On the mechanism of 9-ß-D-arabinofuroanosylguanine resistance in human leukemic cells
(English)Manuscript (preprint) (Other academic)
The present study was undertaken to elucidate the mechanism of resistance to a relatively new nucleoside, 9-ß-D-arabinofuranosylguanine (Ara-G), which is highly toxic to T-cell malignancies. In order to do this we generated two stable resistant cell lines, MOLT-4/AraG500 (100-fold resistant) and MOLT-4/Ara-G900 (180-fold resistant). The presumed limiting step in the activation of Ara-G is catalyzed by both deoxycytidine kinase (dCK) and the mitochondrial deoxyguanosine kinase (dGK). Cross-resistance was noted to analogues such as cytosine arabinoside (Ara-C), cladribine, and fludarabine, but not to difluorodeoxycytidine. HPLC measurements of intracellular triphosphates of Ara-C and Ara-G showed that resistant cells generated significantly lower levels of metabolites. The activity of dGK (MOLT-4/AraG500, 79%, and MOLT-4/Ara-G900, 83%) and dCK (MOLT-4/Ara-G500, 54%, and MOLT- 4/Ara-G900, 73%), as well as protein and mRNA levels were significantly reduced in the resistant cell lines compared to the wild type. The resistant cells also showed decreased activity as well as decreased mRNA expression of the cytosolic 5'-nucleotidase (5'-NT), compared to the wild type. The reduced levels of enzyme activity in the resistant cells may be a consequence of reduced numbers of chromosomes carrying the genes for dGK, dCK and 5 'NT. Unexpectedly, the resistant cells showed higher activity of the mitochondrial enzyme thymidine kinase 2, probably resulting from down-regulation of dGK. No mutations were found in the dCK gene ofMOLT-4/Ara-G500 but in 2 of 13 clones ofMOLT-4/Ara-G900, we found two point mutations, one T to C and other A to G, which gave rise to changes in the amino acid sequence and affected the catalytic activity. The intracellular dNTP levels were determined and a nearly unchanged dCTP pool was observed in the resistant cells, while the other dNTP pools were significantly reduced compared to those in the wild type. These results show that altered activity of the deoxyribonucleoside kinases confer resistance to Ara-G in these cell lines.
Leukemia, 9-ß-D-arabinofuroanosylguanine, deoxycytidine kinase, deoxyguanosine kinase, resistance and cytotoxicity
Medical and Health Sciences
IdentifiersURN: urn:nbn:se:liu:diva-80283OAI: oai:DiVA.org:liu-80283DiVA: diva2:546403