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Biomedical subjects

J Campisi

Publications and source records attributed to J Campisi.

At least 19 recordsLinked to original sources
PubMed1995

Regulation of c-fos expression in senescing Werner syndrome fibroblasts differs from that observed in senescing fibroblasts from normal donors.

The Werner syndrome (WS) is a segmental progeroid syndrome caused by a recessive mutation (WRN) mapped to 8p12. The replicative life spans of somatic cells cultured from WS patients are substantially reduced compared to age-matched controls. Certain molecular concomitants of the replicative decline of normal fibroblast cultures have recently been defined, and it appears that multiple changes in gene expression accompany normal cell senescence. If the mechanisms by which WS cells exit the cell cycle were entirely comparable, the molecular markers of senescence should be identical in normal and WS cells. We find that this is not the case. The constitutive expression of statin, a nuclear protein associated with the nonproliferating state, was comparably expressed in normal and WS senescent cells. Likewise, the steady state levels of p53, a protein known to be involved in the G1 checkpoint of the cell cycle, were similar in early-passage fibroblasts from normal and WS subjects. The levels of p53 were not increased in senescent fibroblasts, whether derived from normal or WS subjects. By contrast, the inducibility of mRNA and protein expression of the c-fos protooncogene is preserved in late-passage WS cells. This is in contrast to what is observed in late-passage fibroblasts from normal subjects. Additional genotypes will have to be examined, however, to determine the specificity of this new aspect of the WS phenotype.

Adult↗
PubMed1995

Suppression of mammary epithelial cell differentiation by the helix-loop-helix protein Id-1.

Cell proliferation and differentiation are precisely coordinated during the development and maturation of the mammary gland, and this balance invariably is disrupted during carcinogenesis. Little is known about the cell-specific transcription factors that regulate these processes in the mammary gland. The mouse mammary epithelial cell line SCp2 grows well under standard culture conditions but arrests growth, forms alveolus-like structures, and expresses beta-casein, a differentiation marker, 4 to 5 days after exposure to basement membrane and lactogenic hormones (differentiation signals). We show that this differentiation entails a marked decline in the expression of Id-1, a helix-loop-helix (HLH) protein that inactivates basic HLH transcription factors in other cell types. SCp2 cells stably transfected with an Id-1 expression vector grew more rapidly than control cells under standard conditions, but in response to differentiation signals, they arrested growth and formed three-dimensional structures similar to those of control cells. Id-1-expressing cells did not, however, express beta-casein. Moreover, 8 to 10 days after receiving differentiation signals, they lost three-dimensional organization, invaded the basement membrane, and then resumed growth. SCp2 cells expressing an Id-1 antisense vector grew more slowly than controls; in response to differentiation signals, they remained stably growth arrested and fully differentiated, as did control cells. We suggest that Id-1 renders cells refractory to differentiation signals and receptive to growth signals by inactivating one or more basic HLH proteins that coordinate growth and differentiation in the mammary epithelium.

Animals↗
PubMed1992

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↗
PubMed1991

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↗
PubMed1991

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↗
PubMed1991

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↗
PubMed1990

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↗
PubMed1990

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↗
PubMed1990

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↗
PubMed1990

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↗
PubMed1989

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↗
PubMed1989

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↗
PubMed1989

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↗