Human thymocyte suspensions were incubated overnight in the presence of dCF to inhibit ADA enzyme activity, and dAdo to mimic the accumulation of substrate under conditions where dAdo is not metabolized by ADA. The AK inhibitor, 5′A5′dAdo, was used to inhibit AK activity [9
], whereas 50μM dCyd was used to inhibit the activity of dCK [10
]. The graphs in show the accumulation of intracellular dATP under ADA-inhibited conditions in both total thymocytes (comprised mainly of double positive thymocytes expressing surface CD4 and CD8; left), and immature CD34+
thymocytes (right). dATP levels were elevated in total thymocytes under ADA-deficient conditions on average at least 20-fold relative to the medium control. The addition of an AK inhibitor reduced the level of accumulated dATP only slightly in total thymocyte suspensions. However, the addition of dCyd substantially reduced the accumulated dATP level in these cells (by approximately 75%). CD34+
thymocytes showed a somewhat unique profile of dATP accumulation under ADA-inhibited conditions. First, they accumulated on average approximately 10-fold higher levels of dATP on a per cell basis than total thymocytes. The reasons for the exaggerated dATP accumulation in immature thymocytes relative to total thymocytes are currently unknown, although it is not due to a higher dAdo kinase activity, at least as measured in cell extracts (data not shown). Second, they displayed a more effective response to the inhibition of AK alone than did total thymocytes, with a 25-30% reduction in dATP. Finally, although the response to dCK inhibition was similar to that in total thymocytes, the additive effect of both inhibitors appeared to be more complete in CD34+
thymocytes than in total thymocytes, normalizing the dATP to the level of the medium control (compared to a remaining nearly 2-fold elevation in total thymocyte suspensions).
Figure 1 Assessment of dATP accumulation and induction of apoptosis in total and CD34+ human thymocytes. A. Total or CD34+ thymocyte suspensions were incubated as described in Methods with the indicated reagents and harvested for dATP analysis by HPLC. Results (more ...)
The amount of induced apoptosis in thymocyte suspensions treated with dCF and dAdo was analyzed by assessment of Annexin-V+ cells compared to controls. The graphs in show the percentage of total thymocytes (left) and CD34+ thymocytes (right) induced to undergo apoptosis under ADA-deficient conditions in the presence and absence of deoxynucleoside kinase inhibitors. In general, in vitro ADA-deficient conditions induced less apoptosis in total thymocyte suspensions than in CD34+ thymocytes, perhaps due to the fact that total thymocytes already have high levels of spontaneous apoptosis. The addition of the AK inhibitor gave no correction to induced apoptosis in either thymocyte preparation, but the addition of the dCK inhibitor reduced the induction of apoptosis substantially in both types of thymocytes (by about 50% in both cases). Interestingly, the addition of both inhibitors had a much more substantial effect on reduction of apoptosis in CD34+ thymocytes, than in total thymocytes. The presence of both inhibitors in immature thymocytes essentially completely abolished the induction of apoptosis under in vitro ADA-deficient conditions, compared with no effect over dCyd alone in total thymocytes. In summary, multiple enzymatic activities appear to be involved in the accumulation of dATP and induction of apoptosis in human thymocytes in vitro. Chimeric human/mouse FTOC studies should help reveal the impact of ADA deficiency on developing human thymocytes, and the contribution of each of the kinase activities assessed here to the toxicity induced by lack of ADA activity.