Ultraviolet A and B differently induce intracellular protein expression in human skin melanocytesa speculation of separate pathways in initiation of melanoma
Hong Zhang1 and
Inger Rosdahl
Department of Dermatology, Institute of Biomedicine and Surgery, Linköping University, SE-581 85 Linköping, Sweden
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Abstract
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Ultraviolet (UV) irradiation has been involved in both initiation and promotion of carcinogenesis in melanoma. Alterations of cellular proliferation proteins, such as p73, Nup88, Id1 and p27 have been considered to play critical roles in melanoma development. However, the molecular mechanisms behind melanoma carcinogenesis are still poorly understood. In this study, we used human skin melanocytes as an experimental model system to investigate effects of UV irradiation on protein expression concerning cellular proliferation. The melanocytes prepared from human foreskin were separately exposed to various doses of UVA or UVB and post-cultivated for 24 or 48 h. Total proteins were isolated from the melanocytes, and expression of p73, Nup88, Id1, p27, bcl-2 and proliferating cell nuclear antigen (PCNA) proteins was examined by western blotting and immunocytochemistry. Results showed that expression of p73 and Nup88 was enhanced by UVA irradiation in a dose- and time-dependent manner. However, expression of Id1, p27, bcl-2 and PCNA proteins was not changed upon exposure to the UVA. Id1 and p27 proteins were over-expressed by exposure to UVB, but expression of p73, Nup88, bcl-2 and PCNA proteins was not changed by the UVB irradiation. The data suggested that UVA and UVB irradiation might lead to alterations of the different intracellular proteins. UVA enhanced protein expression concerning cell growth (p73 and Nup88) and UVB might over-express proteins concerning cellular proliferation (Id1 and p27). UVA and UVB may induce initiation of melanoma via separate intracellular pathways.
Abbreviations: CDK, cyclin-dependent kinase; PBS, phosphate-buffered saline; PCNA, proliferating cell nuclear antigen; UV, ultraviolet
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Introduction
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Ultraviolet (UV) exposure is recognized as a well-known external risk factor for the development of melanoma. Accumulating evidence has shown that both UVA and UVB play critical roles in the aetiology of human melanoma (1,2) and are involved in both initiation and promotion of the multi-step carcinogenesis in melanoma (3). Alterations of cellular proliferation-related proteins, such as p73, Nup88, Id1 and p27 have been considered to play critical roles in melanoma development. UVA irradiation is known to induce oxidative mutagenic DNA base damage (4) and DNA breaks (5). cDNA arrays show that UVA induces over-transcription of the genes involving cell cycle, stress response, apoptosis and transcription factors (6). UVB induces apoptosis in human melanoma cells via down-regulation of bcl-2 and activation of caspases mediated apoptotic pathway (7). In human skin, UVB has been proven to induce phenotypes of atypical melanocytic lesions and melanoma (8). Although both cell cycle change and apoptosis have been suggested to play essential roles in the carcinogenesis of melanoma, the basic mechanisms behind UV-induced melanomas are still not fully clarified.
Over-expression of p73 is associated with vascular invasion, lymph node metastasis and advanced stages (9) and predicts a poor outcome for patients with colorectal carcinoma (10). P73 is highly expressed in metastatic melanoma cells as compared with the primary tumors derived from the same patients (11).
Nup88 is involved in nuclear-cytoplasmic transport and cell growth (12). Over-expression of Nup88 has been found in a wide range of malignant tumors (13). Expression of Nup88 protein is elevated in metastatic melanoma cells as compared with the primary tumors from the same patients (11).
Helixloophelix proteins are a super family of dimeric transcription factors involved in the regulation of cell growth and differentiation (14). Id1, as a member in this family, has been implicated in regulating cellular life span in normal cells (15) and is up-regulated in early melanomas (16).
P27 is a cyclin-dependent kinase (CDK) inhibitor and regulates cellular progression from G1 into S phase by inhibiting the cyclin/CDKs binding (17). P27 is one of the candidates involved in melanoma formation (18) and loss of p27 has been found in metastatic melanoma cells (19).
Bcl-2 is down-regulated by UV irradiation (7,20) and epithelial cells in Bcl-2-deficient mice are susceptible to UV irradiation in respect to cell death and cell cycle progression (21). Expression of proliferating cell nuclear antigen (PCNA) in the melanocytic nevi have been reported to be markedly enhanced after UV irradiation (22). These findings indicate that both bcl-2 and PCNA proteins may be important factors in UV-induced melanoma.
In this study, we have examined the expression of p73, Nup88, p27, Id1, bcl-2 and PCNA proteins in human skin melanocytes after exposure to either UVA or UVB in order to further understand the mechanism behind UV-induced melanoma.
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Materials and methods
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Cell culture medium and compounds
Fetal calf serum (FCS), M199, L-glutamine (200 mM), penicillin (5000 IU/ml), streptomycin (5000 µg/ml), 0.05% trypsin0.02% EDTA were from Life Technologies (Paisley, UK). Dispase II (neutral protease) was from Roche Diagnostics GmbH, Mannheim, Germany. TrisHCl ready gels (12%), PVDF membranes and fat-free milk were from Bio-Rad Laboratories (Hercules, CA). ECLplus kit and Hyper ECL films were from Amersham Pharmacia Biotech (Buckinghamshire, UK). Phosphate-buffered saline (PBS) and PBS-Tween (PBST) tablets were from EC Diagnostics AB (Uppsala, Sweden) and 4% buffered formaldehyde was from Produktion and Laboratorier (Gothenburg, Sweden). Hydrogen peroxide (H2O2) and methanol were from Merck (Darmstadt, Germany) and 3,3-diaminobenzidine tetrahydrochloride (DAB) was from Sigma Chemical Co. (St Louis, MO, USA).
Antibodies and compounds
Monoclonal mouse IgG1, anti-human p27Kip1, bcl-2 and PCNA antibodies were from DAKO A/S (Glostrup, Denmark). Id1 (C-20) rabbit polyclonal IgG and p73 (C-20) goat polyclonal IgG antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-Nup88 polyclonal antibody was a gift from Dr Jose Schneider (Facultad de Ciencias de la Salud, Universidad Rey Juan Carlos, Madrid, Spain). Mouse, rabbit and goat immunoglobulins and peroxidase-anti-peroxidase (PAP) were from DAKO A/S. Goat actin polyclonal IgG antibody and secondary conjugated horseradish peroxidase (HRP) antibodies against mouse and goat were from Santa Cruz Biotechnology. Secondary conjugated HRP antibody against rabbit was from Amersham Pharmacia Biotech.
Separation and cultivation of melanocytes from human foreskins
Human foreskins from white males (115 years old) were carefully washed in cold-buffered penicillin (500 IU/ml) and streptomycin (500 µg/ml). The foreskins were cut into small pieces (
2 x 5 mm) and incubated in DMEM with Dispase II (2 mg/ml) at 4°C for 18 h. The epidermal layer was mechanically separated from the dermis and digested in trypsinEDTA at 37°C for 50 min. The epidermis was aspirated with a plastic pipette every 10 min during the incubation in order to properly dissociate the cells. In brief, the mixed melanocytes and keratinocytes were cultivated in the M199 supplemented with 3% FCS. The melanocytes were separated from the keratinocytes by three to four cycles of trypsinzations with a controlled duration of the incubation. The purified melanocytes were prepared for the experiments.
UV irradiation
After purification, melanocytes (5 x 105/60 mm cell culture dish and 104 cell/microscopy slide) were sub-cultivated under the above mentioned standard cell culture condition for 24 h. The cultures were adjusted to the room temperature and the cell culture medium was replaced by a thin layer of PBS. The melanocytes were directly (without the covers of dishes) exposed to UVA (315400 nm, Hürth, Germany) or UVB (290320 nm, Eindhoven, The Netherlands), respectively. The melanocytes were separately exposed to either UVA (0, 2, 6 or 10 J/cm2) or UVB (0, 30, 60 or 180 mJ/cm2). A Schott WG 305 filter (Mainz, Germany) was used to remove the wavelength <290 nm during the melanocyte exposure to UVA or UVB. After UV exposure the cultures were brought back to the standard cell culture condition and cultivated for 24 or 48 h. The melanocytes in culture were rinsed in cold PBS x3 for western blotting and immunocytochemistry. The controls (un-irradiated groups) went through the same procedure as the experimental groups.
Protein isolation and western blot analysis
After rinsing in cold PBS x3 the cells were lysed in RIPA buffer (150 mM NaCl, 1% Triton X-100, 0.5% NaDOD, 0.1% SDS and 50 mM Tris pH 8.0) on ice for 20 min and centrifuged at 12 000 g at 4°C for 5 min. The total protein concentrations from each sample were measured by a spectrophotometer according to the manufacturer's protocol from Bio-Rad Laboratories. Thereafter, equal amount of total protein was mixed with SDSPAGE sample buffer (2% SDS, 0.03% bromophenol blue, 3% ß-mercaptoethanol, 10% glycerol and 67 mM Tris pH 6.8) and denatured at 95°C for 5 min. The sample of 15 µg protein and 5 µl protein size marker (Bio-Rad Laboratories) was loaded in each lane in 12% TrisHCl ready gels. The gels were run at 150 V on ice for 3060 min (depending on the protein size) and the separated proteins were transferred to PVDF membranes at 100 V for 1.5 h. The membranes were blocked in 5% fat-free milk on an orbital shaker at 4°C overnight. Following washing in PBST x6, the membranes were incubated with primary antibodies (1:2000) on an orbital shaker at 20°C for 2 h and washed in PBST x6. The corresponding secondary antibodies (1:5000) conjugated HRP were applied to the membranes on an orbital shaker at 20°C for 1 h. Followed washing in PBST, the membranes were incubated with ECLplus in the dark for 5 min (according to manufacturer's protocol) and then exposed to Hyper ECL films. Actin was applied as an internal control for equal protein concentrations. The experiments were performed in triplicates and the intensity of the bands were measured by computer. The values were expressed as percentage of controls and data were represented as mean ± SD.
Immunocytochemistry assay
The melanocytes, grown on the microscopy slides for 24 h, were exposed to UVA or UVB under a thin layer of PBS and cultivated in the standard cell culture condition for 24 h. The cultures were carefully washed in PBS x3 and fixed in 4% buffered formaldehyde for 5 min. Following washing in PBS x3, the samples were pre-treated in methanol with 0.5% H2O2 for 5 min and washed in PBS x3, and incubated with the monoclonal primary antibodies at 4°C overnight. Subsequently, the slides were incubated with corresponding immunoglobulins (1:100) in PBS for 60 min, and PAP (1:150) in PBS for 60 min. The slides were washed in PBS x3 between each incubation step and exposed to 0.05% DAB with 0.02% H2O2 in PBS for 8 min. The reaction was stopped by rinsing in running distilled water for 10 min. The cells were briefly counterstained with hematoxylin for 1 min in order to improve nuclear visualization. Following a rinse in running distilled water for 10 min the cells were dehydrated through a serial step-up ethanol to xylene and finally mounted under coverslips (11). All procedures were performed at room temperature unless otherwise indicated. The experiments were performed in triplicate to confirm the immunoreaction. Samples (melanoma cells) known to give positive and negative immunostaining were included in each run as controls. An additional negative control was performed where the primary antibodies were replaced by PBS and then taken through the same procedure. All samples were taken through the procedure in the same run.
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Results
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UVA irradiation induced an increased expression of p73 and Nup88
The normal skin melanocytes in culture were exposed to various doses of UVA (0, 2, 6 or 10 J/cm2, respectively), and post-cultivated in the standard cell culture condition for 24 or 48 h. As shown in Figure 1A and B, melanocytes had a low level of p73 expression in the control (unexposed melanocytes). After exposure to 2 J/cm2 UVA and post-cultivated for 24 h the expression of p73 was increased. When exposed to 10 J/cm2 UVA and cultivated for 48 h p73 was increased as much as 7-fold as compared with the control. The expression of p73 protein in the UVA-exposed melanocytes was confirmed by immunocytochemical assay (Figure 1C). Similarly, Nup88 was expressed at a low level in the control melanocytes. The expression level was increased >3-fold after the cells were exposed to 2 J/cm2 UVA and cultivated for 24 h, and further enhanced >10-fold after the cells were exposed to 10 J/cm2 UVA and cultivated for 48 h. Expression of Nup88 protein in UVA-exposed melanocytes was confirmed by immunocytochemical assay (Figure 1C). The increasing levels of both p73 and Nup88 proteins were UV dose- and post-incubation time-dependent. However, Id1, bcl-2 or PCNA protein was constantly expressed at high levels, while p27 protein was expressed at a low level. Actin as an internal protein control was equally expressed in all the samples (Figure 1A).

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Fig. 1. (A) Expression of p73, Nup88, Id1, p27, bcl-2 and PCNA proteins in human skin melanocytes is detected by western blotting after UVA irradiation at indicated doses (J/cm2) and 24 or 48 h post-incubation in the standard cell culture condition. (B) Expression of p73 and Nup88 proteins was increased in a dose- and time-dependent manner. P73 and Nup88 were increased 7- and 10-fold when the cells were exposed to 10 J/cm2 and post-cultivated for 48 h. The experiments were repeated in triplicates. The values were expressed as percentage of controls and data were represented as mean ± SD. (C) Cellular localization of p73 (left panel) or Nup88 (right panel) proteins as revealed by immunocytochemistry in the melanocytes after exposure to 10 J/cm2 UVA and post-incubation for 24 h. Original magnification, x400.
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UVB irradiation induced an increased expression of Id1 and p27
The normal skin melanocytes in culture were exposed to various doses of UVB (0, 30, 60 and 180 mJ/cm2) and then cultivated in the standard cell culture condition for 24 or 48 h. As shown in Figure 2A and B, Id1 was expressed in the control melanocytes (unexposed to UV). After exposure to 30 mJ/cm2 UVB and post-cultivated for 24 h the expression of Id1 protein was observed to be increased 2.5-fold. When the melanocytes were exposed to 180 mJ/cm2 UVB and post-cultivated for 48 h, Id1 expression was increased to 6.3-fold. The expression of Id1 in the UVB-exposed melanocytes was confirmed by immunocytochemical assay (Figure 2C). P27 protein was observed at a low level in the UV-unexposed melanocyte. The expression of p27 was increased 4.7-fold after exposure to 30 mJ/cm2 UVB and 24 h post-cultivation, and further increased to 9.6-fold after exposure to 180 mJ/cm2 UVB and post-cultivation for 48 h. Expression of p27 in the UVB-exposed melanocytes was confirmed by immunocytochemical assay (Figure 2C). The increased expression of Id1 and p27 proteins was UVB-dose and post cultivation time-dependent. However, p73 was constantly expressed at a low level while Nup88, bcl-2 and PCNA were constantly expressed at similar levels after exposure to UVB and post-cultivation. Actin as an internal protein control was equally expressed in all the samples (Figure 2A).

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Fig. 2. (A) Expression of Id1, p27, p73, Nup88, bcl-2 and PCNA proteins in human skin melanocytes is detected by western blotting after UVB irradiation at indicated doses (mJ/cm2) and 24 or 48 h post-incubation in the standard cell culture condition. (B) Expression of the Id1 and p27 proteins were increased in a dose- and time-dependent manner. Id1 and p27 were increased 6.3- and 9.6-fold when the cells were exposed to 180 mJ/cm2 and post-cultivated for 48 h. The experiments were repeated in triplicates. The values were expressed as percentage of controls and data were represented as mean ± SD. (C) Cellular localization of Id1 (left panel) or p27 (right panel) as revealed by immunocytochemistry in the melanocytes after exposure to 180 mJ/cm2 UVB and post-incubation for 24 h. Original magnification, x400.
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Discussion
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Normal skin melanocytes were exposed to various doses of UVA or UVB and post-cultivated for 24 or 48 h. Expression of Id1, p27, p73, Nup88, bcl-2 and PCNA protein was examined by western blotting and immunocytochemistry. When the normal skin melanocytes were exposed to UVA the expression levels of p73 and Nup88 proteins were increased in a dose- and time-dependent manner. However, UVB did not affect the expression levels of p73 and Nup88 proteins. This indicated that p73 and Nup88 were up-regulated by UVA, but not by UVB. In previous studies, both p73 and Nup88 have been shown to function as cell growth promoters, which are over-expressed in primary and metastatic tumor cells. We have demonstrated recently that the expression of these proteins increased from primary melanoma cells to metastatic cells (11). P73 expression is increased from normal mucosa to carcinoma in the colorectum (10). The present results indicate that effects of UVA and UVB on melanoma cells were through different pathways and further suggest that UVA was involved in carcinogenesis of melanoma via up-regulation of cell growth-involving proteins such as p73 and Nup88.
We found that expression of Id1 and p27 proteins in the melanocytes was up-regulated by exposing the melanocytes to UVB but not to UVA. This response was dose- and time-dependent. The Id1 protein is a negative regulator for the helixloophelix transcription, and inhibits cell proliferation (23), differentiation (15) and DNA-binding (24). Id1 involves the inhibition of E2A bHLH-regulated expression of the gene encoding the CDK inhibitor p21Cip/Waf1. The release of p21 suppression in turn leads to phosphorylation of Rb, resulting in the activation of proteins required for progression into S-phase (25). Expression of Id1 has been found to promote mammary epithelial cells to invade the basement membrane (26). In melanoma, Id1 has been shown to regulate p16 and plays a critical role in the early stages of the tumors (16,27). In the present study, we found a pronounced increase in the expression of Id1 protein after exposure to UVB, but not to UVA, indicating that Id1 protein was involved in the process of cell damage induced by UVB, but not by UVA. In this aspect, the increased levels of Id1 protein may indicate that UVB conducts melanoma through inhibition of the CDK inhibitor p21 in cell cycle to promote cells into S-phase. Further, the over-expression of Id1 might function to reduce cell proliferation and differentiation to minimize the cell damage. Cellular differentiation is related to a decrease in cell proliferation following the increased level of Id1 protein. Reduced cellular differentiation led to an uncontrolled cell growth and cell proliferation.
P27 is an inhibitor for cell cycle and loss of p27 has been associated with a poor prognosis in colorectal patients with Dukes B tumors (19). In a recent paper, we have demonstrated that p27 was expressed in the primary melanoma cells and the expression was reduced in their corresponding metastatic cells (11), supporting the hypothesis that loss of the inhibitory effect by p27 may lead to a tumor progression. In an animal study, p27 protein levels in the epidermis have been increased 2-fold between 3 and 24 h after UVB irradiation, and then decreased to the control levels after 48 h in the standard cell culture condition (28). In this study, the p27 levels were markedly enhanced after the UVB irradiation. This may result in the inhibition of cellular proliferation in a cell cycle, leading to an imbalance cell growth and a tumor.
In this study, we found that the expression levels of bcl-2 and PCNA proteins in the melanocytes were quite high. However, the levels were not significantly changed after exposure to either UVA or UVB, indicating that the effect of UV irradiation did neither involve bcl-2-related apoptosis pathway nor PCNA-related cell growth. These findings do not correspond with previous reports that bcl-2 was down-regulated (7,20) and PCNA was up-regulated (22) by UV irradiation in melanoma cells, HeLa cells and melanocytic nevi. The different results were probably due to the different experimental model systems, such as cell types, UV dose and post-incubation time.
In conclusion, UVA and UVB irradiation-induced alterations of the different intracellular proteins. UVA enhanced the cell growth up-regulators, such as p73 and Nup88, and UVB up-regulated the cell differentiation and cell cycle inhibitors, such as Id1 and p27. UVA and UVB may play different roles in the carcinogenesis of melanoma via different intracellular pathways, leading to cellular proliferation and initiation of melanoma (Figure 3). Therefore, a better strategy to prevent UV-conducted melanoma is to inhibit both UVA and UVB cell damage pathways.

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Fig. 3. A diagram of UVA and UVB differently induced intracellular protein expression in human skin melanocytes. UVA induces over-expression of p73 and Nup88 proteins, and UVB induces over-expression of Id1 and p27 proteins. Increased levels of p73, Nup88 and Id1 result in cellular proliferation. The high level of p27 may induce apoptosis. Imbalance between cellular proliferation and apoptosis leads to the initiation of melanoma.
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Notes
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1 To whom correspondence should be addressed Email: hong.zhang{at}ibk.liu.se 
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Acknowledgments
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We are sincerely thankful to Dr Jose Schneider (Facultad de Ciencias de la Salud, Universidad Rey Juan Carlos, Madrid, Spain) for kindly providing the Nup88 antibody. This work was supported by Swedish Cancer Foundation (3800-B00-05XAB), Welander Finsen Foundation and Swedish Medical Association.
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Received October 24, 2002;
revised September 8, 2003;
accepted September 9, 2003.