Psoralen-Induced DNA Interstrand Cross-Links Block Transcription and Induce p53 in an Ataxia-Telangiectasia and Rad3-Related-Dependent Manner

Abstract

Psoralen plus UVA light (PUVA) is commonly used to treat psoriasis, a common skin disorder associated with rapid proliferation of cells. PUVA exerts its antiproliferative activity through formation of DNA monoadducts and interstrand crosslinks (ICLs). However, this treatment may lead to skin malignancies as a direct result of inducing carcinogenic DNA damage. Inactivation of the p53 tumor suppressor gene is an important event in the development of skin cancer. p53 is rapidly phosphorylated and stabilized in response to DNA damage, and the induction of apoptosis by p53 is an important mechanism by which p53 exerts its tumor-suppressive activity. To better understand the mechanism by which PUVA treatment induces p53, we exposed human skin fibroblasts with PUVA under conditions that differentially produce monoadducts and ICLs and found that psoralen-induced ICLs induced phosphorylation of the Ser-15 site of p53 and apoptosis much more effectively than psoralen-induced monoadducts. The induction of p53 phosphorylation by psoralen ICLs did not require factors believed to be involved in the repair of psoralen ICLs [xeroderma pigmentosum (XP)-A, XP-C, XP-F, Cockayne's syndrome-B, Fanconi anemia] but did require the ataxia-telangiectasia and Rad3-related but not the ataxia-telangiectasia mutated kinase. Psoralen-induced ICLs blocked transcription and replication more efficiently than monoadducts, and induction of p53 and apoptosis correlated with doses causing interference with transcription rather than DNA replication. Our finding that cells underwent apoptosis preferentially during S-phase suggests that the combined blockade of transcription and DNA replication by psoralen ICLs during S-phase elicits a strong apoptotic response.

Embryonic Lethality after Combined Inactivation ofFancd2andMlh1in Mice

“…Since our primary fibroblasts were derived from p53 proficient mice and the immortal cells were obtained from Trp53 mutant mice, p53 status has likely affected the outcome of cell survival and chromosomal breakage assays. Previously, p53 was shown to be involved in cell cycle arrest after ICL induction by psoralen/UVA (39). Accordingly, primary Fancd2 / Trp53 double mutant embryonic fibroblasts showed S-phase progression after ICL induction, while primary Fancd2 mutant p53 proficient cells did not show DNA replication (30).…”

Repair of Laser-localized DNA Interstrand Cross-links in G1 Phase Mammalian Cells

“…In this experimental system, these proteins initiated formation of an unhooking complex additionally containing ERCC1-XPF, the Werner syndrome helicase, replication protein A, and the Pso4 pre-mRNA splicing complex (45). Finally, ICLs block transcription (46), and repair by transcription-coupled repair (47,48) would be independent of early recognition functions.…”

Transcriptional Inhibition by DNA Damage as a Trigger for Cell Death

“…104 Psoralen monoadducts only marginally inhibit RNA polymerase II-mediated transcription while psoralen interstrand crosslinks, which induce a major conformational change of the DNA helix, 105 efficiently inhibit mRNA synthesis. 106 This preferential inhibition of transcription by the interstrand crosslinks correlates with induction of p53 and apoptosis in human fibroblast. 106 Furthermore, the psoralen interstrand crosslinks, but not the monoadducts, are preferentially removed from active genes by TCR.…”

“…106 This preferential inhibition of transcription by the interstrand crosslinks correlates with induction of p53 and apoptosis in human fibroblast. 106 Furthermore, the psoralen interstrand crosslinks, but not the monoadducts, are preferentially removed from active genes by TCR. [107][108][109][110] TCR of psoralen interstrand crosslinks is efficient in RNA polymerase II transcribed genes, while very little repair occurs in ribosomal DNA sequences.…”

“…A similar correlation between transcription blockage, S-phase entry and apoptosis was found following treatment with photoactivated psoralen. 106 Thus, the observation that UV-and psoraleninduced apoptosis is linked to blocked transcription and the finding that treated cells undergo apoptosis preferential when entering S-phase suggest that apoptosis may be triggered as a consequence of complications arising when a replication fork encounters a blocked transcription complex. Since transcription and replication factories are anchored to the nuclear matrix, it is not conceivable that transcription and replication can occur on the same piece of DNA at the same time.…”