Abstract
Most agents that damage DNA act through posttranslational modifications of p53 and activate its downstream targets. However, whether cellular responses to nucleoside analogue-induced DNA damage also operate through p53 posttranslational modification has not been reported. In this study, the relationship between p53 activation and its posttranslational modifications was investigated in the human cancer cell lines A549 and HCT116 in response to 5-aza-2'-deoxycytidine (5-aza-CdR) or cytarabine treatment. 5-Aza-CdR induces p53 posttranslational modifications through activation of an ATM- and Rad3-related (ATR) signaling pathway, and 5-aza-CdR-induced association of replication protein A with chromatin is required for the binding of ATR to chromatin. Upon treatment with 5-aza-CdR, ATR activation is clearly associated with p53 phosphorylation at Ser(15), but not at Thr(18), Ser(20), or Ser(37). This specific p53 phosphorylation at Ser(15) in turn results in acetylation of p53 at Lys(320) and Lys(373)/Lys(382) through transcriptional cofactors p300/CBP-associated factor and p300, respectively. These p53 posttranslational modifications are directly responsible for 5-aza-CdR induced p21(Waf1/Cip1) expression because the binding activity of acetylated p53 at Lys(320)/Lys(373)/Lys(382) to the p21(Waf1/Cip1) promoter, as well as p21(Waf1/Cip1) expression itself are significantly increased after 5-aza-CdR treatment. It is of interest that p53 phosphorylation at Ser(15) and acetylations at Lys(320)/Lys(373)/Lys(382) mutually interact in the 5-aza-CdR induced p21(Waf1/Cip1) expression shown by transfection of artificially mutated p53 expression vectors including S15A, K320R, and K373R/K382R into p53-null H1299 cells. These data taken together show for the first time that 5-aza-CdR activates the ATR signaling pathway, which elicits a specific p53 phosphorylation-acetylation cascade to induce p21(Waf1/Cip1) expression.
Highlights
Tions, p53 is maintained at a low level through its interaction with MDM2 [3, 4], Pirh2 [5], COP1 [6], and ARF-BP1 [7], which mediate both ubiquitination and proteasome-dependent degradation of p53
5-Aza-CdR Activates p21Waf1/Cip1 Expression via ATR Pathway whereas p300/CBP-associated factor (PCAF) acetylates p53 at Lys320 [1, 2, 22, 24]. Another transcription factor, Tip60, was reported to acetylate p53 at Lys120 in response to DNA damage, which is crucial for p53-dependent apoptosis, by selectively affecting the transcription of proapoptotic target genes such as BAX and PUMA [26, 27]
Many different kinds of agents that damage DNA have been determined to act as anticancer agents through activation of the p21Waf1/Cip1 pathway by posttranslational modifications of p53 [1, 12, 14]
Summary
Cell Culture and Nucleoside Analogue Treatment—Human lung cancer cell lines A549 and H1299 were grown in RPMI 1640 supplemented with 10% fetal bovine serum (heat-inactivated at 56 °C for 45 min) and penicillin/streptomycin, in a humidified incubator with 5% CO2 atmosphere at 37 °C. Primers used for mutagenesis had the following sequences: p53-15A-up, 5-CGT CGA GCC CCC TCT GAG(GC) TCA GGA AAC ATT TTC AG-3; p53-15A-down, 5-CTG AAA ATG TTT CCT GACT(GC)CA GAG GGG GCT CGA CG-3; p53–320R-up, 5-CTC TCC CCA GCC AAA GAA(CG) GAA ACC ACT GGA TGG AG-3; p53–320R-down, 5-CTC CAT CCA GTG GT T TCTT(CG)CT TTG GCT GGG GAG AG-3; p53–373R-up, 5-CAC CTG AAG TCC AAA AG(A) G GGT CAG TCT ACC TC-3; p53–373R-down, 5-GA GGT AGA CTG ACC CC(T) TTTT GGA CTT CAG GTG-3; p53–382R-up, 5-CTA CCT CCC GCC ATA AAA G(A)AC TCA TGT TCA AGA-3; and p53–382R-down, 5-TCT TGA ACA TGA GTC(T) TTT TAT GGC GGG AGG TAG-3. Co-immunoprecipitation (Co-IP)—The cells were harvested and lysed in lysis buffer (1% Nonidet P-40, 150 mM NaCl, 50 mM Tris, 0.05% SDS, 1 mM phenylmethylsulfonyl fluoride, and a 1% mixture of protease inhibitors) on ice for 20 min. The samples were analyzed with flow cytometry (BD FACS Calibur) using BD CellQuest software
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
