Resistance to cytotoxic
nucleoside analogues is a major problem in
cancer treatment. The cellular mechanisms involved in this phenomenon have been studied for several years, and some factors have been identified. However, this resistance seems to be multifactorial and more studies are needed to gain better insight into this domain. For this purpose, we developed a
gemcitabine-resistant cell line (MCF7 1K) from the human mammary
adenocarcinoma MCF7 strain by prolonged exposure to
gemcitabine in vitro. MCF7 1K cells are highly resistant to
gemcitabine (533-fold) and cross-resistance is observed with araC (47-fold),
triapine (14-fold), and
hydroxyurea (6.7-fold). Quantitative real-time reverse transcription-PCR and Western blot analysis showed an increase in the gene and
protein expression of the large subunit of
ribonucleotide reductase, R1.
Ribonucleotide reductase activity was also significantly increased in the
gemcitabine-resistant cells. Study of genomic
DNA showed 12-fold increase in R1 gene dosage in MCF7 1K cells. In contrast, the gene and
protein expression of the small subunit of
ribonucleotide reductase, R2, were not modified in this cell line. These results show that
gemcitabine resistance can be associated with genetic modifications of target genes in malignant cells, and suggest that the large subunit of human
ribonucleotide reductase is involved in the cellular response to
gemcitabine.