2-Hydroxyglutarate Production, but Not Dominant Negative Function, Is Conferred by Glioma-Derived NADP+-Dependent Isocitrate Dehydrogenase Mutations
Figure 2
Overexpression of IDH1 R132 and IDH2 R172 mutants does not reduce cellular NADP+-IDH activity.
Total NADP+-IDH reaction rate, which consists of the combined rates of IDH1 and IDH2, was measured in lysates by measuring the rate of conversion of NADP+ to NADPH in the presence of isocitrate. (a) Lysates of stable, clonal HOG cell lines containing an shRNA sequence targeted against IDH1 have lowered NADP+-IDH activity compared to a sister cell line expressing scrambled RNA from the same vector (V). (b) HOG cells were transfected with a vector control, IDH1-WT, an IDH1 R132 mutant, IDH2-WT, or an IDH2 R172 mutant, lysates were collected after 48 h, and total cellular NADP+-IDH reaction rate at 40 µM isocitrate was assayed. (c) The experiment in B was repeated with 293T cells. (d) 293T lysates transfected with vector control, IDH1-R132H, or IDH2-R172K from C were assayed for NADP+-IDH reaction rate at 0 to 80 µM isocitrate.