Gene Expression in Quiescent and Senescent Fibroblasts JUDITH CAMPISI Division of Cell and Molecular Biology Lawrence Berkeley Laboratory University of California Berkeley, California 94720 Senescence: Normal differentiated cells have only a finite capacity for cell division.’V2 This phenomenon has been termed the finite life span phenotype or cellular senescence. The significance of this intrinsic limit to cell proliferation is not yet clearly understood. There are at least two views, not mutually exclusive, regarding the significance of cellular senescence. One holds that senescence, at a cellular level, reflects processes that occur during organismal aging. Another view holds that a finite proliferative life span constitutes a tumor-suppressive mechanism. Support for both points of view was recently received from cellular and molecular biologists. Whatever the case, eventual senescence appears to be an essentially universal feature of differentiated eukaryotic cells in culture and, from a limited body of data that exists, in viva as well. The cellular, biochemical, and molecular biological aspects of cellular senescence were recently r e v i e ~ e d . ” ~ Fibroblasts: Human fibroblasts are perhaps the best characterized and most convenient cell system for studying the finite proliferative life span of cells in culture. Fibroblasts are differentiated cells of mesenchymal origin whose primary function is to provide the cellular and extracellular stroma for epithelial cell layers. During development and in response to injury, fibroblasts are important for remodeling the extracellular matrix and epithelium, and they produce a number of growth factors and cytokines that recruit and activate other cell types. Quiescence: In vivo and in culture, fibroblasts can exist in either of two reversible growth states. In the absence of an anchoring substratum or adequate levels o r types of growth factors, fibroblasts enter a stable, nonproliferating growth state termed quiescence or GO. Inhibitory cytokines and certain cell-cell or cell-matrix contacts can also maintain fibroblasts in GO. In general, when quiescent fibroblasts are provided with an anchoring substratum and growth factors, the cells leave quiescence and enter a proliferating growth state. This transition is commonly referred to as the GO to G1 transition. Once cells have made this transition, they progress through the G1, S, G2, and M phases of the cell cycle. Depending on extracellular conditions, they may then progress through one or more additional cell cycles or return to GO. An important feature of senescent human fibroblasts is that they arrest growth with a G1 DNA content and cannot be stimulated to enter the S phase of the cell cycle by any known combination of physiological extracellular factors (reviewed in refs. 3-6). Thus, a fundamental difference between quiescence and senescence is the reversibility of growth arrest. In early passage cells, the transitions from quiescence to proliferation, and the reverse, can easily and synchronously be induced in culture by depriving fibroblasts of growth factors (commonly supplied as serum) for 48-72 hours and then stimulating them with fresh, serum-containing medium. For early passage human fetal lung or neonatal skin fibroblasts, it takes 1 6 2 0 hours for the average quiescent cell to 195
ANNALS NEW YORK ACADEMY OF SCIENCES
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initiate DNA synthesis in response to growth factor stimulation (reviewed in ref. 6). The temporal organization of GO to G1 transition and the subsequent phases of the cell cycle are shown by FIGURE 1. Growth-related gene expression in quiescent and senescent cells: Over the last several years, many genes have been identified whose expression rises or, less commonly, falls when quiescent fibroblasts are induced to proliferate by growth factor^.^.^ The products of many of these genes are, in fact, differentiated cell products and have little to do with the proliferative response per se. However, the products of some growth factor-inducible genes are related to the proliferative response, and these are generally referred to as growth-related genes. Some growthrelated genes are permissive for cell proliferation, others are necessary but not regulatory, and a few are thought to have regulatory functions. Growth-related genes are often induced at characteristic times during the GO to G1 transition. Some growth-related genes, and the times at which they are induced after growth factor stimulation of quiescent fibroblasts, are shown in FIGURE1.
I
I I I I
I
Rb-unphosphorylated
Rbphosphorylated
FIGURE 1. Temporal organizationof the GO to G1 transition and subsequent cell cycle phases in fibroblasts. Also shown is the timing with which some growth-related genes are induced. See text for complete explanation.
Another important feature of senescent fibroblasts is that they express many of the same genes that are expressed at early passage. Moreover, many genes, including several growth-regulatory protoonocogenes, remain growth factor-inducible in senescent cell^.^,^ These results are consistent with the general belief that growth factor signal transducing mechanisms are essentially intact in senescent cells, and that the failure to proliferate is due to the expression of one or more dominant growth inhibitors. Protooncogenes, oncogenes, and tumor suppressor genes: What types of gene might be expected to control the quiescent and senescent growth states? The best candidates are the protooncogenes and tumor suppressor genes. Protooncogenes are generally positive growth activators that act directly or indirectly to stimulate proliferation. When mutated or inappropriately expressed, they are converted to oncogenes, which stimulate inappropriate growth in a dominant fashion. Most oncogenes present in tumors or transforming retroviruses are derived from normal cell sequences. However, some oncogenic DNA viruses carry one or more oncogenes for which there is no structural cellular homolog (although there may be functional cellular counterparts).
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One such oncogene, the large T antigen of the SV-40 virus, has been studied extensively with regard to cellular senescence. SV-40 T antigen is a multifunctional, 90 kD nuclear phosphoprotein.1° It is essential for the transcription of viral genes and replication of the viral genome. It also induces host cell genes and host cell DNA replication and can immortalize and transform susceptible cells. T antigen is one of the few genes that can induce senescent human fibroblasts to enter the S phase of the cell cycle.11J2How it does so is as yet unknown. In contrast to protooncogenes and oncogenes, tumor suppressor genes are believed to be negative growth regulators (i.e., growth suppressors). Only a few such genes have been identified. Mutations that derange normal tumor suppressor gene function are generally recessive (or act in a dominant negative fashion), because both alleles must be inactivated before inappropriate growth can occur. Recently, T antigen was shown to bind to the retinoblastoma protein (Rb), the product of the retinoblastoma susceptibility gene product.I3 Rb is of particular interest in senescence. It is a -110 kD nuclear phosphoprotein that is present in a relatively underphosphorylated form in quiescent ~e1ls.l~ This is the form to which T antigen binds. In growth factor-stimulated cells, Rb becomes multiply phosphorylated as cells near the G l / S boundary. Thus, phosphorylation or binding to T antigen is believed to inactivate the growth-suppressive effects of Rb. It was recently shown that cell cycle-dependent phosphorylation of Rb fails to occur in senescent human fibroblasts.l5
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RESULTS c-fos protooncogene expression is irreversibly repressed in senescent cells: As noted earlier, several protooncogenes are expressed and growth factor-inducible in senescent human fibroblast^.^^^ These include the c-myc, c-ras-H, and c-jun protooncogenes. A striking exception was the c-fos protoon~ogene.~ c-fos codes for a 60 kD nuclear phosphoprotein that, together with the product of the c-jun protooncogene, acts as a potent transcriptional activator of a number of cellular genes.16 In fibroblasts, c-fos is not expressed in quiescent cells, but it is induced to high levels of expression within an hour after quiescent cells are stimulated by growth factors. Expression then returns to a very low level as cells progress through the G1 phase of the cell cycle. Several lines of evidence indicate that c-fos induction is essential for fibroblasts to leave GO as well as to proliferate exponentially.16 Thus, failure of senescent cells to express c-fos could at least in part explain their inability to proliferate. c-fos expression declined roughly in parallel with the ability of human fibroblasts to enter the S phase of the cell cycle over the course of their proliferative life span in culture. This decline was evident at the level of transcription, detectable by nuclear run-on assays: mRNA abundance, measured by Northern analysis: and nuclear protein, determined by indirect immunofluorescence (TABLE1). Once the cultures reached complete senescence, none of the cells could be induced to express c-fos. This was true whether the cells were stimulated with serum (TABLEI), epidermal growth factor, cholera toxin, or phorbol ester tumor promoters (data not shown). Each of these agents induces c-fos to high levels in early passage quiescent cells.17 T antigen induces c-fos in senescent cells: T antigen and similar genes are the only agents known to induce DNA synthesis in senescent cells, and T antigen most likely acts to stimulate the transcription of cellular genes. Moreover, c-fos expression is essential for entry into S phase, and it is repressed at the level of transcription in senescent cells. We therefore asked if T antigen might induce senescent cells to initiate DNA synthesis through a derepression of the c-fos protooncogene. To test this idea, we microinjected a T-antigen expression plasmid (CMV-T) into
-
ANNALS NEW YORK ACADEMY OF SCIENCES
198
quiescent early passage cells or serum-deprived senescent cells. After 16 hours, 2040% of the injected cells expressed T antigen. At that time, we fixed the cells and stained for nuclear fos protein by indirect immunofluorescence. As expected, none of the uninjected cells stained positive for fos protein. However, a few percentages of the T antigen-injected cells (2-5%) showed moderate to bright nuclear staining. This result suggests that T antigen can induce c-fos expression in at least a small fraction of both quiescent and senescent cells. We next asked if we could synchronize the response of T antigen-injected cells by stimulating them with serum. Serum-deprived cells were injected with CMV-T, allowed to express T antigen, and then stimulated with serum for 60 minutes. In uninjected cells, as expected, 50-70% of the early passage cells stained strongly for fos protein, whereas less than 5% of the senescent cells stained positive. However, in the injected cells, 3 0 4 0 % of both early passage and senescent cells expressed fos protein. Taken together, our results suggest that T antigen derepresses c-fos in senescent human fibroblasts. In both quiescent and senescent cells, it may induce c-fos directly, at least in a small fraction of cells, and in senescent cells it relieves the block to growth factor inducibility. TABLE1. c-Fos Expression during the Proliferative Life Span of Human Fibroblasts" Population Doubline Level
48-Hour Labeling Index
21 32 44 48 52
86 68 28 14
ANNALS NEW YORK ACADEMY OF SCIENCES
196
initiate DNA synthesis in response to growth factor stimulation (reviewed in ref. 6). The temporal organization of GO to G1 transition and the subsequent phases of the cell cycle are shown by FIGURE 1. Growth-related gene expression in quiescent and senescent cells: Over the last several years, many genes have been identified whose expression rises or, less commonly, falls when quiescent fibroblasts are induced to proliferate by growth factor^.^.^ The products of many of these genes are, in fact, differentiated cell products and have little to do with the proliferative response per se. However, the products of some growth factor-inducible genes are related to the proliferative response, and these are generally referred to as growth-related genes. Some growthrelated genes are permissive for cell proliferation, others are necessary but not regulatory, and a few are thought to have regulatory functions. Growth-related genes are often induced at characteristic times during the GO to G1 transition. Some growth-related genes, and the times at which they are induced after growth factor stimulation of quiescent fibroblasts, are shown in FIGURE1.
I
I I I I
I
Rb-unphosphorylated
Rbphosphorylated
FIGURE 1. Temporal organizationof the GO to G1 transition and subsequent cell cycle phases in fibroblasts. Also shown is the timing with which some growth-related genes are induced. See text for complete explanation.
Another important feature of senescent fibroblasts is that they express many of the same genes that are expressed at early passage. Moreover, many genes, including several growth-regulatory protoonocogenes, remain growth factor-inducible in senescent cell^.^,^ These results are consistent with the general belief that growth factor signal transducing mechanisms are essentially intact in senescent cells, and that the failure to proliferate is due to the expression of one or more dominant growth inhibitors. Protooncogenes, oncogenes, and tumor suppressor genes: What types of gene might be expected to control the quiescent and senescent growth states? The best candidates are the protooncogenes and tumor suppressor genes. Protooncogenes are generally positive growth activators that act directly or indirectly to stimulate proliferation. When mutated or inappropriately expressed, they are converted to oncogenes, which stimulate inappropriate growth in a dominant fashion. Most oncogenes present in tumors or transforming retroviruses are derived from normal cell sequences. However, some oncogenic DNA viruses carry one or more oncogenes for which there is no structural cellular homolog (although there may be functional cellular counterparts).
CAMPISI: GENE EXPRESSION IN FIBROBLASTS
197
One such oncogene, the large T antigen of the SV-40 virus, has been studied extensively with regard to cellular senescence. SV-40 T antigen is a multifunctional, 90 kD nuclear phosphoprotein.1° It is essential for the transcription of viral genes and replication of the viral genome. It also induces host cell genes and host cell DNA replication and can immortalize and transform susceptible cells. T antigen is one of the few genes that can induce senescent human fibroblasts to enter the S phase of the cell cycle.11J2How it does so is as yet unknown. In contrast to protooncogenes and oncogenes, tumor suppressor genes are believed to be negative growth regulators (i.e., growth suppressors). Only a few such genes have been identified. Mutations that derange normal tumor suppressor gene function are generally recessive (or act in a dominant negative fashion), because both alleles must be inactivated before inappropriate growth can occur. Recently, T antigen was shown to bind to the retinoblastoma protein (Rb), the product of the retinoblastoma susceptibility gene product.I3 Rb is of particular interest in senescence. It is a -110 kD nuclear phosphoprotein that is present in a relatively underphosphorylated form in quiescent ~e1ls.l~ This is the form to which T antigen binds. In growth factor-stimulated cells, Rb becomes multiply phosphorylated as cells near the G l / S boundary. Thus, phosphorylation or binding to T antigen is believed to inactivate the growth-suppressive effects of Rb. It was recently shown that cell cycle-dependent phosphorylation of Rb fails to occur in senescent human fibroblasts.l5
-
RESULTS c-fos protooncogene expression is irreversibly repressed in senescent cells: As noted earlier, several protooncogenes are expressed and growth factor-inducible in senescent human fibroblast^.^^^ These include the c-myc, c-ras-H, and c-jun protooncogenes. A striking exception was the c-fos protoon~ogene.~ c-fos codes for a 60 kD nuclear phosphoprotein that, together with the product of the c-jun protooncogene, acts as a potent transcriptional activator of a number of cellular genes.16 In fibroblasts, c-fos is not expressed in quiescent cells, but it is induced to high levels of expression within an hour after quiescent cells are stimulated by growth factors. Expression then returns to a very low level as cells progress through the G1 phase of the cell cycle. Several lines of evidence indicate that c-fos induction is essential for fibroblasts to leave GO as well as to proliferate exponentially.16 Thus, failure of senescent cells to express c-fos could at least in part explain their inability to proliferate. c-fos expression declined roughly in parallel with the ability of human fibroblasts to enter the S phase of the cell cycle over the course of their proliferative life span in culture. This decline was evident at the level of transcription, detectable by nuclear run-on assays: mRNA abundance, measured by Northern analysis: and nuclear protein, determined by indirect immunofluorescence (TABLE1). Once the cultures reached complete senescence, none of the cells could be induced to express c-fos. This was true whether the cells were stimulated with serum (TABLEI), epidermal growth factor, cholera toxin, or phorbol ester tumor promoters (data not shown). Each of these agents induces c-fos to high levels in early passage quiescent cells.17 T antigen induces c-fos in senescent cells: T antigen and similar genes are the only agents known to induce DNA synthesis in senescent cells, and T antigen most likely acts to stimulate the transcription of cellular genes. Moreover, c-fos expression is essential for entry into S phase, and it is repressed at the level of transcription in senescent cells. We therefore asked if T antigen might induce senescent cells to initiate DNA synthesis through a derepression of the c-fos protooncogene. To test this idea, we microinjected a T-antigen expression plasmid (CMV-T) into
-
ANNALS NEW YORK ACADEMY OF SCIENCES
198
quiescent early passage cells or serum-deprived senescent cells. After 16 hours, 2040% of the injected cells expressed T antigen. At that time, we fixed the cells and stained for nuclear fos protein by indirect immunofluorescence. As expected, none of the uninjected cells stained positive for fos protein. However, a few percentages of the T antigen-injected cells (2-5%) showed moderate to bright nuclear staining. This result suggests that T antigen can induce c-fos expression in at least a small fraction of both quiescent and senescent cells. We next asked if we could synchronize the response of T antigen-injected cells by stimulating them with serum. Serum-deprived cells were injected with CMV-T, allowed to express T antigen, and then stimulated with serum for 60 minutes. In uninjected cells, as expected, 50-70% of the early passage cells stained strongly for fos protein, whereas less than 5% of the senescent cells stained positive. However, in the injected cells, 3 0 4 0 % of both early passage and senescent cells expressed fos protein. Taken together, our results suggest that T antigen derepresses c-fos in senescent human fibroblasts. In both quiescent and senescent cells, it may induce c-fos directly, at least in a small fraction of cells, and in senescent cells it relieves the block to growth factor inducibility. TABLE1. c-Fos Expression during the Proliferative Life Span of Human Fibroblasts" Population Doubline Level
48-Hour Labeling Index
21 32 44 48 52
86 68 28 14
