma cells. The initiation of c-myc transcription is induced to similar levels after serum stimulation of confluent and subconfluent Balb/c A31 fibroblasts while intragenic pausing within the gene's first exon remains unaffected. Sense c-myc transcription continues unabated for at least 18 hours in subconfluent cells, whereas in confluent cells it rapidly falls to pre-induced levels. Cytoplasmic c-myc mRNAs accumulate within 1-2 hours of serum addition to subconfluent cells and reach a higher level than expected from the degree of induction of sense transcription. However, c-myc mRNA levels fall close to pre-induced levels by 18 hours demonstrating that c-myc expression is initially subject to strong positive and then eventually strong negative post-transcriptional control. Anti-sense transcription within the c-myc locus was found to be constitutive under all these physiological states, thereby demonstrating that c-myc transcriptional control is strand specific. Epidermal growth factor stimulates c-myc transcription in a way different from that of serum: (1) initiation of transcription is not significantly enhanced, but intragenic pausing is significantly abrogated; and (2) post-transcriptional mechanisms do not enhance the degree of c-myc mRNA accumulation. In contrast to our results in fibroblastic cells, differentiating F9 teratocarcinoma cells down-regulate c-myc expression entirely at the post-transcriptional level. Our findings indicate that different cell types preferentially employ different modes of myc control depending on their physiological status.
Animals↗
Regulation of c-myc transcription and mRNA abundance by serum growth factors and cell contact.
We describe effects of serum insufficiency and cell contact on the transcription and abundance of the c-myc proto-oncogene mRNA in BALB/c 3T3 fibroblasts. In exponentially growing cells, withdrawal of serum caused a 10-fold decline in c-myc mRNA within 90 min. At least part of this decline was due to a decrease in the level of myc gene transcription. These cells became quiescent at subconfluence after 36-40 h. Cells made quiescent at subconfluence or confluence contained low levels of c-myc mRNA which rose more than 20-fold 2 h after stimulation of growth by fresh serum. Thereafter, the mRNA level declined. In subconfluent cells, it declined to the level in exponentially growing cells, i.e. nearly 10-fold over the level in quiescent cells. In confluent cells, by contrast, the mRNA returned to near-quiescent levels within 18 h (by mid-S phase). However, c-myc gene transcription was regulated identically in subconfluent and confluent cultures; quiescent cells transcribed c-myc at detectable levels, and stimulation by serum caused a 5-fold increase in 1 h, followed by a decline to about 2-fold over the quiescent level within 18 h. Thus, confluence affected steady state mRNA levels without affecting the level of transcription. Our results suggest that extracellular conditions that modulate cell proliferation (serum and cell contact) exert strong and rapid control over c-myc mRNA by post-transcriptional and transcriptional mechanisms.
Actins↗
Activation of proto-oncogene expression by growth regulatory signals.
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Animals↗