miR-15b/16-2 Regulates Factors That Promote p53 Phosphorylation and Augments the DNA Damage Response following Radiation in the Lung

MicroRNAs (miRNAs) are regulatory RNAs frequently dysregulated in disease and following cellular stress. Investigators have described changes in miR-15b expression following exposure to several stress-inducing anticancer agents, including ionizing radiation (IR), etoposide, and hydrogen peroxide. However, the role for miR-15b as a mediator of cellular injury in organs such as the lung has yet to be explored. In this study, we examined miR-15b expression patterns as well as its potential role in DNA damage and repair in the setting of IR exposure. We showed that miR-15b is up-regulated in a dose- and time-dependent manner in human bronchial epithelial cells following IR. miR-15b expression was highest after 2 h of IR and decreased gradually. Survival rates following IR were also higher in miR-15b/16-2-overexpressing cells. Cell cycle arrest in G₂/M phase and an increased DNA repair response were observed in IR-exposed miR-15b/16-2 stable cells. We observed an up-regulation of components of the ataxia telangiectasia mutated (ATM)/Chek1/p53 pathway in miR-15b/16-2-overexpressing cells after IR. Moreover, a pathway-based PCR expression array of genes demonstrated that miR-15b/16-2 overexpression significantly induced the expression of genes involved in ATM/ataxia telangiectasia and Rad-3-related (ATR) signaling, apoptosis, the cell cycle, and DNA repair pathways. Here we demonstrated a novel biological link between miR-15b and DNA damage and cellular protection in lung cells. We identified Wip1 (PPM1D) as a functional target for miR-15b and determined that miR-15b induction of the DNA damage response is partially dependent upon suppression of Wip1. Our study suggests that miR-15b/Wip1 could be a potential therapeutic target in radiation-induced lung disease.     

Induction of senescence by adenosine suppressing the growth of lung cancer cells

Extracellular adenosine is well reported to suppress tumor growth by induction of apoptosis. However, in this study we found that adenosine treatment results in cellular senescence in A549 lung cancer cells both in vitro and in vivo; adenosine induces cell cycle arrest and senescence in a p53/p21 dependent manner; adenosine elevates the level of phosphor-γH2AX, pCHK2 and pBRCA1, the markers for prolonged DNA damage response which are likely responsible for initiating the cellular senescence. Our study first demonstrates that adenosine suppresses growth of cancer cells by inducing senescence and provides additional evidence that adenosine could act as an effective anticancer agent for targeted cancer therapy.     

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.     

The canonical NF-κB pathway differentially protects normal and human tumor cells from ROS-induced DNA damage

DNA damage responses (DDR) invoke senescence or apoptosis depending on stimulus intensity and the degree of activation of the p53-p21(Cip1/Waf1) axis; but the functional impact of NF-κB signaling on these different outcomes in normal vs. human cancer cells remains poorly understood. We investigated the NF-κB-dependent effects and mechanism underlying reactive oxygen species (ROS)-mediated DDR outcomes of normal human lung fibroblasts (HDFs) and A549 human lung cancer epithelial cells. To activate DDR, ROS accumulation was induced by different doses of H(2)O(2). The effect of ROS induction caused a G2 or G2-M phase cell cycle arrest of both human cell types. However, ROS-mediated DDR eventually culminated in different end points with HDFs undergoing premature senescence and A549 cancer cells succumbing to apoptosis. NF-κB p65/RelA nuclear translocation and Ser536 phosphorylation were induced in response to H(2)O(2)-mediated ROS accumulation. Importantly, blocking the activities of canonical NF-κB subunits with an IκBα super-repressor or suppressing canonical NF-κB signaling by IKKβ knock-down accelerated HDF premature senescence by up-regulating the p53-p21(Cip1/Waf1) axis; but inhibiting the canonical NF-κB pathway exacerbated H(2)O(2)-induced A549 cell apoptosis. HDF premature aging occurred in conjunction with γ-H2AX chromatin deposition, senescence-associated heterochromatic foci and beta-galactosidase staining. p53 knock-down abrogated H(2)O(2)-induced premature senescence of vector control- and IκBαSR-expressing HDFs functionally linking canonical NF-κB-dependent control of p53 levels to ROS-induced HDF senescence. We conclude that IKKβ-driven canonical NF-κB signaling has different functional roles for the outcome of ROS responses in the contexts of normal vs. human tumor cells by respectively protecting them against DDR-dependent premature senescence and apoptosis.     

Downregulation of caveolin-1 affects bleomycin-induced growth arrest and cellular senescence in A549 cells

Bleomycin is an anti-cancer drug that induces both apoptosis and senescence, two processes thought to involve caveolin-1. Here we investigate the role of caveolin-1 in bleomycin-induced senescence. We show that bleomycin-treated A549 cells exhibit: senescence-like cell morphology; a senescence-associated increase in SA-beta-galactosidase activity; cell cycle arrest; and upregulation of p53 and p21. As predicted, we find that caveolin-1 amount increases in response to bleomycin-treatment and that modulation of caveolin-1 affects p21 and p53 levels, cell cycling, and senescence (SA-beta-galactosidase activity). Interestingly, senescence-associated cell cycle arrest via p53 and p21 and SA-beta-galactosidase activity is reduced in young A549 cells when short hairpin RNA specific for caveolin-1 was applied before bleomycin-treatment. Our results support the hypothesis that downregulation of caveolin-1 expression affects bleomycin-induced cell cycle arrest and subsequent cellular senescence that is driven by p53 and p21.     

Two faces of p53: aging and tumor suppression

The p53 tumor suppressor protein, often termed guardian of the genome, integrates diverse physiological signals in mammalian cells. In response to stress signals, perhaps the best studied of which is the response to DNA damage, p53 becomes functionally active and triggers either a transient cell cycle arrest, cell death (apoptosis) or permanent cell cycle arrest (cellular senescence). Both apoptosis and cellular senescence are potent tumor suppressor mechanisms that irreversibly prevent damaged cells from undergoing neoplastic transformation. However, both processes can also deplete renewable tissues of proliferation-competent progenitor or stem cells. Such depletion, in turn, can compromise the structure and function of tissues, which is a hallmark of aging. Moreover, whereas apoptotic cells are by definition eliminated from tissues, senescent cells can persist, acquire altered functions, and thus alter tissue microenvironments in ways that can promote both cancer and aging phenotypes. Recent evidence suggests that increased p53 activity can, at least under some circumstances, promote organismal aging. Here, we discuss the role of p53 as a key regulator of the DNA damage responses, and discuss how p53 integrates the outcome of the DNA damage response to optimally balance tumor suppression and longevity.     

LncRNA CASC11 promoted gastric cancer cell proliferation,migration and invasion in vitro by regulating cell cycle pathway

In this study, we aimed to investigate the effects of lncRNA CASC11 on gastric cancer (GC) cell progression through regulating miR-340-5p and cell cycle pathway. Expressions of lncRNA CASC11 in gastric cancer tissues and cell lines were determined by qRT-PCR. Differentially expressed lncRNAs, mRNAs and miRNAs were screened through microarray analysis. The relationship among CASC11, CDK1 and miR-340-5p was predicted by TargetScan and validated through dual luciferase reporter assay. Western blot assay examined the protein level of CDK1 and several cell cycle regulatory proteins. GO functional analysis and KEGG pathway analysis were used to predict the association between functions and related pathways. Cell proliferation was determined by CCK-8 assays. Cell apoptosis and cell cycle were detected by flow cytometry assay. CASC11 was highly expressed in GC tissues and cell lines. Knockdown of CASC11 inhibited GC cell proliferation, promoted cell apoptosis and blocked cell cycle. KEGG further indicated an enriched cell cycle pathway involving CDK1. QRT-PCR showed that miR-340-5p was down-regulated in GC cells tissues, while CDK1 was up-regulated. Furthermore, CASC11 acted as a sponge of miR-340-5p which directly targeted CDK1. Meanwhile, miR-340-5p overexpression promoted GC cell apoptosis and induced cell cycle arrest, while CDK1 overexpression inhibited cell apoptosis and accelerated cell cycle. Our study revealed the mechanism of CASC11/miR-340-5p/CDK1 network in GC cell line, and suggested that CASC11 was a novel facilitator that exerted a biological effect by activating the cell cycle signaling pathway. This finding provides a potential therapeutic target for GC.     

miR-375 targets the p53 gene to regulate cellular response to ionizing radiation and etoposide in gastric cancer cells

MicroRNAs (miRNAs) offer a new approach for molecular classification and individual therapy of human cancer due to their regulation of oncogenic pathways. In a previous report, elevated miR-375 was found in recurring gastric cancer, and it was predicted that miR-375 may be a regulator of p53 gene. However, its biological role and mechanism of actions remain unknown. In this study, we characterized the expression level of miR-375 in gastric cancer cell lines – BGC823, MGC803, SGC7901, AGS, N87, MKN45 – using RT-PCR. We found that exogenous expression of miR-375 promoted the growth of AGS cells in both liquid and soft agar media. In agreement with the previous report, overexpression of miR-375 in AGS cells reduced the p53 protein expression level. A luciferase assay demonstrated that miR-375 down-regulated p53 expression through an interaction with the 3′ UTR region of p53. In addition, the expression of miR-375 desensitizes cells to ionizing radiation and etoposide. Flow cytometry analyses showed that miR-375 abrogated the cell cycle arrest and apoptosis after DNA damage. These results demonstrate that miR-375 targets p53 to regulate the response to ionizing radiation and etoposide treatment.     

Collaborator of ARF (CARF) Regulates Proliferative Fate of Human Cells by Dose-dependent Regulation of DNA Damage Signaling

Collaborator of ARF (CARF) has been shown to directly bind to and regulate p53, a central protein that controls tumor suppression via cellular senescence and apoptosis. However, the cellular functions of CARF and the mechanisms governing its effect on senescence, apoptosis, or proliferation are still unknown. Our previous studies have shown that (i) CARF is up-regulated during replicative and stress-induced senescence, and its exogenous overexpression caused senescence-like growth arrest of cells, and (ii) suppression of CARF induces aneuploidy, DNA damage, and mitotic catastrophe, resulting in apoptosis via the ATR/CHK1 pathway. In the present study, we dissected the cellular role of CARF by investigating the molecular pathways triggered by its overexpression in vitro and in vivo. We found that the dosage of CARF is a critical factor in determining the proliferation potential of cancer cells. Most surprisingly, although a moderate level of CARF overexpression induced senescence, a very high level of CARF resulted in increased cell proliferation. We demonstrate that the level of CARF is crucial for DNA damage and checkpoint response of cells through ATM/CHK1/CHK2, p53, and ERK pathways that in turn determine the proliferative fate of cancer cells toward growth arrest or proproliferative and malignant phenotypes. To the best of our knowledge, this is the first report that demonstrates the capability of a fundamental protein, CARF, in controlling cell proliferation in two opposite directions and hence may play a key role in tumor biology and cancer therapeutics.     

Cellular senescence induced by p53-ras cooperation is independent of p21waf1 in murine embryo fibroblasts

Oncogenic activation in primary murine fibroblasts initiates a senescence-like cell cycle arrest that depends on the p53 tumor suppressor pathway. Conditional p53 activation efficiently induced a reversible cell cycle arrest but was unable to induce features of senescence. In contrast, coexpression of oncogenic ras with p53 produced an irreversible cell cycle arrest that displayed features of cellular senescence. Introduction of a conditional murine p53 allele (p53val135) into double p53/p21-null mouse embryonic fibroblasts showed that p21waf1 was not required for this effect, since p53-/-;p21-/- double-null cells undergo terminal growth arrest with features of senescence following coexpression of oncogenic Ras and p53. Our results indicate that oncogenic activation of the Ras pathway in murine fibroblasts converts p53 into a senescence inducer through a p21waf1-independent mechanism.