Early p53 alterations in mouse skin carcinogenesis by UVB radiation ...

Proc. Natl. Acad. Sci. USA Vol. 93, pp. 274-278, January 1996 Medical Sciences

Early p53 alterations in mouse skin carcinogenesis by UVB radiation: Immunohistochemical detection of mutant p53 protein in clusters of preneoplastic epidermal cells ROB J. W. BERG*t, HENK J. VAN KRANEN*, HEGGERT G. REBEL*, ANJA DE VRIESt, WILLEM A. VAN VLOTEN*, COEN F. vAN KREIJLt, JAN C. VAN DER LEUN*, AND FRANK R. DE GRUIJL* *Department of Dermatology, University Hospital Utrecht, P.O. Box 85500, 3508 GA Utrecht, The Netherlands; and *Laboratory of Carcinogenesis and Mutagenesis, National Institute of Public Health and Environmental Protection, P.O. Box 1, 3720 BA Bilthoven, The Netherlands

Communicated by Richard B. Setlow, Brookhaven National Laboratory, Upton, NY August 18, 1995 (received for review May 2, 1995)

mation can most directly and unequivocally be established in an animal model in which UV exposure is the only well controlled carcinogenic agent. A robust model is the SKH:HR1 hairless mouse for which the relationship between UVB exposure and carcinogenic response is well established (8, 9) and for which the pathogenesis of UVB-induced SCC shows close similarities with that of human SCC (10). Under physiological circumstances, the wild-type p53 protein has a very short half-life and is present in such small quantities that it is not immunohistochemically detectable (11, 12). There are different pathways that lead to accumulation of the p53 protein up to immunohistochemically detectable levels. (i) DNA damage gives rise to a temporary accumulation of the wild-type p53 protein resulting in an arrest of the cell cycle assumed to prevent replication of damaged DNA (13). (ii) Missense mutations in the p53 gene in general lead to a dramatic increase in half-life of the p53 protein (11). In contrast to the transient accumulation of wild-type p53, the latter can lead to a constitutively high p53 level in the cell. We have recently reported that >75% of UVB-induced murine skin carcinomas show immunostaining with the p53specific polyclonal antiserum CM-5, which was primarily confined to the proliferative compartments of the tumors. A substantial part of the p53-positive staining was associated with point mutations in the conserved domains of the p53 gene (14). Subsequently, we aimed to unravel the timing of these p53 alterations in the course of UV carcinogenesis. We have found that the smallest macroscopically visible skin lesions that arise as benign precursors to the UVB-induced SCCs (actinic keratoses; diameter, 1 mm) already show strong immunoreactivity for the CM-5 antiserum (see Fig. 3A). To exclude transient accumulation of the wild-type p53 protein in these small tumors, we first investigated the epidermal p53 response after a single UVB exposure and subsequently the fading of accumulated wild-type p53 protein after a series of daily UVB exposures. We observed that after longer periods of daily UVB exposure the epidermis contained clusters of cells that strongly stained for the p53 protein. These clusters were detectable long before the appearance of any visible skin lesions, and they remained observable after the wild-type p53 signal had faded in the absence of further UVB exposure, indicating constitutively high levels of the p53 protein. Here we substantiate these findings and report further on the nature of these patches, which appear to show immunoreactivity for an antibody specific for the p53 protein in mutant conformation.

High levels of the p53 protein are immunoABSTRACT histochemically detectable in a majority of human nonmelanoma skin cancers and UVB-induced murine skin tumors. These increased protein levels are often associated with mutations in the conserved domains of the p53 gene. To investigate the timing of the p53 alterations in the process of UVB carcinogenesis, we used a well defined murine model (SKH:HR1 hairless mice) in which the time that tumors appear is predictable from the UVB exposures. The mice were subjected to a series of daily UVB exposures, either for 17 days or for 30 days, which would cause skin tumors to appear around 80 or 30 weeks, respectively. In the epidermis of these mice, we detected clusters of cells showing a strong immunostaining of the p53 protein, as measured with the CM-5 polyclonal antiserum. This cannot be explained by transient accumulation of the normal p53 protein as a physiological response to UVB-induced DNA damage. In single exposure experiments the observed transient CM-5 immunoreactivity lasted for only 3 days and was not clustered, whereas these clusters were still detectable as long as 56 days after 17 days of UVB exposure. In addition, "70%o of these patches reacted with the mutant-specific monoclonal antibody PAb240, whereas transiently induced p53-positive cells did not. In line with indicative human data, these experimental results in the hairless mouse model unambiguously demonstrate that constitutive p53 alterations are causally related to chronic UVB exposure and that they are a very early event in the induction of skin cancer by UVB radiation.

Nonmelanoma skin cancers [i.e., basal cell carcinomas and squamous cell carcinomas (SCCs)] are the most frequent cancers in the United States. Epidemiologic studies have identified solar radiation as the culprit (1), and from animal studies it appeared that the UVB part of the solar spectrum is the most carcinogenic (2). This has been substantiated by detection of mutations in the p53 tumor-suppressor gene in human SCCs (3) and basal cell carcinomas (4) that are characteristic for UVB radiation: i.e., mainly C T transitions TT tandem at dipyrimidine sites among which are CC mutations. There are indications that p53 is involved in the earliest stages of human nonmelanoma skin cancer. Recently, it has been reported that p53 mutations are already present in a benign precursor of SCC, actinic keratosis (5), and in skin adjacent to basal cell carcinomas (6). Furthermore, it has been shown that CC -- TT tandem mutations in the p53 gene are detectable in biopsies from nonneoplastic skin of sun-exposed sites from Australian skin cancer patients (7). The suspected causal relationship between chronic UV exposure and p53 mutation and their relation to tumor for-

MATERIALS AND METHODS Exposure of the Animals. Adult SKH:HR1 hairless mice (Charles River Breeding Laboratories) were exposed to UV

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Abbreviations: SCC, squamous cell carcinoma; MED, minimal erythema dose. tTo whom reprint requests should be addressed.

274

Medical Sciences:

Berg et aL

radiation (1.3 kJ/m2; 250-400 nm) from Philips F40 sunlamps either once or daily for 17 or 30 days. UV doses were determined with a Robertson Berger meter (Solar Light Company, Philadelphia) calibrated against a Kipp 11 thermopile (Kipp, Delft, The Netherlands). This lamp is the successor of the Westinghouse FS40 sunlamp, the lamp that was used in the tumor induction experiments from which the expected tumor appearances were deduced (8, 9). Compared with the latter lamp, the relative energy output of the new lamp is shifted somewhat to the shorter wavelengths and is therefore 20% more carcinogenic, as determined with the SCUP action spectrum for UV carcinogenesis (2). Irradiations took place between 8:30 and 9:00 am. The daily dose is just below the dose causing acute effects such as edema and erythema, the socalled minimal erythema dose (MED). Daily exposures to the UV dose mentioned result in small skin tumors (actinic keratoses with a diameter of - 1 mm) in 50% of the mice in 11 + 1 weeks (8). Discontinuation of the exposures after 17 or 30 days would delay this time point to 76 ± 8 and 30 + 3 weeks, respectively (9). Isolation of Skin Samples. In the single exposure experiment, two mice were euthanized by cervical dislocation after ether anesthesia immediately and 1 h, 3 h, 6 h, 12 h, 24 h, 48 h, 72 h, 96 h, 7 days, and 10 days after exposure. In the 17-day exposure experiment, two mice were euthanized at the same time points after the last exposure as well as at 14, 21, 28, and 56 days. In the 30-day exposure experiment, two mice were sacrificed immediately and at 12 h, 24 h, 48 h, 96 h, 7 days, and 14 days after the last exposure. Small pieces of dorsal skin (approximately 0.7 x 0.7 cm) were taken from every mouse (4 biopsies, or 6-12 biopsies in the 30-day experiment); half of these (i.e., 2 biopsies or 3-6 biopsies in the 30-day experiment) were immediately snap-frozen in liquid nitrogen, and the other half were fixed in 4% buffered formaldehyde. The formaldehyde fixation was for 24 h at 4°C, after which the biopsies were stored in 70% ethanol. From every mouse 4 biopsies were also taken from the (unirradiated) abdominal skin, 2 of which were snap-frozen and 2 of which were immediately fixed in formaldehyde. In addition, in the single exposure experiment at every time point two mice were intraperitoneally injected with 5 mg of BrdUrd solubilized in 0.3 ml of phosphate-buffered saline (PBS) 60 min before they were euthanized. Immunohistochemical Analyses of the Skin. The formaldehyde-fixed biopsies were embedded in paraffin, and the snapfrozen biopies were placed in Tissue-Tek OCT (Miles). Subsequently, 5-gm sections were cut on slides and preserved according to standard procedures (15). Endogenous peroxidase activity was blocked in a H202/methanol/distilled water solution in a 1:50:50 ratio for 20 min. After three washes with PBS/0.5% Tween, the sections were preincubated for 10 min in 10% normal mouse serum in PBS and incubated overnight at 4°C with the rabbit polyclonal antiserum CM-5 (ref. 14; kindly provided by D. Lane) diluted 1:1000 in 10% normal mouse serum in PBS. Incubation in normal mouse serum only was used as a negative control. After three washes in PBS/ 0.5% Tween, the sections were incubated for 1 h at room temperature with biotin-conjugated goat anti-rabbit antibody solution (Vector Laboratories) diluted 1:300 in 5% normal mouse serum in PBS for cryostat sections or 1:50 for paraffin sections. After washing in PBS, staining took place with an avidin/biotin/peroxidase detection system, with diaminobenzidine as the chromogen, as prescribed by the manufacturer (Vectastain Elite ABC kit; Vector Laboratories). Counterstaining was carried out with hematoxylin. Staining against BrdUrd was carried out as described (16) on the paraffinembedded material. Immunostainings with the PAb240 monoclonal antibody (17) were carried out on cryostat sections. After fixation in 4% formaldehyde, the slides were boiled for 15 min in 10 mM citrate buffer (pH 6.0) and subsequently cooled to 37°C (15).

Proc. Natl. Acad. Sci. USA 93 (1996)

275

The sections were washed in PBS, and after incubation in blocking fluid (same as for CM-5) the sections were preincubated for 10 min in 5% normal rabbit serum in PBS/0.2% bovine serum albumine (BSA) and then incubated for 1 h at room temperature with the PAb240 monoclonal antibody (NovoCastra Laboratories, Newcastle, U.K.) in a 1:50 dilution in PBS/0.2% BSA. BSA was used for a negative control. After washing in PBS/0.5% Tween, the sections were incubated for 1 h at room temperature with peroxidase-conjugated rabbit anti-mouse (IgGl) antibody solution (Zymed Laboratories) diluted 1:50 in PBS/0.2% BSA. After three washes in PBS, diaminobenzidine staining took place as prescribed by the manufacturer, and counterstaining was carried out with he-

matoxylin. RESULTS Time Course of Transient Accumulation of Wild-Type p53 Protein After a Single Exposure. Fig. 1 shows murine epidermis stained with the CM-5 antibody after exposure to 1 MED of UVB radiation. The number of cells containing CM-5 immunoreactivity was counted under light microscopy in interfollicular stretches of the epidermal basal layer in a total of 100-160 basal cells per section. The frequencies of p53positive cells at several time points after UV exposure, and in parallel the frequencies of BrdUrd-positive cells (biopsies taken 60 min after BrdUrd injection), are shown in Fig. 2. The highest level of CM-5 immunoreactivity is observed 24 h after

FIG. 1. Immunostaining of mouse skin after a single exposure to 1 MED of UVB radiation with the CM-S polyclonal antibody on paraffin sections. (A) Mouse skin immediately after exposure showing no evidence of p53 immunoreactivity. (B) Twenty-four hours after UVB irradiation, p53 immunoreactivity is found in epidermal keratinocytes, particularly in the basal layer. (X360.)

276

Medical Sciences: Berg et al.

Proc. Natl. Acad. Sci. USA 93

A?> i

AI

-

-~40

(1996)

.9 *.

. a, . ...4

0

20

1

I, -

0.1

4

-----------

1 10 Time after UV exposure, h

I

I

100

-

.;.*

"iV ..4

i/p

.

.j/*

.i4V

S

p

.i

Is

.

.4

Ii's

4 PS

*

FIG. 2. Time course of accumulation of the p53 protein in mouse skin after a single exposure to UVB radiation, measured as the frequency of CM-5-positive epidermal basal cells ( ), and of UVB-induced cell proliferation, measured as the frequency of BrdUrd-positive epidermal basal cells (- - -). Error bars give the SD for two mice per point.

'pp

4.ip'

.4 4

4

4

/'

.1"' .

-

.,#.

B

exposure, which is in agreement with the results of a similar study in human skin (18). The transient time course of the p53 signal (high levels 12-24 h after exposure and signals reduced to background values after 72 h) indicates that this immunostaining reflects the physiological response of the wild-type p53 protein to DNA damage. The increase in the frequency of epidermal cells in S phase peaks 48 h after UVB exposure, 24 h later than the peak in p53-positive cells. This is in line with a wild-type p53-mediated cell cycle arrest by UVB irradiation

4I.p 41 *

's

:''

S;. :.X

(19, 20).

Patches of Cells with Strong CM-5 Immunoreactivity After 17 and 30 Daily Exposures. Clusters of cells with strong p53 immunoreactivity, as measured with the CM-5 polyclonal antiserum, were detected in the biopsies from the mice that had been exposed for 17 days. These cell clusters show more intense immunostaining than basal cells after a single UV exposure. In 30 biopsies from the 17-day exposure experiment, a total of nine such patches with strong CM-5 immunoreactivity were observed; more specifically, they were found in sections from 6 h, 12 h, 24 h, 48 h (two patches), 72 h, 96 h, 14 days, and 56 days after the last exposure. Such patches were not detected in the same number of sections from abdominal skin or in the biopsies from the single exposure experiment. Fig. 3 B and C shows some of the patches of epidermal cells with strong immunostaining of the p53 protein. If these early p53-positive patches are indeed preneoplastic cells on their way to tumor development, one expects their frequency to increase with an increase in the number of irradiation days (i.e., closer to tumor appearance). Therefore, in another experiment we exposed mice for 30 consecutive days. Successive cryostat sections (5 gm) from the skin biopsies were put on separate slides; one section was stained with CM-5 and neighboring sections were stained with other antibodies for comparison. In 66 biopsies from the 14 mice in this latter experiment, 53 patches with strong CM-5 immunoreactivity were found, indicating that the frequency of these patches indeed increases with increasing number of irradiation days. The clusters consisted of 4-35 cells in cross section, with two exceptionally large clusters of >60 cells. Immunoreactivity of the Patches for Antibodies Specific for a Mutant Conformation. We have tested whether the patches show immunoreactivity for the PAb240 monoclonal antibody, the epitope of which is cryptic in the correctly folded, wild-type p53 protein, but is displayed in a distinct aberrant conformation (21). Many different types of point mutations in the conserved domain of the p53 gene lead to this mutant conformation. Exposure of the PAb240 epitope may also occur after denaturation of the wild-type p53 protein (22, 23). As a negative control, this antibody was tested on immunohistochemically detectable levels of the wild-type p53 protein. The

*

.

.1" "fe ....x

?

-.

FIG. 3. (A) Murine actinic keratosis induced by daily exposure to 1 MED of UVB radiation for 12 weeks stained with the CM-S polyclonal antibody against p53 protein. Histological characterization was performed on hematoxylin and eosin-stained sections from the same skin lesion. (X 120.) (B and C) Paraffin sections from the epidermis of mice daily exposed to 1 MED of UVB radiation for 17 days, containing groups of cells with a strong CM-5 immunoreactivity. Mice were killed 12 h (B) and 56 days (C) after the last exposure.

(X240.)

PAb24O antibody did not show any detectable immunoreactivity in epidermal cells of the skin 24 h after a single exposure to 1 MED UVB radiation, at the maximum of the transient accumulation of the wild-type p53 protein. To be sure that under our processing and staining conditions PAb24O does not react with p53 in the wild-type conformation, we overexposed a small dorsal skin

field to 6 MED instead of the 1 MED used

#.o$sM,;+!tai*X'}rj£

Medical Sciences:

Berg et al.

Proc. Natl. Acad. Sci. USA 93 (1996)

in the other experiments. We took a biopsy 24 h after exposure. The epidermis contained nuclei that intensely reacted with the CM-5 polyclonal antiserum, whereas again absolutely no immunoreactivity was detected with the PAb240 monoclonal antibody (Fig. 4 A and B). As a positive control, we have stained skin tumors of hairless mice induced by daily exposures to 1 MED UVB radiation. Of

277

the 24 tumors stained, 22 showed immunoreactivity with the CM-5 polyclonal antiserum, and 20 of those showed immunoreactivity with the PAb240 monoclonal antibody. To further substantiate that this type of immunoreactivity is linked with p53 mutation in these murine tumors, DNA was isolated from 10 PAb240-positive tumors and exons 5, 7, and 8 of the p53 gene were individually sequenced as described earlier (14). Nine of the 10 PAb240-positive tumors revealed a missense mutation in exon 8, of which 8 were C -> T transitions at codon 267, and 1 was a CC -- TT double transition at codon 272 [codon numbers are according to Soussi et al. (24)]. This confirms data based on sequencing of p53 in -160 UVBinduced murine skin tumors, from which 3 clear hotspots emerged including codons 267 and 272 (unpublished data). This result demonstrates that mutant conformations of the murine p53 protein are indeed well detected with the PAb240 monoclonal antibody. Serial cryostat sections of the 30-day exposure experiment were alternately stained with the PAb240 and the CM-5 antibody. Of the 53 cell clusters with strong CM-5 immunoreactivity, 37 showed PAb240 immunoreactivity. These cell clusters comprised 4-25 cells and were in general somewhat smaller than the corresponding CM-5-positive clusters. This is probably a result of the slightly weaker immunostaining with PAb240 than with CM-5. Fig. 4 C and D shows a cell cluster stained with the CM-5 polyclonal antiserum and with the PAb240 monoclonal antibody in the corresponding, neighboring section. The incidences of patches have been summarized in Table 1.

-

...

::::

DISCUSSION

.b'

We have demonstrated that the epidermis of chronically UVB-exposed hairless mice contains cells with aberrant p53 immunostaining-i.e., strong staining in clusters of cells-long before the appearance of skin tumors. These patches appear to increase in number in the course of chronic UV exposure, and they are still detectable at least up to 56 days after the last exposure, whereas the transient accumulation of the wild-type p53 protein triggered by this dose lasts for only 3 days. High cellular levels of the wild-type p53 protein will normally result in cell cycle arrest (13) or even apoptosis (25). Preliminary results indicate that no apoptosis coincided with the p53positive patches (apoptosis detected in situ with an Apoptag kit; Oncor). Hence, these clusters of cells have most likely arisen from clonal expansion of cells with either a mutated p53 allele or an otherwise dysfunctional p53 signal pathway. The PAb240 antibody, which recognizes the p53 protein in a

D:

Table 1. Summary of detection of p53-positive patches in the first and second weeks after discontinuation of daily UVB exposures for 1, 17, and 30 days 4-~~~4

UVB exposure,

days

FIG. 4.

(A

and

B) Neighboring cryostat a single exposure to 6

taken 24 h after

showing immunostaining for the CM-S

sections from the

same

MED of UVB radiation

antibody (A) but

not for the

antibody (B). (X240.) (C and D) Neighboring cryostat sections from the same biopsy taken 24 h after 30 days of daily exposure to 1 MED of UVB radiation showing immunoreactivity for the CM-5 polyclonal antiserum (C) and for the mutant conformation-specific PAb24O

monoclonal

antibody

PAb24O

(D).

(X360.)

t5o, weekst -t 76 ± 8 30 ± 3

0/4 1/6 9/19 (7/19)§ All patches were detected in the exposed dorsal skin; control abdominal skin did not contain any patches. *No. of detected patches/no. of biopsies (3-6 sections per biopsy in the 1-day and 17-day experiments; 1 section per biopsy in the 30-day experiment). 1 17 30

biopsy

No. of p53-positive patches* Week 2 Week 1

0/18 7/18 44/47 (30/47)§

tNo. of weeks for 50% of mice to bear 1-mm skin tumors. tSingle exposure to 1 MED of UVB radiation does not lead to skin tumors within the lifetime of the mice.

§No. of PAb240-positive patches in the corresponding neighboring sections.

278

Medical Sciences: Berg et al.

mutant conformation, stained 70% of these patches, indicating that the majority of patches with strong CM-5 immunoreactivity contain cells with a mutated p53 gene. In relation to the progression of patches to actinic keratoses, it is of interest to know whether the frequency of p53-positive patches remains constant or changes with time after discontinuation of the exposures. UVB-induced mutations in the p53 gene probably arise from replication of DNA containing pyrimidine dimers or pyrimidine-pyrimidone(6-4) photoproducts, which are induced by direct absorption of UVB radiation by the DNA. Therefore, an increase in the frequency of p53-positive patches is not to be expected after discontinuation of exposures. Table 1 shows that the frequency of p53-positive patches had been reduced in the second week compared with the first week after the end of 17 and 30 days of exposure. Functional p53 should force a cell with a heavily UVdamaged genome into apoptosis in order to avoid survival with mutations leading to cell transformation. Cells that are heterozygous or homozygous for p53 mutations have a diminished capacity to undergo apoptosis and therefore show an enhanced survival with persisting DNA damage (5). Thus, UV exposure can exert a selective pressure to the advantage of cells with dysfunctional p53. The decrease in frequency of p53-positive patches after discontinuation of the exposures can be a direct consequence of the removal of this selective pressure. The cells with a dysfunctional p53 then have to compete with the other cells for a place in the epidermal basal cell layer without a competitive edge. Similar to actinic keratoses, these patchespotential precursors of actinic keratoses-will then show enhanced regression in the absence of UVB irradiation. This explanation for the decrease in frequency of p53-positive patches is in line with the protective mechanism against UV carcinogenesis forwarded by Ziegler et al. (5), who expanded the role of p53 as a "guardian of the genome" to that of a "guardian of the tissue." As mentioned before, several studies suggest that p53 mutations are an early event in human nonmelanoma skin cancer. However, studies on chemically induced skin carcinogenesis by dimethylbenzanthracene (DMBA)/phorbol 12-myristate 13acetate (PMA) (initiation/promotion) applications in p53 knockout mice have revealed that the absence of functional p53 has no effect on initiation but greatly enhances malignant progression (26). These results in combination with the present study indicate that the timing of p53 alterations is dependent on the carcinogenic regimen rather than on the species-i.e., on the carcinogen and/or its application (single application of the initiator DMBA followed by chronic application of PMA as tumor promotor vs. chronic UVB exposure). In conclusion, the present study demonstrates that chronic UVB exposure leads to stabilization of the p53 protein in epidermal cells, probably as a result of mutation, leading to constitutive immunohistochemically detectable levels, and that these events occur long before the appearance of macroscopically visible skin lesions. Identifying the type of p53 alterations in the patches with aberrant immunostaining and unraveling the relationship between the induction kinetics of epidermal cells with p53 alterations and the kinetics of the ultimate skin tumors remain to be done.

Proc. Natl. Acad. Sci. USA 93

(1996)

We thank Dr. P. Wester and S. de Waal for help with immunostaining against BrdUrd and p53 and Dr. N. Wikonkal for help with detection of apoptosis. We thank K. Guikers for general assistance and H. Sturkenboom for maintenance of the animals. This work was supported by a grant from the Environment Program of the European Community (EV5V-CT91-0030).

1. Scotto, J. & Fears, T. R. (1982) Incidence ofNonmelanoma Skin Cancer in the United States (Natl. Inst. Health, Bethesda, MD), DHHS Publ. no. (NIH) 82-2433. 2. De Gruijl, F. R., Sterenborg, H. J. C. M., Forbes, P. D., Davies, R. E., Cole, C., van Weelden, H., Slaper, H. & van der Leun, J. C. (1993) Cancer Res. 53, 53-60. 3. Brash, D. E., Rudolph, J. A., Simon, J. A., Lin, A., McKenna, G. J., Baden, H. P., Halperin, A. J. & Ponten, J. (1991) Proc. Natl. Acad. Sci. USA 88,10124-10128. 4. Ziegler, A., Leffell, D. J., Kunala, S., Sharma, H. W., Gailani, M., Simon, J. A., Halperin, A. J., Baden, H. P., Shapiro, P. E., Bale, A. E. & Brash, D. E. (1993) Proc. Natl. Acad. Sci. USA 90, 4216-4220. 5. Ziegler, A., Jonason, A. S., Leffell, D. J., Simon, J. A., Sharma, H. W., Kimmelman, J., Remington, L., Jacks, T. & Brash, D. E. (1994) Nature (London) 372, 773-776. 6. Urano, Y., Asano, T., Yoshimoto, K., Iwahana, H., Kubo, Y., Kato, S., Sasaki, S., Takeuchi, N., Uchida, N., Nakanishi, H., Arase, S. & Itakura, M. (1995) J. Invest. Dermatol. 104, 928-932. 7. Nakazawa, H., English, D., Randell, P. L., Nakazawa, K., Martel, N., Armstrong, B. K. & Yamasaki, H. (1994) Proc. Natl. Acad. Sci. USA 91, 360-364. 8. De Gruijl, F. R., van der Meer, J. B. & van der Leun, J. C. (1983) Photochem. Photobiol. 37, 53-67. 9. De Gruijl, F. R. & van der Leun, J. C. (1991) Cancer Res. 51, 979-984. 10. De Gruijl, F. R. & Forbes, P. D. (1995) BioEssays 17, 651-660. 11. Hall, P. A. & Lane, D. P. (1994) J. Pathol. 172, 1-4. 12. Greenblatt, M. S., Bennet, W. P., Hollstein, M. & Harris, C. C. (1994) Cancer Res. 54, 4855-4878. 13. Kastan, M. B., Onyerkwere, O., Sidransky, D., Vogelstein, B. &

Craig, R. W. (1991) Cancer Res. 51, 6304-6311. 14. Van Kranen, H. J., de Gruijl, F. R., de Vries, A., Sontag, Y., Wester, P. W., Senden, H. C. M. & van Kreijl, C. F. (1995)

Carcinogenesis (London) 16, 1141-1147. 15. Harlow, E. & Lane, D. P. (1988)Antibodies:A Laboratory Manual (Cold Spring Harbor Lab. Press, Plainview NY). 16. Schutte, B., Reynders, M. M. J., Bosman, F. T. & Blijham, G. H.

(1987) J. Histochem. Cytochem. 35, 1343-1345. 17. Gannon, J. V., Greaves, R., Iggo, R. & Lane, D. P. (1990) EMBO

J. 9, 1595-1602. 18. Hall, P. A., McKee, P. H., Menage, H. du P., Dover, R. & Lane, D. P. (1993) Oncogene 8, 203-207. 19. Lane, D. P. (1992) Nature (London) 358, 15-16. 20. Lu, X. & Lane, D. P. (1993) Cell 75, 765-778. 21. Stephen, C. W. & Lane, D. P. (1992) J. Mol. Biol. 225, 577-583. 22. Vojtesek, B., Dolezalova, H., Lauerova, L., Svitakova, M., Havlis, P., Kovarik, J., Midgley, C. A. & Lane, D. P. (1995) Oncogene 10, 389-393. 23. Milner, J. (1995) Trends Biol. Sci. 20, 40-44. 24. Soussi, T., Caron de Fromentel, C. & May, P. (1990) Oncogene 5, 945-952. 25. Yonish-Rouach, E., Resnitzky, D., Lotem, J., Sachs, L., Kimchi, A. & Oren, M. (1991) Nature (London) 352, 345-347. 26. Kemp, C. J., Donehower, L. A., Bradley, A. & Balmain, A. (1993) Cell 74, 813-822.