Interstitial chromosomal deletion of the tuberous sclerosis complex 2 locus is a signature for radiation-associated renal tumors in Eker rats

Abstract

Ionizing radiation can damage DNA and, therefore, is a risk factor for cancer. Eker rats, which carry a heterozygous germline mutation in the tumor-suppressor gene tuberous sclerosis complex 2 (Tsc2), are susceptible to radiation-induced renal carcinogenesis. However, the molecular mechanisms involved in Tsc2 inactivation are unclear. We subjected Fischer 344 × Eker (Long Evans Tsc2^+/- ) F1 hybrid rats to gamma-irradiation (2 Gy) at gestational day 19 (GD19) or postnatal day 5 (PND5) and investigated the patterns of genomic alterations in the Tsc2 allele of renal tumors that developed at 1 year after irradiation (N = 24 tumors for GD19, N = 10 for PND5), in comparison with spontaneously developed tumors (N = 8 tumors). Gamma-irradiation significantly increased the multiplicity of renal tumors. The frequency of LOH at the chromosome 10q12 region, including the Tsc2 locus, was 38%, 29% and 60% in renal carcinomas developed from the nonirradiated, GD19 and PND5 groups, respectively. Array comparative genomic hybridization analysis revealed that the LOH patterns on chromosome 10 in renal carcinomas were classified into chromosomal missegregation, mitotic recombination and chromosomal deletion types. LOH of the interstitial chromosomal deletion type was observed only in radiation-associated carcinomas. Sequence analysis for the wild-type Tsc2 allele in the LOH-negative carcinomas identified deletions (nonirradiated: 26%; GD19: 21%) and base-substitution mutations (GD19: 4%). Reduced expression of Tsc2 was also observed in the majority of the LOH-negative carcinomas. Our results suggest that interstitial chromosomal deletion is a characteristic mutagenic event caused by ionizing radiation, and it may contribute to the assessment of radiation-induced cancer risk.

Keywords: Tsc2; Eker rat; genomic signature; ionizing radiation; renal carcinoma.

Figure 1

Representative histopathological identification of lesions in the kidneys of gamma‐irradiated Tsc2 ^+/− F1 rats. H&E‐stained sections revealed phenotypically altered tubules (A), atypical hyperplasia (B), adenoma (C) and adenocarcinoma (D). A phenotypically altered tubule and atypical hyperplasia are indicated by arrows. Scale bars, in (A) and (B), 50 μm; (C) and (D), 100 μm

Figure 2

LOH patterns reveal different mutational events in spontaneous and radiation‐associated renal tumors from Tsc2 ^+/− F1 rats. A, Polymorphic markers and their positions are shown in the chromosome schematic. Closed circles (●) represent LOH. N, number of cases that have the same LOH pattern. B, Changes in DNA copy number revealed by array comparative genomic hybridization (CGH) analysis. Deleted regions detected by array CGH analysis are shown in green

Figure 3

Distribution of Tsc2 mutations in spontaneous and radiation‐associated rat renal tumors. A schematic depicts the structure of rat Tsc2, including 42 numbered exons and the locations of the functional domains (colored). Hatched boxes indicate the 5′ and 3′ UTR. Arrows indicate the positions of the identified mutations

Figure 4

Quantitative RT‐PCR assessment of Tsc2 expression in normal rat kidney tissues, spontaneous renal tumors and radiation‐associated renal tumors. The data for normal kidney tissues (the mean ± SD of 6 samples) and for renal tumors (the average of two independent experiments, ■ and ▲) are shown. The data for tumors were normalized to the average expression value for normal kidney tissues