Growth properties and growth factor responsiveness in skin fibroblasts from centenarians.

Human fibroblast cultures, which have a finite replicative lifespan in vitro, are the most widely used model for the study of senescence at the cellular level. An inverse relationship between replicative capability and donor age has been reported in human fibroblast strains. We studied the growth capacity of fibroblast primary cultures derived from people whose lifespan was as closer as possible to the expected maximum human lifespan, i.e. people over one hundred. Our data suggest that outgrowth of fibroblasts from biopsies, growth kinetics at different population doubling levels, capability to respond to a classical mitogenic stimulus (such as 20% serum) and a variety of growth factors, were remarkably similar in fibroblasts from centenarians and young controls. On the whole, our data challenge the tenet of a simple and strict relationship between in vivo aging and in vitro proliferative capability of human fibroblasts, at least at the individual level.

c-fos/c-jun expression and AP-1 activation in skin fibroblasts from centenarians.

In vitro replicative senescence is characterized by an irreversible growth arrest due to the inability of the cell to induce some key regulators of cell cycle progression, such as c-fos and AP-1, in response to mitogenic stimuli. In vitro replicative senescence and in vivo aging have been assumed to be two related phenomena, likely controlled by overlapping or interacting genes. As a corollary, fibroblasts from centenarians, which have undergone a long process of senescence in vivo should have very limited proliferative capability. On the contrary, in a previous work we found that fibroblasts from centenarians exhibited the same capacity to respond to different mitogenic stimuli as fibroblasts from young donors. Here we provide evidences that the well preserved proliferative response is likely due to the fact that some pivotal regulators- c-fos, c-jun and AP-1-are still fully inducible, despite a long process of in vivo senescence. Our data therefore suggest that in vivo and in vitro aging are separate phenomena whose possible relationships, if any, have to be ascertained very carefully.

Successful immunosenescence and the remodelling of immune responses with ageing.

In recent decades, major theoretical and technological advances have been achieved in the field of immunology. These have allowed the scientific community to analyse the immune system in a much more sophisticated manner than was possible even 20 years ago. Moreover, great theoretical changes have also occurred in gerontology-in particular, the hypothesis has been put forward that ageing and diseases are two different phenomena, and that successful ageing, i.e. ageing in good psychophysical conditions, is really possible for most humans and animals. Immunosenescence was then carefully investigated, either in selected healthy people of advanced age or in the oldest old people, such as healthy centenarians. The main results showed that most immune parameters are indeed well preserved even at this far advanced age. This paper deals with some of the most important theoretical problems of immunosenescence. An immunological tenet was that the most important phenomenon of immunosenescence is the involution of the thymus. In most textbooks and papers it is taken for granted that the thymus starts its involution immediately after puberty. When people aged 60-65 were considered old, it was not difficult to think that they could live for the rest of their life with a fully involuted thymus. The findings on centenarians challenge this tenet, as they have only a small reduction of T lymphocytes, and a relatively normal number of virgin and memory T cells, together with a functional T cell repertoire. Other observations reported here on centenarians, concerning the activity of B lymphocytes and the cytokine network, as well as those on the well-preserved innate immunity and the cells' capability of undergoing proliferation after appropriate stimuli, suggest that complex immune changes occur with age, but also indicate that we have to modify our attitude, to grasp the new scenario which is emerging. Immunosenescence can no longer be considered as a unidirectional deterioration, and this complex phenomenon is much better described by terms such as 'remodelling', 'reshaping' or 'retuning'.

Interference between DNA binding activities of AP-1 and GR transcription factors in rat thymocytes undergoing dexamethasone-induced apoptosis.

The early molecular events of glucocorticoid-induced apoptosis have been investigated by studying glucocorticoid receptor levels, as well as binding activities to GRE and AP-1 sequences, using nuclear extracts from dexamethasone (Dex)-treated rat thymocytes. When the time-course of glucocorticoid-receptor complexes in nuclei of thymocytes was evaluated by binding studies using the tritiated ligand, we found that nuclear accumulation of radioactive complexes occurred in the first hour of incubation, and was followed by a progressive decline. This trend was confirmed by immunoblotting of nuclear proteins using a monoclonal anti-glucocorticoid receptor antibody. When the kinetics of binding activity to AP-1 and GRE sequences were studied, using nuclear extracts prepared from Dex-treated thymocytes in gel shift assays, we found peaks at 1 and 2 h after Dex treatment, and a return to basal levels in the following hours. Binding specificity was proved by competition studies using non-radioactive sequences, including mutated AP-1. Unexpectedly, however, protein binding to GRE was better competed for by AP-1 sequence than by GRE itself. Data obtained using the super gel shift assay suggested that AP-1/Jun can be responsible for the high affinity for the GRE sequence. Thus, we report here for the first time that an interference between AP-1 and GR in the binding to DNA consensus sequences-previously described in other biological systems-also occurs during apoptosis induced by glucocorticoids in lymphoid cells.

Is polyamine decrease a common feature of apoptosis? Evidence from gamma rays- and heat shock-induced cell death.

Here we report that in rat thymocytes undergoing apoptosis upon two different stimuli, such as heat shock treatment and gamma irradiation, an early mRNA accumulation of ornithine decarboxylase (ODC)--the rate-limiting enzyme of polyamine biosynthesis--was followed by a very marked increase in ODC activity (28-40 and 6-8-fold, respectively). However, polyamine levels started to decrease before the appearance of DNA laddering, being putrescine and spermidine strongly diminished (8-12 hs), and spermine even depleted (12 hs). Taken together with our previous data on another model of apoptosis, i.e., glucocorticoid-induced cell death (Desiderio et al., Cell Growth Differ. 6: 505-513, 1995), these results suggest that an imbalance of polyamine metabolism, i.e., a strong activation of ODC and a paradoxical decrease of the intracellular polyamine content, might be a general feature of the apoptotic process.

Protective effect of N-acetylcysteine in tumor necrosis factor-alpha-induced apoptosis in U937 cells: the role of mitochondria.

The existence of two different pathways for cell death has been postulated. In addition to the passive and traumatic process leading to necrosis, an active program characterized by organelle integrity and called apoptosis has been described. A positive correlation between the apoptotic cell death process and oxidative imbalance has been demonstrated. In fact, the antioxidant N-acetylcysteine (NAC) seems to be capable of impairing the apoptotic program, replenishing intracellular reduced glutathione content in cells exposed to tumor necrosis factor-alpha (TNF) as apoptotic inducer. Moreover, protein synthesis inhibitors such as cycloheximide (CHX) can facilitate apoptotic triggering by TNF, and mitochondrial function was suggested to be essential in the TNF-mediated apoptotic process. With this in mind, a specific analysis using the JC-1 probe, a fluorescent dye which is capable of indicating mitochondrial membrane potential (delta psi m) changes, was carried out. Our results show that TNF exposure is capable of altering the mitochondria and that NAC protection from CHX + TNF-induced apoptosis could be due to a direct effect of the drug on mitochondrial integrity and function.

Massive activation of immune cells with an intact T cell repertoire in acute human immunodeficiency virus syndrome.

In 8 patients with symptomatic, acute primary infection with human immunodeficiency virus (HIV), a dramatic and persistent decrease in CD4+ lymphocytes was seen, accompanied by a marked increase in activated/memory CD8+ T cells (CD38+, CD45R0+, HLA-DR+, with high amounts of cell adhesion molecules), which represented most circulating lymphocytes, but no gross alterations in V beta T cell repertoire. Extremely high plasma levels of proinflammatory cytokines were observed. Three patients were followed for 2-3 years: The number of CD4+ cells, extremely low at first, increased significantly in a few months but decreased rapidly after a short stable period. Cytotoxic T lymphocytes bearing markers of immunologic activation/memory could play an important role in the earliest phases of the disease. It remains to be established how such a dramatic onset could determine the rapid progression of the infection that seems characteristic of patients with acute HIV syndrome.

Lack of selective V beta deletion in CD4+ or CD8+ T lymphocytes and functional integrity of T-cell repertoire during acute HIV syndrome.

OBJECTIVE: To study the V beta T-cell repertoire in peripheral blood lymphocytes (PBL) during acute HIV syndrome by using several anti-V beta monoclonal antibodies (MAb) and to analyse its functionality by stimulating PBL with superantigens (SAg) such as Staphylococcus aureus enterotoxins. METHODS: Cytofluorimetric analysis of V beta T-cell-receptor expression was performed on PBL from eight patients with symptomatic, acute HIV-1 primary infection, showing a dramatic decrease of CD4+ PBL accompanied by a marked increase in activated/memory CD8+ T cells, and on 12 age- and sex-matched healthy controls. PBL were then isolated, stimulated with different SAg, anti-CD3 MAb or phytohaemagglutinin and cultured for 3 days. PBL capability to progress through cell cycle was studied by the classic cytofluorimetric method of bromodeoxyuridine incorporation and DNA staining with propidium iodide. RESULTS: Despite the presence of a few expansions of some V beta families among CD8+ T lymphocytes, no gross alterations in T-cell repertoire were present in patients with acute HIV syndrome. Its functionality was maintained overall, as PBL responsiveness to SAg was well preserved. Interestingly, all CD8+ T cells, although bearing different V beta T-cell receptors, expressed marked signs of activation, i.e., CD45R0, CD38 and major histocompatibility complex class II molecules, and also high amounts of CD11a and CD18. CONCLUSIONS: Our data suggest, at least in the early phases and in the acute form of the infection, that HIV is not likely to act as a SAg. However, further studies are needed to analyse other sites, such as lymph nodes, where HIV could exert other, significant effects, and to study the expression of other V beta families than those investigated here.

Involvement of ornithine decarboxylase and polyamines in glucocorticoid-induced apoptosis of rat thymocytes.

Ornithine decarboxylase (ODC), the first and rate-limiting enzyme of polyamine metabolism, has been shown to be required for entry into and progression through the cell cycle. However, the role of ODC and polyamines in apoptosis remains to be determined. We have examined ODC expression and polyamine levels in thymocytes activated to undergo apoptosis by dexamethasone treatment. We have demonstrated a rapid and reversible induction of ODC (mRNA and activity), as previously reported for the mRNA expression of other "early" genes, c-fos, c-jun, and c-myc, in the same experimental model. Surprisingly, polyamine levels diminished progressively starting at 2-4 h after dexamethasone treatment, and spermine was depleted at 8-12 h. This seemed to be relevant since increasing the intracellular polyamine levels by exogenous spermine administration prevented the DNA "laddering" (2-4 h) and the DNA loss from the nucleus (8-18 h) due to dexamethasone treatment. Moreover, the activities of spermidine/spermine N1-acetyltransferase, which controls the cytosolic polyamine interconversion pathway, and of spermidine N8-acetyltransferase, which regulates the nuclear pool and functions of polyamines, were measured in apoptotic cells. Spermidine/spermine N1-acetyltransferase activity progressively increased and might be responsible for spermidine and spermine excretion as acetyl derivatives. In contrast, spermidine N8-acetyltransferase activity remained unchanged. A completely different scenario was observed in proliferating concanavalin A-treated thymocytes, studied for comparison. In this case, polyamine levels increased, remaining at high values until 12 h. This is likely a consequence of the rapid and prolonged induction of ODC (mRNA and activity), accompanied by that of spermidine/spermine N1-acetyltransferase (mRNA and activity).(ABSTRACT TRUNCATED AT 250 WORDS)

D-ribose and deoxy-D-ribose induce apoptosis in human quiescent peripheral blood mononuclear cells.

In previous papers we reported that D(-)-ribose and 2-deoxy-D-ribose, which rank at the top among reducing sugars, kill a variety of human and animal cells and cell lines. Here we demonstrate that these two sugars induce apoptosis in human quiescent peripheral blood mononuclear cells which are relatively insensitive to apoptosis. Apoptosis was assessed by morphological changes, DNA fragmentation by agarose gel electrophoresis and the appearance of an hypodiploid peak by flow cytometry. 2-deoxy-D-ribose was more potent than D(-)-ribose and apoptosis was evident from 48 hours of culture onwards. 2-deoxy-D-ribose-induced apoptosis was inhibited by N-acetyl-L-cysteine, suggesting that glutathione metabolism and/or oxidative stress are involved in this type of apoptosis. Thus, D(-)-ribose and 2-deoxy-D-ribose can be useful tools to study the cellular and molecular events of apoptosis in human quiescent lymphocytes.

Transcription factors DNA-binding activity in rat thymocytes undergoing apoptosis after heat-shock or dexamethasone treatment.

The onset of apoptosis is generally thought to require the induction of a novel genetic program. Accordingly, we studied the kinetic of DNA-binding activity of several transcription factors in rat thymocytes undergoing apoptosis induced by dexamethasone (DEX) or heat shock (HS) treatment. Here we report that: 1) early activation of AP-1 occurred in both models of apoptosis, even if the intensity of activation and AP-1 complex composition were different and much less evident in HS-treated thymocytes; 2) early NFkB DNA-binding activity was also observed in both types of apoptosis; 3) downregulation of other transcription factors, such as OCT-1 and CREB, with high constitutive activity, was recorded in both models of apoptosis. The fact that some TFs are up-regulated while others are down-regulated suggests that apoptosis is the result of a complex combination of positive and negative signals regulating gene expression.

Studies of the relationship between cell proliferation and cell death. III. AP-1 DNA-binding activity during concanavalin A-induced proliferation or dexamethasone-induced apoptosis of rat thymocytes.

In previous papers we started to investigate the possible relationship existing between cell proliferation and cell death. We reported that in rat thymocytes, undergoing cell proliferation upon stimulation with Concanavalin A or cell death following dexamethasone treatment, an induction of c-fos and c-jun occurs with different but partially overlapping kinetics (Grassilli et al., BBRC, 1992, 188, 1261-1266). To test the hypothesis that, in both processes, the early induction of these nuclear oncogenes is necessary to allow the induction of later-expressed genes regulated at the transcriptional level by AP-1 complex, AP-1 DNA-binding activity was investigated in the aforementioned model. Indeed, we found that AP-1 is activated not only in proliferating but also in apoptotic cells, with a kinetics in accord with our data about c-fos and c-jun mRNA accumulation in the same experimental model. Moreover, our findings suggest that an interaction between AP-1 and glucocorticoid receptor also occurs in our model of dexamethasone-mediated apoptosis.