title: The impact of Cyclosporine A on human epidermal keratinocytes
creator: Schardt, Lisa
subject: ddc-570
subject: 570 Life sciences
description: Increased incidence of cutaneous squamous cell carcinomas (cSCCs) in organ transplant recipients is frequently associated with the immunosuppressive drug Cyclosporine A (CsA). Besides its therapeutic effect on the immune surveillance, evidence is accumulating for an immunity-independent mechanism of CsA on non-immune cells. However, the effects described so far are highly diverse and its long-term influence on human keratinocytes within a tissue context is largely unknown.  Using a 3D organotypic skin culture model (OTC), for long-term treatment of keratinocytes within a physiologically relevant environment, we showed that CsA improved the stratification and differentiation of moderately differentiating HaCaT cells and of poorly differentiating HaCaT-RAS A-5 cells. This was demonstrated by histology and immunofluorescent stainings for differentiation markers, as well as differential RNA expression of several genes associated with the GO-term ‘epidermal development’. Improved differentiation was also demonstrated by an enhanced transepithelial electrical resistance, a trait directly correlated with the epidermal barrier function. However, CsA did not induce enhanced proliferation or invasion of neither HaCaT nor HaCaT-RAS A-5 cells grown in OTCs. In order to determine, whether UV irradiation as a co-carcinogen would enable invasive growth of CsA-treated HaCaT or HaCaT-RAS A-5 cells, OTCs were continuously irradiated with UV in combination with continuous CsA treatment. However, this did not induce tumorigenic transformation. Instead we found that UV irradiation and the combination of UV+CsA induced a similar regulation pattern of ‘epidermal development’ genes as did CsA alone, suggesting a common regulatory mechanism induced by CsA treatment and UV irradiation.  In general, HaCaT epithelia only survive for a restricted time span in OTCs. After 4 to 5 weeks the epithelia become atrophic. Importantly, CsA reproducibly extended the life span of the HaCaT epithelia, by retaining more vital basal and suprabasal keratinocytes. As indicated from an expression array and Ingenuity® Pathway Analysis, CsA reduced cell death in these OTCs, possibly through HGF or Integrin signaling.  Having to use different serum batches for our experiments, we finally discovered that by the addition of a specific fetal calf serum HaCaT cells, in addition to the above described characteristics, became invasive when treated with CsA. Invasion was demonstrated by disruption of the basement membrane and penetration of HaCaT cells into the underlying dermal equivalent. This was associated with a tremendous increase in differentially expressed genes as compared to the serum conditions that did not allow for CsA-induced HaCaT invasion.  Together, our data confirm that in general CsA is not directly transforming keratinocytes into cancer cells under every condition. Instead our data suggest that CsA improves keratinocyte differentiation, in particular in cells with aberrant epidermal differentiation and promotes their longevity. By chance, however, we experienced that, depending on certain serum factors, CsA could also rapidly induce invasion/tumorigenicity – thus simulating a situation typical for a subgroup of transplant recipients, who rapidly develop multiple cSCCs, also termed cutaneous carcinomatous catastrophe. With these findings our study is now suited for an in depth analysis to unravel the pathways sensitizing HaCaT cells for invasive growth. This knowledge may finally help to identify targets to counteract this devastating CsA-dependent side effect.
date: 2018
type: Dissertation
type: info:eu-repo/semantics/doctoralThesis
type: NonPeerReviewed
format: application/pdf
identifier: https://archiv.ub.uni-heidelberg.de/volltextserverhttps://archiv.ub.uni-heidelberg.de/volltextserver/23271/1/Thesis_Lisa%20Schardt.pdf
identifier: DOI:10.11588/heidok.00023271
identifier: urn:nbn:de:bsz:16-heidok-232719
identifier:   Schardt, Lisa  (2018) The impact of Cyclosporine A on human epidermal keratinocytes.  [Dissertation]     
relation: https://archiv.ub.uni-heidelberg.de/volltextserver/23271/
rights: info:eu-repo/semantics/openAccess
rights: http://archiv.ub.uni-heidelberg.de/volltextserver/help/license_urhg.html
language: eng