Self-Renewal Signalling in Presenescent Tetraploid IMR90 Cells

Endopolyploidy and genomic instability are shared features of both stress-induced cellular senescence and malignant growth. Here, we examined these facets in the widely used normal human fibroblast model of senescence, IMR90. At the presenescence stage, a small (2–7%) proportion of cells overcome the 4n-G1 checkpoint, simultaneously inducing self-renewal (NANOG-positivity), the DNA damage response (DDR; γ-H2AX-positive foci), and senescence (p16inka4a- and p21CIP1-positivity) signalling, some cells reach octoploid DNA content and divide. All of these markers initially appear and partially colocalise in the perinucleolar compartment. Further, with development of senescence and accumulation of p16inka4a and p21CIP1, NANOG is downregulated in most cells. The cells increasingly arrest in the 4n-G1 fraction, completely halt divisions and ultimately degenerate. A positive link between DDR, self-renewal, and senescence signalling is initiated in the cells overcoming the tetraploidy barrier, indicating that cellular and molecular context of induced tetraploidy during this period of presenescence is favourable for carcinogenesis

Role of stress-activated OCT4A in the cell fate decisions of embryonal carcinoma cells treated with etoposide

Tumor cellular senescence induced by genotoxic treatments has recently been found to be paradoxically linked to the induction of “stemness.” This observation is critical as it directly impinges upon the response of tumors to current chemo-radio-therapy treatment regimens. Previously, we showed that following etoposide (ETO) treatment embryonal carcinoma PA-1 cells undergo a p53-dependent upregulation of OCT4A and p21Cip1 (governing self-renewal and regulating cell cycle inhibition and senescence, respectively). Here we report further detail on the relationship between these and other critical cell-fate regulators. PA-1 cells treated with ETO display highly heterogeneous increases in OCT4A and p21Cip1 indicative of dis-adaptation catastrophe. Silencing OCT4A suppresses p21Cip1, changes cell cycle regulation and subsequently suppresses terminal senescence; p21Cip1-silencing did not affect OCT4A expression or cellular phenotype. SOX2 and NANOG expression did not change following ETO treatment suggesting a dissociation of OCT4A from its pluripotency function. Instead, ETO-induced OCT4A was concomitant with activation of AMPK, a key component of metabolic stress and autophagy regulation. p16ink4a, the inducer of terminal senescence, underwent autophagic sequestration in the cytoplasm of ETO-treated cells, allowing alternative cell fates. Accordingly, failure of autophagy was accompanied by an accumulation of p16ink4a, nuclear disintegration, and loss of cell recovery. Together, these findings imply that OCT4A induction following DNA damage in PA-1 cells, performs a cell stress, rather than self-renewal, function by moderating the expression of p21Cip1, which alongside AMPK helps to then regulate autophagy. Moreover, this data indicates that exhaustion of autophagy, through persistent DNA damage, is the cause of terminal cellular senescence

Elimination of Senescent Endothelial Cells: Good or Bad Idea?

International audienceCellular senescence has a critical role in many physiopathological contexts. Recent studies highlight the beneficial and adverse effects that eliminating senescent endothelial cells can have on health span, questioning the current development of drugs that induce the death of senescent cells, named senolytics, as a therapeutic strategy

Transcriptional repression of DNA repair genes is a hallmark and a cause of cellular senescence

Abstract Cellular senescence response is (i) activated by numerous stresses, (ii) is characterized by a stable proliferation arrest, and (iii) by a set of specific features. Timely regulated senescence is thought to be beneficial, whereas chronic senescence such as during normal or premature aging is deleterious as it favors most, if not all, age-related diseases. In this study, using in-house or publicly available microarray analyses of transcriptomes of senescent cells, as well as analyses of the level of expression of several DNA repair genes by RT-qPCR and immunoblot, we show that repression of DNA repair gene expression is associated with cellular senescence. This repression is mediated by the RB/E2F pathway and it may play a causal role in senescence induction, as single DNA repair gene repression by siRNA induced features of premature senescence. Importantly, activating RB independently of direct DNA damage also results in repression of DNA repair genes and in the subsequent induction of DNA damage and senescence. The dogma is that DNA damage observed during cellular senescence is directly provoked by DNA lesions following genotoxic attack (UV, IR, and ROS) or by induction of replicative stress upon oncogenic activation. Our in vitro results support a largely unsuspected contribution of the loss of DNA repair gene expression in the induction and the accumulation of the DNA damage observed in most, if not all, kinds of cellular senescence, and thus in the induction of cellular senescence. Further demonstration using in vivo models will help to generalize our findings

DNA methylation of the Oct4A enhancers in embryonal carcinoma cells after etoposide treatment is associated with alternative splicing and altered pluripotency in reversibly senescent cells

Funding Information: Dr. Bogdanova-Jatniece is acknowledged for sharing the sequences for Sox2 RT-qPCR. The authors thank Prof. MS Cragg for reading the manuscript. The study was supported by the Europe Social Fund Project, project No. 2013/0023/1DP/1.1.1.2.0/13/APIA/VIAA/037. The publishing costs are covered by the Riga Stradins University. Funding Information: The study was supported by the Europe Social Fund Project, project No. 2013/0023/1DP/1.1.1.2.0/13/APIA/VIAA/037. The publishing costs are covered by the Riga Stradins University. Publisher Copyright: © 2018 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group. Copyright: Copyright 2018 Elsevier B.V., All rights reserved.The epigenetic mechanisms underlying chemoresistance in cancer cells resulting from drug-induced reversible senescence are poorly understood. Chemoresistant ESC-like embryonal carcinoma PA1 cells treated with etoposide (ETO) were previously found to undergo prolonged G2 arrest with transient p53-dependent upregulation of opposing fate regulators, p21CIP1 (senescence) and OCT4A (self-renewal). Here we report on the analysis of the DNA methylation state of the distal enhancer (DE) and proximal enhancer (PE) of the Oct4A gene during this dual response. When compared to non–treated controls the methylation level increased from 1.3% to 12.5% and from 3% to 19.4%, in the DE and PE respectively. It included CpG and non-CpG methylation, which was not chaotic but presented two patterns in each enhancer. Discorrelating with methylation of enhancers, the transcription of Oct4A increased, however, a strong expression of the splicing form Oct4B was also induced, along with down-regulation of the Oct4A partners of in the pluripotency/self-renewal network Sox2 and Lin28. WB demonstrated disjoining of the OCT4A protein from the chromatin-bound fraction. In survival clones, methylation of the DE was considerably erased, while some remnant of methylation of the PE was still observed. The alternative splicing for Oct4B was reduced, Oct4A level insignificantly decreased, while the expression of Sox2 and Lin28 recovered, all three became proportionally above the control. These findings indicate the involvement of the transient patterned methylation of the Oct4A enhancers and alternative splicing in the adaptive regulation of cell fate choice during the p53-dependant dual state of reversible senescence in ESC-like cancer stem cells.publishersversionPeer reviewe

The cancer aneuploidy paradox: In the light of evolution

Aneuploidy should compromise cellular proliferation but paradoxically favours tumour progression and poor prognosis. Here, we consider this paradox in terms of our most recent observations of chemo/radio-resistant cells undergoing reversible polyploidy. The latter perform the segregation of two parental groups of end-to-end linked dyads by pseudo-mitosis creating tetraploid cells through a dysfunctional spindle. This is followed by autokaryogamy and a homologous pairing preceding a bi-looped endo-prophase. The associated RAD51 and DMC1/γ-H2AX double-strand break repair foci are tandemly situated on the AURKB/REC8/kinetochore doublets along replicated chromosome loops, indicative of recombination events. MOS-associated REC8-positive peri-nucleolar centromere cluster organises a monopolar spindle. The process is completed by reduction divisions (bi-polar or by radial cytotomy including pedogamic exchanges) and by the release of secondary cells and/or the formation of an embryoid. Together this process preserves genomic integrity and chromosome pairing, while tolerating aneuploidy by by-passing the mitotic spindle checkpoint. Concurrently, it reduces the chromosome number and facilitates recombination that decreases the mutation load of aneuploidy and lethality in the chemo-resistant tumour cells. This cancer life-cycle has parallels both within the cycling polyploidy of the asexual life cycles of ancient unicellular protists and cleavage embryos of early multicellulars, supporting the atavistic theory of cancer

The "virgin birth", polyploidy, and the origin of cancer

Recently, it has become clear that the complexity of cancer biology cannot fully be explained by somatic mutation and clonal selection. Meanwhile, data have accumulated on how cancer stem cells or stemloids bestow immortality on tumour cells and how reversible polyploidy is involved. Most recently, single polyploid tumour cells were shown capable of forming spheroids, releasing EMT-like descendents and inducing tumours in vivo. These data refocus attention on the centuries-old embryological theory of cancer. This review attempts to reconcile seemingly conflicting data by viewing cancer as a pre-programmed phylogenetic life-cycle-like process. This cycle is apparently initiated by a meiosis-like process and driven as an alternative to accelerated senescence at the DNA damage checkpoint, followed by an asexual syngamy event and endopolyploid-type embryonal cleavage to provide germ-cell-like (EMT) cells. This cycle is augmented by genotoxic treatments, explaining why chemotherapy is rarely curative and drives resistance. The logical outcome of this viewpoint is that alternative treatments may be more efficacious - either those that suppress the endopolyploidy-associated ‘life cycle’ or, those that cause reversion of embryonal malignant cells into benign counterparts. Targets for these opposing strategies are components of the same molecular pathways and interact with regulators of accelerated senescence

The Cancer Aneuploidy Paradox: In the Light of Evolution

Aneuploidy should compromise cellular proliferation but paradoxically favours tumour progression and poor prognosis. Here, we consider this paradox in terms of our most recent observations of chemo/radio-resistant cells undergoing reversible polyploidy. The latter perform the segregation of two parental groups of end-to-end linked dyads by pseudo-mitosis creating tetraploid cells through a dysfunctional spindle. This is followed by autokaryogamy and a homologous pairing preceding a bi-looped endo-prophase. The associated RAD51 and DMC1/γ-H2AX double-strand break repair foci are tandemly situated on the AURKB/REC8/kinetochore doublets along replicated chromosome loops, indicative of recombination events. MOS-associated REC8-positive peri-nucleolar centromere cluster organises a monopolar spindle. The process is completed by reduction divisions (bi-polar or by radial cytotomy including pedogamic exchanges) and by the release of secondary cells and/or the formation of an embryoid. Together this process preserves genomic integrity and chromosome pairing, while tolerating aneuploidy by by-passing the mitotic spindle checkpoint. Concurrently, it reduces the chromosome number and facilitates recombination that decreases the mutation load of aneuploidy and lethality in the chemo-resistant tumour cells. This cancer life-cycle has parallels both within the cycling polyploidy of the asexual life cycles of ancient unicellular protists and cleavage embryos of early multicellulars, supporting the atavistic theory of cancer