Loss of Emi1-dependent anaphase-promoting complex/cyclosome inhibition deregulates E2F target expression and elicits DNA damage-induced senescence

Expression of the anaphase-promoting complex/cyclosome (APC/C) inhibitor Emi1 is required for the accumulation of APC/C substrates crucial for DNA synthesis and mitotic entry. We show that in vivo Emi1 expression correlates with the proliferative status of the cellular compartment and that cells lacking Emi1 undergo cellular senescence. Emi1 depletion leads to strong decreases in E2F target mRNA and APC/C substrate protein abundances. However, cyclin E mRNA and cyclin E protein levels and associated kinase activities are increased. Cells lacking Emi1 undergo DNA damage, likely explained by replication stress upon deregulated cyclin E- and A-associated kinase activities. Inhibition of ATM kinase prevents induction of senescence, implying that senescence is a consequence of DNA damage. Surprisingly, no senescence or no extensive amount of senescence is evident upon depletion of the Emi1-stabilizing factor Evi5 or Pin1, respectively. Our data suggest that maintenance of a protein stabilization/mRNA expression positive-feedback circuit fueled by Emi1 is required for accurate cell cycle progression, maintenance of DNA integrity, and prevention of cellular senescence.     

TRPC1: Getting physical in space

We tested a hypothesis that activation of growth-promoting pathways is required for cellular senescence. In the presence of serum, induction of p21 caused senescence, characterized by beta-Galactosidase staining, cell hypertrophy, increased levels of cyclin D1 and active TOR (target of rapamycin, also known as mTOR). Serum starvation and rapamycin inhibited TOR and prevented the expression of some senescent markers, despite high levels of p21 and cell cycle arrest. In the presence of serum, p21-arrested cells irreversibly lost proliferative potential. In contrast, when cells were arrested by p21 in the absence of serum, they retained the capacity to resume proliferation upon termination of p21 induction. In normal human cells such as WI38 fibroblasts and retinal pigment epithelial (RPE) cells, serum starvation caused quiescence, which was associated with low levels of cyclin D1, inactive TOR and slim-cell morphology. In contrast, cellular senescence with high levels of TOR activity was induced by doxorubicin (DOX), a DNA damaging agent, in the presence of serum. Inhibition of TOR partially prevented senescent phenotype caused by DOX. Thus growth stimulation coupled with cell cycle arrest leads to senescence, whereas quiescence (a condition with inactive TOR) prevents senescence.     

58-kDa microspherule protein (MSP58) is novel Brahma-related gene 1 (BRG1)-associated protein that modulates p53/p21 senescence pathway

The nucleolar 58-kDa microspherule protein (MSP58) protein is a candidate oncogene implicated in modulating cellular proliferation and malignant transformation. In this study, we show that knocking down MSP58 expression caused aneuploidy and led to apoptosis, whereas ectopic expression of MSP58 regulated cell proliferation in a context-dependent manner. Specifically, ectopic expression of MSP58 in normal human IMR90 and Hs68 diploid fibroblasts, the H184B5F5/M10 mammary epithelial cell line, HT1080 fibrosarcoma cells, primary mouse embryonic fibroblasts, and immortalized NIH3T3 fibroblasts resulted in induction of premature senescence, an enlarged and flattened cellular morphology, and increased senescence-associated β-galactosidase activity. MSP58-driven senescence was strictly dependent on the presence of functional p53 as revealed by the fact that normal cells with p53 knockdown by specific shRNA or cells with a mutated or functionally impaired p53 pathway were effective in bypassing MSP58-induced senescence. At least two senescence mechanisms are induced by MSP58. First, MSP58 activates the DNA damage response and p53/p21 signaling pathways. Second, MSP58, p53, and the SWI/SNF chromatin-remodeling subunit Brahma-related gene 1 (BRG1) form a ternary complex on the p21 promoter and collaborate to activate p21. Additionally, MSP58 protein levels increased in cells undergoing replicative senescence and stress-induced senescence. Notably, the results of analyzing expression levels of MSP58 between tumors and matched normal tissues showed significant changes (both up- and down-regulation) in its expression in various types of tumors. Our findings highlight new aspects of MSP58 in modulating cellular senescence and suggest that MSP58 has both oncogenic and tumor-suppressive properties.     

p21H-ras-induced morphological transformation and increases in c-myc expression are independent of functional protein kinase C.

It has previously been demonstrated that efficient DNA synthesis by oncogenic p21H-ras only occurs in the presence of insulin and is absolutely dependent on functional protein kinase C. Here we show that morphological transformation induced by oncogenic p21H-ras does not require functional protein kinase C. The early phases of protein kinase C-independent morphological transformation do not require de novo protein synthesis. We have also demonstrated that the introduction of p21H-ras into quiescent Swiss 3T3 cells by scrape-loading leads to increased levels of c-myc mRNA similar to those seen following serum stimulation. The increases in c-myc mRNA levels induced by p21H-ras are also independent of functional protein kinase C. Both morphological transformation and the elevation of c-myc mRNA levels do not require insulin. These results demonstrate that p21H-ras is generating protein kinase C-dependent and -independent signals.     

Integrated Stochastic Model of DNA Damage Repair by Non-homologous End Joining and p53/p21- Mediated Early Senescence Signalling

Unrepaired or inaccurately repaired DNA damage can lead to a range of cell fates, such as apoptosis, cellular senescence or cancer, depending on the efficiency and accuracy of DNA damage repair and on the downstream DNA damage signalling. DNA damage repair and signalling have been studied and modelled in detail separately, but it is not yet clear how they integrate with one another to control cell fate. In this study, we have created an integrated stochastic model of DNA damage repair by non-homologous end joining and of gamma irradiation-induced cellular senescence in human cells that are not apoptosis-prone. The integrated model successfully explains the changes that occur in the dynamics of DNA damage repair after irradiation. Simulations of p53/p21 dynamics after irradiation agree well with previously published experimental studies, further validating the model. Additionally, the model predicts, and we offer some experimental support, that low-dose fractionated irradiation of cells leads to temporal patterns in p53/p21 that lead to significant cellular senescence. The integrated model is valuable for studying the processes of DNA damage induced cell fate and predicting the effectiveness of DNA damage related medical interventions at the cellular level.     

Murine coronavirus replication induces cell cycle arrest in G0/G1 phase

Mouse hepatitis virus (MHV) replication in actively growing DBT and 17Cl-1 cells resulted in the inhibition of host cellular DNA synthesis and the accumulation of infected cells in the G₀/G₁ phase of the cell cycle. UV-irradiated MHV failed to inhibit host cellular DNA synthesis. MHV infection in quiescent 17Cl-1 cells that had been synchronized in the G₀ phase by serum deprivation prevented infected cells from entering the S phase after serum stimulation. MHV replication inhibited hyperphosphorylation of the retinoblastoma protein (pRb), the event that is necessary for cell cycle progression through late G₁ and into the S phase. While the amounts of the cellular cyclin-dependent kinase (Cdk) inhibitors p21^Cip1, p27^Kip1, and p16^INK4a did not change in infected cells, MHV infection in asynchronous cultures induced a clear reduction in the amounts of Cdk4 and G₁ cyclins (cyclins D1, D2, D3, and E) in both DBT and 17Cl-1 cells and a reduction in Cdk6 levels in 17Cl-1 cells. Infection also resulted in a decrease in Cdk2 activity in both cell lines. MHV infection in quiescent 17Cl-1 cells prevented normal increases in Cdk4, Cdk6, cyclin D1, and cyclin D3 levels after serum stimulation. The amounts of cyclin D2 and cyclin E were not increased significantly after serum stimulation in mock-infected cells, whereas they were decreased in MHV-infected cells, suggesting the possibility that MHV infection may induce cyclin D2 and cyclin E degradation. Our data suggested that a reduction in the amounts of G₁ cyclin-Cdk complexes in MHV-infected cells led to a reduction in Cdk activities and insufficient hyperphosphorylation of pRb, resulting in inhibition of the cell cycle in the G₀/G₁ phase.     

Arsenite induces premature senescence via p53/p21 pathway as a result of DNA damage in human malignant glioblastoma cells

In this study, we investigate whether arsenite-induced DNA damage leads to p53-dependent premature senescence using human glioblastoma cells with p53-wild type (U87MG-neo) and p53 deficient (U87MG-E6). A dose dependent relationship between arsenite and reduced cell growth is demonstrated, as well as induced γH2AX foci formation in both U87MG-neo and U87MG-E6 cells at low concentrations of arsenite. Senescence was induced by arsenite with senescence-associated β-galactosidase staining. Dimethyl- and trimethyl-lysine 9 of histone H3 (H3DMK9 and H3TMK9) foci formation was accompanied by p21 accumulation only in U87MG-neo but not in U87MG-E6 cells. This suggests that arsenite induces premature senescence as a result of DNA damage with heterochromatin forming through a p53/p21 dependent pathway. p21 and p53 siRNA consistently decreased H3TMK9 foci formation in U87M G-neo but not in U87MG-E6 cells after arsenite treatment. Taken together, arsenite reduces cell growth independently of p53 and induces premature senescence via p53/p21-dependent pathway following DNA damage. [BMB Reports 2014; 47(10): 575-580]     

Signaling of DNA damage is not sufficient to induce p53 response: (re)activation of wt p53 protein strongly depends on cellular context

It is generally accepted that exposure of cells to a variety of DNA-damaging agents leads to up-regulation and activation of wild-type (wt) p53 protein. We investigated the (re)-activation of p53 protein in two human cancer cell lines in which the gene for this tumor suppressor is not mutated: HeLaS(3) cervix carcinoma and MCF-7 breast cancer cells, by induction via different genotoxic and cytotoxic stimuli. Treatment of human cells with the alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) or different anti-cancer drugs resulted in a strong DNA damage as evidenced by Comet assay and a marked increase in site-specific phosphorylation of H2AX. Unlike in MCF-7 cells, in HeLaS(3) cells the expression of p53 protein did not increase after MNNG treatment despite a strong DNA damage. However, other agents for example doxorubicin markedly induced p53 response in HeLaS(3) cells. After exposure of these cells to MNNG, the ATM-dependent effector proteins Chk2 and NBS1 were phosphorylated, thereby evidencing that MNNG-induced DNA breakage was recognized and properly signaled. In HeLaS(3) cells wt p53 protein is not functional due to E6-mediated targeting for accelerated ubiquitylation and degradation. Therefore, the activation of a p53 response to genotoxic stress in HeLaS(3) cells seems to depend on the status of E6 oncoprotein. Indeed, the induction of p53 protein in HeLaS(3) cells in response to distinct agents inversely correlates with the cellular level of E6 oncoprotein. This implicates that the capability of different agents to activate p53 in HeLaS(3) cells primarily depends on their inhibitory effect on expression of E6 oncoprotein.     

miR-300 regulates cellular radiosensitivity through targeting p53 and apaf1 in human lung cancer cells

microRNAs (miRNAs) play a crucial role in mediation of the cellular sensitivity to ionizing radiation (IR). Previous studies revealed that miR-300 was involved in the cellular response to IR or chemotherapy drug. However, whether miR-300 could regulate the DNA damage responses induced by extrinsic genotoxic stress in human lung cancer and the underlying mechanism remain unknown. In this study, the expression of miR-300 was examined in lung cancer cells treated with IR, and the effects of miR-300 on DNA damage repair, cell cycle arrest, apoptosis and senescence induced by IR were investigated. It was found that IR induced upregulation of endogenous miR-300, and ectopic expression of miR-300 by transfected with miR-300 mimics not only greatly enhanced the cellular DNA damage repair ability but also substantially abrogated the G2 cell cycle arrest and apoptosis induced by IR. Bioinformatic analysis predicted that p53 and apaf1 were potential targets of miR-300, and the luciferase reporter assay showed that miR-300 significantly suppressed the luciferase activity through binding to the 3′-UTR of p53 or apaf1 mRNA. In addition, overexpression of miR-300 significantly reduced p53/apaf1 and/or IR-induced p53/apaf1 protein expression levels. Flow cytomertry analysis and colony formation assay showed that miR-300 desensitized lung cancer cells to IR by suppressing p53-dependent G2 cell cycle arrest, apoptosis and senescence. These data demonstrate that miR-300 regulates the cellular sensitivity to IR through targeting p53 and apaf1 in lung cancer cells.     

Expression of human papilloma virus E7 protein causes apoptosis and inhibits DNA synthesis in primary hepatocytes via increased expression of p21(Cip-1/WAF1/MDA6)

The impact of human papilloma virus (HPV16) E7 proteins and retinoblastoma (RB) antisense oligonucleotides upon mitogen-activated protein kinase (MAPK)-mediated inhibition of DNA synthesis via p21(Cip-1/WAF1/MDA6) (p21) was determined in primary hepatocytes. Prolonged activation of the MAPK pathway in p21(+/+) or p21(-/-) hepatocytes caused a large decrease and increase, respectively, in DNA synthesis. Either transfection with RB antisense oligonucleotides, expression of wild type E7, or RB binding mutant E7 (C24S) proteins increased p21 levels and reduced DNA synthesis in p21(+/+) hepatocytes. RB antisense oligonucleotides and E7 proteins increased apoptosis in p21(+/+), but not p21(-/-), hepatocytes. Expression of wild type E7 increased DNA synthesis above control levels in p21(-/-) cells, which was additive with prolonged MAPK activation. In contrast, expression of mutant E7 did not alter DNA synthesis above control levels in p21(-/-) cells and was supra-additive with prolonged MAPK activation. Antisense ablation of RB in p21(-/-) hepatocytes had a weak stimulatory effect upon DNA synthesis itself but enhanced the capacity of mutant E7 protein to stimulate DNA synthesis to the same level observed using wild type E7. The ability of prolonged MAPK activation to stimulate DNA synthesis in the presence of mutant E7 and antisense RB was additive. Collectively, the present data demonstrate that loss of RB function together with loss of p21 function plays an important role in the E7- and MAPK-dependent modulation of apoptosis and DNA synthesis in primary hepatocytes.     

p53 mediates apoptosis induced by c-Myc activation in hypoxic or gamma irradiated fibroblasts

Deregulated c-Myc expression leads to a cellular state where proliferation and apoptosis are equally favored depending on the cellular microenvironment. Since the apoptotic sensitivity of many cells is influenced by the status of the p53 tumor suppressor gene, we investigated whether the induction of apoptosis by DNA damage or non-genotoxic stress are also influenced by the p53 status of cells with altered c-Myc activity. Rat-1 fibroblasts expressing a conditional c-Myc allele (c-MycER), were transfected to express an antisense RNA complimentary to p53 mRNA. Expression of antisense p53 RNA decreased p53 protein levels and delayed p53 accumulation following c-Myc activation. Under hypoxic or low serum conditions, cells expressing antisense p53 were substantially more resistant to c-Myc-induced apoptosis than were control cells. c-Myc activation also sensitized Rat-1 cells to radiation-induced apoptosis. Rat-1 cells expressing antisense p53 RNA were more resistant to apoptosis induced by the combined effects of c-Myc activation and gamma irradiation. In a similar manner, apoptosis induced by c-Myc in serum starved, hypoxic or gamma irradiated fibroblasts was also inhibited by Bcl-2. These data indicate that p53 is involved in c-Myc-mediated apoptosis under a variety of stresses which may influence tumor growth, evolution and response to therapy.     

P21-PARP-1 Pathway Is Involved in Cigarette Smoke-Induced Lung DNA Damage and Cellular Senescence

Persistent DNA damage triggers cellular senescence, which may play an important role in the pathogenesis of cigarette smoke (CS)-induced lung diseases. Both p21^CDKN1A (p21) and poly(ADP-ribose) polymerase-1 (PARP-1) are involved in DNA damage and repair. However, the role of p21-PARP-1 axis in regulating CS-induced lung DNA damage and cellular senescence remains unknown. We hypothesized that CS causes DNA damage and cellular senescence through a p21-PARP-1 axis. To test this hypothesis, we determined the levels of γH2AX (a marker for DNA double-strand breaks) as well as non-homologous end joining proteins (Ku70 and Ku80) in lungs of mice exposed to CS. We found that the level of γH2AX was increased, whereas the level of Ku70 was reduced in lungs of CS-exposed mice. Furthermore, p21 deletion reduced the level of γH2AX, but augmented the levels of Ku70, Ku80, and PAR in lungs by CS. Administration of PARP-1 inhibitor 3-aminobenzamide increased CS-induced DNA damage, but lowered the levels of Ku70 and Ku80, in lungs of p21 knockout mice. Moreover, 3-aminobenzamide increased senescence-associated β-galactosidase activity, but decreased the expression of proliferating cell nuclear antigen in mouse lungs in response to CS. Interestingly, 3-aminobenzamide treatment had no effect on neutrophil influx into bronchoalveolar lavage fluid by CS. These results demonstrate that the p21-PARP-1 pathway is involved in CS-induced DNA damage and cellular senescence.     

HPV 5 and 8 E6 Abrogate ATR Activity Resulting in Increased Persistence of UVB Induced DNA Damage

The role of the E6 oncoprotein from high-risk members of the α human papillomavirus genus in anogenital cancer has been well established. However, far less is known about the E6 protein from the β human papillomavirus genus (β-HPVs). Some β-HPVs potentially play a role in non-melanoma skin cancer development, although they are not required for tumor maintenance. Instead, they may act as a co-factor that enhances the carcinogenic potential of UV damage. Indeed, the E6 protein from certain β-HPVs (HPV 5 and 8) promotes the degradation of p300, a histone acetyl transferase involved in UV damage repair. Here, we show that the expression of HPV 5 and 8 E6 increases thymine dimer persistence as well as the likelihood of a UVB induced double strand break (DSB). Importantly, we provide a mechanism for the increased DNA damage by showing that both extended thymine dimer persistence as well as elevated DSB levels are dependent on the ability of HPV 8 E6 to promote p300 degradation. We further demonstrate that HPV 5 and 8 E6 expression reduces the mRNA and protein levels of ATR, a PI3 kinase family member that plays a key role in UV damage signaling, but that these levels remain unperturbed in cells expressing a mutated HPV 8 E6 incapable of promoting p300 degradation. We confirm that the degradation of p300 leads to a reduction in ATR protein levels, by showing that ATR levels rebound when a p300 mutant resistant to HPV 8 mediated degradation and HPV 8 E6 are co-transfected. Conversely, we show that ATR protein levels are reduced when p300 is targeted for degradation by siRNA. Moreover, we show the reduced ATR levels in HPV 5 and 8 E6 expressing cells results in delayed ATR activation and an attenuated ability of cells to phosphorylate, and as a result accumulate, p53 in response to UVB exposure, leading to significantly reduced cell cycle arrest. In conclusion, these data demonstrate that β-HPV E6 expression can enhance the carcinogenic potential of UVB exposure by promoting p300 degradation, resulting in a reduction in ATR levels, which leads to increased thymine dimer persistence and increased UVB induced DSBs.