Cross-Reactions of Human Lupus Autoantibodies with 8-Methoxypsoralen Photomodified DNA Fragments

DNA fragments of around 200 base pair (average size) have been covalently crosslinked with 8-methoxypsoralen under 365 nm UV light. The photoadduct, induced antibodies in rabbits with a titer of > 1:12,800 by direct bindng ELISA. Binding data showed that the induced antibodies are conformation-specific recognizing restricted conformational change at site of crosslinking. Human autoantibodies against DNA, bound not only to native DNA but to the photomodified DNA fragment as well. In addition, binding patterns of SLE sera obtained from different patients were remarkably similar, indicating the recognition of altered conformation of the modified polymer by naturally occurring SLE anti-DNA autoantibodies.     

Sensitivity of Escherichia coli acrA mutants to psoralen plus near-ultraviolet radiation

The sensitivity to psoralen plus near-ultraviolet radiation (PUVA) was compared in a pair of E. coli strains differing at the acrA locus. Survival was determined for both bacteria and phage lambda. AcrA mutant cells were 40 times more sensitive than wild type to the lethal effect of PUVA. Free lambda phage exposed to PUVA survived as well when plated on acrA mutants as on wild type. In contrast, prophage lambda CI857 ind carried in lysogenic acrA strains was hypersensitive to PUVA. The enhanced sensitivity of bacterial and lambda DNA, when inside acrA cells, was paralleled by an increased photobinding of radiolabelled psoralens in the mutant. Binding was increased specifically to DNA rather than to nucleic acids in general. The difference in psoralen-binding ability determined by the acrA gene persisted after permeabilizing treatment of the cells. The results suggest that the acrA mutation causes an alteration specifically in the environment of the cellular DNA so as to allow increased intercalation and photobinding of psoralens.     

Stress response induced by DNA damage leads to specific, delayed and untargeted mutations

Summary Cells of the mouse T-lymphoma line GRSL13 were treated with 8-methoxy-psoralen plus longwave ultraviolet light (PUVA) under conditions where the biological effects are mainly due to non-persistent DNA crosslinks (PUVA-CL treatment). Fluctuation analysis showed that PUVA-CL treatment resulted in an enhancement of the mutation rate in the progeny of treated cells, which persisted until the eleventh generation after treatment. Since only 5 cross-links are available to account for 52 mutational events observed in the coding region, about 90% of the induced mutational events must have been untargeted. This was confirmed by molecular analysis of these mutations, which showed that 53% of the point mutations arose at sites which are not a target for psoralens. This supports the hypothesis that stress responses may give rise to untargeted mutagenesis. Further support for this hypothesis is provided by the observation that 8-methoxy-psoralen (8-MOP) or UVA alone (both of which are known to induce many pleiotropic effects) each acted as indirect mutagen by enhancing the mutation rate 2–4 fold in the progeny of treated cells.     

Breast cancer cell survival signal is affected by bergapten combined with an ultraviolet irradiation

In this study we have reported that bergapten (B) and bergapten plus UV (PUVA) are able to significantly affect MCF-7, ZR-75 and SKBR-3 breast cancer cell proliferations.B induced a lowering of PI3K/AKT survival signal in MCF-7 cells even in presence of IGF-I stimulation. Furthermore, B and in a higher extent, PUVA up-regulated the p53 mRNA and the protein content. An increased co-association between p21 WAF and proliferating cell nuclear antigen (PCNA) has been observed in PUVA-treated MCF-7 cells, thus inhibiting DNA replication. These results highlight how B, and its photoactivated compound, exert antiproliferative effects and induce apoptotic responses in breast cancer cells.     

维生素C对光化学法诱导皮肤光损伤的光保护作用:OCT定量研究

8-甲氧补骨脂素(8-MOP)联合UVA辐射(即光化学疗法PUVA)因诱导皮肤光损伤的副作用,常被用于实验动物皮肤光损伤模型的构建。为研究维生素C(Vc)对皮肤光损伤的保护效应,本研究在Balb/c小鼠背部皮肤涂抹0.1%8-MOP溶液1h后进行UVA辐照(10 J/cn2)。然后分别涂抹7.5%、15%和30%浓度的Vc溶液的进行光保护治疗,利用光学相干层析成像分析皮肤厚度和光信号衰减的变化,从而评估Vc对皮肤的光保护作用。结果显示,相对于溶媒组,Vc治疗组皮肤厚度减小,光衰减系数增加,其中15%效果尤为明显结果表明,局部涂抹Vc溶液具有一定的光损伤保护效应。     

Modification of extracorporeal photopheresis technology with porphyrin precursors. Comparison between 8-methoxypsoralen and hexaminolevulinate in killing human T-cell lymphoma cell lines in vitro

Background

Extracorporeal photopheresis that exposes isolated white blood cells to 8-methoxypsoralen (8-MOP) and ultraviolet-A (UV-A) light is used for the management of cutaneous T-cell lymphoma and graft-versus-host disease. 8-MOP binds to DNA of both tumor and normal cells, thus increasing the risk of carcinogenesis of normal cells; and also kills both tumor and normal cells with no selectivity after UV-A irradiation. Hexaminolevulinate (HAL)-induced protoporphyrin-IX is a potent photosensitizer that localizes at membranous structures outside of the nucleus of a cell. HAL-mediated photodynamic therapy selectively destroys activated/transformed lymphocytes and induces systemic anti-tumor immunity. The aim of the present study was to explore the possibility of using HAL instead of 8-MOP to kill cells after UV-A exposure.

Methods

Human T-cell lymphoma Jurkat and Karpas 299 cell lines were used to evaluate cell photoinactivation after 8-MOP and/or HAL plus UV-A light with cell proliferation and long term survival assays. The mode of cell death was also analyzed by fluorescence microscopy.

Results

Cell proliferation was decreased by HAL/UV-A, 8-MOP/UV-A or HAL/8-MOP/UV-A. At sufficient doses, the cells were killed by all the regimens; however, the mode of cell death was dependent on the treatment conditions. 8-MOP/UV-A produced apoptotic death exclusively; whereas both apoptosis and necrosis were induced by HAL/UV-A.

Conclusion

8-MOP can be replaced by HAL to inactivate the Jurkat and Karpas 299 T-cell lymphoma cells after UV-A irradiation via apoptosis and necrosis. This finding may have an impact on improved efficacy of photopheresis.     

pBR322DNA与8-甲氧补骨脂素光化学反应部位的碱基顺序特异性

用限制性核酸内切酶酶切试验研究了质粒pBR322 DNA经8-MOP及近紫外线作用后损伤部位的碱基顺序特异性。实验研究发现PUVA损伤的DNA在HindⅢ及RsaⅠ识别位置上酶切反应受到严重抑制,而在SphⅠ,EcoRⅠ,PvuⅡ,BamHI,PstⅠ识到位置上抑制轻微。通过对不同识别位置上碱基顺序及其光化学反应敏感性的分析,推断出DNA的TpA顺序可能是最易接受8-MOP光化学反应的部位。     

Chromosomal damage induced by furocoumarins and UVA in hamster and human cells including cells from patients with ataxia telangiectasia and xeroderma pigmentosum

The comparative photosensitizing effects to near-UV irradiation (UVA) of several naturally occurring furocoumarins, 5-methoxypsoralen (5MOP), psoralen, 8-methoxypsoralen (8MOP) and angelicin in producing chromosome damage in vitro in cells derived from hamster, normal human, ataxia telangiectasia (AT) and xeroderma pigmentosum (XP) patients were studied. In Chinese hamster cells, lethality was greatest with psoralen and least with angelicin; 8MOP and 5MOP were intermediate. 8MOP and 5MOP produced sister-chromatid exchanges with almost equal efficiency and to a larger extent by far than angelicin. In all human cell lines studied 8MOP and 5MOP were similarly effective in the production of sister-chromatid exchanges and chromosomal aberrations. AT and XP cells responded with higher frequencies of sister-chromatid exchanges as well as chromosomal aberrations than normal human cells to 5MOP, 8MOP and angelicin. Evidence is presented which suggests that cell death in Chinese hamster cells following angelicin photosensitization is not clearly related to the production of sister-chromatid exchanges. AT cells were unexpectedly more sensitive to angelicin than normal cells. The presence of 5MOP in some sun-tan preparations is not acceptable in view of the present evidence of its biological activity.     

Photopheresis with UV-A light and 8-methoxypsoralen leads to cell death and to release of blebs with anti-inflammatory phenotype in activated and non-activated lymphocytes

Background: Extracorporeal photopheresis is a therapy for treatment of autoimmune diseases, cutaneous T-cell lymphoma, organ graft rejection as well as graft-versus-host diseases. The exact mechanism how the combination of 8-methoxypsoralen plus UV-A irradiation (PUVA) acts is still unclear. We investigated the cell death of activated and non-activated lymphocytes after PUVA treatment as well as the rate of released blebs and their antigen composition. Results: In presence of 8-MOP, UV-A light highly significantly increased the cell death of activated lymphocytes. The same was observed to a lesser extent in non-activated cells. Blebs derived from activated lymphocytes after PUVA treatment showed the highest surface exposition of phosphatidylserine. These blebs also displayed a high exposure of the antigens CD5 and CD8 as well as a low exposure of CD28 and CD86. Conclusion: PUVA treatment exerts anti-inflammatory effects by inducing apoptosis and apoptotic cell-derived blebs with immune suppressive surface composition.