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Title: Modulation of the ribonucleotide reductase M1-gemcitabine interaction in vivo by N-ethylmaleimide

Abstract

Highlights: {yields} Gemcitabine induces a RRM1 conformational change in tumor cell lines and xenografts. {yields} The 110 kDa RRM1 is unique to gemcitabine interaction among 12 cytotoxic agents. {yields} The 110 kDa RRM1 can be stabilized by the thiol alkylator N-ethylmaleimide. {yields} C218A, C429A, and E431A mutations in RRM1 abolished the conformational change. {yields} The 110 kDa RRM1 may be a specific biomarker of gemcitabine's therapeutic efficacy. -- Abstract: Ribonucleotide reductase M1 (RRM1) is the regulatory subunit of the holoenzyme that catalyzes the conversion of ribonucleotides to 2'-deoxyribonucleotides. Its function is indispensible in cell proliferation and DNA repair. It also serves as a biomarker of therapeutic efficacy of the antimetabolite drug gemcitabine (2',2'-difluoro-2'-deoxycytidine) in various malignancies. However, a mechanistic explanation remains to be determined. This study investigated how the alkylating agent N-ethylmaleimide (NEM) interacts with the inhibitory activity of gemcitabine on its target protein RRM1 in vivo. We found, when cells were treated with gemcitabine in the presence of NEM, a novel 110 kDa band, along with the 90 kDa native RRM1 band, appeared in immunoblots. This 110 kDa band was identified as RRM1 by mass spectrometry (LC-MS/MS) and represented a conformational change resulting from covalent labeling by gemcitabine. Itmore » is specific to gemcitabine/NEM, among 11 other chemotherapy drugs tested. It was also detectable in human tumor xenografts in mice treated with gemcitabine. Among mutations of seven residues essential for RRM1 function, C218A, C429A, and E431A abolished the conformational change, while N427A, C787A, and C790A diminished it. C444A was unique since it was able to alter the conformation even in absence of gemcitabine treatment. We conclude that the thiol alkylator NEM can stabilize the gemcitabine-induced conformational change of RRM1, and this stabilized RRM1 conformation has the potential to serve as a specific biomarker of gemcitabine's therapeutic efficacy.« less

Authors:
; ;  [1];  [1]
  1. Developmental Therapeutics Program, Karmanos Cancer Institute, Detroit, MI (United States)
Publication Date:
OSTI Identifier:
22207509
Resource Type:
Journal Article
Journal Name:
Biochemical and Biophysical Research Communications
Additional Journal Information:
Journal Volume: 413; Journal Issue: 2; Other Information: Copyright (c) 2011 Elsevier Science B.V., Amsterdam, The Netherlands, All rights reserved.; Country of input: International Atomic Energy Agency (IAEA); Journal ID: ISSN 0006-291X
Country of Publication:
United States
Language:
English
Subject:
60 APPLIED LIFE SCIENCES; ALKYLATING AGENTS; BIOLOGICAL MARKERS; CELL PROLIFERATION; CHEMOTHERAPY; CONFORMATIONAL CHANGES; DEOXYCYTIDINE; DNA REPAIR; IN VIVO; MASS SPECTROSCOPY; MICE; MUTATIONS; NEM; NEOPLASMS; PROTEINS; THIOLS; TUMOR CELLS

Citation Formats

Chen, Zhengming, Zhou, Jun, Zhang, Yingtao, and Bepler, Gerold, E-mail: beplerg@karmanos.org. Modulation of the ribonucleotide reductase M1-gemcitabine interaction in vivo by N-ethylmaleimide. United States: N. p., 2011. Web. doi:10.1016/J.BBRC.2011.08.111.
Chen, Zhengming, Zhou, Jun, Zhang, Yingtao, & Bepler, Gerold, E-mail: beplerg@karmanos.org. Modulation of the ribonucleotide reductase M1-gemcitabine interaction in vivo by N-ethylmaleimide. United States. doi:10.1016/J.BBRC.2011.08.111.
Chen, Zhengming, Zhou, Jun, Zhang, Yingtao, and Bepler, Gerold, E-mail: beplerg@karmanos.org. Fri . "Modulation of the ribonucleotide reductase M1-gemcitabine interaction in vivo by N-ethylmaleimide". United States. doi:10.1016/J.BBRC.2011.08.111.
@article{osti_22207509,
title = {Modulation of the ribonucleotide reductase M1-gemcitabine interaction in vivo by N-ethylmaleimide},
author = {Chen, Zhengming and Zhou, Jun and Zhang, Yingtao and Bepler, Gerold, E-mail: beplerg@karmanos.org},
abstractNote = {Highlights: {yields} Gemcitabine induces a RRM1 conformational change in tumor cell lines and xenografts. {yields} The 110 kDa RRM1 is unique to gemcitabine interaction among 12 cytotoxic agents. {yields} The 110 kDa RRM1 can be stabilized by the thiol alkylator N-ethylmaleimide. {yields} C218A, C429A, and E431A mutations in RRM1 abolished the conformational change. {yields} The 110 kDa RRM1 may be a specific biomarker of gemcitabine's therapeutic efficacy. -- Abstract: Ribonucleotide reductase M1 (RRM1) is the regulatory subunit of the holoenzyme that catalyzes the conversion of ribonucleotides to 2'-deoxyribonucleotides. Its function is indispensible in cell proliferation and DNA repair. It also serves as a biomarker of therapeutic efficacy of the antimetabolite drug gemcitabine (2',2'-difluoro-2'-deoxycytidine) in various malignancies. However, a mechanistic explanation remains to be determined. This study investigated how the alkylating agent N-ethylmaleimide (NEM) interacts with the inhibitory activity of gemcitabine on its target protein RRM1 in vivo. We found, when cells were treated with gemcitabine in the presence of NEM, a novel 110 kDa band, along with the 90 kDa native RRM1 band, appeared in immunoblots. This 110 kDa band was identified as RRM1 by mass spectrometry (LC-MS/MS) and represented a conformational change resulting from covalent labeling by gemcitabine. It is specific to gemcitabine/NEM, among 11 other chemotherapy drugs tested. It was also detectable in human tumor xenografts in mice treated with gemcitabine. Among mutations of seven residues essential for RRM1 function, C218A, C429A, and E431A abolished the conformational change, while N427A, C787A, and C790A diminished it. C444A was unique since it was able to alter the conformation even in absence of gemcitabine treatment. We conclude that the thiol alkylator NEM can stabilize the gemcitabine-induced conformational change of RRM1, and this stabilized RRM1 conformation has the potential to serve as a specific biomarker of gemcitabine's therapeutic efficacy.},
doi = {10.1016/J.BBRC.2011.08.111},
journal = {Biochemical and Biophysical Research Communications},
issn = {0006-291X},
number = 2,
volume = 413,
place = {United States},
year = {2011},
month = {9}
}