Effect of administration of caffeine or green tea on the mutation profile in the p53 gene in early mutant p53-positive patches of epidermal cells induced by chronic UVB-irradiation of hairless SKH-1 mice
Pavel Kramata *,
Yao-Ping Lu,
You-Rong Lou,
Julie L. Cohen,
Meir Olcha,
Sandy Liu 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, Piscataway, NJ 08854-8020, USA
* To whom correspondence should be addressed. Tel: +1 732 445 3400 ext. 238; Fax: +1 732 445 0687; Email: kramata{at}rci.rutgers.edu
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Abstract
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Irradiation of SKH-1 mice with UVB light for 20 weeks resulted in a large number of patches of epidermal cells, which was visualized with an antibody that recognizes mutated p53 protein. Oral treatment of mice with caffeine (0.4 mg/ml) or green tea (6 mg tea solids/ml) as the drinking fluid during UVB irradiation decreased the number of patches by
40%. Sequencing analysis of the p53 gene (exons 3 to 9) detected 88, 82 or 39 point mutations in 67, 70 or 29 patches from water, caffeine or tea treated mice, respectively. A major hotspot at codon 270 (Arg
Cys) accounted for 47.7% (water), 70.7% (caffeine) or 46.2% (tea) of all mutations. Patches from caffeine treated mice had fewer types of mutations than patches from mice treated with water or tea. Administration of caffeine or tea during 20 weeks of UVB irradiation eliminated mutations at codons 149 (Pro
Ser) and 210 (Arg
Cys) but increased the frequency of mutations at codon 238 (Ser
Phe). Topical applications of caffeine (1.2 mg in 100 µl acetone) once a day, five times a week for 6 weeks after stopping UVB decreased the number of patches by 63% when compared with mice treated with acetone. DNA sequencing analysis detected 63 and 68 mutations in 48 and 57 patches from acetone or caffeine treated mice, respectively. Although no differences in the frequency, position or types of mutations were observed, the caffeine group harbored less homozygous mutations (12.3% of the total) than the acetone group (31.3% of the total, P = 0.029). In summary, oral treatment of mice with caffeine or green tea during chronic UVB irradiation changed the mutation profile of the p53 gene in early mutant p53 positive epidermal patches, and topical applications of caffeine after discontinuation of chronic UVB irradiation specifically eliminated patches harboring homozygous p53 mutations.
Abbreviations: CPD, cissyn cyclobutane pyrimidine dimers; NER, nucleotide excision repair; SCC, squamous cell carcinomas; TLS, translesion replication synthesis.
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Introduction
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Non-melanoma skin cancer is the most common type of cancer worldwide (1). More than one million cases accounting for
40% of all cancers combined are diagnosed each year in the USA (2). The aging population, increased recreational exposure to sunlight and the depletion of the ozone layer are believed to be major factors contributing to the globally increasing incidence of skin cancer (2,3). The use of common dietary components as chemopreventive agents against skin cancer has been widely studied in animal models and has shown promising results [reviewed in ref. (4)].
In previous studies we found that oral administration of green tea, black tea or caffeine to SKH-1 mice during chronic UVB treatment inhibited UVB-induced skin carcinogenesis, but the decaffeinated teas were inactive (5). Adding caffeine to the decaffeinated teas restored their inhibitory effects indicating that caffeine is a major chemopreventive constituent in tea. In additional studies, we treated SKH-1 mice with a low dose of UVB (30 mJ/cm2) twice a week for 20 weeks and UVB administration was stopped. These tumor-free mice were at a high risk of developing skin tumors during the next several months in the absence of further UVB treatment, and oral or topical administration of caffeine to these high-risk mice inhibited the formation of skin tumors (6,7). Mechanistic studies indicated that administration of caffeine enhanced apoptosis in UVB-induced skin tumors (7) and also in UVB-irradiated normal skin (8,9).
One of the very early changes induced in the skin by chronic UVB irradiation appears to be the formation of patches of epidermal cells that stain with an antibody recognizing an altered conformation of the mutant form of the p53 tumor suppressor protein (10). These patches are common in normal appearing sun-exposed skin in humans (11) and also in the skin of chronically UVB-irradiated mice (12). Sequencing of all exons of the p53 gene in mutant p53 positive patches from mice treated with UVB for 20 weeks demonstrated a close resemblance to the mutational profile observed previously in UVB-induced mouse skin tumors (13). These results along with a study of p53 mutations in human skin patches (14) suggest that mutant p53 positive patches of cells that are present in UVB-exposed skin long before the appearance of tumors are early precursor lesions of tumors.
Application of agents that reduce or alter UVB-induced p53 mutagenesis or create an environment that suppresses the growth of p53 mutant epidermal cell patches may provide a promising chemopreventive strategy as it would likely result in the formation of less invasive tumors and/or in a decrease of the final tumor burden. In a recent study, we found that oral administration of caffeine or green tea together with UVB irradiation for 20 weeks inhibited the UVB-induced formation of mutant p53 positive patches (15). In addition, topical applications of caffeine to high-risk mice after stopping UVB irradiation enhanced the disappearance of these mutant p53 positive patches (15).
The type of p53 mutation in a patch may affect skin tumor development. Most missense p53 mutants exhibit a unique combination of defective functions compared with wild-type p53 (16,17), which likely results in various degrees of inhibition of function of the remaining wild-type p53 allele (dominant-negative effect) and/or differences in promoting tumorigenesis (gain-of-function) (18). To determine whether administration of caffeine or green tea alters the profile of p53 mutations in mutant p53 positive skin patches, the present study examined mutations in the p53 gene (exons 39) in patches from mice treated orally with caffeine or green tea during 20 weeks of UVB irradiation and from high-risk mice treated topically with caffeine for 6 weeks after stopping the UVB treatment of 20 weeks.
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Materials and methods
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Chemicals and animals
Lyophilized green tea was obtained from Unilever-Best Foods (Englewood Cliffs, NJ), caffeine (>99% purity) was from the Sigma Chemical (St Louis, MO), acetone (HPLC grade) and phosphate-buffered formalin were from Fisher Scientific (Springfield, NJ). Water purified by reverse osmosis was used for the preparation of caffeine (0.4 mg/ml water) and green tea (6 mg tea solids/ml water) solutions. Fresh solutions were prepared every 2 days. Female SKH-1 hairless mice (67 weeks old, Charles River Breeding Laboratories, Kingston, NY) were fed Purina Laboratory Chow 5001 diet (Ralston-Purina, St Louis, MO) ad libitum, and they were kept on a 12 h lightdark cycle.
Treatment of mice with UVB light, caffeine or green tea
The UV lamps (FS72T12-UVB-HO; National Biological, Twinsburg, OH) emitted UVB (280320 nm; 7580% of total energy) and UVA (320375 nm; 2025% of total energy). The dose was quantified with a UVB Spectra 305 dosimeter (Daevlin Byran, OH). The radiation was further calibrated with the model IL-1700 research radiometer/photometer (International Light, Neburgport, MA).
Mice were treated with caffeine or green tea solutions as their sole source of drinking fluid (eight and four mice per group, respectively) and eight mice were treated with water as control. The mice were simultaneously irradiated with UVB (30 mJ/cm2) twice a week (Tuesdays and Fridays) for 20 weeks. In another experiment, 35 mice were irradiated with UVB (30 mJ/cm2) twice a week for 20 weeks, and after stopping the UVB treatment 15 mice were treated on their back with 100 µl acetone and 20 mice were treated with caffeine (1.2 mg, 6.2 µmol) in 100 µl acetone once a day, 5 days a week for 6 weeks. At the end of each experimental protocol the mice were killed by cervical dislocation.
Immunohistochemistry
Dorsal skin samples removed from animal trunks (2 x 4 cm) were treated with thermolysin and CaCl2 overnight and fixed in cold acetone. Epidermis was attached to a hybond membrane and epidermal sheets were separated from the dermis with a pair of tweezers. Endogenous peroxidase was blocked with H2O2 in methanol. Non-specific binding of antibody was blocked with normal rabbit serum, bovine serum albumin (BSA) and saponin. The epidermal sheet was incubated either with the monoclonal p53 antibody PAb240 (NCL-p53-240) that recognizes mutant p53 protein (Novocastra, Newcastle, UK) or PAb1620 (OP33) that recognizes wild-type p53 protein (Oncogene Research Products). Following application of biotinylated secondary antibodies and avidin-biotin-peroxidase complex the sheets were stained with 3,3'-diaminobenzidine. The details of the preparation of the epidermal sheets and the staining for mutant p53 are described elsewhere (20).
Microdissection, DNA amplification and sequencing
Samples of approximately 1001000 cells were isolated from the stained clones in epidermal sheets under a microdissecting microscope using a 30-gauge needle (Becton-Dickinson, Rutherford, NJ) and a micromanipulator (Narishige MO-203, Tokyo, Japan). We analyzed 13 ± 2, 10 ± 4 and 10 ± 4 patches per mouse from mice treated orally with water, caffeine or green tea, respectively, and 5 ± 3 patches per mouse from mice treated topically with acetone or caffeine in acetone. The patches were placed into 20 µl of 10 mM TrisHCl (pH 8.5) and 1% Tween-80 and incubated for 16 h at 65°C with 3.2 µg/µl proteinase K (PCR grade, Roche Biochemicals, Indianapolis, IN). Proteinase K was inactivated for 15 min at 95°C and sample aliquots were directly used for DNA amplification (1/10 of the reaction volume). Fragments of 548, 507, 322 and 495 bp (encompassing exons 3 + 4, 5 + 6, 7 and 8 + 9, respectively) were individually amplified in PCR reaction mixtures (25 µl) containing 0.025 U/µl HotStarTaq DNA polymerase (Qiagen, Valencia, CA), Qiagen reaction buffer (1x) with 1.5 mM MgCl2, 200 µM of each dNTP and 0.2 µM of each primer. Cycling conditions were as follows: after the initial 15 min at 94°C there were 40 cycles (94°C, 30 s/55°C, 30 s/72°C, 45 s) and a final 7 min step at 72°C. Primers (IDT, Corralville, IA) located in introns had the following sequences: Ex3,4for: 5'-CCTGGGATAAGTGAGATTCTGTC; Ex3,4rev: 5'-GGCACAGTCTACAGGCTGAAGAG; Ex5,6for: 5'-CCTTGACACCTGATCGTTACTCG; Ex5,6rev: 5'-AGAAAGTCAACATCAGTCTAGGC; Ex7for: 5'-TGTGCCGAACAGGTGGAATATCC; Ex7rev: 5'-ACTCGTGGAACAGAAACAGGCAG; Ex8,9for: 5'-GGCCTAGTTTACACACAGTCAGG; Ex8,9rev: 5'-CACGGCTAGAGATAAAGCCACTG.
Amplified fragments were visualized on a 1.5% agarose gel stained with ethidium bromide. Before sequencing, primers and dNTPs in PCR reaction mixtures were digested with exonuclease I and shrimp alkaline phosphatase (ExoSAP-IT, USB, Cleveland, OH) at 37°C for 15 min and the enzymes were deactivated at 80°C for 15 min. DNA sequencing was performed using the same primers and the BigDye sequencing kit from Applied Biosystems (Foster City, CA) and analyzed using an automatic sequencer ABI Prism 3100 Genetic Analyzer (DNA Sequencing Core Facility, UMDNJ, Piscataway, NJ). Sequencing waveforms were processed using the Chromas2.22 computer program (Technelysium, Tewantin, Australia). A mutation appears as a vertical double signal (peak) at the same position. The position was considered as mutated when one signal reached at least half of the other with a negligible level of noise in the background. Two sequences for each sample (generated with forward and reverse primers) were compared with a standard normal mouse p53 sequence retrieved from the GenBank (AC074149, Mus musculus clone RP23-114M1) using the sequence alignment program MultiAlign that uses a published algorithm (19), and is available online at http://prodes.toulouse.inra.fr/multalin/multalin.html. Mutation changes that appeared on both parallel sequences were scored. The non-stained areas of the epidermis adjacent to patches stained with the antibody that recognizes mutant p53 were also taken for the analysis. No p53 mutations were observed in a total number of 23 control samples taken from non-patch areas of the skin.
Statistical analysis
P-values were calculated from contingency tables using two-tailed Fisher's exact test with 95% confidence intervals. The P-value threshold indicating statistical significance was set to 0.05 (Graph Pad Prism, San Diego, CA).
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Results
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Oral administration of caffeine or green tea during chronic UVB treatment affects the mutation profile of the p53 gene in UVB-induced patches of mutant p53 positive epidermal cells
Irradiation of hairless SKH-1 mice with a low dose of UVB light (30 mJ/cm2) twice a week for 20 weeks induced the formation of cellular patches (>8 adjacent cells per patch) that react with an antibody against the mutated form of p53 (PAb240), but not with an antibody against wild-type p53 (PAb1620) (15). These patches are considered pre-tumorous lesions as they have the potential of developing into squamous cell carcinomas (SCC) several months later without further UVB treatment. We found that SKH-1 mice treated orally with water, caffeine (0.4 mg/ml) or with green tea (6 mg tea solids/ml) as their sole source of drinking fluid together with twice a week UVB irradiation for 20 weeks resulted in an average of 82.6, 51.8 and 46.4 patches of mutant p53 positive cells per cm2, respectively (15). These results indicated that oral administration of caffeine or green tea during chronic UVB irradiation inhibited the formation of these patches by
40%. A more detailed description of effects of orally administered caffeine or green tea on UVB-induced formation of mutant p53 positive patches in mouse epidermis is described elsewhere (15).
We performed a mutation analysis of the p53 gene (exons 39) in 104, 84 and 34 patches from water, caffeine or green tea treated mice, respectively, after 20 weeks of UVB treatment. These patches were isolated with a thin needle and a micromanipulator under a microdissecting microscope and treated as described in the section Materials and methods. A more detailed description of the mutation analysis of the p53 gene of patches isolated from control (water treated) mice is published elsewhere (13). In this study, we report differences in the mutation profile of the p53 gene found in patches from mice treated with caffeine or green tea compared with those from mice treated with water. The incidence, positions and types of base and amino acid substitutions in all p53 mutations detected in these three groups were compared (Table I, oral treatment), and the results are summarized as follows:
- A mutation 270 CGTArg
TGTCys, the major hotspot accounting for 32.1% of all p53 mutations in UVB-induced SCC (20), was also the major hotspot in patches isolated from all three groups. The codon numbering originally reported in ref. (20) did not count the first three N-terminal amino acids of the protein resulting in three-digit lower numbers compared with this study. The proportion of this mutation was significantly higher in the caffeine group (70.7%) than in the water or green tea groups (47.7%, P = 0.003, or 46.2%, P = 0.015, respectively). This result may be a consequence of fewer types of mutations (mutation diversity) found in the caffeine group (14 types, 82 mutations; 17.1% diversity) compared with the water group (27 types, 88 mutations; 30.7% diversity, P = 0.048) or the green tea group (17 types, 39 mutations; 43.6% diversity, P = 0.003), where the percent mutation diversity is defined as the (number of types of mutations/total number of mutations) x100.
- A mutation 149 CCAPro
TCASer, a hotspot accounting for 7.1% of all p53 mutations in UVB-induced SCC (20), was found in 9.1% of all mutations in the water group but was not detected in patches from caffeine or green tea treated mice (P = 0.008 or P = 0.10, respectively). Interestingly, a single mutation at codon 149 detected in a sample from a mouse orally treated with caffeine was CCA
CTA, exchanging Pro for Leu. Similarly in the green tea group, the two mutations found in this position also resulted in a Pro
Leu substitution.
- Mutations at codon 275 (CCTPro
CTTLeu and CCTPro
TCTSer), one of the p53 mutation hotspots present in UVB-induced SCC (20), were missing in samples from caffeine and green tea treated mice, although mutations at this position were much less common in epidermal patches from control mice (4.5% of the total mutations) than previously found in SCC (14.7% of the total mutations) (20).
- A mutation at codon 210 (CGCArg
TGCCys) found in 7.9% of all mutations in patches from water treated mice was not detected in samples from caffeine or green tea treated mice. This mutation was also absent in UVB-induced SCC.
- A new mutation hotspot at codon 238 (TCCSer
TTCPhe) was induced by caffeine or green tea treatment since it was found with much higher frequency in samples from caffeine or green tea treated mice than in control samples (13.4% and 10.3% versus 1.2% of the total mutations, P = 0.005 and P = 0.039, respectively). This mutation was not common in UVB-induced SCC (1.9%) (20).
- The majority of mutations in samples from water, caffeine or green tea treated mice were missense substitutions (9798% of the total mutations), and they contained mostly C
T transitions (89.0%, 97.5% and 83.8% of the total mutations, respectively) located at dipyrimidine sites (97.7%, 98.8%, 94.7%, respectively) on the non-transcribed strand (100, 93.8 and 86.5%, respectively). The higher numbers of dipyrimidine substitutions located on the non-transcribed strand in samples from water treated mice compared with those from caffeine or green tea treated mice (P = 0.027 or P = 0.002, respectively) suggest a possible inhibitory effect of caffeine on transcription-coupled repair. A larger number of samples from green tea treated mice would need to be analyzed to determine whether or not this effect of caffeine is further enhanced by other components of green tea, as the difference between the green tea and caffeine groups was not significant (P = 0.283).
Determination of the distribution of mutations in individual patches showed one or more mutations in 64.4, 83.3 or 85.3% of the patches from water, caffeine or green tea treated mice, respectively. Two green tea samples had three mutations and were the only samples with more than two mutations (Table II). The majority of single mutant samples harbored a mutation hotspot at codon 270 and, similarly, most double-mutants carried this mutation hotspot in combination with another mutation. Studies of the allelic distribution of two p53 mutations in epidermal patches (13) and in skin tumors (21) previously demonstrated that these mutations were mostly located on separate alleles. Thus, similar to a homozygous p53 mutation, the presence of two p53 mutations in a sample likely indicates a loss of the normal p53 allele in the patch. As shown in Table II, caffeine treatment slightly decreased the number of patches harboring two or more mutations compared with the patches from water or green tea treated mice, but the result was not significant (P = 0.15 or P = 0.28, respectively). Also, the number of patches with a homozygous mutation (mutant signal >75% of the total signal at the mutant position, see Figure 1) was very similar in all three groups (Table II). On the other hand, the diversity of patches, i.e. the percentage of samples with a different p53 profile, was significantly lower in the caffeine group (21.4%) than in the water group (41.8%, P = 0.016) or in the green tea group (51.7%, P = 0.004). Accordingly, the proportion of samples harboring the mutation hotspot at codon 270 was significantly higher in caffeine samples (82.9%) than in water or green tea samples (61.2%, P = 0.007, or 62.1%, P = 0.036, respectively). These results suggest that caffeine treatment inhibited the formation of mutations induced by UVB irradiation and/or created an environment that suppressed the growth of patches harboring mutations other than those at codon 270. In contrast, this effect of caffeine was apparently inhibited by treatment with green tea (containing the same amount of caffeine, 0.4 mg/ml).
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Table II. Distribution of p53 mutations in individual UVB-induced epidermal cell patches of hairless mice treated orally with caffeine or green tea in water during chronic UVB irradiation
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In summary, oral administration of caffeine or green tea to mice during UVB irradiation for 20 weeks changed the mutation profile of the p53 gene in UVB-induced epidermal skin patches by eliminating and/or inducing the formation of specific mutation hotspots, and the changes appear to be similar for both treatments. However, treatment with caffeine, in contrast to treatment with green tea, narrowed the spectrum of UVB-induced mutations, which resulted in the formation of patches with lower p53 mutation diversity.
Topical administration of caffeine after stopping chronic UVB treatment decreases the number of epidermal cell patches with a homozygous mutation in the p53 gene
Twice a week irradiation of SKH-1 mice with UVB (30 mJ/cm2) was stopped after 20 weeks and the mice were treated topically with 100 µl of acetone or with caffeine (1.2 mg) in 100 µl of acetone once a day 5 days a week for 6 weeks. As described elsewhere (15), stopping UVB irradiation for 6 weeks results in a 95% decrease in the number of mutant p53 positive patches, and treating the mice topically with caffeine decreases the number of patches by an additional 63% (15). The average number of p53 positive patches in the UVB-pretreated mice after stopping UVB and topical acetone or caffeine treatment for 6 weeks was earlier found to be 4.1 and 1.5 patches/cm2, respectively (15).
Here, we analyzed the sequence of the p53 gene in 67 and 87 patches that persisted in the epidermis of acetone and caffeine treated mice, respectively, using the same methodology as described above, and we compared the incidence, positions and types of base and amino acid substitutions of all detected p53 mutations between these two groups (Table I, topical treatment). Similar to the patches that were present before stopping UVB treatment in control mice, the major mutation hotspot was found at codon 270, and all minor hotspots at codons 149, 210, 238 and 275 were also present in both groups. As shown in Table I, no significant differences in the mutational profile were observed between the two groups.
The distribution of mutations in individual mutant p53 positive patches showed one or two mutations in 72.1 and 65.5% of the patches from acetone and caffeine treated mice, respectively, with a majority of samples harboring a single mutation in both the groups (Table III). Importantly, a significantly lower percentage of samples from caffeine treated mice than from acetone treated mice harbored homozygous mutations (12.3 versus 31.3%, respectively, P=0.029) suggesting that cellular patches lacking the normal p53 allele may be preferentially eliminated by topical treatment with caffeine. A lower number of double mutant samples was also observed in patches from the caffeine group compared with the acetone group, although the difference was not significant (19.3 and 30.6%, respectively, P=0.258).
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Table III. Distribution of p53 mutations in individual UVB-induced epidermal cell patches from SKH-1 hairless mice treated topically with acetone or caffeine after stopping chronic UVB irradiation
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Collectively, these results demonstrated that topical applications of caffeine on mouse skin for 6 weeks after the discontinuation of chronic UVB irradiation selectively decreased the number of cellular patches with a homozygous mutation in the p53 gene, but this treatment had no selectivity towards patches harboring a specific p53 mutation hotspot.
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Discussion
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Oral administration of caffeine or green tea during chronic UVB irradiation, or topical administration of caffeine after stopping the UVB irradiation induced significant changes in the mutation profile of the p53 gene in potentially pre-cancerous patches of mutant p53-immunoreactive epidermal cells in SKH-1 hairless mice. Although each of these treatments decreased the number of early patches in the skin (15) and ultimately resulted in a similar reduction of UVB-induced skin tumors (5,7), the mechanism of their effects may be quite different. Differences between the effects of orally and topically administrated caffeine and also between the effects of orally administered caffeine and green tea to modify the UVB-induced mutation profile of the p53 gene were observed and may help identify mechanisms of chemoprevention by caffeine and green tea during UVB-induced skin carcinogenesis.
Oral administration of caffeine or green tea during the course of UVB treatment eliminated the formation of two UVB-induced mutation hotspots in the p53 gene of skin patches (codons 149 and 210) and stimulated the formation of a mutation hotspot at position 238. Interestingly, these changes were seen in both caffeine and green tea treated mice indicating that caffeine may be the major modifying factor in green tea in this model. The majority of UVB-induced mutations arises from unrepaired cissyn cyclobutane pyrimidine dimers (CPDs) (22) by a mutagenic bypass of the lesion with a translesion replication synthesis (TLS) DNA polymerase and/or by spontaneous deamination of 5-methylcytosine within the pyrimidine dimer (23). Possible explanations for the observed effects of caffeine may include its interaction with (i) an enzyme(s) participating in nucleotide excision repair (NER), a pathway removing UV-induced lesions from DNA (24); (ii) a TLS DNA polymerase affecting the fidelity of replicative bypass of UV-induced lesions during DNA replication; and (iii) processes involved with UVB-induced formation of pyrimidine dimers at methylated CpG sequences and/or deamination of 5-methylcytosine within the pyrimidine dimers. In addition to these possible effects, caffeine may also suppress or stimulate the growth of cells harboring specific p53 mutations and thus select patches of cells with a specific p53 mutation profile. It is also important to point out that orally administered caffeine is metabolized, and its metabolites may contribute to the final effect. Besides caffeine (1,3,7-trimethylxanthine), we have detected significant levels of theophylline (1,3-dimethylxanthine), 1,7-dimethylxanthine and theobromine (3,7-dimethylxanthine) in the plasma and epidermis of mice treated orally with caffeine (unpublished observations).
Although caffeine was shown to inhibit the gene-specific NER of CPDs in UV-irradiated CHO cells (25), there is no direct evidence of an interaction between caffeine and enzymes that participate in the NER complex, such as those from the well characterized complementation group (XPA to G) of xeroderma pigmentosum, a human genetic disorder associated with an extreme sensitivity to sunburn and an early onset of skin cancer (26). The mutation spectrum of the p53 gene in UV-induced tumors in mice with a knock-out of the XPA (27) or XPC (28,29) gene shows no discernible similarities to the caffeine- or tea-induced changes in the p53 mutation profile observed in our study indicating that the changes we observed after treatment with caffeine or green tea were not caused by an interaction of caffeine with XPA or XPC.
The only known DNA polymerase that efficiently bypasses CPDs in vitro is pol
(30). This enzyme is likely a major polymerase that bypasses UV-induced lesions in vivo since a defect in the pol
gene results in a variant form of xeroderma pigmentosum (31) associated with a high incidence of skin cancer, skin sunlight hypersensitivity (32) and UV-induced hypermutability (33). Caffeine is highly toxic to UV-irradiated cells from XP-V patients (34), but the effect is eliminated by the overexpression of pol
(35) indicating a protective effect of pol
in UV-irradiated cells that are treated with caffeine. A recent study revealed the existence of a caffeine sensitive polymerase responsible for the bypass of UV-induced lesions in coordination with pol
(36), possibly pol
(37) or pol
(38). Whether or not the inhibition of this polymerase by caffeine could potentially lead to the elimination of several mutation hotspots observed in this study is not known. Alternatively, caffeine may induce increased levels of DNA polymerases that bypass the lesion with high fidelity.
In contrast to the effect of oral administration of caffeine or green tea during UVB treatment on the p53 mutational profile, the effect of caffeine applied topically after stopping chronic UVB treatment to decrease the number of patches with homozygous p53 mutations and also to decrease tumorigenicity cannot be explained by any of the mechanisms discussed above. An important difference between the topical and oral administration of caffeine is its local concentration. The local concentration of topically applied caffeine in the epidermis may reach substantially higher levels than the 31 µM detected in the epidermis of orally treated mice (unpublished observations). Assuming that the dorsal area of the mouse skin treated with 1.2 mg (6.2 µmol) of caffeine is 10 cm2 and the epidermis is
30 µm thick (3 cell layers), the total volume of the exposed epidermal tissue would be
30 µl. The presence of one-quarter to one-half of the caffeine dose (3.1 µmol) in the epidermis would result in a 50100 mM concentration of caffeine in the treated epidermis. Although this calculation is a very rough estimate and there may be substantial absorption into the blood, it illustrates the possibility of millimolar concentrations of caffeine in the epidermis shortly after the topical application of caffeine.
Mutant p53 positive patches from mice treated topically with caffeine for 6 weeks after stopping 20 weeks of twice weekly UVB irradiation did not exhibit any differences in the types and frequencies of p53 mutations compared with acetone treated controls. However, they harbored considerably fewer homozygous p53 mutations than patches from acetone treated mice indicating that patches with a loss of the normal p53 allele may be preferentially eliminated by the caffeine treatment. Since the decrease in the number of homozygous mutations was observed mostly at codon 270, the patches harboring homozygous mutation at this codon may be particularly sensitive to elimination by caffeine treatment. Whether there is a difference among cells harboring various homozygous p53 mutations in their sensitivity to caffeine remains to be investigated.
Several cell culture studies found that p53-deficient or p53-mutated cells were more sensitive to caffeine-induced apoptosis following DNA damage than their p53-normal counterparts (3941). Although the mechanism of this phenomenon is not entirely clear, it was reported that caffeine abrogates the G2 checkpoint by the inhibition of ATR protein kinase and induces premature chromatin condensation preferentially in DNA-damaged cells that have lost normal p53 function (42). Since these cells fail to activate the G1 checkpoint in response to DNA damage, but have an intact G2 checkpoint and accumulate in the G2 phase of the cell cycle (43), they are particularly sensitive to abrogation of the G2 checkpoint that leads to lethal mitosis. The concentration of caffeine that inhibits the kinase activity of ATR by 50% in vitro is 3 mM (44), and 1 mM concentration increased premature chromatin condensation as a result of inhibition of ATR kinase (42). These concentrations are achievable in the epidermis of mice treated topically with caffeine, but they are at least 30-fold higher than the peak concentration of caffeine found in the epidermis of mice treated with caffeine orally. The concentration of caffeine in the epidermis may be critical for eliminating patches with a homozygous p53 mutation since patches from mice treated orally with caffeine or green tea did not exhibit a lower incidence of homozygous p53 mutations compared with controls. Whether caffeine inhibits ATR or other enzymes of the G2 checkpoint signaling pathway in vivo and whether such an effect can explain the selectivity of caffeine against cells with a homozygous p53 mutation needs further investigation.
It is important to note that in the mouse epidermis and in cell culture studies DNA damage was essential for the apoptotic effect of caffeine. In our study with topically applied caffeine the use of UVB as the source of DNA damage was stopped before the start of caffeine applications, and the mice were not exposed to any other external source of DNA damage during the caffeine treatment. If the mechanism of caffeine were the same as in the cell culture studies, patches of cells with a homozygous p53 mutation should have been eliminated by caffeine shortly after the last dose of UVB. Although we performed the p53 mutation analysis only in patches harvested after 6 weeks of caffeine treatment, we counted the numbers of patches at weekly intervals after stopping UVB and found that the major decrease in numbers of patches in caffeine treated mice relative to acetone treated mice indeed occurred during the first week of caffeine treatment, and the difference between the two groups remained approximately constant until the termination of the experiment at 8 weeks (15). Therefore, application of caffeine immediately after discontinuation of UVB treatment might be critical for the observed effect. In other studies, topical applications of caffeine enhanced apoptosis in UVB-induced tumors, but there was no effect of caffeine in non-tumor areas of the skin (7). It would be of interest to see whether there is a selective stimulatory effect of caffeine or tea on apoptosis in tumors and pre-tumorous patches with specific homozygous mutations in the p53 gene.
In summary, our study demonstrated that oral administration of caffeine or green tea during the course of UVB irradiation for 20 weeks altered the profile of mutations in the p53 gene in mutant p53 positive patches of epidermal cells, but these treatments did not decrease the proportion of homozygous mutations. In contrast, topical applications of caffeine to mice pretreated with UVB had no effect on the profile of mutations in the p53 gene but decreased the number of patches with homozygous p53 mutations.
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Acknowledgments
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A.H.C. is the M.William and Myrle W.Garbe Professor of Cancer and Leukemia Research. This research was supported in part by NIH Grants CA80759 and CA88961. Conflict of Interest Statement: None declared.
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Received April 13, 2005;
revised June 2, 2005;
accepted June 15, 2005.