Deoxyguanosine-resistant leukemia L1210 cells. Loss of specific deoxyribonucleoside kinase activity

Abstract

A mouse leukemia L1210 cell line was selected for resistance to deoxyguanosine. The deoxyguanosine-resistant cells (dGuo-R) were 126-fold less sensitive to deoxyguanosine than the wild-type cells. The IC50 values for araC and araG were increased, but only 10-12-fold in the dGuo-R cells when compared with the wild-type cells. The dGuo-R cell line showed an increased level of resistance to 2-fluoro-2'-deoxyadenosine and 2-fluoroadenine arabinoside (11-14-fold), but essentially no increase in resistance to deoxyadenosine or adenine arabinoside. Deoxyribonucleoside kinase activity was decreased only slightly (19%) when deoxycytidine was utilized as substrate; when cytosine arabinoside or deoxyguanosine was used as the substrate, the kinase activity in the extracts from the dGuo-R cells was only 10% of the enzyme activity in the extracts from the wild-type cells. The determination of the kinetic parameters, Km and Vmax, indicated that there were marked decreases in the Vmax values for deoxyguanosine and cytosine arabinoside as substrates, but not for deoxycytidine as substrate; the Km values for deoxycytidine and cytosine arabinoside were increased in the extracts from the dGuo-R cells. By use of high-performance liquid chromatography, the kinase activities in the extracts from the wild-type and resistant cells could be resolved. There was the specific loss of kinase activity toward cytosine arabinoside and deoxyguanosine as substrates. These data indicate that the dGuo-R cells have decreased levels of a specific deoxyribonucleoside kinase activity.

Fig. 1. Deoxyribonucleoside kinase activities in fractions separated by HPLC

The dCyd kinase (•), araC kinase (○), and dGuo kinase (▪) activities were determined in the fractions off the HPLC column. Equal amounts of extract protein from the wild-type and dGuo-R cells were put on the column. A shows the kinase activities in the extract from the wild-type cells; B shows the kinase activities in the extract from the dGuo-R cells; C shows the UV profile of the protein eluting from the column (the extracts from the wild-type and dGuo-R cells gave the same profile). Note that the y axis for A and B is a log plot. Extracts from the wild-type cells were run over the HPLC column in four different experiments; extracts from the dGuo-R cells were run over the HPLC column in two separate experiments. For dCyd, dGuo, and araC as substrates, 10,000 dpm of product on the filter paper were equivalent to 0.012, 0.027, and 0.013 nmol/h.