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PubMed1986

Culture media variation as related to in vitro aging of human fibroblasts: I. Effects on population doubling, nuclear volume and nuclear morphology.

The relative effect of five commonly used culture media (MEM, BME, McCoy's 5A, M199 and HMEM) on the population doubling level (PDL), nuclear volume and nuclear morphology was examined during in vitro senescence of WI-38 human fetal fibroblasts. Statistical analyses showed that cells grown in M199 had a significantly lower PDL than that of cells cultured in any other medium. The PDL in McCoy's 5A was significantly lower compared to that in BME, MEM and HMEM. Cells grown in BME, MEM and HMEM showed similar PDL. It was found that the nuclei of aged cells grown in M199 were significantly larger in volume than cells aged in any other medium. The average increases in nuclear volume of cells during aging in BME, MEM and McCoy's 5A were statistically equivalent. The increase in nuclear volume in HMEM was significantly smaller than that of cells aging in M199 and was longer than that of cells aging in BME or MEM. Linear regression analysis showed that there was a linear increase in nuclear volume as a function of PDL for cells aged in all five media. However, the rate of increase in nuclear volume with increasing PDL varied from medium to medium. There was no significant difference between media on induction of abnormal nuclear morphology as related to PDL. The relative effects of all five media were not uniform on the three cellular parameters investigated during in vitro aging of WI-38 cells. It is, therefore, suggested that one should keep this medium differential in mind to allow meaningful comparison of possible changes in various morphological parameters during in vitro senescence of diploid human fibroblasts such as WI-38.

Cell Line↗

PubMed2002

The shortened replicative life span of prohibitin mutants of yeast appears to be due to defective mitochondrial segregation in old mother cells.

Prohibitin proteins have been implicated in cell proliferation, aging, respiratory chain assembly and the maintenance of mitochondrial integrity. The prohibitins of Saccharomyces cerevisiae, Phb1 and Phb2, have strong sequence similarity with their human counterparts prohibitin and BAP37, making yeast a good model organism in which to study prohibitin function. Both yeast and mammalian prohibitins form high-molecular-weight complexes (Phb1/2 or prohibitin/BAP37, respectively) in the inner mitochondrial membrane. Expression of prohibitins declines with senescence, both in mammalian fibroblasts and in yeast. With a total loss of prohibitins, the replicative (budding) life span of yeast is reduced, whilst the chronological life span (the survival of stationary cells over time) is relatively unaffected. This effect of prohibitin loss on the replicative life span is still apparent in the absence of an assembled respiratory chain. It also does not reflect the production of extrachromosomal ribosomal DNA circles (ERCs), a genetic instability thought to be a major cause of replicative senescence in yeast. Examination of cells containing a mitochondrially targeted green fluorescent protein indicates this shortened life span is a reflection of defective mitochondrial segregation from the mother to the daughter in the old mother cells of phb mutant strains. Old mother phb mutant cells display highly aberrant mitochondrial morphology and, frequently, a delayed segregation of mitochondria to the daughter. They often arrest growth with their last bud strongly attached and with the mitochondria adjacent to the septum between the mother and the daughter cell.

Cell Division↗

PubMedDate not supplied

Effect of in vitro aging on the modulation of protein and fibronectin biosynthesis by the elastin-laminin receptor in human skin fibroblasts.

The 67-kD elastin-laminin receptor (ELR) subunit which carries the recognition site for elastin peptides (EP) is a lectin. Its binding with galactosides can modulate the kinetics of its interaction with its ligand, EP. In this study the biosynthesis of proteins, collagen and fibronectin were evaluated in the presence of agonists and antagonists of the receptor on human skin fibroblasts. The biosynthesis of total proteins determined by 3H-proline incorporation and of fibronectin (by immunoprecipitation) were shown to increase with passage number. The presence of 1 microg/ml kappa-elastin (EP) in the culture medium increased both total proteins and fibronectin biosynthesis. Melibiose, an agonist of the receptor at 5 microg/ml (140 microM), decreased both proteins and fibronectin biosynthesis in the culture medium of human skin fibroblast at the 10th and 15th passage. These results show that the ELR can control the biosynthetic mechanisms of some of the macromolecular constituents of extracellular matrix such as fibronectin and moderate its age and passage-dependent upregulation.

Cells, Cultured↗

PubMed2002

Regions and activities of simian virus 40 T antigen that cooperate with an activated ras oncogene in transforming primary rat embryo fibroblasts.

Prolonged expression of a ras oncogene in primary cells accelerates the natural process of senescence. This ras-induced permanent growth arrest is bypassed in cells expressing the simian virus 40 large T antigen. Previously we showed that two regions of T antigen, a region consisting of the N-terminal 147 amino acids and a region consisting of amino acids 251 to 708 (T251-708), independently overcome ras-induced senescence. Coexpression of either T-antigen fragment and Ras results in the appearance of dense foci of transformed cells. Using a series of mutants that produce shorter T-antigen fragments, we show that the C-terminal limit of the N-terminal T-antigen fragment that cooperates with Ras lies between amino acids 83 and 121. The N-terminal limit of the C-terminal T-antigen fragment lies between amino acids 252 and 271. In addition, we present evidence that cooperation between the N-terminal fragment and Ras depends upon an intact T-antigen J domain and the ability of the T antigen to bind and inactivate the growth-suppressive effect of the tumor suppressor Rb. Introduction of specific amino acid substitutions surrounding residue 400 into T251-708 prevented the fragment from cooperating with Ras. T251-708 proteins with these same substitutions inhibited the transcriptional transactivating potential of p53 as effectively as did the wild-type protein. Thus, at least one activity contained within T251-708, other than inactivating p53 as a transcriptional transactivator, is likely to be required to bypass Ras-induced senescence.

Amino Acid Sequence↗

PubMed1998

Life span of skin fibroblasts in patients with systemic sclerosis.

BACKGROUND: It remains unknown whether the life span of systemic sclerosis (SSc) skin fibroblasts (FB) and that of normals are the same or not, though SSc skin FB have been shown to present abnormalities such as elevated collagen synthesis. OBJECTIVE: To find out whether the life span of SSc skin FB and that of normals are the same or not. METHODS: Cell generation and colony-forming efficiency were calculated in 10 normal and 7 SSc FB, and total life spans were studied. RESULTS: Colony-forming efficiency decreased from 30 to 5% until the cells reached senescence. The cell generations of normal and SSc FB showed no significant difference. CONCLUSION: SSc FB is senescent and may indicate that the senescence of SSc FB is independent of its abnormal features.

Adult↗

PubMed1991

Age-related differences in promoter-induced extension of in vitro proliferative life span of Syrian hamster fibroblasts.

The proliferative capacity of Syrian hamster dermal fibroblasts has been previously shown to be inversely related to the age of the donor (Mech. Ageing and Dev., 34 (1986) 151). The present study demonstrates an inverse correlation between in vivo age and the in vitro morphological and proliferative response of Syrian hamster dermal fibroblasts to the tumor promoter phorbol-12,13-didecanoate. Treatment of fetal fibroblasts with promoter increased the proliferative life span of the cultures by approximately 2-fold, but did not increase the frequency of conversion to established cell lines. Neonatal and young adult fibroblasts exhibited intermediate responses to promoter treatment, showing 54.9% and 33.1% extension, respectively. In contrast, promoter treatment had no significant effect on aged adult fibroblasts. Maximal extension required continual treatment beginning in primary culture or at passage 1. Promoter-induced extension of proliferative life span appears to be mediated through the prolonged maintenance of small, highly proliferative cells that are present in primary cultures of these cells.

Aging↗

PubMed1999

Further evidence for the rejuvenating effects of the dipeptide L-carnosine on cultured human diploid fibroblasts.

We have confirmed and extended previous results on the beneficial effects of L-carnosine on growth, morphology, and longevity of cultured human fibroblasts, strains MRC-5 and HFF-1. We have shown that late-passage HFF-1 cells retain a juvenile appearance in medium containing 50 mM carnosine, and revert to a senescent phenotype when carnosine is removed. Switching cells between medium with and without carnosine also switches their phenotype from senescent to juvenile, and the reverse. The exact calculation of fibroblast lifespans in population doublings (PDs) depends on the proportion of inoculated cells that attach to their substrate and the final yield of cells in each subculture. We have shown that carnosine does not affect cell attachment, but does increase longevity in PDs. However, the plating efficiency of MRC-5 cells seeded at low density is strongly increased in young and senescent cells by carnosine, as shown by the growth of individual colonies. We have also demonstrated that very late-passage MRC-5 cells (with weekly change of medium without subculture) remain attached to their substrate much longer in medium containing carnosine in comparison to control cultures, and also retain a much more normal phenotype. Carnosine is a naturally occurring dipeptide present at high concentration in a range of human tissues. We suggest it has an important role in cellular homeostasis and maintenance.

Carnosine↗

PubMed2000

Senescence bypass screen identifies TBX2, which represses Cdkn2a (p19(ARF)) and is amplified in a subset of human breast cancers.

To identify new immortalizing genes with potential roles in tumorigenesis, we performed a genetic screen aimed to bypass the rapid and tight senescence arrest of primary fibroblasts deficient for the oncogene Bmi1. We identified the T-box member TBX2 as a potent immortalizing gene that acts by downregulating Cdkn2a (p19(ARF)). TBX2 represses the Cdkn2a (p19(ARF)) promoter and attenuates E2F1, Myc or HRAS-mediated induction of Cdkn2a (p19(ARF)). We found TBX2 to be amplified in a subset of primary human breast cancers, indicating that it might contribute to breast cancer development.

Adenocarcinoma↗

PubMed1988

Culture media variation as related to in vitro aging of human fibroblasts: II. Effects on nucleolar number/cell, volume/nucleolus and total nucleolar volume/cell.

The relative effect of five commonly used culture media (MEM, BME, McCoy's 5A, M199 and HMEM) on the average nucleolar number/cell, the average volume/nucleolus and the total nucleolar volume/cell was examined during in vitro senescence of WI-38 human fetal fibroblasts. Statistical analyses show that cells aging in MEM show a higher number of nucleoli/cell than that of cells aging in any other medium. For cells aging in the other four media, there are no significant differences in the average number of nucleoli/cell. Linear regression analysis shows that in all cases there is a linear decrease in the average number of nucleoli/cell as a function of PDL. Statistical analyses show that the average volume/nucleolus is significantly greater for cells aging in M199 than in any other medium. Cells aging in HMEM show smaller average nucleolar volume than cells aging in M199, but display larger volumes than that of cells aged in BME, McCoy's 5A, or MEM. Cells aging in BME and McCoy's 5A media show no significant difference among each other in terms of average nucleolar volume, but a difference in this parameter is noted in cells aging in BME and MEM. A linear regression analysis shows that the average volume/nucleolus increases linearly as a function of age for cells grown in all five media. Analysis of the total nucleolar volume/cell in the five media shows that cells aging in M199 and HMEM are not significantly different from each other in terms of this variable, but show significantly larger volumes than those of cells aging in BME, McCoy's 5A and MEM. Cells aging in BME, McCoy's 5A and MEM display no significant difference with regard to this parameter. Linear regression analysis shows a positive linear relationship between the PDL and the total nucleolar volume/cell. The relative effects of all five media are not the same on the three cellular variables studied during in vitro aging of WI-38 cells. We, therefore, suggest that one should note this medium differential in order to allow meaningful comparison of results on possible changes in various morphological parameters during in vitro senescence of diploid human fibroblasts.

Cell Division↗

PubMed2003

Fibroblasts: what's new in cellular biology?

This paper briefly examines the fibroblast network with particular emphasis on the exceptionally complex pattern of specific interactions and their effects on dermal integrity and homeostasis regulation systems. It will be some time before we have a full understanding of the cellular biology mechanisms involved in the operation of lasers, flashlamps, peels, mechanical dermabrasions, fillers or topicals on the skin.

Cellular Senescence↗

PubMed2003

Developmental differences in the immortalization of lung fibroblasts by telomerase.

The role of ambient (21%) and physiological oxygen (2-5%) in the immortalization of fetal vs. adult human lung fibroblasts was examined. Growth in low oxygen and antioxidants extended the lifespan of both fetal and adult strains. As the ectopic expression of telomerase could immortalize adult lung fibroblasts cultured in ambient oxygen, the lifespan-shortening effects of 21% oxygen must have been largely limited to telomeres. By contrast, fetal lung fibroblasts could not be immortalized in ambient oxygen in spite of telomere elongation by telomerase, suggesting more widespread oxidative damage. The long-term culture requirements for the immortalization of WI-38 fetal lung fibroblasts included supplementation with N-(tert) butyl hydroxylamine, dexamethasone, zinc and vitamin B12, in addition to growth in physiological oxygen. The mechanisms regulating telomere shortening remain controversial. The present results suggest that both end-replication and oxidative damage events contribute to telomere shortening in lung fibroblasts in vitro. These observations emphasize the need for better analytical techniques to distinguish whether the correlation of short telomeres with disease and mortality in humans reflects the consequences of increased proliferation, telomere shortening as a result of oxidative damage or some combination of these processes.

Adult↗

PubMed2006

Bmi-1 is a novel molecular marker of nasopharyngeal carcinoma progression and immortalizes primary human nasopharyngeal epithelial cells.

The Bmi-1 oncoprotein regulates proliferation and oncogenesis in human cells. Its overexpression leads to senescence bypass in human fibroblasts and immortalization of human mammary epithelial cells. In this study, we report that compared with normal nasopharyngeal epithelial cells (NPEC), Bmi-1 is overexpressed in nasopharyngeal carcinoma cell lines. Importantly, Bmi-1 was also found to be overexpressed in 29 of 75 nasopharyngeal carcinoma tumors (38.7%) by immunohistochemical analysis. In contrast to nasopharyngeal carcinoma, there was no detectable expression of Bmi-1 in noncancerous nasopharyngeal epithelium. Moreover, high Bmi-1 expression positively correlated with poor prognosis of nasopharyngeal carcinoma patients. We also report that the overexpression of Bmi-1 leads to bypass of senescence and immortalization of NPECs, which normally express p16(INK4a) and exhibit finite replicative life span. Overexpression of Bmi-1 in NPECs led to the induction of human telomerase reverse transcriptase activity and reduction of p16(INK4a) expression. Mutational analysis of Bmi-1 showed that both RING finger and helix-turn-helix domains of it are required for immortalization of NPECs. Our findings suggest that Bmi-1 plays an important role in the development and progression of nasopharyngeal carcinoma, and that Bmi-1 is a valuable marker for assessing the prognosis of nasopharyngeal carcinoma patients. Furthermore, this study provides the first cellular proto-oncogene immortalized nasopharyngeal epithelial cell line, which may serve as a cell model system for studying the mechanisms involved in the tumorigenesis of nasopharyngeal carcinoma.

Biomarkers, Tumor↗

PubMed1993

The impaired transcription factor AP-1 DNA binding activity in lymphocytes derived from subjects with some symptoms of premature aging.

The study of human disorders known as premature aging syndromes may provide insight into the mechanisms of cellular senescence. The main feature of cellular senescence in vitro is cessation of cell proliferation. Down syndrome (DS) and neuronal ceroid-lypofuscinosis (NCL) are clinically characterized by the premature onset of numerous features normally associated with human aging. Phytohemagglutinin stimulated lymphocytes derived from DS subjects showed a statistically significant diminished proliferation capacity in comparison with lymphocytes derived from NCL and healthy individuals. We demonstrated, by applying the electrophoretic mobility shift assay, slightly impaired AP-1 DNA binding activity in NCL lymphocytes and strong in DS ones. Our results showed that the same molecular mechanisms of proliferation cessation could exist in fibroblasts characterized by replicative senescence and in lymphocytes derived from individuals with premature aging syndromes (Down).

Adolescent↗

PubMed1995

The effect of low oxygen tension on the in vitro-replicative life span of human diploid fibroblast cells and their transformed derivatives.

Human diploid fibroblasts (HDF) IMR90, starting at various population doubling levels (PDL), were serially cultured under four different oxygen conditions; the conventional atmospheric 20% O2 condition and three lower oxygen conditions (1, 6, and 12% O2). All cultures from different PDLs showed that a longer replicative life span was achieved under the lower oxygen conditions. When the starting culture PDL was between 33 and 62, the increased life span rate was constant (average 22%) at 1% O2, but the rate decreased when the starting PDL was higher. The growth advantage under the 1% O2 condition was also observed in the very late passages of cultures to some extent, but terminal cultures with senescent cells were not stimulated by the low oxygen condition. When cultures at an extended PDL under the 1% O2 condition were shifted back to the 20% O2 condition, the cells rapidly senesced. HDF from a subject with Werner syndrome, a premature aging disease, which are known to have reduced replicative potential in vitro, also showed 43% increase in life span under the lowest oxygen conditions. When SV40 large T-transformed IMR90 cells at preimmortal stages were tested, no significant growth differences were observed under different oxygen conditions, and all the cultures died out at a similar PDL. These results suggest: (1) The atmospheric 20% oxygen tension hastens HDF senescence. (2) Young cells are more resistant to oxygen tension than old cells. (3) SV40 large T transformation abolishes the oxygen effect on cellular aging.

Cell Division↗

PubMed2003

Targeting efficiency of a-1,3-galactosyl transferase gene in pig fetal fibroblast cells.

Animal cloning technology with somatic cells provides an alternative tool to conventional methods for producing transgenic animals. Gene targeting in animals is made feasible using somatic cells with homologous recombination procedure that is a major technique in embryonic stem cells for knocking-out genes. Homologous recombination events in somatic cells are relatively inefficient as compared to those in ES cells, suggesting the need for establishment of efficient gene targeting system in somatic cells. To investigate the efficiency of positive and negative selection for gene targeting in pig fetal fibroblast cells, pig alpha-1,3-galactosyl transferase (13-GT) gene was used for gene targeting. The neomycin phosphotransferase (Neo(r)) and herpes simplex virus-thymidine kinase (HSV-tk) genes were used as positive and negative selection markers in this experiment. Following transfection with targeting DNA construct, the pig fetal fibroblast cells were selected against resistance of G418 and gancyclovir. In DMEM medium containing 5 to 10% serum, Pig fetal fibroblast cells failed to proliferate during drug selection. Increasing serum concentration to 15% of medium yielded less senescent colonies of pig fetal fibroblast cells following drug selection that allowed enough cell colonies to screen genomic DNA. The frequency of gene targeting in pig fetal fibroblast cells with double drug selection was more than 10-fold efficient compared to that with G418 single selection. Double selection method with Neo' and HSV-tk genes could be useful for gene targeting in somatic cells for production of cloned animals carrying targeted endogenous genes.

Animals↗

PubMed2005

Profiling molecular targets of TGF-beta1 in prostate fibroblast-to-myofibroblast transdifferentiation.

The development of age-related proliferative disorders of the prostate gland is supported by transdifferentiation and cellular senescence processes in the stroma. Both processes are involved in remodeling of stromal tissue, as observed in benign prostatic hyperplasia (BPH), and in "reactive stroma" adjacent to prostate cancer (PCa). It has been assumed that TGF-beta1 plays a key role in the aging prostate by inducing premature senescence and favoring myofibroblast differentiation. Therefore, we evaluated the stromal cell phenotypes of human primary adult prostatic fibroblasts (n=3) and the molecular and cellular mechanisms of growth arrest after treatment with TGF-beta1 and of in vitro cellular senescence. Microarray analysis, quantitative PCR, immunofluorescence and western blot revealed that cellular senescence and transdifferentiation of fibroblasts have distinct underlying mechanisms, pathways and gene and protein expression profiles in human PrSCs. In clear contrast to senescent cells, TGF-beta1-treated cells morphologically transdifferentiated into myofibroblasts with dense cytoskeletal fibers and increased expression of smooth muscle cell alpha-actin, calponin and tenascin. TGF-beta1 induced neither expression of senescence-associated markers nor genes involved in terminal growth arrest, such as senescence-associated beta-galactosidase and cyclin-dependent kinase (cdk) inhibitors p16(Ink4A) and p21(Cip1) but increased p15(Ink4B) protein expression. Differentiation inhibitor (Id-1) protein level down-regulation was observed under both conditions. Genes specifically up-regulated by transdifferentiation but not by cellular senescence of PrSCs were metalloproteinase 1 tissue inhibitor (Timp1), transgelin (Tagln), gamma 2 actin (Actg2), plasminogen activator inhibitor 1 (Serpinel), insulin-like growth factor binding protein 3 (Igfbp3), parathyroid hormone-like hormone (Pthlp), Tgfb-1, four and a half LIM domains 2 (Fhl-2), hydrogen peroxide-inducible clone 5 (Hic5) and cartilage oligomeric matrix protein (Comp). Other genes, such as Cdc28 protein kinase 1 (Cks1b), v-myb myeloblastosis viral oncogene homolog (MybL2), pyruvate kinase, muscle 2 (Pkm2) and Forkhead box M1 (FoxM1), were down-regulated only upon TGF-beta1 treatment but not by cellular senescence. Pyruvate dehydrogenase kinase 3 (Pdk3) and connective tissue growth factor (Ctgf) were up-regulated and hyaluronan synthase 3 (Has3) down-regulated under both conditions. Moreover, GageC1, a prostate/testis-specific protein overexpressed in symptomatic BPH and PCa was induced in transdifferentiated stromal cells. Genes such as GageC1 could be promising targets for therapeutic inhibitors of stromal tissue remodeling and progression of BPH and PCa.

Cell Differentiation↗

PubMed1992

The cultured diploid fibroblast as a model for the study of cellular aging.

The limited proliferative potential of the cultured human diploid fibroblast is now well established. A number of biological correlates suggest that this culture system is a model for the study of aging at the cellular level. The mechanism(s) that causes the loss of proliferative activity is unknown; the results of some recent studies indicate that specific genes may play a pivotal role in cellular aging in vitro. The extent to which changes in proliferative functions are causally related to aging in vivo is currently under investigation.

Cells, Cultured↗

PubMed1995

Senescence-dependent regulation of type 1 plasminogen activator inhibitor in human vascular endothelial cells.

Type 1 plasminogen activator inhibitor (PAI-1) is the primary inhibitor plasminogen activator and has been found to be increased in a number of clinical conditions generally defined as prothrombotic. Since in aging and in atherosclerosis the changes observed in the endothelium resemble those of in vitro aged endothelial cells, we have examined the expression of PAI-1 in cells at different population doublings. In senescent endothelial cells, PAI-1 mRNA and protein are constitutively high, but uninducible by exogenous interleukin 1 alpha as well as by the phorbol ester TPA. Interestingly the increase of PAI-1 levels correlates with the upregulation of interleukin 1 alpha, which characterizes endothelial cell senescence. Since PAI-1 expression is not increased in young cells made nondividing by contact inhibition, we anticipate that PAI-1 expression can be used as an appropriate marker of endothelial senescence. Moreover, PAI-1 was not upregulated in senescent or in progeric human fibroblasts, which do not overexpress interleukin 1 alpha, thus suggesting that multiple pathways may exist to regulate aging of human fibroblasts and endothelial cells.

Blotting, Northern↗