title: In vivo induction of resistance to gemcitabine results in increased expression of ribonucleotide reductase subunit M1 as the major determinant.
abstract: Gemcitabine is a deoxycytidine (dCyd) analogue with activity against several solid cancers. Gemcitabine is activated by dCyd kinase (dCK) and interferes, as its triphosphate dFdCTP, with tumor growth through incorporation into DNA. Alternatively, the metabolite gemcitabine diphosphate (dFdCDP) can interfere with DNA synthesis and thus tumor growth through inhibition of ribonucleotide reductase. Gemcitabine can be inactivated by the enzyme dCyd deaminase (dCDA). In most in vitro models, resistance to gemcitabine was associated with a decreased dCK activity. In all these models, resistance was established using continuous exposure to gemcitabine with increasing concentrations; however, these in vitro models have limited clinical relevance. To develop in vivo resistance to gemcitabine, we treated mice bearing a moderately sensitive tumor Colon 26-A (T/C = 0.25) with a clinically relevant schedule (120 mg/kg every 3 days). By repeated transplant of the most resistant tumor and continuation of gemcitabine treatment for >1 year, the completely resistant tumor Colon 26-G (T/C = 0.96) was created. Initial studies focused on resistance mechanisms known from in vitro studies. In Colon 26-G, dCK activity was 1.7-fold decreased; dCDA and DNA polymerase were not changed; and Colon 26-G accumulated 1.5-fold less dFdCTP, 6 hours after a gemcitabine injection, than the parental tumor. Based on in vitro studies, these relative minor changes were considered insufficient to explain the completely resistant phenotype. Therefore, an expression microarray was done with Colon 26-A versus Colon 26-G. Using independently grown nonresistant and resistant tumors, a striking increase in expression of the RRM1 subunit gene was found in Colon 26-G. The expression of RRM1 mRNA was 25-fold increased in the resistant tumor, as measured by real-time PCR, which was confirmed by Western blotting. In contrast, RRM2 mRNA was 2-fold decreased. However, ribonucleotide reductase enzyme activity was only moderately increased in Colon 26-G. In conclusion, this is the first model with in vivo induced resistance to gemcitabine. In contrast to most in vitro studies, dCK activity was not the most important determinant of gemcitabine resistance. Expression microarray identified RRM1 as the gene with the highest increase in expression in the Colon 26-G, which might clarify its complete gemcitabine-resistant phenotype. This study is the first in vivo evidence for a key role for RRM1 in acquired gemcitabine resistance.
authors.affiliation: Department of Medical Oncology, VU University Medical Center, Amsterdam, The Netherlands.
authors.forename: Andries M
authors.initials: AM
authors.lastname: Bergman
authors.forename: Paul P
authors.initials: PP
authors.lastname: Eijk
authors.forename: Veronique W T
authors.initials: VW
authors.lastname: Ruiz van Haperen
authors.forename: Kees
authors.initials: K
authors.lastname: Smid
authors.forename: Gijsbert
authors.initials: G
authors.lastname: Veerman
authors.forename: Isabelle
authors.initials: I
authors.lastname: Hubeek
authors.forename: Paul
authors.initials: P
authors.lastname: van den Ijssel
authors.forename: Bauke
authors.initials: B
authors.lastname: Ylstra
authors.forename: Godefridus J
authors.initials: GJ
authors.lastname: Peters
mesh_terms: D000818:Animals; D015153:Blotting, Western; D003110:Colonic Neoplasms / Q000188:drug therapy* / Q000201:enzymology* / Q000235:genetics; D003564:Cytidine Deaminase; D003841:Deoxycytidine / Q000031:analogs & derivatives* / Q000494:pharmacology; D003842:Deoxycytidine Kinase / Q000096:biosynthesis / Q000235:genetics; D003845:Deoxycytosine Nucleotides / Q000378:metabolism; D019008:Drug Resistance, Neoplasm; D005260:Female; D020869:Gene Expression Profiling; D051379:Mice; D008807:Mice, Inbred BALB C; D009700:Nucleoside Deaminases / Q000096:biosynthesis / Q000235:genetics; D020411:Oligonucleotide Array Sequence Analysis; D021122:Protein Subunits; D012264:Ribonucleotide Reductases / Q000096:biosynthesis* / Q000235:genetics; D000093542:Gemcitabine
chemical_list: D003845:Deoxycytosine Nucleotides; D021122:Protein Subunits; D003841:Deoxycytidine; C024107:2'-deoxycytidine 5'-triphosphate; D012264:Ribonucleotide Reductases; D003842:Deoxycytidine Kinase; D009700:Nucleoside Deaminases; D003564:Cytidine Deaminase; C011530:deoxycytidine deaminase; D000093542:Gemcitabine