An increase of cytochrome C oxidase mediated disruption of gemcitabine incorporation into DNA in a resistant KB clone.
Mechanistic aberrations leading to Gemcitabine (2',2'-dFdCyd,2,2-difluorodeoxycytidine, Gem) resistance may include alteration in its transport, metabolism and incorporation into DNA. To explore the mechanism of Gem resistance, the restriction fragment differential display PCR (RFDD-PCR) was employed to compare the mRNA expression patterns of KBGem (Gem resistant), KBHURs (hydroxyurea resistant) and KBwt (parental KB cell). Nine gene fragments were overexpressed specifically in the KBGem clone. Sequencing and BLAST results showed that three fragments represent cytochrome C oxidase (CCOX, respiration complex IV) subunit III (CCOX3). The cDNA microarray confirmed that the mRNAs of CCOX and ATP synthase subunits were upregulated in KBGem as compared to KBwt and KBHURs. The increase in CCOX1 protein and activity led to the increase of free ATP concentration, which is consistent with the gene expression profile of KBGem. Furthermore, the sensitivity to Gem could be reversed by sodium azide, a CCOX inhibitor. Following the treatment of sodium azide, the cellular accumulation of [3H]-Gem increased in a dose (of azide)-dependent manner, which is associated with increase of [3H]-Gem incorporation into DNA in KBGem. In summary, an increase of CCOX activity and free ATP level may reduce the transport, metabolism and DNA incorporation of Gem, resulting in Gem resistance.
Liu
Xiyong
X
Department of Clinical & Molecular Pharmacology, City of Hope National Medical Center, 1500 E. Duarte Road, Duarte, CA 91010-3000, United States.
Zhou
Bingsen
B
Mi
Shu
S
Xue
Lijun
L
Shih
Jennifer
J
Lee
Janice
J
Chau
Jennifer
J
Un
Frank
F
Yen
Yun
Y
D000255:Adenosine Triphosphate / Q000032:analysis; D000964:Antimetabolites, Antineoplastic / Q000494:pharmacology*; D015153:Blotting, Western; D002999:Clone Cells; D004247:DNA / Q000235:genetics / Q000378:metabolism*; D003841:Deoxycytidine / Q000031:analogs & derivatives* / Q000494:pharmacology; D019008:Drug Resistance, Neoplasm; D003576:Electron Transport Complex IV / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D007624:KB Cells; D020411:Oligonucleotide Array Sequence Analysis; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000378:metabolism; D019810:Sodium Azide / Q000494:pharmacology; D000093542:Gemcitabine
D000964:Antimetabolites, Antineoplastic; D004791:Enzyme Inhibitors; D012333:RNA, Messenger; D003841:Deoxycytidine; D000255:Adenosine Triphosphate; D004247:DNA; D019810:Sodium Azide; D003576:Electron Transport Complex IV; D000093542:Gemcitabine