Role of epigenetic effectors in maintenance of the long-term persistent bystander effect in spleen in vivo

Abstract

Radiation therapy is a primary treatment modality for brain tumors, yet it has been linked to the increased incidence of secondary, post-radiation therapy cancers. These cancers are thought to be linked to indirect radiation-induced bystander effect. Bystander effect occurs when irradiated cells communicate damage to nearby, non-irradiated 'bystander' cells, ultimately contributing to genome destabilization in the non-exposed cells. Recent evidence suggests that bystander effect may be epigenetic in nature; however, characterization of epigenetic mechanisms involved in bystander effect generation and its long-term persistence has yet to be defined. To investigate the possibility that localized X-ray irradiation induces persistent bystander effects in distant tissue, we monitored the induction of epigenetic changes (i.e. alterations in DNA methylation, histone methylation and microRNA (miRNA) expression) in the rat spleen tissue 24 h and 7 months after localized cranial exposure to 20 Gy of X-rays. We found that localized cranial radiation exposure led to the induction of bystander effect in lead-shielded, distant spleen tissue. Specifically, this exposure caused the profound epigenetic dysregulation in the bystander spleen tissue that manifested as a significant loss of global DNA methylation, alterations in methylation of long interspersed nucleotide element-1 (LINE-1) retrotransposable elements and down-regulation of DNA methyltransferases and methyl-binding protein methyl CpG binding protein 2 (MeCP2). Further, irradiation significantly altered expression of miR-194, a miRNA putatively targeting both DNA methyltransferase-3a and MeCP2. This study is the first to report conclusive evidence of the long-term persistence of bystander effects in radiation carcinogenesis target organ (spleen) upon localized distant exposure using the doses comparable with those used for clinical brain tumor treatments.

Paternal cranial irradiation induces distant bystander DNA damage in the germline and leads to epigenetic alterations in the offspring

“…The same shielding was used in the published studies on the bystander effect in mouse skin and in mouse and rat spleen. [14][15][16] We also considered the fact that even though the shielding of the animal body is complete, the X-rays can be reflected as they pass through the tissue, thus forming a small 'scatter' dose in the protected tissue. 16 To check if a possible small scatter dose could contribute to the generation of bystander effects in testes, we exposed a separate cohort of animals to a scatter dose of ~0.0012 Gy calculated according to the Monte Carlo simulation and directly measured by RAD-CHECK TM .…”

“…16 To check if a possible small scatter dose could contribute to the generation of bystander effects in testes, we exposed a separate cohort of animals to a scatter dose of ~0.0012 Gy calculated according to the Monte Carlo simulation and directly measured by RAD-CHECK TM . [14][15][16] Sham treated animals served as a control group.…”

“…It has been shown previously that the accumulation of DNA damage is accompanied by the loss of DNA methylation, [24][25][26] and the bystander effects X-irradiation may be epigenetically mediated. [11][12][13][14][15]27 DNA methylation is an important epigenetic mechanism that safeguards genome stability of the cells, regulates gene expression and chromatin structure. [28][29][30] The altered DNA methylation has deleterious consequences for the sperm cells.…”

“…6,13 Recent studies have shown that bystander effects also operate in vivo, and that body part or cranial irradiation may exert deleterious changes in the distant protected somatic tissues. [14][15][16] However, the potential of distant bystander damage to the germline has never been studied.…”

DNA damage-induced upregulation of miR-709 in the germline downregulates BORIS to counteract aberrant DNA hypomethylation

“…The miRNA microarray analysis was performed by LC Sciences (Houston, TX) as described before. 6,[22][23][24] microRNA fluorescence in situ hybridization (FISH) using locked nucleic acid (LNA) probes and tyramide signal amplification. Tissue fixation of miRNA FISH was conducted as recommended.…”