Changes of serum-induced ornithine decarboxylase activity and putrescine content during aging of IMR-90 human diploid fibroblasts

The roles of ornithine decarboxylase (ODC, EC 4.1.1.17) and polyamines in cellular aging were investigated by examining serum-induced changes of these parameters in quiescent IMR-90 human diploid fibroblasts as a function of their population doubling level (PDL) and in human progeria fibroblasts. Serum stimulation caused increases of ODC and DNA synthesis in IMR-90 human diploid fibroblasts, with maximal values occurring, respectively, 10 hr and 22 hr after serum stimulation. Both serum-induced ODC activity and DNA synthesis in IMR-90 cells were found to be inversely related to their PDL. Maximal ODC activity and DNA synthesis in young cells (PDL = approximately 18-22) were, respectively, five-fold and six-fold greater than that in old cells (PDL = approximately 50-55), which in turn were comparable or slightly higher than that in progeria fibroblasts. Polyamine contents (putrescine, spermidine, and spermine) in quiescent IMR-90 cells did not show significant PDL-dependency. The putrescine and spermine contents in quiescent progeria cells were comparable to those in quiescent IMR-90 cells. The spermidine content in quiescent progeria cells, however, was extremely low, less than half of that in quiescent IMR-90 cells. Serum stimulation caused a marked increase in putrescine content in young cells but not in old cells or in progeria cells. The spermidine and the spermine content in IMR-90 cells, either young or old, and in progeria cells did not change significantly after serum stimulation. Our study indicated that aging of IMR-90 human diploid fibroblasts was accompanied by specific changes of polyamine metabolism, namely, the serum-induced ODC activity and putrescine accumulation. These changes were also observed in progeria fibroblasts derived from patients with Hutchinson-Gilford syndrome.     

Periodontal ligament cell kinetics following orthodontic tooth movement.

The population of periodontal ligament (PDL) fibroblasts examined in this study may include osteogenic progenitor cells. PDL fibroblast and osteoblast kinetics in the periodontal ligament of the rat were measured following orthodontic stimulation of bone formation. Both single and multiple injections of tritiated thymidine (3H-TdR) were used. In single injection experiments, the peak percentage of PDL fibroblasts labeled with 3H-TdR is 15% at 22 hr post-stimulation. In multiple injection experiments, the total percentage of fibroblasts in the PDL which respond by synthesizing DNA is 50%. 3H-TdR-labeled osteoblasts appear at the same rate as, but with a time delay after, the labeled fibroblasts. Following stimulation, the most likely source of osteoblasts at the bbone forming site is not only fibroblasts which make DNA, divide, then differentiate, but also fibroblasts which either are differentiated to osteoblasts without DNA synthesis and cell division, or are released from G2 block by the orthodontic stimulation.     

PERIODONTAL LIGAMENT CELL KINETICS FOLLOWING ORTHODONTIC TOOTH MOVEMENT

The population of periodontal ligament (PDL) fibroblasts examined in this study may include osteogenic progenitor cells. PDL fibroblast and osteoblast kinetics in the periodontal ligament of the rat were measured following orthodontic stimulation of bone formation. Both single and multiple injections of tritiated thymidine (³H-TdR) were used. In single injection experiments, the peak percentage of PDL fibroblasts labeled with ³H-TdR is 15% at 22 hr post-stimulation. In multiple injection experiments, the total percentage of fibroblasts in the PDL which respond by synthesizing DNA is 50%. ³H-TdR-Iabeled osteoblasts appear at the same rate as, but with a time delay after, the labeled fibroblasts. Following stimulation, the most likely source of osteoblasts at the bone-forming site is not only fibroblasts which make DNA, divide, then differentiate, but also fibroblasts which either are differentiated to osteoblasts without DNA synthesis and cell division, or are released from G₂ block by the orthodontic stimulation.     

Stiffening of human skin fibroblasts with age

Changes in mechanical properties are an essential characteristic of the aging process of human skin. Previous studies attribute these changes predominantly to the altered collagen and elastin organization and density of the extracellular matrix. Here, we show that individual dermal fibroblasts also exhibit a significant increase in stiffness during aging in vivo. With the laser-based optical cell stretcher we examined the viscoelastic biomechanics of dermal fibroblasts isolated from 14 human donors aged 27 to 80. Increasing age was clearly accompanied by a stiffening of the investigated cells. We found that fibroblasts from old donors exhibited an increase in rigidity of ∼60% with respect to cells of the youngest donors. A FACS analysis of the content of the cytoskeletal polymers shows a shift from monomeric G-actin to polymerized, filamentous F-actin, but no significant changes in the vimentin and microtubule content. The rheological analysis of fibroblast-populated collagen gels demonstrates that cell stiffening directly results in altered viscoelastic properties of the collagen matrix. These results identify a new mechanism that may contribute to the age-related impairment of elastic properties in human skin. The altered mechanical behavior might influence cell functions involving the cytoskeleton, such as contractility, motility, and proliferation, which are essential for reorganization of the extracellular matrix.     

Altered regulation of platelet-derived growth factor A-chain and c-fos gene expression in senescent progeria fibroblasts

The study of human genetic disorders known as premature aging syndromes may provide insight into the mechanisms of cellular senescence. These diseases are clinically characterized by the premature onset and accelerated progression of numerous features normally associated with human aging. Previous studies have indicated that fibroblasts derived from premature aging syndrome patients have in vitro growth properties similar to senescent fibroblasts from normal individuals. As an initial approach to determine whether gene expression is altered in premature aging syndrome fibroblasts, RNA was prepared from various cell strains and used for gel blot hybridization experiments. Although normal fibroblasts only express platelet-derived growth factor (PDGF) A-chain mRNA for a brief period following mitogenic stimulation, one strain of Hutchinson-Gilford (progeria) syndrome fibroblasts, AG3513, constitutively expresses PDGF A-chain mRNA and PDGF-AA homodimers. The PDGF A-chain gene does not appear to be amplified or rearranged in these fibroblasts. AG3513 progeria fibroblasts have properties characteristic of senescent cells, including an altered morphology and a diminished mitogenic response to growth promoters. The diminished response of AG3513 progeria fibroblasts to PDGF stimulation was examined in some detail. Studies using 125I-PDGF-BB, which binds with high affinity to both A- and B-type PDGF receptors, indicate that normal and AG3513 progeria fibroblasts have a similar number of PDGF receptors. Although receptor autophosphorylation occurs normally in PDGF-stimulated AG3513 progeria fibroblasts, c-fos mRNA induction does not. The senescent phenotype of AG3513 fibroblasts is probably unrelated to their constitutive PDGF A-chain gene expression; further studies are necessary in order to directly address this issue. Also, additional analysis of this progeria fibroblast strain may provide information on the control of mitogen-inducible gene expression in normal cells.     

In vitro studies of age-associated diseases.

We have carried out studies on cultured human fibroblasts in an attempt to trace the origins of age-dependent disorders to the cellular and molecular levels. Three interrelated areas are discussed. First, skin donors with diabetes mellitus (a disease complex that features inappropriate hyperglycemia) produce cultured fibroblasts with a moderate reduction in growth capacity, while two inherited disorders of inappropriate hyperglycemia and premature aging, progeria and Werner syndrome, yield fibroblast cultures with more severely impaired growth capacity. Second, there is a decreased response of progeria level and donor age; evidence is presented that this defective hormone responsiveness in aging cells may reside at the hormone receptor on the surface membrane, the cyclic AMP system, the intracellular enzymatic machinery, or all of these sites. Third, tissue factor, a procoagulant that activates the extrinsic clotting mechanism, is more abundant in cells from the premature aging syndromes of progeria and Werner syndrome. Fibroblast aging in vitro may help to explain various concomitants of normal aging and diabetes mellitus including cell dropout, impairment of hormone responsiveness, and increased atherothrombosis.     

Isolation and characterization of cultured human periodental ligament fibroblast-specific cDNAs

The molecular mechanisms that control the function of periodontal ligament (PDL) fibroblasts remain unclear. We speculated that the character of differentiating PDL fibroblasts is defined by the altered expansion of specific genes not found in neighboring gingival fibroblasts in the periodontium. To expand this set, subtractive hybridization was applied between cultured human PDL and gingival fibroblasts to identify genes differentially expressed in PDL. Consequently five candidate clones, PDLs (periodontal ligament specific) 5, -17, -22, -25, and -31 were identified and characterized by homology search, Northern analysis, and in situ hybridization. Although the mRNAs of these clones were expressed by bone marrow cells and rarely by gingival fibroblasts, the highest expression was detected in the PDL cells, which were uniformly distributed throughout the whole PDL. Amongst the five candidate clones, we focused on PDLs17, because it is a hypothetical protein whose biological function has not been reported yet in the database. Polyclonal antiserum raised against PDLs17 peptide was made, and stained the PDL fibroblasts, osteoblast-like cells and stromal cells in the bone marrow, but not gingival fibroblasts. The results suggest that clones, PDLs5, -17, -22, -25, and -31 may be used as PDL fibroblast-specific markers, and that PDLs17 could act as an important factor in the differentiation process of PDL fibroblasts.     

Delayed kinetics of DNA double-strand break processing in normal and pathological aging

Accumulation of DNA damage may play an essential role in both cellular senescence and organismal aging. The ability of cells to sense and repair DNA damage declines with age. However, the underlying molecular mechanism for this age-dependent decline is still elusive. To understand quantitative and qualitative changes in the DNA damage response during human aging, DNA damage-induced foci of phosphorylated histone H2AX (γ-H2AX), which occurs specifically at sites of DNA double-strand breaks (DSBs) and eroded telomeres, were examined in human young and senescing fibroblasts, and in lymphocytes of peripheral blood. Here, we show that the incidence of endogenous γ-H2AX foci increases with age. Fibroblasts taken from patients with Werner syndrome, a disorder associated with premature aging, genomic instability and increased incidence of cancer, exhibited considerably higher incidence of γ-H2AX foci than those taken from normal donors of comparable age. Further increases in γ-H2AX focal incidence occurred in culture as both normal and Werner syndrome fibroblasts progressed toward senescence. The rates of recruitment of DSB repair proteins to γ-H2AX foci correlated inversely with age for both normal and Werner syndrome donors, perhaps due in part to the slower growth of γ-H2AX foci in older donors. Because genomic stability may depend on the efficient processing of DSBs, and hence the rapid formation of γ-H2AX foci and the rapid accumulation of DSB repair proteins on these foci at sites of nascent DSBs, our findings suggest that decreasing efficiency in these processes may contribute to genome instability associated with normal and pathological aging.     

The susceptibility of Werner's syndrome and other human skin fibroblasts to SV40-induced transformation and immortalization

In attempts to transform and immortalize human cell cultures, skin fibroblasts from normal donors of different ages, from patients with the premature ageing diseases Werner's syndrome (WS) and progeria (PR), and from donors with the cancer-prone diseases ataxia telangiectasia (AT), Bloom's syndrome (BS) and Fanconi's anaemia (FA), were infected with SV40 virus and their growth monitored thereafter. Lesch-Nyhan (LN) fibroblasts were also infected. SV40-infected cultures from two normal and from WS, AT and LN donors attained a spectrum of transformed properties, high mitotic activity at confluence, presence of T-antigen, anchorage independence and altered morphology. Most of these pretransformed cultures died in the crisis period. However, two cultures from the WS and LN patients survived the crisis period and have now been grown to more than 200 passages. For the LN culture the crisis period was at least 200 days. Both permanent lines retain the properties of pretransformed cells, but differ in their modal chromosome number and ability to grow in methionine-free medium. It can be concluded from these experiments that transformation by SV40 to permanent lines is a rare event in human skin fibroblasts, even when these cells were taken from patients predisposed to form cancers.     

Increased monocytic adhesion by senescence in human umbilical vein endothelial cells

We investigated whether replicative senescence of endothelial cells contributed to the pathogenesis of atherosclerosis in human umbilical vein endothelial cells (HUVECs). HUVECs at a population-doubling level of 30 (PDL30) divided much more slowly than those at PDL9. The percentage of SA-β-Gal-positive cells and the mRNA expression levels of PAI-1 and p21 at PDL30 were significantly higher than those at PDL9. The changes induced by aging were evaluated according to the mRNA expression level of genes related to the endothelial cell function. The expression level of many adhesion molecules promoting monocytic adhesion was significantly increased, and monocytic adhesion on HUVECs was found to be significantly promoted by aging. Monocytic adhesion is an essential early event in the development of atherosclerosis, and our results suggest that replicative senescence of the vascular endothelial cells induced increased expression of adhesion molecules. The consequent increase in monocytic adhesion may then promote the pathogenesis of atherosclerosis.     

Changes in chromatin structure during aging of human skin fibroblasts

Human skin fibroblasts from embryo, 16-, 30- and 60-year-old adults were cultivated and passaged in vitro. Their chromatin structures were examined by the sensitivity to micrococcal nuclease and by electron microscopy. When the mode of DNA degradation by the nuclease was analysed during in vitro aging of the embryo skin fibroblasts, the discrete ladder of nucleosomal DNA became obscure in old cells. Analogous change of chromatin structure was also observed even in young cells as their donor ages increased. From the observation with electron microscopy, it became clear that chromatin of fibroblasts from 30-year-old adults does not have regularly spaced nucleosomes, compared with chromatin from embryo. These results suggest that the length of the linker DNA which connects core particles becomes to be heterogeneous by aging, both in vivo and in vitro in human skin fibroblasts.     

Evaluation of the reorganization in the high-order structure of DNA occurring during cell senescence

Electron microscopic pictures of loosened chromatin obtained from human fibroblasts aged in vitro were analyzed with an image processor. It was found that the density of the 30-nm chromatin fibers decreased progressively through the cell population lifespan. The spacing between the fibers increased abruptly during the last divisions (phase IV). The evolution of these 2 parameters, density and spacing, was identical, respectively, with that of the decline in the rate of initiating DNA synthesis after seeding the cells and with that of the maximal percent of cells synthesizing DNA during a 24-h period between seeding and resting phase. Cells from 2 human donors (30 and 60 years old) with Werner's syndrome were compared, using the same methodology, with those of 9 normal donors ranging from 29 to 98 years old. The spacing between chromatin fibers in the Werner's cells was significantly increased as compared to the age-matched normal controls while the density was within the normal range. The results are discussed with regard to other modifications in DNA structure previously reported in the same cell system.     

Expression of connective tissue growth factor, a biomarker in senescence of human diploid fibroblasts, is up-regulated by a transforming growth factor-beta-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 beta-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-beta (TGF-beta), in view of the high levels of TGF-beta 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-beta1 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-beta/Smad pathway functions in the induction of CTGF, a novel biomarker protein of cellular senescence in human fibroblasts.     

Increased Monocytic Adhesion by Senescence in Human Umbilical Vein Endothelial Cells

We investigated whether replicative senescence of endothelial cells contributed to the pathogenesis of atherosclerosis in human umbilical vein endothelial cells (HUVECs). HUVECs at a population-doubling level of 30 (PDL30) divided much more slowly than those at PDL9. The percentage of SA-β-Gal-positive cells and the mRNA expression levels of PAI-1 and p21 at PDL30 were significantly higher than those at PDL9. The changes induced by aging were evaluated according to the mRNA expression level of genes related to the endothelial cell function. The expression level of many adhesion molecules promoting monocytic adhesion was significantly increased, and monocytic adhesion on HUVECs was found to be significantly promoted by aging. Monocytic adhesion is an essential early event in the development of atherosclerosis, and our results suggest that replicative senescence of the vascular endothelial cells induced increased expression of adhesion molecules. The consequent increase in monocytic adhesion may then promote the pathogenesis of atherosclerosis.     

Senescent phenotypes of skin fibroblasts from patients with Tangier disease

Tangier disease (TD) is characterized by a deficiency of high density lipoprotein (HDL) in plasma and patients with TD have an increased risk for coronary artery disease (CAD). Recently, we reported that fibroblasts from TD exhibited large and flattened morphology, which is often observed in senescent cells. On the other hand, data have accumulated to show the relationship between cellular senescence and development of atherosclerotic CAD. The aim of the present study was to investigate whether TD fibroblasts exhibited cellular senescence. The proliferation of TD fibroblasts was gradually decreased at population doubling level (PDL) approximately 10 compared with control cells. TD cells practically ceased proliferation at PDL approximately 30. DNA synthesis was markedly decreased in TD fibroblasts. TD cells exhibited a higher positive rate for senescence-associated beta-galactosidase (SA-beta-gal), which is one of the biomarkers of cellular senescence in vitro. These data showed that TD cells reached cellular senescence at an earlier PDL compared with controls. Although, there was no difference in the telomere length of fibroblasts between TD and controls at the earlier passage (PDL 6), the telomere length of TD cells was shorter than that of controls at the late passage (PDL 25). Taken together, the current study demonstrates that the late-passaged TD fibroblasts showed senescent phenotype in vitro, which might be related to the increased cardiovascular manifestations in TD patients.     

Gram Staining Applied to Human Spermatozoa: a Simple Method for Studying Chromatin Condensation Status

Gram staining applied to human spermatozoa from fertile donors is described. The stain revealed populations of Gram-positive and Gram-negative spermatozoa. Data showed a significant and progressive decrease in the percentage of Gram-positive spermatozoa at different times during the chromatin decondensation procedure (SDS-BSA and SDS-EDTA). No significant correlation could be found between Gram staining and other functional tests used for spermatozoa; only the aniline blue staining test showed a poor correlation. Our study demonstrates that normal spermatozoa with regular chromatin condensation appear Gram-positive, while spermatozoa with altered chromatin condensation appear Gramnegative.     

Enhanced nuclear protein export in premature aging and rescue of the progeria phenotype by modulation of CRM1 activity

The study of Hutchinson–Gilford progeria syndrome (HGPS) has provided important clues to decipher mechanisms underlying aging. Progerin, a mutant lamin A, disrupts nuclear envelope structure/function, with further impairment of multiple processes that culminate in senescence. Here, we demonstrate that the nuclear protein export pathway is exacerbated in HGPS, due to progerin‐driven overexpression of CRM1, thereby disturbing nucleocytoplasmic partitioning of CRM1‐target proteins. Enhanced nuclear export is central in HGPS, since pharmacological inhibition of CRM1 alleviates all aging hallmarks analyzed, including senescent cellular morphology, lamin B1 downregulation, loss of heterochromatin, nuclear morphology defects, and expanded nucleoli. Exogenous overexpression of CRM1 on the other hand recapitulates the HGPS cellular phenotype in normal fibroblasts. CRM1 levels/activity increases with age in fibroblasts from healthy donors, indicating that altered nuclear export is a common hallmark of pathological and physiological aging. Collectively, our findings provide novel insights into HGPS pathophysiology, identifying CRM1 as potential therapeutic target in HGPS.     

Ultrastructural organization of nucleoproteins during aging of cultured human embryonic fibroblasts

Analysis of nucleoproteins in resting human embryonic fibroblasts in vitro at different population doubling levels (PDL) using electron microscopy revealed the disappearance of non-nucleolar ribonucleoprotein structures at high PDL, the nucleoli became larger and the filamentous masses containing the nascent nucleolar RNA displayed a fibrillo-granular pattern which has never been described previously. In addition, conventional fixation revealed the disappearance of most of the stainable chromatin whose threads were unusually spaced and shortened specially at the nuclear surface after loosening. We interpret these changes in chromatin organization as the consequence of the alkali-sensitive sites that accumulate during senescence.     

Kinetics of chick embryo cell types in culture

The growth kinetics and population doubling limits of chick embryonic fibroblasts, chondroblasts, and retinal pigment cells were compared. Chondroblasts were found to have a cumulative population doubling level (37 +/- 3 PDL) similar (p = 0.05) to that of control fibroblasts (42 +/- 2 PDL), in individual and pooled clones. While both cell types have similar doubling potential, the proportion of tritium-labeled nuclei decreases, and differs significantly as doubling level increases. This age-associated decline is due to an extension in the population doubling time. Direct cell-cycle analysis shows this increase to occur in the G1 phase. Furthermore, cartilage colonies maintain their phenotypic expression (metachromasia) throughout their lifespan under conditions of subcloning at sparse density. When fibroblasts derived from 15 day chick embryos are compared with fibroblasts from 10 day embryos (41 +/- 2 PDL) there is no significant difference (p = 0.05) in cumulative PDL or percent labeled nuclei, indicating that fibroblasts of different embryonic age have similar potential. The addition of hydrocortisone and insulin to the medium significantly shortens (25 +/- 2 PDL) the lifespan of 10 day chick fibroblasts. Kinetics of retinal pigment cells show a population doubling potential (29 +/- 1 PDL) different from fibroblasts and chondroblasts, suggesting that different cell types may not have similar limits on doubling potential when first determined in embryogenesis. Hydrocortisone and insulin have no effect on the growth kinetics or lifespan of retinal pigment cells in culture.