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Role of tumor necrosis factor-alpha
in the regulation of keratinocyte
cell cycle and DNA repair
after ultraviolet-B radiation



Annesofie Faurschou  


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Accepted by: Faculty of Health Sciences Copenhagen University
Defended on: September 4, 2009
Official opponents: Lone Skov , Bent Deleuran , Olle Larkö
Tutors: Hans Christian Wulf , Robert Gniadecki

Published in the PhD Database: September 11, 2009


English abstract
This PhD thesis is based on three studies conducted at the Department of Dermatology, Bispebjerg Hospital, Copenhagen.

Tumor necrosis factor-α (TNF-α) is a proinflammatory cytokine produced in the skin in response to ultraviolet B radiation (UVB). TNF-α facilitates UVB-induced apoptosis and probably contributes to removal of damaged cells. Surprisingly, murine TNF-α-knockout models have demonstrated that TNF-α is necessary for the early stages of skin carcinogenesis and development of squamous cell carcinoma.

In the present PhD thesis, we examined the effects of TNF-α on DNA repair and cell cycle regulation in UVB-irradiated keratinocytes. In the model of premalignant keratinocytes (HaCaT), TNF-α abolished the UVB-induced G2/M checkpoint and diminished the DNA repair despite induction of apoptosis. TNF-α activated the protein kinase B/Akt and regulation of its downstream targets, mTOR, Bad and FoxO3a. This effect was dependent on atypical protein kinase C species (aPKC) since a specific peptide blocking the activity of the PKC and ι/λ abrogated the activation of Akt by TNF-α. The aPKC-Akt axis was likely to be responsible for the TNF-α-induced decrease in DNA repair since blocking of Akt activity restored DNA repair.

Since anti-tumor necrosis factor-α (TNF-α) approaches are increasingly used in the therapy of autoimmune diseases and one of the safety concerns is the potential enhancement of skin carcinogenesis, we investigated the effect of the chimeric monoclonal anti-TNF-α antibody infliximab on UVB-irradiated HaCaT cells. Cells treated with infliximab had significantly increased levels of DNA damage despite enhanced G2/M checkpoint arrest, increased apoptosis and inhibition of Akt.

In conclusion, we identified a possible novel mechanism by which TNF-α promotes UVB-induced skin carcinogenesis. This depends on aPKC-Akt activation and inhibition of DNA repair. TNF-α-treated cells are prone to escape checkpoint control and are possibly more likely to accumulate mutations, which may constitute a relevant mechanism enhancing tumor development. The effect of anti-TNF-α therapy on skin carcinogenesis warrants further investigation as our study indicates that, in contrast to what had been expected, infliximab may impair DNA repair.



Danish abstract

Denne ph.d. afhandling er baseret på tre studier udført på dermatologisk afdeling, Bispebjerg Hospital.

Tumor nekrose faktor-α (TNF-α) er et proinflammatorisk cytokin, som blandt andet dannes i huden ved bestråling med kortbølget ultraviolet sollys (UVB). TNF-α faciliterer UVB-induceret apoptose og bidrager hermed sandsynligvis til elimination af skadede hudceller. Forsøg på TNF-α-knockout mus har imidlertid overraskende vist, at TNF-α er nødvendig for de tidlige stadier af hudkræft og udviklingen af pladecelle carcinom.

Formålet med ph.d. afhandlingen var at undersøge effekten af TNF-α på DNA reparation og cellecyklus regulation i UVB-belyste keratinocytter. I præmaligne keratinocytter (HaCaT) hæmmede TNF-α det UVB-inducerede G2/M checkpoint og svækkede DNA reparationen på trods af øget apoptose. TNF-α aktiverede protein kinase B/Akt og dennes regulering af målproteinerne mTOR, Bad and FoxO3a. Effekten af TNF-α på Akt var afhængig af atypiske protein kinase C arter (aPKC). Et specifikt peptid, der blokerede aktiviteten af aPKC, hæmmede således TNF-α¿s aktivering af Akt. aPKC-Akt signaleringsaksen var ansvarlig for den TNF-α-inducerede hæmning af DNA reparationen, da blokering af Akt førte til normalisering af celle responset.

Anti-TNF-α terapi benyttes i stigende grad til behandling af autoimmune sygdomme, og en af bekymringerne er en potentiel risiko for progression af hudkræft. Vi undersøgte derfor effekten af det kimæriske monoklonale anti-TNF-α antistof infliximab på UVB-belyste HaCaT celler. Celler, som var behandlet med infliximab, havde signifikant øget niveau af DNA beskadigelse til trods for øget aktivering af G2/M checkpoint kontrollen, øget apoptose og hæmning af Akt.

Konklusionen på afhandlingen er, at vi identificerede en mulig ny mekanisme ved hvilken TNF-α bidrager til udviklingen af UVB-fremkaldt hudkræft. Denne er afhængig af aPKC-Akt aktivering og hæmning af DNA reparationen. TNF-α behandlede celler undslipper G2/M checkpoint kontrollen og akkumulerer herved sandsynligvis mutationer, som kan føre til udvikling af hudkræft. Effekten af anti-TNF-α behandling på hudkræft kræver yderligere studier, da infliximab mod vores forventning viste sig at kunne hæmme DNA reparationen.