Measurement of UVB-Induced DNA damage and its consequences in models of immunosuppression.
Exposure to UVB results in formation of cyclobutane pyrimidine dimers (CPDs) and 6-4 photoproducts in DNA. These can be quantified by a variety of techniques including alkaline gel electrophoresis, ELISAs, Southwestern blotting, and immunohistochemistry. Damage to DNA results in activation of damage response pathways, as indicated by Western blotting using antibodies specific for p53 and breast cancer-associated gene 1 (BRCA1) phosphorylation. The signal from DNA damage to activation of these response pathways appears to be mediated by FKBP12-rapamycin-associated protein (FRAP), since these phosphorylation events are blocked by rapamycin. UVB-induced DNA damage also leads to induction of immunosuppressive cytokines including tumor necrosis factor alpha (TNF-alpha) and interleukin (IL)-10 in skin. Induction of TNF-alpha by UVB is readily detectable in cultured normal human epidermal keratinocytes (NHEKs) using ELISA, while induction of IL-10 is readily detectable in cultured mouse keratinocytes but not in NHEKs. Induction of DNA damage by liposome-encapsulated HindIII results in induction of immunosuppressive responses similar to UVB. Clinical testing shows that liposome-encapsulated T4 endonuclease V or photolyase stimulates repair of CPDs in the skin of human subjects, and prevents UVB-induced immunosuppression. Stimulation of repair and prevention of immunosuppression have been linked to prevention of skin cancer by liposome-encapsulated T4 endonuclease V in repair-deficient xeroderma pigmentosum patients.
Yarosh
Daniel B
DB
AGI Dermatics, Freeport, NY 11520, USA. danyarosh@agiderm.com
Boumakis
Stavroula
S
Brown
Anne B
AB
Canning
Matthew T
MT
Galvin
Jason W
JW
Both
Dawn M
DM
Kraus
Eliyahu
E
O'Connor
Adrienne
A
Brown
David A
DA
D000818:Animals; D020552:Comet Assay; D004247:DNA / Q000528:radiation effects*; D004249:DNA Damage; D004260:DNA Repair; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D007108:Immune Tolerance / Q000528:radiation effects*; D007150:Immunohistochemistry; D016753:Interleukin-10 / Q000096:biosynthesis; D008081:Liposomes; D011740:Pyrimidine Dimers / Q000032:analysis; D014409:Tumor Necrosis Factor-alpha / Q000096:biosynthesis; D014466:Ultraviolet Rays
D008081:Liposomes; D011740:Pyrimidine Dimers; D014409:Tumor Necrosis Factor-alpha; D016753:Interleukin-10; D004247:DNA