长期传代后HL-60细胞内脱氧胞苷激酶与阿糖胞苷耐药性的关系

The association of deoxycytidine kinase and Ara-C resistance in long-term subcultured HL-60 cells

目的通过观察人早幼粒白血病细胞系(HL-60)及其耐药细胞株(HL-60/R)经过长期传代后细胞内脱氧胞苷激酶(deoxycytidine kinase,dck)mRNA表达及其酶活性的变化规律,探讨dck与阿糖胞苷(Ara-C)疗效及耐药现象发生的相关性。方法分别采用实时PCR方法和核素液闪法测定HL-60和HL-60/R细胞内dck的mRNA表达水平及其酶活性水平。通过体外培养传代,观察2种细胞株经长期传代(5、10、20、30、40、50代)后细胞内dck的mRNA表达及酶活性的变化规律。结果传代初期结果显示,HL-60和HL-60/R两组细胞株之间的mRNA表达水平分别为0.37±0.03和0.17±0.03,活性值分别为0.069±0.006nmol.min-1.mg-1 protein和0.019±0.007nmol.min-1.mg-1 protein,HL-60的dck mRNA表达水平和酶活性均明显高于HL-60/R(P<0.01)。并且两组细胞株之间的显著性差异(P<0.01)持续存在于各传代过程中。HL-60和HL-60/R两组各传代节点(10、20、30、40和50代)的dck mRNA表达水平与酶活性与本组第5代细胞相比,则无明显变化(P>0.05)。结论 HL-60及HL-60/R两组细胞株间的dck mRNA表达和酶活性在传代初期存在显著差异,且两组间的差异持续存在。但与各自的初期传代相比,长期传代后的dck mRNA表达和酶活性变化并不明显,提示dck仅与Ara-C的初期疗效明显相关,而与Ara-C耐药现象的发生可能无关。

Objective To investigate the association of intracellular deoxycytidine kinase （dck） with the efficacy and resistance of cytosine arabinoside （Ara-C） by evaluating the mRNA levels of dck and its enzyme activities in the long-term subcultured human leukemia HL-60 cell line and its resistant variant （HL-60/R）, respectively. Methods The mRNA levels of dck were evaluated by real-time PCR and its enzyme activities by nuclide scintillation method in the long-term subcultured generations （5th, 10th, 20th, 30th, 40th, 50th ） of both HL-60 and HL-60/R cell lines, respectively. Results Significant differences were found between the HL-60 and HL-60/R cell lines in the initial passage （5＇h）, in terms of the mRNA levels of dck （0.37＋0.03 vs. 0.17±0. 03,P〈0. 01） and its enzymeactivities （0. 069±0. 006 nmol·min^-1·mg^-1 protein vs. 0. 019±0. 007 nmol· min^-1·mg^-1 protein, P%0.001）. Similar results were observed between both cell lines in each different passages （10th ,20th, 30th ,40th ,50th） （all P〈0.01）. However,there was no difference in the mRNA expression levels or the activities of dck between the initial passage （5th） and its long-term subcultured generations （10th ,20th, 30th,40th, 50th） in the HL-60 and HL-60/R cell lines （all P〉 0.05）. Conclusions The significant differences in the mRNA levels and the enzyme activities of dck between HL-60 and HL-60/R cell lines in the initial cultures and their long-term passage cultures indicated that dck might be highly related to the Ara-C early sensitivity. However,no significant differences were detected between the 5th passage and their long-term passage cultures in either HL-60 or HL-60/R cell line,indicating there may have no relationship between the dck and occurrence of Ara-C resistance.