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Download Normal Cytosine Base in DNA Abnormal 5-chloro
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The Occurrence of Reactive Oxygen and Nitrogen Species in Normal Endometrial Tissues Might Explain Signature Hotspot Mutations Found in the PTEN Gene in Endometrial Adenocarcinoma Michaela Huynh MD/PhD Candidate, University of Texas Medical Branch MD/PhD Combined Degree Program Outline • Endometrial adenocarcinoma • Reactive Oxygen and Nitrogen Species • Findings • Implications • Future directions MD/PhD Combined Degree Program Endometrial Cancer Type I vs Type II Type I: •PTEN mutated in ~80% EC type I Chalhoub N, Baker S. 2009. Annu. Rev. Pathol. Mech. Dis. MD/PhD Combined Degree Program Endometrial Cancer Type I vs Type II PTEN Mutation Spectrum Type I: 225 R130 200 175 Transition (C > T; G > A) 150 Count •PTEN mutated in ~80% EC type I •PTEN truncation mutations •Hotspot at codon 130 •Origin of mutation is unknown Other 125 Transversion (C > G) 100 75 50 25 0 0 25 50 75 100 125 150 175 200 225 250 275 300 325 350 375 400 Codon COSMIC MD/PhD Combined Degree Program Potential Mechanism of Mutation Promoter vs Exonic methylation PTEN c.388C->T Cl- Spontaneous Deamination DNMT T Normal Cytosine Base in DNA Abnormal 5chloro-cytosine MD/PhD Combined Degree Program 5-methylcytosine Thymine mC is observed in benign endometrium Presence of mC: • can spontaneously deaminate to T • increases the likelihood of damage to adjacent G Methylation Status of Codon 130 and 142 in exon 5 of hPTEN in endomyometrial tissue Percent methylation 100% 80% 60% pt 1 40% pt 2 pt 3 20% pt4 0% 130 coding 130 noncoding 142 coding Codon MD/PhD Combined Degree Program 142 noncoding Potential mechanism of Mutation PTEN c388C->G C G -> GC -> C G -> C G -> G G -> GG oxG C -> Gh/Sp C -> Gh/Sp C -> CG MD/PhD Combined Degree Program ROS and RNS L-arginine NADPH + O2 NADPH Oxidase NO + O2 - proteins ONOO8-oxo-G MD/PhD Combined Degree Program 3-nitrotyrosine Spiroiminodihydantoin (Sp), Guanidinohydantoin (Gh), etc. Nitrotyrosine is observed in benign endometrium proteins 3-nitrotyrosine ONOO- S t a in in g in t e n s it y o f e n d o m e t r ia l g la n d s in t h e b a s a lis * In te n s ity S tr o n g M o d e ra te W eak P r o lif e r a t iv e S e c re to ry M e n s tr u a l c y c le p h a s e MD/PhD Combined Degree Program Nitrated peptides are identified by LC-MS/MS Identified peptide unique to ACTBL2 Manually verified the identity and the nitration site MD/PhD Combined Degree Program ACTBL2 and Nitrotyrosine colocalize DAPI Nitrotyrosine ACTBL2 Merge DAPI Nitrotyrosine ACTBL2 Merge 20X 63X MD/PhD Combined Degree Program Implications for DNA damage mC is detectable in exonic regions of PTEN contribute to C->T mutations Peroxynitrite damage is detectable in benign endometrial tissue Peroxynitrite can also damage DNA MD/PhD Combined Degree Program Future Directions Determine physiological role of ACTBL2 Query proteomics data for DNA damage repair proteins Determine significance of mC mediated C->T mutations in other genes mutated in endometrial cancer (i.e. FGFR2) Determine significance of peroxynitrite damage in other uterine pathologies (i.e. leiomyomata) MD/PhD Combined Degree Program Acknowledgements Sowers’ Lab Members MD/PhD Combined Degree Program Claiborne Fant, MS •Pooja Patel, MD BMB program •Jason Herring, PhD Whitehurst •Jay Patel, PhD •Jacob Theruvathu, PhD Dr. James Lee, Dr. Tracy Toliver, JoAlice Surgical Pathology Department, especially residents Dr. Eduardo Eyzaguirre, pathology residents, administrative staff Histology Core •Jyothi Dhuguru, PhD Ken Escobar, Kerry Graves, Natalie Dobias Optical Microscopy Core Adrianna Palucci, PhD Dr. Sandra Hatch, MD MS Core Bill Russell, PhD Cheryl Lichti, PhD Funding MD/PhD Combined Degree Program NIH Mendoza Distinguished Chair in Radiation Oncology Ruth Levy Kempner Professorship in Radiation Oncology Truman G. Blocker Endowment for the MD/PhD Program ROS and RNS L-arginine NADPH + O2 NADPH Oxidase NO + O2 Superoxide Dismutase proteins ONOO8-oxo-G H2O2 + O2 MPO, Cl- Catalase HOCl H2O + O2 macromolecules 3-Chlorotyrosine 5-chloro-cytosine MD/PhD Combined Degree Program 3-nitrotyrosine 5-methyl-cytosine Spiroiminodihydantoin (Sp), Guanidinohydantoin (Gh), etc.
