Effect of AZT on thymidine phosphorylation in cultured H9c2, U-937, and Raji cell lines

Abstract

3'-azido-3'-deoxythymidine (AZT) has been shown to be a potent inhibitor of thymidine kinase 2 in work from this laboratory. Inhibition results in decreased salvage of thymidine to TTP, which may lead to depletion of the TTP pool and result in the mitochondrial dysfunction and mt-DNA depletion observed with AZT toxicity. The effect of AZT on thymidine phosphorylation in growing cells expressing thymidine kinase 1 has not been shown. Three cell lines were used in these experiments: H9c2, derived from rat cardiomyoblasts; U-937, derived from human monocytes; and Raji, derived from human lymphoblasts. AZT inhibited growth in a concentration-dependent manner in U-937 cells, but not the other cell lines. The phosphorylation of [3H]-thymidine or [3H]-AZT was determined during log growth. All cell lines salvaged and phosphorylated thymidine to TTP, with TTP the major product. The U-937 cells had a much more active salvage pathway than the other cells. All cell lines phosphorylated AZT to the triphosphate, but the major product was AZTMP. The AZT inhibition of growth of the U-937 cells did not correlate with levels of the phosphorylated AZT. In contrast, pro-drug AZT was shown to inhibit thymidine phosphorylation in all lines with 50% inhibition concentrations (IC50) ranging from 4.4 to 21.9muM. Since the U-937 cells expressed higher activity of the salvage pathway than the other cell lines, the U-937 cells may rely more heavily on the salvage pathway for TTP synthesis, accounting for AZT inhibition of growth.

Figure 1. Time course of thymidine phosphorylation in cultured cells

Flasks of cells (U-937, Raji, and H9c2) were incubated for up to 90 minutes in the presence of growth media with a total concentration of [methyl-³H]-thymidine of 0.12 μM for U-937 and Raji cells (1550 DPM/pmol) and 1.4 μM for H9c2 cells (4000 DPM/pmol). Samples were prepared as described in Materials and Methods and analyzed by reverse phase HPLC with an in-line scintillation counter. The DPM data obtained from each experiment was converted to pmoles by dividing by the specific radioactivity of the thymidine pool for that experiment and normalized to 1 μM thymidine. Data represent the mean ± SEM of three to four independent trials. The sums of TDP and TTP from the cellular extracts are shown as pmol per 10⁶ cells versus minutes of incubation. TMP levels were negligible.

Figure 2. Time course of AZT phosphorylation in cultured cells

Flasks of cells (U-937, Raji, and H9c2) were incubated for up to 48 hours with the addition of 1 μM [methyl-³H]-AZT (2200 DPM/pmol) to the growth media. Samples were prepared as described in Materials and Methods and the DPM s obtained converted to pmoles by dividing by the specific radioactivity of the AZT pool for that experiment. Data represent the mean ± SEM of three to four trials. AZTMP (left) and the sums of AZTDP and AZTTP (right) from the cellular extracts are shown as pmol per 10⁶ cells versus hours of incubation.

Figure 3. Effect of AZT on thymidine phosphorylation in cultured cells

Flasks of cells were incubated in growth media with unlabeled AZT (0–200 μM) and a total [methyl-³H]-thymidine concentration of 0.12 μM in U-937 and Raji (1550 DPM/pmol) and 1.4 μM in H9c2 (4000 DPM/pmol). Cellular extracts were prepared after 10 min of incubation as described in Materials and Methods. Data represents the mean ± SEM of three to four trials, shown as the percent of the 0 μM AZT control versus AZT concentration. Total TNP represents the sum of TMP, TDP, and TTP. Both TTP and TMP were detected in the U-937 (left) and Raji (center) trials. However, TTP was the only phosphorylated form of thymidine seen with the H9c2 cells (right), so only the Total TNP line is shown. The 50% inhibitory concentration (IC₅₀) for AZT inhibition of total thymidine phosphorylation was calculated from the best fit line (dashed lines) (SigmaPlot 10).

Figure 4. Effect of AZT on cell growth

Flasks of U-937 (left) and Raji (right) cells were incubated in growth media containing AZT (0–200 μM). Cells were counted every 24 hours through 7 days of incubation. Data represents the mean of three trials ± SEM. The CD₅₀ for AZT on growth of U-937 cells was calculated for each of the 3 through 7 day periods (Sigma Plot 10) and averaged giving a value of 54 ± 8 μM. The lack of effect of AZT on growth of H9c2 cells is described in the text.