Thapsigargin has similar effect on p53 protein response to benzo[a]pyreneDNA adducts as TPA in mouse skin
Raisa Serpi1,2,
Johanna Piispala1,
Matti Järvilehto2 and
Kirsi Vähäkangas1,3
1 Department of Pharmacology and Toxicology, University of Oulu,PO Box 5000, FIN-90401 Oulu and
2 Department of Biology,University of Oulu, PO Box 333, FIN-90571 Oulu, Finland
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Abstract
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The level of p53 tumor suppressor protein increases in response to DNA damage caused by benzo[a]pyrene (B[a]P). The most used tumor promoter in the two step mouse skin carcinogenesis model, 12-O-tetradecanoylphorbol-13-acetate (TPA) decreases this response in mouse skin. In this study the effect of another promoter, thapsigargin was tested on B[a]P-induced p53 response using immunohistochemistry, western blotting and immunoelectron microscopy. We also studied the localization of p53 protein after treatments with BP and TPA or thapsigargin. Thapsigargin had a TPA-like effect on the acute induction of p53 protein related to benzo[a]pyrene-7,8-diol-9,10-epoxideDNA adducts in the skin of C57BL/6 mouse. After B[a]P treatment, there was slightly more putatively wild-type p53 protein in nuclei than in cytoplasm of the cells. Neither TPA nor thapsigargin affected the localization of p53 protein. Since both compounds increase the level of intracellular calcium, the inhibition of the p53 response may depend on the level of intracellular calcium. Inhibition of the putatively genome-protecting increase in p53 protein may be one of the critical effects of tumor promoters.
Abbreviations: B[a]P, benzo[a]pyrene; BPDE, benzo[a]pyrene-7,8-diol-9,10-epoxide; IEM, immunoelectron microscopy; PKC, protein kinase C; SFS, synchronous fluorescence spectrophotometry; TPA, 12-O-tetradecanoylphorbol-13-acetate.
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Introduction
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The tumor suppressor gene, p53, has been shown to be the most commonly mutated gene in human tumors (13). Its product is a multi-functional nuclear phosphoprotein that carries out at least some of its functions by binding to specific sites on DNA and inducing the transcription factors of other genes (4,5). The p53 protein is able to block the cell cycle at G1 and G2/M phases. G1 block is mediated through transcriptional activation of WAF1/CIP1, the product of which, p21 protein, is suggested to be the mediator of tumor suppressing activity of p53 protein (6,7). In addition to cell-cycle arrest, which may give DNA time to repair before replication, there are implications that p53 is more directly involved in DNA repair (5,8). The p53 protein can also trigger apoptosis, i.e. kill the damaged cells, which produces the integrity of the genome after exposure to DNA-damaging agents (9,10). Loss of function of p53 protein can result from either a mutation of the p53 gene or binding of the protein to other proteins.
Mutations in the p53 gene are rare in papillomas, but are detected in carcinomas in experimental mouse skin carcinogenesis (11,12). According to Kemp et al. (13), loss of the p53 protein and its function may be more relevant to malignant conversion than promotion in epidermal carcinogenesis. 12-O-tetradecanoylphorbol-13-acetate (TPA) and thapsigargin belong to the large group of known tumor promoters in the two-step mouse skin carcinogenesis model (for a review see 14). The mechanism of promotion by TPA, which is not well understood, can be based on the activation of protein kinase C (PKC). This can cause a rise in intracellular Ca2+ levels (15). Thapsigargin is a weak promoter that originates from a Mediterranean plant Thapsia garganica, and is known to be highly toxic (16). Thapsigargin, having some structural similarities to TPA, prevents the accumulation of intracellular Ca2+ in endoplasmic reticulum by inhibiting Ca2+-ATPase and thus increases the Ca2+ level in the cytosol. Benzo[a]pyrene (B[a]P) is a classic complete carcinogen, which means that it can act both as an initiator and promoter in skin carcinogenesis. Like other polycyclic aromatic hydrocarbons, B[a]P requires metabolic activation to benzo[a]pyrene-7,8-diol-9,10-epoxide (BPDE) in order to exert its carcinogenic effects. BPDE induces DNA damage by binding covalently to DNA (17).
DNA damage by chemicals as well as radiation are known to cause accumulation of p53 protein within cells (8). Low-dose X-ray irradiation has been shown to cause p53 accumulation in various mouse organs, including skin (18). In our previous study, using CM5 antibody in immunohistochemistry, we found an increase in nuclear p53 protein by B[a]P treatment of mouse skin. TPA after B[a]P decreased this p53 response which was in correlation with the formation of BPDEDNA adducts (19,20). In this study, we have analyzed the localization of p53 protein after B[a]P-treatment by immunoelectron microscopy (IEM) and compared the effect of thapsigargin to that of TPA and also investigated the effect of thapsigargin on X-ray-induced p53 accumulation to test the hypothesis of p53 protein being involved in the mechanism of tumor promotion by these substances.
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Materials and methods
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Animals and treatments
Male C57BL/6 mice (920 weeks of age) were maintained in plastic cages at a constant 25°C with a 12 h lightdark cycle and had free access to food (Standard rodent pellets, Special Diets Services, Essex, UK) and water. The backs of the mice were shaved 3 days before the treatments and only mice with the fur in the non-growing phase were selected. In the B[a]P-treatment groups (Table I
), 500 µg B[a]P (Sigma, St Louis, MO) were applied topically in 100 µl of acetone by dropping slowly on the shaved backs of the mice. After 24 h the mice were either killed (positive controls) or treated twice on 2 consecutive days with thapsigargin (Sigma) or TPA (Sigma), again in 100 µl of acetone. Control animals were treated on the first day with 100 µl of acetone and the following treatments with the promoters were as described (groups 4 and 5). Twenty-four hours after the last applications, skin samples from the exposed area (2x2 cm) were collected. The treated skin was divided for immunohistochemistry, BPDEDNA adduct measurements, western blotting and IEM. As controls, two mice were treated by X-ray irradiation (50 cGy, 120 kV, 10 mA, 38 s), one of which was also treated twice by thapsigargin; first treatment 17 h before and the second 3 h after the X-ray treatment. These timepoints were selected because the effect of X-ray treatment on p53 in mouse skin appears and fades away quicker than the effect of B[a]P (18,19).
Synchronous fluorescence spectrophotometry (SFS) for BPDEDNA adducts
Samples for DNA isolation were treated in 50°C water for 30 s and the epidermis was then scraped off with a surgical blade to test tubes and stored at 20°C in SDSEDTA buffer. DNA was isolated from the epidermis using a phenol extraction/ethanol precipitation method (21). After hydrolysis of 100 µg of DNA in 0.1 M HCl at 90°C for 3 h, the BPDEDNA adducts were measured as described previously (21). In SFS at an excitation wavelength of 345 nm, the height of the peak is linearly correlated with the level of adducts when scanned synchronously with a wavelength difference of 34 nm. The sensitivity of SFS is ~1 adduct/107 nucleotides and one fluorescence unit equals ~1.1 fmol BPDE/µg DNA.
Immunohistochemistry
For immunohistochemistry, the skin sections were fixed in neutral 10% formalin, embedded in paraffin and cut into 5 µm sections and mounted on slides coated with poly-L-lysin. The sections were deparaffinized in xylene and dehydrated in graded ethanol. To block endogenous peroxidases, the sections were treated with 0.1% hydrogen peroxide in absolute methanol. Non-specific binding was blocked by incubating the sections in 20% fetal calf serum in phosphate-buffered saline (PBS). The avidinbiotin complex method was used for immunohistochemical detection of p53 protein. The sections were incubated for 12 h with polyclonal antibody CM5 (a gift from Prof. D.Lane, University of Dundee, UK), originally raised against murine wild-type p53 similarly to CM1 (22). Samples treated otherwise similarly, excluding CM5 treatment, were used as negative controls. Then the sections were treated with secondary anti-rabbit immunoglobulin and the avidinbiotin complex (both from Dako, Denmark). The colour was developed with diaminobenzidine and the samples were lightly counterstained with hematoxylin. The nucleus stains brown in a positive case and blue in a negative case. The percentages of p53 protein immunopositive keratinocytes were counted in five high power (40x objective) fields including at least 500 keratinocytes per case.
Western blotting
For immunoblotting analysis, the treated skin was removed, subcutaneous fat was removed on ice and the outermost part of the skin with hairs were scraped off. The skin sample was homogenized for 15 min in 1.5 ml of cytoplasmic lysis buffer (containing 20% glycerol, 20 mM HEPES, 10 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 1 mM DTT, 0.1% NP-40, 100 µg/ml PMSF, 1 µg/ml aprotinin, 1 µg/ml pepstatin A and 1 µg/ml antipain) on ice, then centrifuged for 4 min at 2000 r.p.m. To the pellet, 1 ml of nuclear lysis buffer was added (500 mM instead of 10 mM NaCl, for whole-cell extract skin sample was homogenized in this) and incubated for 30 min in an ice bath. The samples were then centrifuged for 15 min at 15 000 r.p.m. at 4°C and the supernatant (nuclear extract) was collected. Proteins were separated electrophoretically for 45 min in a 10% polyacrylamide gel (Bio-Rad Power Pac 200; Bio-Rad, Hercules, CA) and transferred for 1 h to nitrocellulose filter (Optitran Ba-S 85; Schleicher & Schuell, Germany). To avoid unspecific binding, the filter was incubated for 1 h at 20°C with 5% Tris-buffered saline (TBS) containing 0.5% cow's milk powder. The samples were then treated for 1 h at 20°C with p53 antibody CM5 (dilution 1:2000) in 0.5% TBSmilk and overnight at 4°C with horseradish peroxidase labeled anti-rabbit secondary antibody (401315; Calbiochem; dilution 1:4000). The complex formed by p53 protein and the antibodies was detected by chemiluminescent ECL reaction according to the manufacturer's instructions (RPN 2106; Amersham).
Immunoelectron microscopy (IEM)
Samples for IEM were collected from the treated skin with a 2 mm diameter stance. Samples were fixed in 4 or 2% paraformaldehyde, the latter with 0.2% glutaraldehyde, in 0.1 M PBS, pH 7.4, at 20°C for 2 h, dehydrated through a graded ethanol series and propylenoxide twice for 15 min and embedded in araldite (Araldite 502 Kit; Electron Microscopy Sciences) with overnight polymerization at 60°C. Thin sections (6090 nm) were cut with a Reichert Ultracut E ultramicrotome and collected in copper and nickel grids. Sections were first etched with 10% H2O2 for 10 min, then treated with glycine (4% paraformaldehyde as fixative) or 0.1% borohydride (2% paraformaldehyde and 0.2% glutaraldehyde as fixatives) for 10 min and 8% bovine serum albumin (BSA) in PBS, pH 7.4, for 15 min, then labeled with primary antibody CM5 (dilutions 1:2000 and 1:10 000) in 1.5% BSA, 0.8% fish skin gelatine (FSG), 0.05% Tween-20 in PBS for 2 h at 20°C. The p53 proteinantibody complexes were coupled with 10 nm gold particles (Prot-A-Gold; Sigma) diluted in 1.5% BSA, 0.15% FSG, 0.1% Tween-20 in PBS for 45 min at 20°C. Finally, the sections were double stained with uranylacetate and Pb-citrate for 10 and 5 min. Sections were screened and photographed in a transmission electron microscope (Jeol JEM-100 CX II, Japan). Evaluation of the photographic samples from different treatment groups was done with blind test and independently by three people.
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Results
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There was no statistically significant difference in SFS analysis for BPDEDNA adducts between the different treatment groups, indicating that tumor promoters thapsigargin and TPA do not affect the level of BPDEDNA adducts (Table II
). B[a]P-induced p53 protein was acutely more pronounced at 24 h than at 72 h. The p53 protein detected in this study is most likely an increase of wild-type protein because the timeframe used (2472 h) does not allow clonal expansion of mutated cells to such an extent that would be detectable by the methodologies used.
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Table II. The effect of acetone, TPA or thapsigargin on the level of BPDEDNA adducts and p53 immunohistochemistry in B[a]P-treated C57BL/6 mice
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However, there was a clear difference between different groups in the percentage of p53-protein-positive nuclei in immunohistochemistry (Figure 1
). Both TPA and thapsigargin decreased the B[a]P-induced number of p53-positive nuclei (Table II
). There was some variation between individual mice, and the effect of TPA was more pronounced than that of thapsigargin.
The decrease of p53 protein by the promoters was confirmed in western blot analysis where a clear difference could also be seen between different groups. Both thapsigargin and TPA decreased the amount of p53 protein induced by BPDEDNA adducts, the effect of TPA being greater than that of thapsigargin (Figure 2A
). Also, radiation-induced p53 was decreased by thapsigargin (Figure 2B
). A weaker band ~35 kDa in size was seen in B[a]P and thapsigargin treated whole-cell extract sample and seemed to be more marked compared with the B[a]P and acetone control (data not shown).

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Fig. 2. Western blotting of p53 protein after B[a]P or X-ray treatment in C57BL/6 mouse skin. (A) Lane 1, 500 µg B[a]P (positive control); lane 2, 500 µg B[a]P and 20 µg TPA twice on 3 consecutive days; lane 3, 500 µg B[a]P and acetone twice; lane 4, 500 µg B[a]P and 5.5 µg thapsigargin twice; and lane 5, 500 µg B[a]P and acetone twice. (B) Lane 1, 500 µg B[a]P (positive control); lane 2, 50 cGy X-ray irradiation and 5.5 µg thapsigargin twice; lane 3, 50 cGy X-ray irradiation and acetone twice.
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IEM was used to study the localization of p53 protein. In the positive control p53 proteinantibodygold complexes can clearly be seen as black particles (Figure 3AC
). The particle density was strongest in the positive control, slightly decreased in B[a]P and acetone treated tissue sample, weaker in B[a]P and thapsigargin treated tissue samples and clearly weakest in B[a]P and TPA treated tissue samples. The numbers of particles indicate slightly more p53 protein in the nuclei of cells than in cytoplasm. No p53 protein was found in mitochondria, whereas it seems to be present in all other cell organelles.
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Discussion
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In our previous studies, immunohistochemically detectable p53 protein was induced by B[a]P in mouse skin and this response was inhibited by the treatment with TPA (19,20). We have now confirmed the effect of B[a]P and TPA on p53 with western blotting and IEM in addition to the previous immunohistochemistry. The p53 protein induced is most likely wild-type because the short time from treatment with B[a]P to collection of samples does not allow clonal expansion of any mutated cells.
Kemp et al. (13) have shown that p53 gene null mice lacking both alleles of the p53 gene undergo malignant progression faster than mice with a normal p53 gene, whereas no effect on promotion was noticed. Our own results show that TPA and thapsigargin decrease the carcinogen-induced p53 protein response. The weakened function of wild-type p53 protein could make cells more susceptible to gene mutations including p53 mutations which then speed cells quickly through the transition phase from the stage of tumor promotion to the stage of progression.
Wild-type p53 protein is not usually detectable by immunohistochemistry, but binding to viral or cellular proteins or a mutation may increase its normally very short half-life, making p53 protein detectable. The level of nuclear p53 protein has been shown to increase due to both physical and chemical DNA damage in vitro and in vivo. Accumulated p53 protein has been detected after exposure of cultured cells and human skin in vivo to UV radiation (23 and references therein), cytostatic drugs (24) and exposure to different carcinogens both in cell culture and in whole animals (20,25,26). There has been no change in the level of mRNA in the cells in most of these experimental models. The mechanism for the accumulation of p53 protein is, thus, not transcriptional (24,27). Other possible mechanisms for the increase in p53 protein after DNA damage include stabilization of the p53 protein through phosphorylation by a DNA-activated protein kinase (28,29), inhibition of degradation, which occurs by ubiquitin (30) and calpain (31,32) systems, and effects on the localization of p53 within subcellular structures (33).
It is clear that the effect of phosphorylation on the function of the p53 protein is more complex than a simple on-and-off system (34). Gross changes in phosphorylation status may have effects on p53 transactivation potential that single changes to the same direction cannot have. DNA damage can lead to phosphorylation of p53 protein and this phosphorylation may be involved in induction and activation of p53 (35). Ionizing-radiation-induced phosphorylation at Ser15, which is catalyzed by DNAPKC (34) may regulate both stability and functions of the p53 protein (35). PKC has been shown to phosphorylate recombinant murine p53 protein (36). On the other hand, Milne et al. (37) found that phosphorylation of murine p53 through PKC was not stimulated by the addition of the PKC activator TPA to the cells. This treatment, however, led to a 4-fold stimulation of p53 phosphorylation by a MAP kinase. Thapsigargin, although not studied in p53 phosphorylation before, has been shown to increase the phosphorylation of GLUT-4 (38). It is possible that thapsigargin also affects p53 phosphorylation. Whether and which changes in phosphorylation are involved in B[a]P-induced p53 response and its inhibition by TPA and thapsigargin, remains to be seen.
The effect of thapsigargin, an inhibitor of Ca2+-ATPase in endoplasmic reticulum, on BPDEDNA adduct-related induction of p53 protein was similar to that by TPA. Thapsigargin has some structural similarities with TPA but does not bind to phorbol ester receptor in the particulate fraction of mouse skin (39). Thapsigargin prevents the accumulation of intracellular calcium into endoplasmic reticulum and increases the calcium level in cytosol (40). TPA, by activation of PKC, also leads to a rise in intracellular calcium level through Ca2+ release from intracellular stores and Ca2+ entry into cells (16). In glioma cells, TPA induces a slow increase in intracellular calcium while thapsigargin produces a rapid increase (15). The p53 is a substrate for cleavage by the calcium-activated protease, calpain, suggesting that calpain cleavage plays a role in the regulation of p53 stability (31). Through calpain-activation the effect of TPA and thapsigargin on intracellular calcium levels may be linked to the inhibition of p53 response. One plausible mechanism by which they could decrease the carcinogen-induced p53 protein is the activation of Ca2+-dependent calpain by the increase of Ca2+ leading to a more efficient proteolysis of p53. The smaller 35 kDa band seen in our western blots resembles the putatively calpain-cleaved product of p53 protein seen in the study of Pariat et al. (32). It has to be kept in mind, however, that B[a]P and its metabolites can also increase intracellular Ca2+ in vitro, albeit in high concentrations (41).
The p53 protein has to be localized in the nucleus for its function as a transcription factor. In tumors, one possible derangement in p53 protein is the inhibition of the entrance to the nucleus (33). We used IEM to see if there is any difference in p53 protein concentrations in different cell organelles after the various treatments. Neither TPA nor thapsigargin had any detectable effects on the localization of p53 in B[a]P-treated mouse skin in vivo.
In conclusion, we have shown that tumor promoters TPA and thapsigargin inhibit the B[a]P-induced increase in wild-type p53 and that thapsigargin also inhibits X-ray induced p53 in mouse skin. Inhibition of the putatively genome protecting effect of p53 against chemical-induced DNA damage deserves attention as a potential factor in tumor promotion.
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Acknowledgments
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We gratefully acknowledge the technical assistance of senior laboratory technicians Kirsi Salo, Riitta Harjula and senior animal technicians Tuula Inkala and Ulla Hirvonen. We thank Dr Paavo Pääkkö and his group (Department of Pathology, University of Oulu) for the help in immunohistochemical analysis, and Ms Judith A.Welsh for critical reading of the manuscript. We also acknowledge the generous gift of p53 antibody CM5 from Prof. David Lane (University of Dundee, Dundee, UK). This study was financially supported by grants from the Finnish Cancer Societies and University of Oulu to K.V.
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Notes
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3 To whom correspondence should be addressedEmail: kirsi.vahakangas{at}oulu.fi 
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Received July 3, 1998;
revised March 15, 1999;
accepted June 9, 1999.