Silencing of Testin expression is a frequent event in spontaneous lymphomas from Trp53-mutant mice

Abstract

The tumour suppressor gene, TES, is frequently methylated in many human tumours. Previously, we demonstrated that TES promoter methylation and transcriptional silencing was the most common molecular abnormality detected in childhood acute lymphoblastic leukaemia (ALL). Trp53-mutant mouse models predominantly develop B- and T-cell lymphomas, which are widely considered equivalent to childhood T and B ALL. In this study, we examined expression of Tes transcript and Testin protein in spontaneous tumours obtained from three Trp53-mutant mouse models. Using immunohistochemistry, we report that 47% of lymphomas lacked Testin protein compared to only 7% of non-lymphoid tumours. Further examination of the lymphomas from Trp53-null and Trp53-mΔpro homozygous mutant mice revealed that 63% and 69% respectively of the isolated lymphomas were Testin negative, which is similar to reported rates in childhood T-ALL. Surprisingly, lymphomas from Trp53-Δ122 mice were frequently Testin positive (> 60%), suggesting that the presence of the Trp53-Δ122 protein appeared to mitigate the requirement for Tes silencing in lymphomagenesis. Quantitative RT-PCR results confirmed that this lack of Testin protein was due to Tes transcriptional silencing, although bisulfite sequencing demonstrated that this was not due to promoter methylation. These results are consistent with the Testin protein having lymphoid tumour suppressor activity in both mice and humans.

Figure 1

Testin immunohistochemistry of lymphomas. (A) Examples of immunohistochemistry results showing Testin-positive (right) and Testin-negative (left) lymphoma cells in tumours in the spleen (upper) and thymus (lower). (B) Pie-charts showing numbers of mice with lymphomas with respect to Testin status. (i) All lymphomas. (ii) B- or T-cell lymphomas. (iii) Trp53-mutant genotype of lymphomas. (Testin status: blue box negative; orange box positive; grey box mixed).

Figure 2

Tes expression and promoter methylation in Trp53-mutant lymphomas. (A) Table showing the genotype for the Trp53-mutant lymphomas tested. (B) Quantitative RT-PCR results showing Tes mRNA expression levels calculated relative to Rps29 and β2m expression and normalised to spleen expression level for lymphomas and normal mouse tissues. Relative Tes expression levels are shown. (C) Methylation lollipop plots for normal tissues and lymphomas showing only the unique methylation patterns obtained. The observed frequency for each unique clone pattern is shown.