Platelet-activating factor is crucial in psoralen and ultraviolet A-induced immune suppression, inflammation, and apoptosis.

Psoralen plus UVA (PUVA) is used as a very effective treatment modality for various diseases, including psoriasis and cutaneous T-cell lymphoma. PUVA-induced immune suppression and/or apoptosis are thought to be responsible for the therapeutic action. However, the molecular mechanisms by which PUVA acts are not well understood. We have previously identified platelet-activating factor (PAF), a potent phospholipid mediator, as a crucial substance triggering ultraviolet B radiation-induced immune suppression. In this study, we used PAF receptor knockout mice, a selective PAF receptor antagonist, a COX-2 inhibitor (presumably blocking downstream effects of PAF), and PAF-like molecules to test the role of PAF receptor binding in PUVA treatment. We found that activation of the PAF pathway is crucial for PUVA-induced immune suppression (as measured by suppression of delayed type hypersensitivity to Candida albicans) and that it plays a role in skin inflammation and apoptosis. Downstream of PAF, interleukin-10 was involved in PUVA-induced immune suppression but not inflammation. Better understanding of PUVA's mechanisms may offer the opportunity to dissect the therapeutic from the detrimental (ie, carcinogenic) effects and/or to develop new drugs (eg, using the PAF pathway) that act like PUVA but have fewer side effects.

Molecular mechanism of PUVA-induced apoptosis in mouse epidermal cells

A combination of psoralens and ultraviolet-A radiation referred to as PUVA, is widely used in the treatment of psoriasis. PUVA therapy is highly effective in killing hyperproliferative cells, but its mechanism of action has not been fully elucidated. Psoralen binds to DNA, and upon photoactivation by UVA, it forms monofunctional adducts and interstrand cross-links. PUVA treatment has been shown to be mutagenic and to produce tumors in animals. In addition, epidemiological studies have reported a 10 to 15 percent increased risk of developing squamous cell carcinoma in individuals treated chronically with PUVA. However, it remains a treatment for skin disorders such as psoriasis because its benefits outweigh its risks. The widespread use of PUVA therapy and its associated cancer risk requires us to understand the molecular mechanisms by which PUVA induces cell death. Immortalized JB6 mouse epidermal cells, p53−/− mice, and Fas Ligand−/− (gld) mice were used to investigate the molecular mechanism by which PUVA kills cells. Treatment of JB6 cells with 10 μg/ml 8-methoxypsoralen followed by irradiation with 20 kJ/m2 UVA resulted in cell death. The cells exhibited morphological and biochemical characteristics of apoptosis such as chromatin condensation, DNA ladder formation, and TUNEL-positivity. PUVA treatment stabilized and phosphorylated p53 leading to its activation, as measured by nuclear localization and induction of p21Waf/Cip1, a transcriptional target of p53. Subsequent in vivo studies revealed that there was statistically significantly less apoptosis in p53 −/− mice than in p53+/+ mice at 72 hours after PUVA. In addition, immunohistochemical analysis revealed more Fas and FasL expression in p53+/+ mice than in p53−/− mice, suggesting that p53 is required to transcriptionally activate Fas, which in turn causes the cells to undergo apoptosis. Studies with gld mice confirmed a role for Fas/FasL interactions in PUVA-induced apoptosis. There was statistically significantly less apoptosis in gld mice compared with wild-type mice 24, 48, and 72 hours after PUVA. These results demonstrate that PUVA-induced apoptosis in mouse epidermal cells requires p53 and Fas/FasL interactions. These findings may be important for designing effective treatments for diseases such as psoriasis without increasing the patient's risk for skin cancer. ^

Roles of mismatch repair proteins in the recognition and processing of DNA interstrand crosslinks in human cells

DNA interstrand crosslinks (ICLs) are among the most toxic type of damage to a cell. Many ICL-inducing agents are widely used as therapeutic agents, e.g. cisplatin, psoralen. A bettor understanding of the cellular mechanism that eliminates ICLs is important for the improvement of human health. However, ICL repair is still poorly understood in mammals. Using a triplex-directed site-specific ICL model, we studied the roles of mismatch repair (MMR) proteins in ICL repair in human cells. We are also interested in using psoralen-conjugated triplex-forming oligonucleotides (TFOs) to direct ICLs to a specific site in targeted DNA and in the mammalian genomes. ^ MSH2 protein is the common subunit of two MMR recognition complexes, and MutSα and MutSβ. We showed that MSH2 deficiency renders human cell hypersensitive to psoralen ICLs. MMR recognition complexes bind specifically to triplex-directed psoralen ICLs in vitro. Together with the fact that psoralen ICL-induced repair synthesis is dramatically decreased in MSH2 deficient cell extracts, we demonstrated that MSH2 function is critical for the recognition and processing of psoralen ICLs in human cells. Interestingly, lack of MSH2 does not reduce the level of psoralen ICL-induced mutagenesis in human cells, suggesting that MSH2 does not contribute to error-generating repair of psoralen ICLs, and therefore, may represent a novel error-free mechanism for repairing ICLs. We also studied the role of MLH1, anther key protein in MMR, in the processing of psoralen ICLs. MLH1-deficient human cells are more resistant to psoralen plus UVA treatment. Importantly, MLH1 function is not required for the mutagenic repair of psoralen ICLs, suggesting that it is not involved in the error-generating repair of this type of DNA damage in human cells. ^ These are the first data indicating mismatch repair proteins may participate in a relatively error-free mechanism for processing psoralen ICL in human cells. Enhancement of MMR protein function relative to nucleotide excision repair proteins may reduce the mutagenesis caused by DNA ICLs in humans. ^ In order to specifically target ICLs to mammalian genes, we identified novel TFO target sequences in mouse and human genomes. Using this information, many critical mammalian genes can now be targeted by TFOs.^

Involvement of HMGB1 in the repair of DNA adducts and the responses to DNA damage in mammalian cells

High mobility group protein B1 (HMGB1) is a multifunctional protein with roles in chromatin structure, transcription, V(D)J recombination, and inflammation. HMGB1 also binds to and bends damaged DNA, but the biological consequence of this interaction is not clearly understood. We have shown previously that HMGB1 binds cooperatively with nucleotide excision repair (NER) damage recognition proteins XPA and RPA to triplex-directed psoralen DNA interstrand crosslinks (ICLs). Based on this we hypothesized that HMGB1 is enhancing the repair of DNA lesions, and through this role, is affecting DNA damage-induced mutagenesis and cell survival. Because HMGB1 is also a chromatin protein, we further hypothesized that it is acting to facilitate chromatin remodeling at the site of the DNA damage, to allow access of the repair machinery to the DNA lesion. We demonstrated here that HMGB1 could bind to triplex-directed psoralen ICLs in a complex with NER proteins XPC-RAD23B, XPA and RPA, which occurred in the presence or absence of DNA. Supporting these findings, we demonstrated that HMGB1 enhanced repair of triplex-directed psoralen ICLs (by nucleotide incorporation), as well as removal of UVC irradiation-induced DNA lesions from the genome (by radioimmunoassay). We also explored HMGB1's role in chromatin remodeling upon DNA damage. Immunoblotting demonstrated that, in contrast to HMGB1 proficient cells, cells lacking HMGB1 showed no increase in histone acetylation after UVC irradiation. Additionally, purified HMGB1 protein enhanced chromatin formation in an in vitro chromatin assembly system. However, HMGB1 also has a role in DNA repair in the absence of chromatin, as shown by measuring UVC-induced nucleotide incorporation on a naked substrate. Upon exploration of HMGB1's effect on several cellular outcomes of DNA damage, we found that mammalian cells lacking HMGB1 were hypersensitive to DNA damage induced by psoralen plus UVA irradiation or UVC radiation, showing less survival and increased mutagenesis. These results reveal a new role for HMGB1 in the error-free repair of DNA lesions in a chromosomal context. As strategies targeting HMGB1 are currently in development for treatment of sepsis and rheumatoid arthritis, our findings draw attention to potential adverse side effects of anti-HMGB1 therapy in patients with inflammatory diseases. ^

Involvement of {\it p53\/} in PUVA-induced skin cancers

A combination of psoralen and ultraviolet-A radiation, commonly referred to as "PUVA," is widely used in the treatment of psoriasis. However, PUVA treatment increases the risk of developing skin cancer in psoriasis patients and induces skin cancer in mice. It is, however unknown whether the increased incidence of skin cancer in PUVA treated psoriasis patients is due to the carcinogenic effects of PUVA therapy or due to an indirect effect such as immunosuppression, which can permit the growth of tumors induced by UVB radiation. In this study, we used the p53 tumor suppressor gene as a molecular marker to determine whether PUVA-induced mouse skin cancers contain unique mutations in p53 that are different from UV-induced mutations, and if so, determine whether skin cancers from PUVA treated patients have PUVA-type or UV-type p53 mutations. Since the DNA lesions induced by PUVA are quite different from those induced by UV, we hypothesize that p53 mutations induced by PUVA may also be different from those induced by UV.^ Analysis of PUVA-induced murine skin cancers for p53 mutations revealed that 14 of 15 (93%) missense mutations detected in these cancers were localized at 5$\sp\prime$-TA/5$\sp\prime$-TAT sites, potential sites of psoralen photoadditions. Mutations at these sequences are exceedingly rare in UV-induced murine skin cancers. In addition, PUVA-induced murine skin cancers did not contain UV signature (C $\to$ T or CC $\to$ TT transitions) mutations in p53. These results suggest that PUVA induces unique mutations in p53 that can be distinguished from those induced by UV.^ Next we determined whether SCCs arising in PUVA treated psoriasis patients have PUVA-type or UV-type p53 mutations. The results indicated that 16 of 25 (64%) missense p53 mutations detected in SCCs from PUVA treated patients were located at 5$\sp\prime$-TG, 5$\sp\prime$-TA and 5$\sp\prime$-TT sites, putative sites of psoralen photobinding. Interestingly, about 32% of p53 mutations detected in SCCs from PUVA treated patients had the UV signature. Taken together these results suggest that both PUVA and UVB play a role in the development of SCCs in psoriasis patients undergoing PUVA therapy. ^