Pharmacogenetic variants in TPMT alter cellular responses to cisplatin in inner ear cell lines

Abstract

Cisplatin is a highly-effective and widely-used chemotherapeutic agent that causes ototoxicity in many patients. Pharmacogenomic studies of key genes controlling drug biotransformation identified variants in thiopurine methyltransferase (TPMT) as predictors of cisplatin-induced ototoxicity, although the mechanistic basis of this interaction has not been reported. Expression constructs of TPMT*3A, *3B and *3C variants were generated and monitored in cultured cells. Cellular TPMT*3A levels were detected at >20-fold lower amounts than the wild type confirming the unstable nature of this variant. The expression of wild type TPMT (TPMT*1) in two murine ear cell lines, HEI-OC1 and UB/OC-1, significantly mitigated their susceptibility to cisplatin toxicity. Cisplatin treatment induced Tlr4 gene expression in HEI-OC1 cells and this response was blunted by the expression of wild type TPMT but not TPMT*3A. In line with the significant mitigation of TPMT*1-expressing cells to cisplatin cytotoxicity, these findings demonstrate a drug-gene interaction between increased TPMT activity and decreased susceptibility to cisplatin-induced toxicity of inner ear cells.

Fig 1. Pharmacogenetic variants in TPMT show differential stability in cell culture.

Top, Representative western blot of HEK293T cells expressing indicated TPMT variants. TPMT was specifically detected using an HA-epitope tag. Bottom, quantification of West blot analysis, normalizing HA signal intensity to corresponding GAPDH signal intensity. Shown are results from two independent experiments. TPMT*3A levels are 4% of wild type TPMT levels.

Fig 2. TPMT variation influences cisplatin cytotoxicity in murine inner ear cell lines.

HEI-OC1 or UB/OC-1 cells expressing TPMT*1 or TPMT*3A (as indicated) were treated with varying concentrations of cisplatin, quantified by MTT assay and the best-fit IC₅₀ values were determined using a non-linear log(inhibitor) vs. normalized response model. Dose response curves are shown in S3 Fig and curve fitting parameters are listed in S2 Table. Data are presented as the mean and standard error of the mean. * and ** denote P < 0.05 and P < 0.01, respectively using Extra sum-of-squares F test.

Fig 3. TPMT influences response of a cisplatin biosensor in a murine inner ear cell line.

A, Tlr4 expression was quantified in HEI-OC1 cells treated with the indicated concentrations of cisplatin. Tlr4 expression increased with increasing cisplatin and served as a cisplatin biosensor. B, HEI-OC1 cells expressing TPMT*1 or TPMT*3A were treated with 25 μM cisplatin or left untreated and relative expression of the cisplatin biosensor was determined. Data are presented as the mean and standard error of the mean for nine replicates for untreated cells (3 independent experiments) and 6 replicates for cisplatin treated cells (2 independent experiments). *, ** and ns denote P < .05, P < .01 and not significant, respectively using one-way ANOVA with Tukey post-test analyses.