- Email: [email protected]
The induction of p53 after PUVA photochemotherapy - in vitro and ex vivo
ESDR I JSID I SID Abstracts
s222
1327
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INFRARED RADIATION INDUCES MATRIX METALLOPROTEINASE-1 EXPRESSION IN HUMAN DERMAL FlBROBLA8T.9. Weleer -Ruzich. Clic. & EXD.photodermatol., Dept. of Dermatol., Univ. of Diisseldorf, Diisscldorf, Germany.
TNF-o, INHIBITS THE INITIATION STAGE OF THE CUTANEOUS CHEMICAL CARCINOGENESIS PATHWAY. D Rotiauti. CY Anderson. D Semler. KX Tubesing. H Mukhtar. S Balasubamanian. CA Elmets. Depts of Dermatology. Case Western Reserve University, Cleveland, OH and University of Alabama at Birmingham, AL. Atthough cytokioes play a key role in the host response t0 CtianeOUS kImOr% there is little information on their impact on eady stages in skin cancer development, To investigate the influence of cytokines on the initiation stage Of cutaneous chemical carcinogenesis paihway. panels of C3HmeN mice ware inJected with neutralizing antibodies (Ab) to IL-1~. IL-lp. 11-10, IL-12 and TNF-a. They were then placed on a dimethylbenz(a)anvlracene (DMBA) initiation, TPA promotion skin carcinogenesis protocol. Animals treated with Ab to IL-la, IL-lj3, IL10 and IL-12 had tumor responses that were identical to control Ab traated mice. In contrast. mice treated with TNFa Ab developed significantly mom tumors/mouse than contmls (9.67f1.34 vs 6.64 i 1.05). DMBA binding to epidermal DNA, which correlates witi the mutation frequency of chemical carcinogens, was increased in the TNFa Ab treated group compared to controls (7600 cpm vs 3175 cpm). Pentoxifylline (PTX) inhibits TNF-o transcription. Experiments were therefore conducted to determine whether it aI% augmented DMBA tumorigenesis. In contrast to TNF-a Ab. PTX did not augment the development Of Skin tumors and may even have inhibited it. It did suppress elicitation of call-mediated immunity to DMBA. The results suggest that TNF-a protects against DMBA skin carcinogenesis by radudng the number of mutant cells that can avolva into wtaneow tumors. The fad that PTX did not augment DMEA tumorigenesie may relate to the fact that pm-formed TNF-a is suffident for lhis to occur.
in fibroblasts from &erotic ns, matrix netalloprotcinase-1 (MMP-1; = to be reduced. Synthesis of MMP-I can be wllancnase II exoression has been increased by ‘in&o w.posurc of human: dermal fibroblasts~to ultraviolet Al radiation
(UVAl; 34lk4C0 nm), and recent rcpckts UVAl phototherapy can be effectively uscd u, treat patients with lesions such as in localited sclerodcrma. The irradiation devices used these in-vitro and in-vivo studies do not exclusively emit LJVAlR, but also infrared ‘radiation (IR). It is currently not known whether the contribution Qf IR to tbc therapeutic efficacy of WAl-phototberapy of localized sclerodcna is adCerseor beneficial in nature. ‘Ibcreforcs, in the present study, normal human dcrmal fibmblasts were exposed to IR in the range of 681%7ooOnm (maximum of 2800 am at 1083 Kelvin) and subsequentlyanalyzed for MMP-1 expression. Exposure of cells to sublethal doses of IR induced MMP-1 mRNA expression (differential RT-FCR) in a dose- sod time-dependent ntarmcr. Increased MMP-1 mRNA cxprcSSion was ohscrvcd 8 hou1s after cxpdsure and found to be maximal 24 hours a&x irradiation with approx. 400 I/cm*. In-viva exposure of normal human skin to III also significantly upregulated MMP-1 mRNA expression. Infrared radiation-induced uprcgulationaf MMP-1 mRNA expression may cause matrix degradation, because mRNA expression of tissue inhibitor of mctalloprotcinasc-1 (TIMP-1) in IR.exposcd tibroblasts remained unaltered. These SNdics demonstrate that IR induces MMP-1 expression in human dermsl tibroblasts in vitro and in-viva. Contaminating IR in UVAl phototherapy may thus be of benefit for patients with connective tissue diseases.
1331
1328 PHOTOACITVATED HYPEIUCIN INHlF3lTSPROLIFERATION AND INDUCES A HIGH BATE OF APOITOTIC DEATH OF NORMAL AND h4ALIGNfl TLYMPHOCYTES FROM PATIENTS WITH CTCL. m ,Q.i&& Dept. of Dermatology, University of Pennsylvania, Philadelphia, PA and VIMRX Pbarmcceuticals,Wilmington, DE. Hypcricin is a photiynsmic compound that is activated by c&a visible (4CQ700 run) or UVA (320.400 cm) light, and has been demonstrated to inhibit the growth of s vaziety of ncop&ic cell lyres. H@cin was found to inhibit pmlifcmtive responses of malignant T-cells derived from blood of patients with cutzncous T-cell lymphoma (CrCL). Control cells included peripbcml blood mononuclear cells (PBMC)
from normal voluntccn
or Epstein-Ban
Viis
(EBV)-transformed
lymphocytes. cells were incubated with hypzicia or I-mcthoxypsomIen &MOP) then s!imolated with the mitogen ConA (10 u&l). Culhucs were prepared in the dark to minimize photoactivation. Hypericin, photoactivated with 1.1 3.3 J/cm2 visible light, inhibited cclhdsr proliferation of malignant T-cells with IC50 values from 0.34. 0.53 uM, normal PBMC with ICSOof 0.110.76 uM and EBV-usnsformcd cells with ICXI of 0.75-3.2 uM. UVA-photoactivated hypxicin (0.5 - 2.0 J/cm2 could also inhibit proliferation with ICSOvalues of 0.57-1.8 uhf, 0.7-4.6 uM and 2.0-3.7 oh4 for malignant, normal or EBV-transformed ceils. rcspectivcly. Hypcricin, photoactivated with either WA or visible light could induce near complete apoptosis (94%) in mslignam CTCL T-cells whereas lower levels of apeptosis (37-88%) were induced in normal PBMC. These dats indicate that hypericin inhibits mitogen-induced proliferation of malignant T-cells from patients with CTCL, PBMC fmm normal individuals, as well as BBV-transformedlymphocytes and that inhibition of cell proliferation is depcodent on the concentmtion of hypcricin used and dose of light rcquircd to photoactivate the compound. Induction of apqtosis is, in patt, one mechanism by which photosctivated hypcricin inhibits malignant T-cxUpmliferation.
Rcchcxhc SUI 18 &au,
MSERM U312.li6piti
Saint Louis, 1 ax Claude Vellcfsux 75010 Paris @It)
Endogenous antioxidant defense systems. including glutatbionc (OSH) and glutatbionc pcmxidase (GSHPx). play an important role in the UVAdeletcrious effects which an related to the formation of active oxygen and radical s&es (ROS). We invtstigatcd tbc consequences of a modulation of Lhe reduced GSH lcvcl and/or OSHPn activity in human skin cultured fibmblasls on dx extend of lipid peroxidation (thiobarbituric acid naetive substances assay) and on the Lethality (neutral red way performed immediatly after UVA exposure) induced in these cells upon exposure to UVA light. GSHPx activity was modulated in skin fibmblasts by selenium-depletion and repletion of dx medium of cultured cells. Culture in a medium containing 2% (2 FCS) instead of 10% v/v of foetal calf scmm (10 FCS) leads to a decrease in the GSHPx activity which is prevented by addition of sodium sclcnitc in tbc 2 PCS culture medium. In these conditions, GSHPx activity varies from a factor two to six. This dccrcasc in tbc intracellular OSHPx activity leads to s slight increase in UVA-induced lipid pemxidation sad to s moderate increase of UVA-induced tcthstitby of tibmblasts. The cnhaneement of these UVAdeleterious effects is abolished when sclenite is added in the 2 FCS medium during the culture of cells. The treatment of cultured tibmblasts by buthionine-sulfoximine (BSO),an inhibitor of glutatbione synthesis. leads to a dewcase in the endogcnous eontent of reduced GSH in fibmhlasts. When the decrease in intracellular OSH is at least of a factor tive. UVAinduced lipid peroxidation is slightly enhanced without great moditications of UVAinduced ktbality. Experiments on skin tibmblssts exhibiting both a reduced GSHPn activity and a reduced GSH level allow to further dclincatc the role of GSH as a cofactor of OSHPx. In conclusion, we show that the decrcssc in the intracellular GSH level sod/or the GSHPx activity in cultured human skin tibmblasts can stigthly amplify tbe UVA-induced lipid pemxidation and lethality. ll~ese effects remain moderste when physiological variations of fhc GSH level and ffie GSHPx activity are consider&.
1329
1332
SUPPRESSION OF IRON CATACYZED FREE RADICAL GENERATION BY AMINO ACID CONJUGATES WITH SALICYL ALDEHYDE
THE INDUCTION OF pi3 AFTER PUVA PHOTOCHEhtOTHERAPV - IN “!TRO AND Ex “I”0 P. Donaffi’. 0 Bethea’.E. Vw,dxb&KM. Knabler’w, FP. u, Deptr. ofDcmnto,ogy, ‘ThomasJefferson Univ. and ~Allegheny Univ., Philadelphia PA, ‘Univ. ofVim Vienna, Austria
Central Research Laboratories, Ajinamoto Co., Inc. Kawasaki, Japan. Generation of &eeeradicals by W light accelerates skin aging known as photoaging. Cutaneous iron catalyzes the generation of the free radicals. We designed novel a&oxidants which suppress the iron catalyzed free radical generation by mimicking the binding site of iron sequestering proteins. These antioxidants, N-(Z-Hydro~-benzyl)amino acids, were prepared by the reaction of amino acids such as serine or glycine with salicyl aldehyde. The iron binding capacity of the compounds was assessed by measuring the increased absorbance at 466nm due to thecomplex formation. We evaluated the suppression of iron catalyzed hydroxyl radical formation by FSR spin trapping technique. Ultraviolet induced lipid pemtidation was assessed by the determination of malondialdehyde reactive substance in UV eqosed mouse 3T3 cell homogenate. The compounds formed 2:l ligand to iron complexes. N-(2-Hydroxy-benzyl) swine suppressed the hydmxyl radical generation by 82.9+9.6% (meanfSD, n=3) when the compound was added in two-hold excess to imn. It suppressed UV induced lipid peroxydation by 57.0+8.4% (mean*SD n=lO) relative to the contml. These results indicate that W induced oddative stress can be diminished by these compounds. The iron sequestering proteins model introduces novel approaches to the molecular design of antioxidants for skin photoaging.
s222
1327
1330
INFRARED RADIATION INDUCES MATRIX METALLOPROTEINASE-1 EXPRESSION IN HUMAN DERMAL FlBROBLA8T.9. Weleer -Ruzich. Clic. & EXD.photodermatol., Dept. of Dermatol., Univ. of Diisseldorf, Diisscldorf, Germany.
TNF-o, INHIBITS THE INITIATION STAGE OF THE CUTANEOUS CHEMICAL CARCINOGENESIS PATHWAY. D Rotiauti. CY Anderson. D Semler. KX Tubesing. H Mukhtar. S Balasubamanian. CA Elmets. Depts of Dermatology. Case Western Reserve University, Cleveland, OH and University of Alabama at Birmingham, AL. Atthough cytokioes play a key role in the host response t0 CtianeOUS kImOr% there is little information on their impact on eady stages in skin cancer development, To investigate the influence of cytokines on the initiation stage Of cutaneous chemical carcinogenesis paihway. panels of C3HmeN mice ware inJected with neutralizing antibodies (Ab) to IL-1~. IL-lp. 11-10, IL-12 and TNF-a. They were then placed on a dimethylbenz(a)anvlracene (DMBA) initiation, TPA promotion skin carcinogenesis protocol. Animals treated with Ab to IL-la, IL-lj3, IL10 and IL-12 had tumor responses that were identical to control Ab traated mice. In contrast. mice treated with TNFa Ab developed significantly mom tumors/mouse than contmls (9.67f1.34 vs 6.64 i 1.05). DMBA binding to epidermal DNA, which correlates witi the mutation frequency of chemical carcinogens, was increased in the TNFa Ab treated group compared to controls (7600 cpm vs 3175 cpm). Pentoxifylline (PTX) inhibits TNF-o transcription. Experiments were therefore conducted to determine whether it aI% augmented DMBA tumorigenesis. In contrast to TNF-a Ab. PTX did not augment the development Of Skin tumors and may even have inhibited it. It did suppress elicitation of call-mediated immunity to DMBA. The results suggest that TNF-a protects against DMBA skin carcinogenesis by radudng the number of mutant cells that can avolva into wtaneow tumors. The fad that PTX did not augment DMEA tumorigenesie may relate to the fact that pm-formed TNF-a is suffident for lhis to occur.
in fibroblasts from &erotic ns, matrix netalloprotcinase-1 (MMP-1; = to be reduced. Synthesis of MMP-I can be wllancnase II exoression has been increased by ‘in&o w.posurc of human: dermal fibroblasts~to ultraviolet Al radiation
(UVAl; 34lk4C0 nm), and recent rcpckts UVAl phototherapy can be effectively uscd u, treat patients with lesions such as in localited sclerodcrma. The irradiation devices used these in-vitro and in-vivo studies do not exclusively emit LJVAlR, but also infrared ‘radiation (IR). It is currently not known whether the contribution Qf IR to tbc therapeutic efficacy of WAl-phototberapy of localized sclerodcna is adCerseor beneficial in nature. ‘Ibcreforcs, in the present study, normal human dcrmal fibmblasts were exposed to IR in the range of 681%7ooOnm (maximum of 2800 am at 1083 Kelvin) and subsequentlyanalyzed for MMP-1 expression. Exposure of cells to sublethal doses of IR induced MMP-1 mRNA expression (differential RT-FCR) in a dose- sod time-dependent ntarmcr. Increased MMP-1 mRNA cxprcSSion was ohscrvcd 8 hou1s after cxpdsure and found to be maximal 24 hours a&x irradiation with approx. 400 I/cm*. In-viva exposure of normal human skin to III also significantly upregulated MMP-1 mRNA expression. Infrared radiation-induced uprcgulationaf MMP-1 mRNA expression may cause matrix degradation, because mRNA expression of tissue inhibitor of mctalloprotcinasc-1 (TIMP-1) in IR.exposcd tibroblasts remained unaltered. These SNdics demonstrate that IR induces MMP-1 expression in human dermsl tibroblasts in vitro and in-viva. Contaminating IR in UVAl phototherapy may thus be of benefit for patients with connective tissue diseases.
1331
1328 PHOTOACITVATED HYPEIUCIN INHlF3lTSPROLIFERATION AND INDUCES A HIGH BATE OF APOITOTIC DEATH OF NORMAL AND h4ALIGNfl TLYMPHOCYTES FROM PATIENTS WITH CTCL. m ,Q.i&& Dept. of Dermatology, University of Pennsylvania, Philadelphia, PA and VIMRX Pbarmcceuticals,Wilmington, DE. Hypcricin is a photiynsmic compound that is activated by c&a visible (4CQ700 run) or UVA (320.400 cm) light, and has been demonstrated to inhibit the growth of s vaziety of ncop&ic cell lyres. H@cin was found to inhibit pmlifcmtive responses of malignant T-cells derived from blood of patients with cutzncous T-cell lymphoma (CrCL). Control cells included peripbcml blood mononuclear cells (PBMC)
from normal voluntccn
or Epstein-Ban
Viis
(EBV)-transformed
lymphocytes. cells were incubated with hypzicia or I-mcthoxypsomIen &MOP) then s!imolated with the mitogen ConA (10 u&l). Culhucs were prepared in the dark to minimize photoactivation. Hypericin, photoactivated with 1.1 3.3 J/cm2 visible light, inhibited cclhdsr proliferation of malignant T-cells with IC50 values from 0.34. 0.53 uM, normal PBMC with ICSOof 0.110.76 uM and EBV-usnsformcd cells with ICXI of 0.75-3.2 uM. UVA-photoactivated hypxicin (0.5 - 2.0 J/cm2 could also inhibit proliferation with ICSOvalues of 0.57-1.8 uhf, 0.7-4.6 uM and 2.0-3.7 oh4 for malignant, normal or EBV-transformed ceils. rcspectivcly. Hypcricin, photoactivated with either WA or visible light could induce near complete apoptosis (94%) in mslignam CTCL T-cells whereas lower levels of apeptosis (37-88%) were induced in normal PBMC. These dats indicate that hypericin inhibits mitogen-induced proliferation of malignant T-cells from patients with CTCL, PBMC fmm normal individuals, as well as BBV-transformedlymphocytes and that inhibition of cell proliferation is depcodent on the concentmtion of hypcricin used and dose of light rcquircd to photoactivate the compound. Induction of apqtosis is, in patt, one mechanism by which photosctivated hypcricin inhibits malignant T-cxUpmliferation.
Rcchcxhc SUI 18 &au,
MSERM U312.li6piti
Saint Louis, 1 ax Claude Vellcfsux 75010 Paris @It)
Endogenous antioxidant defense systems. including glutatbionc (OSH) and glutatbionc pcmxidase (GSHPx). play an important role in the UVAdeletcrious effects which an related to the formation of active oxygen and radical s&es (ROS). We invtstigatcd tbc consequences of a modulation of Lhe reduced GSH lcvcl and/or OSHPn activity in human skin cultured fibmblasls on dx extend of lipid peroxidation (thiobarbituric acid naetive substances assay) and on the Lethality (neutral red way performed immediatly after UVA exposure) induced in these cells upon exposure to UVA light. GSHPx activity was modulated in skin fibmblasts by selenium-depletion and repletion of dx medium of cultured cells. Culture in a medium containing 2% (2 FCS) instead of 10% v/v of foetal calf scmm (10 FCS) leads to a decrease in the GSHPx activity which is prevented by addition of sodium sclcnitc in tbc 2 PCS culture medium. In these conditions, GSHPx activity varies from a factor two to six. This dccrcasc in tbc intracellular OSHPx activity leads to s slight increase in UVA-induced lipid pemxidation sad to s moderate increase of UVA-induced tcthstitby of tibmblasts. The cnhaneement of these UVAdeleterious effects is abolished when sclenite is added in the 2 FCS medium during the culture of cells. The treatment of cultured tibmblasts by buthionine-sulfoximine (BSO),an inhibitor of glutatbione synthesis. leads to a dewcase in the endogcnous eontent of reduced GSH in fibmhlasts. When the decrease in intracellular OSH is at least of a factor tive. UVAinduced lipid peroxidation is slightly enhanced without great moditications of UVAinduced ktbality. Experiments on skin tibmblssts exhibiting both a reduced GSHPn activity and a reduced GSH level allow to further dclincatc the role of GSH as a cofactor of OSHPx. In conclusion, we show that the decrcssc in the intracellular GSH level sod/or the GSHPx activity in cultured human skin tibmblasts can stigthly amplify tbe UVA-induced lipid pemxidation and lethality. ll~ese effects remain moderste when physiological variations of fhc GSH level and ffie GSHPx activity are consider&.
1329
1332
SUPPRESSION OF IRON CATACYZED FREE RADICAL GENERATION BY AMINO ACID CONJUGATES WITH SALICYL ALDEHYDE
THE INDUCTION OF pi3 AFTER PUVA PHOTOCHEhtOTHERAPV - IN “!TRO AND Ex “I”0 P. Donaffi’. 0 Bethea’.E. Vw,dxb&KM. Knabler’w, FP. u, Deptr. ofDcmnto,ogy, ‘ThomasJefferson Univ. and ~Allegheny Univ., Philadelphia PA, ‘Univ. ofVim Vienna, Austria
Central Research Laboratories, Ajinamoto Co., Inc. Kawasaki, Japan. Generation of &eeeradicals by W light accelerates skin aging known as photoaging. Cutaneous iron catalyzes the generation of the free radicals. We designed novel a&oxidants which suppress the iron catalyzed free radical generation by mimicking the binding site of iron sequestering proteins. These antioxidants, N-(Z-Hydro~-benzyl)amino acids, were prepared by the reaction of amino acids such as serine or glycine with salicyl aldehyde. The iron binding capacity of the compounds was assessed by measuring the increased absorbance at 466nm due to thecomplex formation. We evaluated the suppression of iron catalyzed hydroxyl radical formation by FSR spin trapping technique. Ultraviolet induced lipid pemtidation was assessed by the determination of malondialdehyde reactive substance in UV eqosed mouse 3T3 cell homogenate. The compounds formed 2:l ligand to iron complexes. N-(2-Hydroxy-benzyl) swine suppressed the hydmxyl radical generation by 82.9+9.6% (meanfSD, n=3) when the compound was added in two-hold excess to imn. It suppressed UV induced lipid peroxydation by 57.0+8.4% (mean*SD n=lO) relative to the contml. These results indicate that W induced oddative stress can be diminished by these compounds. The iron sequestering proteins model introduces novel approaches to the molecular design of antioxidants for skin photoaging.