Figure 2

Cisplatin decreases glucose incorporation into DNA and RNA via the non-oxidative pentose phosphate pathway, reduces PRPP synthetase activity by reducing intracellular phosphate, and increases protein PARylation: effects of amino acid deprivation; PARP1 Knock-down Increases PRPP Availability. (A) Carbon 1 of glucose (depicted by the filled circle) is oxidized to CO₂ on glucose transit through the oxidative pentose phosphate pathway, whereas it can be incorporated into PRPP and subsequently into DNA and RNA after transit through the non-oxidative pentose phosphate pathway. (B-D,F-I,K) Dih10 cells were incubated for 6 h in complete or lysine-deficient medium in the absence or presence of 100 µM cisplatin. (B,C) [1-¹⁴C]-glucose oxidation to CO₂ (B) or incorporation into DNA and RNA (C) was measured during the last 60 min of the incubation. (D,F) Cells were extracted, and PRPP synthetase activity was measured in the extracts in the presence of 40 mM (D) or 1 mM (F) NaPO4, pH, 7.4 added to the assay buffer. (E) Dih10 cells were incubated in lysine-deficient medium, and at the indicated times, PRPP synthetase activity was measured in cell extracts in the presence of 40 mM NaPO4, pH 7.4. (G) Inorganic phosphate in cell extracts was measured using a colorimetric assay. (H–J) Cells were extracted, and proteins were analyzed by SDS-PAGE/immunoblotting. The density of the indicated protein bands was quantified using a Li-COR Odyssey Scanner, and normalized to β-actin. The experiments were repeated three times, with the numbers below the blots showing the mean density ± SD for each of the four conditions (H,I) or for the indicated lanes (J). Full blots are shown. (H) An antibody that detects poly(ADP-ribose) polymers was used. The sum of the density of the two bands corresponding to molecular weights of approximately 38 and 33 kDA was quantified; β-actin was imaged on a separate immunoblot. (I) A PARP1 antibody was used. (J) Clonally-derived Dih10 cells that had been infected with either a control shRNA vector or a PARP1 shRNA vector were extracted, and a PARP1 antibody was used. Each lane corresponds to an independently-derived clone. (K) PRPP availability was measured in the three independently-derived clones shown in Panel J, as described in the Fig. 1 legend. Clones infected with control virus are shown in black-outlined boxes, and clones infected with the PARP1 shRNA viral vector are shown in grey-outlined boxes. In Panels B–D, and F and G, each symbol in the bar graphs is the mean of duplicate samples from an independent experiment, with the bar height representing the mean value of all experiments and the error bars indicating the standard deviation. In Panel E, the symbols are the mean of three independent experiments performed in duplicate, and the error bars are the standard deviation. In Panel K, the boxes show the full range of the three independently-derived clones, each measured in duplicate, and the horizontal line in the box is the mean value. *, **, ***, and **** indicate p < 0.05, < 0.01, < 0.001, and < 0.0001, respectively, for the indicated comparisons, or in Panel H to the control condition. CDDP, cisplatin; PARP1, poly(ADP-ribose) polymerase-1; PPP, pentose phosphate pathway; PRPP, phosphoribosyl-pyrophosphate.