Carcinogenesis vol.11 no.2 pp.229-234, 1990
in kmase C
Mary Steidl Matsui and Vincent A.OeLeo Department of Dermatology, Columbia University, New York, NY 10032, USA
Introduction Five to six hundred thousand new cases of skin cancer are diagnosed each year in the United States, and skin cancers represent 25 % of all cancers which occur in the Southwestern states. UV radiation plays a significant role in the development of basal and squamous cell skin cancer (1 - 3 ) . Experimentally, UV radiation has been capable of both initiating and promoting tumors in vivo (4,5). UV radiation of mid and short wavelengths, UVB (290-320 nm*) and UVC (100-290 nm), as well as long wave UV light (UVA, 320—400 nm), has been shown to damage DNA directly and result in DNA lesions such as pyrimidine dimers, single strand breaks, and protein—DNA crosslinks (6-8), and induction of oncogene expression, stimulation of unscheduled DNA repair and asynchronous polyoma DNA replication (9,10). Although UVB is more efficient at inducing acute changes in skin exposed to sunlight, UVA can produce •Abbreviations: AB, assay buffer; DMSO, dimethylsulfoxide; DMEM, Dulbecco's modified Eagle's medium; EGF, epidermal growth factor; NC, nitrocellulose membrane; PMSF, phenylmethylsulfonyl fluoride; PDBu, phorbol dibutyrate; PBS, phosphate-buffered saline; PKC, protein kinase C; PS, phosphatidyl serine; SDS, sodium dodeoylsulfate; TPA, 12-O-tetradecanoyl phorbol-13-acetate; UVA, ultraviolet A, 320-400 nm; UVB, ultraviolet B, 290-320 nm; UVC, ultraviolet C, 100-290 nm. © Oxford University Press
Materials and methods Cell culture Mouse fibroblast C3H10T1/2 cells were used for these studies because we have previously reported the effects of UVB on phorbol ester binding, EGF binding and PKC activity in this cell line (15). Cells were used between passages 9 and 15 and were grown to subconfluence in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% calf serum, penicillin (75 U/ml) and streptomycin (25 /ig/ml). Chemicals and biochemical* [I25I]EGF (125-165 /iCi/jig), [3H]phorbol dibutyrate (PDBu) (16 Ci/mmol) and [7-32P]ATP (30 Ci/mmol) were purchased from New England Nuclear Corp., Boston, MA. Receptor grade EGF and PDBu, histone IU-S, ATP, bovine serum albumin, phenylmethylsulfonyl fluoride (PMSF), Tris-HCl and DEAE-Sephacel were purchased from Sigma Chemical Co., St Louis, MO. Hydrofluor scintillation fluid was obtained from National Diagnostics, Highland Park, NJ. Phosphocellulose paper (Whatman grade P81) was from Fischer, Springfield, NJ, phosphatidylserine (PS) was from Avanti Polar Lipids, Birmingham, AL, and TPA was from LC Services, Lafayette, CA. Leupeptin was a generous gift of the US-Japan Cooperative Cancer Program. Tissue culture media, antibiotics and buffers were obtained from Gibco Laboratories, Grand Island, NY, calf serum and plastic tissue culture dishes from Flow Laboratories, McLean, VA. Light source and irradiation UVA radiation was obtained from a 2000 W high pressure Hg vapor lamp with iron and gallium iodide, and filtered through a Schott WG 320 nm filter (Honle
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Exposure to ultraviolet (UV) radiation has been well correlated with skin cancer incidence. Long wave UV radiation (320-400 nm, UVA) is a major component of natural sunlight and cosmetic tanning 'salon' light, and has been shown not only to damage DNA and to act as a complete carcinogen, but also to promote ultraviolet B (280—320 nm, UVB) carcinogenesis. The mechanism by which the latter occurs is unknown, but it is believed to be related to the inflammation and irritation which results from UV exposure. In order to examine the possibility that UVA stimulates the same signalling pathway as do the phorbol esters, a class of much more thoroughly characterized skin tumor promoters, we exposed cells in culture to UVA radiation and measured cellular responses related to protein kinase C (PKC) activation. The data presented here demonstrate that a low, physiologic dose of UVA inhibits epidermal growth factor binding and increases PKC activity in cultured mammalian fibroblasts. The increase in cytosolic activity is not completely translocated to the membrane, and can be partially suppressed by puromycin and cycloheximide but not by actinomycin D. These observations are the first evidence to suggest that a protein which has been strongly linked to chemical tumor promotion may also be a critical mediator for UV-induced promotion. The response of cells to UVA is also unique, in that it does not cause a 12-0-tetradecanoyl phorbol-13-acetate-like rapid redistribution of PKC activity followed by down regulation.
erythema and pigmentation in normal skin and is thought to play a major role in photoaging and wrinkling (11,12). Information as to the identity and role of endogenous chromophores and about the mechanisms of photodamage is very meagre (13). This is so even though UVA radiation is known to penetrate Caucasian skin at least to the dermis and to irradiate significant numbers of cells in the peripheral circulation (14). Increased interest in effects of UV on skin cancer development has developed recently, in part because of increased human exposure to UVA made possible through the use of sunscreens which block predominantly UVB radiation and through the use of artificial light sources producing massive doses of UVA radiation in cosmetic sun tanning parlors. We have previously determined that UV radiation of 280 — 320 nm (UVB, mid wave UV) shares a number of biologic effects with the phorbol ester tumor promoter 12-O-tetradecanoyl phorbol-13-acetate (TPA) (15). Both UVB and TPA induce the stimulation of arachidonic acid release, prostaglandin E2 production, the inhibition of epidermal growth factor (EGF) binding and cellular proliferation in cultured cells (16—18). However, UVB treatment had no effect on protein kinase C (PKC) activity or phorbol ester binding (15). In the series of experiments described here, we wished to explore the possibility that UVA- and TPA-induced promotion of epidermal tumors share a common pathway by examining the effect of UVA on the subcellular distribution of the phorbol ester receptor, PKC. The hypothesis we wished to test was that UVA acts as an epidermal tumor promoter by stimulating PKC activation, as does TPA, initiating a cascade of events which result in phosphorylation of and inhibition of ligand binding to the EGF receptor, and initial translocation and subsequent down regulation of PKC, ultimately resulting in permanent phenotypic changes in an initiated cell.
M.S.Matsui and V.A.DeLeo
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UVA EXPOSURE ( J / c m 2 ) Fig. 1. Changes in specific binding of [3H]PDBu (O—O) or (I25I]EGF (O O) to C3H10T1/2 cells exposed to doses of UVA radiation from 0 to 10 J/cm2. Irradiation was performed and specific binding determined as described in Materials and methods. Each point represents the average of data obtained from triplicate dishes for total and non-specific binding. The experiment was repeated in its entirety with similar results. actinomycin D (6 ^g/ml) was added to growth media. After irradiation, cells were incubated at 37°C for 45 min and then placed on ice and harvested. The percent stimulation or inhibition of UVA-induced PKC activity was calculated using the following formula: UVD-D D
_x 100 = % inhibition or stimulation
UV-Con Con where UVD represents pmol 32P transferred/^g protein/min by cells irradiated and treated with drug; D, drug-treated sham-irradiated cells; UV, irradiated cells; Con, sham-irradiated cells. Immunoblotting Semi-purified protein extracts were refiltered through Amicon filters and resuspended in loading buffer (143 mM Tris at ph 6.8, 4.5% SDS, 3% 2-mercaptoethanol and 7.5% glycerol). Equal amounts of protein from each sample were subjected to discontinuous polyacrylamide gel electrophoresis in the presence of SDS (SDS-PAGE) (20) in which the stacking gel was 4% acrylamide:bis (50:1) and the resolving gel was 8%. The separated proteins were then electrophoretically transferred overnight to a nitrocellulose membrane (NC) (21). The NC was incubated at 4°C overnight with 0.5% Carnation instant milk in PBS and then incubated for 4 h in monoclonal anti-PKC (Amersham, Arlington Heights, IL). NC was washed and immunodetection performed by the horseradish peroxidase method or by incubation of the NC with 1 /jCi/filter donkey [l25I]antimouse antibody (Amersham, Arlington Heights, IL) followed by autoradiography. Densitometric analysis of autoradiographs was achieved by the use of a Molecular Dynamics 300A computing densitometer (Molecular Dynamics, Sunnyvale, CA).
Results Changes in PDBu and EGF binding following UVA irradiation Activation of PKC by TPA has previously been shown to result in phosphorylation of the EGF receptor and inhibition of EGF binding (22), so we examined the effect of UVA radiation on
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in kmase C
Mary Steidl Matsui and Vincent A.OeLeo Department of Dermatology, Columbia University, New York, NY 10032, USA
Introduction Five to six hundred thousand new cases of skin cancer are diagnosed each year in the United States, and skin cancers represent 25 % of all cancers which occur in the Southwestern states. UV radiation plays a significant role in the development of basal and squamous cell skin cancer (1 - 3 ) . Experimentally, UV radiation has been capable of both initiating and promoting tumors in vivo (4,5). UV radiation of mid and short wavelengths, UVB (290-320 nm*) and UVC (100-290 nm), as well as long wave UV light (UVA, 320—400 nm), has been shown to damage DNA directly and result in DNA lesions such as pyrimidine dimers, single strand breaks, and protein—DNA crosslinks (6-8), and induction of oncogene expression, stimulation of unscheduled DNA repair and asynchronous polyoma DNA replication (9,10). Although UVB is more efficient at inducing acute changes in skin exposed to sunlight, UVA can produce •Abbreviations: AB, assay buffer; DMSO, dimethylsulfoxide; DMEM, Dulbecco's modified Eagle's medium; EGF, epidermal growth factor; NC, nitrocellulose membrane; PMSF, phenylmethylsulfonyl fluoride; PDBu, phorbol dibutyrate; PBS, phosphate-buffered saline; PKC, protein kinase C; PS, phosphatidyl serine; SDS, sodium dodeoylsulfate; TPA, 12-O-tetradecanoyl phorbol-13-acetate; UVA, ultraviolet A, 320-400 nm; UVB, ultraviolet B, 290-320 nm; UVC, ultraviolet C, 100-290 nm. © Oxford University Press
Materials and methods Cell culture Mouse fibroblast C3H10T1/2 cells were used for these studies because we have previously reported the effects of UVB on phorbol ester binding, EGF binding and PKC activity in this cell line (15). Cells were used between passages 9 and 15 and were grown to subconfluence in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% calf serum, penicillin (75 U/ml) and streptomycin (25 /ig/ml). Chemicals and biochemical* [I25I]EGF (125-165 /iCi/jig), [3H]phorbol dibutyrate (PDBu) (16 Ci/mmol) and [7-32P]ATP (30 Ci/mmol) were purchased from New England Nuclear Corp., Boston, MA. Receptor grade EGF and PDBu, histone IU-S, ATP, bovine serum albumin, phenylmethylsulfonyl fluoride (PMSF), Tris-HCl and DEAE-Sephacel were purchased from Sigma Chemical Co., St Louis, MO. Hydrofluor scintillation fluid was obtained from National Diagnostics, Highland Park, NJ. Phosphocellulose paper (Whatman grade P81) was from Fischer, Springfield, NJ, phosphatidylserine (PS) was from Avanti Polar Lipids, Birmingham, AL, and TPA was from LC Services, Lafayette, CA. Leupeptin was a generous gift of the US-Japan Cooperative Cancer Program. Tissue culture media, antibiotics and buffers were obtained from Gibco Laboratories, Grand Island, NY, calf serum and plastic tissue culture dishes from Flow Laboratories, McLean, VA. Light source and irradiation UVA radiation was obtained from a 2000 W high pressure Hg vapor lamp with iron and gallium iodide, and filtered through a Schott WG 320 nm filter (Honle
229
Downloaded from http://carcin.oxfordjournals.org/ at Harvard Library on July 4, 2015
Exposure to ultraviolet (UV) radiation has been well correlated with skin cancer incidence. Long wave UV radiation (320-400 nm, UVA) is a major component of natural sunlight and cosmetic tanning 'salon' light, and has been shown not only to damage DNA and to act as a complete carcinogen, but also to promote ultraviolet B (280—320 nm, UVB) carcinogenesis. The mechanism by which the latter occurs is unknown, but it is believed to be related to the inflammation and irritation which results from UV exposure. In order to examine the possibility that UVA stimulates the same signalling pathway as do the phorbol esters, a class of much more thoroughly characterized skin tumor promoters, we exposed cells in culture to UVA radiation and measured cellular responses related to protein kinase C (PKC) activation. The data presented here demonstrate that a low, physiologic dose of UVA inhibits epidermal growth factor binding and increases PKC activity in cultured mammalian fibroblasts. The increase in cytosolic activity is not completely translocated to the membrane, and can be partially suppressed by puromycin and cycloheximide but not by actinomycin D. These observations are the first evidence to suggest that a protein which has been strongly linked to chemical tumor promotion may also be a critical mediator for UV-induced promotion. The response of cells to UVA is also unique, in that it does not cause a 12-0-tetradecanoyl phorbol-13-acetate-like rapid redistribution of PKC activity followed by down regulation.
erythema and pigmentation in normal skin and is thought to play a major role in photoaging and wrinkling (11,12). Information as to the identity and role of endogenous chromophores and about the mechanisms of photodamage is very meagre (13). This is so even though UVA radiation is known to penetrate Caucasian skin at least to the dermis and to irradiate significant numbers of cells in the peripheral circulation (14). Increased interest in effects of UV on skin cancer development has developed recently, in part because of increased human exposure to UVA made possible through the use of sunscreens which block predominantly UVB radiation and through the use of artificial light sources producing massive doses of UVA radiation in cosmetic sun tanning parlors. We have previously determined that UV radiation of 280 — 320 nm (UVB, mid wave UV) shares a number of biologic effects with the phorbol ester tumor promoter 12-O-tetradecanoyl phorbol-13-acetate (TPA) (15). Both UVB and TPA induce the stimulation of arachidonic acid release, prostaglandin E2 production, the inhibition of epidermal growth factor (EGF) binding and cellular proliferation in cultured cells (16—18). However, UVB treatment had no effect on protein kinase C (PKC) activity or phorbol ester binding (15). In the series of experiments described here, we wished to explore the possibility that UVA- and TPA-induced promotion of epidermal tumors share a common pathway by examining the effect of UVA on the subcellular distribution of the phorbol ester receptor, PKC. The hypothesis we wished to test was that UVA acts as an epidermal tumor promoter by stimulating PKC activation, as does TPA, initiating a cascade of events which result in phosphorylation of and inhibition of ligand binding to the EGF receptor, and initial translocation and subsequent down regulation of PKC, ultimately resulting in permanent phenotypic changes in an initiated cell.
M.S.Matsui and V.A.DeLeo
36 1
LLJ
32
O
"o
28
Q_ O
24
BOUN
Q"
20 16
Q Z
12 8 4 1
0
1
2
1
1
4
1
1
6
1
1
8
1
1
10
UVA EXPOSURE ( J / c m 2 ) Fig. 1. Changes in specific binding of [3H]PDBu (O—O) or (I25I]EGF (O O) to C3H10T1/2 cells exposed to doses of UVA radiation from 0 to 10 J/cm2. Irradiation was performed and specific binding determined as described in Materials and methods. Each point represents the average of data obtained from triplicate dishes for total and non-specific binding. The experiment was repeated in its entirety with similar results. actinomycin D (6 ^g/ml) was added to growth media. After irradiation, cells were incubated at 37°C for 45 min and then placed on ice and harvested. The percent stimulation or inhibition of UVA-induced PKC activity was calculated using the following formula: UVD-D D
_x 100 = % inhibition or stimulation
UV-Con Con where UVD represents pmol 32P transferred/^g protein/min by cells irradiated and treated with drug; D, drug-treated sham-irradiated cells; UV, irradiated cells; Con, sham-irradiated cells. Immunoblotting Semi-purified protein extracts were refiltered through Amicon filters and resuspended in loading buffer (143 mM Tris at ph 6.8, 4.5% SDS, 3% 2-mercaptoethanol and 7.5% glycerol). Equal amounts of protein from each sample were subjected to discontinuous polyacrylamide gel electrophoresis in the presence of SDS (SDS-PAGE) (20) in which the stacking gel was 4% acrylamide:bis (50:1) and the resolving gel was 8%. The separated proteins were then electrophoretically transferred overnight to a nitrocellulose membrane (NC) (21). The NC was incubated at 4°C overnight with 0.5% Carnation instant milk in PBS and then incubated for 4 h in monoclonal anti-PKC (Amersham, Arlington Heights, IL). NC was washed and immunodetection performed by the horseradish peroxidase method or by incubation of the NC with 1 /jCi/filter donkey [l25I]antimouse antibody (Amersham, Arlington Heights, IL) followed by autoradiography. Densitometric analysis of autoradiographs was achieved by the use of a Molecular Dynamics 300A computing densitometer (Molecular Dynamics, Sunnyvale, CA).
Results Changes in PDBu and EGF binding following UVA irradiation Activation of PKC by TPA has previously been shown to result in phosphorylation of the EGF receptor and inhibition of EGF binding (22), so we examined the effect of UVA radiation on
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