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

Journal Article · Fri Sep 23 04:00:00 EDT 2011 · Biochemical and Biophysical Research Communications

DOI:https://doi.org/10.1016/J.BBRC.2011.08.111· OSTI ID:22207509

Chen, Zhengming; Zhou, Jun; Zhang, Yingtao ^[1]; Bepler, Gerold ^[1]

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1. Developmental Therapeutics Program, Karmanos Cancer Institute, Detroit, MI (United States)

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.

• OSTI ID: 22207509
• Journal Information: Biochemical and Biophysical Research Communications, Journal Name: Biochemical and Biophysical Research Communications Journal Issue: 2 Vol. 413; ISSN BBRCA9; ISSN 0006-291X
• Country of Publication: United States
• Language: English

Related Subjects

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