Human in vitro skin organ culture as a model system for evaluating DNA repair

Abstract

Background UV-exposures result in accumulation of genetic lesions that facilitate the development of skin cancer. Numerous pharmacologic agents are currently under development to both inhibit formation of DNA lesions and enhance repair. Drugs must be evaluated in vitro, currently performed in cell culture systems, before being tested on humans. Current systems do not account for the architecture and diverse cellularity of intact human skin. Objective To establish a novel, functionally viable, and reproducible in vitro skin organ culture system for studying the effects of various pharmacologic agents on DNA repair. Methods Human skin was obtained from neonatal foreskins. Intact skin punches derived from foreskins were cultured in vitro prior to exposure to UV-irradiation, and evaluated for DNA-damage using a DNA dot blot. Serial skin biopsies were obtained from patients with actinic keratoses treated with topical imiquimod. Expression of immune-stimulating and DNA repair genes was evaluated in ex vivo and in vitro samples. Results DNA dot blots revealed active repair of UV induced lesions in our in vitro skin organ culture. The photo-protective effect of sunscreen was detected, while imiquimod treatment did not enhance DNA repair in vitro. The DNA repair molecules XPA and XPF were up-regulated in the skin of imiquimod treated patients with actinic keratoses and imiquimod treated bone marrow-derived cell lines, but not keratinocytes. Conclusion Our in vitro human skin organ culture model detected repair of UV-induced DNA lesions, and may be easily adapted to investigate various photo-protective drugs intended to prevent or treat skin cancer.

A new hair follicle-derived human epidermal model for the evaluation of sunscreen genoprotection

“…Moreover, determination of SPF is a tedious and time-consuming process that requires participation of human volunteers. Numerous works have been devoted to the development of in vitro techniques as complementary tools to SPF [6][7][8][9][10][11][12][13][14][15]. These approaches mostly rely on the induction of UVB and/or UVA-activated molecular pathways in cutaneous cells as endpoints for the evaluation of the photoprotection afforded by sunscreens following topical application.…”

Human Organ Culture: Updating the Approach to Bridge the Gap from In Vitro to In Vivo in Inflammation, Cancer, and Stem Cell Biology

“…In HOCs of skin, topical imiquimod, a pharmacological agent that facilitate the development of skin cancer through accumulation of genetic lesions failed to enhance DNA repair after UV-exposure ( 43 ). In contrast to imiquimod-treated epidermal bone marrow-derived cells (BMDCs) ( 43 ), attributing these differences to the KCs in the epidermal layer lacking TLR7/8 while BMDCs such as LCs express TLR7/8 ( 44 ).…”

“…In HOCs of skin, topical imiquimod, a pharmacological agent that facilitate the development of skin cancer through accumulation of genetic lesions failed to enhance DNA repair after UV-exposure ( 43 ). In contrast to imiquimod-treated epidermal bone marrow-derived cells (BMDCs) ( 43 ), attributing these differences to the KCs in the epidermal layer lacking TLR7/8 while BMDCs such as LCs express TLR7/8 ( 44 ). A differential response to imiquimod has also been observed in stimulated KCs monolayers (HaCaT cells, immortalized human KCs and primary KCs), which failed to show DNA repair in contrast to stimulated KG-1, a monocytic cell line ( 43 ).…”

“…In contrast to imiquimod-treated epidermal bone marrow-derived cells (BMDCs) ( 43 ), attributing these differences to the KCs in the epidermal layer lacking TLR7/8 while BMDCs such as LCs express TLR7/8 ( 44 ). A differential response to imiquimod has also been observed in stimulated KCs monolayers (HaCaT cells, immortalized human KCs and primary KCs), which failed to show DNA repair in contrast to stimulated KG-1, a monocytic cell line ( 43 ). These data suggested that LCs activated by imiquimod might potentially influence neighboring KCs and thus provide an indirect protection against UV damage.…”

Monitoring UV‐induced signalling pathways in an ex vivo skin organ culture model using phospho‐antibody array

“…Skin explants can be maintained for 7 days with preserved viability and morphology . While a few studies have investigated some aspects of skin response to UV irradiation in human skin explants, the investigation of multiple signalling pathways in response to UV exposure in this model has not been reported.…”