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Proc. Natl. Acad. Sci. USA Vol. 82, pp. 6158-6162, September 1985 Cell Biology A possible mechanism of psoralen phototoxicity not involving direct interaction with DNA (receptors/psoriasis/vitiligo/photocheniistry) JEFFREY D. LASKIN*, EDMUND LEE, EDWARD J. YURKOW, DEBRA L. LASKIN, AND MICHAEL A. GALLO Department of Environmental and Community Medicine, University of Medicine and Dentistry of New Jersey-Rutgers Medical School and Department of Pharmacology and Toxicology, Rutgers University, Piscataway, NJ 08854 Communicated by Gerald N. Wogan, May 23, 1985 ABSTRACT Psoralens in combination with ultraviolet OCH3 light (UVA; 320-400 nm) are used in the photochemical 0 0 0 treatment of a variety of skin diseases including vitiligo, a skin depigmentational disorder, and psoriasis, a disease of acceler- ated epidermal cell proliferation. Although it is generally assumed that the major site of action of the psoralens is DNA, MeOPso we have obtained evidence that another site may be the primary target for these compounds. We have identified specific, saturable, high-affinity binding sites for 8-methoxypsoralen on HeLa cells and have detected specific binding of 8- methoxypsoralen to four other human cell lines and five mouse cell lines. In HeLa cells, specific binding is reversible and independent of the ability of the compound to intercalate into DNA. In addition, binding sites become covalently modified by Me3Pso the psoralen after UVA exposure. Specific binding of 8- [methoxy-l3Hlmethoxypsoralen constitutes 79% of the label FIG. 1. Structure of MeOPso and Me3Pso. bound to the cells. Scatchard analysis indicated two classes of psoralen binding sites: high-affinity sites with a Kd of 19 x 10-9 treatment of psoriasis. Stimulation of pigment production by M (1.8 x 105 sites per cell) and low-affinity sites with a Kd of psoralen plus UVA in melanocytes induces skin tanning. The 4 x 10-6 M (7.1 x 106 sites per cell). Four structurally related increase in melanocyte proliferation after this therapy ame- psoralen analogs block 8-methoxypsoralen binding in a manner liorates the symptoms of vitiligo. To understand the mech- that parallels their biological activity. Based on these findings, anism of action of psoralen/UVA therapy, these two oppos- we hypothesize that specific binding sites for psoralens on ing effects must be taken into account. Although alkylation of mammalian cells mediate, at least in part, psoralen-induced DNA by photoactivated psoralens may be involved in the phototoxicity. therapeutic responses observed with these drugs, it is likely that other mechanisms contribute to the diverse actions of Psoralens are the best-characterized of the photosensitizing psoralen plus UVA. Here we provide evidence that, in chemicals. These compounds, also known as furocoumarins, mammalian cells, a site other than DNA is a major target for occur naturally in more than two dozen plant species. Two of the psoralens. We have identified specific, saturable, high- the psoralen analogs, 8-methoxypsoralen (MeOPso) and affinity binding sites for the psoralens on a number of 4,5',8-trimethylpsoralen (Me3Pso) (Fig. 1), are used clinically different in in the photochemotherapy of skin diseases such as psoriasis, cell types culture. Binding of psoralens to these mycosis fungoides, vitiligo, and eczema (see ref. 1 for sites is distinct from their intercalation into DNA. In addition, review). Typically, patients are administered MeOPso orally binding sites can be alkylated by psoralens after UVA light or Me3Pso topically and are then exposed to a measured dose treatment. Based on our results, we hypothesize that the of ultraviolet (UVA; 320-400 nm) irradiation. It has been interaction of photoactivated psoralens with binding sites on hypothesized that the actions of psoralen in the skin are due different cell types contributes, at least in part, to the to their ability to form DNA adducts following UVA light mechanism of psoralen/UVA photochemotherapy. irradiation (2). Psoralens are known to intercalate into DNA in a dark reaction. After exposure to UVA light, they form MATERIALS AND METHODS mono- and bifunctional adducts with pyrimidine bases in DNA. This results in crosslinking of psoralen between Reagents. All-trans-retinoic acid, psoralen, 43-phorbol base-paired strands of DNA (1, 2). 12-myristate 13-acetate (PMA), dexamethasone, and In the skin, psoralen plus UVA treatment produces two methotrexate were from Sigma; MeOPso and anthralin (1,8- clinically important yet opposing effects on different epider- dihydroxy-9-anthrone), from Elder Pharmaceuticals; 8- mal cell populations. Photochemotherapy with psoralen plus [methoxy-3H]methoxypsoralen ([3H]MeOPso, >99% pure, UVA results in inhibition of basal cell division and, at the 67.9 Ci/mmol; 1 Ci = 37 GBq), from New England Nuclear; same time, stimulation of melanocyte proliferation and dif- and benzoyl peroxide, from Aldrich. 5-Methylangelicin was ferentiation (3). Inhibition of basal cell division causes a generously provided by M. Pathak (Harvard Medical School, decreased rate of keratinocyte growth and maturation and Boston, MA). partly explains the efficacy of psoralen plus UVA in the Abbreviations: MeOPso, 8-methoxypsoralen; Me3Pso, 4,5',8- The publication costs of this article were defrayed in part by page charge trimethylpsoralen; PMA, 4,B-phorbol 12-myristate 13-acetate; UVA, payment. This article must therefore be hereby marked "advertisement" ultraviolet light of 320-400 nm wavelength. in accordance with 18 U.S.C. §1734 solely to indicate this fact. *To whom reprint requests should be addressed. 6158 Downloaded by guest on September 29, 2021 Cell Biology: Laskin et al. Proc. Natl. Acad. Sci. USA 82 (1985) 6159 Cell Cultures. HeLa and KB cells were from Flow Labo- ratories. G-361 melanoma cells were from the American Type Culture Collection (ATCC CRL 1424). PAM 212 cells were obtained from S. Yuspa (National Institutes of Health). KM, HF-A, and 3T3 cells were obtained from J. Hirsh (Rutgers University, New Brunswick, NJ). S-180, B16, and L cells were obtained as described (4, 5). For binding assays, all cells 0L were maintained in monolayer culture at 370C in a 5% CO2 atmosphere in Dulbecco's modified Eagle's medium supple- CL mented with 10% newborn calf serum. E Binding Assay. The binding assay was performed at 40C. Confluent cells (for HeLa cells, -2 x 105 cells per cm2 in 60-mm dishes) were washed three times with binding buffer 0 15 30 45 (Earle's salts solution with 25 mM Hepes at pH 7.2). The cells min were then incubated with 2 ml of binding buffer containing 2.3 Time, nM [3H]MeOPso (67.9 Ci/mmol). Nonspecific binding was FIG. 2. Time course of [3H]MeOPso binding to HeLa cells. determined by incubating cells with binding buffer containing [3H]MeOPso (2.3 nM, 67.9 Ci/mmol) was incubated with HeLa cells the radioligand and excess unlabeled MeOPso (10 ,g/ml). for various times to determine total binding. Duplicate plates After 30 min the reaction was terminated by aspirating the received [3H]MeOPso and an excess of unlabeled MeOPso (10 binding buffer from the cells and washing them with ice-cold gg/ml, 46 AM) during the same time periods to determine nonspecific Dulbecco's phosphate-buffered saline (6 x 5 ml). The cells binding at each point. Specific binding was determined by subtracting then were solubilized with 2 ml of0.2 M NaOH, and duplicate nonspecific binding from total binding. The solid line represents 0.5-ml aliquots were taken for scintillation counting. Specific specific binding of [3H]MeOPso with increasing incubation times. In some assays, unlabeled MeOPso (10 Ag/ml, 46 AM) was added binding was obtained by subtracting nonspecifically bound directly to the incubation medium after 30 min (arrow) to prevent material from the total. Each data point presented represents rebinding of dissociated [3H]MeOPso. Specific binding under these the average of duplicate cell culture plates. To determine conditions was also determined by subtracting nonspecific binding saturability, the assay was performed with binding buffer from the total after the addition of MeOPso. The dashed line containing [3H]MeOPso (2.3 nM, 67.9 Ci/mmol) and various represents the reversal of specific binding of [3H]MeOPso. amounts of unlabeled MeOPso. Nonspecific binding was not saturable and increased linearly with increasing concentra- ing HeLa cells with various concentrations of [3H]MeOPso; tions of [3H]MeOPso. In addition, specific binding increased binding was saturable and Scatchard analysis of the binding in proportion to the number of cells on the plates and no data produced a curvilinear plot (Fig. 3). This was resolved binding was observed in the absence of cells. into two linear components, each characterizing a putative Measurement of Covalent Binding of [3H]MeOPso to Cells binding site (8-10): a high-affinity binding site, with a and DNA. Cells were incubated for 30 min with 2 ml ofbinding dissociation constant (Kd) of 19 x 10-9 M and 1.8 X 105 buffer containing [3H]MeOPso as described above for bind- binding sites per cell, and a low-affinity site, with a Kd of 4 ing assays. The cells then were exposed to UVA emitted from x 10-6 M and 7.1 x 106 binding sites per cell. To determine a BLB fluorescent tube (F40 BL, Sylvania). The incident whether the heterogeneity of binding was due to the presence light on the culture plates was 2.4 mW/cm2 as measured with of more than one cell type in the population of cultured cells, an International Light UV-radiometer, Model IL 442A. Cells we isolated subpopulations of HIeLa cells by two successive were exposed to 1.28 J/cm2 of UVA. After irradiation, cells were rinsed with phosphate-buffered saline and solubilized with NaOH for liquid scintillation counting as described above to determine total cell-associated radioactivity.

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