Remi-Martin Laberge

Senescent cells promote their own recognition and removal through the immune system by generating a bioactive secretome called the senescence-associated secretory phenotype (SASP). Sturmlechner et al. report that the cell cycle regulator p21 directs an early form of the SASP, which they call the p21-activated secretory phenotype (PASP) (see the Perspective by Reen and Gil). As part of the PASP, the chemokine CXCL14 attracts macrophages, which monitor stressed cells expressing elevated p21. If… Show more

Senescent cells promote their own recognition and removal through the immune system by generating a bioactive secretome called the senescence-associated secretory phenotype (SASP). Sturmlechner et al. report that the cell cycle regulator p21 directs an early form of the SASP, which they call the p21-activated secretory phenotype (PASP) (see the Perspective by Reen and Gil). As part of the PASP, the chemokine CXCL14 attracts macrophages, which monitor stressed cells expressing elevated p21. If stressed cells recuperate and p21 levels return to normal within 4 days, then macrophages disengage from their targets. Otherwise, macrophages recruit cytotoxic T cells that facilitate target cell removal. Other cell cycle regulators such as p16 can induce many factors overlapping with the PASP, but p21 uniquely drives this CXCL14-mediated “timer” mechanism of senescent cell immunosurveillance. —STS Show less

Cellular senescence is a cell fate response characterized by a permanent cell cycle arrest driven primarily the by cell cycle inhibitor and tumor suppressor proteins p16Ink4a and p21Cip1/Waf1. In mice, the p21Cip1/Waf1 encoding locus, Cdkn1a, is known to generate two transcripts that produce identical proteins, but one of these transcript variants is poorly characterized. We show that the Cdkn1a transcript variant 2, but not the better-studied variant 1, is selectively elevated during natural… Show more

Cellular senescence is a cell fate response characterized by a permanent cell cycle arrest driven primarily the by cell cycle inhibitor and tumor suppressor proteins p16Ink4a and p21Cip1/Waf1. In mice, the p21Cip1/Waf1 encoding locus, Cdkn1a, is known to generate two transcripts that produce identical proteins, but one of these transcript variants is poorly characterized. We show that the Cdkn1a transcript variant 2, but not the better-studied variant 1, is selectively elevated during natural aging across multiple mouse tissues. Importantly, mouse cells induced to senescence in culture by genotoxic stress (ionizing radiation or doxorubicin) upregulated both transcripts, but with different temporal dynamics: variant 1 responded nearly immediately to genotoxic stress, whereas variant 2 increased much more slowly as cells acquired senescent characteristics. Upon treating mice systemically with doxorubicin, which induces widespread cellular senescence in vivo, variant 2 increased to a larger extent than variant 1. Variant 2 levels were also more sensitive to the senolytic drug ABT-263 in naturally aged mice. Thus, variant 2 is a novel and more sensitive marker than variant 1 or total p21Cip1/Waf1 protein for assessing the senescent cell burden and clearance in mice. Show less

We elucidated the molecular cross-talk between cartilage and synovium in osteoarthritis, the most widespread arthritis in the world, using the powerful tool of single-cell RNA-sequencing. Multiple cell types were identified based on profiling of 10,640 synoviocytes and 26,192 chondrocytes: 12 distinct synovial cell types and 7 distinct articular chondrocyte phenotypes from matched tissues. Intact cartilage was enriched for homeostatic and hypertrophic chondrocytes, while damaged cartilage was… Show more

We elucidated the molecular cross-talk between cartilage and synovium in osteoarthritis, the most widespread arthritis in the world, using the powerful tool of single-cell RNA-sequencing. Multiple cell types were identified based on profiling of 10,640 synoviocytes and 26,192 chondrocytes: 12 distinct synovial cell types and 7 distinct articular chondrocyte phenotypes from matched tissues. Intact cartilage was enriched for homeostatic and hypertrophic chondrocytes, while damaged cartilage was enriched for prefibro- and fibro-, regulatory, reparative and prehypertrophic chondrocytes. A total of 61 cytokines and growth factors were predicted to regulate the 7 chondrocyte cell phenotypes. Based on production by > 1% of cells, 55% of the cytokines were produced by synovial cells (39% exclusive to synoviocytes and not expressed by chondrocytes) and their presence in osteoarthritic synovial fluid confirmed. The synoviocytes producing IL-1beta (a classic pathogenic cytokine in osteoarthritis), mainly inflammatory macrophages and dendritic cells, were characterized by co-expression of surface proteins corresponding to HLA-DQA1, HLA-DQA2, OLR1 or TLR2. Strategies to deplete these pathogenic intra-articular cell subpopulations could be a therapeutic option for human osteoarthritis. Show less

Chronological age represents the single greatest risk factor for human disease. One plausible explanation for this correlation is that mechanisms that drive ageing might also promote age-related diseases. Cellular senescence, which is a permanent state of cell cycle arrest induced by cellular stress, has recently emerged as a fundamental ageing mechanism that also contributes to diseases of late life, including cancer, atherosclerosis and osteoarthritis. Therapeutic strategies that safely… Show more

Chronological age represents the single greatest risk factor for human disease. One plausible explanation for this correlation is that mechanisms that drive ageing might also promote age-related diseases. Cellular senescence, which is a permanent state of cell cycle arrest induced by cellular stress, has recently emerged as a fundamental ageing mechanism that also contributes to diseases of late life, including cancer, atherosclerosis and osteoarthritis. Therapeutic strategies that safely interfere with the detrimental effects of cellular senescence, such as the selective elimination of senescent cells (SNCs) or the disruption of the SNC secretome, are gaining significant attention, with several programmes now nearing human clinical studies. Show less

Senescent cells (SnCs) accumulate in many vertebrate tissues with age and contribute to age-related pathologies, presumably through their secretion of factors contributing to the senescence-associated secretory phenotype (SASP). Removal of SnCs delays several pathologies and increases healthy lifespan. Aging and trauma are risk factors for the development of osteoarthritis (OA), a chronic disease characterized by degeneration of articular cartilage leading to pain and physical disability… Show more

Senescent cells (SnCs) accumulate in many vertebrate tissues with age and contribute to age-related pathologies, presumably through their secretion of factors contributing to the senescence-associated secretory phenotype (SASP). Removal of SnCs delays several pathologies and increases healthy lifespan. Aging and trauma are risk factors for the development of osteoarthritis (OA), a chronic disease characterized by degeneration of articular cartilage leading to pain and physical disability. Senescent chondrocytes are found in cartilage tissue isolated from patients undergoing joint replacement surgery, yet their role in disease pathogenesis is unknown. To test the idea that SnCs might play a causative role in OA, we used the p16-3MR transgenic mouse, which harbors a p16INK4a (Cdkn2a) promoter driving the expression of a fusion protein containing synthetic Renilla luciferase and monomeric red fluorescent protein domains, as well as a truncated form of herpes simplex virus 1 thymidine kinase (HSV-TK). This mouse strain allowed us to selectively follow and remove SnCs after anterior cruciate ligament transection (ACLT). We found that SnCs accumulated in the articular cartilage and synovium after ACLT, and selective elimination of these cells attenuated the development of post-traumatic OA, reduced pain and increased cartilage development. Intra-articular injection of a senolytic molecule that selectively killed SnCs validated these results in transgenic, non-transgenic and aged mice. Selective removal of the SnCs from in vitro cultures of chondrocytes isolated from patients with OA undergoing total knee replacement decreased expression of senescent and inflammatory markers while also increasing expression of cartilage tissue extracellular matrix proteins. Collectively, these findings support the use of SnCs as a therapeutic target for treating degenerative joint disease. Show less

Senescent cells (SCs) accumulate with age and after genotoxic stress, such as total-body irradiation (TBI). Clearance of SCs in a progeroid mouse model using a transgenic approach delays several age-associated disorders, suggesting that SCs play a causative role in certain age-related pathologies. Thus, a 'senolytic' pharmacological agent that can selectively kill SCs holds promise for rejuvenating tissue stem cells and extending health span. To test this idea, we screened a collection of… Show more

Senescent cells (SCs) accumulate with age and after genotoxic stress, such as total-body irradiation (TBI). Clearance of SCs in a progeroid mouse model using a transgenic approach delays several age-associated disorders, suggesting that SCs play a causative role in certain age-related pathologies. Thus, a 'senolytic' pharmacological agent that can selectively kill SCs holds promise for rejuvenating tissue stem cells and extending health span. To test this idea, we screened a collection of compounds and identified ABT263 (a specific inhibitor of the anti-apoptotic proteins BCL-2 and BCL-xL) as a potent senolytic drug. We show that ABT263 selectively kills SCs in culture in a cell type- and species-independent manner by inducing apoptosis. Oral administration of ABT263 to either sublethally irradiated or normally aged mice effectively depleted SCs, including senescent bone marrow hematopoietic stem cells (HSCs) and senescent muscle stem cells (MuSCs). Notably, this depletion mitigated TBI-induced premature aging of the hematopoietic system and rejuvenated the aged HSCs and MuSCs in normally aged mice. Our results demonstrate that selective clearance of SCs by a pharmacological agent is beneficial in part through its rejuvenation of aged tissue stem cells. Thus, senolytic drugs may represent a new class of radiation mitigators and anti-aging agents. Show less

The TOR (target of rapamycin) kinase limits longevity by poorly understood mechanisms. Rapamycin suppresses the mammalian TORC1 complex, which regulates translation, and extends lifespan in diverse species, including mice. We show that rapamycin selectively blunts the pro-inflammatory phenotype of senescent cells. Cellular senescence suppresses cancer by preventing cell proliferation. However, as senescent cells accumulate with age, the senescence-associated secretory phenotype (SASP) can… Show more

The TOR (target of rapamycin) kinase limits longevity by poorly understood mechanisms. Rapamycin suppresses the mammalian TORC1 complex, which regulates translation, and extends lifespan in diverse species, including mice. We show that rapamycin selectively blunts the pro-inflammatory phenotype of senescent cells. Cellular senescence suppresses cancer by preventing cell proliferation. However, as senescent cells accumulate with age, the senescence-associated secretory phenotype (SASP) can disrupt tissues and contribute to age-related pathologies, including cancer. MTOR inhibition suppressed the secretion of inflammatory cytokines by senescent cells. Rapamycin reduced IL6 and other cytokine mRNA levels, but selectively suppressed translation of the membrane-bound cytokine IL1A. Reduced IL1A diminished NF-κB transcriptional activity, which controls much of the SASP; exogenous IL1A restored IL6 secretion to rapamycin-treated cells. Importantly, rapamycin suppressed the ability of senescent fibroblasts to stimulate prostate tumour growth in mice. Thus, rapamycin might ameliorate age-related pathologies, including late-life cancer, by suppressing senescence-associated inflammation. Show less

With the global aging population, Alzheimer's disease, Parkinson's disease and mild cognition impairment are increasing in prevalence. The success of rapamycin as an agent to extend lifespan in various organisms, including mice, brings hope that chronic mTOR inhibition could also refrain age-related neurodegeneration. Here we review the evidence suggesting that mTOR inhibition - mainly with rapamycin - is a valid intervention to delay age-related neurodegeneration. We discuss the potential… Show more

With the global aging population, Alzheimer's disease, Parkinson's disease and mild cognition impairment are increasing in prevalence. The success of rapamycin as an agent to extend lifespan in various organisms, including mice, brings hope that chronic mTOR inhibition could also refrain age-related neurodegeneration. Here we review the evidence suggesting that mTOR inhibition - mainly with rapamycin - is a valid intervention to delay age-related neurodegeneration. We discuss the potential mechanisms by which rapamycin may facilitate neurodegeneration prevention or restoration of cognitive function. We also discuss the known side effects of rapamycin and provide evidence to alleviate exaggerated concerns regarding its wider clinical use. We explore the small molecule alternatives to rapamycin and propose future directions for their development, mainly by exploring the possibility of targeting the downstream effectors of mTOR: S6K1 and especially S6K2. Finally, we discuss the strengths and weaknesses of the models used to determine intervention efficacy for neurodegeneration. We address the difficulties of interpreting data using the common way of investigating the efficacy of interventions to delay/prevent neurodegeneration by observing animal behavior while these animals are under treatment. We propose an experimental design that should isolate the variable of aging in the experimental design and resolve the ambiguity present in recent literature. Show less

DNA damage has been implicated in ageing, but direct evidence for a causal relationship is lacking, owing to the difficulty of inducing defined DNA lesions in cells and tissues without simultaneously damaging other biomolecules and cellular structures. Here we directly test whether highly toxic DNA double-strand breaks (DSBs) alone can drive an ageing phenotype using an adenovirus-based system based on tetracycline-controlled expression of the SacI restriction enzyme. We deliver the adenovirus… Show more

DNA damage has been implicated in ageing, but direct evidence for a causal relationship is lacking, owing to the difficulty of inducing defined DNA lesions in cells and tissues without simultaneously damaging other biomolecules and cellular structures. Here we directly test whether highly toxic DNA double-strand breaks (DSBs) alone can drive an ageing phenotype using an adenovirus-based system based on tetracycline-controlled expression of the SacI restriction enzyme. We deliver the adenovirus to mice and compare molecular and cellular end points in the liver with normally aged animals. Treated, 3-month-old mice display many, but not all signs of normal liver ageing as early as 1 month after treatment, including ageing pathologies, markers of senescence, fused mitochondria and alterations in gene expression profiles. These results, showing that DSBs alone can cause distinct ageing phenotypes in mouse liver, provide new insights in the role of DNA damage as a driver of tissue ageing. Show less

Cellular senescence suppresses cancer by halting the growth of premalignant cells, yet the accumulation of senescent cells is thought to drive age-related pathology through a senescence-associated secretory phenotype (SASP), the function of which is unclear. To understand the physiological role(s) of the complex senescent phenotype, we generated a mouse model in which senescent cells can be visualized and eliminated in living animals. We show that senescent fibroblasts and endothelial cells… Show more

Cellular senescence suppresses cancer by halting the growth of premalignant cells, yet the accumulation of senescent cells is thought to drive age-related pathology through a senescence-associated secretory phenotype (SASP), the function of which is unclear. To understand the physiological role(s) of the complex senescent phenotype, we generated a mouse model in which senescent cells can be visualized and eliminated in living animals. We show that senescent fibroblasts and endothelial cells appear very early in response to a cutaneous wound, where they accelerate wound closure by inducing myofibroblast differentiation through the secretion of platelet-derived growth factor AA (PDGF-AA). In two mouse models, topical treatment of senescence-free wounds with recombinant PDGF-AA rescued the delayed wound closure and lack of myofibroblast differentiation. These findings define a beneficial role for the SASP in tissue repair and help to explain why the SASP evolved. Show less

DPPE, a tamoxifen derivative with antihistamine activity, was previously shown to potentiate the toxicity of chemotherapeutic drugs. Recently, a Phase III clinical study using doxorubicin with DPPE demonstrated significant increase in the overall survival of breast cancer patients. In this study we examined the effects of DPPE alone on the growth of drug sensitive and P-gp positive CHO cell line. Our results demonstrate DPPE is selectively toxic to P-gp positive cells and the sensitivity to… Show more

DPPE, a tamoxifen derivative with antihistamine activity, was previously shown to potentiate the toxicity of chemotherapeutic drugs. Recently, a Phase III clinical study using doxorubicin with DPPE demonstrated significant increase in the overall survival of breast cancer patients. In this study we examined the effects of DPPE alone on the growth of drug sensitive and P-gp positive CHO cell line. Our results demonstrate DPPE is selectively toxic to P-gp positive cells and the sensitivity to DPPE alone correlated with the levels of P-gp expression. Moreover, in MDR cells, DPPE-induced apoptosis was significantly reduced with Bcl2 overexpression and in the presence of P-gp ATPase inhibitor, PSC833. Furthermore, knockdown of P-gp expression in MDR cells with P-gp-siRNA reversed DPPE sensitivity and increased their sensitivity to doxorubicin and taxol but not to cisplatin. The addition of DPPE to membrane fractions led to dose-dependent increase in P-gp ATPase that was inhibited with PSC833. Moreover, incubation of P-gp positive cells with DPPE led to a significant increase in superoxide levels and a drop in cellular ATP and GSH pools that were reversible with inhibitors of P-gp ATPase. The combined presence of DPPE and the mitochondria electron transport complex III inhibitor, antimycin A, synergized in their effects on the growth of MDR cells but had no effect on the growth of parental drug sensitive cells. Collectively, the results of this study provide a possible mechanism that may be relevant to the clinical results of DPPE in breast cancer trial and demonstrates DPPE as P-gp collateral sensitivity drug. Show less

The overexpression of P-glycoprotein (P-gp, ABCB1) in cancer cells often leads to multidrug resistance (MDR) through reduced drug accumulation. However, certain P-gp-positive cells display hypersensitivity, or collateral sensitivity, to certain compounds that are believed to induce Pgp-dependent oxidative stress. We have previously reported that MDR P-gp-positive CHO cells are collaterally sensitive to verapamil (VRP; Laberge et al. (2009) [1]). In this report we extend our previous findings… Show more

The overexpression of P-glycoprotein (P-gp, ABCB1) in cancer cells often leads to multidrug resistance (MDR) through reduced drug accumulation. However, certain P-gp-positive cells display hypersensitivity, or collateral sensitivity, to certain compounds that are believed to induce Pgp-dependent oxidative stress. We have previously reported that MDR P-gp-positive CHO cells are collaterally sensitive to verapamil (VRP; Laberge et al. (2009) [1]). In this report we extend our previous findings and show that drug resistant CHO cells are also collaterally sensitive to physiologic levels of progesterone (PRO) and deoxycorticosterone (DOC). Both PRO and DOC collateral sensitivities in CH(R)C5 cells are dependent on P-gp-expression and ATPase, as knockdown of P-gp expression with siRNA or inhibition of P-gp-ATPase with PSC833 reverses PRO- and DOC-induced collateral sensitivity. Moreover, the mitochondrial complexes I and III inhibitors (antimycin-A and rotenone, respectively) synergize with PRO and DOC-induced collateral sensitivity. We also show that VRP inhibits PRO and DOC collateral sensitivity, consistent with earlier findings relating to the VRP's modulation of PRO and DOC-stimulation of P-gp ATPase. The findings of this study demonstrate a P-gp-dependent collateral sensitivity of MDR cells in the presence of physiologically achievable concentrations of progesterone and deoxycorticosterone. Show less

Here we describe a pseudoegene that is activated by pro-inflammatory signals that acts to modulate the activity of NF-kB by interacting with RelA to inhibit DNA binding and target gene activation.

Senescence is a cellular response to damage and stress. The senescence response prevents cancer by suppressing the proliferation of cells with a compromised genome and contributes to optimal wound healing in normal tissues. Persistent senescent cells are also thought to drive aging and age-associated pathologies through their secretion of inflammatory factors that modify the tissue microenvironment and alter the function of nearby normal or transformed cells. Understanding how senescent cells… Show more

Senescence is a cellular response to damage and stress. The senescence response prevents cancer by suppressing the proliferation of cells with a compromised genome and contributes to optimal wound healing in normal tissues. Persistent senescent cells are also thought to drive aging and age-associated pathologies through their secretion of inflammatory factors that modify the tissue microenvironment and alter the function of nearby normal or transformed cells. Understanding how senescent cells alter the microenvironment would be aided by the ability to induce or eliminate senescent cells at will in vivo. Here, we combine the use of the synthetic nucleoside analog ganciclovir (GCV) with herpes simplex virus thymidine kinase (HSVtk) activity to create or eliminate senescent human cells. We show that low concentrations of GCV induce senescence through the accumulation of nuclear DNA damage while higher concentrations of GCV, similar to those used in vivo, kill non-dividing senescent cells via mitochondrial DNA (mtDNA) damage and caspase-dependent apoptosis. Using this system, we effectively eliminated xenografted normal human senescent fibroblasts or induced senescence in human breast cancer cells in vivo. Thus, cellular senescence and mtDNA damage are outcomes of synthetic nucleoside analog treatment, indicating that the GCV-HSVtk combination can be used effectively to promote the targeted formation or eradication of senescent cells. Show less

Exposure to environmental toxins is associated with a variety of age-related diseases including cancer and neurodegeneration. For example, in Parkinson's disease (PD), chronic environmental exposure to certain toxins has been linked to the age-related development of neuropathology. Neuronal damage is believed to involve the induction of neuroinflammatory events as a consequence of glial cell activation. Cellular senescence is a potent anti-cancer mechanism that occurs in a number of… Show more

Exposure to environmental toxins is associated with a variety of age-related diseases including cancer and neurodegeneration. For example, in Parkinson's disease (PD), chronic environmental exposure to certain toxins has been linked to the age-related development of neuropathology. Neuronal damage is believed to involve the induction of neuroinflammatory events as a consequence of glial cell activation. Cellular senescence is a potent anti-cancer mechanism that occurs in a number of proliferative cell types and causes the arrest of proliferation of cells at risk of malignant transformation following exposure to potentially oncogenic stimuli. With age, senescent cells accumulate and express a senescence-associated secretory phenotype (SASP; that is the robust secretion of many inflammatory cytokines, growth factors and proteases). Whereas cell senescence in peripheral tissues has been causally linked to a number of age-related pathologies, little is known about the induction of cellular senescence and the SASP in the brain. On the basis of recently reported findings, we propose that environmental stressors associated with PD may act in part by eliciting senescence and the SASP within non neuronal glial cells in the ageing brain, thus contributing to the characteristic decline in neuronal integrity that occurs in this disorder. Show less

Depending on the cell type and tissue environment, epithelial and mesenchymal cell phenotypes are not static and can be highly dynamic. Epithelial-mesenchymal transitions (EMTs) and reverse EMTs provide flexibility during embryogenesis. While EMTs are a critical normal process during development and wound healing, properties of the EMT have been implicated in human pathology, particularly cancer metastasis. A normal undamaged epithelium does not typically exhibit features of an EMT. However… Show more

Depending on the cell type and tissue environment, epithelial and mesenchymal cell phenotypes are not static and can be highly dynamic. Epithelial-mesenchymal transitions (EMTs) and reverse EMTs provide flexibility during embryogenesis. While EMTs are a critical normal process during development and wound healing, properties of the EMT have been implicated in human pathology, particularly cancer metastasis. A normal undamaged epithelium does not typically exhibit features of an EMT. However, particularly under the influence of the surrounding microenvironment, cancer cells may reactivate developmental phenotypes out of context in the adult. This reactivation, such as the EMT, can facilitate tumor cell invasion and metastasis, and therefore is a major mechanism of tumor progression. Conversely, cellular senescence, which is associated with aging, is a process by which cells enter a state of permanent cell cycle arrest, thereby constituting a potent tumor suppressive mechanism. However, accumulating evidence shows that senescent cells can have deleterious effects on the tissue microenvironment. The most significant of these effects is the acquisition of a senescence-associated secretory phenotype (SASP) that turns senescent fibroblasts into pro-inflammatory cells having the ability to promote tumor progression, in part by inducing an EMT in nearby epithelial cells. Here, we summarize the potential impacts of SASP factors, particularly interleukins, on tissue microenvironments and their ability to stimulate tumor progression through induction of an EMT. Show less

Cellular senescence suppresses cancer by arresting the proliferation of cells at risk for malignant transformation. Recently, senescent cells were shown to secrete numerous cytokines, growth factors, and proteases that can alter the tissue microenvironment and may promote age-related pathology. To identify small molecules that suppress the senescence-associated secretory phenotype (SASP), we developed a screening protocol using normal human fibroblasts and a library of compounds that are… Show more

Cellular senescence suppresses cancer by arresting the proliferation of cells at risk for malignant transformation. Recently, senescent cells were shown to secrete numerous cytokines, growth factors, and proteases that can alter the tissue microenvironment and may promote age-related pathology. To identify small molecules that suppress the senescence-associated secretory phenotype (SASP), we developed a screening protocol using normal human fibroblasts and a library of compounds that are approved for human use. Among the promising library constituents was the glucocorticoid corticosterone. Both corticosterone and the related glucocorticoid cortisol decreased the production and secretion of selected SASP components, including several pro-inflammatory cytokines. Importantly, the glucocorticoids suppressed the SASP without reverting the tumor suppressive growth arrest and were efficacious whether cells were induced to senesce by ionizing radiation or strong mitogenic signals delivered by oncogenic RAS or MAP kinase kinase 6 overexpression. Suppression of the prototypical SASP component IL-6 required the glucocorticoid receptor, which, in the presence of ligand, inhibited IL-1α signaling and NF-κB transactivation activity. Accordingly, co-treatments combining glucocorticoids with the glucocorticoid antagonist RU-486 or recombinant IL-1α efficiently reestablished NF-κB transcriptional activity and IL-6 secretion. Our findings demonstrate feasibility of screening for compounds that inhibit the effects of senescent cells. They further show that glucocorticoids inhibit selected components of the SASP and suggest that corticosterone and cortisol, two FDA-approved drugs, might exert their effects in part by suppressing senescence-associated inflammation. Show less

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… Show more

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. Show less

P-glycoprotein (or P-gp1, ABCB1) expression in tumor cells is causative of multidrug resistance through the active efflux of drugs across the cell membrane. However, the over-expression of P-glycoprotein in some tumor cells has been associated with increased sensitivity, or "collateral sensitivity", of multidrug resistant cells to specific drugs, including the calcium channel blocker verapamil. We previously demonstrated that collateral sensitivity to verapamil correlates with the effect of… Show more

P-glycoprotein (or P-gp1, ABCB1) expression in tumor cells is causative of multidrug resistance through the active efflux of drugs across the cell membrane. However, the over-expression of P-glycoprotein in some tumor cells has been associated with increased sensitivity, or "collateral sensitivity", of multidrug resistant cells to specific drugs, including the calcium channel blocker verapamil. We previously demonstrated that collateral sensitivity to verapamil correlates with the effect of this drug on P-gp1 ATPase, and is reversed by inhibitors of P-gp1 ATPase (e.g., PSC 833 and Ivermectin). In this report, we expand on our earlier study and demonstrate that P-gp1 expression in drug-resistant cells modulates collateral sensitivity. Using P-gp1-specific siRNA, P-gp1 expression in the multidrug resistant CH(R)C5 cells was significantly down-regulated beginning on day 2 post-transfection of siRNA. Furthermore, down-regulation of P-gp1 led to increased sensitivity of CH(R)C5 cells to paclitaxel and doxorubicin, but not to cis-platinum, due to inhibition of P-gp1 drug efflux pump. Down-regulation of P-gp1 expression completely reversed collateral sensitivity to verapamil. Moreover, known inhibitors of ETC, rotenone and antimycin A which cause an increase in reactive oxygen species, synergized with verapamil-induced collateral sensitivity leading to increased cell death as determined by MTT cell survival assay. Similarly, the addition of hydrogen peroxide also synergized with verapamil. Taken together, the results of this study demonstrate a direct link between P-gp1 expression and collateral sensitivity of drug-resistant cells, possibly due to an increase in reactive oxygen species. Show less

The over-expression of ABCC1 transmembrane protein has been shown to cause multidrug resistance in tumor cell lines. ABCC1 is a member of the ABC transmembrane proteins that function as efflux pumps with diverse substrate specificity. Several endogenous cell metabolites, including the leukotriene C4 (LTC(4)) and glutathione (GSH) are substrates for ABCC1 protein. ABCC1 expression in certain tumor cells was demonstrated to confer hypersensitivity to glutathione modulating agents. In this report… Show more

The over-expression of ABCC1 transmembrane protein has been shown to cause multidrug resistance in tumor cell lines. ABCC1 is a member of the ABC transmembrane proteins that function as efflux pumps with diverse substrate specificity. Several endogenous cell metabolites, including the leukotriene C4 (LTC(4)) and glutathione (GSH) are substrates for ABCC1 protein. ABCC1 expression in certain tumor cells was demonstrated to confer hypersensitivity to glutathione modulating agents. In this report we have investigated the mechanism of collateral sensitivity seen in tumor cells over-expressing ABCC1 protein. The results of this study show that ABCC1 expression in tumor cells correlates with their hypersensitivity to various glutathione modulating agents, as demonstrated in H69AR-drug selected and HeLa/ABCC1-transfectant cells. This effect was triggered either through inhibition of GSH synthesis with BSO or by increasing ABCC1-mediated GSH transport with verapamil or apigenin. In addition, our results show that the hypersensitivity of ABCC1-expressing cells to BSO, verapamil or apigenin was preceded by an increase in reactive oxygen species (or ROS). A decrease in GSH level is also observed prior the increase in ROS. In addition, we show that hypersensitivity to the BSO, verapamil or apigenin leads to tumor cell death by apoptosis. Together, the results of this study demonstrate that ABCC1 potentiates oxidative stress in tumor cells through reductions in cellular GSH levels. Show less

There is a growing interest regarding the use of camptothecins (CPTs) for the management of ovarian cancer. Since topoisomerase I has been established as a prime target of these drugs in other experimental models, it was important to determine whether sensitivity to CPTs in ovarian cancer cells is also correlated with the cellular level of this enzyme. Despite the 7-fold increase in topoisomerase expression achieved by adenovirus-mediated expression, the sensitivity to a CPT derivative… Show more

There is a growing interest regarding the use of camptothecins (CPTs) for the management of ovarian cancer. Since topoisomerase I has been established as a prime target of these drugs in other experimental models, it was important to determine whether sensitivity to CPTs in ovarian cancer cells is also correlated with the cellular level of this enzyme. Despite the 7-fold increase in topoisomerase expression achieved by adenovirus-mediated expression, the sensitivity to a CPT derivative (topotecan), was not improved compared with control cells harboring an endogenous level of the enzyme. This observation is in accordance with the similar level of topoisomerase I activity found in control and overexpressing cells and suggests that these cells may efficiently regulate the enzyme activity. Indeed, topoisomerase I overexpressing cells are characterized by a lack of alkaline phosphatase sensitivity and elimination of the hyperphosphorylated form of the protein. Taken together, these observations strongly suggest that an alteration in the phosphorylation state of topoisomerase I could limit its activity and prevent improvement of CPT response in ovarian cancer cells. In addition, a limited extent of topoisomerase I phosphorylating activity was found in nuclear extract of OVCAR-3 cells. Hence, providing enhancement in topoisomerase I expression may not result in improvement of CPT response in ovarian cancer cells because of an efficient control of the phosphorylation state of the enzyme. Show less

Several causes of male infertility remain idiopathic. Recently, the condensed state of the sperm head has been demonstrated as a discriminating parameter for the assessment of male infertility. Altered DNA condensation is associated with an increase in DNA strand breakage so the genetic integrity of the male gamete is threatened. The origin of the DNA strand breaks in unknown. However, transient DNA strand breaks appear in the whole population of elongating spermatids during mid-spermiogenesis… Show more

Several causes of male infertility remain idiopathic. Recently, the condensed state of the sperm head has been demonstrated as a discriminating parameter for the assessment of male infertility. Altered DNA condensation is associated with an increase in DNA strand breakage so the genetic integrity of the male gamete is threatened. The origin of the DNA strand breaks in unknown. However, transient DNA strand breaks appear in the whole population of elongating spermatids during mid-spermiogenesis steps. Most likely, these transient breaks are required to support the change in DNA topology associated with chromatin remodeling at these steps. Histones hyperacetylation is also coincident with the DNA strand breakage steps. Hyperacetylation of histones may represent a necessary condition for strand breakages to form allowing access to the yet unknown enzymatic activity involved in the removal of DNA supercoils. A better characterization of this enzyme activity at these steps is necessary as this may represent a very sensitive process where altercations in the genetic integrity of the male gamete may arise and persist up to the mature spermatozoa. During the chromatin remodeling in spermatids, the combined DNA-condensing activities provides by the basic transition proteins and protamines may optimize the strand repair process emphasizing the link between altered sperm DNA condensation and DNA fragmentation. The mutagenic potential of these events may have been overlooked as it may result in fertility and/or developmental problems. Show less

Transient DNA strand breaks are generated in the whole population of elongating spermatids and are perfectly coincident with histone H4 hyperacetylation at chromatin-remodeling steps. Given the limited DNA repair capacity of elongating spermatids, chromatin remodeling may present a threat to genetic integrity of the male gamete. The nature of the DNA strand breakage, the enzymes involved, and the role of H4 hyperacetylation in the process must be determined to further investigate the potential… Show more

Transient DNA strand breaks are generated in the whole population of elongating spermatids and are perfectly coincident with histone H4 hyperacetylation at chromatin-remodeling steps. Given the limited DNA repair capacity of elongating spermatids, chromatin remodeling may present a threat to genetic integrity of the male gamete. The nature of the DNA strand breakage, the enzymes involved, and the role of H4 hyperacetylation in the process must be determined to further investigate the potential mutagenic consequences of this important transition. We used the metachromatic dye acridine orange in combination with fluorescence-activated cell sorting to achieve separation of spermatids according to their condensation state. Using single-cell electrophoresis (comet assay), in both alkaline and neutral conditions, we demonstrated that double-stranded breaks account for most of the DNA fragmentation observed in purified elongating spermatids. DNA strand breaks were generated in round spermatids as a result of de novo histone hyperacetylation induced by trichostatin A, whereas an increase in endogenous DNA strand breaks was observed in elongating spermatids. Using a short-term culture of testicular cells, we demonstrated that DNA strand breaks in spermatids were abolished on incubation with two functionally different topoisomerase II inhibitors. Hence, topoisomerase II appears as the unique enzyme responsible for the transient double-stranded breaks in elongating spermatids but depends on histone hyperacetylation for its activity. Show less