Coordination between Cell Cycle Progression and Cell Fate Decision by the p53 and E2F1 Pathways in Response to DNA Damage
2010; Elsevier BV; Volume: 285; Issue: 41 Linguagem: Inglês
10.1074/jbc.m110.134650
ISSN1083-351X
AutoresXiao Peng Zhang, Feng Liu, Wei Wang,
Tópico(s)Epigenetics and DNA Methylation
ResumoAfter DNA damage, cells must decide between different fates including growth arrest, DNA repair, and apoptosis. Both p53 and E2F1 are transcription factors involved in the decision process. However, the mechanism for cross-talk between the p53 and E2F1 pathways still remains unclear. Here, we proposed a four-module kinetic model of the decision process and explored the interplay between these two pathways in response to ionizing radiation via computer simulation. In our model the levels of p53 and E2F1 separately exhibit pulsatile and switching behaviors. Upon DNA damage, p53 is first activated, whereas E2F1 is inactivated, leading to cell cycle arrest in the G1 phase. We found that the ultimate decision between cell life and death is determined by the number of p53 pulses depending on the extent of DNA damage. For repairable DNA damage, the cell can survive and reenter the S phase because of the activation of E2F1 and inactivation of p53. For irreparable DNA damage, growth arrest is overcome by growth factors, and activated p53 and E2F1 cooperate to initiate apoptosis. We showed that E2F1 promotes apoptosis by up-regulating the proapoptotic cofactors of p53 and procaspases. It was also revealed that deregulated E2F1 by oncogene activation can make cells sensitive to DNA damage even in low serum medium. Our model consistently recapitulates the experimental observations of the intricate relationship between p53 and E2F1 in the DNA damage response. This work underscores the significance of E2F1 in p53-mediated cell fate decision and may provide clues to cancer therapy. After DNA damage, cells must decide between different fates including growth arrest, DNA repair, and apoptosis. Both p53 and E2F1 are transcription factors involved in the decision process. However, the mechanism for cross-talk between the p53 and E2F1 pathways still remains unclear. Here, we proposed a four-module kinetic model of the decision process and explored the interplay between these two pathways in response to ionizing radiation via computer simulation. In our model the levels of p53 and E2F1 separately exhibit pulsatile and switching behaviors. Upon DNA damage, p53 is first activated, whereas E2F1 is inactivated, leading to cell cycle arrest in the G1 phase. We found that the ultimate decision between cell life and death is determined by the number of p53 pulses depending on the extent of DNA damage. For repairable DNA damage, the cell can survive and reenter the S phase because of the activation of E2F1 and inactivation of p53. For irreparable DNA damage, growth arrest is overcome by growth factors, and activated p53 and E2F1 cooperate to initiate apoptosis. We showed that E2F1 promotes apoptosis by up-regulating the proapoptotic cofactors of p53 and procaspases. It was also revealed that deregulated E2F1 by oncogene activation can make cells sensitive to DNA damage even in low serum medium. Our model consistently recapitulates the experimental observations of the intricate relationship between p53 and E2F1 in the DNA damage response. This work underscores the significance of E2F1 in p53-mediated cell fate decision and may provide clues to cancer therapy. IntroductionThe tumor suppressor p53 has a crucial role in preventing tumorigenesis (1Meek D.W. Nat. Rev. Cancer. 2009; 9: 714-723Crossref PubMed Scopus (487) Google Scholar). Upon various stresses, p53 is stabilized and activated to function primarily as a transcription factor, regulating the expression of a large number of genes involved in cell cycle arrest, DNA repair, or apoptosis (2Murray-Zmijewski F. Slee E.A. Lu X. Nat. Rev. Mol. Cell Biol. 2008; 9: 702-712Crossref PubMed Scopus (331) Google Scholar). Thus, p53 is at the hub of numerous signaling pathways triggered by various stresses. Previously, it was proposed that cell fate after DNA damage is governed by p53 levels, i.e. a low level of p53 leads to transient growth arrest and cell survival, whereas a high level promotes irreversible apoptosis (3Vousden K.H. Lane D.P. Nat. Rev. Mol. Cell Biol. 2007; 8: 275-283Crossref PubMed Scopus (1707) Google Scholar). Recently, it has been reported that p53 levels can exhibit oscillations in response to DNA damage induced by ionizing radiation (IR) (4Lev Bar-Or R. Maya R. Segel L.A. Alon U. Levine A.J. Oren M. Proc. Natl. Acad. Sci. U.S.A. 2000; 97: 11250-11255Crossref PubMed Scopus (479) Google Scholar, 5Lahav G. Rosenfeld N. Sigal A. Geva-Zatorsky N. Levine A.J. Elowitz M.B. Alon U. Nat. Genet. 2004; 36: 147-150Crossref PubMed Scopus (804) Google Scholar). Whereas damped oscillations of p53 levels were observed at the population level (4Lev Bar-Or R. Maya R. Segel L.A. Alon U. Levine A.J. Oren M. Proc. Natl. Acad. Sci. U.S.A. 2000; 97: 11250-11255Crossref PubMed Scopus (479) Google Scholar), a series of undamped pulses was observed at the single-cell level (5Lahav G. Rosenfeld N. Sigal A. Geva-Zatorsky N. Levine A.J. Elowitz M.B. Alon U. Nat. Genet. 2004; 36: 147-150Crossref PubMed Scopus (804) Google Scholar). In such a digital mode, it is the number of p53 pulses rather than their amplitudes and duration that is related to the extent of DNA damage and determines cell fate (5Lahav G. Rosenfeld N. Sigal A. Geva-Zatorsky N. Levine A.J. Elowitz M.B. Alon U. Nat. Genet. 2004; 36: 147-150Crossref PubMed Scopus (804) Google Scholar). p53 pulses can be generated by negative feedback loops with time delay (6Ma L. Wagner J. Rice J.J. Hu W. Levine A.J. Stolovitzky G.A. Proc. Natl. Acad. Sci. U.S.A. 2005; 102: 14266-14271Crossref PubMed Scopus (291) Google Scholar, 7Geva-Zatorsky N. Rosenfeld N. Itzkovitz S. Milo R. Sigal A. Dekel E. Yarnitzky T. Liron Y. Polak P. Lahav G. Alon U. Mol. Syst. Biol. 2006; 2: 0033Crossref PubMed Scopus (484) Google Scholar, 8Batchelor E. Mock C.S. Bhan I. Loewer A. Lahav G. Mol. Cell. 2008; 30: 277-289Abstract Full Text Full Text PDF PubMed Scopus (335) Google Scholar) or coupled positive and negative feedback loops (9Ciliberto A. Novak B. Tyson J.J. Cell Cycle. 2005; 4: 488-493Crossref PubMed Scopus (197) Google Scholar, 10Zhang T. Brazhnik P. Tyson J.J. Cell Cycle. 2007; 6: 85-94Crossref PubMed Scopus (109) Google Scholar).How stressed cells exploit p53 pulses to translate various stresses into different cellular outcomes is not completely understood. Several studies have explored the functional roles of p53 pulses in response to DNA damage. Tyson and co-workers (10Zhang T. Brazhnik P. Tyson J.J. Cell Cycle. 2007; 6: 85-94Crossref PubMed Scopus (109) Google Scholar) classified active p53 into three distinct forms according to its phosphorylation status and showed that p53 pulses subserve the decision between cell cycle arrest/repair and apoptosis. We developed an integrated model of the p53 signaling network to reveal the whole process from the generation of DNA damage to the choice of cell fate, stressing that two forms of phosphorylated p53 play distinct roles in cell fate decision (11Zhang X.P. Liu F. Cheng Z. Wang W. Proc. Natl. Acad. Sci. U.S.A. 2009; 106: 12245-12250Crossref PubMed Scopus (181) Google Scholar). Batchelor et al. (12Batchelor E. Loewer A. Lahav G. Nat. Rev. Cancer. 2009; 9: 371-377Crossref PubMed Scopus (179) Google Scholar) proposed that p53 pulses can allow for a wide variety of temporal expression patterns of target genes. These studies suggest that the pulsatile response of p53 may represent a flexible and efficient mechanism by which cellular responses can be organized coherently. Notably, the different roles played by p53 are associated with its specific post-translational modifications.Although p53 is pivotal to cell fate decision, cross-talk between p53 and other transcription factors also has important roles. Among them, the E2F family is best known for its ability to regulate entry into and progression through S phase of the cell cycle (13Wu L. Timmers C. Maiti B. Saavedra H.I. Sang L. Chong G.T. Nuckolls F. Giangrande P. Wright F.A. Field S.J. Greenberg M.E. Orkin S. Nevins J.R. Robinson M.L. Leone G. Nature. 2001; 414: 457-462Crossref PubMed Scopus (489) Google Scholar). Specifically, E2F1 can promote both cell cycle progression and apoptosis (14Hallstrom T.C. Nevins J.R. Proc. Natl. Acad. Sci. U.S.A. 2003; 100: 10848-10853Crossref PubMed Scopus (133) Google Scholar), and deregulated E2F1 can cooperate with p53 to trigger apoptosis (15Wu X. Levine A.J. Proc. Natl. Acad. Sci. U.S.A. 1994; 91: 3602-3606Crossref PubMed Scopus (808) Google Scholar, 16Qin X.Q. Livingston D.M. Kaelin Jr., W.G. Adams P.D. Proc. Natl. Acad. Sci. U.S.A. 1994; 91: 10918-10922Crossref PubMed Scopus (691) Google Scholar, 17DeGregori J. Leone G. Miron A. Jakoi L. Nevins J.R. Proc. Natl. Acad. Sci. U.S.A. 1997; 94: 7245-7250Crossref PubMed Scopus (601) Google Scholar). E2F1 can induce production of several p53 cofactors including p53DINP1, 3The abbreviations used are: p53DINP1p53-dependent damage inducible nuclear protein 1ATMataxia telangiectasia mutatedRbretinoblastoma proteinWip1wild-type p53-induced phosphatase 1ASPPapoptosis-stimulating protein of p53p53AIP1p53-regulated apoptosis-inducing protein 1Apaf-1apoptotic protease activating factor-1GygrayDSBdouble-strand breakDSBCDSB complexCycEcyclin ECytoCcytochrome cCaspcaspase. ASPP1, and ASPP2 (ASPP1/2 are collectively referred to as ASPP thereafter) (18Hershko T. Chaussepied M. Oren M. Ginsberg D. Cell Death Differ. 2005; 12: 377-383Crossref PubMed Scopus (90) Google Scholar). p53DINP1 promotes apoptosis by phosphorylating p53 at Ser-46 (19Okamura S. Arakawa H. Tanaka T. Nakanishi H. Ng C.C. Taya Y. Monden M. Nakamura Y. Mol. Cell. 2001; 8: 85-94Abstract Full Text Full Text PDF PubMed Scopus (283) Google Scholar), while ASPP proteins enhance binding of p53 to the promoters of proapoptotic genes (20Samuels-Lev Y. O'Connor D.J. Bergamaschi D. Trigiante G. Hsieh J.K. Zhong S. Campargue I. Naumovski L. Crook T. Lu X. Mol. Cell. 2001; 8: 781-794Abstract Full Text Full Text PDF PubMed Scopus (559) Google Scholar). On the other hand, the activation of p53 first induces G1 phase arrest by inhibiting E2F1 via p21 (21He G. Siddik Z.H. Huang Z. Wang R. Koomen J. Kobayashi R. Khokhar A.R. Kuang J. Oncogene. 2005; 24: 2929-2943Crossref PubMed Scopus (195) Google Scholar). Therefore, E2F1 competes with p53 for cell cycle control but cooperates with p53 in apoptosis induction. Moreover, E2F1 levels can behave like a bistable switch when driven by growth factors (22Yao G. Lee T.J. Mori S. Nevins J.R. You L. Nat. Cell Biol. 2008; 10: 476-482Crossref PubMed Scopus (291) Google Scholar). An issue thus arises concerning how the combination of p53 pulsing and E2F1 switching contributes to the decision between cell survival and death after DNA damage. It is also interesting to explore the effect of p53 cofactors on cellular outcomes, which was seldom considered in the above theoretical models for p53 pulses.Motivated by the above considerations, we developed an integrated model to explore how cell cycle progression and cell fate decision are well coordinated by p53 and E2F1 in the DNA damage response. The model is composed of four modules: a DNA repair module, an ATM sensor, a p53 pulse generator, and a cell fate decision module. The model can characterize the process from the generation of DNA damage to the choice of cell fate. We found that the cell fate is determined by the number of p53 pulses, which depends on the extent of DNA damage for each fixed concentration of growth factor. E2F1 potentiates p53-dependent apoptosis in two ways: 1) it induces p53 cofactors to bias p53 activity toward apoptosis, and 2) it up-regulates the levels of procaspases to make cells sensitive to death stimuli. We concluded that activation of E2F1 or p53 alone results in S-phase entry and G1 arrest, respectively, whereas concomitant activation of p53 and E2F1 initiates apoptosis.EXPERIMENTAL PROCEDURESThe cellular response to DNA damage can be considered as a signal transduction process, and the signaling pathways involved are rather complicated. It is difficult to obtain all precise data to characterize in detail the whole process of DNA damage response. Here, we focus on exploring the essential mechanisms for cell fate decision. With limited experimental data available, our model was constructed based on established biological facts and reasonable simplifications. We also plotted schematic diagrams to depict cross-talk between the p53 and E2F1 pathways (23Elkon R. Vesterman R. Amit N. Ulitsky I. Zohar I. Weisz M. Mass G. Orlev N. Sternberg G. Blekhman R. Assa J. Shiloh Y. Shamir R. BMC Bioinformatics. 2008; 9: 110Crossref PubMed Scopus (50) Google Scholar). The key points of the model are addressed as follows.An Integrated Model for Cell Signaling Network in Response to DNA DamageWe developed an integrated model for the p53 network composed of four modules (Fig. 1). DNA damage is produced in cells exposed to IR. The DNA repair module characterizes the generation and repair of DNA damage, which is essentially stochastic. Upon IR, ATM is activated by autophosphorylation, acting as a sensor of DNA damage (6Ma L. Wagner J. Rice J.J. Hu W. Levine A.J. Stolovitzky G.A. Proc. Natl. Acad. Sci. U.S.A. 2005; 102: 14266-14271Crossref PubMed Scopus (291) Google Scholar, 24Bakkenist C.J. Kastan M.B. Nature. 2003; 421: 499-506Crossref PubMed Scopus (2652) Google Scholar). Subsequently, p53 is activated by phosphorylation (25Stommel J.M. Wahl G.M. EMBO J. 2004; 23: 1547-1556Crossref PubMed Scopus (312) Google Scholar), and interlinked positive and negative feedback loops involving p53 and Mdm2 underlie p53 pulses (10Zhang T. Brazhnik P. Tyson J.J. Cell Cycle. 2007; 6: 85-94Crossref PubMed Scopus (109) Google Scholar). Active p53 is distinguished between p53 arrester and p53 killer, which contribute to cell cycle arrest and apoptosis, respectively (10Zhang T. Brazhnik P. Tyson J.J. Cell Cycle. 2007; 6: 85-94Crossref PubMed Scopus (109) Google Scholar, 11Zhang X.P. Liu F. Cheng Z. Wang W. Proc. Natl. Acad. Sci. U.S.A. 2009; 106: 12245-12250Crossref PubMed Scopus (181) Google Scholar). Once activated by growth factors, E2F1 can promote the transition from the G1 to S phase and cooperate with p53 killer to induce apoptosis. Moreover, p53-inducible Wip1 feeds back to inhibit ATM activity (8Batchelor E. Mock C.S. Bhan I. Loewer A. Lahav G. Mol. Cell. 2008; 30: 277-289Abstract Full Text Full Text PDF PubMed Scopus (335) Google Scholar), enclosing a negative feedback loop from the fourth to the second module. In the following we present the details of each module sequentially.DNA Repair ModuleA double-strand break (DSB) is generally considered the typical form of DNA damage induced by IR (26Fei P. El-Deiry W.S. Oncogene. 2003; 22: 5774-5783Crossref PubMed Scopus (410) Google Scholar). According to experimental observations, 1 Gy of IR may induce 25–40 DSBs per cell (27Al Rashid S.T. Dellaire G. Cuddihy A. Jalali F. Vaid M. Coackley C. Folkard M. Xu Y. Chen B.P. Chen D.J. Lilge L. Prise K.M. Bazett Jones D.P. Bristow R.G. Cancer Res. 2005; 65: 10810-10821Crossref PubMed Scopus (91) Google Scholar). DSB repair proceeds in a stochastic way. The stochasticity in the generation and repair of DSBs is transmitted downstream of this module (6Ma L. Wagner J. Rice J.J. Hu W. Levine A.J. Stolovitzky G.A. Proc. Natl. Acad. Sci. U.S.A. 2005; 102: 14266-14271Crossref PubMed Scopus (291) Google Scholar); that is, there exists variability in cellular responses to the same stress signal. Non-homologous end joining is the predominant pathway for DSB repair, especially in the G1 phase (28Burma S. Chen B.P. Chen D.J. DNA Repair. 2006; 5: 1042-1048Crossref PubMed Scopus (301) Google Scholar). We simplified the repair process into a three-state process that is characterized by reversible binding of repair proteins to DSB, forming a complex (DSBC), and by an irreversible repair process from the complex to fixed DNA. Specifically, we adopted the two-lesion-kinetic model (29Stewart R.D. Radiat. Res. 2001; 156: 365-378Crossref PubMed Scopus (73) Google Scholar) and applied the Monte Carlo method proposed by Ma et al. (6Ma L. Wagner J. Rice J.J. Hu W. Levine A.J. Stolovitzky G.A. Proc. Natl. Acad. Sci. U.S.A. 2005; 102: 14266-14271Crossref PubMed Scopus (291) Google Scholar) to mimic the repair process (see supplemental Method S1 and Fig. S1).In simulations, we considered a population of 2000 cells that are exposed to the same IR. Because Poisson distribution is typically used to characterize the random induction of DSBs (30Bonner W.M. Proc. Natl. Acad. Sci. U.S.A. 2003; 100: 4973-4975Crossref PubMed Scopus (131) Google Scholar, 31Rief N. Löbrich M. J. Biol. Chem. 2002; 277: 20572-20582Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar), the initial numbers of DSBs are assumed to obey the Poisson distribution with a mean of 35 DSBs per Gy per cell. Because repair proteins are much fewer than DSBs in most cases (6Ma L. Wagner J. Rice J.J. Hu W. Levine A.J. Stolovitzky G.A. Proc. Natl. Acad. Sci. U.S.A. 2005; 102: 14266-14271Crossref PubMed Scopus (291) Google Scholar), it is assumed that there are 20 repair proteins in each cell. p53 certainly plays a role in DNA repair, but its regulatory role in non-homologous end joining is complicated and controversial (32Sengupta S. Harris C.C. Nat. Rev. Mol. Cell Biol. 2005; 6: 44-55Crossref PubMed Scopus (436) Google Scholar). Some studies revealed that p53 can promote the rejoining of DNA with lesions (33Yang T. Namba H. Hara T. Takmura N. Nagayama Y. Fukata S. Ishikawa N. Kuma K. Ito K. Yamashita S. Oncogene. 1997; 14: 1511-1519Crossref PubMed Scopus (81) Google Scholar, 34Tang W. Willers H. Powell S.N. Cancer Res. 1999; 59: 2562-2565PubMed Google Scholar), whereas inhibitory effects of p53 on non-homologous end joining were also reported (35Akyüz N. Boehden G.S. Süsse S. Rimek A. Preuss U. Scheidtmann K.H. Wiesmüller L. Mol. Cell. Biol. 2002; 22: 6306-6317Crossref PubMed Scopus (151) Google Scholar, 36Bill C.A. Yu Y. Miselis N.R. Little J.B. Nickoloff J.A. Mutat. Res. 1997; 385: 21-29Crossref PubMed Scopus (39) Google Scholar). For simplicity, we did not consider the effect of p53 on DNA repair.ATM SensorThe role of ATM as a sensor of DNA damage has been widely recognized (24Bakkenist C.J. Kastan M.B. Nature. 2003; 421: 499-506Crossref PubMed Scopus (2652) Google Scholar, 37Lee J.H. Paull T.T. Science. 2005; 308: 551-554Crossref PubMed Scopus (1056) Google Scholar). In unstressed cells, ATM exists as a dimer, and its kinase activity is sequestered. Upon IR, ATM can be recruited by repair proteins, and intermolecular phosphorylation leads to rapid disassociation of dimers into monomers (37Lee J.H. Paull T.T. Science. 2005; 308: 551-554Crossref PubMed Scopus (1056) Google Scholar). There exists a positive feedback loop in which active ATM (i.e. phosphorylated monomer) further promotes the phosphorylation of inactive ATM (24Bakkenist C.J. Kastan M.B. Nature. 2003; 421: 499-506Crossref PubMed Scopus (2652) Google Scholar). Thus, the activation of ATM is traditionally characterized by a switch (38Mouri K. Nacher J.C. Akutsu T. PLoS. ONE. 2009; 4e5131Crossref PubMed Scopus (23) Google Scholar, 39Chickarmane V. Ray A. Sauro H.M. Nadim A. SIAM J. Appl. Dyn. Syst. 2007; 6: 61-78Crossref Scopus (26) Google Scholar). However, pulses of ATM levels have recently been observed in human breast cancer MCF-7 cells, and a recurrent initiation mechanism was proposed based on the negative feedback loop between ATM and p53 via Wip1 (8Batchelor E. Mock C.S. Bhan I. Loewer A. Lahav G. Mol. Cell. 2008; 30: 277-289Abstract Full Text Full Text PDF PubMed Scopus (335) Google Scholar). Here, we consider three forms of ATM: ATMd (inactive dimer), ATM (inactive monomer), and ATM* (active monomer). The total level of ATM is assumed to be constant (6Ma L. Wagner J. Rice J.J. Hu W. Levine A.J. Stolovitzky G.A. Proc. Natl. Acad. Sci. U.S.A. 2005; 102: 14266-14271Crossref PubMed Scopus (291) Google Scholar) (see supplemental Fig. S2).The dynamics of this module are characterized by Equations 1–3 in supplemental Method S2. The phosphorylation and dephosphorylation of ATM can be considered as enzyme-catalyzed reactions and assumed to follow the Michaelis-Menten kinetics (40Kholodenko B.N. Nat. Rev. Mol. Cell Biol. 2006; 7: 165-176Crossref PubMed Scopus (1003) Google Scholar). Due to the positive and negative feedback loops, the phosphorylation rate of ATM should be positively correlated with the number of DSBCs and ATM* levels, whereas the dephosphorylation of ATM* is promoted by Wip1. It is assumed that the dimerization rate of ATM is far smaller than its undimerization rate, so that ATM dimers are predominant in unstressed cells.p53 Pulse GeneratorAlthough a single negative feedback loop with time delay can produce periodic oscillations (6Ma L. Wagner J. Rice J.J. Hu W. Levine A.J. Stolovitzky G.A. Proc. Natl. Acad. Sci. U.S.A. 2005; 102: 14266-14271Crossref PubMed Scopus (291) Google Scholar), it has been proposed that coupled negative and positive feedback loops can make oscillations more robust (9Ciliberto A. Novak B. Tyson J.J. Cell Cycle. 2005; 4: 488-493Crossref PubMed Scopus (197) Google Scholar, 10Zhang T. Brazhnik P. Tyson J.J. Cell Cycle. 2007; 6: 85-94Crossref PubMed Scopus (109) Google Scholar). Indeed, several negative and positive feedback loops have been identified in the p53 pathway (41Harris S.L. Levine A.J. Oncogene. 2005; 24: 2899-2908Crossref PubMed Scopus (1486) Google Scholar). The negative feedback between p53 and Mdm2 is the basis for p53 oscillation, while a double-negative feedback loop involving p53, Akt, and Mdm2 also has a role (9Ciliberto A. Novak B. Tyson J.J. Cell Cycle. 2005; 4: 488-493Crossref PubMed Scopus (197) Google Scholar, 10Zhang T. Brazhnik P. Tyson J.J. Cell Cycle. 2007; 6: 85-94Crossref PubMed Scopus (109) Google Scholar). Akt promotes the nuclear translocation of Mdm2 to degrade p53 and inhibit its activity, whereas p53 can indirectly inactivate Akt through PTEN (42Gottlieb T.M. Leal J.F. Seger R. Taya Y. Oren M. Oncogene. 2002; 21: 1299-1303Crossref PubMed Scopus (379) Google Scholar, 43Mayo L.D. Donner D.B. Proc. Natl. Acad. Sci. U.S.A. 2001; 98: 11598-11603Crossref PubMed Scopus (941) Google Scholar). Moreover, as mentioned above, ATM* activates p53, but p53 inhibits ATM* via Wip1.In our model the p53 pulse generator is composed of three coupled feedback loops (marked by lines with different colors in Fig. 2): 1) the negative feedback loop between p53 and Mdm2, 2) the positive feedback loop involving p53, Akt and Mdm2, and 3) the negative feedback loop between ATM and p53 via Wip1. Nuclear p53 is distinguished between inactive p53 (p53) and active p53 (p53*), whereas cytoplasmic p53 is ignored. Three forms of Mdm2 are introduced, namely Mdm2c (unphosphorylated cytoplasmic form), Mdm2cp (phosphorylated cytoplasmic form), and Mdm2n (nuclear form). Akt in the cytoplasm is differentiated between Akt (unphosphorylated form) and Aktp (phosphorylated form). For simplicity, the effect of nuclear Akt is not considered.FIGURE 2p53 pulse generator model. There exist three feedback loops: the negative feedback loop between p53 and Mdm2 (red and purple), the positive feedback loop involving p53, Akt, and Mdm2 (blue and purple), and the negative feedback loop between ATM and p53 via Wip1 (green).View Large Image Figure ViewerDownload Hi-res image Download (PPT)The dynamics of this module are characterized by Equations 4–13 in supplemental Method S2. The regulated transcription of Mdm2 by p53 is incorporated into the regulation of protein synthesis. The basal production rate of Mdm2 is set to be much smaller than the maximal p53-induced production rate (44Landers J.E. Cassel S.L. George D.L. Cancer Res. 1997; 57: 3562-3568PubMed Google Scholar). The Hill coefficient is set to 4 considering the cooperative binding of tetrameric p53 to DNA (45Jeffrey P.D. Gorina S. Pavletich N.P. Science. 1995; 267: 1498-1502Crossref PubMed Scopus (435) Google Scholar). Note that ATM can promote Mdm2 degradation and p53 activation by phosphorylation, and the two processes can be considered as enzyme-catalyzed reactions (25Stommel J.M. Wahl G.M. EMBO J. 2004; 23: 1547-1556Crossref PubMed Scopus (312) Google Scholar, 46Prives C. Cell. 1998; 95: 5-8Abstract Full Text Full Text PDF PubMed Scopus (627) Google Scholar). Consequently, both the degradation rate of Mdm2 and the activation rate of p53 are ATM-dependent and described in the form of the Michaelis-Menten function. Similarly, the degradation of p53 by Mdm2 is characterized by the Michaelis-Menten kinetics because Mdm2 acts as an E3 ubiquitin ligase in the ubiquitination of p53 (47Brooks C.L. Gu W. Mol. Cell. 2006; 21: 307-315Abstract Full Text Full Text PDF PubMed Scopus (683) Google Scholar).Because there is no remarkable variation in the total level of Akt after irradiation (42Gottlieb T.M. Leal J.F. Seger R. Taya Y. Oren M. Oncogene. 2002; 21: 1299-1303Crossref PubMed Scopus (379) Google Scholar), it is assumed to be constant. The phosphorylation of Akt should be phosphatidylinositol 3,4,5-trisphosphate (PIP3)-dependent (48Vivanco I. Sawyers C.L. Nat. Rev. Cancer. 2002; 2: 489-501Crossref PubMed Scopus (5077) Google Scholar), and the effect of PIP3 is reflected in the parameter kakt in the model (49Wee K.B. Aguda B.D. Biophys. J. 2006; 91: 857-865Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). Similarly, the dephosphorylation of Aktp is phosphatidylinositol 4,5-biphosphate (PIP2)-dependent (48Vivanco I. Sawyers C.L. Nat. Rev. Cancer. 2002; 2: 489-501Crossref PubMed Scopus (5077) Google Scholar), and this effect is incorporated into the parameters kakts and k1akts, which are separately the rate constants of basal and p53-dependent dephosphorylation of Akt (49Wee K.B. Aguda B.D. Biophys. J. 2006; 91: 857-865Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). Because p53-inducible PTEN can promote the conversion from PIP3 to PIP2 as well as Aktp dephosphorylation, this effect is simplified into the modulation of Aktp dephosphorylation by p53 with the rate constant of k1akts (49Wee K.B. Aguda B.D. Biophys. J. 2006; 91: 857-865Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). We set kakts < kakt < k1akts to ensure that Aktp is predominant in unstressed cells and Akt becomes dominant in stressed cells. Moreover, we chose the values of other parameters in this module to ensure that the period of p53 pulses is between 4 and 7 h (5Lahav G. Rosenfeld N. Sigal A. Geva-Zatorsky N. Levine A.J. Elowitz M.B. Alon U. Nat. Genet. 2004; 36: 147-150Crossref PubMed Scopus (804) Google Scholar, 7Geva-Zatorsky N. Rosenfeld N. Itzkovitz S. Milo R. Sigal A. Dekel E. Yarnitzky T. Liron Y. Polak P. Lahav G. Alon U. Mol. Syst. Biol. 2006; 2: 0033Crossref PubMed Scopus (484) Google Scholar).Cell Fate Decision ModuleCell fate can be governed by signaling pathways involving p53 and E2F1. Both p53 and E2F1 function as transcription factors. It has been recognized that post-translational modifications and cofactors regulate the promoter selectivity of p53 (2Murray-Zmijewski F. Slee E.A. Lu X. Nat. Rev. Mol. Cell Biol. 2008; 9: 702-712Crossref PubMed Scopus (331) Google Scholar). E2F1 can up-regulate the levels of p53 cofactors and proapoptotic proteins (18Hershko T. Chaussepied M. Oren M. Ginsberg D. Cell Death Differ. 2005; 12: 377-383Crossref PubMed Scopus (90) Google Scholar, 50Nahle Z. Polakoff J. Davuluri R.V. McCurrach M.E. Jacobson M.D. Narita M. Zhang M.Q. Lazebnik Y. Bar-Sagi D. Lowe S.W. Nat. Cell Biol. 2002; 4: 859-864Crossref PubMed Scopus (362) Google Scholar). The schematics of this module are shown in Fig. 3, and its key points are addressed as follows.FIGURE 3Cell fate decision module model. Active p53 is divided into p53 arrester and p53 killer. p53 arrester is a primarily phosphorylated form of p53 on Ser-15 and Ser-20, whereas p53 killer is a further phosphorylated form of p53 on Ser-46. p53 arrester induces cell cycle arrest in the G1 phase by inducing p21, which inhibits E2F1 activity, whereas p53 killer induces expression of proapoptotic genes. The conversion between p53 arrester and p53 killer is controlled by Wip1 and p53DINP1. E2F1 can be activated by growth factors. p53 killer and E2F1 cooperate to transactivate p53DINP1. With the help of E2F1-induced ASPP, p53 killer induces expression of p53AIP1 and Bax, which results in mitochondrial outer membrane permeabilization (MOMP). Moreover, E2F1 promotes apoptosis by up-regulating several key proapoptotic factors including Apaf-1 and procaspase-9 and -3.View Large Image Figure ViewerDownload Hi-res image Download (PPT)First, the phosphorylation of p53 modulates its selective expression of target genes (51Bode A.M. Dong Z. Nat. Rev. Cancer. 2004; 4: 793-805Crossref PubMed Scopus (1012) Google Scholar). Based on phosphorylation on different residues, active p53 is divided into two forms in our model, p53 arrester and p53 killer, which promote cell cycle arrest and apoptosis, respectively (see Fig. 3 and supplemental Fig. S3). Here, p53 arrester refers to p53 primarily phosphorylated at Ser-15 and Ser-20 by ATM, whereas p53 killer is p53 further phosphorylated at Ser-46 by p53DINP1 and HIPK2 (homeodomain interacting protein kinase) (52Tomasini R. Samir A.A. Carrier A. Isnardon D. Cecchinelli B. Soddu S. Malissen B. Dagorn J.C. Iovanna J.L. Dusetti N.J. J. Biol. Chem. 2003; 278: 37722-37729Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar). p53 arrester alone induces
Referência(s)