Overexpression of ribonucleotide reductase as a mechanism of resistance to 2,2-difluorodeoxycytidine in the human KB cancer cell line

Yih Gang Goan, Bingsen Zhou, Edward Hu, Shu Mi, Yun Yen

研究成果: 雜誌貢獻文章

196 引文 (Scopus)

摘要

In this study, human oropharyngeal epidermoid carcinoma KB cells that were resistant to 2,2-difluorodeoxycytidine (dFdCyd) were selected and designated the KB-Gem clone. The KB parental cell line IC50 was 0.3 μM dFdCyd, as compared with the KB-Gem clone IC50 of 32 μM dFdCyd. The KB-Gem clone demonstrated overexpression of ribonucleotide reductase (RR) M2 subunit mRNA (9-fold) and overexpression of M2 protein (2-fold); RR activity was 2.3- fold higher than the KB parental cell line. Both the dATP and dCTP pools of the KB-Gem clone increased 2-fold over the parental cell line, with no change in the dGTP and dTTP pools. Reverse transcriptase-PCR was used to clone the cDNA of deoxycytidine kinase (DCK). Resulting sequences revealed two silent mutations in the KB-Gem clone. The amino acid sequence of the DCK protein and mRNA expression remained unchanged. The KB-Gem clone's DCK enzyme activity was 56% of that of the parental cell line. After the endogenous dNTPs were removed with a G-25 column, no difference was evident between the enzyme activities of the KB-Gem clone and parental cells. Thus, contrary to previous hypotheses, DCK deficiency does not play the primary role in the resistance mechanism of dFdCyd, accepting a secondary role to the overexpression of the target gene, RR, and pool expansion.

原文英語
頁(從 - 到)4204-4207
頁數4
期刊Cancer Research
59
發行號17
出版狀態已發佈 - 九月 1 1999
對外發佈Yes

指紋

KB Cells
Ribonucleotide Reductases
Clone Cells
Deoxycytidine Kinase
Cell Line
Neoplasms
Inhibitory Concentration 50
Messenger RNA
Enzymes
Reverse Transcriptase Polymerase Chain Reaction
Amino Acid Sequence
Squamous Cell Carcinoma
Proteins
Complementary DNA

ASJC Scopus subject areas

  • Cancer Research
  • Oncology

引用此文

Overexpression of ribonucleotide reductase as a mechanism of resistance to 2,2-difluorodeoxycytidine in the human KB cancer cell line. / Goan, Yih Gang; Zhou, Bingsen; Hu, Edward; Mi, Shu; Yen, Yun.

於: Cancer Research, 卷 59, 編號 17, 01.09.1999, p. 4204-4207.

研究成果: 雜誌貢獻文章

Goan, Yih Gang ; Zhou, Bingsen ; Hu, Edward ; Mi, Shu ; Yen, Yun. / Overexpression of ribonucleotide reductase as a mechanism of resistance to 2,2-difluorodeoxycytidine in the human KB cancer cell line. 於: Cancer Research. 1999 ; 卷 59, 編號 17. 頁 4204-4207.
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abstract = "In this study, human oropharyngeal epidermoid carcinoma KB cells that were resistant to 2,2-difluorodeoxycytidine (dFdCyd) were selected and designated the KB-Gem clone. The KB parental cell line IC50 was 0.3 μM dFdCyd, as compared with the KB-Gem clone IC50 of 32 μM dFdCyd. The KB-Gem clone demonstrated overexpression of ribonucleotide reductase (RR) M2 subunit mRNA (9-fold) and overexpression of M2 protein (2-fold); RR activity was 2.3- fold higher than the KB parental cell line. Both the dATP and dCTP pools of the KB-Gem clone increased 2-fold over the parental cell line, with no change in the dGTP and dTTP pools. Reverse transcriptase-PCR was used to clone the cDNA of deoxycytidine kinase (DCK). Resulting sequences revealed two silent mutations in the KB-Gem clone. The amino acid sequence of the DCK protein and mRNA expression remained unchanged. The KB-Gem clone's DCK enzyme activity was 56{\%} of that of the parental cell line. After the endogenous dNTPs were removed with a G-25 column, no difference was evident between the enzyme activities of the KB-Gem clone and parental cells. Thus, contrary to previous hypotheses, DCK deficiency does not play the primary role in the resistance mechanism of dFdCyd, accepting a secondary role to the overexpression of the target gene, RR, and pool expansion.",
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