Self-rated health in individuals with and without disease is associated with multiple biomarkers representing multiple biological domains.

Self-rated health (SRH) is one of the most frequently used indicators in health and social research. Its robust association with mortality in very different populations implies that it is a comprehensive measure of health status and may even reflect the condition of the human organism beyond clinical diagnoses. Yet the biological basis of SRH is poorly understood. We used data from three independent European population samples (N approx. 15,000) to investigate the associations of SRH with 150 biomolecules in blood or urine (biomarkers). Altogether 57 biomarkers representing different organ systems were associated with SRH. In almost half of the cases the association was independent of disease and physical functioning. Biomarkers weakened but did not remove the association between SRH and mortality. We propose three potential pathways through which biomarkers may be incorporated into an individual's subjective health assessment, including (1) their role in clinical diseases; (2) their association with health-related lifestyles; and (3) their potential to stimulate physical sensations through interoceptive mechanisms. Our findings indicate that SRH has a solid biological basis and it is a valid but non-specific indicator of the biological condition of the human organism.

Design, recruitment, logistics, and data management of the GEHA (Genetics of Healthy Ageing) project.

In 2004, the integrated European project GEHA (Genetics of Healthy Ageing) was initiated with the aim of identifying genes involved in healthy ageing and longevity. The first step in the project was the recruitment of more than 2500 pairs of siblings aged 90 years or more together with one younger control person from 15 areas in 11 European countries through a coordinated and standardised effort. A biological sample, preferably a blood sample, was collected from each participant, and basic physical and cognitive measures were obtained together with information about health, life style, and family composition. From 2004 to 2008 a total of 2535 families comprising 5319 nonagenarian siblings were identified and included in the project. In addition, 2548 younger control persons aged 50-75 years were recruited. A total of 2249 complete trios with blood samples from at least two old siblings and the younger control were formed and are available for genetic analyses (e.g. linkage studies and genome-wide association studies). Mortality follow-up improves the possibility of identifying families with the most extreme longevity phenotypes. With a mean follow-up time of 3.7 years the number of families with all participating siblings aged 95 years or more has increased by a factor of 5 to 750 families compared to when interviews were conducted. Thus, the GEHA project represents a unique source in the search for genes related to healthy ageing and longevity.

Ageing research in Greece.

Ageing research in Greece is well established. Research groups located in universities, research institutes or public hospitals are studying various and complementary aspects of ageing. These research activities include (a) functional analysis of Clusterin/Apolipoprotein J, studies in healthy centenarians and work on protein degradation and the role of proteasome during senescence at the National Hellenic Research Foundation; (b) regulation of cell proliferation and tissue formation, a nationwide study of determinants and markers of successful ageing in Greek centenarians and studies of histone gene expression and acetylation at the National Center for Scientific Research, Demokritos; (c) work on amyloid precursor protein and Presenilin 1 at the University of Athens; (d) oxidative stress-induced DNA damage and the role of oncogenes in senescence at the University of Ioannina; (e) studies in the connective tissue at the University of Patras; (f) proteomic studies at the Biomedical Sciences Research Center Alexander Fleming; (g) work on Caenorhabditis elegans at the Foundation for Research and Technology; (h) the role of ultraviolet radiation in skin ageing at Andreas Sygros Hospital; (i) follow-up studies in healthy elderly at the Athens Home for the Aged; and (j) socio-cultural aspects of ageing at the National School of Public Health. These research activities are well recognized by the international scientific community as it is evident by the group's very good publication records as well as by their direct funding from both European Union and USA. This article summarizes these research activities and discuss future directions and efforts towards the further development of the ageing field in Greece.

Clusterin/apolipoprotein J is a novel biomarker of cellular senescence that does not affect the proliferative capacity of human diploid fibroblasts.

Normal human fibroblasts have a limited replicative potential in culture and eventually reach a state of irreversible growth arrest, termed senescence. In a previous study aiming to identify genes that are differentially regulated during cellular senescence we have cloned clusterin/apolipoprotein J (Apo J), a 80 kDa secreted glycoprotein. In the current report we pursue our studies and show that senescence of human diploid fibroblasts is accompanied by up-regulation of both Apo J mRNA and protein levels, but with no altered biogenesis, binding partner profile or intracellular distribution of the two Apo J forms detected. To analyze the causal relationship between senescence and Apo J protein accumulation, we stably overexpressed the Apo J gene in primary as well as in SV40 T antigen-immortalized human fibroblasts and we showed no alteration of the proliferative capacity of the transduced cells. Despite previous reports on tumor-derived cell lines, overexpression of Apo J in human fibroblasts did not provide protection against apoptosis or growth arrest induced by hydrogen peroxide. Overall, our results suggest that Apo J overexpression does not induce senescence but it is rather a secondary consequence of the senescence phenotype. To our knowledge this is the first report that provides a functional analysis of human Apo J during replicative senescence.

Over-expression of CDKIs p15INK4b, p16INK4a and p21CIP1/WAF1 genes mediate growth arrest in human osteosarcoma cell lines.

Human somatic cells cultured in vitro exhibit a limited number of divisions. In contrast immortal cells have lost their growth regulatory mechanisms and, thus, continue to divide indefinitely. Cyclin-dependent kinase inhibitors (CDKIs) represent one of the most important regulatory factors in both mortality and immortality, as they are over-expressed in senescent cells and are down-regulated in a number of human cancers. In the present study we determined the effect of CDKIs on the proliferation ability of human osteosarcoma cell lines. Transient expression of various CDKIs (p15INK4b, p16INK4a and p21CIP1/WAF1) in KHOS cells resulted in growth arrest and the cells failed to enter the S-phase of the cell cycle as shown by a DNA synthesis inhibition assay. In addition, stable transfection of p21CIP1/WAF1 and p16INK4a genes in two osteosarcoma cell lines (KHOS and U2-OS cells) showed that p21CIP1/WAF1 was able to repress the immortal phenotype in both cell lines, whereas temporary over-expression of p16INK4a reversibly inhibited the cell growth. Therefore this study indicates that CDKIs mediate growth arrest in human osteosarcoma cell lines and provides further evidence of the existence of molecular links between cellular mortality and immortality.

v-FBR-fos oncogene fails to rescue mammalian cells from growth arrest but affects the responses of human fibroblasts to heparin.

The effects of v-fos oncogene on the proliferation of mammalian cells were studied using several approaches. Constitutive overexpression of v-FBR-fos in normal human fibroblasts (MRC-5) and of v-FBR-fos in human chondrocytes (HAC21) failed to immortalise them, extend their in vitro lifespan, increase their growth rates or induce cellular transformation. Further, v-FBR-fos did not render MRC-5 growth factor-independent or alter their responsivenness to serum, but it markedly suppressed their heparin-induced proliferation. A conditionally immortalized, temperature-sensitive rat embryo fibroblast cell line (tsa14) which undergoes growth arrest upon inactivation of a thermolabile SV40 large T antigen by a temperature shift producing a phenotype that mimmicks the senescent phenotype, was also used to study the effects of v-FBR-fos on cell proliferation. Whereas a wild-type SV40 large T antigen rescued tsa14 from a temperature-dependent growth arrest, v-FBR-fos failed to do so. Hence, v-FBR-fos was not sufficient to, at least, complement the tsa14 growth defect. There was no change in the expression of c-jun and junB, members of the AP-1 transcriptional complex in MRC-5v-fos cells. These data show that v-FBR-fos is not sufficient to rescue mammalian cells from senescence but it can affect the responses of human fibroblasts to heparin suggesting a role of fos in cell proliferation.

Correlation of in vitro cytotoxicity and clinical response to chemotherapy in ovarian and breast cancer patients.

During the last years, a number of assays have been developed aiming at predicting the most effective chemotherapy regimen for each individual, avoiding possible toxicity of ineffective drugs. In the present study we have used an in vitro chemosensitivity/chemoresistance assay in order to evaluate cytotoxic treatment in ovarian and breast cancer patients. The assay was applied in 77 ovarian and breast cancer samples and the observed in vitro responses to various chemotherapeutic drugs or combinations of drugs were then correlated to the in vivo responses and the overall clinical data of the examined patients. Direct comparison was possible for 25 cases. The overall positive predictive value of the assay was 50% and the negative predictive value was 57%. However, it was observed that the positive predictive value for ovarian patients was 69% and that the negative predictive value for breast patients was 100%. Therefore this study indicates that although in vitro chemosensitivity/chemoresistance is a valuable assay, further analysis and implications of other factors are required for a general evaluation of cytotoxic treatment for patients with ovarian and breast cancer.

Evaluation of cytotoxic treatment of patients with osteosarcoma by an in vitro chemoresistance assay.

During the previous two decades several assays have been developed aiming to select the most effective chemotherapy regimen for each individual, avoiding the possible toxicity of ineffective drugs. In order to evaluate cytotoxic treatment for patients with osteosarcoma, we applied an in vitro chemoresistance assay by culturing tumour cells and determining their in vitro survival rates after exposure to various chemotherapeutic drugs. The conditions of the assay were optimised in two established osteosarcoma cell lines (KHOS and U2-OS), as compared with data derived after treatment of primary normal adult osteoblasts. Chemotherapeutic drugs (cisplatin, adriamycin or methotrexate or combinations) concentrations were chosen in a range that has been reported to induce tumour cell death in the plasma patients' The method applied successfully in 6 cell cultures originated from biopsies of 7 patients with osteosarcoma and the in vitro response to chemotherapeutic drugs was correlated with the clinical outcome. Such analysis revealed both positive and negative correlation of the in vitro data to the patients clinical responses. Therefore, this study indicated that, although in vitro chemoresistance is a valuable assay, additional analysis and implications of other factors are required for a general evaluation of cytotoxic treatment for patients with osteosarcoma.

Fibroblast cultures from healthy centenarians have an active proteasome.

Healthy centenarians represent the best example of successful ageing. Various studies have shown that centenarians have escaped the major age-associated diseases, they have several well-conserved immune parameters and at least one gene allele has been identified and linked with their increased longevity. During ageing there is an accumulation of oxidised proteins, a phenomenon that has been related to an impaired function of the 20S proteasome in aged cells. We have, therefore, analysed the expression and the proteolytic activity of the proteasome in centenarian cells. Four fibroblast cultures derived from healthy centenarians were studied and compared with cultures derived from adult donors of different ages. Analysis of several proteasome subunits RNA expression levels, determination of one peptidase activity and identification of oxidised proteins in these samples revealed that centenarian cultures have a functional proteasome. In addition, it was found that the centenarian cultures exhibit characteristics similar to the younger rather than the older control donors derived cultures in all three assays. These data indicate that centenarian cells may be different from elderly donors cells, thus opening up new dimensions for the identification and characterisation of factors that are linked with longevity.

Aging and longevity. A paradigm of complementation between homeostatic mechanisms and genetic control?

Aging is a universal and inevitable phenomenon that affects nearly all animal species. It can be considered the product of an interaction between genetic, environmental, and lifestyle factors, which in turn influence longevity that varies between and within species. It has been proposed not only that the aging process is under genetic control, but that it can also be considered a result of the failure of homeostasis due to the accumulation of damage. This review article discusses these issues, focusing on the function of genes that associate with aging and longevity, as well as on the molecular mechanisms that control cell survival and maintenance during aging.

Genetics of aging: lessons from centenarians.

Aging is a universal phenomenon that affects nearly all animal species. It can be considered as the product of an interaction between genetic, environmental and lifestyle factors, which in turn influence longevity that varies between and within species. Several studies have been focused in healthy centenarians, because these exceptional individuals represent the best example of successful aging. These studies have shown that centenarians have escaped the major age-associated diseases, they have well conserved several immune parameters, and at least one gene allele has been identified and linked with longevity. In parallel, studies at cellular level have identified several genes that influence, positively or negatively, normal replicative in vitro life-span. The ability of these genes to regulate aging in vitro, in conjunction with the telomeres shortening hypothesis have raised the intriguing question of the existence of a molecular clock that counts and thus may modulates human aging and longevity. This review article will discuss these issues, focusing in the nature of the genetic factors that associate with these phenomena.

Induction of replicative senescence biomarkers by sublethal oxidative stresses in normal human fibroblast.

We tested the long-term effects of sublethal oxidative stresses on replicative senescence. WI-38 human diploid fibroblasts (HDFs) at early cumulative population doublings (CPDs) were exposed to five stresses with 30 microM tert-butylhydroperoxide (t-BHP). After at least 2 d of recovery, the cells developed biomarkers of replicative senescence: loss of replicative potential, increase in senescence-associated beta-galactosidase activity, overexpression of p21(Waf-1/SDI-1/Cip1), and inability to hyperphosphorylate pRb. The level of mRNAs overexpressed in senescent WI-38 or IMR-90 HDFs increased after five stresses with 30 microM t-BHP or a single stress under 450 microM H(2)O(2). These corresponding genes include fibronectin, osteonectin, alpha1(I)-procollagen, apolipoprotein J, SM22, SS9, and GTP-alpha binding protein. The common 4977 bp mitochondrial DNA deletion was detected in WI-38 HDFs at late CPDs and at early CPDs after t-BHP stresses. In conclusion, sublethal oxidative stresses lead HDFs to a state close to replicative senescence.

Different functions are required for initiation and maintenance of immortalization of rat embryo fibroblasts by SV40 large T antigen.

We have used two different, but complementary assays to characterize functions of SV40 T antigen that are necessary for its ability to immortalize rat embryo fibroblasts. In accordance with previous work, we found that several functions were required. These include activities that map to the p53 binding domain and the amino terminal 176 amino acids which contain the J domain as well as the CR1 and CR2 domain required for binding and sequestering the RB family of pocket proteins. Moreover, we found that even though activities dependent only upon the amino terminus were sufficient for immortalization they were unable to maintain it. This suggests that immortalization by these amino terminal functions requires either additional events or immortalization of a subset of cells within the heterogeneous rat embryo fibroblast population. We further found that an activity dependent upon amino acids 17 - 27 which remove a portion of the CR1 domain and the predicted alpha-1 helix of the J domain was not necessary to maintain growth but was required for direct immortalization suggesting that at least one of the functions required initially was not required to maintain the immortal state. This represents the first demonstration that some of the functions required for maintenance of the immortal state differ from those required for initiation of immortalization.

Telomere length in fibroblasts and blood cells from healthy centenarians.

Several lines of evidence indicate that telomere shortening during in vitro aging of human somatic cells plays a causal role in cellular senescence. A critical telomere length seems to be associated with the replicative block characterizing senescent cells. In this paper we analyzed the mean length of the terminal restriction fragments (TRF) in fibroblast strains from 4 healthy centenarians, that is, in cells aged in vivo, and from 11 individuals of different ages. No correlation between mean TRF length and donor age was found. As expected, telomere shortening was detected during in vitro propagation of centenarian fibroblasts, suggesting that in fibroblasts aged in vivo telomeres can be far from reaching a critical length. Accordingly, chromosome analysis did not show the presence of telomeric associations in early passage centenarian fibroblasts. In blood cells from various individuals, the expected inverse correlation between mean TRF length and donor age was found. In particular, a substantial difference (about 2 kb) between telomere length in the two cell types was observed in the same centenarian. Expression analysis of three senescence-induced genes, i.e., fibronectin, apolipoprotein J, and p21, revealed for only the fibronectin expression levels a clear positive correlation with donor age. Our results suggest that (1) telomere shortening could play a different role in the aging of different cell types and (2) the characteristics of fibroblasts aged in vitro might not be representative of what occurs in vivo.

Expression of the growth arrest specific genes in rat embryonic fibroblasts undergoing senescence.

Replicative senescence is an in vitro biological phenomenon that results the irreversible growth arrest of mammalian cells. In contrast, cellular quiescence is reversible upon the addition of mitogens or replating. Quiescence is associated with the expression of a particular gene family--the growth arrest specific (GAS) genes. I have determined whether gas genes expression also associates with senescence, as well as whether serum stimulation of senescent cells has the same inhibiting effect as in quiescent cells. Analysis of RNA of cultures of rat embryonic fibroblasts suggests that gas-1 and gas-6 gene expression is associated with the irreversible growth arrest of senescence. However, serum was found to have a different effect upon gas-1 and gas-6 expression in quiescent and senescent cells, because its stimulation of quiescent cells results in a dramatic decrease of their RNA levels, but it had no effect in senescent cells. Finally, analysis of the data is raising the possibility that the gas-1 and gas-6 genes expression may also be associated with conditions of cellular stress.

Cloning and identification of genes that associate with mammalian replicative senescence.

Cellular senescence and limited proliferative capacity of normal diploid cells has a dominant phenotype over immortality of cancerous cells, suggesting its regulation by the expression of a set of genes. In order to isolate the genes that associate with senescence, we have employed a clonal system of conditional SV40 T antigen rat embryo fibroblast cell lines which undergo senescence upon T antigen inactivation. Construction of cDNA libraries from two conditional cell lines and application of differential screening and subtractive hybridization techniques have resulted in the cloning of eight senescence-induced genes (SGP-2/Apo J, alpha 1-procollagen, osteonectin, fibronectin, SM22, cytochrome C oxidase, GTP-alpha, and a novel gene) and a senescence-repressed gene (FRS-2). Three of these genes encode for extracellular matrix proteins, others are involved in the calcium-dependent signal transduction pathways, while the SGP-2/Apo J gene may have a cellular protective function. RNA analysis has shown that the senescence-associated genes are overexpressed in both normal rat embryonic fibroblasts and human osteoblasts cell cultures undergoing aging in vitro. In comparison, the expression of these genes in a rat fibroblast immortalized cell line (208F cells) was down-regulated after both its partial and its full transformation by ras oncogenes. Thus, cloning of senescence-associated genes opens up new ways to elucidate and/or to modulate aging and cancer.

Molecular links between cellular mortality and immortality (review).

Normal diploid cells cultivated in vitro exhibit limited division potential while undergoing ageing during serial passaging. In contrast, cells that have been genetically transformed appear to have lost the regulatory mechanisms of limited growth potential and may continue to divide indefinitely. While cellular mortality is characterised by a progressive cessation of cell growth manifested in cell culture by senescence, immortalisation is the escape from senescence as a result of multiple mechanisms involving the inactivation of tumour suppressor genes, the elevated expression of oncogenes, as well as other genetic and epigenetic changes. The mechanisms governing mortality and immortality are fundamentally linked. The physiological and biochemical features which characterise cellular mortality are examined, herein in the search for markers and timing mechanisms of mortality. The genetic elements involved in the control of mortality and immortality are also discussed, and the fundamental similarities between the molecular and genetic aspects which govern the determination of the phenotypes manifesting the two processes are underlined.

Rat embryo fibroblasts immortalized with simian virus 40 large T antigen undergo senescence upon its inactivation.

Introduction of simian virus 40 T antigen into rodent fibroblasts gives rise to cells that can proliferate indefinitely but are dependent upon it for maintenance of their growth once the normal mitotic life span has elapsed. Inactivation of T antigen in these immortalized cells causes rapid and irreversible cessation of growth. To determine whether this growth arrest is associated with entry into senescence, we have undertaken a genetic and biological analysis of conditionally immortal (tsa) cell lines derived by immortalizing rat embryo fibroblasts with the thermolabile tsA58 T antigen. This analysis has identified the following parallels between the tsa cells after inactivation of T antigen and senescent rat embryo fibroblasts: (i) growth arrest is irreversible; (ii) it occurs in G1 as well as G2; (iii) the G1 block can be partially overcome by stimulation with 20% fetal calf serum, but the G2 block cannot be overcome; (iv) 20% fetal calf serum induces c-fos, but c-myc is unaltered; and (v) fibronectin and p21(Waf1/Cip1/Sdi1) are upregulated upon growth arrest. These results suggest that T-antigen-immortalized fibroblasts are committed to undergo senescence but are prevented from undergoing this process by T antigen. Inactivation of T antigen removes this block and results in senescence of the cells. Thus, these cell lines may represent a powerful system for study of the molecular basis of entry into senescence.