DNMT2/TRDMT1 gene knockout compromises doxorubicin-induced unfolded protein response and sensitizes cancer cells to ER stress-induced apoptosis.

The acidic, hypoxic and nutrient-deprived tumor microenvironment may induce endoplasmic reticulum (ER) stress and the unfolded protein response (UPR) may exert an important cytoprotective role by promoting folding of newly synthesized proteins and cancer cell survival. The lack of DNMT2/TRDMT1 methyltransferase-mediated C38 tRNA methylation compromises translational fidelity that may result in the accumulation of misfolded and aggregated proteins leading to proteotoxic stress-related cell death. In the present study, DNMT2/TRDMT1 gene knockout-mediated effects were investigated during doxorubicin (DOX)-induced ER stress and PERK-, IRE1- and ATF6-orchestrated UPR in four genetically different cellular models of cancer (breast and cervical cancer, osteosarcoma and glioblastoma cells). Upon DOX stimulation, DNMT2/TRDMT1 gene knockout impaired PERK activation and modulated NSUN and 5-methylcytosine RNA-based responses and microRNA profiles. The lack of DNMT2/TRDMT1 gene in DOX-treated four cancer cell lines resulted in decreased levels of four microRNAs, namely, miR-23a-3p, miR-93-5p, miR-125a-5p and miR-191-5p involved in the regulation of several pathways such as ubiquitin-mediated proteolysis, amino acid degradation and translational misregulation in cancer. We conclude that DNMT2/TRDMT1 gene knockout, at least in selected cellular cancer models, affects adaptive responses associated with protein homeostasis networks that during prolonged ER stress may result in increased sensitivity to apoptotic cell death.

Knockout of TRDMT1 methyltransferase affects DNA methylome in glioblastoma cells.

PURPOSE: We have previously shown that TRDMT1 methyltransferase is a regulator of chemotherapy-associated responses in glioblastoma cells. Despite the fact that glioblastoma, a common and malignant brain tumor, is widely characterized in terms of genetic and epigenetic markers, there are no data on TRDMT1-related changes in 5-methylcytosine pools in the genome. In the present study, the effect of TRDMT1 gene knockout (KO) on DNA methylome was analyzed. METHODS: CRISPR-based approach was used to obtain TRDMT1 KO glioblastoma cells. Total 5-methylcytosine levels in DNA, DNMT1 pools and DNMT activity were studied using ELISA. Reduced representation bisulfite sequencing (RRBS) was considered to comprehensively evaluate DNA methylome in glioblastoma cells with TRDMT1 KO. RESULTS: TRDMT1 KO cells were characterized by decreased levels of total 5-methylcytosine in DNA and DNMT1, and DNMT activity. RRBS-based methylome analysis revealed statistically significant differences in methylation-relevant DMS-linked genes in control cells compared to TRDMT1 KO cells. TRDMT1 KO-associated changes in DNA methylome may affect the activity of several processes and pathways such as telomere maintenance, cell cycle and longevity regulating pathway, proteostasis, DNA and RNA biology. CONCLUSIONS: TRDMT1 may be suggested as a novel modulator of gene expression by changes in DNA methylome that may affect cancer cell fates during chemotherapy. We postulate that the levels and mutation status of TRDMT1 should be considered as a prognostic marker and carefully monitored during glioblastoma progression.

Evaluation of Anticancer and Antibacterial Activity of Four 4-Thiazolidinone-Based Derivatives.

Heterocycles are commonly known for their unique features, e.g., antibacterial or anticancer properties. Although many synthetic heterocycles, such as 4-thiazolidinone (4-TZD), have been synthesized, their potential applications have not yet been fully investigated. However, many researchers have reported relevant results that can be a basis for the search for new potential drugs. Therefore, the aim of this study was to evaluate the cytotoxic, cytostatic, and antibacterial effects of certain 4-thiazolidinone-based derivatives, Les-3166, Les-5935, Les-6009, and Les-6166, on human fibroblasts (BJ), neuroblastoma (SH-SY5Y), epithelial lung carcinoma (A549), and colorectal adenocarcinoma (CACO-2) cell lines in vitro. All tested compounds applied in a concentration range from 10 to 100 µM were able to decrease metabolic activity in the BJ, A549, and SH-SY5Y cell lines. However, the action of Les-3166 was mainly based on the ROS-independent pathway, similarly to Les-6009. In turn, Les-5935 and Les-6166 were able to promote ROS production in BJ, A549, and SH-SY5Y cells, compared to the control. Les-3166, Les-6009, and Les-6166 significantly increased the caspase-3 activity, especially at the concentrations of 50 µM and 100 µM. However, Les-5935 did not induce apoptosis. Only Les-5935 showed a minor cytostatic effect on SH-SY5Y cells. Additionally, the antibacterial properties of the tested compounds against P. aeruginosa bacterial biofilm can be ranked as follows: Les-3166 > Les-5935 > Les-6009. Les-6166 did not show any anti-biofilm activity. In summary, the study showed that Les-5935, Les-6009, and Les-6166 were characterized by anticancer properties, especially in the human lung cancer cell. In cases of BJ, SH-SY5Y, and CACO-2 cells the anticancer usage of such compounds is limited due to effect visible only at 50 and 100 µM.

Altered dynamics in the circadian oscillation of clock genes in serum-shocked NIH-3T3 cells by the treatment of GYY4137 or AOAA.

BACKGROUND AND PURPOSE: Several members of the core clock mechanism are equipped with a Per-Arnt-Sim (PAS) domain through which they can bind haem [Fe(II)]. Haem is a ligand for the orphan receptors REV-ERBα/ß (NR1D1/2), which regulate circadian rhythm and metabolism. The ability to bind haem sensitises these clock components to the action of small molecule gases, including NO, CO and H2S. Studies conducted with European hamsters revealed that during winter sleep, key clock genes stop oscillating. At the same time, H2S, when administered at subtoxic concentrations, can induce a hypometabolic state in the cell. We suppose that core clock components, including the nuclear receptors REV-ERBs, neuronal PAS domain protein 2 (nPAS2) and PER2, can be H2S targets. The general objective of this study was to investigate the effect of the H2S system on the expression profile of the core clock genes in cells in vitro. EXPERIMENTAL APPROACH: We analysed the expression of Per1, Per2, Per3, Bmal1, Cry1, Cry2, Nr1d1, Nfil-3 and Dbp messenger RNA (mRNA) in serum-shocked NIH-3T3 cells treated with a slow-releasing H2S donor (GYY4137) or the cystathionine beta-synthase (CBS) inhibitor (AOAA) cultured under constant darkness and collected during 3 days in 3 h interval. KEY RESULTS AND CONCLUSIONS AND IMPLICATIONS: We found that pharmacological CBS inhibition increased the general expression and dynamics of several clock genes. On the other hand, increased H2S decreased Per2 expression. These data suggest that CBS can affect circadian clock and effect on clock-controlled transcription output.

The Roles of Host 5-Methylcytosine RNA Methyltransferases during Viral Infections.

Eukaryotic 5-methylcytosine RNA methyltransferases catalyze the transfer of a methyl group to the fifth carbon of a cytosine base in RNA sequences to produce 5-methylcytosine (m5C). m5C RNA methyltransferases play a crucial role in the maintenance of functionality and stability of RNA. Viruses have developed a number of strategies to suppress host innate immunity and ensure efficient transcription and translation for the replication of new virions. One such viral strategy is to use host m5C RNA methyltransferases to modify viral RNA and thus to affect antiviral host responses. Here, we summarize the latest findings concerning the roles of m5C RNA methyltransferases, namely, NOL1/NOP2/SUN domain (NSUN) proteins and DNA methyltransferase 2/tRNA methyltransferase 1 (DNMT2/TRDMT1) during viral infections. Moreover, the use of m5C RNA methyltransferase inhibitors as an antiviral therapy is discussed.

A Non-Vector Approach to Increase Lipid Levels in the Microalga Planktochlorella nurekis.

Microalgae are freshwater and marine unicellular photosynthetic organisms that utilize sunlight to produce biomass. Due to fast microalgal growth rate and their unique biochemical profiles and potential applications in food and renewable energy industries, the interest in microalgal research is rapidly increasing. Biochemical and genetic engineering have been considered to improve microalgal biomass production but these manipulations also limited microalgal growth. The aim of the study was the biochemical characterization of recently identified microalgal strain Planktochlorella nurekis with elevated cell size and DNA levels compared to wild type strain that was achieved by a safe non-vector approach, namely co-treatment with colchicine and cytochalasin B (CC). A slight increase in growth rate was observed in twelve clones of CC-treated cells. For biochemical profiling, several parameters were considered, namely the content of proteins, amino acids, lipids, fatty acids, ß-glucans, chlorophylls, carotenoids, B vitamins and ash. CC-treated cells were characterized by elevated levels of lipids compared to unmodified cells. Moreover, the ratio of carotenoids to chlorophyll a and total antioxidant capacity were slightly increased in CC-treated cells. We suggest that Planktochlorella nurekis with modified DNA levels and improved lipid content can be considered to be used as a dietary supplement and biofuel feedstock.

Snake venoms promote stress-induced senescence in human fibroblasts.

Snake venoms are widely studied in terms of their systemic toxicity and proteolytic, hemotoxic, neurotoxic, and cytotoxic activities. However, little is known about snake-venom-mediated effects when used at low, noncytotoxic concentrations. In the current study, two human fibroblast cell lines of different origin, namely WI-38 fetal lung fibroblasts and BJ foreskin fibroblasts were used to investigate snake-venom-induced adaptive response at a relatively noncytotoxic concentration (0.01 µg/ml). The venoms of Indochinese spitting cobra ( Naja siamensis), western green mamba ( Dendroaspis viridis), forest cobra ( Naja melanoleuca), and southern copperhead ( Agkistrodon contortrix) were considered. Snake venoms promoted FOXO3a-mediated oxidative stress response and to a lesser extent DNA damage response, which lead to changes in cell cycle regulators both at messenger RNA and protein levels, limited cell proliferation and migration, and induced cellular senescence. Taken together, we have shown for the first time that selected snake venoms may also exert adverse effects when used at relatively noncytotoxic concentrations.

Activation of transposable elements and genetic instability during long-term culture of the human fungal pathogen Candida albicans.

It has been repeatedly reported that transposable elements (TE) become active and/or mobile in the genomes of replicatively and stress-induced senescent mammalian cells. However, the biological role of senescence-associated transposon activation and its occurrence and relevance in other eukaryotic cells remain to be elucidated. In the present study, Candida albicans, a prevalent opportunistic fungal pathogen in humans, was used to analyze changes in gene copy number of selected TE, namely Cirt2, Moa and Cmut1 during long-term culture (up to 90 days). The effects of stress stimuli (fluconazole, hydrogen peroxide, hypochlorite) and ploidy state (haploid, diploid, tetraploid cells) were also considered. An increase in copy number of Cirt2 and Moa was the most accented in tetraploid cells after 90 days of culture that was accompanied by changes in karyotype patterns and slightly more limited growth rate compared to haploid and diploid cells. Stress stimuli did not potentiate TE activity. Elevation in chromosomal DNA breaks was also observed during long-term culture of cells of different ploidy, however this was not correlated with increased TE activity. Our results suggest that increased TE activity may promote genomic diversity and plasticity, and cellular heterogeneity during long-term culture of C. albicans cells.

FTIR and Raman Spectroscopy-Based Biochemical Profiling Reflects Genomic Diversity of Clinical Candida Isolates That May Be Useful for Diagnosis and Targeted Therapy of Candidiasis.

Despite the fact that Candida albicans is documented to be the main cause of human candidiasis, non-C. albicans Candida (NCAC) species, such as Candida glabrata and Candida tropicalis, are also suggested to be implicated in the etiopathogenesis of opportunistic fungal infections. As biology, epidemiology, pathogenicity, and antifungal resistance of NCAC species may be affected as a result of genomic diversity and plasticity, rapid and unambiguous identification of Candida species in clinical samples is essential for proper diagnosis and therapy. In the present study, 25 clinical isolates of C. albicans, C. glabrata, and C. tropicalis species were characterized in terms of their karyotype patterns, DNA content, and biochemical features. Fourier transform infrared (FTIR) spectra- and Raman spectra-based molecular fingerprints corresponded to the diversity of chromosomal traits and DNA levels that provided correct species identification. Moreover, Raman spectroscopy was documented to be useful for the evaluation of ergosterol content that may be associated with azole resistance. Taken together, we found that vibrational spectroscopy-based biochemical profiling reflects the variability of chromosome patterns and DNA content of clinical Candida species isolates and may facilitate the diagnosis and targeted therapy of candidiasis.

Plant-Derived Molecules α-Boswellic Acid Acetate, Praeruptorin-A, and Salvianolic Acid-B Have Age-Related Differential Effects in Young and Senescent Human Fibroblasts In Vitro.

Testing and screening of plant-derived molecules on normal human cells in vitro is a widely used approach for discovering their eventual health beneficial effects for human ageing and longevity. As little is known about age-associated differential effects of such molecules, here we report that young (<25% replicative lifespan completed) and near-senescent (>90% replicative lifespan completed) human skin fibroblasts exposed for 1-15 days to a wide range of concentrations (0.1-100 µM) of the three selected phytochemicals, namely α-boswellic acid acetate (ABC), praeruptorin-A (PTA), and salvianolic acid-B (SAB) had age-related differential effects. The parameters studied were the metabolic activity (MTT assay), cellular morphological phenotype, one-step growth characteristics, expression of genes involved in the cell cycle regulation and cytokine network genes, protein levels of p53, cytosolic superoxide dismutase (SOD1) and microtubule-associated protein 1A/1B-light chain 3 (LC3), and the extent of protein carbonylation and protein aggregation as a sign of oxidative stress. All three compounds showed biphasic hormetic dose response by stimulating cell growth, survival and metabolic activity at low doses (up to 1 µM), while showing inhibitory effects at high doses (>10 µM). Furthermore, the response of early passage young cells was different from that of the late passage near-senescent cells, especially with respect to the expression of cell cycle-related and inflammation-related genes. Such studies have importance with respect to the use of low doses of such molecules as health-promoting and/or ageing-interventions through the phenomenon of hormesis.

Downregulation of methyltransferase Dnmt2 results in condition-dependent telomere shortening and senescence or apoptosis in mouse fibroblasts.

Dnmt2 is a highly conserved methyltransferase of uncertain biological function(s). As Dnmt2 was considered as a driver of fruit fly longevity and a modulator of stress response, we decided to evaluate the role of Dnmt2 during stress-induced premature senescence in NIH3T3 mouse fibroblasts. Stable knockdown of Dnmt2 resulted in hydrogen peroxide-mediated sensitivity and apoptosis, whereas in the control conditions, senescence was induced. Cellular senescence was accompanied by elevated levels of p53 and p21, decreased telomere length and telomerase activity, increased production of reactive oxygen species and protein carbonylation, and DNA damage. Dnmt2 silencing also promoted global DNA and RNA hypermethylation, and upregulation of methyltransferases, namely Dnmt1, Dnmt3a, and Dnmt3b. Taken together, we show for the first time that Dnmt2 may promote lifespan in the control conditions and survival during stress conditions in mouse fibroblasts.

Ursolic acid-mediated changes in glycolytic pathway promote cytotoxic autophagy and apoptosis in phenotypically different breast cancer cells.

Plant-derived pentacyclic triterpenotids with multiple biological activities are considered as promising candidates for cancer therapy and prevention. However, their mechanisms of action are not fully understood. In the present study, we have analyzed the effects of low dose treatment (5-20 µM) of ursolic acid (UA) and betulinic acid (BA) on breast cancer cells of different receptor status, namely MCF-7 (ER+, PR+/-, HER2-), MDA-MB-231 (ER-, PR-, HER2-) and SK-BR-3 (ER-, PR-, HER2+). UA-mediated response was more potent than BA-mediated response. Triterpenotids (5-10 µM) caused G0/G1 cell cycle arrest, an increase in p21 levels and SA-beta-galactosidase staining that was accompanied by oxidative stress and DNA damage. UA (20 µM) also diminished AKT signaling that affected glycolysis as judged by decreased levels of HK2, PKM2, ATP and lactate. UA-induced energy stress activated AMPK that resulted in cytotoxic autophagy and apoptosis. UA-mediated elevation in nitric oxide levels and ATM activation may also account for AMPK activation-mediated cytotoxic response. Moreover, UA-promoted apoptosis was associated with decreased pERK1/2 signals and the depolarization of mitochondrial membrane potential. Taken together, we have shown for the first time that UA at low micromolar range may promote its anticancer action by targeting glycolysis in phenotypically distinct breast cancer cells.

Helicobacter pylori-induced premature senescence of extragastric cells may contribute to chronic skin diseases.

Helicobacter pylori, one of the most frequently observed bacterium in the human intestinal flora, has been widely studied since Marshall and Warren documented a link between the presence of H. pylori in the gastrointestinal tract and gastritis and gastric ulcers. Interestingly, H. pylori has also been found in several other epithelial tissues, including the eyes, ears, nose and skin that may have direct or indirect effects on host physiology and may contribute to extragastric diseases, e.g. chronic skin diseases. More recently, it has been shown that H. pylori cytotoxin CagA expression induces cellular senescence of human gastric nonpolarized epithelial cells that may lead to gastrointestinal disorders and systemic inflammation. Here, we hypothesize that also chronic skin diseases may be promoted by stress-induced premature senescence (SIPS) of skin cells, namely fibroblasts and keratinocytes, stimulated with H. pylori cytotoxins. Future studies involving cell culture models and clinical specimens are needed to verify the involvement of H. pylori in SIPS-based chronic skin diseases.

Chronic exposure to rapamycin and episodic serum starvation modulate ageing of human fibroblasts in vitro.

Mild stress-induced activation of stress response (SR) pathways, such as autophagy, heat shock response, oxidative SR, DNA damage response, and inflammatory response, can be potentially health beneficial. Using the model system of cellular ageing and replicative senescence in vitro, we have studied the ageing modulatory effects of the two conditions, rapamycin and serum starvation. Chronic exposure to 0.1, 1 and 10 nM rapamycin positively modulated the survival, growth, morphology, telomere length, DNA methylation levels, 8-oxo-dG level in DNA, N6-methyl-adenosine level in RNA, and ethanol stress tolerance of serially passaged normal human skin fibroblasts. Furthermore, episodic (once a week) serum starvation of human skin fibroblasts extended their replicative lifespan by about 22%, along with the maintenance of early passage youthful morphology even in late passage cultures. Although the results of this study may be considered preliminary, it can be inferred that intermittent and episodic induction of SR, rather than chronic up-regulation of SR, is more effective and applicable in the practice of hormesis for healthy ageing and longevity.

Affected chromosome homeostasis and genomic instability of clonal yeast cultures.

Yeast cells originating from one single colony are considered genotypically and phenotypically identical. However, taking into account the cellular heterogeneity, it seems also important to monitor cell-to-cell variations within a clone population. In the present study, a comprehensive yeast karyotype screening was conducted using single chromosome comet assay. Chromosome-dependent and mutation-dependent changes in DNA (DNA with breaks or with abnormal replication intermediates) were studied using both single-gene deletion haploid mutants (bub1, bub2, mad1, tel1, rad1 and tor1) and diploid cells lacking one active gene of interest, namely BUB1/bub1, BUB2/bub2, MAD1/mad1, TEL1/tel1, RAD1/rad1 and TOR1/tor1 involved in the control of cell cycle progression, DNA repair and the regulation of longevity. Increased chromosome fragility and replication stress-mediated chromosome abnormalities were correlated with elevated incidence of genomic instability, namely aneuploid events-disomies, monosomies and to a lesser extent trisomies as judged by in situ comparative genomic hybridization (CGH). The tor1 longevity mutant with relatively balanced chromosome homeostasis was found the most genomically stable among analyzed mutants. During clonal yeast culture, spontaneously formed abnormal chromosome structures may stimulate changes in the ploidy state and, in turn, promote genomic heterogeneity. These alterations may be more accented in selected mutated genetic backgrounds, namely in yeast cells deficient in proper cell cycle regulation and DNA repair.

Relationships between rDNA, Nop1 and Sir complex in biotechnologically relevant distillery yeasts.

Distillery yeasts are poorly characterized physiological group among the Saccharomyces sensu stricto complex. As industrial yeasts are under constant environmental stress during fermentation processes and the nucleolus is a stress sensor, in the present study, nucleolus-related parameters were evaluated in 22 commercially available distillery yeast strains. Distillery yeasts were found to be a heterogeneous group with a variable content and length of rDNA and degree of nucleolus fragmentation. The levels of rDNA were negatively correlated with Nop1 (r = -0.59, p = 0.0038). Moreover, the protein levels of Sir transcriptional silencing complex and longevity regulators, namely Sir1, Sir2, Sir3 and Fob1, were studied and negative correlations between Sir2 and Nop1 (r = -0.45, p = 0.0332), and between Sir2 and Fob1 (r = -0.49, p = 0.0211) were revealed. In general, S. paradoxus group of distillery yeasts with higher rDNA pools and Sir2 level than S. bayanus group was found to be more tolerant to fermentation-associated stress stimuli, namely mild cold/heat stresses and KCl treatment. We postulate that rDNA state may be considered as a novel factor that may modulate a biotechnological process.

Fatty Acid Profile and Biological Activities of Linseed and Rapeseed Oils.

It has been postulated that fatty acids found in edible oils may exert beneficial health effects by the modulation of signaling pathways regulating cell differentiation and proliferation, especially in the treatment of cardiovascular diseases. In the present study, the biological effects of selected edible oils--linseed (LO) and rapeseed (RO) oils--were tested in vitro on fibroblast cells. The fatty acid profile of the oils was determined using gas chromatography and FTIR spectroscopy. LO was found to be rich in α-linolenic acid (ALA), whereas oleic acid was the most abundant species in RO. Fatty acids were taken up by the cells and promoted cell proliferation. No oxidative stress-mediated cytotoxic or genotoxic effects were observed after oil stimulation. Oils ameliorated the process of wound healing as judged by improved migration of fibroblasts to the wounding area. As ALA-rich LO exhibited the most potent wound healing activity, ALA may be considered a candidate for promoting the observed effect.

Assessment of yeast chromosome XII instability: single chromosome comet assay.

The tools and techniques used in single-cell analysis of DNA damage in yeast Saccharomyces cerevisiae are limited. In this study, we modified the single cell gel electrophoresis assay, namely, the single chromosome comet assay based on DNA break analysis, at the chromosomal level. We studied the largest yeast chromosome XII, which contains the rDNA locus, and we investigated its instability using cell cycle checkpoint-, DNA damage- and antioxidative defence-deficient, and lifespan-deregulated yeast mutant strains. Moreover, we compared chromosome XII instability with the variability of nucleolar rDNA fluorescence signals. Three single-gene-deletion strains, cells lacking single-stranded DNA endonuclease, Rad1p; NAD(+)-dependent histone deacetylase, Sir2p; and gamma glutamylcysteine synthetase, Gsh1p, were more prone to chromosome XII instability compared to corresponding wildtype strains, indicating that DNA damage repair machinery, chromatin silencing and redox homeostasis may contribute to genome stability. Elevation in the number of DNA breaks was correlated with a high variability in the levels of nucleolar rDNA in the Δrad1 background, while unaffected chromosome XII and low variability in nucleolar rDNA fluorescence signals were observed in the Δtor1 longevity mutant. Taken together, the single chromosome comet assay may be successfully used to study DNA damage at the chromosomal level, which might be overlooked using whole population analysis on DNA breaks with PFGE separation.

Links between nucleolar activity, rDNA stability, aneuploidy and chronological aging in the yeast Saccharomyces cerevisiae.

The nucleolus is speculated to be a regulator of cellular senescence in numerous biological systems (Guarente, Genes Dev 11(19):2449-2455, 1997; Johnson et al., Curr Opin Cell Biol 10(3):332-338, 1998). In the budding yeast Saccharomyces cerevisiae, alterations in nucleolar architecture, the redistribution of nucleolar protein and the accumulation of extrachromosomal ribosomal DNA circles (ERCs) during replicative aging have been reported. However, little is known regarding rDNA stability and changes in nucleolar activity during chronological aging (CA), which is another yeast aging model used. In the present study, the impact of aberrant cell cycle checkpoint control (knock-out of BUB1, BUB2, MAD1 and TEL1 genes in haploid and diploid hemizygous states) on CA-mediated changes in the nucleolus was studied. Nucleolus fragmentation, changes in the nucleolus size and the nucleolus/nucleus ratio, ERC accumulation, expression pattern changes and the relocation of protein involved in transcriptional silencing during CA were revealed. All strains examined were affected by oxidative stress, aneuploidy (numerical rather than structural aberrations) and DNA damage. However, the bub1 cells were the most prone to aneuploidy events, which may contribute to observed decrease in chronological lifespan. We postulate that chronological aging may be affected by redox imbalance-mediated chromosome XII instability leading to both rDNA instability and whole chromosome aneuploidy. CA-mediated nucleolus fragmentation may be a consequence of nucleolus enlargement and/or Nop2p upregulation. Moreover, the rDNA content of chronologically aging cells may be a factor determining the subsequent replicative lifespan. Taken together, we demonstrated that the nucleolus state is also affected during CA in yeast.

A comparison of replicative senescence and doxorubicin-induced premature senescence of vascular smooth muscle cells isolated from human aorta.

Senescence of vascular smooth muscle cells (VSMCs) contributes to aging as well as age-related diseases of the cardiovascular system. Senescent VSMCs have been shown to be present in atherosclerotic plaques. Both replicative (RS) and stress-induced premature senescence (SIPS) accompany cardiovascular diseases. We aimed to establish the signature of RS and SIPS of VSMCs, induced by a common anticancer drug, doxorubicin, and to discover the so far undisclosed features of senescent cells that are potentially harmful to the organism. Most of the senescence hallmarks were common for both RS and SIPS; however, some differences were observed. 32 % of doxorubicin-treated cells were arrested in the G2/M phase of the cell cycle, while 73 % of replicatively senescing cells were arrested in the G1 phase. Moreover, on the basis of alkaline phosphatase activity measurements, we show that a 7-day treatment with doxorubicin (dox), does not cause precocious cell calcification, which is a characteristic feature of RS. We did not observe calcification even though after 7 days of dox-treatment many other markers characteristic for senescent cells were present. It can suggest that dox-induced SIPS does not accelerate the mineralization of vessels. We consider that detailed characterization of the two types of cellular senescence can be useful in in vitro studies of potential anti-aging factors.