Early Population Doubling Level Research Articles

Early loss of proliferative potential of human peritoneal mesothelial cells in culture: the role of p16INK4a-mediated premature senescence

Much has been learned about the mechanisms underlying cellular senescence. The pathways leading to senescence appear to vary, depending on the cell type and cell culture conditions. In this respect, little is known about senescence of human peritoneal mesothelial cells (HPMC). Previous studies have significantly differed in the reported proliferative lifespan of HPMC. Therefore, in the present study, we have examined how HPMC enter state of senescence under conditions typically used for HPMC culture. HPMC were isolated from omentum and grown into senescence. The cultures were assessed for the growth rate, the presence of senescence markers, activation of cell-cycle inhibitors, and the oxidative stress. HPMC were found to reach, on average, six population doublings before senescence. The terminal growth arrest was associated with decreased expression of Ki67 antigen, increased percentage of cells in the G1 phase, reduced early population doubling level cDNA-1 mRNA expression, and the presence of senescence-associated beta-galactosidase. Compared with early-passage cells, the late-passage HPMC exhibited increased expression of p16INK4a but not of p21Cip1. In addition, these cells generated more reactive oxygen species and displayed increased presence of oxidatively modified DNA (8-hydroxy-2'-deoxyguanosine). These results demonstrate that early onset of senescence in omentum-derived HPMC may be associated with oxidative stress-induced upregulation of p16INK4a.

Expression of connective tissue growth factor, a biomarker in senescence of human diploid fibroblasts, is up-regulated by a transforming growth factor-β-mediated signaling pathway

Molecular changes associated with cellular senescence in human diploid fibroblasts (HDF), IMR-90, were analyzed by two-dimensional differential proteome analysis. A high percentage of replicative senescent cells were positive for senescence-associated β-galactosidase activity, and displayed elevated levels of p21 and p53 proteins. Comparison of early population doubling level (PDL) versus replicative senescent cells among the 1000 spots resolved on gels revealed that the signal intensities of six spots were increased fivefold, whereas those of four spots were decreased. Proteome analysis data demonstrated that connective tissue growth factor (CTGF) is an age-associated protein. Up-regulation of CTGF expression in senescent cells was further confirmed by Western blotting and RT-PCR. We postulate that CTGF expression is controlled, in part, by transforming growth factor-β (TGF-β), in view of the high levels of TGF-β isoforms as well as type I and II receptors detected only in late PDL of HDF cells. To verify this hypothesis, we stimulated early PDL cells with TGF-β1 as well as stress inducing agents such as hydrogen peroxide. As expected, CTGF expression and Smad protein phosphorylation were dramatically increased up to observed levels in normal replicative senescent cells. In vivo experiments disclosed that CTGF, pSmad, and p53 were constitutively expressed at basal levels in up to 18-month-old rat liver, and expression was significantly up-regulated in 24-month-old rat tissue. However, expression patterns were not altered at all periods examined in livers of caloric-restricted rats. In view of both in vitro and in vivo data, we propose that the TGF-β/Smad pathway functions in the induction of CTGF, a novel biomarker protein of cellular senescence in human fibroblasts.

Loss of EPC-1/PEDF Expression During Skin Aging In Vivo

EPC-1/PEDF (early population doubling level cDNA-1/retinal pigmented epithelium-derived factor) is a single-copy, quiescence-specific gene that is transcribed into a 1.5 kb mRNA and then translated into a 50 kDa secreted protein that is a potent inhibitor of angiogenesis. EPC-1 expression has been detected in a number of cultured cell lines, including lung and skin fibroblasts, retinal pigmented epithelial cells, and endometrial stromal fibroblasts. Furthermore, its expression has been shown to decline during replicative aging of these cells in culture. In this report, we describe our examination of the age-related changes in EPC-1 expression in situ in skin sections from donors of different ages. EPC-1 mRNA is detected primarily in the dermal layer of the skin and its expression declines with increasing donor age. This decline is statistically significant between young (less than 31 years old) and middle-aged (between 30 and 60 years old) donors, with the decline becoming less dramatic at older ages. This age-related decline in the expression of an angiogenic inhibitor contributes to the imbalance of angiogenic modulators that is observed during aging. In fact, this decline may reflect a compensatory change to help reverse the decline of angiogenesis marked by reduced abundance of microvessels. This downregulation of an angiogenesis inhibitor may, in turn, play a critical role in the development of diseases caused by abnormal vascularization. The potential role of the age-associated decline in EPC-1 expression in tissue remodeling and in the development of skin diseases with excessive angiogenesis may provide new insights into disease prevention.

Putative role for EPC‐1/PEDF in the G0 growth arrest of human diploid fibroblasts

EPC-1/PEDF expression is closely associated with reversible growth arrest in normal human diploid fibroblast-like (HDF) cells and is diminished with proliferative senescence in vitro. EPC-1 expression in HDF cells is induced under conditions of density-dependent contact inhibition and growth factor deprivation. Antiserum generated against EPC-1 recognizes a secreted protein of approximately 50 kDa from medium conditioned by early passage HDF cells, but not from senescent cells. The addition of EPC-1 antiserum to early population doubling level (PDL) cultures near the plateau phase of growth significantly increases the number of cells entering DNA synthesis. Affinity purified EPC-1 antibodies alone enhance the ability of near plateau-phase early PDL WI-38 cells to synthesize DNA by as much as threefold. Further, the addition of recombinant EPC-1 (rEPC-1) to logarithmically growing cells resulted in a marked decrease in the ability of these cells to enter DNA synthesis. We also demonstrate the loss of EPC-1 expression in WI-38 and IMR-90 HDF cell lines with both senescence and simian virus 40 (SV40) transformation. The loss of EPC-1 expression with SV40 transformation occurs at the level of steady-state mRNA and protein accumulation with genomic EPC-1 sequences grossly intact. Taken together, these results suggest that EPC-1 may play a role in the entry of early passage fibroblasts into a G(0) state or the maintenance of such a state once reached.

Extension of cell life-span and telomere length in animals cloned from senescent somatic cells.

The potential of cloning depends in part on whether the procedure can reverse cellular aging and restore somatic cells to a phenotypically youthful state. Here, we report the birth of six healthy cloned calves derived from populations of senescent donor somatic cells. Nuclear transfer extended the replicative life-span of senescent cells (zero to four population doublings remaining) to greater than 90 population doublings. Early population doubling level complementary DNA-1 (EPC-1, an age-dependent gene) expression in cells from the cloned animals was 3.5- to 5-fold higher than that in cells from age-matched (5 to 10 months old) controls. Southern blot and flow cytometric analyses indicated that the telomeres were also extended beyond those of newborn (<2 weeks old) and age-matched control animals. The ability to regenerate animals and cells may have important implications for medicine and the study of mammalian aging.

Regulation of EPC-1/PEDF in normal human fibroblasts is posttranscriptional.

The EPC-1 (early population doubling level cDNA-1) gene, also known as pigment epithelium-derived factor, encodes a protein belonging to the serine protease inhibitor (serpin) superfamily that has been reported to inhibit angiogenesis and proliferation of several cell types. We have previously reported that the EPC-1 mRNA and the secreted EPC-1 protein are expressed at levels more than 100-fold higher in early passage, G(0), WI-38 cells compared to either proliferating or senescent WI-38 fibroblasts. To examine the molecular mechanisms that regulate changes in EPC-1 gene expression in WI-38 cells, we isolated and characterized the human EPC-1 gene and determined the mRNA cap site. Transcriptional assays showed no change in the transcription rates of EPC-1 between young proliferating, quiescent, and senescent WI-38 cells. These results suggest posttranscriptional regulation of the EPC-1 gene. Reverse transcriptase polymerase chain reaction measurements (of hnRNA) indicate regulation at the hnRNA level. The regulation of the EPC-1 gene at the level of hnRNA can explain the observed slow increase in the steady-state EPC-1 mRNA levels when cells become quiescent. The reduction of EPC-1 mRNA levels that occurs when cells exit G(0) and are induced to proliferate can be accounted for by a reduction of the EPC-1 mRNA stability in stimulated cells as compared to quiescent cells.

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.

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 G0cultures. 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 G0upon 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.

Immortalization of immunologically committed Epstein-Barr virus-transformed human B-lymphoblastoid cell lines accompanied by a strong telomerase activity

The immunological characteristics and immortalization processes of three EBV-transformed human B-lymphoblastoid cell lines, N0003, N0005 and N6803, with strong telomerase and infinitively proliferating activities are described. The three cell lines were apparently immortalized: they developed a strong telomerase activity at the population doubling levels (PDLs) between 11 and 135, and continued proliferation over 250 PDLs. All the cell lines expressed CD22, CD19 and CD20 antigens. They were uniformly stained with IgM (N0005), IgG (N6803) or IgA (N0003) at early PDLs between 17 and 20, and they secreted the corresponding class of Ig into the medium; the N6803 and N0003 cell lines continued to secrete each class of Ig at decreased levels while the N0005 cell line expressed or secreted virtually no Ig after immortalization. Karyotype analysis of the immortalized cell lines showed that they were derived from a single cell because they shared a set of abnormal chromosomes within each cell population, and two of the cell lines attained clonal characteristics before they developed a strong telomerase activity. These results indicate that the three immortalized cell lines with a strong telomerase activity correspond to the intermediate stages of B-cell differentiation naturally committed to a specific Ig class, and suggest that they were derived from a B-lymphoblastoid cell committed to a specific class of Ig with poor telomerase activity, rather than from a strongly telomerase-positive B-lymphoblastoid cell either committed or multipotential.

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 αI (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.

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.

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.

Modulation of purine synthesis and phosphoribosyl-pyrophosphate content in human fibroblasts at different population doublings

Human diploid fibroblasts of embryonic origin which display the limited lifespan phenomenon, respond to purine base deprivation with a large accumulation of phosphoribosylpyrophosphate when studied at early population doubling levels. This suggests that the purine salvage enzymes are the main consumers of phosphoribosylpyrophosphate in such cells. There is a strong positive correlation between the rate of de novo purine synthesis and the phosphoribosylpyrophosphate content, consistent with its role in the first and rate-limiting reaction of de novo purine synthesis. With increasing population doublings, the cells become less responsive to purine base deprivation; the phosphoribosylpyrophosphate does not increase significantly and a lower rate of de novo purine synthesis is observed. As the rate of cell replication decreases with serial passages, it becomes apparent that such cells appropriately adjust the concentration of phosphoribosylpyrophosphate, thereby controlling the rate of purine synthesis according to their needs for cellular growth.

Comparative studies between microbiological culture and uptake of uridine/uracil to detect mycoplasmal infection of cell cultures

Controlled, prospective studies were performed to compare detection of cell culture mycoplasmas by ratio of uptake of tritiated uridine (UdR) to tritiated uracil (U) and by microbiological culture. Culture was by standard agar and broth inoculation with aerobic and anaerobic incubation; immunofluorescent staining of indicator cell cultures was used to detect M. hyorhinis. The ratio of uptake of UdR to U (UdRU) and interpretation of test results were by standard published methods and performed in triplicate. 115 cell cultures were simultaneously assayed by the two techniques. 84 cultures (73.1%) yielded agreement between the 2 methods; 2 cultures (1.7%) yielded conflicting results, and 29 cultures (25.2%) yielded UdRU results in the questionable range. Conflicting results consisted of two negative UdRU tests in mouse cell cultures infected with M. orale. In separate studies, 3T-6 cultures freshly infected with M. orale yielded negative UdRU results 3 days after infection, questionable results after 10 days and a positive UdRU 17 days after infection. UdRU detected infection in fibroblast, epithelial, and lymphocyte cell cultures. Highest UdRU ratios were detected in human skin fibroblasts at early population doubling levels (PDLs), 4064 in one culture at PDL4. UdRU was determined for IMR-90, a human diploid fibroblast at 12 different PDLs using the same lot of media. UdRU gradually decreased throughout the life of the culture, from 2 125 at PDL6 to 340 at PDL36. Cultures in phase III and others exhibiting poor growth frequently yielded questionable or false-positive UdRU results and were not included in tabulations of these results. UdRU determined in endothelial cell cultures decreased as population density increased. In a representative experiment performed over a 4-day period, the UdRU values were 1 808, 955 and 356 when the number of endothelial cells in culture were 5.3 × 105, 6.6 × 105 and 1.1 × 106, respectively.

Proliferative response of human diploid fibroblasts to intermittent light exposure.

Three- to four-hour exposure to fluorescent light, one to three times weekly, reproducibly enhanced the proliferation rate of human diploid fibroblasts. This enhancement was observed in WI-38 and a line from whole embryo mince at late population doubling level (PDL) as well as in a line from adult skin at early PDL. Single or multiple exposures of short duration stimulated proliferation, whereas exposures of long duration were cytotoxic. This proliferative response is reversible, and is mediated through the culture medium, Dulbecco Vogt's supplemented with 10% fetal bovine serum. Apparently light produces some mitogenic substance(s) in the culture medium that accumulates in the cells and is toxic or growth-stimulatory depending on its concentration per cell. Another possibility is that light produces in the medium both cytotoxic and growth-stimulatory substances.

DNA-binding proteins in young and senescent normal human fibroblasts

DNA-binding proteins (DBP) from normal human diploid cells, strain WI38, were isolated by DNA-cellulose chromatography using undenatured calf thymus DNA. The DBP in the 0.15 M NaCl eluate were fractionated by polyacrylamide gel electrophoresis. Comparisons of the amounts of the DBP in different cell populations were made by labelling the cells with either 3H- or 14C-amino acid precursors for 40 h prior to pooling the cells for co-isolation of their DBP. When WI38 cells in the replicative and stationary phases were compared, five proteins, P5b (87 000 D), P6a (50 000 D), P8 (33 000 D), P9 (28 000 D) and P10 (25 000 D) were labelled to a greater extent in the replicating cells and two proteins, P5c (72 000 D) and P12 (18 000 D) were labelled to a greater extent in the stationary phase cells. In addition, several high molecular weight DBP, partially characterized as collagen and protocollagen, were preferentially labelled in the stationary phase cells. Stationary phase senescent WI38 cells at or near the end of their in vitro lifespan characteristically showed an increased proportion of protein component P8 (33 000 D) relative to stationary phase WI38 cells at early population doubling levels. Further characterization of WI38-P8 showed that it binds preferentially to single-stranded DNA and amounts to greater than 1% of the total soluble protein in young cells in growth phase. Thus WI38-P8 appears to be comparable to the P8 protein studied by Tsai & Green [27] in mouse 3T6 and human SB cells. The component which is increased in senescent or terminal phase non-dividing cell populations is judged to be the P8 protein by its position in SDS-gels and its preferential binding to single-stranded DNA.