Administration of green tea or caffeine enhances the disappearance of UVB-induced patches of mutant p53 positive epidermal cells in SKH-1 mice

Yao-Ping Lu, You-Rong Lou, Jie Liao, Jian-Guo Xie, Qing-Yun Peng, Chung S. Yang and Allan H. Conney *

Susan Lehman Cullman Laboratory for Cancer Research, Department of Chemical Biology, Ernest Mario School of Pharmacy, Rutgers, The State University of New Jersey, 164 Frelinghuysen Road, Piscataway, NJ 08854-8020, USA

* To whom correspondence should be addressed. Tel: +1 732 445 4940; Fax: +1 732 445 0687; Email: aconney{at}rci.rutgers.edu


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Irradiation of female SKH-1 hairless mice with UVB (30 mJ/cm2) twice a week for 10–20 weeks resulted in the formation of a large number of cellular patches (>8 adjacent cells/patch) that are recognized with an antibody (Pab240) which recognizes mutated but not wild-type p53 protein. These patches are not recognized by an antibody (Pab1620) to wild-type p53 protein. The patches, which are considered putative early cellular markers of the beginning of tumor formation, started appearing after 4–6 weeks of UVB treatment, and multiple patches were observed after treatment for 10 weeks. The number and size of the patches increased progressively with continued UVB treatment. Discontinuation of UVB for 4 weeks resulted in an 80–90% decrease in the number of these patches. The number of the remaining patches did not decrease any further but remained relatively constant for at least 4–9 weeks. Oral administration of green tea (6 mg tea solids/ml) or caffeine (0.4 mg/ml) as the sole source of drinking fluid during irradiation with UVB, twice a week for 20 weeks, inhibited UVB-induced formation of mutant p53 positive patches by ~40%. Oral administration of green tea (6 mg tea solids/ml) as the sole source of drinking fluid or topical applications of caffeine (6.2 µmol) once a day 5 days a week starting immediately after discontinuation of UVB treatment enhanced the rate and extent of disappearance of the mutant p53-positive patches. Topical applications of caffeine to the dorsal skin of mice pretreated with UVB for 20 weeks resulted in enhanced apoptosis selectively in focal basal cell hyperplastic areas of the epidermis (putative precancerous lesions), but not in areas of the epidermis that only had diffuse hyperplasia. Our studies indicate that the chemopreventive effect of caffeine or green tea may occur by a proapoptotic effect preferentially in early precancerous lesions.


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Skin carcinogenesis is a complex sequential process that probably involves mutagenic changes in many genes including protooncogenes and tumor suppressor genes. The p53 gene, encoding a transcriptional transactivator participating in the activation of genes that induce cell-cycle arrest, DNA repair and/or apoptosis following DNA damage, is the most commonly mutated gene in human cancers (1,2). The high frequency of p53 mutations in human tumors highlights the importance of p53-dependent functions in suppressing cancer development and/or progression. Missense ‘UV-signature’ mutations at dipyrimidine sites in the p53 gene are found in the majority of non-melanoma skin cancers (35). The detection of patches of p53-mutated keratinocytes in sun-exposed human skin (6) and the formation of p53-mutated patches on chronically UVB-irradiated mouse skin within a few weeks (7,8) indicate that p53 mutations may occur early during the development of skin tumors.

Earlier studies in our laboratory demonstrated that topical application of caffeine to the skin of mice immediately after exposure to UVB enhanced UVB-induced apoptosis, but had no effect on apoptosis in non-UVB-treated normal epidermis (9). Topical applications of caffeine 5 days a week for 20 weeks to tumor-free ‘high risk mice’ previously treated with UVB for 20 weeks inhibited the subsequent formation of non-malignant tumors and squamous cell carcinomas, and these treatments had a selective proapoptotic effect in the tumors but not in non-tumor areas of the epidermis (10). Since most UVB-induced tumors have one or more p53 mutations we suspected that caffeine could induce apoptosis in DNA-damaged cells by a p53-independent mechanism. In a recent study, we found that topical application of caffeine enhances UVB-induced apoptosis in p53 knockout mice by a p53 independent mechanism (11). These studies suggest that caffeine treatment inhibits skin carcinogenesis by enhancing apoptosis selectively in UVB-induced DNA-damaged cells or in tumors with a p53 mutation, but not in normal cells.

The aim of this present study was to measure the kinetics of UVB-induced formation of patches of cells (>8 adjacent cells per patch) that stain for mutant p53 protein during and after stopping chronic UVB exposure in the epidermis of SKH-1 hairless mice. In addition, we investigated the effects of administration of green tea or caffeine on the formation and elimination of patches of mutant p53 positive cells.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Chemicals and animals
Acetone (HPLC grade) and 10% phosphate-buffered formalin were obtained from Fisher Scientific (Springfield, NJ), caffeine (>99% pure) was obtained from Sigma Chemical (St Louis, MO). Lyophilized green tea was obtained from Unilever-Best Foods (Englewood Cliffs, NJ).

Female SKH-1 hairless mice (6–7 weeks old) were purchased from the Charles River Breeding Laboratories (Kingston, NY), and the animals were kept in our animal facility for at least 1 week before use. Mice were given water and Purina Laboratory Chow 5001 diet from the Ralston-Purina (St Louis, MO) ad libitum, and they were kept on a 12 h light/12 h dark cycle.

Treatment of mice with UVB
SKH-1 mice were irradiated with UVB (30 mJ/cm2) twice a week for 10 or 20 weeks as described elsewhere (10). [Although we refer to the UV exposure as UVB, the radiation source emits 75–80% UVB (280–320 nm) and 20–25% UVA (>320 nm).] In some experiments, the mice were treated with 0.6% green tea (6 mg tea solids/ml) or 0.04% caffeine (0.4 mg/ml) as their sole source of drinking fluid during the treatment or after stopping the treatment with UVB. In other experiments, the mice were treated topically with 100 µl acetone or with caffeine (1.2 mg, 6.2 µmol) in 100 µl acetone after stopping treatment with UVB as described in the tables.

Preparation of epidermal sheets for measuring patches of p53 mutated cells
A rectangular portion of dorsal skin, ~30 mm x 40 mm (from head to tail), was removed and floated in thermolysin (100 µg/ml) in phosphate buffered saline (PBS) containing 5 mM CaCl2, overnight at 4°C. The skin sample was fixed in cold acetone (–20°C) twice for 20 min each time, and it was then placed epidermis side down on a ‘hybond membrane’, which was used as a support for helping to separate dermis from the epidermis. The epidermis was stapled on hybond membrane and put into a chamber slide (2 mm x 5 mm with the orientation of head to tail) for immunohistochemical staining. Endogenous peroxidase was blocked with methanol containing 1.5% H2O2, and non-specific binding was blocked with 10% normal rabbit serum, 0.2% bovine serum albumin and 0.1% saponin in PBS for 20 min at room temperature. The sheets were incubated with a 1:25 dilution of monoclonal p53 primary antibody PAb240 (NCL-p53-240; Novocastra, Newcastle, UK) or PAb1620 (OP33, Oncogene Research Product) in PBS overnight at 4°C, and unbound antibody was removed by washing four times for 5 min in each wash in PBS/0.5% Tween-20. The sheets were then incubated with secondary antibody (rabbit anti-mouse IgG1 (Zymed; San Francisco, CA) (1:50 dilution) in PBS for 1 h at room temperature, followed by incubation with avidin–biotin peroxidase complex and stained with 3,3'-diaminobenzidine. The sheets were dehydrated and mounted on glass. PAb1620 recognizes p53 (wild-type conformation), whereas PAb240 is specific for recognizing p53 protein in a mutant conformation in our non-denatured tissue sections. The procedure used is an improved modification of an earlier method for the preparation of epidermal sheets (7).

Determination of apoptosis by caspase 3 immunostaining
Caspases play a pivotal role in the initiation and execution of apoptosis, and caspase 3 (active form) is found in cells undergoing apoptosis by proteolysis. Affinity-purified polyclonal rabbit antibody that reacts with the mouse p20 subunit of caspase 3 but not with the precursor form was purchased from R&D (Catalog No. AF835; Minneapolis, MN). Skin sections used for the measurement of caspase 3 were stained by the horseradish peroxidase-conjugated-avidin method (12) with some modification as described by our laboratory (9). In brief, endogenous peroxidase was blocked by incubating tissue sections in 3% hydrogen peroxide in methanol for 30 min at room temperature. Sections were then treated with 0.01 M sodium citrate buffer (pH 6.0) in a microwave oven at high setting and temperature for 10 min. The sections were incubated with a protein block (normal goat serum) for 10 min, followed by avidin D for 15 min and biotin blocking solution for 15 min (Avidin–Biotin blocking kit from Vector Laboratory, Burlingame, CA) at room temperature. The sections were then incubated with caspase 3 primary antibody (1:2000 dilution) for 30 min at room temperature followed by incubation with a biotinylated anti-rabbit secondary antibody for 30 min and incubated with conjugated-avidin solution (ABC ellite kit purchased from Vector Laboratory) for 30 min. Color development was achieved by incubation with 0.02% 3,3'-diaminobenzidine tetrahydrochloride containing 0.02% hydrogen peroxide for 10 min at room temperature. The slides were then counterstained with hematoxylin, dehydrated and coverslips added for permanent mounting. A positive reaction was shown as a light brown to dark brown precipitate in the cytoplasmic and/or perinuclear portion of the cells. The percentage of caspase 3 positive cells in the epidermis was calculated from the number of caspase 3 stained cells divided by the total number of epidermal cells counted from the entire length (about 25 mm) of epidermis x100 for each skin section.

Determination of apoptosis by morphology of the cells
The identification of apoptotic cells was based morphologically on cell membrane shrinkage and nuclear condensation attributable to fragmentation of the cells (13,14). Apoptotic cells were identified in the epidermis as cells with a homogeneous, densely-staining glossy eosinophilic cytoplasm and a small hyperchromatic condensed pyknotic nucleus that can readily be seen with routine H&E-stained histological sections of the skin using light microscopy. The percentage of apoptotic cells in the epidermis (basal plus suprabasal layer) was calculated from the number of these cells per 100 cells counted from the entire 25-mm length of epidermis for each skin section as done routinely in this laboratory (9,11).


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Specificity of antibodies for recognizing wild-type and mutated p53 proteins in mouse epidermis
Our early time course study showed that irradiation of SKH-1 hairless mice with a single high dose of UVB (180 mJ/cm2) resulted in a rapid increase in the number of epidermal cells with wild-type p53 protein, and the peak increase in the number of wild-type p53 positive epidermal cells (>200-fold higher than for control non-irradiated mice) occurred at 8–12 h after exposure to UVB (15). The p53 positive cells were seen primarily in the basal cell layer, but some were also observed in the suprabasal layer of the epidermis near the basal layer. In the present study, in order to characterize the antibodies, mice were treated with a single dose of UVB (180 mJ/cm2) and killed 10 h after UVB. Immunohistochemical staining in epidermal whole sheets showed that a large number of scattered p53 positive cells were detected by an antibody (Pab1620) to wild-type p53 (Figure 1A), but not with an antibody (Pab240) to mutated type p53 (Figure 1B). In another experiment, mice were treated with UVB (30 mJ/cm2) twice a week for 20 weeks and killed 3 days after the last dose of UVB. Immunohistochemical staining in epidermal whole sheets showed that no nuclear p53 positive cells were found with an antibody (Pab1620) to wild-type p53 (Figure 1C), but a large number of cellular patches (>8 cells/patch) were observed with an antibody (Pab240) to mutated p53. A large representative patch is shown in Figure 1D. These results indicated that whole epidermal sheets fixed in cold acetone provided native proteins suitable for differential immunohistochemical detection with an antibody (Pab240) that specifically recognizes the mutant form of p53 protein (with an altered conformation) or with an antibody (Pab1620) that recognizes the wild-type p53 protein.



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Fig. 1. Selectivity of staining of epidermal sheets with antibodies to wild-type and mutant p53 proteins. (A and B) SKH-1 mice were treated with UVB (180 mJ/cm2) once, and the mice were killed 10 h later to obtain large numbers of epidermal cells with high levels of wild-type p53 protein. In another study, (C and D) the mice were treated with UVB (30 mJ/cm2) twice a week for 20 weeks, and the mice were killed at 3 days after the last irradiation with UVB to obtain patches of hyperproliferating epidermal cells with the mutated form of p53 protein. Epidermal sheets were prepared and stained with an antibody (PAb1620) that recognizes wild-type p53 protein (A and C) or a mutant form of p53 protein (PAb240) (B and D). Large darkly-stained hair follicles (non-specifically stained) were observed in all samples. The magnification used was 100x.

 
Time course for UVB-induced formation of patches of epidermal cells with a mutant form of p53 protein and effect of discontinuation of UVB
Irradiation of hairless SKH-1 mice with UVB (30 mJ/cm2) twice a week for 20 weeks resulted in the formation of a large number of cellular patches (>8 adjacent cells/patch) in epidermal whole sheets with an antibody (Pab240) that specifically recognizes mutated p53 protein. These patches started appearing after treatment with UVB twice a week for 4–6 weeks (Figure 2). The earliest mutant p53 positive epidermal patch was observed at 4 weeks after UVB and contained eight adjacent cluster cells (Figure 3A), and multiple patches were observed after 10 weeks of UVB treatment (Figure 2). Their number and size increased progressively with the duration of UVB treatment. After 20 weeks of UVB treatment, many large patches were observed (Figure 3B) and the number of mutant p53 positive patches/cm2/mouse was >80. The large number of patches observed after 20 weeks of UVB treatment made it difficult to count them accurately. Discontinuation of UVB after 10 or 20 weeks resulted in an 80–90% decrease in the number of patches by 4 weeks (Figure 2, Table I). The disappearance of most of the patches after discontinuation of UVB was associated with scar-like shadows under a light microscope, and was composed of a weak, faded or diffuse mutant p53 staining, and small areas of some patches still displayed clusters of cells with moderate to strong mutant p53 nuclear staining (Figure 3C and D). The number of remaining patches did not decrease further but remained relatively constant during the next 4–8 weeks after stopping UVB (Figure 2, Table I). It is likely that patches of mutant p53 positive cells that remained after discontinuation of UVB represented clones of precancer cells.



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Fig. 2. Time course for UVB-induced formation of patches of epidermal cells with a mutated form of p53 protein and effect of discontinuation of UVB. SKH-1 mice were treated with UVB (30 mJ/cm2) twice a week for 20 weeks. After 10 weeks, UVB exposure was stopped in some of the mice. In other mice, UVB treatment was stopped after 20 weeks. Epidermal sheets were prepared and stained with an antibody that recognizes a mutant form of p53 protein (PAb240). The number of patches (>8 cells/patch) was quantified for each mouse. Each value represents the mean ± SE from 5 mice.

 


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Fig. 3. Morphology of patches of cells that express mutant p53 protein after discontinuation of UVB. (A) SKH-1 mice were treated with UVB (30 mJ/cm2) twice a week for 4 weeks. (B) Mice were treated with UVB twice a week for 20 weeks. (C) Mice were treated with UVB (30 mJ/cm2) twice a week for 20 weeks and UVB was discontinued for 1 week. (D) Mice were treated twice a week with UVB (30 mJ/cm2) for 20 weeks and UVB was discontinued for 4 weeks. Patches of epidermal cells that express mutant p53 protein were determined with an antibody to mutant p53 protein (PAb240). (C) and (D) show diffuse staining with the PAb240 antibody and suggest the break up or dissolution of patches of cells that express mutant p53 protein after stopping UVB. The magnification used was 100x.

 

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Table I. Stimulatory effect of oral administration of green tea or topical applications of caffeine on the disappearance of patches of cells with a mutant form of p53 protein in whole sheets of the epidermis in SKH-1 mice previously treated with UVB for 10 or 20 weeks

 
Effect of reexposure to UVB after its discontinuation on the formation of patches of epidermal cells with a mutant form of p53 protein
Although discontinuation of UVB after 20 weeks of twice a week irradiation resulted in an 80–90% decrease in the number of cellular patches that express the mutant form of p53, retreatment of these mice with UVB once rapidly restored some of these patches in whole sheets of the epidermis. In this study, mice were treated with UVB (30 mJ/cm2 twice a week) for 20 weeks and UVB was stopped. Six weeks later, the mice were treated with 90 mJ/cm2 of UVB once, and sheets of the epidermis were prepared and patches of cells that express the mutant form of p53 were determined by immunohistochemistry at various times after reexposure to UVB. The results showed that the number of patches of cells that express mutant form of p53 in whole sheets of the epidermis was very low before reexposure to UVB and was increased by 188% at 24 h and 288% at 36 h after the single irradiation with UVB (Table II). The results suggest that some of the marked decrease in the number of mutant patches of p53 positive cells observed after discontinuation of UVB results from decreased formation of the mutant form of p53 protein rather than from the loss of these cells (e.g. some patches of cells capable of expressing mutant p53 protein are present but are not expressing the protein in the absence of UVB).


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Table II. Retreatment of high risk SKH-1 mice with UVB after its discontinuation induces partial reappearance of patches of mutant p53 positive cells in whole sheets of the epidermis

 
Effect of oral administration of green tea on the disappearance of patches of epidermal cells with a mutant form of p53 protein after stopping UVB treatment in mice treated with UVB for 10 weeks
In order to determine whether tea has an effect on the disappearance of early patches of epidermal cells with a mutant form of p53 protein after stopping UVB, SKH-1 mice were treated with UVB (30 mJ/cm2) twice a week for 10 weeks to obtain a substantial number of patches of cells that express a mutant form of p53 protein, and UVB was then discontinued. The disappearance of patches was measured in mice given either water or 0.6% green tea (6 mg/ml) as their sole source of drinking fluid for 2, 4, 7, 10 and 13 weeks after discontinuation of UVB. In both groups, the number of mutant p53 positive patches decreased markedly for 4 weeks after discontinuation of UVB and the number of mutant p53 positive patches plateaued at a low and approximately constant level for the next 9 weeks (Figure 2, Table I). The results indicated that treatment of the animals with green tea after discontinuation of UVB enhanced the rate of decrease of mutant p53 positive patches when compared with animals that received only water, and the average extent of decrease of the patches was 48% more in the green tea-treated animals than in the water group at the various times after the start of treatment with green tea (Table I).

Effect of topical applications of caffeine on the disappearance of patches of epidermal cells with a mutant form of p53 protein after stopping UVB treatment in mice treated with UVB for 20 weeks (high risk mice)
In order to determine whether caffeine has an effect on the disappearance of late patches of epidermal cells with a mutant form of p53 protein after stopping UVB, SKH-1 mice were treated with UVB (30 mJ/cm2) twice a week for 20 weeks to obtain mice without tumors but with a large number of mutant p53 positive patches and a high risk of developing tumors during the next several months (high risk mice) (10). The disappearance of mutant p53 positive patches was measured after discontinuation of UVB. The results indicated that topical applications of caffeine (1.2 mg) in 100 µl of acetone 5 days a week immediately after stopping UVB enhanced the rate of disappearance and the extent of decrease in the number of mutant p53 positive patches (Table I). The number of mutant p53 positive patches in the caffeine treated mice was 52–63% less than in the acetone-treated vehicle control group between 1–8 weeks after discontinuation of UVB (Table I).

Inhibitory effect of oral administration of green tea or caffeine together with UVB irradiation for 20 weeks on UVB-induced formation of patches of epidermal cells with a mutant form of p53 protein
Treatment of SKH-1 mice with UVB (30 mJ/cm2) twice a week for 20 weeks resulted in no tumors but resulted in 82.6 ± 5.0 patches of cells that express the mutant form of p53 protein/cm2/mouse and this effect was inhibited by ~40% in mice that received green tea (6 mg tea solids/ml) or caffeine (0.4 mg/ml) as their sole source of drinking fluid during the 20 week irradiation period (Table III).


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Table III. Effect of oral administration of green tea or caffeine in SKH-1 mice on UVB-induced formation of patches of epidermal cells with mutant p53 protein

 
Effect of oral administration of green tea or topical application of caffeine in mice treated with UVB for 20 weeks (high risk mice) on apoptosis in areas of the epidermis with focal basal cell hyperplasia
Since topical applications of caffeine to the dorsal skin of mice previously treated with UVB for 20 weeks resulted in an enhanced disappearance of mutant p53 positive patches of epidermal cells, we determined whether topical applications of caffeine to the high risk mice after stopping UVB enhanced apoptosis selectively in precancerous cells, such as focal basal cell hyperplastic areas, which are considered an early stage of tumor formation. Examples of focal basal cell hyperplasia and diffuse hyperplastic areas of the epidermis are shown in Figure 4. In this study, mice were treated with UVB (30 mJ/cm2) twice a week for 20 weeks and UVB treatment was stopped. The animals were then treated topically with 100 µl acetone or 6.2 µmol caffeine in 100 µl acetone twice a day (9 a.m. and 4 p.m.) for 3 or 14 days and the animals (8 mice/group) were killed 2 h after the morning application of caffeine. The results showed that topical applications of caffeine selectively increased apoptosis as measured by caspase 3 positive cells by 68–74% in focal basal cell hyperplastic areas of the epidermis when compared with acetone-treated control mice (Table IV). No caffeine-induced increase in apoptosis was observed in areas of the epidermis that had diffuse hyperplasia (Table IV). Oral administration of 0.6% green tea to UVB-pretreated high risk mice for 2 weeks stimulated apoptosis by 89% in focal basal cell hyperplastic areas of the skin but not in areas with diffuse hyperplasia (data not shown). In contrast to these studies, topical applications of (–)-epigallocatechin gallate (6.2 µmol) twice a day for 3 or 14 days failed to increase apoptosis in focal basal cell hyperplastic areas of the epidermis as measured immunohistochemically with an antibody for caspase 3 (active form) (data not shown).



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Fig. 4. Morphology of focal basal cell hyperplasia and diffuse hyperplasia of the epidermis. The upper panel shows a diffuse hyperplasia area of the epidermis and the lower panel shows a focal basal cell hyperplasia area. The magnification used was 100x.

 

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Table IV. Stimulatory effect of caffeine administration on caspase 3 (active form) in focal basal cell hyperplastic areas of the epidermis in high risk SKH-1 mice previously treated with UVB for 20 weeks

 
In a time course study, UVB-pretreated high risk mice were treated topically once with caffeine (6.2 µmol) in 100 µl acetone or acetone alone 1 or 4 weeks after discontinuing UVB, and morphologically identified apoptotic cells were determined at various times after caffeine administration. The results indicated that topical application of caffeine increased apoptotic cells by 281 or 191% at 6 h after application of caffeine in the two studies, and these effects were observed specifically in basal cell focal hyperplastic areas of the epidermis but not in areas of the epidermis that have diffuse hyperplasia (Table V). Topical application of caffeine selectively increased apoptosis by 346% in focal basal cell hyperplastic areas of the epidermis when compared with acetone-treated control mice (Table V). The caffeine-induced increase in apoptosis occurred at 6 h after a single topical application of caffeine but not at 10, 16 or 24 h after caffeine treatment (Table V).


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Table V. Stimulatory effect of a single topical application of caffeine on apoptosis in the epidermis of high risk SKH-1 mice previously treated with UVB for 20 weeks

 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In the present study, we found that treatment of the dorsal skin of SKH-1 hairless mice with a low dose of UVB (30 mJ/cm2) twice a week for 20 weeks induced the formation of patches of adjacent cells (>8 cells/patch) that stain for mutant p53 protein. The dose of UVB used in these studies was within the range of common human exposure (1618). The first appearance of patches of cells with mutant p53 protein occurred after 4 weeks of UVB treatment, and with continued treatment the number and size of these patches increased. The formation of patches of cells that express p53 mutant protein after chronic UVB treatment have been described by others and is thought to represent the formation of hyperproliferating cells that are early precursors of tumors observed many months later (7,8,19). DNA sequencing of the entire coding region of the p53 gene (exons 2–11) from 104 patches of epidermal cells that express the mutant form of p53 protein after 20 weeks of UVB treatment revealed that only 64% of these patches had a mutation in the coding region of the p53 gene (20). The reasons and physiological significance of patches of cells that express p53 protein with a mutant conformation but without a mutation in the coding region of the gene is unknown. Other less extensive studies have also failed to find p53 mutations in some UVB-induced patches of mutant p53 positive epidermal cells (21) or in UVB-induced tumors (22). The reason(s) for this interesting observation is unknown, but the effect appears to be heritable.

The number of mutant p53 positive patches decreased markedly after discontinuation of UVB and then remained constant for at least 9 weeks (Figure 2, Table I). A decrease in the number of patches after discontinuation of UVB has also been seen by others (8). It is likely that the low constant level of mutant p53 positive patches that was observed after discontinuation of UVB represents permanently altered cells that have a capacity to become tumors. It is of interest that reexposure of these mice to a single irradiation of UVB several weeks after its discontinuation resulted in the restoration of a fraction of these patches (again expressing the mutant form of p53 protein) by 16–36 h after reexposure to UVB (Table II). These results indicate that the disappearance of at least some of the p53 positive patches that occurred after discontinuing UVB resulted from decreased expression of the mutant form of p53 protein rather than by a loss of the cells that express the protein.

Oral administration of green tea or topical applications of caffeine starting immediately after discontinuation of UVB treatment enhanced the rate and extent of disappearance of the mutant p53 positive patches (Table I) as well as enhancing apoptosis in focal basal cell hyperplastic areas of the epidermis (Table IV). Although in the present study we did not quantify differences in the number of cells expressing the mutant form of p53 between focal basal cell hyperplastic areas and diffuse cell hyperplastic areas, our preliminary data suggest that more mutated p53 type positive cells were present in the focal basal cell hyperplastic areas than those in diffuse cell hyperplastic areas. The selective proapoptotic effects of the administration of green tea or caffeine on early precancerous lesions described here resemble the effects of tea and caffeine to (i) enhance acute UVB-induced apoptosis in the epidermis (but not in normal non-UVB treated skin) (9,11,23) and (ii) to increase apoptosis in skin tumors (but not in the surrounding epidermis) (10,24). These proapoptotic effects may represent mechanisms for the cancer chemopreventive action of tea and caffeine when administered during the course of chronic UVB treatment or after stopping UVB irradiation.

Several studies indicate that cancer cells with a disrupted G1 checkpoint (loss of p53 function) are sensitized to ATR/Chk1 inhibition thereby causing premature chromatin condensation and lethal mitosis (25,26). Caffeine sensitizes cultured cancer cells to the toxic effects of DNA-damaging chemotherapeutic agents possibly by blocking normal checkpoint control of the cell cycle and allowing lethal mitosis of the caffeine-treated cells (27). Caffeine-induced override of the G2 checkpoint may be caused by an inhibitory effect of caffeine on ATR/Chk1 kinase activities (2830). Combinations of caffeine and {gamma} radiation or caffeine and UVB radiation caused a synergistic effect on apoptosis in p53 defective cells via a p53-independent pathway (11,31). Cancer cells with defective G1 checkpoint control appear to be selectively affected by ATR/Chk1 inhibitors and suggest that ATR/Chk1 inhibitors that are more potent than caffeine may be useful adjuvant agents for cancer therapy. The concept of the selectivity of ATR/Chk1 inhibitors for p53 defective cells may help explain why caffeine treatment enhanced the rate and extent of disappearance of mutant p53-positive patches in the present study and why caffeine has a selective apoptotic effect in focal basal cell hyperplastic areas of the epidermis and in UVB-induced skin tumors (shown to have p53 mutations) but not in non-tumor areas of the epidermis (10). The preferential sensitization of cells that lack functional p53 to DNA damage-induced apoptosis raises hopes that adjuvant therapy with G2 checkpoint inhibitors will increase the therapeutic efficacy of DNA-damaging therapies in the large population of cancer patients whose tumor cells lack functional p53.


    Acknowledgments
 
We thank Florence Florek for her excellent help in the preparation of this paper. This work was supported in part by NIH grants No. CA80759 and CA88961.

Conflict of Interest Statement: None declared.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received December 12, 2004; revised March 23, 2005; accepted March 25, 2005.





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