Identification of potentially cytotoxic lesions induced by UVA photoactivation of DNA 4-thiothymidine in human cells

摘要

Photochemotherapy—in which a photosensitizing drug is combined with ultraviolet or visible radiation—has proven therapeutic effectiveness. Existing approaches have drawbacks, however, and there is a clinical need to develop alternatives offering improved target cell selectivity. DNA substitution by 4-thiothymidine (S⁴TdR) sensitizes cells to killing by ultraviolet A (UVA) radiation. Here, we demonstrate that UVA photoactivation of DNA S⁴TdR does not generate reactive oxygen or cause direct DNA breakage and is only minimally mutagenic. In an organotypic human skin model, UVA penetration is sufficiently robust to kill S⁴TdR-photosensitized epidermal cells. We have investigated the DNA lesions responsible for toxicity. Although thymidine is the predominant UVA photoproduct of S⁴TdR in dilute solution, more complex lesions are formed when S⁴TdR-containing oligonucleotides are irradiated. One of these, a thietane/S⁵-(6-4)T:T, is structurally related to the (6-4) pyrimidine:pyrimidone [(6-4) Py:Py] photoproducts induced by UVB/C radiation. These lesions are detectable in DNA from S⁴TdR/UVA-treated cells and are excised from DNA more efficiently by keratinocytes than by leukaemia cells. UVA irradiation also induces DNA interstrand crosslinking of S⁴TdR-containing duplex oligonucleotides. Cells defective in repairing (6-4) Py:Py DNA adducts or processing DNA crosslinks are extremely sensitive to S⁴TdR/UVA indicating that these lesions contribute significantly to S⁴TdR/UVA cytotoxicity.