2′,3′-Dideoxycytidine metabolism in a new drug-resistant cell line

Abstract

2',3'-Dideoxycytidine (ddC) is a nucleoside analogue that inhibits human immunodeficiency virus type 1 (HIV-1) replication in vitro and is currently used in the therapy of acquired immune deficiency syndrome (AIDS). This compound exerts a delayed cytotoxicity due to inhibition of mitochondrial DNA (mDNA) synthesis. Long-term exposure of U937 human monoblastoid cells to ddC resulted in a time- and concentration-dependent decrease in mDNA content and Rhodamine 123 fluorescence. However, after 2 months on 0.1 microM ddC, a drug-resistant cell line (U937-R) with 66% of the normal amount of mDNA was isolated. ddC transport in U937 and U937-R cell lines was similar. In contrast, U937-R accumulated ddC phosphorylated derivatives at a much lower rate and to a reduced concentration into acid-soluble material. The rate of 2',3'-dideoxycytidine 5'-triphosphate (ddCTP) formation in U937-R cells was almost one-third of that measured in normal cells, although the rate of ddCTP catabolism was similar in both cell lines. Dideoxyliponucleotide (ddCDP-choline and ddCDP-ethanolamine) formation was also much slower (between one-half and one-third as fast) in U937-R than in control cells, although catabolism occurred at similar rates. ddC was phosphorylated by a cytoplasmic deoxycytidine kinase in both cell lines. This enzyme showed Km values for ddC of 80 +/- 7 and 140 +/- 9 microM in U937 and U937-R cells respectively. Furthermore, Vmax was 12 +/- 1.1 and 7.8 +/- 0.5 pmol/min per mg of protein in U937 and U937-R. Thus resistance to ddC toxicity may be due to cells' decreased ability to accumulate intracellular ddC anabolites, which may depend on cytoplasmic deoxycytidine kinase.

The Inhibitory Effect of ddC on Human Immunodeficiency Virus Replication Diminishes in Cells that are Chronically Exposed to the Drug

“…One possible mechanism of drug resistance, which has already been documented in cells exposed to antitumour nucleoside analogues, concerns the inactivation of the enzyme responsible for drug phosphorylation, namely deoxycytidine kinase (Ruiz van Harperen & Peters, 1994). This was not the case in the drug-resistant cells described in this paper, although deoxycytidine kinase had a reduced affinity and reduced Vmax for ddC (Magnani et al, 1995a). ddC-sensitive and ddC-resistant cells were indistinguishable by flow cytometry in term of surface CD4 receptors.…”

“…Isolation of the ddC-resistant cell line (U937-R) was obtained after exposure of U937 cells to 0.1 flM ddC (Fluka, Switzerland) for at least 1 month, as reported previously (Magnani et al, 1995a). HIV-1 (lIIB strain) (Popovic et al, 1984) was obtained by coculturing CEM-SS cells pretreated with Polybrene (8 ug mL-l; Sigma) for 12 h with chronically infected H9 cells (3:1 ratio).…”

“…This phenomenon is less known and has been poorly investigated. Recently we reported that U937 human monoblastoid cells, when exposed to therapeutically relevant concentrations of ddC for long periods of time, became resistant to drug mitochondrial toxicity (Magnani et al, 1995a). We suggested that this phenomenon was associated with a reduced ability to accumulate 2 1,3 1 -dideoxycytidine 5 1-triphosphate (ddCTP, the active form of ddC) (Magnani et al, 1995a,b).…”

Metabolism, mitochondrial uptake and toxicity of 2′,3′-dideoxycytidine

“…Furthermore, cells exposed to ddCyd undergo a time-dependent depletion of mtDNA [18]. The biochemical basis of this event is poorly understood.…”

“…In human cells, ddCyd is rapidly converted into ddCTP and dideoxyliponucleotides (mainly ddCDP-choline) [17][18][19]. Furthermore, cells exposed to ddCyd undergo a time-dependent depletion of mtDNA [18].…”