In the present study, we developed a new cell line variant of the T lymphoblastic leukemia CCRF-CEM cell line, which was resistant to ara-G, an active compound of nelarabine (Figures
1 and
2, Table
1), and investigated its mechanism of drug resistance. Reduced transporter hENT1 transcript level and decreased dCK and dGK protein levels (Figure
4) resulted in decreased ara-GTP production (Figure
1) in CEM/ara-G cells. The subsequent incorporation of ara-G into nuclear and mitochondrial DNA was reduced (Figure
5), and unable to inhibit DNA synthesis in both fractions of CEM/ara-G cells (Figure
5). Importantly, the cytotoxic effect of ara-G was almost unchanged on CEM cells that were depleted of mitochondrial DNA (Figure
6, Table
2), suggesting that mitochondrial DNA damage was unlikely to contribute greatly to ara-G cytotoxicity. Thus, the reduced triphosphate production (Figure
1) and the subsequent reduction of drug incorporation into nuclear DNA (Figure
5) were closely associated with the development of ara-G resistance in CEM/ara-G cells. The anti-apoptotic nature was also related to the drug resistance in this cell line (Figure
7).
Previously, 3 independent studies investigated the mechanisms of ara-G resistance in leukemic cell lines. Shewach et al. first developed an ara-G-resistant leukemic clone from T lymphoblastic leukemia MOLT-4 cells and demonstrated decreased production of intracellular ara-GTP [
29]. However, they did not determine the mechanisms for the reduced ara-GTP production. Curbo et al. generated 2 ara-G-resistant CEM subclones that were 132-fold and 260-fold more ara-G resistant than CEM [
30]. They demonstrated a decrease in ara-G incorporation into mitochondrial DNA and loss of dCK activity. However, they showed that the drug incorporation into mitochondrial DNA was not associated with the acute cytotoxicity induced by ara-G in their later study [
31]. Their latest study further demonstrated that the depletion of mitochondria DNA does not attenuate the cytotoxicity of ara-G in MOLT-4 cells [
32]. They concluded that the loss of dCK activity is the critical factor responsible for ara-G resistance. Our study demonstrated that ara-G inhibited both nuclear and mitochondrial DNA synthesis in CEM cells (Figure
5). However, the result showing that ρ
0CEM cells were similarly sensitive to ara-G (Figure
6) suggests that the critical event should be the inhibition of nuclear DNA synthesis not mitochondrial DNA damage. Lotfi et al. developed 2 ara-G-resistant MOLT-4 variants that were 108-fold and 184-fold more ara-G resistant than MOLT-4 [
33]. They showed that dGK deficiency was the most prominent change in these cells and that a dCK defect was associated with increased ara-G resistance [
33]. They further identified increases in Bcl-xL in these ara-G-resistant clones [
34]. The alteration of the kinases and anti-apoptotic Bcl-xL indicate a possible contribution of these factors to ara-G resistance, which is consistent with our present findings. Nevertheless, apart from these reports, we clearly showed all of the successive changes in the transporter hENT1, kinases (dCK and dGK), ara-GTP production, ara-G incorporation into nuclear and mitochondrial DNA, inhibition of DNA synthesis, and induction of mitochondria-mediated apoptosis. Thus, unlike previous studies, the present study was comprehensive and systematic in investigating the mechanism of resistance to ara-G in leukemic cells.
CEM/ara-G cells demonstrated cross-resistance to F-ara-A and ara-C. However, the resistance to the purine analog F-ara-A was much greater than that to the pyrimidine analog ara-C (Table
1). Because F-ara-A and ara-C share an identical pathway for their intracellular activation, the difference in resistance might be due to a structural difference between the 2 agents, but this possibility was not investigated in detail here. Nevertheless, one strategy to overcome ara-G resistance may be a high-dose ara-C therapy that can achieve 50-fold higher plasma ara-C concentrations than regular-dose ara-C, which would surpass the level of cross-resistance to ara-C [
35,
36].