Untersuchung des Einflusses ionisierender Strahlung auf die Expression der Histonvariante H2A.J in Keratinozyten der präputialen Epidermis

Abstract Hintergrund: Zelluläre Seneszenz beschreibt den terminalen Proliferationstopp von weiterhin metabolisch aktiven Zellen, der durch eine Vielzahl von Stimuli ausgelöst werden kann. Dabei spielt Seneszenz eine ambivalente Rolle, da sie einerseits an der embryonalen Entwicklung, der Wundheilung sowie der Tumorsuppression beteiligt ist und andererseits relevante Prozesse im Rahmen des physiologischen Alterungsprozesses begleitet. Ein möglicher Auslöser prämaturer Seneszenz stellt ionisierende Strahlung dar, deren DNA-schädigende Wirkung im Rahmen der Radiotherapie zur Tumorkontrolle eingesetzt wird. In vorangegangenen Publikationen wurde bereits untersucht, ob durch die Exposition von muriner und humaner Epidermis mit ionisierender Strahlung eine veränderte Expression der Histonvariante H2A.J nachweisbar ist und ob diese mit altersabhängiger Seneszenz assoziiert werden kann. Darüber hinaus zeigten ex-vivo Untersuchungen eine Korrelation zwischen H2A.J-Expression und strahleninduzierter Seneszenz in der humanen, abdominellen Epidermis von erwachsenen Probanden. In dieser Arbeit wurde basierend auf den bereits publizierten Ergebnissen untersucht, ob ein Zusammenhang zwischen der veränderten Expression der Histonvariante H2A.J und zellulärer Seneszenz nach der Exposition mit ionisierender Strahlung auch in der infantilen präputialen Epidermis nachweisbar ist. Material und Methoden: Im Rahmen elektiver Zirkumzisionen wurden 19 Hautproben männlicher Patienten ohne relevante Grunderkrankungen im Alter von 1 - 10 Jahren akquiriert. Die Epidermis wurde mit einer definierten Dosis bestrahlt, anschließend erfolgte die Markierung der Histonvariante H2A.J und proliferations-assoziiertem Ki67 mittels fluoreszenz-gekoppelten Antikörpern und die Visualisierung der H2A.J- und/oder Ki67-positiver Zellen mit Hilfe eines Fluoreszenzmikroskopes. Ergebnisse: Die quantitative Analyse der H2A.J- und Ki67-positiven Zellen ergab keinen signifikanten Unterschied im Vergleich zur Kontrollgruppe. Es konnte jedoch ein bestrahlungsunabhängiger Anstieg an H2A.J-positiven Zellen, ausgehend vom Stratum germinativum hin zum Stratum granulosum, nachgewiesen werden. Schlussfolgerung: Es konnten kein Zusammenhang zwischen der applizierten Dosis und einer veränderten H2A.J-Expression in seneszenten Zellen der präputialen Epidermis von pädiatrischen Probanden festgestellt werden. Die bestrahlungsunabhängige schichtspezifische Verteilung H2A.J-positiver Zellen könnte ein Hinweis auf einen möglichen Zusammenhang zwischen H2A.J und der Differenzierung von Keratinozyten sein.Summary Background: Cellular senescence describes the terminal proliferation arrest of still metabolically active cells, which can be triggered by a variety of stimuli. Senescence plays an ambivalent role, as it is involved in embryonic development, wound healing and tumor suppression on the one hand and accompanies relevant processes in the physiological aging process on the other hand. A possible trigger of premature senescence is ionizing radiation, whose DNA-damaging effect is used in radiotherapy for tumor control. Previous publications have investigated whether exposure of murine and human epidermis to ionizing radiation results in altered expression of the histone variant H2A.J and whether this can be associated with age-related senescence. Furthermore, ex-vivo studies showed a correlation between H2A.J expression and radiation-induced senescence in the human abdominal epidermis of adult volunteers. In this work, based on the previously published results, we investigated whether a correlation between altered expression of the histone variant H2A.J and cellular senescence after exposure to ionizing radiation is also detectable in infantile preputial epidermis. Material and Methods: Nineteen skin samples from male patients without relevant underlying diseases aged 1 - 10 years were acquired during elective circumcisions. The epidermis was irradiated with a defined dose, followed by labeling of histone variant H2A.J and proliferation-associated Ki67 using fluorescence-coupled antibodies and visualization of H2A.J- and/or Ki67-positive cells using a fluorescence microscope. Results: Quantitative analysis of H2A.J- and Ki67-positive cells revealed no significant difference compared with the control group. However, an irradiation-independent increase of H2A.J-positive cells, starting from the stratum germativum to the stratum granulosum, could be detected. Conclusion: No correlation between the applied dose and altered H2A.J expression in senescent cells of the preputial epidermis of pediatric subjects could be detected. The irradiation-independent layer-specific distribution of H2A.J-positive cells may indicate a possible link between H2A.J and keratinocyte differentiation

Cultured Human Foreskin as a Model System for Evaluating Ionizing Radiation-Induced Skin Injury

Purpose: Precise molecular and cellular mechanisms of radiation-induced dermatitis are incompletely understood. Histone variant H2A.J is associated with cellular senescence and modulates senescence-associated secretory phenotype (SASP) after DNA-damaging insults, such as ionizing radiation (IR). Using ex vivo irradiated cultured foreskin, H2A.J was analyzed as a biomarker of radiation-induced senescence, potentially initiating the inflammatory cascade of radiation-induced skin injury. Methods: Human foreskin explants were collected from young donors, irradiated ex vivo with 10 Gy, and cultured in air-liquid interphase for up to 72 h. At different time-points after ex vivo IR exposure, the foreskin epidermis was analyzed for proliferation and senescence markers by immunofluorescence and immunohistochemical staining of sectioned tissue. Secretion of cytokines was measured in supernatants by ELISA. Using our mouse model with fractionated in vivo irradiation, H2A.J expression was analyzed in epidermal stem/progenitor cell populations localized in different regions of murine hair follicles (HF). Results: Non-vascularized foreskin explants preserved their tissue homeostasis up to 72 h (even after IR exposure), but already nonirradiated foreskin epithelium expressed high levels of H2A.J in all epidermal layers and secreted high amounts of cytokines. Unexpectedly, no further increase in H2A.J expression and no obvious upregulation of cytokine secretion was observed in the foreskin epidermis after ex vivo IR. Undifferentiated keratinocytes in murine HF regions, by contrast, revealed low H2A.J expression in non-irradiated skin and significant radiation-induced H2A.J upregulations at different time-points after IR exposure. Based on its staining characteristics, we presume that H2A.J may have previously underestimated the importance of the epigenetic regulation of keratinocyte maturation. Conclusions: Cultured foreskin characterized by highly keratinized epithelium and specific immunological features is not an appropriate model for studying H2A.J-associated tissue reactions during radiation-induced dermatitis

Cultured Human Foreskin as a Model System for Evaluating Ionizing Radiation-Induced Skin Injury

Purpose: Precise molecular and cellular mechanisms of radiation-induced dermatitis are incompletely understood. Histone variant H2A.J is associated with cellular senescence and modulates senescence-associated secretory phenotype (SASP) after DNA-damaging insults, such as ionizing radiation (IR). Using ex vivo irradiated cultured foreskin, H2A.J was analyzed as a biomarker of radiation-induced senescence, potentially initiating the inflammatory cascade of radiation-induced skin injury. Methods: Human foreskin explants were collected from young donors, irradiated ex vivo with 10 Gy, and cultured in air-liquid interphase for up to 72 h. At different time-points after ex vivo IR exposure, the foreskin epidermis was analyzed for proliferation and senescence markers by immunofluorescence and immunohistochemical staining of sectioned tissue. Secretion of cytokines was measured in supernatants by ELISA. Using our mouse model with fractionated in vivo irradiation, H2A.J expression was analyzed in epidermal stem/progenitor cell populations localized in different regions of murine hair follicles (HF). Results: Non-vascularized foreskin explants preserved their tissue homeostasis up to 72 h (even after IR exposure), but already non-irradiated foreskin epithelium expressed high levels of H2A.J in all epidermal layers and secreted high amounts of cytokines. Unexpectedly, no further increase in H2A.J expression and no obvious upregulation of cytokine secretion was observed in the foreskin epidermis after ex vivo IR. Undifferentiated keratinocytes in murine HF regions, by contrast, revealed low H2A.J expression in non-irradiated skin and significant radiation-induced H2A.J upregulations at different time-points after IR exposure. Based on its staining characteristics, we presume that H2A.J may have previously underestimated the importance of the epigenetic regulation of keratinocyte maturation. Conclusions: Cultured foreskin characterized by highly keratinized epithelium and specific immunological features is not an appropriate model for studying H2A.J-associated tissue reactions during radiation-induced dermatitis