Genotoxic stress-mediated cell cycle activities for the decision of ...

Perspective

Perspective

Cell Cycle 10:19, 3239-3248; October 1, 2011; © 2011 Landes Bioscience

Genotoxic stress-mediated cell cycle activities for the decision of cellular fate Adnan Erol Erol Project Development House for the Disorders of Energy Metabolism; Istanbul, Turkey

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enomic integrity maintenance is critical for prevention of a wide variety of adverse cellular effects including apoptosis, cellular senescence and malignant cell transformation. Coupled with normal replication, the local intracellular and extracellular stresses cause damage to cellular DNA that is recognized and repaired by the DNA damage response (DDR) pathway. p53 induces the transcription of genes that negatively regulate progression of the cell cycle in response to DNA damage, and thus participates in maintaining genome stability. p53 and many other anti-proliferative factors such as TGFβ regulate the expression of different cyclin-dependent kinase inhibitors (CDKIs). Paradoxically, one of the cellular proliferative factors, c-Myc proto-oncogene also controls the expression of these CDKIs and modulates the fate of cell in response to DNA damage. Furthermore, involvement of numerous other proteins in the DDR and crosstalk between them are likely to substantiate the DDR as one of the genome’s most extensive signaling networks. Versatile protein kinases in this network affect the decision about four basic cellular fates, which are quiescence, apoptosis, oncogenesis and senescence, in response to DNA damage.

unfavorably on physiological functions, increasing entropy.1 The effect of DNA damage on organismal aging becomes apparent when DNA damage rapidly accumulates early in life as a consequence of defects in genome maintenance system. DNA repair systems protect not only against cancer but also equally against premature aging.2

DNA Damage Response (DDR) ©201 1L andesBi os c i enc e . different types of DNA lesions Donotdi s t r i but e.Although are repaired or tolerated through the

Key words: c-Myc, JNK, mTORC1, mTORC2, p53, senescence, TGFb Abbreviations: ATM, ataxia telangiectasia mutated; ATR, ATM and Rad3related; FOXO, the O subfamily of forkhead transcription factors; G6PDH, glucose-6 phosphate dehydrogenase; GSK3, glycogen synthase kinase 3; Hdm2, human mdm2 (double minute 2); IGFBP-3, insulin-like growth factorbinding protein 3; IRS1, insulin receptor substrate 1; JNK, c-Jun N-terminal kinase; PARP, poly (ADP-ribose) polymerase; PTEN, phosphatase and tensin homolog Submitted: 07/21/11 Revised: 07/31/11 Accepted: 08/01/11 DOI: 10.4161/cc.10.19.17460 Correspondence to: Adnan Erol; Email: [email protected]

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The integrity of genomic and mitochondrial DNA and the transmission of the information are important for the survival of individuals. Damage to nuclear and mitochondrial DNA can decrease the molecular fidelity and subsequently increase the accumulation of defective cellular components, which impact

actions of different sets of proteins, DDR generally has several common features. One major characteristic of the DDR is slowing down or arrest of cell cycle progression through checkpoint signaling. Additional features involve transcriptional regulation of a variety of genes, post-translational modification of DNA repair proteins, and changes in chromatin structure surrounding damaged DNA.3 The recruitment of DNA damage signaling and repair proteins to sites of genomic damage constitutes a primary event triggered by DNA damage.4 Genomic damage events result in activation of the phosphoinositide-3-kinase (PI3K) related kinases (PIKKs), including ATM, ATR and DNA-dependent protein kinase (DNA-PK). Consequently, they amplify and convey the signal from the damaged DNA to DNA-repair and cell cycle machineries, such as the p53 pathway.4 In response to DNA damage, cell cycle checkpoints (G1, S, G2 /M) are activated and stop cell cycle progression to allow time for repair thereby preventing transmission of damaged or incompletely

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©201 1L andesBi os c i enc e. Donotdi s t r i but e.

Figure 1. Canonical insulin-IRS1-PI3K signaling toward mTORC1/S6K activation is outlined in (A). In response to genotoxic stress creating double strand break (DSB), Ku proteins accumulate on the DNA fragment and activate DNA-PK. Nuclear PDK1 and DNA-PK-mediated full activation of nuclear Akt inhibits transcriptional activity of FOXO proteins and translocate into cytosol. Fully activated nuclear Akt stimulates transcription of p21 directly and indirectly through inhibition of Hdm2 and activation of p53. Nuclear p53, in addition to its DNA repairing activity, may inhibit glucose transport and proximal insulin signaling pathway. DNA-PK may inhibit mTORC2, impairing cytosolic Akt S473 phosphorylation, while stimulating nuclear Akt S473 phosphorylation (B). In (C), extracellular glucose and integrin-mediated activation of TGFβ in response to chronic cellular stress is depicted. Blue arrows indicate stimulation, red arrows for the inhibition.

replicated chromosomes. Cdk2/cyclin E complexes are inhibited to arrest cells at the G1/S checkpoint whereas Cdk1/ cyclin B complexes are inhibited to arrest the cells at G2 /M phase of the cell cycle. The G1 cell cycle checkpoint is primarily responsible for preventing damaged DNA from being replicated. During G1/S DNA damage checkpoint arrest, ATM/ATRmediated activation of p53 stimulates one of its downstream targets, p21, which binds to and inhibits CDKs, thereby arresting cells at the G1/S transition.5,6 In the absence of Cdk2, Cdk1 is responsible

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for driving the cells through the G1/S transition, which in turn is inhibited by p21 in response to DNA damage to maintain the G1/S checkpoint.7 Cdk2 also acts upstream in the DNA damage pathway in the activation of DNA repair proteins that is independent of its downstream role in cell cycle progression.8 DNA double-strand breaks (DSBs) are the most threatening type of DNA damage. DSBs are detected by the Ku70/80 antigen complex. DNA-PK is recruited to the DSB via its interaction with Ku complex that simulates the protein kinase

activity of DNA-PK, promoting the interaction of DNA-PK with nuclear Akt.3,4 Akt is activated by several stimuli, including hormones, growth factors, cytokines and also by DNA damage. Deregulation of Akt is implicated in degenerative processes such as carcinogenesis and diabetes9 (Fig. 1B). Canonical vs. Non-Canonical Akt Signaling Pathways Akt activation canonically is regulated by phosphorylation at two critical sites:

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threonine 308 (T308) and serine 473 (S473). Phosphorylation of both residues is required for the full activation and maximum kinase activity. Phosphoinositidedependent protein kinase 1 (PDK1) has been identified as a primary kinase that will phosphorylate Akt at T308 in its activation loop. Surprisingly, however, several enzymes referred to as PDK2 have been proposed as candidates to mediate the phosphorylation of Akt-S473 at the hydrophobic motif.10 The well-described PDK2 is mTORC2, which phosphorylates Akt at S473 in response to mitogenic stimulations.11 Mammalian target of rapamycin (mTOR) protein, one of the PIKKs, can be found in two distinct complexes; unique accessory proteins raptor and rictor distinguish these complexes as mTORC1 and mTORC2, respectively.10,12 In response to growth factors, PI3K activation leads to full activation of Akt, which then phosphorylate tuberous sclerosis protein 2 (TSC2), a component of TSC1/TSC2 complex. The latter is a tumor suppressor complex that transmits growth factor and energy signals to mTORC1 by regulating the GTP-loading state of Rheb, a Ras-related GTP-binding protein. mTORC1 is important for the regulation of protein translation, ribosome biogenesis and cell growth through phosphorylation of its substrates S6-kinase (S6K) and 4E-BP1.10,12 Critically, S6K mediates inhibitory IRS1 serine phosphorylation, which disrupts IRS1 interaction with insulin receptor, leading to its degradation.13 Similarly, S6K phosphorylates rictor and inhibits mTORC2 and Akt signaling, providing a regulatory link between the two mTOR complexes (Fig. 1A).14 Akt signaling regulates a wide range of cellular processes through phosphorylation of a variety of downstream target substrates, including mTOR, FOXO proteins, Hdm2, Bad, p21 and GSK3 etc. The mechanism for Akt to recognize its downstream substrates is dependent on the phosphorylation status of the S473 or the ratio between the phosphorylation status of S473 and T308. Phosphorylation of FOXO proteins, but not TSC2, GSK3 and Hdm2 depends on Akt S473 phosphorylation. This could suggest that S473-deficient Akt retains enough

catalytic activity to phosphorylate most of its substrates.15-17 In genotoxic stress conditions, on the other hand, a non-canonical nuclear Akt activation may occur following DNA damage.3 A nuclear PDK1, DNA-PK and Akt pool arising from DNA damage may provide an increased concentration of all three proteins, which are clearly a prerequisite and a stimulus for phosphorylation events to take place.4 Both T308 by PDK1 and S473 phosphorylations by DNA-PK instead of mTORC2 appear to be essential for the activation of Akt in response to DNA damage in the nucleus.3,4,9,18 After DNA damage, nuclear active Akt appears to affect transcription, in particular of p53-regulated genes. This is due to p53 stabilization that is dependent on the activation of DNA-PK and Akt-mediated inactivation of Hdm2.19 Thus, fully activated nuclear Akt may phosphorylate the downstream targets such as FOXO4, p21 and Hdm2.19 Interestingly, FOXO4, but not FOXO3a, is phosphorylated after DNA damage when Akt activation is dependent on DNA-PK.3 FOXO4 carries three Akt-dependent phosphorylation sites by which FOXO4 is negatively regulated, denoting its nuclear export and inactivation. FOXO4 can only function as a tumor suppressor and control apoptosis if it is located in the nucleus.20 Activation of nuclear Akt also induces the upregulation of the cyclin-dependent kinase inhibitor p21, which induces cell cycle arrest and promotes cellular survival (Fig. 1B). Recent evidence indicates a crosstalk between the two Akt S473 kinases, DNA-PK and mTORC2. Elevated Akt S473 phosphorylation and PKCα activity, which is also phosphorylated at hydrophobic motif by mTORC2, are observed in DNA-PK deficient cell.9 Furthermore, mammalian Tel2 (mTel2) is required for the stability of all PIKKs. mTel2 is also important for the incorporation of newly synthesized mTOR into mTORC1 and mTORC2 complexes.21 Thus, mTel2 may function as a coordinator among PIKKs, suggesting the existence of crosstalk between different PIKKs where alteration of one PIKK may influence the other.9 Consequently, increased DNA-PK activity may lead to decreased Akt signaling

mediated by mTORC2 associated with the tissue-specific disruption of the Akt/ FOXO pathway 9 (Fig. 1B). Transforming growth factor β (TGFβ) is a critical cytokine for mammalian development and homeostasis, playing crucial roles in the pathogenesis of a variety of diseases. The highly conserved core of the canonical TGFβ signaling is a simple linear cascade that involves the TGFβ ligand, two types of receptors (type I and II) and the signal transducers, Smads. On activation, the receptor complex phosphorylates the receptor-regulated Smad proteins (R-Smads), Smad2 and Smad3. Activated R-Smads interact with common partner Smad4, and accumulate in the nucleus, where the Smad complex directly binds defined elements on the DNA and regulates target gene expression together with numerous other factors.22,23 The suppression of cell proliferation by TGFβ is modulated by the Smads-mediated transcriptional downregulation of c-Myc and upregulation of p15, p21, p27 CDKIs24 (Fig. 2). It is now clear that, in addition to the Smad pathway, the TGFβ receptor complex is capable of inducing nonSmad signals, which may be dependent or independent of Smads. Integration of Smad and non-Smad signaling pathways ultimately determines the nature of the cellular response.23 Of these non-Smad effectors, there is mounting evidence that PI3K plays a critical role in regulating both the inhibitory and stimulatory TGFβ response. TGFβ can rapidly activate PI3K, which is implicated in TGFβmediated actin filament reorganization and cell migration.25 Through this IRS1independent and non-canonical PI3K activation, Akt is also regulated by two phosphorylation events, which include the modifications of T308 by PDK1 and S473 by mTORC2. Subsequently, mTORC1 activation occurs via a canonical PI3KAkt-TSC2-dependent pathway.26 This activates downstream substrates including S6K.27 mTORC1 and S6K exert inhibitory serine phosphorylation of IRS1.13 More importantly, TGFβ may induce a decrease in the IRS1 protein and phosphorylation levels, implying a potential role of IRS1 in TGFβ-mediated biological function28 (Fig. 2).



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©201 1L andesBi os c i enc e. Donotdi s t r i but e. Figure 2. After stimulation by TGFβ, Smad2 and Smad3 (RSmads) become phosphorylated by the activated TGFβ receptor kinases, oligomerize with Smad4 forming Smad complex (SmadC). This complex translocates to the nucleus and regulates the expression of TGFβ target genes with close interaction of FOXO transcription factors, leading p15 and p21-induced cell cycle arrest. TGFβ receptor kinase also activates PI3K non-canonically, inducing full activation of Akt. Subsequent mTORC1/S6K activation stimulates cellular growth promoting pathways. mTORC1/S6K signaling pathway also results in negative phosphorylations on IRS1 and mTORC2, inhibiting their activities. Thus, T308-mediated partial activation of Akt-mTORC1/S6K leads persistent inhibition of IRS1. In addition, TGFβ-mediated inhibition of IRS1 expression potentiates existing cellular insulin resistance. Consequently, stress-induced autoregulatory TGFβ loop regulates cellular senescence by persistent and strong cell cycle arrest, unregulated mTORC1 activity and p53 inhibition. Blue arrows indicate stimulation and red arrows for the inhibition.

Stress-Induced Inverse Activities of JNK1 and JNK2 in the Regulation of Cell Fate JNK, one of the stress-activated kinases, is the proximal stress sensor for the most of the cellular stresses. Various stress signals, proinflammatory stimuli, and some mitogenic signals induce JNK activity, which in turn phosphorylates its substrates, such as c-Jun, c-Myc and ATF2, as well as nontranscription factors, such as members of the Bcl-2 family proteins.29-32 JNK activation was shown to either induce apoptosis or stimulate

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cellular proliferation and transformation.33 Among three members of the JNK family, JNK1 and JNK2 are expressed ubiquitously.34 Different JNK isozymes may have evolved for specific biological functions, probably depending on the activating stimuli and responding cell.29 c-Jun has been shown to be essential for efficient transition through the G1 phase of the cell cycle. JNK1 and JNK2 differentially regulate c-Jun stability and phosphorylation, thereby differentially affecting cellular proliferation.29,35 JNK levels and activity are tightly controlled during the cell cycle to ensure

seamless entry into mitosis under normal growth conditions. Elevated JNK expression or activity could be due to increased transcription or impaired degradation.36 In unstimulated cells, the basal physiological JNK1 function shows exclusive cytosolic localization.31,37 By contrast, c-Jun is mostly bound to JNK2, which appears to be responsible for c-Jun ubiquitination and degradation.29,34 In the cellular response to DNA damage, JNK2 is released from c-Jun, thereby giving more access to active JNK1, resulting in increased JNK1mediated c-Jun phosphorylation, stability and activity.29,34


©201 1L andesBi os c i enc e. Donotdi s t r i but e. Figure 3. Genotoxic stress-induced activation of p53 initiates reversible cell cycle arrest by inducing p21 activation and translocation of FOXO into cytosol (cFOXO: cytosolic FOXO). p21 inhibits p53-dependent transcription of proapoptotic proteins (PUMA and Bax). Finally, inhibition of mTORC1 by p53 leads to cellular quiescence (QUIESCENCE). In case of unrepairable damage or second hit of cellular stress, JNK isomers are activated. JNK1 increases the expression of c-Myc, leading to C-Myc-Miz-1-mediated anti-apoptotic Bcl-2 and p21 suppression. JNK1-induced activation of JNK2 phosphorylates cytosolic FOXO and translocates it into nucleus. Nuclear FOXO (nFOXO) stimulates translocation of p53 into cytosol. Subsequently, cytosolic p53 (c-p53) interacts with Bax on the outer membrane of mitochondria and opens mitochondrial permeability transition pore (MPTP), releasing cytochrome c into cytosol that initiates apoptosis (APOPTOSIS). If the apoptosis-prone same cell had genetically mutant p53 or JNK2 inability to stabilize and translocate p53 into cytosol, JNK1-induced irrepressible c-Myc activity may reason in uncontrolled proliferation (ONCOGENESIS). Activation of TGFβ due to chronic stress stimulates both canonical Smad and non-canonical PI3K/Akt-mTORC1 pathways. Smad-induced c-Myc suppression and SmadC-Miz-1 interaction causes irreversible cell cycle arrest through induction of CDKIs. TGFβ-PI3K pathway may also phosphorylate JNK1, which ultimately increases SmadC transcription activity. Thus, TGFβ signaling-mediated irreversible cell cycle arrest, mTORC1 activation and p53 inhibition may engage a senescence program (SENESCENCE).

JNKs participate in a feedback mechanism with p53 to regulate the apoptotic process, and p53 is oppositely regulated by JNK1 and JNK2.38 In response to oxidative stress, JNK1 promotes cellular survival through regulation of c-Myc and p21 expression and phosphorylation of JNK2.32,35 In addition to p53 phosphorylation, activated JNK2 is a positive regulator of p53 expression.35 JNK2 also phosphorylates FOXO family members


and triggers their relocalization from cytosol to the nucleus (Fig. 3).39-41 p53-Regulated Alterations in Response to Genotoxic Stress Once a cell has been damaged and the DDR and p53 are activated, a complex signaling network is engaged to result in a long-term cell fate decision.42,43 It seems that whenever the cell gets into trouble,

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p53 is called upon to help deal with the problem. Remarkably, the range of possible outcomes upon p53 activation is also extremely diverse and includes the ability to control the pathways of energy metabolism and modulate damaging stresses.44,45 Cell cycle arrest, which is a common response to the tumor suppressor protein p53, protects cells from DNA damage. Activation of p53 through the DDR usually leads to either proper repair of

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the lesion or elimination of the damaged cell from the proliferative cell pool.42 Furthermore, although p53 has also long been thought of as an inducer of senescence, it has been shown that p53 can act as a suppressor of cellular senescence.46 Nuclear p53 regulates energy metabolism through transcriptional activities to facilitate the cellular repairing activities. p53 inhibits the expression of the glucose transporters GLUT1 and GLUT4,47 and moderates the glycolytic activity, diverting glucose to the pentose phosphate pathway (PPP), which leads to the production of NADPH.48 In addition, p53 also inhibits the expression of insulin and IGF1 receptors, while increasing the expression of IGFBP-3, all of which dampen insulin/ IGF1 signaling.49 Furthermore, p53 is able to diminish the activity of mTORC1 following genotoxic stress; thus, p53mediated cell cycle arrest remains reversible as long as p53 inhibits mTORC1.5055 These endocrine responses seem to be highly conserved mechanism, which is likely evolved to cope with stress, and conserve energy resources for maintenance and repair rather than utilizing them for proliferation and growth30,49 (Fig. 1B). p53 has been shown to have dual mechanisms for inducing cell death. After genotoxic stress, nuclear accumulation of p53 causes activation of downstream proapoptotic gene expression (e.g., PUMA, Bax and Noxa) to induce cell death.56 However, in addition to increased amount of these proapoptotic proteins, translocation of p53 to the cytosol is requisite to execute the mitochondrial death pathway. p53 can act as a BH3 protein to induce Bax activation if forced to translocate into cytosol57 (Fig. 3). JNK-activated and relocated FOXO protein into nucleus has a negative effect on nuclear p53 activation and causes translocation of p53 to cytosol, even in the presence of DNA damaging signals.58 Thus, p53 accumulates in the cytosol and directly interacts with Bcl-2 family members.56,59 In addition to p53, FOXO protein could also regulate PUMA at transcriptional level independent of p53.57,60 This FOXO-induced PUMA behaves as a secondary death signal and binds to Bcl-x L , releasing cytosolic p53 to directly activate Bax and induces mitochondrial outer

membrane permeabilization (Fig. 3).59,61,62 Moreover, an important portion of cytosolic p53 also binds to G6PD through a direct protein-protein interaction and inhibits the glucose flux through the PPP, suppressing the production of NADPH, one of the major cellular antioxidants.63 The activation of the p53 protein and its network of genes sets in motion an elaborate process of autoregulatory-positive or autoregulatory-negative feedback loops, which connect the p53 pathway to other signal transduction pathways in the cell, and through this broader communication permits the completion of the reversal of the p53 programmed responses to stress.64 At this point a crucially important question arises: how these disparate regulatory functions of p53 are handled and managed in stressed-cells? The answer most probably resides in the subcellular localization of p53. Nuclear localization and transcriptional activities of p53 in DNA damage bring to mind the performance of Dr. Jekyll, leading to the cell choice of repair and live. However, under the enforced translocation to the cytosol in this stressful situation, p53 easily disguises itself as Mr. Hyde and drags the cell to die.

apoptosis is not achieved, suggesting that p21 expression can protect from apoptosis without altering p53 activity.67 When the stress-induced damage is too severe for the cell to recover, p53 initiates programmed cell death, thus eliminating cells that may have acquired irreparable and potentially oncogenic alterations (Fig. 3).67 At this point, the question is what determines the choice of response to p53? The proto-oncogene c-Myc, which is a central regulator of cellular proliferation and cell growth, can either activate or repress the expression of specific target genes associated with various biological functions.68,69 c-Myc represses a number of anti-proliferative genes, including p15, p21 and p27. With the exception of the p27 promoter, c-Myc does not directly contact the DNA of promoters in repressed target genes. Instead, c-Myc is recruited to these promoters by binding to other, promoterassociated proteins such as Miz-1, Sp1 and YY1.68,70 Although the p21 promoter is highly responsive to p53, Miz-1 also plays a critical role in regulating p21 expression.67 Thus, c-Myc via interaction with Miz1 suppresses p21 induction by p53 and thus switches the p53 response from cytostatic to apoptotic.67,71 Furthermore, expression of c-Myc also inhibits the transcription of p15, which is associated with cellular senescence, by forming a complex with Miz-1 (Fig. 3).68 Miz-1 also has a crucial role in the cellular response to TGFβ, which releases Miz-1 from Miz-1-Myc complex. Free Miz-1 synergizes with Smad proteins and activates CDKI expression.72 TGFβ induces cell cycle arrest through the cooperative action with p15, p21 and other CDKIs (Fig. 3).73,74 Significantly, TGFβ-induced activation of p21 transcription occurs via a p53-independent mechanism.74


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p21: Traffic Cop at the Crossroads of Cell Fate-Determining Molecules p53, c-Myc, Miz-1 and TGFβ Activation of p53 inhibits its nuclear export and degradation, thus resulting in increased levels of p53. p53 then activates one of its downstream targets, p21, which binds to and inhibits Cdk2/cyclin E complexes, arresting cells at the G1/S checkpoint.7 Thus, p53 can effectively cause cells to stop proliferating, which allows for the repair of any damaged DNA, preventing mutations from being passed on the daughter cells.65 Even when both cell cycle arrest and apoptotic target genes are induced by p53, the resultant response may still be cell cycle arrest. Under these conditions, apoptosis ensues only following selective inhibition of p53 target genes that encode a survival function, like p21.66 In the presence of p21, p53 can still bind to the pro-apoptotic promoters and induce the accumulation of its product, but

Cellular Fate Determination After Genotoxic Stress Cell cycle checkpoints activated in response to genotoxic stress prevent replication of damaged DNA presumably to allow time for the decision about cellular fate. As loss of these checkpoints result in cell transformation, the executive DDR machinery impacts the cell fate decisions


in different ways as a tumorigenesis barrier. In the light of above-mentioned molecular mechanisms, four different cell fates are: Quiescence. In response to DNA damage, p53 induces temporary cell cycle arrest or quiescence, which allows cells to recover from whatever stress is inducing p53. Quiescent cells neither grow in size nor progress through the cell cycle.55,75 In the absence of mitogenic stimuli or oncogene activation, p53-induced p21 cause cell cycle arrest, while inhibiting apoptosis.66 In quiescent cells, there happens a reversible and complete silencing of cytosolic Akt downstream targets through DNA-PK-induced mTORC2 inhibition and p53-inhibited mTORC1 pathway.9,44,76 These cells could resume cycling after p53-mediated rehabilitation.59 However, in p53-“locked” quiescent cells, which cannot proliferate, stimulation with growth factors, hormones and nutrients may cause their senescence through activation of mTORC1 signaling (Fig. 3).75,77 Apoptosis. Under some circumstances, p53 can take out cells attempting to proliferate beyond their defined limits by inducing cell death, thereby completely eliminating the errant cells.65 p53-induced apoptosis does not only require activation of proapoptotic target genes but also involves transcription-independent functions of p53 in the cytosol.78 After p53 stabilization in response to DNA damage, a second hit of cellular stress is sensed by JNK molecules. Stress-activated JNK1 specifically may increase c-Myc expression, and thus may repress the transcription of Miz-1-regulated Bcl-2 and p21 even in the presence of activated p53.35,67,69 Consequently, c-Myc removes p21- and Bcl2-mediated protection against apoptosis, enabling cell death (Fig. 3).35,69 Furthermore, stress-induced JNK1 activation also phosphorylates JNK2,79 which results in phosphorylation and stabilization of p53.35,80 JNK2 activation also phosphorylates and causes translocation of FOXO proteins into nucleus,41,80 where JNK2-phosphroylated FOXO may enforce nuclear p53 to translocate into cytosol.57,58 Cytosolic p53 interacts with antiapoptotic Bcl-2 family members, and awaits a secondary death signal.56,59 Furthermore, cytosolic JNK2 promotes

Bax translocation to mitochondria by phosphorylation-dependent release of Bax from 14-3-3, a cytoplasmic anchor for the latter.81 Oncogenesis. In a p53-mutated or JNK2 activation-hindered cell, uncontrollable stress-induced nuclear JNK1 activity may increase c-Jun and c-Myc expression with the potent suppression of CDKIs, leading to unregulated cellular proliferation.32,82 Recent data show genetic loss of JNK2 leads to earlier and more frequent tumorigenesis.83 Furthermore, JNK1induced c-Jun activity downregulates PTEN expression in a p53-independent manner, leading to upregulation of the Akt survival pathway.84 Remarkably, c-Jun induction concomitantly results in Akt S473 phosphorylation and full activation of Akt that is often activated in many cancers.84 Moreover, activated JNK1 translocates to the outer mitochondrial membrane, where indirectly inactivates pyruvate dehydrogenase that may create a shift from aerobic metabolism of pyruvate in mitochondrion to its anaerobic reduction to lactate in cytosol (Warburg effect).85 Senescence. Even low levels of persistent DNA damage in transcribed regions of genome can result in sustained somatotropic attenuation. As a consequence of this attenuation, cells become resistant to insulin/IGF1 signaling.86 Chronic genotoxic stress may increase the extracellular glucose load due to p53-mediated inhibition of GLUT transporters and insulin/IGF1 signaling pathway.45,47 High extracellular glucose concentrations may enhance TGFβ activity through rapid externalization of TGFβ receptors and metalloproteinase-mediated activation of latent TGFβ.87,88 Glucose may increase both Smad-dependent signaling and Smad-independent activation of PI3K/ AKT-mTOR pathway.87,89 In addition to p53-regulated extracellular glucosemediated indirect effects on the activation of TGFβ signaling pathway, p53 and the activated Smad complex may bind to each other and to their cognate sites on DNA, leading to a synergistic transcriptional activation.90,91 Moreover, persistent impairment of insulin/IGF1-IRS1-mediated PI3K activation may stimulate integrins by physically coupling them to insulin/

IGF1 receptors. This in turn may initiate an energy-dependent, integrin-mediated inside-out signaling mechanism,92 which is one of the major activators of latent TGFβ (Fig. 1C).93 Activation of TGFβ receptor serine/ threonine kinase initiates PI3K/Akt/ mTORC1 signaling toward increased S6K activity.94 Both S6K and mTORC1 reason in inhibitory serine phosphorylation of IRS1,13 which further potentiates existing cellular insulin resistance. Increased mTORC1/S6K activities also inhibit mTORC2 and phosphorylation of Akt at S473,14,94 which is required for phosphorylation of FOXO proteins.11,95 Thus, unphosphorylated FOXO translocates into nucleus and functions as key partners of Smad3 and Smad4 in TGFβdependent formation of transactivation complex.96 However, in case of defective S473 phosphorylation, other Akt targets are unaffected; thus, activation of Hdm2 downregulates p53-mediated activities.10,97 TGFβ can directly stimulate activation of JNK1 via phosphorylation dependent mechanism through PI3K signaling pathway.98 JNK1, but not JNK2, in turn has been linked to phosphorylation of Smad2/3, and is required for TGFβinduced transcriptional activation.98,99 Notably, in JNK1 deficient cells, TGFβinduced Smad phosphorylation and nuclear translocation were intact, but that binding of Smad complexes to the Smad DNA response element is diminished.98 In the absence of TGFβ, c-Myc prevents p15, p21 and p57 transcription by forming a complex with Miz1. Stimulation with TGFβ relieves the repression of these CDKIs by downregulating c-Myc and promotes transcriptional activation of them by the induction of nuclear Smad complex that may contact with the free Miz-1, forming an activator complex.73,100 Thus, when the expression of c-Myc is switched off, TGFβ reasons in a ATM-p53-independent strong cell cycle arrest.100,101 Altogether, TGFβ-dependent autocrine regulatory circuit fulfills all components of a complete senescence activating program, which are irreversible cell cycle arrest, mTORC1 activation, and suppression of p53.78 Consequently, it is tempting to speculate that stressinduced senescence may be persistent



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TGFβ-induced and T308-mediated partial Akt activation state (Figs. 2 and 3). Effects of DNA Damage Response on Terminally Differentiated Cells Terminally differentiated postmitotic cells are believed to stay in an ‘extended G0 phase,’ locked by CDKIs.102,103 What will happen to those differentiated and permanently quiescent cells when they encounter with genotoxic stimulation? Unlike proliferating cells where DNA damage typically triggers cell cycle checkpoints, postmitotic cells activate their cell cycle machinery and result in Cdk5-ATMmediated phosphorylation of p53,104,105 which may lead a proper scan and repair of DNA.105 It is most likely that the inability to repair DNA using the ATM-p53 system may expose postmitotic cells to two degenerative choices. In case of new wave of acute and strong stress hit would lead these cells to JNK-dependent apoptosis,104,106,107 similar to mitotically active cells. However, in chronic low grade stress conditions, second choice could operate through TGFβ-mediated persistent signaling that may lead to overstimulation of mTORC1 pathway. Both of these two irreversible processes cause degenerative diseases through irreplaceable apoptotic losses or maladaptive overactivation of postmitotic cells.12,103,108,109

and the maintenance of the senescent characteristics of the cell. In this sense, in a full agreement with Blagosklonny and colleagues,46 senescence suppressive influence of p53 is mechanistically has been overviewed. Another issue, importance of subcellular localization of signaling molecules, such as Akt, FOXO, JNK and p53 are underscored for their cell cycle-linked proper functions. Even small changes of these proteins in activation states or subcellular localizations could affect the cellular fate from repair to the choice “between a rock and a hard place”: apoptosis or carcinogenesis. In this context, pathogenesis of senescence, fourth choice of the cell, has been detailed in a novel approach. Furthermore, the relation of Akt with mTOR and the importance of its activation state whether full or partial have been underscored as being in the very heart of the cell cycle determination process. Thus, persistent mTOR-driven signaling regulations may lead purposeless cellular aging, which supports the “quasi-program” of aging theory of Blagosklonny.111 Organisms maintain life against the inevitable influence of the second law of thermodynamics, which dictates that entropy must increase over time. As long as the organism performs its life functions, entropy continue to be generated.112 A gene gains nothing by going off selfishly on its own; therefore, even the selfish oncogenes cooperate with altruistic tumor suppressor genes in physiologic conditions, protecting multicellularity. However, after DNA damage, the conflict between these putatively distinct sets of genes increases the genomic entropy. Consequently, there may be no specific aging genes or program; instead, when the resisting capability of organism to maintain genomic stability is exhausted, surrendering to universal thermodynamic laws, leads a process of aging toward the final end.

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Concluding Remarks Longevity is regulated by the joint actions of many maintenance and repair pathways. Each pathway guards against the age-related accumulation of particular kinds of damage before the relevant lesions build up to a level of that threatens survival.110 Quite a few other molecules related with these pathways, such as PARP and sirtuins,12,75,108 which have also important roles in DDR, are not mentioned in order not to further complicate comprehensibility. Instead, some very well-known stress-linked molecules such as p53, JNK and TGFβ have been characterized with their diverse functions. Accordingly, p53 is assumed as the executor of an agonizing choice; repair and live or die. p53 activation may also lead to the induction of senescence, but not to the progression

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