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

Abstract

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.

Photoinduced Damage to Cellular DNA: Direct and Photosensitized Reactions†

“…This leads to a high phototoxicity of the cells upon UVA-irradiation. This was rationalized in terms of oxygenindependent formation of DNA photoproducts including an expected S5 thietane derivative, a 6-4PP analogue (227), through photocycloaddition with vicinal thymine and putative ICLs (228) that however remain to be characterized. Attempts to search for an increase in the level of 8-oxoGua in cellular S4Thy DNA were unsuccessful despite the fact that S4Thy is an efficient generator of 1O2 upon UVA irradiation (229).…”

Oxidatively-generated damage to DNA and proteins mediated by photosensitized UVA

“…Small amounts of thymidine sulfenate (TSO) and a dimeric form, tentatively identified as T-S-T, are also produced. dT is not a significant photoproduct of DNA S 4 dT, however, and it is not detected in irradiated double-stranded oligonucleotides [62]. Degradation of DNA-embedded S 4 T is heavily dependent on sequence context.…”

“…Monoubiquitination of the FANCD2 protein is a key step in FA pathway activation and provides a sensitive indicator of the presence of ICLs. UVA irradiation of cells treated with 6-TG, S 4 dT or the halothiopyrimidine deoxynucleosides induces monoubiquitination of FANCD2 and FA cells are hypersensitive to all these drug/UVA combinations [13], [36], [62]. 6-Thioguanine UVA induces efficient crosslinking of double-stranded oligonucleotides heavily substituted with 6-TG [66]. In these model substrates, most ICLs are formed via disulfide bridges and crosslinking is significantly enhanced by closely opposed 6-TGs.…”

“…Their formation can also be inferred from the chromosome aberrations present in the karyotypes of treated cells. The observed radial chromosomes and chromosome breaks are characteristically associated with aberrantly resolved ICLs [66]. Thiothymidine UVA also crosslinks duplex oligonucletides containing S 4 T. ICL formation is largely independent of sequence context although it is less efficient when S 4 T has a flanking T, most likely because of the preferential formation of intrastrand photoproducts [62]. UVA-induced crosslinking is between S 4 T and the complementary A.…”

5-Iodo-4-thio-2′-deoxyuridine: Synthesis, Structure, and Cytotoxic Activity

“…It undergoes UVA-mediated conversion to potentially cytotoxic lesions including inter-and intrastrand DNA crosslinks. 12 In the absence of UVA, cells can accumulate significant levels of 4-thioTdR without detrimental effects on proliferation. 13,14 The combined use of 4-thioTdR and UVA offers a novel approach to selectively kill rapidly proliferating cells, such as cancer cells, while causing minimal damage to normal tissues.…”