Development and biochemical characterization of a 2′- C-cyano-2′-deoxy-1- β- d-arabino-pentofuranosylcytosine (CNDAC)-resistant variant of the human fibrosarcoma cell line HT-1080

Abstract

2′- C-Cyano-2′-deoxy-1- β- d-arabino-pentofuranosylcytosine (CNDAC) is an antitumor nucleoside with a novel chemical structure that exerts potent antitumor activity against various human tumor cells in vitro and in vivo. In order to be active it needs to be phosphorylated by deoxycytidine (dCyd) kinase. We induced resistance to CNDAC in the human fibrosarcoma cell line HT-1080 by exposure to increasing concentrations of CNDAC. The resistant cells showed over 560 times higher resistance as compared to that of the parental HT-1080 cells and were cross-resistant to the other 2′-deoxycytidine derivatives. The dCyd kinase mRNA expression of the resistant cells decreased and there was the expression of aberrant mRNA of dCyd kinase which contained a 116-nucleotide deletion within the coding region, corresponding to the fifth exon of the gene. The dCyd kinase enzymatic activity of the resistant cells was deficient. The initial uptake of CNDAC into the resistant cells was similar to that of the parental cells. However, the incorporation of CNDAC into the DNA fraction of the resistant cells was significantly less than that of the parent cells. These results led us to conclude that the acquired resistance to CNDAC can be attributed to a deficiency of dCyd kinase activity, which should be based on a remarkable decrease in mRNA expression and genetic mutation of the dCyd kinase gene, but not on cellular CNDAC accumulation.