title: DNA double helix unwinding triggers transcription block-dependent apoptosis: a semiquantitative probe of the response of ATM, RNAPII, and p53 to two DNA intercalators.
abstract: We have previously shown the binding modes of two DNA interacting analogues (1)a {3-(4-methyl-piperazin)-8-oxo-8H-acenaphtho[1,2-b]pyrrole-9-carbonitrile} and (3)a {3-(3-dimethylamino-propylamino)-8-oxo-8H-acenaphtho[1,2-b]pyrrole-9-carbonitrile} with the DNA double helix. In this study, we have determined the notably different DNA damage signal pathway elicited by (1)a and (3)a due to the different extents to which they unwind the DNA double helix. First, we have identified that ataxia-telangiectasia-mutated (ATM) protein kinase can respond to DNA double helix unwinding caused by both (1)a and (3)a. In addition, the amount of ATM activation is consistent with the degree to which the DNA double helix was unwound. Consequently, we used (1)a and (3)a to semiquantitatively probe the response of RNA polymerase II (RNAPII) and p53 toward DNA double helix unwinding in vivo. By means of flow cytometry, immunocytochemistry, ChIP, quantitative real-time polymerase chain reaction, and Western blot analyses, we measured the level of p53 and RNAPII phosphorylation, in addition to the dynamics of the RNAPII distribution along the c-Myc gene. These results provided novel evidence for the impact of subtle DNA structural changes on the activity of RNAPII and p53. Moreover, DNA double helix conformational damage-dependent apoptosis was studied for the first time. These results indicated that (1)a can induce transcriptional blockage following a shift of the unphosphorylated IIa form of RNAPII to the phosphorylated IIo form, while (3)a is unable to induce the same effect. Subsequently, p53 accumulation and phosphorylation events occur that lead to apoptosis in the case of (1)a exposure. This suggests that the transcriptional blockage is also correlated to the degree of double helix unwinding. Furthermore, we found that the degree of DNA conformational damage determines whether or not apoptosis occurs through transcriptional blockage. Under our experimental conditions, ATM does not participate in the downstream events even when it has been activated. Thus, p53-mediated apoptosis may be independently triggered by transcriptional blockage.
authors.affiliation: State Key Laboratory of Fine Chemicals, Dalian University of Technology, Dalian 116012, People's Republic of China. zczhang@dlut.edu.cn
authors.forename: Zhichao
authors.initials: Z
authors.lastname: Zhang
authors.forename: Yuanyuan
authors.initials: Y
authors.lastname: Wang
authors.forename: Ting
authors.initials: T
authors.lastname: Song
authors.forename: Jin
authors.initials: J
authors.lastname: Gao
authors.forename: Guiye
authors.initials: G
authors.lastname: Wu
authors.forename: Jing
authors.initials: J
authors.lastname: Zhang
authors.forename: Xuhong
authors.initials: X
authors.lastname: Qian
mesh_terms: D000073:Acenaphthenes / Q000737:chemistry / Q000633:toxicity*; D017209:Apoptosis / Q000235:genetics; D064007:Ataxia Telangiectasia Mutated Proteins; D018797:Cell Cycle Proteins / Q000378:metabolism*; D002460:Cell Line; D004247:DNA / Q000737:chemistry; D004268:DNA-Binding Proteins / Q000378:metabolism*; D016259:Genes, myc; D006801:Humans; D007364:Intercalating Agents / Q000737:chemistry / Q000633:toxicity*; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D011760:Pyrrolidinones / Q000737:chemistry / Q000633:toxicity*; D012319:RNA Polymerase II / Q000378:metabolism*; D015398:Signal Transduction / Q000235:genetics; D014158:Transcription, Genetic; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism*; D025521:Tumor Suppressor Proteins / Q000378:metabolism*
chemical_list: C553473:3-(3-dimethylaminopropylamino)-8-oxo-8H-acenaphtho(1,2-b)pyrrole-9-carbonitrile; C553472:3-(4-methylpiperazin)-8-oxo-8H-acenaphtho(1,2-b)pyrrole-9-carbonitrile; D000073:Acenaphthenes; D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D007364:Intercalating Agents; D011760:Pyrrolidinones; D016159:Tumor Suppressor Protein p53; D025521:Tumor Suppressor Proteins; D004247:DNA; C576324:ATM protein, human; D064007:Ataxia Telangiectasia Mutated Proteins; D017346:Protein Serine-Threonine Kinases; D012319:RNA Polymerase II