Cutting edge: Chk1 directs senescence and mitotic catastrophe in recovery from G2 checkpoint arrest

Besides the well‐understood DNA damage response via establishment of G₂ checkpoint arrest, novel studies focus on the recovery from arrest by checkpoint override to monitor cell cycle re‐entry. The aim of this study was to investigate the role of Chk1 in the recovery from G₂ checkpoint arrest in HCT116 (human colorectal cancer) wt, p53^–/– and p21^–/– cell lines following H₂O₂ treatment. Firstly, DNA damage caused G₂ checkpoint activation via Chk1. Secondly, overriding G₂ checkpoint led to (i) mitotic slippage, cell cycle re‐entry in G₁ and subsequent G₁ arrest associated with senescence or (ii) premature mitotic entry in the absence of p53/p21^WAF1 causing mitotic catastrophe. We revealed subtle differences in the initial Chk1‐involved G₂ arrest with respect to p53/p21^WAF1: absence of either protein led to late G₂ arrest instead of the classic G₂ arrest during checkpoint initiation, and this impacted the release back into the cell cycle. Thus, G₂ arrest correlated with downstream senescence, but late G₂ arrest led to mitotic catastrophe, although both cell cycle re‐entries were linked to upstream Chk1 signalling. Chk1 knockdown deciphered that Chk1 defines long‐term DNA damage responses causing cell cycle re‐entry. We propose that recovery from oxidative DNA damage‐induced G₂ arrest requires Chk1. It works as cutting edge and navigates cells to senescence or mitotic catastrophe. The decision, however, seems to depend on p53/p21^WAF1. The general relevance of Chk1 as an important determinant of recovery from G₂ checkpoint arrest was verified in HT29 colorectal cancer cells.     

Characterization of the p53-Dependent Postmitotic Checkpoint following Spindle Disruption

The p53 tumor suppressor gene product is known to act as part of a cell cycle checkpoint in G₁ following DNA damage. In order to investigate a proposed novel role for p53 as a checkpoint at mitosis following disruption of the mitotic spindle, we have used time-lapse videomicroscopy to show that both p53^+/+ and p53^−/− murine fibroblasts treated with the spindle drug nocodazole undergo transient arrest at mitosis for the same length of time. Thus, p53 does not participate in checkpoint function at mitosis. However, p53 does play a critical role in nocodazole-treated cells which have exited mitotic arrest without undergoing cytokinesis and have thereby adapted. We have determined that in nocodazole-treated, adapted cells, p53 is required during a specific time window to prevent cells from reentering the cell cycle and initiating another round of DNA synthesis. Despite having 4N DNA content, adapted cells are similar to G₁ cells in that they have upregulated cyclin E expression and hypophosphorylated Rb protein. The mechanism of the p53-dependent arrest in nocodazole-treated adapted cells requires the cyclin-dependent kinase inhibitor p21, as p21^−/− fibroblasts fail to arrest in response to nocodazole treatment and become polyploid. Moreover, p21 is required to a similar extent to maintain cell cycle arrest after either nocodazole treatment or irradiation. Thus, the p53-dependent checkpoint following spindle disruption functionally overlaps with the p53-dependent checkpoint following DNA damage.     

The control of CD4<Superscript>+</Superscript>CD25<Superscript>+</Superscript>Foxp3<Superscript>+</Superscript>regulatory T cell survival

   

CD4⁺CD25⁺Foxp3⁺ regulatory T (T_reg) cells are believed to play an important role in suppressing autoimmunity and maintaining peripheral tolerance. How their survival is regulated in the periphery is less clear. Here we show that T_reg cells express receptors for gamma chain cytokines and are dependent on an exogenous supply of these cytokines to overcome cytokine withdrawal apoptosis in vitro. This result was validated in vivo by the accumulation of T_reg cells in Bim^-/- and Bcl-2 tg mice which have arrested cytokine deprivation apoptosis. We also found that CD25 and Foxp3 expression were down-regulated in the absence of these cytokines. CD25⁺ cells from Scurfy mice do not depend on cytokines for survival demonstrating that Foxp3 increases their dependence on cytokines by suppressing cytokine production in T_reg cells. Our study reveals that the survival of T_reg cells is strictly dependent on cytokines and cytokine producing cells because they do not produce cytokines. Our study thus, demonstrates that different gamma chain cytokines regulate T_reg homeostasis in the periphery by differentially regulating survival and proliferation. These findings may shed light on ways to manipulate T_reg cells that could be utilized for their therapeutic applications.     

Absence of p53-dependent apoptosis leads to UV radiation hypersensitivity,enhanced immunosuppression and cellular senescence

Genotoxic stress triggers the p53 tumor suppressor network to activate cellular responses that lead to cell cycle arrest, DNA repair, apoptosis or senescence. This network functions mainly through transactivation of different downstream targets, including cell cycle inhibitor p21, which is required for short-term cell cycle arrest or long-term cellular senescence, or proapoptotic genes such as p53 upregulated modulator of apoptosis (PUMA) and Noxa. However, the mechanism that switches from cell cycle arrest to apoptosis is still unknown. In this study, we found that mice harboring a hypomorphic mutant p53, R172P, a mutation that abrogates p53-mediated apoptosis while keeping cell cycle control mostly intact, are more susceptible to ultraviolet-B (UVB)-induced skin damage, inflammation and immunosuppression than wild-type mice. p53^R172P embryonic fibroblasts (MEFs) are hypersensitive to UVB and prematurely senesce after UVB exposure, in stark contrast to wild-type MEFs, which undergo apoptosis. However, these mutant cells are able to repair UV-induced DNA lesions, indicating that the UV-hypersensitive phenotype results from the subsequent damage response. Mutant MEFs show an induction of p53 and p21 after UVR, while wild-type MEFs additionally induce PUMA and Noxa. Importantly, p53^R172P MEFs failed to downregulate anti-apoptotic protein Bcl-2, which has been shown to play an important role in p53-dependent apoptosis. Taken together, these data demonstrate that in the absence of p53-mediated apoptosis, cells undergo cellular senescence to prevent genomic instability. Our results also indicate that p53-dependent apoptosis may play an active role in balancing cellular growth.Key words: UVB irradiation, p53, DNA damage, DNA damage responses, apoptosis, senescence     

Absence of p53-dependent apoptosis leads to UV radiation hypersensitivity,enhanced immunosuppression and cellular senescence

Genotoxic stress triggers the p53 tumor suppressor network to activate cellular responses that lead to cell cycle arrest, DNA repair, apoptosis or senescence. This network functions mainly through transactivation of different downstream targets, including cell cycle inhibitor p21, which is required for short-term cell cycle arrest or long-term cellular senescence, or proapoptotic genes such as p53 upregulated modulator of apoptosis (PUMA) and Noxa. However, the mechanism that switches from cell cycle arrest to apoptosis is still unknown. In this study, we found that mice harboring a hypomorphic mutant p53, R172P, a mutation that abrogates p53-mediated apoptosis while keeping cell cycle control mostly intact, are more susceptible to ultraviolet-B (UVB)-induced skin damage, inflammation, and immunosuppression than wild-type mice. p53^R172P embryonic fibroblasts (MEFs) are hypersensitive to UVB and prematurely senesce after UVB exposure, in stark contrast to wild-type MEFs, which undergo apoptosis. However, these mutant cells are able to repair UV-induced DNA lesions, indicating that the UV hypersensitive phenotype results from the subsequent damage response. Mutant MEFs show an induction of p53 and p21 after UVR, while wild-type MEFs additionally induce PUMA and Noxa. Importantly, p53^R172P MEFs failed to downregulate anti-apoptotic protein Bcl-2, which has been shown to play an important role in p53-dependent apoptosis. Taken together, these data demonstrate that in the absence of p53-mediated apoptosis, cells undergo cellular senescence to prevent genomic instability. Our results also indicate that p53-dependent apoptosis may play an active role in balancing cellular growth.     

Damage-induced cell–cell communication in different cochlear cell types via two distinct ATP-dependent Ca<Superscript>2+</Superscript> waves

Intercellular Ca²⁺ waves can coordinate the action of large numbers of cells over significant distances. Recent work in many different systems has indicated that the release of ATP is fundamental for the propagation of most Ca²⁺ waves. In the organ of hearing, the cochlea, ATP release is involved in critical signalling events during tissue maturation. ATP-dependent signalling is also implicated in the normal hearing process and in sensing cochlear damage. Here, we show that two distinct Ca²⁺ waves are triggered during damage to cochlear explants. Both Ca²⁺ waves are elicited by extracellular ATP acting on P2 receptors, but they differ in their source of Ca²⁺, their velocity, their extent of spread and the cell type through which they propagate. A slower Ca²⁺ wave (14 μm/s) communicates between Deiters’ cells and is mediated by P2Y receptors and Ca²⁺ release from IP₃-sensitive stores. In contrast, a faster Ca²⁺ wave (41 μm/s) propagates through sensory hair cells and is mediated by Ca²⁺ influx from the external environment. Using inhibitors and selective agonists of P2 receptors, we suggest that the faster Ca²⁺ wave is mediated by P2X₄ receptors. Thus, in complex tissues, the expression of different receptors determines the propagation of distinct intercellular communication signals.     

Dynamics of <Superscript>18</Superscript>O Incorporation from H <Subscript>2</Subscript><Superscript>18</Superscript>O into Soil Microbial DNA

Heavy water (H₂¹⁸O) has been used to label DNA of soil microorganisms in stable isotope probing experiments, yet no measurements have been reported for the ¹⁸O content of DNA from soil incubated with heavy water. Here we present the first measurements of atom% ¹⁸O for DNA extracted from soil incubated with the addition of H₂¹⁸O. Four experiments were conducted to test how the atom% ¹⁸O of DNA, extracted from Ponderosa Pine forest soil incubated with heavy water, was affected by the following variables: (1) time, (2) nutrients, (3) soil moisture, and (4) atom% ¹⁸O of added H₂O. In the time series experiment, the atom% ¹⁸O of DNA increased linearly (R ² = 0.994, p < 0.01) over the first 72 h of incubation. In the nutrient addition experiment, there was a positive correlation (R ² = 0.991, p = 0.006) between the log₁₀ of the amount of tryptic soy broth, a complex nutrient broth, added to soil and the log₁₀ of the atom% ¹⁸O of DNA. For the experiment where soil moisture was manipulated, the atom% ¹⁸O of DNA increased with higher soil moisture until soil moisture reached 30%, above which ¹⁸O enrichment of DNA declined as soils became more saturated. When the atom% ¹⁸O for H₂O added was varied, there was a positive linear relationship between the atom% ¹⁸O of the added water and the atom% ¹⁸O of the DNA. Results indicate that quantification of ¹⁸O incorporated into DNA from H₂¹⁸O has potential to be used as a proxy for microbial growth in soil.     

Antitumor activity of a self-adjuvanting glyco-lipopeptide vaccine bearing B cell,CD4<Superscript>+</Superscript> and CD8<Superscript>+</Superscript> T cell epitopes

Molecularly defined synthetic vaccines capable of inducing both antibodies and cellular anti-tumor immune responses, in a manner compatible with human delivery, are limited. Few molecules achieve this target without utilizing external immuno-adjuvants. In this study, we explored a self-adjuvanting glyco-lipopeptide (GLP) as a platform for cancer vaccines using as a model MO5, an OVA-expressing mouse B16 melanoma. A prototype B and T cell epitope-based GLP molecule was constructed by synthesizing a chimeric peptide made of a CD8⁺ T cell epitope, from ovalbumin (OVA_257–264) and an universal CD4⁺ T helper (Th) epitope (PADRE). The resulting CTL–Th peptide backbones was coupled to a carbohydrate B cell epitope based on a regioselectively addressable functionalized templates (RAFT), made of four α-GalNAc molecules at C-terminal. The N terminus of the resulting glycopeptides (GP) was then linked to a palmitic acid moiety (PAM), obviating the need for potentially toxic external immuno-adjuvants. The final prototype OVA-GLP molecule, delivered in adjuvant-free PBS, in mice induced: (1) robust RAFT-specific IgG/IgM that recognized tumor cell lines; (2) local and systemic OVA_257–264-specific IFN-γ producing CD8⁺ T cells; (3) PADRE-specific CD4⁺ T cells; (4) OVA-GLP vaccination elicited a reduction of tumor size in mice inoculated with syngeneic murine MO5 carcinoma cells and a protection from lethal carcinoma cell challenge; (5) finally, OVA-GLP immunization significantly inhibited the growth of pre-established MO5 tumors. Our results suggest self-adjuvanting glyco-lipopeptide molecules as a platform for B Cell, CD4⁺, and CD8⁺ T cell epitopes-based immunotherapeutic cancer vaccines. Both I. Bettahi and G. Dasgupta have contributed equally to this work.     

Inhibitory role of TGIF in the As<Subscript>2</Subscript>O<Subscript>3</Subscript>-regulated p21<Superscript><Emphasis Type="Italic">WAF1/CIP1</Emphasis></Superscript> expression

Although arsenic is an infamous carcinogen, it has been effectively used to treat acute promyelocytic leukemia, and can induce cell cycle arrest or apoptosis in human solid tumors. Previously, we had demonstrated that opposing effects of ERK1/2 and JNK on p21 expression in response to arsenic trioxide (As₂O₃) are mediated through the Sp1 responsive elements of the p21 promoter in A431 cells. Presently, we demonstrate that Sp1, and c-Jun functionally cooperate to activate p21 promoter expression through Sp1 binding sites (−84/−64) by using DNA affinity binding, chromatin immunoprecipitation, and promoter assays. Surprisingly, As₂O₃-induced c-Jun(Ser63/73) phosphorylation can recruit TGIF/HDAC1 to the Sp1 binding sites and then suppress p21 promoter activation. We suggest that, after As₂O₃ treatment, the N-terminal domain of c-Jun phosphorylation by JNK recruits TGIF/HDAC1 to the Sp1 sites and then represses p21 expression. That is, TGIF is involved in As₂O₃-inhibited p21 expression, and then blocks the cell cycle arrest.     

Involvement of p53 and p21 in Cellular Defects and Tumorigenesis in Atm−/− Mice

Disruption of the mouse Atm gene, whose human counterpart is consistently mutated in ataxia-telangiectasia (A-T) patients, creates an A-T mouse model exhibiting most of the A-T-related systematic and cellular defects. While ATM plays a major role in signaling the p53 response to DNA strand break damage, Atm^−/− p53^−/− mice develop lymphomas earlier than Atm^−/− or p53^−/− mice, indicating that mutations in these two genes lead to synergy in tumorigenesis. The cell cycle G₁/S checkpoint is abolished in Atm^−/− p53^−/− mouse embryonic fibroblasts (MEFs) following γ-irradiation, suggesting that the partial G₁ cell cycle arrest in Atm^−/− cells following γ-irradiation is due to the residual p53 response in these cells. In addition, the Atm^−/− p21^−/− MEFs are more severely defective in their cell cycle G₁ arrest following γ-irradiation than Atm^−/− and p21^−/− MEFs. The Atm^−/− MEFs exhibit multiple cellular proliferative defects in culture, and an increased constitutive level of p21 in these cells might account for these cellular proliferation defects. Consistent with this notion, Atm^−/− p21^−/− MEFs proliferate similarly to wild-type MEFs and exhibit no premature senescence. These cellular proliferative defects are also rescued in Atm^−/− p53^−/− MEFs and little p21 can be detected in these cells, indicating that the abnormal p21 protein level in Atm^−/− cells is also p53 dependent and leads to the cellular proliferative defects in these cells. However, the p21 mRNA level in Atm^−/− MEFs is lower than that in Atm^+/+ MEFs, suggesting that the higher level of constitutive p21 protein in Atm^−/− MEFs is likely due to increased stability of the p21 protein.     

Cyclin-dependent kinase inhibitor p21<Superscript>Waf1</Superscript>: Contemporary view on its role in senescence and oncogenesis

p21^Waf1 was identified as a protein suppressing cyclin E/A-CDK2 activity and was originally considered as a negative regulator of the cell cycle and a tumor suppressor. It is now considered that p21^Waf1 has alternative functions, and the view of its role in cellular processes has begun to change. At present, p21^Waf1 is known to be involved in regulation of fundamental cellular programs: cell proliferation, differentiation, migration, senescence, and apoptosis. In fact, it not only exhibits antioncogenic, but also oncogenic properties. This review provides a contemporary understanding of the functions of p21^Waf1 depending on its intracellular localization. On one hand, when in the nucleus, it serves as a negative cell cycle regulator and tumor suppressor, in particular by participating in the launch of a senescence program. On the other hand, when p21^Waf1 is localized in the cytoplasm, it acts as an oncogene by regulating migration, apoptosis, and proliferation.     

Alpha-santalol,a chemopreventive agent against skin cancer,causes G<Subscript>2</Subscript>/M cell cycle arrest in both p53-mutated human epidermoid carcinoma A431 cells and p53 wild-type human melanoma UACC-62 cells

Background

α-Santalol, an active component of sandalwood oil, has shown chemopreventive effects on skin cancer in different murine models. However, effects of α-santalol on cell cycle have not been studied. Thus, the objective of this study was to investigate effects of α-santalol on cell cycle progression in both p53 mutated human epidermoid carcinoma A431 cells and p53 wild-type human melanoma UACC-62 cells to elucidate the mechanism(s) of action.

Methods

MTT assay was used to determine cell viability in A431 cells and UACC-62; fluorescence-activated cell sorting (FACS) analysis of propidium iodide staining was used for determining cell cycle distribution in A431 cells and UACC-62 cells; immunoblotting was used for determining the expression of various proteins and protein complexes involved in the cell cycle progression; siRNA were used to knockdown of p21 or p53 in A431 and UACC-62 cells and immunofluorescence microscopy was used to investigate microtubules in UACC-62 cells.

Results

α-Santalol at 50-100 μM decreased cell viability from 24 h treatment and α-santalol at 50 μM-75 μM induced G₂/M phase cell cycle arrest from 6 h treatment in both A431 and UACC-62 cells. α-Santalol altered expressions of cell cycle proteins such as cyclin A, cyclin B1, Cdc2, Cdc25c, p-Cdc25c and Cdk2. All of these proteins are critical for G₂/M transition. α-Santalol treatment up-regulated the expression of p21 and suppressed expressions of mutated p53 in A431 cells; whereas, α-santalol treatment increased expressions of wild-type p53 in UACC-62 cells. Knockdown of p21 in A431 cells, knockdown of p21 and p53 in UACC-62 cells did not affect cell cycle arrest caused by α-santalol. Furthermore, α-santalol caused depolymerization of microtubules similar to vinblastine in UACC-62 cells.

Conclusions

This study for the first time identifies effects of α-santalol in G₂/M phase arrest and describes detailed mechanisms of G₂/M phase arrest by this agent, which might be contributing to its overall cancer preventive efficacy in various mouse skin cancer models.

     

Cell-Free Culture Supernatant of Probiotic Lactobacillus fermentum Protects Against H2O2-Induced Premature Senescence by Suppressing ROS-Akt-mTOR Axis in Murine Preadipocytes

Information regarding cellular anti-senescence attributes of probiotic bacteria vis-à-vis modulation of senescence-associated secretory phenotype (SASP) and mTOR signaling is very limited. The present study assessed anti-senescence potential of secretory metabolites of probiotic Lactobacillus fermentum (Lact. fermentum) using H₂O₂-induced model of senescence in 3T3-L1 preadipocytes. Application of H₂O₂-induced cellular senescence characterized by increased cell size and SA-β-gal activity, activation of SASP and reactive oxygen species (ROS), DNA damage response and induction of cell cycle inhibitors (p53/p21^WAF1/p16^INK4a). Further, a robust stimulation of the PI3K/Akt/mTOR pathway and AMPK signaling was also observed in H₂O₂-treated cells. However, exposure of cells to cell-free supernatant of Lact. fermentum significantly attenuated phosphorylation of PI3K/Akt/mTOR pathway and alleviated senescence markers p53, p21^WAF1, SA-β-gal, p38MAPK, iNOS, cox-2, ROS, NF-κB, and DNA damage response. These results provide evidence that secretory metabolites of Lact. fermentum can mitigate the development as well as severity of stress-induced senescence thereby indicating its utility for use as anti-aging or age-delaying agent.

     

Monoamine oxidase‐A is a novel driver of stress‐induced premature senescence through inhibition of parkin‐mediated mitophagy

Cellular senescence, the irreversible cell cycle arrest observed in somatic cells, is an important driver of age‐associated diseases. Mitochondria have been implicated in the process of senescence, primarily because they are both sources and targets of reactive oxygen species (ROS). In the heart, oxidative stress contributes to pathological cardiac ageing, but the mechanisms underlying ROS production are still not completely understood. The mitochondrial enzyme monoamine oxidase‐A (MAO‐A) is a relevant source of ROS in the heart through the formation of H₂O₂ derived from the degradation of its main substrates, norepinephrine (NE) and serotonin. However, the potential link between MAO‐A and senescence has not been previously investigated. Using cardiomyoblasts and primary cardiomyocytes, we demonstrate that chronic MAO‐A activation mediated by synthetic (tyramine) and physiological (NE) substrates induces ROS‐dependent DNA damage response, activation of cyclin‐dependent kinase inhibitors p21^cip, p16^ink4a, and p15^ink4b and typical features of senescence such as cell flattening and SA‐β‐gal activity. Moreover, we observe that ROS produced by MAO‐A lead to the accumulation of p53 in the cytosol where it inhibits parkin, an important regulator of mitophagy, resulting in mitochondrial dysfunction. Additionally, we show that the mTOR kinase contributes to mitophagy dysfunction by enhancing p53 cytoplasmic accumulation. Importantly, restoration of mitophagy, either by overexpression of parkin or inhibition of mTOR, prevents mitochondrial dysfunction and induction of senescence. Altogether, our data demonstrate a novel link between MAO‐A and senescence in cardiomyocytes and provides mechanistic insights into the potential role of MAO‐dependent oxidative stress in age‐related pathologies.