Psoralen photobiology: the relationship between DNA damage, chromatin structure, transcription, and immunogenic effects.
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Animals↗
Biomedical subjects
F P Gasparro
Publications and source records attributed to F P Gasparro.
Rectal suppositories of 8-methoxsalen produce fewer gastrointestinal side effects than the oral formulation.
BACKGROUND: Gastrointestinal side effects are associated with the oral ingestion of 8-methoxsalen (8-MOP), including the liquid and crystalline formulations. OBJECTIVE: The objective of this study was to determine whether the gastrointestinal symptoms associated with 8-MOP could be decreased by altering the route of administration. METHODS: In an open pilot study, 8-MOP rectal suppositories were given to six patients with psoriasis vulgaris who had significant nausea or abdominal pain with the oral liquid form of the drug. RESULTS: On a scale of 0 to 5, this group of patients reported a mean score of 4.4 for nausea, 0.3 for vomiting, 2.1 for abdominal pain, and 1.3 for headaches with oral 8-MOP. With the suppository form, the mean scores were 0 for nausea, 0 for vomiting, 0 for abdominal pain, and 0 for headaches. These latter values represent scores for the entire treatment period. Clinical severity scores of psoriasis improved from a mean of 6.5 (maximum possible score = 9) at the start of the trial to a mean of 1 at its conclusion. Plasma 8-MOP levels of more than 100 ng/ml were observed in all patients who received the suppositories; in only one patient were the 8-MOP plasma levels significantly higher with the oral form than with the rectal form. CONCLUSION: Rectal suppositories of 8-MOP were associated with significantly fewer gastrointestinal side effects than the oral form of the drug; this was accomplished without compromising clinical efficacy.
Abdominal Pain↗
Apoptosis induction of ultraviolet light A and photochemotherapy in cutaneous T-cell Lymphoma: relevance to mechanism of therapeutic action.
The anti-tumor action of many chemotherapeutic agents has recently been attributed to the induction of apoptosis in the malignant cell population. In this study, we investigated the ability of extracorporeal photopheresis (ExP) and in vitro PUVA (8-methoxy-psoralen + ultraviolet A) therapy to induce apoptosis in peripheral blood mononuclear cells from Sezary syndrome patients and normal controls. Flow cytometric analysis of ExP- or PUVA-treated peripheral blood lymphocytes demonstrated two distinct cell populations within 24 h of treatment. One population was similar to untreated controls with the other exhibiting characteristics of apoptotic cell death, i.e., a loss of cell volume and an accompanying increase in cell density. This latter population was comprised of cells with DNA strand breaks as determined by the Tdt-mediated deoxyuridine triphosphate-biotin nick end labeling assay. Apoptosis was also confirmed morphologically by fluorescent and electron microscopy as well as by demonstration of characteristic DNA strand breaks (laddering) using gel electrophoresis. Apoptosis was not observed with 8-methoxypsoralen (< or = 300 ng per ml) alone; however, ultraviolet A alone at doses > or = 2 J per cm2 induced apoptosis in lymphocytes. Peripheral blood T-cell subpopulations of Sezary syndrome patients, including the malignant clone, were equally susceptible to apoptosis subsequent to either photopheresis or PUVA treatment. In contrast, monocytes (CD14+/CD45+) appear to be resistant to apoptosis induction by ExP or PUVA treatment. Moreover, ExP-treated and untreated monocytes phagocytized apoptotic, but not untreated, peripheral blood mononuclear cells. ExP and PUVA have been shown to be efficacious and well-tolerated therapies in the treatment of dermatologic diseases and transplant rejection. These data suggest that induction of apoptosis may be an important event for therapeutic efficacy.
Apoptosis↗
8-methoxypsoralen and ultraviolet a radiation activate the human elastin promoter in transgenic mice: in vivo and in vitro evidence for gene induction.
Treatment of skin diseases with the combination of 8-methoxypsoralen and ultraviolet A radiation (PUVA) results in clinical alterations in treated skin that resemble those observed in chronically photodamaged skin. The PUVA-treated patients develop nonmelanoma skin cancers, pigmentary alterations and wrinkling characteristic of sun-induced changes. The major alteration in the dermis of sun-damaged skin is the deposition of abnormal elastic fibers, termed solar elastosis. Up-regulation of elastin promoter activity in dermal fibroblasts explains the excess elastic tissue but not the reason for the aberrant morphology of the elastotic material. In order to study photoaging in an experimental system, we utilized a transgenic mouse line that expresses the human elastin promoter/chloramphenicol acetyltransferase construct in a tissue-specific and developmentally regulated manner. Although UVB radiation has been demonstrated to increase promoter activity in vitro, UVA fails to demonstrate a similar effect at the doses utilized. In this study, we demonstrate the ability of PUVA treatment to up-regulate elastin promoter activity both in vitro and in vivo. These data help to explain the development of photoaging in sun-protected PUVA-treated skin. We attribute the up-regulation of elastin promoter activity in response to PUVA to the formation of DNA photoadducts, which do not occur in response to UVA radiation alone.
Animals↗
Triplex-mediated, in vitro targeting of psoralen photoadducts within the genome of a transgenic mouse.
Light-activated psoralens can covalently modify DNA and are widely used to study nucleic acid secondary structure and mutagenesis. Sequence specificity can be added to the photoaddition reaction by attaching the psoralen to an oligonucleotide designed to recognize a double-stranded DNA binding site through formation of a triple helix. We have previously used this strategy to study targeted psoralen modification of a triplex binding site within the bacterial supF gene carried in viral genomes. In the present work we report the targeting of psoralen photoadducts in vitro to a specific site in the genome of a transgenic mouse. Both 10 base and 16 base oligonucleotide-psoralen conjugates were capable of sequence-specific modification of genomic mouse DNA, while a truncated 8 base conjugate was not. Light activation was necessary, and a dose dependence was demonstrated for target site modification and mutagenesis. The 10 base conjugate rapidly found its target, with sequence-specific binding occurring after just 10 min incubation in the presence of mouse DNA. The ability to target psoralen photoadducts within mammalian genomes may prove useful in the study of chromatin structure and DNA repair. Moreover, this work may lead to potential in vivo applications of targeted psoralen modification.
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Psoralen photobiology: recent advances.
Clinical efficacy may arise from psoralen-induced photodamage by the induction of a panoply of biomolecules. In cellular studies with UV or photoactivatable agents, the activation of NF-kappa B, probably the most widely studied transcription factor, occurs after its release from an inhibiting factor, I kappa B. The activation of transcription factors has also been correlated with the UV absorption spectrum of DNA (35). Furthermore, it was shown that the photoadducts do not need to be processed or repaired because transcription factor induction is observed in repair-deficient cells. In these latter cells lower UV doses are required to induce these transcription factors. As a result of transcription factor induction, treated cells may have a greater number of class I molecules on their surface and an altered cytokine profile. It is possible (and likely) that these effects occur in different cells to different extents. The important message is that activational events may occur that could alter the cell's (or cells') ability to regulate a disease process. Many studies have shown that the time frame for this type of damage-induced event may range from seconds to minutes (36). Clearly 8-MOP/UVA-induced events such as those described above could be initiated as the result of a single photochemotherapy session.
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Kinetic analysis of apoptosis induction in human cell lines by UVA and 8-MOP.
Whereas previous studies have indicated that DNA damage as a result of 8-methoxypsoralen (8-MOP) and UVA treatment leads to cell death, this study establishes the minimum concentrations of 8-MOP and UVA necessary to induce apoptosis in human T-lymphocytic and monocytic cell lines. In order to asses apoptosis, we used fluorescent microscopy to examine changes in light scattering as well as internucleosomal DNA fragmentation. Generation of a dose response curve showed that the minimum combination of UVA and 8-MOP that was necessary to induce greater than background levels of apoptosis within 24 h of treatment was 0.5 J/cm2 UVA and 12.5 ng/mL of 8-MOP. A striking observation was that UVA alone at doses > or = 1.0 J/cm2, but not 8-MOP alone (0-300 ng/mL), induced significant apoptosis in the Sup-T1 cells induced by UVA alone was not as great as that of 8-MOP and UVA in combination, a highly significant correlation between the product of the concentration of 8-MOP (ng/mL) times the dose of UVA (J/cm2) and the percentage of apoptotic cells was observed. This correlation provides an important tool for studying the relationship of UVA-induced DNA damage to apoptosis induction. moreover, it will provide a means by which early events in the apoptotic pathway can be dissected.
Apoptosis↗
The lack of efficacy of 4,6,6'-trimethylangelicin to induce immune suppression in an animal model for photopheresis: a comparison with 8-MOP.
Photopheresis is an extracorporeal form of photochemotherapy with 8-methoxypsoralen (8-MOP) and UVA (PUVA). Patients ingest 8-MOP and then a psoralen-rich buffy coat is obtained by centrifugation and mixed with saline. This mixture is recirculated through a UVA radiation field and then reinfused. Photopheresis appears to be effective for several T cell-mediated disorders, because the treatment results in a specific immune response against the pathogenic clone of T cells involved. With PUVA therapy, the whole body of the patient is exposed to UVA, after ingestion of 8-MOP. Upon UVA exposure 8-MOP binds to, amongst others, DNA and induces DNA monoadducts and interstrand cross-links. As a result of these photoadducts photocarcinogenicity is a risk in PUVA. In PUVA for psoriasis, it proved that angular furocoumarins, although almost incapable of inducing DNA cross-links (less carcinogenic), are still effective. In order to determine if monoadducts induced by photopheresis could also be effective we used, specifically, 4,6,4'-trimethylangelicin (TMA). In this report, we compare the photodegradation of both TMA and 8-MOP under conditions relevant to the in vivo situation, as well as the effect both compounds have on the viability of rat lymphocytes as measured with the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. We show that TMA did not induce immunosuppression in vivo, even after extensive irradiation. In addition a dose dependency of 8-MOP/UVA versus the induced immune suppression was carried out. It was shown that there is a log dose/response correlation of r=0.9205.
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Mutagenesis by 8-methoxypsoralen and 5-methylangelicin photoadducts in mouse fibroblasts: mutations at cross-linkable sites induced by offoadducts as well as cross-links.
Psoralens are used clinically in the treatment of several skin diseases, including psoriasis, vitiligo, and cutaneous T cell lymphoma. However, psoralen treatment has been associated with an increased risk of squamous cell carcinoma of the skin. To elucidate molecular events that may play a role in the psoralen-related carcinogenesis, we examined psoralen-induced mutagenesis in a mouse fibroblast cell line carrying a recoverable, chromosomally integrated lambda phage shuttle vector. Using the supF gene as a mutation reporter gene, we determined the spectrum of mutations induced by photoactivation of 8-methoxypsoralen and of 5-methylangelicin. Both psoralens generated predominately T:A to A:T and some T:A to G:C transversions. Most of the mutations occurred at either 5' TpA or 5' ApT sites, both of which are conducive to interstrand cross-link formation. However, 5-methylangelicin produces only monoadducts, whereas 8-methoxypsoralen generated 20% cross-links and 80% monoadducts under the conditions of our experiments, as measured by direct HPLC analysis of the DNA from the treated cells. Although most of the mutations occurred at potentially cross-linkable sites, these results implicate monoadducts, as well as cross-links, as critical premutagenic lesions in psoralen-treated mammalian cells. These findings may help in the identification of carcinogenic changes induced by psoralen, and they may aid in the improved design of psoralen-based treatment regimens in the future.
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Psoralen-protein photochemistry--a forgotten field.
8-Methoxypsoralen in combination with long wavelength ultraviolet light is employed for the treatment of several cutaneous disorders, such as psoriasis, vitiligo and mycosis fungoides. It is common to attribute the efficacy of the photochemotherapy to the formation of psoralen DNA photoadducts. Thus, the main research effort has been directed towards the elucidation of nucleic acid photochemistry and related subsequent events (mutagenicity, toxicity). However, psoralens have been shown to undergo photoaddition reactions with other cellular components. In this review the status of psoralen-DNA photobiology is briefly summarized. The main focus, however, is on a survey of psoralen photochemical modification of proteins and the ways by which these additional photobiological events can impact the antigenicity and potentially immunogenicity of treated cells. Some preliminary results show the extent of psoralen-amino acid photoadduct formation and their impact on enzymatic processing.
Binding Sites↗
Treatment with 8-MOP and UVA enhances MHC class I synthesis in RMA cells: preliminary results.
The response of psoriasis and cutaneous T-cell lymphoma to treatment with 8-methoxypsoralen (8-MOP) and long wavelength ultraviolet light (UVA) is only partly understood. Psoralens form photoadducts within the DNA after activation by UVA and this damage leads to the inhibition of DNA synthesis. Additionally, it has been shown that different forms of DNA damage can induce a stress response, leading to upregulation of selected products. Among these are the major histocompatibility complex (MHC) class I genes. Thus the aim of the present study was to assess the rate of synthesis of MHC class I proteins in murine T-cell lymphoma cells (RMA) after treatment with 8-MOP and UVA. RMA cells were treated with 8-MOP (50-200 ng ml-1) and UVA (1 J.cm-2) and metabolically labelled with 35S-methionine 4 and 24 h after treatment. MHC class I synthesis was determined by immunoprecipitation of the cell lysates with an anti-Kb monoclonal antibody, Y3. After 4 h, treated and untreated cells demonstrated no differences in the rate of MHC class I synthesis. However, after 24 h a dose-dependent increase in MHC class I synthesis was observed. This increase in MHC class I expression could be responsible, at least partly, for the responses observed in patients treated with photopheresis.
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Increased surface expression of class I MHC molecules on immunogenic cells derived from the xenogenization of P815 mastocytoma cells with 8-methoxypsoralen and long-wavelength ultraviolet radiation.
In a previous study we demonstrated that the treatment of the highly tumorigenic cell line, P815, with 8-methoxypsoralen and long-wavelength ultraviolet radiation resulted in the production of several immunogenic clones (tum-). Mice inoculated with the tum- cells survived much longer than mice inoculated with the original tumorigenic cells (tum+). It was suggested that the increased survival of mice treated with the tum- clones arose as a result of an increased antigenicity derived from the phototreatment. In this report we show that the tum- cells have a greater density of class I MHC molecules on their surface (50-157% compared to P815). Class I MHC density on the cell surface is required to elicit targeted cytotoxic responses. These results can be considered in terms of human class I MHC assays which show that many human tumor cells have a reduced expression of class I MHC. Because other DNA damaging agents have also been shown to enhance class I expression, it is suggested that in addition to the cytotoxic effects of these agents, other pleiotropic effects must be considered. Photochemotherapy may phenotypically alter cells so that the enhanced expression of class I MHC molecules on the surface of phototreated cells may be associated with the clinical responses observed in cutaneous T cell lymphoma patients.
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8-Methoxypsoralen photoadduct formation in complementary oligonucleotides containing a cross-linkable site.
The complete profile of 8-methoxypsoralen photoadduct formation in complementary oligonucleotides (5'-GAGTATGAG and 5'-CATAC) has been determined. Equimolar solutions of the oligonucleotides were irradiated at 4 degrees C in order to stabilize the mini-double helix. Photomodified oligonucleotides were separated by reversed phase chromatography on a Vydac C4 column. Photoadduct formation favored the 5'TAT site in the 9mer over the 5'ATA site in the 5mer by a factor of two. Split-dose studies showed that the monoadducts formed on GAGTATGAG were preferentially converted to cross-links by an additional UVA exposure.
Adenine↗
5-Methoxypsoralen photoadduct formation: conversion of monoadducts to crosslinks.
5-Methoxypsoralen is often substituted for 8-methoxypsoralen in the photochemotherapy of psoriasis even though the nature of the resulting photadducts in cellular DNA has not been determined. A recent molecular mechanics study with a model oligonucleotide predicted that intercalated 5-methoxypsoralen molecules would tend to favor the preferential formation of 3,4-monoadducts. Such a result would be contrary to the photoadduct patterns observed with other psoralens. In this study we show that 5-methoxypsoralen photoadducts formation is, in fact, very similar to that for other psoralens, i.e., the primary photoadduct is the 4',5'-monoadduct which can be quantitatively converted to crosslink.
5-Methoxypsoralen↗
Site-specific targeting of psoralen photoadducts with a triple helix-forming oligonucleotide: characterization of psoralen monoadduct and crosslink formation.
A polypurine tract in the supF gene of bacteriophage lambda (base pairs 167-176) was selected as the target for triple helix formation and targeted mutagenesis by an oligopurine (5'-AGGAAGGGGG-3') containing a chemically linked psoralen derivative (4'-hydroxymethyl-4,5',8-trimethylpsoralen) at its 5' terminus (psoAG10). The thymines at base pairs 166 and 167, a 5'ApT site, were targeted for photomodification. Exposure of the triple helical complex to long wavelength ultraviolet radiation led to the covalent binding of psoAG10 to the targeted region in the supF gene and to the induction of site-specific mutations. We report here experiments to characterize the photomodification of the targeted region of the supF gene in the context of triple helix formation. An electrophoretic mobility-shift assay showed that, at low radiation doses, monoadducts at base pair 166 were the major photoadducts. At higher doses the monoadducts were converted to crosslinks between base pairs 166 and 167. HPLC analysis of enzymatically hydrolyzed photoreaction mixtures was used to confirm the electrophoresis results. A strong strand preference for specific photoadduct formation was also detected.
Bacteriophage lambda↗
The specific effects of 8-methoxypsoralen photoadducts on cell growth: HPLC analysis of monoadduct and crosslink formation in cells exposed to split-dose treatment.
The effects of 8-methoxypsoralen (8-MOP) monoadducts and crosslinks on growth and viability of mastocytoma cells were investigated. To induce monoadduct formation (4',5'-monoadducts and 3,4-monoadducts), the cells were incubated with 8-MOP (1 microgram ml-1) and exposed to 419 nm radiation, resulting in the formation of more than 96% monoadducts. After washing and resuspension, the cells were exposed to a small dose of long-wavelength UV radiation (UVA, 2 J cm-2) to convert monoadducts into crosslinks. Similar adduct levels were obtained after either 8-MOP plus visible light treatment or 8-MOP plus split-dose protocol. Cells treated with 419 nm light resumed normal growth rates more rapidly than cells which also received the UVA dose. High performance liquid chromatography (HPLC) analysis of DNA obtained from each group of cells showed that the UVA step resulted in an increase in crosslinks from 3.2% after 419 nm radiation to 56.5% after UVA irradiation.
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4'-Aminomethyl-4,5',8-trimethylpsoralen photochemistry: the effect of concentration and UVA fluence on photoadduct formation in poly(dA-dT) and calf thymus DNA.
The photochemistry of 4'-aminomethyl-4,5',8-trimethylpsoralen (AMT) with poly(dA-dT) and calf thymus DNA was studied. The extent of photoadduct formation and the distribution of photoadducts (3,4- and 4',5'-monoadducts and crosslinks) were determined by liquid scintillation analysis and HPLC, respectively. The adducts were characterized on the basis of their UV absorption spectra and mass spectral analysis. The high DNA binding constant for AMT (1.5 x 10(5) M-1) led to a high fraction of intercalated molecules, which contributed to the high level of AMT photoadduct formation, as many as 102 adducts per kilobase pair. In addition, there is a distinct difference in the adduct distribution compared to the previously studied 8-methoxypsoralen (8-MOP). Under the conditions employed for the photochemical studies, virtually all of the AMT molecules in solution are intercalated, occupying 25% of the base pair sites. Under similar conditions, 8-MOP molecules occupied 10 times fewer sites. Thus, for AMT, DNA base pair sites other than 5'TA, the well-characterized strong binding for psoralens in general, are an additional target for photomodification, which results in the formation of a higher percentage of monoadducts. The proportion of photoadducts formed was virtually independent of AMT concentration and UVA (320-400 nm radiation) fluence.
Animals↗
Inhibition of smooth muscle cell proliferation by visible light-activated psoralen.
The present study was designed to evaluate the effect of 8-methoxypsoralen (8-MOP) activated with visible light (419 nm) on the suppression of smooth muscle cell (SMC) proliferation in vitro. We hypothesize that if visible light (VL) instead of UVA is used to photoactivate 8-MOP, cytotoxic 8-MOP-DNA cross-link formation can be minimized. Bovine aorta SMCs (2 x 10(4)/cm2) were incubated with 8-MOP (1 micrograms/mL) for 30 minutes (in the dark) and exposed to a range of VL (2 to 69 J/cm2) to determine the dose of VL that inhibits SMC proliferation with minimal toxicity. The results show that 8-MOP in combination with 2 to 12 J/cm2 VL reversibly inhibited SMC proliferation for up to 5 days after treatment. SMC viability was confirmed by trypan blue exclusion. 8-MOP in combination with 23- or 69-J/cm2 VL irreversibly inhibited SMC proliferation. In cell cycle studies, 12-J/cm2 VL was used to activate 8-MOP. A phase-specific G2 blockade that correlated temporally with recovery of SMC replication was observed. Photoadduct repair studies showed that cell proliferation rates recovered when 60% of the adducts had been removed. These results demonstrate for the first time the possibility of using VL to activate 8-MOP to inhibit cell proliferation and suggest that 8-MOP/VL photochemotherapy can be used to control SMC growth.
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