Interrelation between cellular rRNA content and regulation of the cell cycle of normal and transformed mouse cell lines

The relation between cellular rRNA content, as a measure of cell size, and the regulation of the cell cycle has been investigated for swiss 3T3 and the spontaneously transformed swiss 3T6 cell line. It is shown that the characteristic of percent of quiescent cells stimulated into the cell cycle versus cellular rRNA content is basically different for 3T3 and 3T6 cells: 3T3 cells do not enter the cell cycle below a certain threshold of cellular rRNA content, whereas 3T6 cells start proliferation without a substantial increase of rRNA. These data are interpreted as consistent with transformation of 3T6 cells being in essence their uncoupling from the requirement of normal cells of passing over a threshold of cellular rRNA content (cell size) before initiating DNA-replication.     

Cellular content of ribosomal RNA in relation to the progression and competence signals governing proliferation of 3T3 and SV40-3T3 cells

The method for differential fluorescence staining of cellular RNA and DNA by acridine orange (AO) was optimized for 3T3 and SV40-3T3 cells. Cellular contents of DNA and of ribosomal RNA (rRNA) were determined by dual-channel flow cytometry during cell-density-dependent proliferation and after stimulation of quiescent cells. With increasing density of 3T3 cells, cellular content of rRNA decreases by about 60%, whereas SV40-3T3 cells do not exhibit a significant dependence of rRNA content on cell density. 3T3 cells stimulated early after becoming quiescent resume reaccumulation of rRNA after a delay of only 4 h, whereas cells maintained at quiescence for several days exhibit a delay of about 12 h before a significant rise of rRNA is observed. The extent of rise of cellular rRNA content after different regimens of stimulation of quiescent 3T3 cells does not correlate well with the fraction of cells entering the cell cycle. These and other reported instances of discordance between rRNA content and stimulation into the cell cycle are resolved by showing that of the two signals governing entry into the cell cycle only the progression signal, but not the competence signal is associated with reaccumulation of cellular rRNA. The present results are consistent with the progression function being in essence the achievement of a threshold number of ribosomes per cell, which in conjunction with the competence signal is sufficient for initiation of the cell cycle.     

Accumulation of oxidative DNA damage, 8-oxo-2'-deoxyguanosine, and change of repair systems during in vitro cellular aging of cultured human skin fibroblasts

Effects of in vitro cellular aging on the content of 8-oxo-2'-deoxyguanosine, a typical oxidation product of DNA bases, were examined in cultured human skin fibroblasts. The 8-oxo-2'-deoxyguanosine content in the DNA of TIG-3S cells established from skin tissues of a fetal donor increased immediately before the cessation of proliferation. TIG-114 and TIG-104 cells established from skin tissues of adult and aged donors, respectively, showed similar changes in 8-oxo-2'-deoxyguanosine content during in vitro cellular aging. The accumulation of 8-oxo-2'-deoxyguanosine in late-passage cells was dependent on the number of cell divisions, and not on the cultivation time. Increases in the activities of superoxide dismutase and glutathione peroxidase were observed prior to the increase in 8-oxo-2'-deoxyguanosine content, while the catalase activity decreased gradually during in vitro cellular aging at late-passage. Furthermore, the activities of 8-oxo-2'-deoxyguanosine endonuclease and DNA polymerases decreased with the progression of proliferation. These results indicate that defense systems against oxidative stress in late-passage cells remain sufficiently active before the cessation of cell division, but that repair systems against oxidative damage decay at late-passage. Oxidative stress beyond the antioxidant capacity and/or repair activity seems to result in an accumulation of 8-oxo-2'-deoxyguanosine in late-passage cells.     

Gastroprotective and ulcer-healing effect of new solidagenone derivatives in human cell cultures

The labdane diterpene solidagenone 1 and its semisynthetic and biotransformation derivatives 2-10 were assessed for gastroprotective and ulcer-healing effect using human epithelial gastric cells (AGS) and human lung fibroblasts (MRC-5). The ability of the compounds to protect the AGS cells against the damage induced by sodium taurocholate (NaT), to stimulate the cellular reduced glutathione (GSH), prostaglandin E(2) content, enhance AGS and MRC-5 cell proliferation and to scavenge superoxide anion in vitro was studied. The cytotoxicity of the compounds was assessed towards MRC-5 fibroblasts and AGS cells. A significant reduction of cell damage after NaT incubation was observed when the AGS cells were pretreated with compounds 2 and 6. Treatment with compounds 4-6, 8 and 9 significantly stimulated the GSH content in AGS cell cultures. None of the studied compounds was active as a superoxide anion scavenger. In AGS cells treated with compounds 1-10, only compound 5 was able to increase prostaglandin content. Concerning the proliferation assays, a significant stimulating effect was observed for compounds 2, 8, 9 on AGS cells and for 5, 7-9 on MRC-5 fibroblasts. Regarding cytotoxicity, solidagenone showed higher toxicity while compounds 4 and 7 were the less toxic. Our results showed that most of the studied compounds act in vitro as gastroprotectors increasing the cellular GSH content. Additionally, some derivatives exhibited in vitro ulcer-healing effect stimulating the cell proliferation.     

The cytotoxic effects of a novel IH636 grape seed proanthocyanidin extract on cultured human cancer cells

This study was conducted to investigate the effects of aging on collagen and collagenase expression by human dermal fibroblasts. To evaluate this effect, the expression of these ECM was determined and compared between either fetal and adult fibroblasts or dermal fibroblasts at various passages. A total of 13 cell strains, 8 fetal foreskin and 5 adult dermal fibroblasts, were grown to 80-90% confluency and their rates of cell proliferation and expression of mRNA for collagenase (MMP-1) and pro 1(I) chain of type I collagen was determined and compared. Fetal cells had a significantly higher rate of proliferation relative to adult fibroblasts evaluated within 10 days of culture. Northern analysis was used to evaluate the steady state levels of mRNA in these cells. The result of these experiments revealed a significantly greater expression of mRNA for collagenase (58.6 ± 7.7 vs. 9.9 ± 1.5, p < 0.05) in strains of adult fibroblasts. This was consistent with collagenase activity of conditioned medium derived from adult cells relative to fetal fibroblasts. However the expression of pro 1(I) chain of type I collagen mRNA was not significantly (56.2 ± 5.2 vs. 58.5 ± 3.5) different between adult and fetal fibroblasts. This finding was confirmed by measuring total collagen production present in conditioned medium of these cells using hydroxyproline as an index for collagen production. The cellular response to IGF-1 and IFN-2b as representatives of fibrogenic and anti-fibrogenic factors were also evaluated. When expression of collagenase was used as an indication for cellular response, the degree of this response to IGF-1 but not IFN-2b was significantly greater in fetal relative to adult cells. Serial passage was also used as an in vitro model for aging fibroblasts and found a gradual reduction in pro 1(I) chain of type I collagen mRNA and hydroxyproline formation due to passaging. In conclusion, a slower rate of proliferation, a greater collagenase activity and expression of collagenase mRNA by aging fibroblasts could be some of the main reasons for attenuation of wound healing in elderly patients.     

Accelerated methylation of ribosomal RNA genes during the cellular senescence of Werner syndrome fibroblasts.

Ribosomal DNA (rDNA) metabolism has been implicated in cellular and organismal aging. The role of rDNA in premature and normal human aging was investigated by measuring rDNA gene copy number, the level of rDNA methylation, and rRNA expression during the in vitro senescence of primary fibroblasts from normal (young and old) donors and from Werner syndrome (WS) patients. In comparison to their normal counterparts, WS fibroblasts grew slowly and reached senescence after fewer doublings. The rDNA copy number did not change significantly throughout the life span of both normal and WS fibroblasts. However, in senescent WS and normal old fibroblasts, we detected rDNA species with unusually slow electrophoretic mobility. Cellular aging in Saccharomyces cerevisiae is accompanied by the formation and accumulation of rDNA circles. Our analysis revealed that the rDNA species observed in this study were longer, linear rDNA molecules attributable to the inhibition of ECO:RI cleavage by methylation. Furthermore, isoschizomeric restriction analysis confirmed that in vitro senescence of fibroblasts is accompanied by significant increases in cytosine methylation within rDNA genes. This increased methylation is maximal during the abbreviated life span of WS fibroblasts. Despite increased methylation of rDNA in senescent cells, the steady-state levels of 28S rRNA remained constant over the life span of both normal and WS fibroblasts.     

An analysis of replicative senescence in dermal fibroblasts derived from chronic leg wounds predicts that telomerase therapy would fail to reverse their disease-specific cellular and proteolytic phenotype

The accumulation of senescent fibroblasts within tissues has been suggested to play an important role in mediating impaired dermal wound healing, which is a major clinical problem in the aged population. The concept that replicative senescence in wound fibroblasts results in reduced proliferation and the failure of refractory wounds to respond to treatment has therefore been proposed. However, in the chronic wounds of aged patients the precise relationship between the observed alteration in cellular responses with aging and replicative senescence remains to be determined. Using assays to assess cellular proliferation, senescence-associated staining beta-galactosidase, telomere length, and extracellular matrix reorganizational ability, chronic wound fibroblasts demonstrated no evidence of senescence. Furthermore, analysis of in vitro senesced fibroblasts demonstrated cellular responses that were distinct and, in many cases, diametrically opposed from those exhibited by chronic wound fibroblasts. Forced expression of telomerase within senescent fibroblasts reversed the senescent cellular phenotype, inhibiting extracellular matrix reorganizational ability, attachment, and matrix metalloproteinase production and thus produced cells with impaired key wound healing properties. It would appear therefore that the distinct phenotype of chronic wound fibroblasts is not simply due to the aging process, mediated through replicative senescence, but instead reflects disease-specific cellular alterations of the fibroblasts themselves.     

Increased glutathione synthesis associated with platelet-derived growth factor stimulation of NIH3T3 fibroblasts

Previous data show a relation between GSH content and proliferation of normal and tumour cells. We recently demonstrated a specific involvement of GSH in the autophosphorylation activity of the platelet-derived growth factor (PDGF) receptor in NIH3T3 fibroblasts. In this study we demonstrate that the stimulation by PDGF of serum-starved NIH3T3 cells increases cellular GSH content, while no change in oxidized GSH content was measured. Experiments performed with actinomycin, cycloheximide and buthionine sulfoximide, a specific inhibitor of the rate-limiting enzyme of the de novo synthesis of GSH gamma-glutamylcysteine synthetase (gamma-GCS), confirm PDGF induction of GSH synthesis. These results provide the first demonstration that PDGF mediated transduction signals seem strictly related to mechanisms involved in the increase of gamma-GCS activity associated with increased gamma-GCS heavy subunit mRNA levels. In fact, serum and epidermal growth factor (EGF) stimulation of quiescent NIH3T3 and NIH3T3, which overexpress EGF receptor, does not affect GSH content or its synthesis. These data may be related to a possible GSH role in the redox regulation of cell proliferation mediated by PDGF.     

Collagen peptides modulate the metabolism of extracellular matrix by human dermal fibroblasts derived from sun‐protected and sun‐exposed body sites

Clinical data published in recent years have demonstrated positive effects of collagen hydrolysate (CH) on skin aging clinical signs. CH use as food supplement has a long history; however, few studies have addressed the underlying purpose of CH on the cellular and molecular biology of skin cells that could elucidate clinical improvement findings. Wide diversity of characteristics has been reported for dermal fibroblasts derived from different body sites and it is unknown whether collagen peptides could modulate differently cells from chronological aged and photoaged skin areas. This study investigated the influence of CH on the extracellular matrix metabolism and proliferation of human dermal fibroblasts (HDFs) derived from chronological aged (sun‐protected) and photoaged (sun‐exposed) body sites. CH treatment did not affect cellular proliferation of either cell cultures, but notably modulated cell metabolism in monolayer model, increasing the content of dermal matrix precursor and main protein, procollagen I and collagen I, respectively. These effects were confirmed in the human dermal equivalent model. The increase in collagen content in the cultures was attributed to stimulation of biosynthesis and decreased collagen I metabolism through inhibition of metalloproteinase activity (MMP) 1 and 2. Modulation of CH in dermal metabolism did not differ between cells derived from sun‐protected and sun‐exposed areas, although lower concentrations of CH seemed to be enough to stimulate sun‐exposed‐derived HDFs, suggesting more pronounced effect in these cells. This study contributes to understanding the biological effects of CH on skin cells and viability of its use as a functional ingredient in food supplements.     

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.     

Donor age reflects the replicative lifespan of human fibroblasts in culture

Human fibroblasts, which have a finite lifespan in cultures, have been widely used as a model system for cellular aging, and frequently used as one model of human aging. But whether cellular aging contributes to organismal aging has been controversial. To reinvestigate this question, we cultured human fibroblasts from the skin of one individual volunteer collected at different ages. Over a period of 27 years (donor age 36 years to 62 years), we obtained skin cells four times at appropriate intervals, and established eight fibroblast lines. These human fibroblasts have presented evidence for a correlation between donor age and proliferative lifespan in vitro . This result parallels the fact that telomeric DNA size cultured fibroblasts decrease with the increase in donor age. These cell lines had a normal diploid human chromosome constitution and will be useful in studies of human biology including aging.     

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.     

Correlation between contractility and proliferation in human fibroblasts

The contractile power of human fibroblasts was checked through their life span in vitro, using a plasma clot retraction test. It was found to decline with a pattern analogous to that of the different phases identifiable by the study of the kinetics of proliferation of these cells. The capacity to retract a plasma clot was higher in cells harvested during active growth than in cells harvested in resting phase. The decreased ability to retract during aging becomes apparent when cells are harvested in resting phase. Decreased retractile activity was also observed in postnatal cells as compared with embryonic cells. The results support a correlation between the initiation of DNA synthesis and the turnover of cytoskeletal elements. The data fit our previous results showing that the early proliferative disturbance during cellular senescence consists of a decline in the probability of initiating the division cycle linked to impaired cell attachment and spreading.     

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.     

Novel monoclonal antibodies identify antigenic determinants unique to cellular senescence.

Normal human diploid fibroblasts exhibit a limited lifespan in vitro and are used as a model to study in vivo aging. Monoclonal antibodies were generated against partially purified surface membranes from human diploid fibroblasts at the end of their lifespan (senescent). Three hybridomas were isolated that secreted antibodies reacting to cellular determinants expressed specifically on senescent human fibroblasts of different origin, including neonatal foreskin, embryonic lung, and adult skin punch biopsy, but not expressed on matched young cells. The antibodies did not bind to immortal human cells and normal young cells made reversibly nondividing, indicating the antigens are not expressed in cells that are not senescent. The antibodies identified senescent cells in a mixed cell population and expression of the senescent cell antigens correlated strongly with the cells inability to synthesize DNA at the onset of senescence. The antigens appeared to be cell surface or extracellular matrix associated, and the epitopes were destroyed by mild trypsin treatment. Western analysis indicated all three antibodies reacted with fibronectin. Though the antigenic determinants on the fibronectin molecule were not accessible in the intact young cell, the epitopes were present in fibronectin extracted from both senescent and young cells, as well as purified human plasma fibronectin. These antibodies and the senescent specific expression of the antigens provide powerful tools to investigate the mechanisms leading to in vitro senescence. This may enable us to investigate directly the relationship between cellular aging and aging of the individual.     

Accelerated telomere erosion is associated with a declining immune function of caregivers of Alzheimer's disease patients

Caregivers of Alzheimer's disease patients endure chronic stress associated with a decline of immune function. To assess the psychological and immunological changes of caregivers, we compared depressive symptoms, PBMC composition, in vitro activation-induced proliferation and cytokine production, and telomere length and telomerase activity of 82 individuals (41 caregivers and 41 age- and gender-matched controls). We found depressive symptoms were significantly higher in caregivers than in controls (p < 0.001). Correspondingly, caregivers had significantly lower T cell proliferation but higher production of immune-regulatory cytokines (TNF-alpha and IL-10) than controls in response to stimulation in vitro. We examined the impact of these changes on cellular replicative lifespan and found that caregivers had significantly shorter telomere lengths in PBMC than controls (6.2 and 6.4 kb, respectively, p < 0.05) with similar shortening in isolated T cells and monocytes and that this telomere attrition in caregivers was not due to an increase of shorter telomere possessing T cell subsets in PBMC. Finally, we showed that basal telomerase activity in PBMC and T cells was significantly higher in caregivers than in controls (p < 0.0001), pointing to an unsuccessful attempt of cells to compensate the excessive loss of telomeres in caregivers. These findings demonstrate that chronic stress is associated with altered T cell function and accelerated immune cell aging as suggested by excessive telomere loss.     

Heterogeneity of fibroblast response in host-tumor cell-cell interactions in metastatic tumors

The spread and invasion of tumor cells into host tissues are associated with the release of elevated levels of collagenolytic activity of both host and tumor cell origins. However, the mechanisms of regulation of the enzyme activity is still unresolved. Histological examination of human and animal tumors revealed morphological changes in stromal fibroblasts and mast cells at the tumor periphery. Numerous mast cells appeared at microfoci along the tumor: host tissue junction and mast cell degranulation were associated with collagenolysis. In vitro studies, using rat mammary adenocarcinoma and human lung adenocarcinoma cells, showed that both tumor cells and host fibroblasts participate in matrix degradation. Tumor-associated stromal fibroblasts released higher levels of enzyme activity than normal fibroblasts and were more responsive to stimulation by tumor-conditioned media and soluble mast cell products. Host fibroblasts appear to be heterogeneous populations of responsive and nonresponsive subpopulations based on their response to tumor- or mast-cell-mediated stimulation of collagenase release. Fibroblast subpopulations were obtained by density fractionation of serum-deprived, synchronized confluent fibroblasts on discontinuous Percoll gradient. Density-fractionated fibroblast subpopulations differed in their response to stimulation by mast cell products and tumor-cell-conditioned media. The stimulatory activity of tumor-cell-conditioned media also varied as a function of the metastatic potential of the tumor cells. The data suggest that cellular interactions between tumor cells and select subpopulations of host fibroblasts at the tumor periphery play a key role in host tissue degradation. However, heterogeneity of stromal fibroblasts may determine the site and extent of the tissue damage at foci of tumor invasion.