Lysosome-mediated processing of chromatin in senescence

Cellular senescence is a stable proliferation arrest, a potent tumor suppressor mechanism, and a likely contributor to tissue aging. Cellular senescence involves extensive cellular remodeling, including of chromatin structure. Autophagy and lysosomes are important for recycling of cellular constituents and cell remodeling. Here we show that an autophagy/lysosomal pathway processes chromatin in senescent cells. In senescent cells, lamin A/C–negative, but strongly γ-H2AX–positive and H3K27me3-positive, cytoplasmic chromatin fragments (CCFs) budded off nuclei, and this was associated with lamin B1 down-regulation and the loss of nuclear envelope integrity. In the cytoplasm, CCFs were targeted by the autophagy machinery. Senescent cells exhibited markers of lysosomal-mediated proteolytic processing of histones and were progressively depleted of total histone content in a lysosome-dependent manner. In vivo, depletion of histones correlated with nevus maturation, an established histopathologic parameter associated with proliferation arrest and clinical benignancy. We conclude that senescent cells process their chromatin via an autophagy/lysosomal pathway and that this might contribute to stability of senescence and tumor suppression.     

Remodeling of chromatin structure in senescent cells and its potential impact on tumor suppression and aging

Cellular senescence is an important tumor suppression process, and a possible contributor to tissue aging. Senescence is accompanied by extensive changes in chromatin structure. In particular, many senescent cells accumulate specialized domains of facultative heterochromatin, called Senescence-Associated Heterochromatin Foci (SAHF), which are thought to repress expression of proliferation-promoting genes, thereby contributing to senescence-associated proliferation arrest. This article reviews our current understanding of the structure, assembly and function of these SAHF at a cellular level. The possible contribution of SAHF to tumor suppression and tissue aging is also critically discussed.     

Rb-mediated heterochromatin formation and silencing of E2F target genes during cellular senescence

Cellular senescence is an extremely stable form of cell cycle arrest that limits the proliferation of damaged cells and may act as a natural barrier to cancer progression. In this study, we describe a distinct heterochromatic structure that accumulates in senescent human fibroblasts, which we designated senescence-associated heterochromatic foci (SAHF). SAHF formation coincides with the recruitment of heterochromatin proteins and the retinoblastoma (Rb) tumor suppressor to E2F-responsive promoters and is associated with the stable repression of E2F target genes. Notably, both SAHF formation and the silencing of E2F target genes depend on the integrity of the Rb pathway and do not occur in reversibly arrested cells. These results provide a molecular explanation for the stability of the senescent state, as well as new insights into the action of Rb as a tumor suppressor.     

Lessons from senescence: Chromatin maintenance in non-proliferating cells

Cellular senescence is an irreversible proliferation arrest, thought to contribute to tumor suppression, proper wound healing and, perhaps, tissue and organismal aging. Two classical tumor suppressors, p53 and pRB, control cell cycle arrest associated with senescence. Profound molecular changes occur in cells undergoing senescence. At the level of chromatin, for example, senescence associated heterochromatic foci (SAHF) form in some cell types. Chromatin is inherently dynamic and likely needs to be actively maintained to achieve a stable cell phenotype. In proliferating cells chromatin is maintained in conjunction with DNA replication, but how non-proliferating cells maintain chromatin structure is poorly understood. Some histone variants, such as H3.3 and macroH2A increase as cells undergo senescence, suggesting histone variants and their associated chaperones could be important in chromatin structure maintenance in senescent cells. Here, we discuss options available for senescent cells to maintain chromatin structure and the relative contribution of histone variants and chaperones in this process. This article is part of a Special Issue entitled: Histone chaperones and chromatin assembly.     

DNA double strand break responses and chromatin alterations within the aging cell

Cellular senescence is a state of permanent replicative arrest that allows cells to stay viable and metabolically active but resistant to apoptotic and mitogenic stimuli. Specific, validated markers can identify senescent cells, including senescence-associated β galactosidase activity, chromatin alterations, cell morphology changes, activated p16- and p53-dependent signaling and permanent cell cycle arrest. Senescence is a natural consequence of DNA replication-associated telomere erosion, but can also be induced prematurely by telomere-independent events such as failure to repair DNA double strand breaks. Here, we review the molecular pathways of senescence onset, focussing on the changes in chromatin organization that are associated with cellular senescence, particularly senescence-associated heterochromatin foci formation. We also discuss the altered dynamics of the DNA double strand break response within the context of aging cells. Appreciating how, mechanistically, cellular senescence is induced, and how changes to chromatin organization and DNA repair contributes to this, is fundamental to our understanding of the normal and premature human aging processes associated with loss of organ and tissue function in humans.     

Fez1/Lzts1 a new mitotic regulator implicated in cancer development

The retinoblastoma (Rb) tumor suppressor gene product, pRb, has an established role in the implementation of cellular senescence, the state of irreversible G1 cell cycle arrest provoked by diverse oncogenic stresses. In murine cells, senescence cell cycle arrest can be reversed by subsequent inactivation of pRb, indicating that pRb is required not only for the onset of cellular senescence, but also for the maintenance of senescence program in murine cells. However, in human cells, once pRb is fully activated by p16^INK4a, senescence cell cycle arrest becomes irreversible and is no longer revoked by subsequent inactivation of pRb, suggesting that p16^INK4a/Rb-pathway activates an alternative mechanism to irreversibly block the cell cycle in human senescent cells. Here, we discuss the molecular mechanism underlying the irreversibility of senescence cell cycle arrest and its potential towards tumor suppression.     

Chromatin maintenance and dynamics in senescence: a spotlight on SAHF formation and the epigenome of senescent cells

Senescence is a stable proliferation arrest characterized by profound changes in cellular morphology and metabolism as well as by extensive chromatin reorganization in the nucleus. One particular hallmark of chromatin changes during senescence is the formation of punctate DNA foci in DAPI-stained senescent cells that have been called senescence-associated heterochromatin foci (SAHF). While many advances have been made concerning our understanding of the effectors of senescence, how chromatin is reorganized and maintained in senescent cells has remained largely elusive. Because chromatin structure is inherently dynamic, senescent cells face the challenge of developing chromatin maintenance mechanisms in the absence of DNA replication in order to maintain the senescent phenotype. Here, we summarize and review recent findings shedding light on SAHF composition and formation via spatial repositioning of chromatin, with a specific focus on the role of lamin B1 for this process. In addition, we discuss the physiological implication of SAHF formation, the role of histone variants, and histone chaperones during senescence and also elaborate on the more general changes observed in the epigenome of the senescent cells.     

Senescent phenotype achieved in vitro is indistinguishable, with the exception of Bcl-2 content, from that attained during the in vivo aging process

Senescent phenotype can be attained by diverse agents, thus suggesting that there might be molecular differences between the senescence achieved in vivo and the senescence-like state attained in vitro under culture conditions. In this study we compare the senescent phenotype reached by cells derived from young animals when cultured in vitro with the one associated with the in vivo aging process. Several in vitro senescence parameters, including MTT reduction, proliferation rate, DNA synthesis, SA-beta-gal staining, and both in vivo and in vitro Bcl-2 content, were determined. Alterations in DNA electrophoretic mobility were evaluated to test differences in bulk chromatin structure. Our results indicate that although it is possible to achieve a senescent phenotype with cells derived from young animals aged in culture, this phenotype differs from the one observed in older animals, due to lack of in vivo damage inducers to which cells are being exposed during natural aging.     

Lamin B1 loss is a senescence-associated biomarker

Cellular senescence is a potent tumor-suppressive mechanism that arrests cell proliferation and has been linked to aging. However, studies of senescence have been impeded by the lack of simple, exclusive biomarkers of the senescent state. Senescent cells develop characteristic morphological changes, which include enlarged and often irregular nuclei and chromatin reorganization. Because alterations to the nuclear lamina can affect both nuclear morphology and gene expression, we examined the nuclear lamina of senescent cells. We show here than lamin B1 is lost from primary human and murine cell strains when they are induced to senesce by DNA damage, replicative exhaustion, or oncogene expression. Lamin B1 loss did not depend on the p38 mitogen-activated protein kinase, nuclear factor-κB, ataxia telangiectasia-mutated kinase, or reactive oxygen species signaling pathways, which are positive regulators of senescent phenotypes. However, activation of either the p53 or pRB tumor suppressor pathway was sufficient to induce lamin B1 loss. Lamin B1 declined at the mRNA level via a decrease in mRNA stability rather than by the caspase-mediated degradation seen during apoptosis. Last, lamin B1 protein and mRNA declined in mouse tissue after senescence was induced by irradiation. Our findings suggest that lamin B1 loss can serve as biomarker of senescence both in culture and in vivo.     

Quantitative identification of senescent cells in aging and disease

Senescent cells are present in premalignant lesions and sites of tissue damage and accumulate in tissues with age. In vivo identification, quantification and characterization of senescent cells are challenging tasks that limit our understanding of the role of senescent cells in diseases and aging. Here, we present a new way to precisely quantify and identify senescent cells in tissues on a single‐cell basis. The method combines a senescence‐associated beta‐galactosidase assay with staining of molecular markers for cellular senescence and of cellular identity. By utilizing technology that combines flow cytometry with high‐content image analysis, we were able to quantify senescent cells in tumors, fibrotic tissues, and tissues of aged mice. Our approach also yielded the finding that senescent cells in tissues of aged mice are larger than nonsenescent cells. Thus, this method provides a basis for quantitative assessment of senescent cells and it offers proof of principle for combination of different markers of senescence. It paves the way for screening of senescent cells for identification of new senescence biomarkers, genes that bypass senescence or senolytic compounds that eliminate senescent cells, thus enabling a deeper understanding of the senescent state in vivo.     

CDK inhibitors (p16/p19/p21) induce senescence and autophagy in cancer-associated fibroblasts, “fueling” tumor growth via paracrine interactions,without an increase in neo-angiogenesis

Here, we investigated the compartment-specific role of cell cycle arrest and senescence in breast cancer tumor growth. For this purpose, we generated a number of hTERT-immortalized senescent fibroblast cell lines overexpressing CDK inhibitors, such as p16(INK4A), p19(ARF) or p21(WAF1/CIP1). Interestingly, all these senescent fibroblast cell lines showed evidence of increased susceptibility toward the induction of autophagy (either at baseline or after starvation), as well as significant mitochondrial dysfunction. Most importantly, these senescent fibroblasts also dramatically promoted tumor growth (up to ~2-fold), without any comparable increases in tumor angiogenesis. Conversely, we generated human breast cancer cells (MDA-MB-231 cells) overexpressing CDK inhibitors, namely p16(INK4A) or p21(WAF1/CIP1). Senescent MDA-MB-231 cells also showed increased expression of markers of cell cycle arrest and autophagy, including β-galactosidase, as predicted. Senescent MDA-MB-231 cells had retarded tumor growth, with up to a near 2-fold reduction in tumor volume. Thus, the effects of CDK inhibitors are compartment-specific and are related to their metabolic effects, which results in the induction of autophagy and mitochondrial dysfunction. Finally, induction of cell cycle arrest with specific inhibitors (PD0332991) or cellular stressors [hydrogen peroxide (H?O?) or starvation] indicated that the onset of autophagy and senescence are inextricably linked biological processes. The compartment-specific induction of senescence (and hence autophagy) may be a new therapeutic target that could be exploited for the successful treatment of human breast cancer patients.     

Dynamic assembly of chromatin complexes during cellular senescence: implications for the growth arrest of human melanocytic nevi

The retinoblastoma (RB)/p16(INK4a) pathway regulates senescence of human melanocytes in culture and oncogene-induced senescence of melanocytic nevi in vivo. This senescence response is likely due to chromatin modifications because RB complexes from senescent melanocytes contain increased levels of histone deacetylase (HDAC) activity and tethered HDAC1. Here we show that HDAC1 is prominently detected in p16(INK4a)-positive, senescent intradermal melanocytic nevi but not in proliferating, recurrent nevus cells that localize to the epidermal/dermal junction. To assess the role of HDAC1 in the senescence of melanocytes and nevi, we used tetracycline-based inducible expression systems in cultured melanocytic cells. We found that HDAC1 drives a sequential and cooperative activity of chromatin remodeling effectors, including transient recruitment of Brahma (Brm1) into RB/HDAC1 mega-complexes, formation of heterochromatin protein 1 beta (HP1 beta)/SUV39H1 foci, methylation of H3-K9, stable association of RB with chromatin and significant global heterochromatinization. These chromatin changes coincide with expression of typical markers of senescence, including the senescent-associated beta-galactosidase marker. Notably, formation of RB/HP1 beta foci and early tethering of RB to chromatin depends on intact Brm1 ATPase activity. As cells reached senescence, ejection of Brm1 from chromatin coincided with its dissociation from HP1 beta/RB and relocalization to protein complexes of lower molecular weight. These results provide new insights into the role of the RB pathway in regulating cellular senescence and implicate HDAC1 as a likely mediator of early chromatin remodeling events.