Transgenerational inheritance of enhanced susceptibility to radiation-induced medulloblastoma in newborn Ptch1⁺/⁻ mice after paternal irradiation

Abstract

The hypothesis of transgenerational induction of increased cancer susceptibility after paternal radiation exposure has long been controversial because of inconsistent results and the lack of a mechanistic interpretation. Here, exploiting Ptch1 heterozygous knockout mice, susceptible to spontaneous and radiation-induced medulloblastoma, we show that exposure of paternal germ cells to 1 Gy X-rays, at the spermatogonial stage, increased by a considerable 1.4-fold the offspring susceptibility to medulloblastoma induced by neonatal irradiation. This effect gained further biological significance thanks to a number of supporting data on the immunohistochemical characterization of the target tissue and preneoplastic lesions (PNLs). These results altogether pointed to increased proliferation of cerebellar granule cell precursors and PNLs cells, which favoured the development of frank tumours. The LOH analysis of tumor DNA showed Ptch1 biallelic loss in all tumor samples, suggesting that mechanisms other than interstitial deletions, typical of radiation-induced medulloblastoma, did not account for the observed increased cancer risk. This data was supported by comet analysis showing no differences in DNA damage induction and repair in cerebellar cells as a function of paternal irradiation. Finally, we provide biological plausibility to our results offering evidence of a possible epigenetic mechanism of inheritance based on radiation-induced changes of the microRNA profile of paternal sperm.

Keywords: epigenetic inheritance; medulloblastoma; microRNA; patched1 knockout mice; transgenerational carcinogenesis.

Figure 1. Medulloblastoma induction

A. Representative histological image of medulloblastoma. Bar = 1000 μm. B. Kaplan-Meier kinetic analysis of medulloblastoma incidence obtained in unirradiated or P2-irradiated progeny of unirradiated [P-0Gy (F1–0Gy) and P-0Gy (F1–1Gy)] and irradiated fathers [P-1Gy (F1–0Gy) and P-1Gy (F1–1Gy)]. C. Representative analysis of chromosome-13 LOH in radiation-induced medulloblastomas. Solid squares indicate no LOH; open squares denote loss of signal from one allele (LOH); cM = centiMorgan.

Figure 2. Comet assay and immunohistochemistry results in cerebellum cells from P2 mice

A. Dose-response curves for comet tail intensity in cerebellum cells from irradiated offsprings of irradiated (P-1Gy) or unirradiated (P-0Gy) fathers. On the same graphic, open symbols represent Tail Intensity values in cells irradiated with 1Gy x-ray on slides. Tail Intensity values were statistically increased in all irradiated groups over matched unirradiated groups (P < 0.0001). B. DNA repair in the progeny of irradiated (P-1Gy) or unirradiated (P-0Gy) fathers. Induced damage (1 Gy T0, P < 0.0001) and residual damage 1 hour after irradiation (1 Gy T 1 h) are shown. C–D. Representative image of EGL immunostained with anti-PCNA antibody from 2-weeks old unirradiated progeny of non-irradiated (C) and irradiated fathers (D) E. Rate of GCPs proliferation index, expressed as percentage of PCNA positive cells over the total number of cells counted in each mouse cerebellum, and F. relative EGL thickness (n = 6). P < 0.0001. Bars = 10 μm.

Figure 3. Genomic instability in adult progeny of irradiated (P-1Gy) or unirradiated (P-0Gy) fathers

Background levels of comet tail intensity in bone marrow A. and spleen B. cells. The comparison of Tail Intensities did not show any effect of parental irradiation on the endogenous level of DNA damage in these organs. Background and radiation-induced frequencies of bone marrow micronucleated PCEs C. confirmed no increase of spontaneous genomic instability due to paternal irradiation and demonstrated that sensitivity to radiation-induced chromosome damage was similarly unaffected. * = P < 0.005

Figure 4. Medulloblastoma preneoplastic lesions in the irradiated progeny of unirradiated and irradiated fathers

A–C. Histologic appearance of PNLs (arrow) at 4 weeks of age, their incidence (B) and dimensional analysis (C) D–G. At 8 weeks of age, in the cerebella of irradiated-F1 progeny, PNLs appear as small nodules (D) or microtumors (E), with no difference in total incidence between groups (F) Graphic representation of PNLs distribution by size (pie charts) and average areas (histogram) in both experimental group; MT: microtumors, SN: small nodules (G) Bars = 200 μm.

Figure 5. Paternal irradiation drives PNLs towards a more proliferative condition

A–D. Representative images of PNLs (area < 5 × 10⁵ μm²) immunostained with anti-PCNA (A and C) and anti-NeuN (B and D). E. Immunohistochemical analysis showed significantly higher PCNA positive cells (n = 6; P < 0.05) along with lower NeuN expression (P < 0.05) in PNLs from irradiated fathers compared to unirradiated counterpart (n = 6). ML = Molecular layer; IGL = internal granule layer; PNL = Preneoplastic Lesion. Bars = 100 μm.

Figure 6. Microarray analysis of microRNA expression in paternal spermatozoa

Panel A. fluorescent signal recorded indicating the high call-response rate despite the minimal microRNA amount used. Panel B. PCA results; each dot indicates the overall microRNA expression profile in a single mouse either unexposed (yellow dots) or exposed (red dots). Panel C. Scatter plot analysis; each dot indicates the expression intensity of a single microRNA in control (horizontal axis) and in radiation exposed mice (vertical axis). Dots located outside the 2-fold variation interval (diagonal green lines) are altered by treatment. Colors indicate the level of expression of each microRNA (blue, low; yellow intermediate; red, high).