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R J Pignolo

Publications and source records attributed to R J Pignolo.

At least 19 recordsLinked to original sources
PubMed1999

Effects of donor age on the expression of a marker of replicative senescence (EPC-1) in human dermal fibroblasts.

EPC-1 (early population doubling level cDNA-1) is a quiescence-specific gene expressed at high levels by early passage WI-38 fibroblasts under conditions of either density-dependent growth arrest or serum deprivation. Late passage WI-38 cells lose the ability to express EPC-1 under all conditions tested. The decline in EPC-1 mRNA is gradual during the replicative life span and correlates inversely with the population doubling level (PDL) of the cells. The objective of this study was to determine whether the decline in EPC-7 mRNA abundance observed during proliferative senescence also occurs in cultures derived from donors of different ages. To address this question, we examined the abundance of EPC-1 mRNA in 28 skin fibroblast lines established from healthy donors of different ages ranging from 12 fetal weeks to 94 years. EPC-1 expression was measured, under conditions of growth arrest, prior to the end of the replicative life span of the cultures. Despite some variability in steady-state transcript levels among the cell lines, EPC-1 expression was significantly lower in cells derived from the fetal donor group (12-20 gestational weeks) than in cells derived from adult donors. An in vitro age-dependent decline in EPC-1 expression was observed in all the skin lines examined, independent of donor age; however, no significant difference was observed between the young adult donor group (17-33 years) and the old adult donor group (78-94 years). Thus, expression of EPC-1 is linked to the replicative age of the cells and whether the cells are derived from fetal skin or adult skin. In adults, EPC-1 expression is independent of donor age.

Adolescent↗
PubMed1998

Relationship between donor age and the replicative lifespan of human cells in culture: a reevaluation.

Normal human diploid fibroblasts have a finite replicative lifespan in vitro, which has been postulated to be a cellular manifestation of aging in vivo. Several studies have shown an inverse relationship between donor age and fibroblast culture replicative lifespan; however, in all cases, the correlation was weak, and, with few exceptions, the health status of the donors was unknown. We have determined the replicative lifespans of 124 skin fibroblast cell lines established from donors of different ages as part of the Baltimore Longitudinal Study of Aging. All of the donors were medically examined and were declared "healthy," according to Baltimore Longitudinal Study of Aging protocols, at the time the biopsies were taken. Both long- and short-lived cell lines were observed in all age groups, but no significant correlation between the proliferative potential of the cell lines and donor age was found. A comparison of multiple cell lines established from the same donors at different ages also failed to reveal any significant trends between proliferative potential and donor age. The rate of [3H]thymidine incorporation and the initial rates of growth during the first few subcultivations were examined in a subset of cell lines and were found to be significantly greater in fetal lines than in postnatal lines. Cell lines established from adults did not vary significantly either in initial growth rate or in [3H]thymidine incorporation. These results clearly indicate that, if health status and biopsy conditions are controlled, the replicative lifespan of fibroblasts in culture does not correlate with donor age.

Adolescent↗
PubMed1998

Senescent WI-38 fibroblasts overexpress LPC-1, a putative transmembrane shock protein.

We have recently reported the isolation of cDNAs for a number of genes that are differentially expressed between nonproliferating early (young) and late (senescent) population doubling level (PDL) WI-38 human, fetal lung-derived, fibroblast-like cells. We now demonstrate that one of these isolates, LPC-1 (Late PDL cDNA-1), derives from an approximately 2.9-kb mRNA species that is expressed at a two- to fivefold higher level in serum-starved, confluent, senescent versus similarly treated young WI-38 cells. Nucleotide sequence analysis of this cDNA confirms its identity with that of a cDNA encoding a marker (p63) for the rough endoplasmic recticulum and a related swine hepatic cardiogenic shock protein. We show that LPC-1 expression in early PDL WI-38 cells is strictly cell cycle-regulated and its expression peaks 9-12 h after serum stimulation of G0 cultures. The steady state levels of LPC-1 transcript in early PDL cells preceeding and following its peak expression are low, reflecting basal levels seen in G0 upon removal of serum. Late PDL cells, however, seem to have lost this tight cell cycle regulation seen in early PDL cells and inappropriately express high levels of the transcript after serum stimulation. Specific antiserum detects a protein of approximately 63 kDa by Western analysis and elicits intense cytoplasmic staining of senescent fibroblasts by immunohistochemistry. Related genomic sequences are found in all mammalian species examined as well as in the chicken. These findings are consistent with the hypothesis that senescent WI-38 cells exhibit a state of growth arrest fundamentally distinct from that of quiescent (G0) young cells.

Animals↗
PubMed1997

Development and age-associated differences in electron transport potential and consequences for oxidant generation.

We determined the activities of NADH dehydrogenase (ND), succinate dehydrogenase, and cytochrome c oxidase (COX) in 29 skin fibroblast lines established from donors ranging in age from 12 gestational weeks to 94 years. The results of this study demonstrate that all three of the enzyme activities examined are greater in adult-derived fibroblasts than in the fetal cell lines. The ratio of enzyme activities that control electron entry into and exit from the electron transport chain varied directly with lucigenin-detected chemiluminescence (an indicator of .O2- generation) and inversely with H2O2 generation. These results indicate a clear difference in the predominant oxidant species generated during fetal and adult stages of life. We also examined the mRNA abundances of different components of the electron transport chain complexes. We observed higher abundances of mitochondrial encoded mRNAs (COX 1 and ND 4) in cell lines established from adults than in fetal cells. No differences in the mRNA abundances of the nuclear encoded sequences (COX 4 and ND 51) were observed in fetal and postnatal-derived lines. Succinate dehydrogenase mRNA abundance was greater in cell lines established from postnatal donors than in fetal cell lines. No significant differences between cell lines established from young and old adults were detected in any of the parameters examined.

Adult↗
PubMed1997

Cloning of the human twist gene: its expression is retained in adult mesodermally-derived tissues.

We have previously reported on the isolation of several differentially expressed genes derived from young and senescent non growing WI-38 human fetal lung-derived fibroblasts. A 0.8-kb cDNA clone, isolate EPC-A2 (early population doubling cDNA-A2), encodes the 3' end of the human homolog of the Twist protein. Twist genes encode basic helix-loop-helix DNA-binding transcription factors that play crucial roles in mesoderm development. Here, we report the cloning and sequencing of the genomic human twist gene. It encodes a protein of 201 amino acids with 96% amino acid sequence identity to mouse Twist, and 100% sequence conservation in the DNA-binding region among all species in which it has been characterized. We further show that expression of human twist is retained in mesodermally-derived tissues and cell lines derived from adult donors.

Adult↗
PubMed1995

Analysis of EPC-1 growth state-dependent expression, specificity, and conservation of related sequences.

The transcript for EPC-1 (early population doubling level (PDL) cDNA-1) is induced under conditions of growth arrest due to density-dependent contact inhibition and/or serum deprivation in early-passage but not in senescent WI-38 fibroblasts. We have characterized the EPC-1 transcript with respect to its cell-cycle regulation, tissue specificity, and interspecies conservation of related genomic sequences. In low density, quiescent (serum-deprived), early-passage fibroblasts that are stimulated to proliferate with fresh serum, steady-state EPC-1 transcript levels are steadily reduced until they reach a basal level approximately 24 h after stimulation. However, when early-passage fibroblasts are made quiescent by both serum deprivation and density-dependent contact inhibition and then stimulated with serum, steady-state EPC-1 transcript levels remain relatively constant throughout a 36 h period following serum stimulation. Senescent WI-38 cells (> 95% life span completed) do not express EPC-1 under these conditions. We show that differences in the regulation of EPC-1 transcript levels in early-passage cells are due to differences in growth state rather than changes in cell density or contact. We also show that expression of the EPC-1 transcript is limited to specific cell types and that related genomic sequences are found in all mammalian species examined as well as in the chicken.

Blotting, Northern↗
PubMed1995

Alterations in the molecular response to DNA damage during cellular aging of cultured fibroblasts: reduced AP-1 activation and collagenase gene expression.

Transcriptional activation of c-fos in response to both serum stimulation and DNA damage requires the serum response element. The inability of in vitro aged or senescent fibroblasts to proliferate in response to serum has been shown to be associated with repressed c-fos expression and reduced AP-1 binding activity. In contrast, we have observed similar levels of c-fos mRNA and protein expression in young (early passage) and old (late passage) cells following their treatment with ultraviolet (UV) irradiation or methyl methanesulfonate (MMS). Thus, the early events in the signal transduction pathway leading to transcriptional activation of c-fos following DNA damage are distinct from those mediating the gene's expression in response to mitogenic stimulation. Despite normal levels of c-fos expression, we observed a reduced level of AP-1 binding activity in old cells relative to young cells treated with UV irradiation or MMS. Reduced AP-1 binding activity is associated with reduced expression of the AP-1-dependent gene, collagenase, in old cells treated with DNA damaging agents. Since other DNA damage-inducible genes also contain an AP-1 regulatory element presumed to play a role in their expression, reduced AP-1 binding activity is likely to have a major impact on the old cell's ability to respond appropriately to DNA damage.

Base Sequence↗
PubMed1995

Identification of proteins differentially expressed in quiescent and proliferatively senescent fibroblast cultures.

We have examined the differential expression of proteins in quiescent (nongrowing) early versus late population doubling level (PDL) WI-38 human diploid fibroblasts. Age-associated changes in gene expression at the level of both secreted and total cellular protein were identified using high-resolution, two-dimensional gel electrophoresis followed by N-terminal sequencing or Western blot analysis. A comparison of secreted proteins revealed the senescence-specific absence of all forms of EPC-1, a protein associated with early PDL cells in the G0 state. This comparison also revealed the absence of a processed (cleaved) form of alpha I (type III) procollagen in senescent cells, suggesting a distinctive remodeling of the extracellular matrix. A comparison of total cellular protein between early passage and proliferatively aged fibroblasts identified an altered form of the enzyme ubiquitin carboxyl-terminal hydrolase in senescent cells, a result that could explain the increase in ubiquitinated proteins in these cells. These findings further elucidate the phenotype of fibroblasts aged in vitro and support the concept that senescent cells are arrested in a state fundamentally different from that of G0 early PDL cells.

Amino Acid Sequence↗
PubMed1994

Alterations in contact and density-dependent arrest state in senescent WI-38 cells.

Normal human WI-38 fibroblast-like cells in culture undergo a process of senescence, one feature of which is a gradual decline in proliferative capacity. As these cells reach the end of their replicative life span they exhibit decreases in the fraction of cells able to synthesize DNA, in the number of doublings per passage (constant seeding density), and in the cell harvest and saturation densities. They also display increased average cell cycle times, largely at the expense of longer G1 intervals. These alterations are accompanied by morphologic changes, including cell enlargement. Before the end of the replicative life span or phase-out, there is a highly reproducible (55/58 sublines) cell loss of approximately 50%; however, a stable population survives that can exist in a viable yet nonproliferative state for many months. This stable population maintains an extremely low saturation density, representing < 5% of that achieved by early passage cultures. Further, we show that maximum harvest densities achieved by senescent cells are lower, irrespective of seeding densities, i.e. when placed at cell densities higher than those normally achieved by senescent cultures they display a net decline in cell number. This decline continues until the cell density approximates the density that would have been achieved had the cultures been seeded at standard density (1 x 10(4) cells/cm2).(ABSTRACT TRUNCATED AT 250 WORDS)

Biomarkers↗
PubMed1994

Molecular biology of aging.

Aging is an extremely complex biologic phenomenon of immense importance. Currently we have only a poor and incomplete understanding of the fundamental molecular mechanisms involved. Despite numerous observations and diverse theories, no unifying or proven hypotheses have emerged. It is reasonable to conclude, however, that aging is a multifactorial process composed of both genetic and environmental components. Each physiologic system within an organism, each tissue within a system, and each cell type with a tissue appears to have its own trajectory of aging. Thus, aging must be studied as parts of a whole and understood as the sum of its parts. Cellular "clocks" exist and operate in the absence of higher-order "clocks". However, higher-order clocks are certainly in place in vivo, but their relationship to cellular clocks is not well understood. All aging changes have a cellular basis, and aging is perhaps best studied, fundamentally, at the cellular level under defined and controlled environmental conditions. Aging changes at the cellular level must be viewed, however, as components of a hierarchical, dynamic, and interacting network whose functional integrity progressively deteriorates with time. The powerful tools of molecular biology are now being applied by scientists to evaluate the leading hypotheses. The results of these studies should serve to advance our understanding of aging and to focus future research efforts. This work should provide the scientific foundation to enhance the quality of life for people suffering the failings of age.

Aging↗
PubMed1993

Senescent WI-38 cells fail to express EPC-1, a gene induced in young cells upon entry into the G0 state.

Recently we reported the isolation of cDNAs for a number of genes that are differentially expressed between nonproliferating early (young) and late (senescent) population doubling level (PDL) WI-38 human, fetal lung-derived, fibroblast-like cells. We now demonstrate that one of these isolates, EPC-1 (early PDL cDNA-1), derives from an approximately 1.4-kilobase mRNA species that is expressed at a > or = 100-fold higher level in serum-starved, confluent, young versus similarly treated senescent WI-38 cells. Complete nucleotide sequence analysis of this cDNA confirms its identity with that of a cDNA encoding a secreted, retinal pigmented epithelium differentiation factor as well as similarity of the encoded protein with a number of mammalian serine protease inhibitors. We show that EPC-1 expression is associated with G0 growth arrest in WI-38 cells. The mRNA readily accumulates in density-arrested and/or serum-starved young cells but not in log phase, low density young cells. In contrast, EPC-1 transcript abundance and expression of the encoded, secreted protein are both negligible in senescent WI-38 cells under all culture conditions tested. These findings support the hypothesis that senescent WI-38 cells exhibit a state of growth arrest fundamentally distinct from that of quiescent (G0) young cells.

Amino Acid Sequence↗
PubMed1993

Replicative senescence of human fibroblast-like cells in culture.

The life history of fibroblast and fibroblast-like cells includes an initial stage of outgrowth and establishment in culture; a period of vigorous proliferation which has a variable length, depending on the tissue of origin, age of the donor, etc.; a period of declining proliferative vigor which includes substantial cell death; and finally, the emergence of an (apparently) long-lived population which is unable to proliferate in response to growth factors. During the phase of declining proliferative vigor, the cells acquire characteristics, some of which are similar to the characteristics of cells in older individuals. Eventually the culture completely loses proliferative capacity. A comparable life history has been described for glial cells, keratinocytes, vascular smooth muscle cells, endothelial cells, and lymphocytes which suggests that this life history is characteristic of those cell types that, in vivo, retain the capacity for proliferation throughout the life span. Numerous studies have shown a correlation between the age of the tissue donor and the replicative life span of the cells in culture. In addition, for a small sample of species, there is a direct correlation between fibroblast replicative life span in vitro and maximum life span potential of the species. The period in the life history that is usually referred to as the "senescent phase" is probably more complicated than was originally thought, since studies with life span modulators suggest that there is a "conditionally" senescent state from which cells can be rescued for one or more additional rounds of DNA synthesis. Finally, the cells enter an "obligatory" arrested state in which only SV40 infection can reverse the block to DNA synthesis but not the block to mitosis. The modern era of aging research in tissue culture is just over 30 years old. The inception of the field really began with the recognition by Hayflick and Moorhead (109) that the phenomenon of senescence in vitro paralleled, in some of its characteristics, cell aging in vivo and thus provided a model that could be used to study the cellular mechanisms underlying senescence in controlled environmental conditions. The research in this area began with a detailed characterization and comparison of young versus senescent cell morphology and physiology. These studies provided the basis for a wide variety of subsequent studies that addressed possible mechanisms underlying cell senescence. These included studies on DNA repair, protein synthetic errors, chromatin structure and function, and mechanisms for modulating replicative life span.(ABSTRACT TRUNCATED AT 400 WORDS)

Biomarkers↗
PubMed1992

Renewed DNA synthesis in senescent WI-38 cells by expression of an inducible chimeric c-fos construct.

As normal human cells approach the end of their proliferative lifespan in vitro they lose responsiveness to a variety of growth factors, to which they respond with DNA replication when they are young. Recently it has been reported that the protooncogene c-fos is not expressed in senescent cells (Seshadri and Campisi, 1990). In this study we have found that both c-jun and jun B, partners of c-fos in heterodimeric transactivating complexes, are equivalently expressed in young and senescent cells at both early (1-6 hr) and late (12 or 16 hr) time points following serum stimulation of quiescent cells. We have also investigated the effect of the enforced expression of c-fos in senescent WI-38 cells using an inducible construct carrying the murine c-fos gene under the control of the sheep metallothionein promoter. We have found that the transient transfection and subsequent activation of the conditional promoter with Zn++ stimulated DNA synthesis in a significant fraction of senescent cells which had completed 90%-95% of their proliferative lifespan. However, populations which had completed 100% of their proliferative life span and nondividing cultures which had been selected with BrdU did not respond to the expression of the c-fos gene. These results demonstrate that one of the primary events associated with senescence in human cells is the suppression of c-fos gene expression, but additional phenotypic changes must also occur in order to explain the ultimate loss of proliferative responsiveness of these cells.

Aging↗
PubMed1992

Overexpression of the two-chain form of cathepsin B in senescent WI-38 cells.

We have examined differential protein expression in serum-stimulated young and senescent WI-38 human fetal lung-derived cells in culture using high-resolution two-dimensional gel electrophoresis. Overexpression of a protein with an approximate M(r) of 29,000 and pI of 5.8 was observed in senescent cells during the G0 and throughout the G1 stage of the cell cycle. Automated amino-terminal sequencing of the peptide from polyvinylidene difluoride electroblots showed 100% sequence identity to cathepsin B or pre-procathepsin B in a 12-amino acid overlap, beginning at residue 48 or 129, respectively. The 29-kDa peptide corresponds to the heavy chain of the two-chain enzyme form. Cathepsin B activity was found to be decreased in cells aged in vitro in comparison to that in young controls. Changes in the steady-state levels of both the 4.0- and the 2.2-kb cathepsin B transcripts between young and senescent cells cannot account for the overexpression of the two-chain form of the enzyme. These results suggest that increased proteolysis of a conformationally more labile single-chain form and/or decreased turnover and accumulation of a less active form of this lysosomal protease occur in senescent fibroblasts and may account for the observed decreased cathepsin B activity in senescent cells in culture.

Amino Acid Sequence↗
PubMed1992

Skin fibroblasts from aged Fischer 344 rats undergo similar changes in replicative life span but not immortalization with caloric restriction of donors.

We have compared the in vitro replicative life span and characteristics of immortalization of skin fibroblast cultures derived from ad libitum-fed and caloric-restricted Fischer 344 rats of 6, 24, and 29 months of age. Cells from all 6-, 24-, and 29-month-old animals showed a gradual decline in proliferative potential as evidenced by decreases in harvest density, in the fraction of cells initiating DNA synthesis, and in the number of population doublings per passage. These declines were accompanied by morphological changes including cell enlargement. The replicative life span prior to immortalization decreased significantly with donor age (P less than 0.0001), while caloric restriction had no effect on the cumulative population doubling level. Prior to immortalization mitotic cells from all cultures showed a normal rat karyotype. Postcrisis cultures tended to have more polyploid cells but there were no characteristic or specific chromosomal changes found in the cells with an immortalized phenotype. Interestingly, fibroblasts derived from caloric-restricted animals had a significantly slower growth rate through the tenth week after immortalization (P less than 0.005). When these cultures were seeded at one-quarter the normal seeding density, to favor the outgrowth of the fastest growing cells, a population with a more "transformed" phenotype emerged.

Age Factors↗
PubMed1992

Differential gene expression between young and senescent, quiescent WI-38 cells.

To investigate age-related changes in gene expression in WI-38 cells, we isolated RNA from young and senescent, quiescent cultures and made subtracted cDNA libraries. Density-arrested cells were incubated in serum-free MCDB-104 for 3 days. RNA was then isolated and subtracted cDNA libraries were made in the phagemid vector pCDM8. Both by picking clones at random from these subtracted libraries and by differential hybridization screening with subtracted cDNA probes from young and senescent cells, we have identified a total of 11 genes for which RNA is expressed differentially in these quiescent young and senescent WI-38 cultures. Two genes, EPC-1 and EPC-A2, with elevated RNA levels in young cells, have sequences which have not previously been identified. Two of the genes with elevated RNA expression in the senescent cells are the mitochondria-coded genes for NADH dehydrogenase subunit 4 and for cytochrome b. We also identified seven other genes with elevated RNA levels in senescent cells. Three of these, LPC-1, LPC-14 and LPC-24, have been partially sequenced and have not previously been identified. These studies show that density-arrested, serum-deprived, quiescent young and senescent cells express a number of genes differentially. These differences are not growth-dependent, but are age-dependent. Our studies also show that the methods employed here, which include careful regulation of the cell cultures and subtraction of the libraries, result in libraries from which differentially expressed genes can be identified, either by random selection or by differential hybridization screening with subtracted probes.

Aging↗
PubMed1990

A member of the ras gene superfamily is expressed specifically in T, B and myeloid hemopoietic cells.

A 1.26 kb murine cDNA having 31% homology with human ras and 55% homology with human rho proteins was isolated using an oligonucleotide probe homologous to catalytic subdomain of a tyrosine kinase subfamily. Northern blot analysis indicates that the expression of the murine gene is restricted to the cells of hemopoietic lineages and the mRNA levels increase with the terminal differentiation of hemopoietic cells into granulocytes.

Amino Acid Sequence↗