Revisiting sensitivity of senescent cells to BH3 mimetics.

B cell leukemia/lymphoma 2 (BCL2) homology domain 3 (BH3) mimetics were reported to selectively kill senescent cells and improve age-related diseases. Defining why these cells show increased sensitivity to these molecules will help to identify new pharmacological compounds with senolytic activity. Here, we discuss how recent research findings provide new clues to understand this vulnerability.

Cholesterol biosynthetic pathway induces cellular senescence through ERRα.

Cellular senescence is a cell program induced by various stresses that leads to a stable proliferation arrest and to a senescence-associated secretory phenotype. Accumulation of senescent cells during age-related diseases participates in these pathologies and regulates healthy lifespan. Recent evidences point out a global dysregulated intracellular metabolism associated to senescence phenotype. Nonetheless, the functional contribution of metabolic homeostasis in regulating senescence is barely understood. In this work, we describe how the mevalonate pathway, an anabolic pathway leading to the endogenous biosynthesis of poly-isoprenoids, such as cholesterol, acts as a positive regulator of cellular senescence in normal human cells. Mechanistically, this mevalonate pathway-induced senescence is partly mediated by the downstream cholesterol biosynthetic pathway. This pathway promotes the transcriptional activity of ERRα that could lead to dysfunctional mitochondria, ROS production, DNA damage and a p53-dependent senescence. Supporting the relevance of these observations, increase of senescence in liver due to a high-fat diet regimen is abrogated in ERRα knockout mouse. Overall, this work unravels the role of cholesterol biosynthesis or level in the induction of an ERRα-dependent mitochondrial program leading to cellular senescence and related pathological alterations.

RSK3 switches cell fate: from stress-induced senescence to malignant progression.

BACKGROUND: TGFß induces several cell phenotypes including senescence, a stable cell cycle arrest accompanied by a secretory program, and epithelial-mesenchymal transition (EMT) in normal epithelial cells. During carcinogenesis cells lose the ability to undergo senescence in response to TGFß but they maintain an EMT, which can contribute to tumor progression. Our aim was to identify mechanisms promoting TGFß-induced senescence escape. METHODS: In vitro experiments were performed with primary human mammary epithelial cells (HMEC) immortalized by hTert. For kinase library screen and modulation of gene expression retroviral transduction was used. To characterize gene expression, RNA microarray with GSEA analysis and RT-qPCR were used. For protein level and localization, Western blot and immunofluorescence were performed. For senescence characterization crystal violet assay, Senescence Associated-ß-Galactosidase activity, EdU staining were conducted. To determine RSK3 partners FLAG-baited immunoprecipitation and mass spectrometry-based proteomic analyses were performed. Proteosome activity and proteasome enrichment assays were performed. To validate the role of RSK3 in human breast cancer, analysis of METABRIC database was performed. Murine intraductal xenografts using MCF10DCIS.com cells were carried out, with histological and immunofluorescence analysis of mouse tissue sections. RESULTS: A screen with active kinases in HMECs upon TGFß treatment identified that the serine threonine kinase RSK3, or RPS6KA2, a kinase mainly known to regulate cancer cell death including in breast cancer, reverted TGFß-induced senescence. Interestingly, RSK3 expression decreased in response to TGFß in a SMAD3-dependent manner, and its constitutive expression rescued SMAD3-induced senescence, indicating that a decrease in RSK3 itself contributes to TGFß-induced senescence. Using transcriptomic analyses and affinity purification coupled to mass spectrometry-based proteomics, we unveiled that RSK3 regulates senescence by inhibiting the NF-κΒ pathway through the decrease in proteasome-mediated IκBα degradation. Strikingly, senescent TGFß-treated HMECs display features of epithelial to mesenchymal transition (EMT) and during RSK3-induced senescence escaped HMECs conserve EMT features. Importantly, RSK3 expression is correlated with EMT and invasion, and inversely correlated with senescence and NF-κΒ in human claudin-low breast tumors and its expression enhances the formation of breast invasive tumors in the mouse mammary gland. CONCLUSIONS: We conclude that RSK3 switches cell fate from senescence to malignancy in response to TGFß signaling.

Expression of the Calcium-Binding Protein CALB1 Is Induced and Controls Intracellular Ca2+ Levels in Senescent Cells.

In response to many stresses, such as oncogene activation or DNA damage, cells can enter cellular senescence, a state of proliferation arrest accompanied by a senescence-associated secretory phenotype (SASP). Cellular senescence plays a key role in many physiopathological contexts, including cancer, aging and aging-associated diseases, therefore, it is critical to understand how senescence is regulated. Calcium ions (Ca2+) recently emerged as pivotal regulators of cellular senescence. However, how Ca2+ levels are controlled during this process is barely known. Here, we report that intracellular Ca2+ contents increase in response to many senescence inducers in immortalized human mammary epithelial cells (HMECs) and that expression of calbindin 1 (CALB1), a Ca2+-binding protein, is upregulated in this context, through the Ca2+-dependent calcineurin/NFAT pathway. We further show that overexpression of CALB1 buffers the rise in intracellular Ca2+ levels observed in senescent cells. Finally, we suggest that increased expression of Ca2+-binding proteins calbindins is a frequent mark of senescent cells. This work thus supports that, together with Ca2+channels, Ca2+-binding proteins modulate Ca2+ levels and flux during cellular senescence. This opens potential avenues of research to better understand the role of Ca2+ and of Ca2+-binding proteins in regulating cellular senescence.

NF-κB-dependent secretome of senescent cells can trigger neuroendocrine transdifferentiation of breast cancer cells.

Cellular senescence is characterized by a stable proliferation arrest in response to stresses and the acquisition of a senescence-associated secretory phenotype, called SASP, composed of numerous factors including pro-inflammatory molecules, proteases, and growth factors. The SASP affects the environment of senescent cells, especially during aging, by inducing and modulating various phenotypes such as paracrine senescence, immune cell activity, and extracellular matrix deposition and organization, which critically impact various pathophysiological situations, including fibrosis and cancer. Here, we uncover a novel paracrine effect of the SASP: the neuroendocrine transdifferentiation (NED) of some epithelial cancer cells, evidenced both in the breast and prostate. Mechanistically, this effect is mediated by NF-κB-dependent SASP factors, and leads to an increase in intracellular Ca2+ levels. Consistently, buffering Ca2+ by overexpressing the CALB1 buffering protein partly reverts SASP-induced NED, suggesting that the SASP promotes NED through a SASP-induced Ca2+ signaling. Human breast cancer dataset analyses support that NED occurs mainly in p53 WT tumors and in older patients, in line with a role of senescent cells and its secretome, as they are increasing during aging. In conclusion, our work, uncovering SASP-induced NED in some cancer cells, paves the way for future studies aiming at better understanding the functional link between senescent cell accumulation during aging, NED and clinical patient outcome.

Low expression of PCK2 in breast tumors contributes to better prognosis by inducing senescence of cancer cells.

Cell cycle arrest, one of the main characteristics of cellular senescence, has been described as a crucial barrier that needs to be bypassed for cancer progression. Typically, cellular senescence can be induced by multiple stresses including telomere shortening, oncogenic activation as well as therapy treatment, and contributes to the inhibition of epithelial-mesenchymal transition (EMT), tumor suppression or progression depending on the senescence-associated secretory phenotype (SASP) components. However, the mechanisms underlying cancer cell senescence remain partially understood. Here, according to METABRIC database, we identified that patients with senescent-like breast tumors show better short-term survival, lower tendency of neoplasm histological grades, lower tumor stages, and negative status of estrogen receptor (ER) and progesterone receptor (PR) compared with non-senescent ones. Interestingly, Kyoto encyclopedia of genes and genomes (KEGG) analysis identified insulin signaling was significantly repressed in senescent breast tumors. Further verification in cultured breast cancer cells indicated that phosphoenolpyruvate carboxykinase 2 (PCK2) was significantly inhibited after therapy treatment. In addition, knockdown of PCK2 induced a senescent phenotype of breast cancer cells. Moreover, comparing with the non-senescent group, the senescent breast cancers displayed lower EMT capacity both in patients and breast cancer cell lines after knocking down PCK2. In conclusion, we described for the first time that low expression level of PCK2 may contribute to better prognosis via triggering senescent phenotype and thereby inhibiting EMT capacity in breast cancers.

The mitochondrially-localized nucleoside diphosphate kinase D (NME4) is a novel metastasis suppressor.

BACKGROUND: Mitochondrial nucleoside diphosphate kinase (NDPK-D, NME4, NM23-H4) is a multifunctional enzyme mainly localized in the intermembrane space, bound to the inner membrane. RESULTS: We constructed loss-of-function mutants of NDPK-D, lacking either NDP kinase activity or membrane interaction and expressed mutants or wild-type protein in cancer cells. In a complementary approach, we performed depletion of NDPK-D by RNA interference. Both loss-of-function mutations and NDPK-D depletion promoted epithelial-mesenchymal transition and increased migratory and invasive potential. Immunocompromised mice developed more metastases when injected with cells expressing mutant NDPK-D as compared to wild-type. This metastatic reprogramming is a consequence of mitochondrial alterations, including fragmentation and loss of mitochondria, a metabolic switch from respiration to glycolysis, increased ROS generation, and further metabolic changes in mitochondria, all of which can trigger pro-metastatic protein expression and signaling cascades. In human cancer, NME4 expression is negatively associated with markers of epithelial-mesenchymal transition and tumor aggressiveness and a good prognosis factor for beneficial clinical outcome. CONCLUSIONS: These data demonstrate NME4 as a novel metastasis suppressor gene, the first localizing to mitochondria, pointing to a role of mitochondria in metastatic dissemination.

Loss of the Metastasis Suppressor NME1, But Not of Its Highly Related Isoform NME2, Induces a Hybrid Epithelial-Mesenchymal State in Cancer Cells.

Epithelial-mesenchymal transition (EMT) is important for the initial steps of metastasis. Although it is well accepted that the nucleoside diphosphate kinase NME1 is a metastasis suppressor, its effect on EMT remains poorly documented, as does that of its closely related isoform, NME2. Here, by using gene silencing, inactivation and overexpression strategies in a variety of cellular models of cancer, we show that NME1 is a powerful inhibitor of EMT. Genetic manipulation of NME2, by contrast, had no effect on the EMT phenotype of cancer cells, indicating a specific function of NME1 in EMT regulation. Loss of NME1 in epithelial cancer cells resulted in a hybrid phenotype intermediate between epithelial and mesenchymal cells, which is known to be associated with cells with a highly metastatic character. Conversely, overexpression of NME1 in mesenchymal cancer cells resulted in a more epithelial phenotype. We found that NME1 expression was negatively associated with EMT markers in many human cancers and was reduced in human breast tumor cell lines with the aggressive 'triple-negative' phenotype when compared to human breast tumor cell lines positive for estrogen receptor. We show that NME1, but not NME2, is an inhibitor of essential concerted intracellular signaling pathways involved in inducing EMT, including the AKT and MAPK (ERK, p38, and JNK) pathways. Additionally, NME1 depletion considerably altered the distribution of E-cadherin, a gatekeeper of the epithelial phenotype, shifting it from the plasma membrane to the cytosol and resulting in less E-cadherin on the cell surface than in control cells. Functional aggregation and dispersion assays demonstrated that inactivation of NME1 decreases E-cadherin-mediated cell-cell adhesion. We conclude that NME1, but not NME2, acts specifically to inhibit EMT and prevent the earliest stages of metastasis.

Elimination of Senescent Endothelial Cells: Good or Bad Idea?

Cellular 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.

PLA2R1 promotes DNA damage and inhibits spontaneous tumor formation during aging.

Although aging is a major risk factor for most types of cancers, it is barely studied in this context. The transmembrane protein PLA2R1 (phospholipase A2 receptor) promotes cellular senescence, which can inhibit oncogene-induced tumor initiation. Functions and mechanisms of action of PLA2R1 during aging are largely unknown. In this study, we observed that old Pla2r1 knockout mice were more prone to spontaneously develop a wide spectrum of tumors compared to control littermates. Consistently, these knockout mice displayed increased Parp1, a master regulator of DNA damage repair, and decreased DNA damage, correlating with large human dataset analysis. Forced PLA2R1 expression in normal human cells decreased PARP1 expression, induced DNA damage and subsequent senescence, while the constitutive expression of PARP1 rescued cells from these PLA2R1-induced effects. Mechanistically, PARP1 expression is repressed by a ROS (reactive oxygen species)-Rb-dependent mechanism upon PLA2R1 expression. In conclusion, our results suggest that PLA2R1 suppresses aging-induced tumors by repressing PARP1, via a ROS-Rb signaling axis, and inducing DNA damage and its tumor suppressive responses.

Genetic screens reveal mechanisms for the transcriptional regulation of tissue-specific genes in normal cells and tumors.

The proper tissue-specific regulation of gene expression is essential for development and homeostasis in metazoans. However, the illegitimate expression of normally tissue-restricted genes-like testis- or placenta-specific genes-is frequently observed in tumors; this promotes transformation, but also allows immunotherapy. Two important questions are: how is the expression of these genes controlled in healthy cells? And how is this altered in cancer? To address these questions, we used an unbiased approach to test the ability of 350 distinct genetic or epigenetic perturbations to induce the illegitimate expression of over 40 tissue-restricted genes in primary human cells. We find that almost all of these genes are remarkably resistant to reactivation by a single alteration in signaling pathways or chromatin regulation. However, a few genes differ and are more readily activated; one is the placenta-expressed gene ADAM12, which promotes invasion. Using cellular systems, an animal model, and bioinformatics, we find that a non-canonical but druggable TGF-ß/KAT2A/TAK1 axis controls ADAM12 induction in normal and cancer cells. More broadly, our data show that illegitimate gene expression in cancer is an heterogeneous phenomenon, with a few genes activatable by simple events, and most genes likely requiring a combination of events to become reactivated.

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.

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

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.

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.

Nucleolar aggresomes mediate release of pericentric heterochromatin and nuclear destruction of genotoxically treated cancer cells.

The role of the nucleolus and autophagy in maintenance of nuclear integrity is poorly understood. In addition, the mechanisms of nuclear destruction in cancer cells senesced after conventional chemotherapy are unclear. In an attempt to elucidate these issues, we studied teratocarcinoma PA1 cells treated with Etoposide (ETO), focusing on the nucleolus. Following treatment, most cells enter G2 arrest, display persistent DNA damage and activate p53, senescence, and macroautophagy markers. 2-5 µm sized nucleolar aggresomes (NoA) containing fibrillarin (FIB) and damaged rDNA, colocalized with ubiquitin, pAMPK, and LC3-II emerge, accompanied by heterochromatin fragments, when translocated perinuclearly. Microscopic counts following application of specific inhibitors revealed that formation of FIB-NoA is dependent on deficiency of the ubiquitin proteasome system coupled to functional autophagy. In contrast, the accompanying NoAs release of pericentric heterochromatin, which exceeds their frequency, is favored by debilitation of autophagic flux. Potential survivors release NoA in the cytoplasm during rare mitoses, while exit of pericentric fragments often depleted of H3K9Me3, with or without encompassing by NoA, occurs through the nucleolar protrusions and defects of the nuclear envelope. Foci of LC3-II are accumulated in the nucleoli undergoing cessation of rDNA transcription. As an origin of heterochromatin fragmentation, the unscheduled DNA synthesis and circular DNAs were found in the perinucleolar heterochromatin shell, along with activation and retrotransposition of ALU elements, colocalized with 45S rDNA in NoAs. The data indicate coordination of the basic nucleolar function with autophagy regulation in maintenance of the integrity of the nucleolus associated domains secured by inactivity of retrotransposons.

Somatic polyploidy is associated with the upregulation of c-MYC interacting genes and EMT-like signature.

The dependence of cancer on overexpressed c-MYC and its predisposition for polyploidy represents a double puzzle. We address this conundrum by cross-species transcription analysis of c-MYC interacting genes in polyploid vs. diploid tissues and cells, including human vs. mouse heart, mouse vs. human liver and purified 4n vs. 2n mouse decidua cells. Gene-by-gene transcriptome comparison and principal component analysis indicated that c-MYC interactants are significantly overrepresented among ploidy-associated genes. Protein interaction networks and gene module analysis revealed that the most upregulated genes relate to growth, stress response, proliferation, stemness and unicellularity, as well as to the pathways of cancer supported by MAPK and RAS coordinated pathways. A surprising feature was the up-regulation of epithelial-mesenchymal transition (EMT) modules embodied by the N-cadherin pathway and EMT regulators from SNAIL and TWIST families. Metabolic pathway analysis also revealed the EMT-linked features, such as global proteome remodeling, oxidative stress, DNA repair and Warburg-like energy metabolism. Genes associated with apoptosis, immunity, energy demand and tumour suppression were mostly down-regulated. Noteworthy, despite the association between polyploidy and ample features of cancer, polyploidy does not trigger it. Possibly it occurs because normal polyploidy does not go that far in embryonalisation and linked genome destabilisation. In general, the analysis of polyploid transcriptome explained the evolutionary relation of c-MYC and polyploidy to cancer.

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.

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.

Volume increase and spatial shifts of chromosome territories in nuclei of radiation-induced polyploidizing tumour cells.

The exposure of tumour cells to high doses of ionizing radiation can induce endopolyploidization as an escape route from cell death. This strategy generally results in mitotic catastrophe during the first few days after irradiation. However, some cells escape mitotic catastrophe, polyploidize and attempt to undergo genome reduction and de-polyploidization in order to create new, viable para-diploid tumour cell sub-clones. In search for the consequences of ionizing radiation induced endopolyploidization, genome and chromosome architecture in nuclei of polyploid tumour cells, and sub-nuclei after division of bi- or multi-nucleated cells were investigated during 7 days following irradiation. Polyploidization was induced in p53-function deficient HeLa cells by exposure to 10Gy of X-irradiation. Chromosome territories #1, #4, #12 and centromeres of chromosomes #6, #10, #X were labelled by FISH and analysed for chromosome numbers, volumes and spatial distribution during 7 days post irradiation. The numbers of interphase chromosome territories or centromeres, respectively, the positions of the most peripherally and centrally located chromosome territories, and the territory volumes were compared to non-irradiated controls over this time course. Nuclei with three copies of several chromosomes (#1, #6, #10, #12, #X) were found in the irradiated as well as non-irradiated specimens. From day 2 to day 5 post irradiation, chromosome territories (#1, #4, #12) shifted towards the nuclear periphery and their volumes increased 16- to 25-fold. Consequently, chromosome territories returned towards the nuclear centre during day 6 and 7 post irradiation. In comparison to non-irradiated cells (∼500µm(3)), the nuclear volume of irradiated cells was increased 8-fold (to ∼4000µm(3)) at day 7 post irradiation. Additionally, smaller cell nuclei with an average volume of about ∼255µm(3) were detected on day 7. The data suggest a radiation-induced generation of large intra-nuclear chromosome territories and their repositioning prior to genome reduction.