Effect of eflornithine on mutation frequency in temozolomide-treated U87MG cells

Abstract

Treatment of infiltrative glioma presents a number of unique challenges due to poor penetration of typical chemotherapeutic agents into the infiltrating edge of tumors. The current chemotherapy options include nitrosoureas (e.g., lomustine) and the imidazotetrazine-class monofunctional DNA alkylating agent, temozolomide (TMZ). Both classes of drugs alkylate DNA and have relatively unrestricted passage from blood into brain where infiltrative tumor cells reside. Recent research indicates that secondary mutations detected in the RB and AKT-mTOR signaling pathways are linked to characteristics of recurrent tumors specific to TMZ-treated patients. It has been hypothesized that a decrease in rate of secondary mutations may result in delay of tumor recurrence. To that end, this study was designed to test viability of decreasing secondary mutations by disrupting the cell division cycle using eflornithine, a specific inhibitor of ornithine decarboxylase. U87MG glioblastoma cell line characterized by chromosomal abnormalities commonly attributed to primary cancers was used as a model for this study. The cells were subjected to TMZ treatment for 3 days followed by eflornithine (DFMO) treatment for 4 or 11 days. It was shown that TMZ significantly increased the frequency of mutations in U87MG glioblastoma cells while DFMO-treated cells showed mutation frequency statistically similar to that of the untreated cells on the respective treatment days. The findings of this study provide evidence to support the hypothesis that DFMO may inhibit progression of DNA mutations caused by alkylating chemotherapy agents, such as TMZ.

Keywords: chemotherapy; eflornithine; glioma; secondary mutations; temozolomide.

Figure 1. Mutation frequency in untreated U87MG glioma cells over the course of experiment: Exon-Seq analysis of the complete data set.

Figure 2. Mutation frequency in subset of nucleotides of U87MG glioma cells susceptible to TMZ-induced mutations: not-treated cells vs. cells treated with TMZ 40, 80 and 200 μM concentrations on day 3 of the treatment.

Figure 3. Trends in mutation frequency in nucleotides susceptible to TMZ -induced mutations.

Mean mutation frequency increase observed after 3 days of treatment with TMZ at three different concentrations (40, 80 and 200 μM) is in the range of 34–37%. Subsequent treatment with 50, 100 and 200 μM DFMO yields mutation frequency statistically equivalent to that of untreated cells.

Figure 4. Means/ANOVA analysis of mutation frequency in U87MG glioma cell nucleotides susceptible to TMZ-induced mutations.

Statistically significant increase of mutation frequency demonstrated for TMZ-treated vs. untreated cells on Day 3. Subsequent treatment with DFMO at each level of TMZ showed significantly lower level of mutation frequency on days 7 and 14 (day 7 and 14 data shown in aggregate)¹. ¹Note: the top and bottom of each diamond represent the confidence interval for each group. The mean line across the middle of each diamond represents the group mean. The multiple comparison test is illustrated by a comparison circles plot on the right of the graph and the degree of intersection of the circles shows whether the group means are significantly different. Non-intersecting circles show statistically significant differences.

Figure 5. Examples of TMZ-induced mutation frequency increase and subsequent mutation-inhibiting action of DFMO for genes involved in carcinogenesis.

(A) Changes in C/T mutation frequency in TP53BP1 gene. (B) Changes in G/A mutation frequency in ADAM32 gene. (C) Changes in C/T mutation in GPR116 gene. (D) Changes in T/A mutation in MUC16 gene.