Mutational response of Fanconi anaemia cells to shuttle vector site-specific psoralen cross-links.

Fanconi anaemia (FA) is a hereditary tumour-prone disorder. FA cells exposed to DNA crosslinking agents show an increased frequency of chromosome aberrations and of deletion type mutations. The molecular basis of FA presumably is a deficiency in cellular repair of DNA adducts. In this work a shuttle vector plasmid was treated with 8-methoxypsoralen + a split dose of UVA (leading to crosslink induction), and transfected into FA lymphoblasts. The supF gene of the vector showed a mutation frequency similar to that of normal cells; however, the number of base substitutions was relatively low, whereas a high level (50%) of deletions was seen. With both normal and FA cells these deletions varied greatly in size and were randomly distributed within the supF gene. DNA cross-links were also induced using a triple helix forming 22-mer oligonucleotide linked to a psoralen molecule and being complementary to part of supF, leading to a > 30-fold increase of mutations, which were mainly position 167 single-base substitutions and showed a pattern identical to that of the normal cells. This normal response of FA cells to the site-specific DNA cross-links may reflect that not all gene sequences of FA cells are subjected to abnormal DNA repair. Alternatively, it may reflect a lower than normal genome-overall activity of a DNA cross-link repair complex, fully capable of efficiently repairing only molecules carrying relatively few adducts.

Effects of in vitro PUVA on human leukocyte function.

Human polymorphonuclear and monomorphonuclear leukocytes (PMNL and MMNL) were exposed in vitro to 8-methoxypsoralen (8-MOP, 0.1-80 micrograms/ml) and/or UV-A radiation (0.03-2 J/cm2) and then analysed for the following functions: chemotaxis, bactericidal activity and proliferation in response to mitogen stimulation. The functions of PMNL became depressed only at a high PUVA dose level (about 20 micrograms/ml of 8-MOP plus 2 J/cm2 of UV-A), whereas with MMNL chemotaxis was inhibited at 1 microgram/ml of 8-MOP plus 2 J/cm2 of UV-A and lymphocyte proliferation was diminished at 0.1 microgram/ml plus 0.1 J/cm2. Since with the MMNL, as compared with the PMNL, a longer time period was present between PUVA exposure and analysis, and since no difference between these cell types in trypan blue exclusion could be seen, the relative sensitivity of the MMNL functions was taken as evidence of DNA damage being a mechanism for the observed PUVA-induced effects.

Studies of DNA and chromosome damage in skin fibroblasts and blood lymphocytes from psoriasis patients treated with 8-methoxypsoralen and UVA irradiation.

Exposure of human lymphocytes and skin fibroblasts in vitro to a single, clinically used dose of PUVA, i.e., 0.1 micrograms/ml of 8-methoxypsoralen (8-MOP) plus 0.9-4 J/cm2 of longwave ultraviolet radiation (UVA), lead to the formation of DNA damage as determined by alkaline elution, and to chromosome aberrations and sister chromatid exchanges (SCE). When lymphocyte-enriched plasma was obtained from psoriasis patients 2 h after oral intake of 8-MOP and then UVA irradiated (1.8-3.6 J/cm2) in vitro, an increased frequency of chromosome aberrations and SCE was observed. Normal levels of chromosome aberrations and SCE were found in lymphocytes of psoriasis patients after 3-30 weeks of PUVA treatment in vivo. A small but statistically significant increase in the SCE frequency was observed in the lymphocytes of psoriasis patients treated for 1-6 years with PUVA (mean 18.0 SCE/cell) as compared with before PUVA (mean 15.8, p less than 0.05). Skin fibroblasts of psoriasis patients analyzed 5 years after the start of PUVA treatment showed a normal number of SCE but a high fraction of filter-retained DNA in the alkaline elution assay, suggesting the presence of cross-linked DNA.

Sister chromatid exchange in human populations: the effect of smoking, drug treatment, and occupational exposure.

Increased rate of sister chromatid exchange (SCE) in peripheral lymphocytes has been observed in smokers as compared to nonsmokers and in patients receiving certain cytostatic drugs. The increased SCE frequency in smokers was shown to depend on the number of cigarettes smoked per day, as well as on the duration of smoking. DNA cross-links caused by photochemotherapy against psoriasis, 8-methoxypsoralen plus UVA irradiation (PUVA), as well as by the anti-cancer chemotherapeutic agent CCNU, were shown to be more effective at inducing SCE's than other types of DNA damage caused by these treatments. These observations suggest that SCE analysis may be used as an indicator of genotoxic exposure in vivo, provided that the various types of DNA damage caused by genotoxic agents and the dose, as well as the time of exposure in relation to the time of sampling, are considered.

DNA damage in human skin fibroblasts exposed to UVA light used in clinical PUVA treatment.

Human skin fibroblasts were irradiated with a clinically used UVA light source. The doses (1.1 and 3 J/cm2) were similar to those reaching the dermis during clinical PUVA treatment of psoriasis. DNA strand breaks, as determined by alkaline elution, were formed in a dose-dependent way and disappeared within 1 hr of postincubation at 37 degrees C. These findings have clinical implications since UVA-induced DNA damage may be accompanied by mutagenic and tumor promoting effects.

Genetic aspects of psoriasis: mode of inheritance and action of PUVA on DNA.

The results of some family and experimental studies related to psoriasis are summarized. Complex segregation analysis of Lomholt's classical family material of psoriasis from the Faroe Islands gave clear evidence of a major locus (additive gene with a frequency of 0.07) plus a strong polygenic component (genetic heritability 0.87). An analysis of another family material showed complete linkage between the major locus for psoriasis and the HLA region. Treatment of cells with 8-methoxypsoralene plus a small dose of UVA induces monoadducts, some of which appear to remain in the DNA for at least 7 days of post-treatment incubation. These monoadducts can be activated to form DNA cross-links by a second, larger UVA dose. 8-Methoxypsoralene plus UVA-induced DNA cross-links can be modified by a repair process which involves the formation of DNA breaks. This process in not observed in XPA cells.