Ewa Sikora

Impact of cellular senescence signature on ageing research

Cellular senescence as the state of permanent inhibition of cell proliferation is a tumour-suppressive mechanism. However, due to the associated secretory phenotype senescence can also contribute to cancer and possibly other age-related diseases, such as obesity, diabetes, atherosclerosis and Alzheimers disease. There are two major mechanisms of cellular senescence; replicative senescence depends on telomere erosion or dysfunction whilst stress-induced premature senescence (SIPS) is telomere-independent and also includes oncogene-induced senescence (OIS). The senescence phenotype is characterised by altered cellular morphology, increased activity for senescence-associated-β-galactosidase (SA-β-GAL), increased formation of senescence-associated heterochromatin foci (SAHF) and promyelocytic leukemia protein nuclear bodies (PML NBs), permanent DNA damage, chromosomal instability and an inflammatory secretome. Some of these markers have been identified in cells from age-related pathologies. However, to improve our understanding of the contribution of cellular senescence to organismal ageing and age-related disease, it is imperative to define an unequivocal signature of cellular senescence that is functionally connected with normal and pathological ageing. Herein, we describe the processes leading to senescence, and the current biomarkers of cellular senescence, with particular emphasis on the causal role of DNA damage responses involved in the process. We highlight the gaps in our knowledge both of the processes leading to senescence, and the signature of cellular senescence both in vitro and in vivo. A well-defined set of senescence biomarkers for ageing and age-related disease would have a strong impact on the diagnosis, staging and predicted outcomes of age-related disease, providing the basis for a pharmacological intervention to postpone ageing and age-related disease.

Genome-wide linkage analysis for human longevity: Genetics of Healthy Aging Study

Clear evidence exists for heritability of human longevity, and much interest is focused on identifying genes associated with longer lives. To identify such longevity alleles, we performed the largest genome‐wide linkage scan thus far reported. Linkage analyses included 2118 nonagenarian Caucasian sibling pairs that have been enrolled in 15 study centers of 11 European countries as part of the Genetics of Healthy Aging (GEHA) project. In the joint linkage analyses, we observed four regions that show linkage with longevity; chromosome 14q11.2 (LOD = 3.47), chromosome 17q12‐q22 (LOD = 2.95), chromosome 19p13.3‐p13.11 (LOD = 3.76), and chromosome 19q13.11‐q13.32 (LOD = 3.57). To fine map these regions linked to longevity, we performed association analysis using GWAS data in a subgroup of 1228 unrelated nonagenarian and 1907 geographically matched controls. Using a fixed‐effect meta‐analysis approach, rs4420638 at the TOMM40/APOE/APOC1 gene locus showed significant association with longevity (P‐value = 9.6 × 10−8). By combined modeling of linkage and association, we showed that association of longevity with APOEε4 and APOEε2 alleles explain the linkage at 19q13.11‐q13.32 with P‐value = 0.02 and P‐value = 1.0 × 10−5, respectively. In the largest linkage scan thus far performed for human familial longevity, we confirm that the APOE locus is a longevity gene and that additional longevity loci may be identified at 14q11.2, 17q12‐q22, and 19p13.3‐p13.11. As the latter linkage results are not explained by common variants, we suggest that rare variants play an important role in human familial longevity.

Apoptosis-like, reversible changes in plasma membrane asymmetry and permeability, and transient modifications in mitochondrial membrane potential induced by curcumin in rat thymocytes

Curcumin (diferuoylmethane) is a natural compound with anticarcinogenic activities which is able to exert either proapoptotic or antiapoptotic effects in different cell types. This paper focuses on the sequence and extent of primary events induced by curcumin, in comparison with those occurring during dexamethasone‐induced apoptosis in rat thymocytes. It also presents annexin VI‐FITC as a new probe for studying membrane asymmetry. Curcumin readily penetrates into the cytoplasm, and is able to accumulate in membranous structures such as plasma membrane, endoplasmic reticulum and nuclear envelope. Curcumin‐treated cells exhibit typical features of apoptotic cell death, including shrinkage, transient phosphatidylserine exposure, increased membrane permeability and decrease in mitochondrial membrane potential. However, nuclei morphology, DNA fragmentation, the extent and time‐course of membrane changes are different from those observed during dexamethasone‐induced apoptosis, suggesting that, despite many similarities, the mode of action and the events triggered by curcumin are different from those occurring during typical apoptosis.

What evidence is there for the existence of individual genes with antagonistic pleiotropic effects

Classical evolutionary theory predicts the existence of genes with antagonistic effects on longevity and various components of early-life fitness. Quantitative genetic studies have provided convincing evidence that such genes exist. However, antagonistic pleiotropic effects have rarely been attributed to individual loci. We examine several classes of longevity-assurance genes: those involved in regulation of the gonad; the insulin-like growth factor pathway; free-radical scavenging; heat shock proteins and apoptosis. We find initial evidence that antagonistic pleiotropic effects are pervasive in each of these classes of genes and in various model systems--although most studies lack explicit studies of fitness components. This is particularly true of human studies. Very little is known about the early-life fitness effects of longevity loci. Given the possible medical importance of such effects we urge their future study.

Inhibition of proliferation and apoptosis of human and rat T lymphocytes by curcumin, a curry pigment

Curcumin (diferuoylmethane), the yellow pigment in the rhizome of tumeric (Curcuma longa), an ingredient of curry spice, is known to exhibit a variety of pharmacological effects including antitumor, antiinflammatory, and antiinfectious activities. Although its precise mode of action remains elusive, curcumin has been shown to suppress the activity of the AP-1 transcription factor in cells stimulated to proliferate. In this study, we observed that curcumin (50 microM) inhibited proliferation of rat thymocytes stimulated with concanavalin A (Con A) as well as that of human Jurkat lymphoblastoid cells in the logarithmic growth phase. The pigment also inhibited apoptosis in dexamethasone-treated rat thymocytes and in UV-irradiated Jurkat cells as judged by DNA ladder formation, cellular morphological changes, and flow cytometry analysis. The inhibition of apoptosis by curcumin in rat thymocytes was accompanied by partial suppression of AP-1 activity. Complete suppression of AP-1 activity was observed in Con A-treated, proliferating thymocytes. The capacity of curcumin to inhibit both cell growth and death strongly implies that these two biological processes share a common pathway at some point and that curcumin affects a common step, presumably involving a modulation of the AP-1 transcription factor.

Induction of senescence with doxorubicin leads to increased genomic instability of HCT116 cells

Induction of senescence has been proposed as a possible in vivo tumor response to anticancer treatment. Senescent cancer cells are often polyploid, however, their route to polyploidy is poorly recognized (endoreduplication versus aberrant mitoses). We showed that after treatment of HCT116 cells with a low dose of doxorubicin most of them stopped proliferation as documented by SA-beta-galactosidase activity and the lack of Ki67 expression. Increased expression of other common senescence markers, p53, p21 and cyclin D1, was also observed. The cells became giant, polyploid and polymorphic, with multinucleated cells comprising a substantial fraction. The vast majority of the doxorubicin-treated cells did not enter mitoses, as evidenced by mitotic index analysis, as well as by the predominantly cytoplasmic localization of cyclin B1 and a lack of separation of multiplied centrosomes. This allowed us to conclude that doxorubicin-treated HCT116 cells underwent endoreduplication. However, the rare events of aberrant mitoses of polyploid cells observed by us led to aneuploid progeny as was documented by cytogenetic analysis of survivors. Thus, a senescence-inducing treatment of HCT116 cancer cells had a dual effect-it stopped the proliferation of the majority of the cells, but also led to the appearance of proliferating aneuploid ones.

Genetics of healthy aging in Europe: The EU-integrated project GEHA (GEnetics of Healthy Aging)

Abstract:  The aim of the 5‐year European Union (EU)‐Integrated Project GEnetics of Healthy Aging (GEHA), constituted by 25 partners (24 from Europe plus the Beijing Genomics Institute from China), is to identify genes involved in healthy aging and longevity, which allow individuals to survive to advanced old age in good cognitive and physical function and in the absence of major age‐related diseases. To achieve this aim a coherent, tightly integrated program of research that unites demographers, geriatricians, geneticists, genetic epidemiologists, molecular biologists, bioinfomaticians, and statisticians has been set up. The working plan is to: (a) collect DNA and information on the health status from an unprecedented number of long‐lived 90+ sibpairs (n= 2650) and of younger ethnically matched controls (n= 2650) from 11 European countries; (b) perform a genome‐wide linkage scannning in all the sibpairs (a total of 5300 individuals); this investigation will be followed by linkage disequilibrium mapping (LD mapping) of the candidate chromosomal regions; (c) study in cases (i.e., the 2650 probands of the sibpairs) and controls (2650 younger people), genomic regions (chromosome 4, D4S1564, chromosome 11, 11.p15.5) which were identified in previous studies as possible candidates to harbor longevity genes; (d) genotype all recruited subjects for apoE polymorphisms; and (e) genotype all recruited subjects for inherited as well as epigenetic variability of the mitochondrial DNA (mtDNA). The genetic analysis will be performed by 9 high‐throughput platforms, within the framework of centralized databases for phenotypic, genetic, and mtDNA data. Additional advanced approaches (bioinformatics, advanced statistics, mathematical modeling, functional genomics and proteomics, molecular biology, molecular genetics) are envisaged to identify the gene variant(s) of interest. The experimental design will also allow (a) to identify gender‐specific genes involved in healthy aging and longevity in women and men stratified for ethnic and geographic origin and apoE genotype; (b) to perform a longitudinal survival study to assess the impact of the identified genetic loci on 90+ people mortality; and (c) to develop mathematical and statistical models capable of combining genetic data with demographic characteristics, health status, socioeconomic factors, lifestyle habits.

Curcumin Affects Components of the Chromosomal Passenger Complex and Induces Mitotic Catastrophe in Apoptosis-Resistant Bcr-Abl-Expressing Cells

The Bcr-Abl oncoprotein plays a major role in the development and progression of chronic myeloid leukemia and is a determinant of chemotherapy resistance occurring during the blast crisis phase of the disease. The aim of this article was to investigate the possibility of combating the resistance to apoptosis caused by Bcr-Abl by inducing an alternative cell death process. As a model of chronic myeloid leukemia, we employed Bcr-Abl-transfected mouse progenitor 32D cells with low and high Bcr-Abl expression levels corresponding to drug-sensitive and drug-resistant cells, respectively. The drug curcumin (diferuloylmethane), a known potent inducer of cell death in many cancer cells, was investigated for efficacy with Bcr-Abl-expressing cells. Curcumin strongly inhibited cell proliferation and affected cell viability by inducing apoptotic symptoms in all tested cells; however, apoptosis was a relatively late event. G2-M cell cycle arrest, together with increased mitotic index and cellular and nuclear morphology resembling those described for mitotic catastrophe, was observed and preceded caspase-3 activation and DNA fragmentation. Mitosis-arrested cells displayed abnormal chromatin organization, multipolar chromosome segregation, aberrant cytokinesis, and multinucleated cells—morphologic changes typical of mitotic catastrophe. We found that the mitotic cell death symptoms correlated with attenuated expression of survivin, a member of the chromosomal passenger complex, and mislocalization of Aurora B, the partner of survivin in the chromosomal passenger complex. Inhibition of survivin expression with small interfering RNA exhibited similar mitotic disturbances, thus implicating survivin as a major, albeit not the only, target for curcumin action. This study shows that curcumin can overcome the broad resistance to cell death caused by expression of Bcr-Abl and suggests that curcumin may be a promising agent for new combination regimens for drug-resistant chronic myeloid leukemia. (Mol Cancer Res 2006;4(7):457–69)

Curcumin induces permanent growth arrest of human colon cancer cells: Link between senescence and autophagy

Curcumin, a natural polyphenol derived from the rhizome of Curcuma longa, is a potent anticancer agent, which restricts tumor cell growth both in vitro and in vivo. Thus far curcumin was shown to induce death of cancer cells. This study reports the induction of cellular senescence of human colon cancer cells HCT116 upon curcumin treatment. The SA-β-galactosidase activation was observed both in p53+/+ and p53-/- cells, however the latter ones were less sensitive to the prosenescent activity of curcumin. Upregulation of p53 and p21 proteins was observed in p53+/+ HCT116, while p53-independent induction of p21 was noticed in p53-/- HCT116. Moreover, the senescence of HCT116 cells was accompanied by autophagy, that was confirmed by electron microscopy observations of autophagosomes in the curcumin-treated cells as well as LC3-II expression, punctue staining of LC3 and increased content of acidic vacuoles. Inhibition of autophagy, due to the diminished expression of ATG5 by RNAi decreased the number of senescent cells induced by curcumin, but did not lead to increased cell death. Altogether, we demonstrated a new antitumor activity of curcumin leading to cancer cell senescence and revealed the presence of a functional link between senescence and autophagy in curcumin-treated cells.

Effect of glutathione depletion on caspase-3 independent apoptosis pathway induced by curcumin in Jurkat cells

Curcumin, a yellow pigment from Curcuma longa, exhibits anti-inflammatory, antitumor, and antioxidative properties. Although its precise mode of action has not been elucidated so far, numerous studies have shown that curcumin may induce apoptosis in normal and cancer cells. Previously, we showed that in Jurkat cells curcumin induced nontypical apoptosis-like pathway, which was independent of mitochondria and caspase-3. Now we show that the inhibition of caspase-3 by curcumin, which is accompanied by attenuation of internucleosomal DNA fragmentation, may be due to elevation of glutathione, which increased in curcumin-treated cells to 130% of control. We have demonstrated that glutathione depletion does not itself induce apoptosis in Jurkat cells; though, it can release cytochrome c from mitochondria and caspase-3 from inhibition by curcumin, as shown by Western blot. The level of Bcl-2 protein was not affected by glutathione depletion even upon curcumin treatment. Altogether, our results show that in Jurkat cells curcumin prevents glutathione decrease, thus protecting cells against caspase-3 activation and oligonucleosomal DNA fragmentation. On the other hand, it induces nonclassical apoptosis via a still-unrecognized mechanism, which leads to chromatin degradation and high-molecular-weight DNA fragmentation.