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PubMed2001

Role of p14(ARF) in replicative and induced senescence of human fibroblasts.

Following a proliferative phase of variable duration, most normal somatic cells enter a growth arrest state known as replicative senescence. In addition to telomere shortening, a variety of environmental insults and signaling imbalances can elicit phenotypes closely resembling senescence. We used p53(-/-) and p21(-/-) human fibroblast cell strains constructed by gene targeting to investigate the involvement of the Arf-Mdm2-p53-p21 pathway in natural as well as premature senescence states. We propose that in cell types that upregulate p21 during replicative exhaustion, such as normal human fibroblasts, p53, p21, and Rb act sequentially and constitute the major pathway for establishing growth arrest and that the telomere-initiated signal enters this pathway at the level of p53. Our results also revealed a number of significant differences between human and rodent fibroblasts in the regulation of senescence pathways.

Aging↗

PubMed1994

Expression of terminin, a senescence-related cytoplasmic protein, in the aging rat brain.

Terminin is a cytoplasmic protein expressed in irreversibly growth-arrested senescent fibroblast cultures and in terminally differentiated cells of various epithelia. In the present study, terminin was identified by immunohistochemistry in the cytoplasm of neurons and glia of aging rat brain using the monoclonal antibody (Mab) 1.2. Few terminin-positive neurons were observed in 3-month-old brain. At 18 months, terminin immunoreactivity was noted in dentate gyrus granule cells, in hippocampal fibre projections and in neuronal perikarya of deep cerebellar nuclei (but not in cerebellar cortex). In 33-month brain, terminin immunoreactivity in the dentate gyrus was more intense than at 18 months but immunoreactive fibre bundles in the hippocampus were no longer seen. At 33 months, the cerebellar granule cell layer contained terminin-positive horizontal interneurons and received immunoreactive axonal projections not seen in the younger preparations. In addition, ubiquitous low-level terminin expression was noted in neurons of the cerebral cortex, hippocampus and cerebellum of the 33-month-old animals. Thus, in the rat CNS, an increase in terminin appears to be a physiologic marker of neuronal aging. Small numbers of terminin-positive neuroglia were present in gray and white matter of 3-month-old brain and became increasingly more abundant in the 18- and 33-month-old animals. However, even in senescent brains, terminin-positive neuroglia represented a very small fraction of the entire glial pool. Terminin expression in slowly renewing neuroglial populations may identify those cells in which degeneration and death are imminent.(ABSTRACT TRUNCATED AT 250 WORDS)

Aging↗

PubMed1992

Loss of transcription factor AP-1 DNA binding activity during lymphocyte aging in vivo.

The main feature of cellular senescence is cessation of cell proliferation. Protooncogene c-fos, which is required for the cell to enter into DNA synthesis, is repressed in senescent fibroblasts. Diminished expression of c-fos and impaired formation of AP-1, which is a complex of c-Fos and c-Jun proteins acting as a transcription factor, was found in lymphocytes derived from old (> 18 months) mice and stimulated with Con A. There were no differences in c-jun expression and formation of other transcription factors (AP-2 and AP-3) between lymphocytes isolated from old and young mice.

Animals↗

PubMed2005

Inactivation of imprinted genes induced by cellular stress and tumorigenesis.

Cellular proliferation under stressful conditions may result in permanent genetic and epigenetic changes. Using primary mouse embryonic fibroblasts, we have completed a screening test to identify gene expression changes triggered when cells proliferate under stress. In this manner, we have discovered a novel phenomenon that consists of the rapid and coordinated silencing of genes subject to imprinting, including Cdkn1c, Igf2, H19, Ndn1, Grb10, and Meg3. This generalized silencing of imprinted genes is independent of the stress-responsive tumor suppressors p53, p19(Arf), and p16(Ink4a), and it is also independent of the oxidative culture conditions and the stress response known as "mouse embryonic fibroblast senescence". In the case of Cdkn1c and H19, their silencing is associated with unscheduled de novo methylation of the normally expressed allele at their corresponding CpG island promoters, thus resulting in biallelic methylation. Finally, we provide evidence for frequent de novo methylation of Cdkn1c in a variety of murine cancer types. Altogether, our data support the concept that silencing of imprinted genes, including methylation of Cdkn1c, constitutes an epigenetic signature of cellular stress and tumorigenesis.

Animals↗

PubMed2000

Causes and effects of the chronic inflammation in venous leg ulcers.

The pathogenesis of venous leg ulcers is multifactorial. In this review article new physiological, molecular and cellular abnormalities in venous ulcers related to the chronic inflammation are presented and discussed. Venous hypertension causes disturbed microcirculation and pathological changes of the capillaries, which eventually locks the condition in a self-amplifying, detrimental cascade with persistent elevated levels and activities of pro-inflammatory cytokines and proteases preventing progress into a healing phase. As a consequence fibroblasts senescence and become less responsive to growth factors the older the ulcers become. Current data imply there is no deficiency but rather an unfavorable distribution of growth factors in venous ulcers. An imbalance in proteolytic enzymes and their endogenous inhibitors is a common finding in chronic venous leg ulcers. Variation in disease severity and concomitant ailments in this heterogeneous patient group may explain the contradictory results in the literature. Thus, to advance the areas of research further, longitudinal studies involving larger number of patients are required to identify the major pathogenic factors.

Chronic Disease↗

PubMed1982

Clonal selection in cultured human fibroblasts: role of protein synthetic errors.

Protein synthetic error frequency, determined in cell-free extracts as delta leu/delta phe incorporation following poly(U) stimulation, has been found to decrease progressively in several strains of human diploid fibroblasts during their limited replicative lifespan. To explore the basis of this phenomenon, we followed a mass (uncloned) culture of one normal strain at 13 stages of its replicative lifespan. We found a progressive tenfold decline in error frequency that was inversely correlated with passage level (r = -.93, p less than .001). This could not be ascribed to the slow rates of replication associated with fibroblast senescence because slowing of growth by serum deprivation did not change error frequency. Additionally, terminal mass cultures maintained for 16 wk at saturation density to minimize cell selection did not change error frequency over this time. Error frequencies in 12 individual clones purified from the parental culture did not decline on repeated passage, either remaining constant or, in two clones, rising abruptly three- to five-fold after initial assays. Error frequencies of clones showed a weak inverse correlation with growth vigor but not with the maximum doubling number. We conclude that selective pressures favor more vigorously dividing clones with low protein synthetic error frequencies leading to their predominance in mass cultures.

Blood↗

PubMed1985

Autoradiographic detection of diphtheria toxin resistant mutants in human diploid fibroblasts.

An autoradiographic procedure for the detection of diphtheria toxin (DT) resistant (DipR) mutants in human diploid fibroblast (HDF) cells has been developed. The assay is based on the observation that when HDFs from confluent cultures are seeded in medium containing 0.01 flocculating units/ml or higher concentration of DT, protein synthesis in sensitive cells is severely inhibited by 4-6 hr. If at this or later time, a radiolabeled protein precursor (eg, 3H-leucine) is added to the culture, it is almost exclusively incorporated into the resistant cells, which are then readily identified by autoradiography. The DipR cells can also be identified by labeling in the presence of 3H-thymidine, although a higher background is observed in these experiments. Reconstruction experiments using DipS and DipR HDFs show that the frequency of heavily labeled cells that are detected by autoradiography show an excellent correlation with the number of DipR cells added and to the number of DipR cells as detected by conventional colony forming assay. These studies provide strong evidence that the labeled cells identified by autoradiography are bona fide DipR mutants. The detection of DipR cells by autoradiography is apparently not affected by the presence of the sensitive cells in the mixtures. The spontaneous frequency of DipR cells in HDFs has been found to be in the range of 1-5 X 10(-6), and this increases in a dose dependent manner upon treatment with the mutagen ethyl methanesulfonate. These results indicate that the autoradiographic assay could be used for quantitative mutagenesis. Since the autoradiographic assay does not depend on cell division, it may prove useful in estimating the incidence of pre-existing mutations in cell populations that either do not divide or have very limited growth potential (eg, lymphocytes, muscle cells, neurons, senescent fibroblasts, etc).

Autoradiography↗

PubMed2000

Regulation of the quiescence-induced genes: quiescin Q6, decorin, and ribosomal protein S29.

The transition from growth to quiescence is deeply deranged in cancer cells. Expression of the quiescence-induced genes, quiescin Q6, decorin, and S29, was examined in important physiological states and in several cell types. Senescent fibroblasts expressed neither Q6 nor decorin mRNAs. The quiescins were induced in serum-deprived cultures. Trypsinized cells, which rapidly reattached to the culture dish, expressed Q6, S29, and decorin mRNAs at reduced levels, compared to those that remained in suspension. Expression of Q6 and S29 mRNAs in endothelial cells was low in growth phase and high in quiescent cells. Q6 and S29 mRNAs were found in a large variety of human tissues. The quiescin Q6 protein was detected in WI38 cell extracts and in conditioned medium from quiescent cells. A complex regulation of the quiescins by growth and attachment status in specific cell types may be of importance in pathological growth regulation and the development of cancer.

Amino Acid Sequence↗

PubMed2005

G1P3, an interferon inducible gene 6-16, is expressed in gastric cancers and inhibits mitochondrial-mediated apoptosis in gastric cancer cell line TMK-1 cell.

Expression of an interferon inducible gene 6-16, G1P3, increases not only in type I interferon-treated cells but also in human senescent fibroblasts. However, the function of 6-16 protein is unknown. Here we report that 6-16 is 34 kDa glycosylated protein and localized at mitochondria. Interestingly, 6-16 is expressed at high levels in gastric cancer cell lines and tissues. One of exceptional gastric cancer cell line, TMK-1, which do not express detectable 6-16, is sensitive to apoptosis induced by cycloheximide (CHX), 5-fluorouracil (5-FU) and serum-deprivation. Ectopic expression of 6-16 gene restored the induction of apoptosis and inhibited caspase-3 activity in TMK-1 cells. Thus 6-16 protein has anti-apoptotic function through inhibiting caspas-3. This anti-apoptotic function is expressed through inhibition of the depolarization of mitochondrial membrane potential and release of cytochrome c. By two-hybrid screening, we found that 6-16 protein interacts with calcium and integrin binding protein, CIB/KIP/Calmyrin (CIB), which interacts with presenilin 2, a protein involved in Alzheimer's disease. These protein interactions possibly play a pivotal role in the regulation of apoptosis, for which further detailed analyses are need. These results overall indicate that 6-16 protein may have function as a cell survival protein by inhibiting mitochondrial-mediated apoptosis.

Apoptosis↗

PubMed1986

Disappearance of statin, a protein marker for non-proliferating and senescent cells, following serum-stimulated cell cycle entry.

Statin, a protein of 57,000 D, is present in the nuclei of quiescent or senescent fibroblasts (Wang, E, J cell biol 100 (1985) 545), but is absent in their young replicating counterparts. Immunohistochemical survey of a variety of tissues demonstrates that the presence of statin is a marker for cells that are no longer involved in proliferation, i.e. those cells that are terminally differentiated. Statin expression was examined by immunofluorescence microscopy in serum-starved cultures whose replication had been reinitiated by raising the serum concentration from 0.5 to 10%. Prior to serum addition, more than 85% of the cells stained positively for statin. After stimulation with serum, the expression of statin disappeared rapidly within the first 12-14 h. On the other hand, an increase in the level of DNA synthesis, signifying entry into S phase, was observed initially at 18 h after serum stimulation, and reached maximal levels 6 h later. Immunoprecipitation of statin derived from cells harvested at different intervals after serum stimulation revealed that the level of statin synthesis was reduced by 4 h and was hardly detectable at 8 h. These results demonstrate that the synthesis of statin occurs primarily when cells are in a quiescent state, and declines rapidly when cells are induced to proliferate; this decline precedes the transition from G1 to S phase.

Cell Cycle Proteins↗

PubMed2000

Human sphingosine kinase: molecular cloning, functional characterization and tissue distribution.

Sphingosine-1-phosphate (SPP), the product of sphingosine kinase, is an important signaling molecule with intra- and extracellular functions. The cDNA for the mouse sphingosine kinase has recently been reported. In this paper we describe the cloning, expression and characterization of the human sphingosine kinase (huSPHK1). Sequence analysis comparison revealed that this kinase is evolutionarily very conserved, having a high degree of homology with the murine enzyme, and presenting several conserved regions with bacteria, yeast, plant, and mammalian proteins. Expressed huSPHK1 cDNA specifically phosphorylates D-erythro-sphingosine and, to a lesser extent, D, L-erythro-dihydrosphingosine, and not at all the 'threo' isoforms of dihydrosphingosine; hydroxy-ceramide or non-hydroxy-ceramide; diacylglycerol (DAG); phosphatidylinositol (PI); phosphatidylinositol-4-phosphate (PIP); or phosphatidylinositol-4, 5-bisphosphate (PIP(2)). huSPHK1 shows typical Michaelis-Menten kinetics (V(max)=56microM and K(m)=5microM). The kinase is inhibited by D,L-threo-dihydrosphingosine (K(i)=3microM), and by N, N-dimethyl-sphingosine (K(i)=5microM). Northern blots indicate highest expression in adult lung and spleen, followed by peripheral blood leukocyte, thymus and kidney, respectively. It is also expressed in brain and heart. In addition, database searches with the stSG2854 sequence indicate that huSPHK1 is also expressed in endothelial cells, retinal pigment epithelium, and senescent fibroblasts.

Amino Acid Sequence↗

PubMedDate not supplied

Proteolyzed matrix as a template for the regulation of tumor progression.

Pericellular proteolysis plays a pivotal function in cell invasion, a hallmark of tumor growth and metastasis. The minidegradome constituted of two matrix metalloproteinases (MMP), i.e. MMP-2 and MT1-MMP, associated with tissue inhibitor of metalloprotease-2 (TIMP-2) and integrin (alpha(v)beta(3)) or CD(44), is mainly involved in such invasive program. It catalyzes matrix degradation but, alternatively, proteolytic exposure of matricryptic sites or matrikines liberation by those enzymes regulates either positively or negatively tumor cell migration. That applies to types I and IV collagens, elastin, laminin 5, as described here, but such phenomenon might be extended to other matrix macromolecules. The development of tumors from epithelium origin is related to aging. Senescent fibroblasts are characterized by increased expression of MMPs, (particularly collagenase-1 (MMP-1) and stromelysin-1 (MMP-3)) and deposited matrix by those aged cells was shown to favor cancer cell growth. Thus, compositional variation of matrix-surrounding tumor cells, with formation of matricryptic sites and matrikines, can be considered as one main epigenetic factor contributing to tumor progression. A matrix-directed pharmacological approach in cancer is now emerging.

Animals↗

PubMed2003

Genetic ablation of the tumor suppressor menin causes lethality at mid-gestation with defects in multiple organs.

Patients suffering from multiple endocrine neoplasia type 1 (MEN1) are predisposed to multiple endocrine tumors. The MEN1 gene product, menin, is expressed in many embryonic, as well as adult tissues, and interacts with several proteins in vitro and in vivo. However, the biological function of menin remains largely unknown. Here we show that disruption of the Men1 gene in mice causes embryonic lethality at E11.5-E13.5. The Men1 null mutant embryos appeared smaller in size, frequently with body haemorrhages and oedemas, and a substantial proportion of them showed disclosure of the neural tube. Histological analysis revealed an abnormal development of the nervous system and heart hypotrophy in some Men1 null embryos. Furthermore, Men1 null livers generally displayed an altered organization of the epithelial and hematopoietic compartments associated with enhanced apoptosis. Chimerism analysis of embryos generated by injection of Men1 null ES cells, showed that cells lacking menin do not seem to have a general cell-autonomous defect. However, primary Men1 null embryonic fibroblasts entered senescence earlier than their wild-type counterparts. Despite normal proliferation ability, Men1 null ES cells exhibited a deficiency to form embryoid bodies, suggesting an impaired differentiation capacity in these cells. The present study demonstrates that menin plays an important role in the embryonic development of multiple organs in addition to its proposed role in tumor suppression.

Alleles↗

PubMed1997

Proliferative lifespan checkpoints: cell-type specificity and influence on tumour biology.

Lifespan checkpoints are viewed here as intrinsic mechanisms which desensitise cells to external growth signals as a programmed response to proliferative age, as distinct from externally-triggered differentiation. This review focuses on the role of tumour suppressor gene products as essential mediators of cell cycle arrest at lifespan checkpoints, concentrating in particular on p53. Although drawing inevitably on fibroblast senescence and telomere erosion paradigms, other lifespan clocks and signal pathways are discussed. Particular emphasis is placed on cell-type diversity in the nature, number and timing of lifespan checkpoints and its importance for tumour biology. Breast and thyroid cancer are used to illustrate the concept that the "choice" of checkpoint(s) in a given normal cell may have a determining influence on the mutational spectrum and clinical behaviour of its tumours.

Animals↗

PubMed2001

Crystal structure of human PEDF, a potent anti-angiogenic and neurite growth-promoting factor.

Pigment epithelium-derived factor (PEDF), a noninhibitory member of the serpin superfamily, is the most potent inhibitor of angiogenesis in the mammalian ocular compartment. It also has neurotrophic activity, both in the retina and in the central nervous system, and is highly up-regulated in young versus senescent fibroblasts. To provide a structural basis for understanding its many biological roles, we have solved the crystal structure of glycosylated human PEDF to 2.85 A. The structure revealed the organization of possible receptor and heparin-binding sites, and showed that, unlike any other previously characterized serpin, PEDF has a striking asymmetric charge distribution that might be of functional importance. These results provide a starting point for future detailed structure/function analyses into possible mechanisms of PEDF action that could lead to development of therapeutics against uncontrolled angiogenesis.

Amino Acid Sequence↗

PubMed2003

Down-regulation of Myc as a potential target for growth arrest induced by human polynucleotide phosphorylase (hPNPaseold-35) in human melanoma cells.

Terminal differentiation and senescence share several common properties, including irreversible cessation of growth and changes in gene expression profiles. To identify molecules that converge in both processes, an overlapping pathway screening was employed that identified old-35, which is human polynucleotide phosphorylase (hPNPaseold-35), a 3',5'-exoribonuclease. We previously demonstrated that hPNPaseold-35 is a type I interferon-inducible gene that is also induced in senescent fibroblasts. In vitro RNA degradation assays confirmed its exoribonuclease properties, and overexpression of hPNPaseold-35 resulted in growth suppression in HO-1 human melanoma cells. The present study examined the molecular mechanism of the growth-arresting property of hPNPaseold-35. When overexpressed by means of a replication-incompetent adenoviral vector (Ad.hPNPaseold-35), hPNPaseold-35 inhibited cell growth in all cell lines tested. Analysis of cell cycle revealed that infection of HO-1 cells with Ad.hPNPaseold-35 resulted in arrest in the G1 phase and eventually apoptosis accompanied by marked reduction in the S phase. Infection with Ad.hPNPaseold-35 resulted in reduction in expression of the c-myc mRNA and Myc protein and modulated the expression of proteins regulating G1 checkpoint and apoptosis. In vitro mRNA degradation assays revealed that hPNPaseOLD-35 degraded c-myc mRNA. Overexpression of Myc partially but significantly protected HO-1 cells from Ad.hPNPaseold-35-induced growth arrest, indicating that Myc down-regulation might directly mediate the growth-inhibitory properties of Ad.hPNPaseold-35. Inhibition of hPNPaseold-35 by an antisense approach provided partial but significant protection against interferon-beta-mediated growth inhibition, thus demonstrating the biological significance of hPNPaseold-35 in interferon action.

Antineoplastic Agents↗

PubMed2004

Stimulation of quiescent cells by individual polypeptide growth factors is limited to one cell cycle.

Since little is known about the function of polypeptide growth factors as regulators of multiple cell cycles, we compared the ability of FGF1, PDGF-AB and serum to induce a second round of DNA synthesis in Swiss 3T3 cells previously exposed to either FGF1, PDGF-AB or serum during the first cell cycle using [14C]- and [3H]thymidine in a double labeling system to distinguish between the first and second cell cycles. Surprisingly, we observed that cells exposed to either FGF1 or PDGF-AB in the first cell cycle were unable to synthesize DNA in response to FGF1 or PDGF-AB in the second cell cycle; yet these cells responded well to serum as a second cycle mitogen. Interestingly, while cells exposed to either FGF1 or PDGF-AB in the second cycle displayed normal receptor-mediated signaling and expressed cyclin D and E, they, like senescent fibroblasts and endothelial cells, failed to express cyclin A, and the continuous exposure of cells to either FGF1 or PDGF-AB resulted in a decrease in the kinase activity of the cyclin E/cdk2 complex. In addition, an increased association of this complex was observed with p21 CIP in an FGF1-dependent manner as well as with p27 KIP in a PDGF-AB-dependent manner. Lastly, the downregulation of p21 expression using an antisense strategy was able to partially rescue the replicative response of Swiss 3T3 cells to FGF1 in the second cycle. These data suggest that (i) FGF1 and PDGF-AB may limit their mitogenic effect to a single cell cycle, (ii) entry into the second round of replication is serum dependent and (iii) the self-limiting nature of FGF1 and PDGF-AB correlates with the accumulation of the cdk inhibitors, p21 and p27, respectively.

Animals↗

PubMed1979

SV40-transformed human diploid cells that remain transformed throughout their limited lifespan.

Of ninety-one subcultured foci of SV40-transformed WI-38 human diploid fibroblasts, none yielded cells that grew indefinitely, but all cells in each subculture continued to produce T antigen and to look morphologically transformed throughout their lifespan. These results are consistent with the commitment theory of fibroblast senescence, but predict that a special transformation event is necessary to accout for the rare survivors.

Antigens, Neoplasm↗