title: HSP27 regulates p53 transcriptional activity in doxorubicin-treated fibroblasts and cardiac H9c2 cells: p21 upregulation and G2/M phase cell cycle arrest.
abstract: Treatment of cancer patients with anthracyclin-based chemotherapeutic drugs induces congestive heart failure by a mechanism involving p53. However, it is not known how p53 aggravates doxorubicin (Dox)-induced toxicity in the heart. On the basis of in vitro acute toxicity assay using heat shock factor-1 (HSF-1) wild-type (HSF-1(+/+)) and HSF-1-knockout (HSF-1(-/-)) mouse embryonic fibroblasts and neonatal rat cardiomyocyte-derived H9c2 cells, we demonstrate a novel mechanism whereby heat shock protein 27 (HSP27) regulates transcriptional activity of p53 in Dox-treated cells. Inhibition of p53 by pifithrin-alpha (PFT-alpha) provided different levels of protection from Dox that correlate with HSP27 levels in these cells. In HSF-1(+/+) cells, PFT-alpha attenuated Dox-induced toxicity. However, in HSF-1(-/-) cells (which express a very low level of HSP27 compared with HSF-1(+/+) cells), there was no such attenuation, indicating an important role of HSP27 in p53-dependent cell death. On the other hand, immunoprecipitation of p53 was found to coimmunoprecipitate HSP27 and vice versa (confirmed by Western blotting and matrix-assisted laser desorption/ionization time of flight), demonstrating HSP27 binding to p53 in Dox-treated cells. Moreover, upregulation of p21 was observed in HSF-1(+/+) and H9c2 cells, indicating that HSP27 binding transactivates p53 and enhances transcription of p21 in response to Dox treatment. Further analysis with flow cytometry showed that increased expression of p21 results in G(2)/M phase cell cycle arrest in Dox-treated cells. Overall, HSP27 binding to p53 attenuated the cellular toxicity by upregulating p21 and prevented cell death.
authors.affiliation: The Ohio State University, Columbus, OH 43210, USA.
authors.forename: C D
authors.initials: CD
authors.lastname: Venkatakrishnan
authors.forename: Kathy
authors.initials: K
authors.lastname: Dunsmore
authors.forename: Hector
authors.initials: H
authors.lastname: Wong
authors.forename: Sashwathi
authors.initials: S
authors.lastname: Roy
authors.forename: Chandan K
authors.initials: CK
authors.lastname: Sen
authors.forename: Altaf
authors.initials: A
authors.lastname: Wani
authors.forename: Jay L
authors.initials: JL
authors.lastname: Zweier
authors.forename: Govindasamy
authors.initials: G
authors.lastname: Ilangovan
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000903:Antibiotics, Antineoplastic / Q000633:toxicity*; D052160:Benzothiazoles / Q000494:pharmacology; D015153:Blotting, Western; D002453:Cell Cycle / Q000187:drug effects*; D002455:Cell Division; D002460:Cell Line; D050759:Cyclin-Dependent Kinase Inhibitor p21 / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004317:Doxorubicin / Q000633:toxicity*; D005347:Fibroblasts / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D005434:Flow Cytometry; D016195:G2 Phase; D055551:HSP27 Heat-Shock Proteins; D000076249:Heat Shock Transcription Factors; D006360:Heat-Shock Proteins / Q000378:metabolism*; D047468:Immunoprecipitation; D051379:Mice; D018345:Mice, Knockout; D008969:Molecular Sequence Data; D032383:Myocytes, Cardiac / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D009363:Neoplasm Proteins / Q000378:metabolism*; D011485:Protein Binding; D051381:Rats; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014050:Toluene / Q000031:analogs & derivatives / Q000494:pharmacology; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism; D014158:Transcription, Genetic / Q000187:drug effects; D015533:Transcriptional Activation / Q000187:drug effects; D016159:Tumor Suppressor Protein p53 / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D015854:Up-Regulation
chemical_list: D000903:Antibiotics, Antineoplastic; D052160:Benzothiazoles; C493957:Cdkn1a protein, mouse; C493958:Cdkn1a protein, rat; D050759:Cyclin-Dependent Kinase Inhibitor p21; D004268:DNA-Binding Proteins; D055551:HSP27 Heat-Shock Proteins; D000076249:Heat Shock Transcription Factors; D006360:Heat-Shock Proteins; C490017:Hsf1 protein, mouse; C486114:Hspb1 protein, rat; C486699:Hspb2 protein, mouse; D009363:Neoplasm Proteins; D014157:Transcription Factors; D016159:Tumor Suppressor Protein p53; D014050:Toluene; D004317:Doxorubicin; C121565:pifithrin