The loss of the tumor suppressor gene product p16 in melanoma is well documented, although the normal physiological function of p16 in skin melanocytes is unknown. In this report, we demonstrate that when human skin was irradiated with suberythemal doses of UV radiation, levels of p16 were dramatically increased by 16 h postirradiation, peaking at 24 h, and declining by 72 h. p16 was expressed in the nucleus and cytoplasm of melanocytes and keratinocytes within the epidermis, and the pattern of p16 expression within the epidermis was dependent on the penetrative ability of the different UV wavebands. The existence of a UV-induced response pathway involving up-regulated p16 expression may provide a mechanism linking the loss of p16 and UV exposure with the development of melanoma.

CDKN2A, encoding the cdk3 inhibitor protein p16, has been identified as a tumor suppressor gene, and mutations have been reported in the germ line of members of familial melanoma kindreds and in approximately 10% of sporadic melanoma cases (reviewed in Refs. 1) and 2). CDKN2A has also been found to be deleted, mutated, or hypermethylated in a high proportion of melanoma cell lines (3), as well as in a variety of other tumor types (4).

The p16 protein binds to and inhibits cdk4 and cdk6, thus preventing cdk4- and cdk6-dependent phosphorylation of Rb and resulting in a block during the G1-S phase transition of the cell cycle. By their nature, cdk inhibitors must increase in level to function, and in the majority of reports, this Rb-dependent p16 function has been demonstrated by ectopic overexpression of p16 in a range of cell types (5, 6, 7). However, the cellular roles of endogenous p16 are still relatively poorly understood. p16 has been demonstrated to play a role in replicative senescence, with levels accumulating with increasing numbers of population doublings (8, 9), and the loss of p16 expression is a critical event in the immortalization of a range of cell types (10, 11). Small increases in p16 levels have also been reported during G1-S and S-G2 phases of proliferating cells, although this is not associated with a cell cycle delay (12, 13). We previously reported that levels of p16 increased in response to low-dose UV in epidermal cell lines, and that this correlated with a G2 phase cell cycle delay (14, 15).

The UV region of the electromagnetic spectrum is subdivided into three wavebands: UVC (200–290 nm), which is absorbed by the earth’s atmosphere, whereas UVB (290–320 nm) and UVA (320–400 nm) both penetrate the earth’s ozone layer. Although all three types of UV induce DNA damage, the nature and amount of damage is wavelength-dependent with pyrimidine dimers of the cyclobutane type and 6–4 photoadducts (which are more closely associated with the carcinogenic potential of UV) predominant at shorter wavelengths and single-strand breaks and DNA protein cross-linking at longer wavelengths (16). The anatomical organization of skin and the differential penetration characteristics of UVC and UVB mean that conclusions from in vitro studies may not be directly transferable to human skin because the carcinogenic potential of a particular wavelength in vivo will be a function of both the dose received by the stem cells in the basal layer (related to penetration characteristics) and the effectiveness of the radiation in inducing DNA damage (17).

Here we have used neonatal foreskins in a limited organ culture to investigate the consequences of UV irradiation on p16 levels. Neonatal foreskins are a widely used source of cells of dermal and epidermal origin, and the skin itself has a fully developed architecture and a relatively high density of melanocytes (18). The foreskin model also has the benefits of being UV-naive, and the exposure and culture conditions can be controlled and more easily sampled, and a much larger area is available than skin punch biopsies. Using this experimental system, we present evidence of elevated levels of p16 in human skin associated with suberythemal UV exposure (at doses that are not sufficient to elicit an erythemic response) in a large sample. Using the UVC waveband produces pyrimidine dimers of the cyclobutane type and 6-4 photoadducts, which are also the primary products of the physiologically relevant UVB waveband. We demonstrate elevated levels of p16 in target cell type for melanoma, i.e., melanocytes, and also epidermal keratinocytes. The potential consequences of the elevated p16 levels are discussed.

Organ Cultures.

Organ cultures were established from neonatal foreskins and maintained in MCDB 153 medium (Sigma), supplemented with 60 μg/ml penicillin, 100 μg/ml streptomycin, and 5 μg/ml fungizone. Briefly, foreskin tissue was obtained after circumcision and placed in RPMI 1640 supplemented with 10% serum supreme (Biowhittaker), 60 μg/ml penicillin, 100 μg/ml streptomycin, 40 μg/ml gentamicin, and 5 μg/ml fungizone, for transportation. Samples were then rinsed in sodium hypochlorite for 1–2 min, then immersed in versene for 1–2 min, and rinsed in PBS (pH 7.4). The fatty tissue was removed from the dermal side of the skin, and samples were placed flat with epidermal surface up in 50-mm tissue culture plates. Base medium (approximately 1 ml) was added. The epidermal surface of explants remained uncovered by culture medium. Organ cultures were incubated at 37°C in a humidified incubator.

Before irradiation, samples were rinsed in PBS and irradiated in a minimal amount of PBS (approximately 300 μl). Irradiation with doses of 10 and 20 J m−2 UVC were performed using a UVS-52 mineralite lamp (UV Products, San Gabriel, CA), with a maximal output at 254 nm as described previously (19). For UVB irradiation, doses of 150 Jm−2, 250 Jm−2 and 875 Jm−2 (one minimal erythemal dose), were used via a single FS20T12 UVB lamp (Light Sources) with maximal output at 313 nm. After irradiation, the PBS was removed, and basal medium was replaced. Ionizing γ radiation (10 Gy) from a 137Cs γ emitter, which emitted at 4 Gy/min, was used as a control.

Immunohistochemistry.

Skin samples were fixed and embedded in paraffin wax, and sections (4 μm) were placed on Menzel superfrost plus adhesive slides and were subsequently dewaxed and rehydrated to water through descending graded alcohols, then transferred to PBS (pH 7.4). Antigen retrieval was performed by immersing the slides in 10 mm citric acid (pH 6.0) and autoclaving on a 20-min cycle, then allowed to cool for 30 min, and washed with PBS. Endogenous peroxidase activity was quenched by incubating the sections with 3% hydrogen peroxide in absolute methanol for 10 min. After washing in PBS, the slides were placed in a Sequenza vertical staining system (Shandon). The primary antibody was a p16 polyclonal antibody that was developed in rabbits immunized with a synthetic peptide sequence of 15 amino acids on the COOH terminus of human p16 and affinity-purified before use (20); this was incubated on the sections at 1:300 for 1 h at room temperature. A second monoclonal p16 antibody (PharMingen 13251a) was used to confirm specificity. A Zymed Histostain-Plus kit (Zymed, CA) was used to reveal the presence of p16 within the tissue using the substrate AEC to produce a red end product. B8G3, directed against epitopes on the melanocyte-specific protein tyrosinase-related protein 1, was used to identify melanocytes within the basal layer (21), and a CD1a antibody was used to identify Langerhan’s cells (1590 Immunotech, France). A mouse monoclonal antibody was used to detect p53 (DO-1, Santa Cruz). Sections were lightly counterstained with Mayer’s hematoxylin for 1–2 min. Because the AEC product is solvent soluble, the sections were covered with Clearmount aqueous permanent mounting medium (Zymed), and heated to 75°C for 10–15 min, then allowed to cool before being dipped in xylene and mounted with Histomount Mounting Medium (Zymed). With each immunohistochemical run, a positive control and a negative control, omitting the primary antibody, were run. Other controls included a peptide block, in which an immunogen peptide was preincubated with the primary antibody before addition to the tissue. Apoptotic cells were identified using TUNEL staining with an In Situ Cell Death Detection kit, POD (Boehringer Mannheim).

Evaluation of p16 Expression.

There were a total of 129 samples collected and analyzed, consisting of: (a) 47 unirradiated controls; (b) 8 receiving UVC treatment (6 = 10 Jm−2; 2 = 20 Jm−2); and (c) 74 receiving UVB treatment (38 = 150 Jm−2; 23 = 250 Jm−2; 13 = 875 Jm−2). A cell was considered to be immunolabeled and positive for p16 expression if there was a visibly detectable signal within the nucleus, cytoplasm, or both nucleus and cytoplasm of a cell. A time course was undertaken on a number of samples and were harvested at 0, 8, 12, 16, 24, 48, and 72 h in organ culture for irradiated and unirradiated control samples. A field on each slide representative of the entire sample was selected, and the number of cells expressing p16 was measured and reported as a percentage of the total number of cells counted in each field. From the fields analyzed, there was an average of 171 cells counted per field. A Zeiss Axioskop microscope was used for all of the observations, and images were captured using a MTI digital camera. Images were analyzed using Image-Pro Plus Software, Version 3.0 (Media Cybernetics).

Statistical Methods.

The level of p16 expression in cells was related to the UV dose, epidermal localization and distribution, and the duration of response in relation to p16 induction. All of the statistical analyses were performed using the SPSS version 8 program. The Mann-Whitney U test was used on nonparametric data to determine whether two independent groups (control versus UV-irradiated samples) were significantly different.

In control (unirradiated skin), p16 was not detectable or was expressed at low levels (Fig. 1,A). Skin samples exposed to low doses of UVB or UVC (150–250 Jm−2 UVB and 10–20 Jm−2 UVC) contained increased p16 levels in all cases examined (n = 82; Fig. 1, B and C). The specificity of staining for p16 was demonstrated by preincubating the antibody with the immunogen peptide; this completely blocked staining in the irradiated samples (Fig. 1 D). Similar staining patterns were also observed using a second, monoclonal antibody against p16 (see “Materials and Methods;” data not shown). The induction of p16 appeared to be a specific response to UV radiation, inasmuch as the exposure of foreskins to ionizing radiation (10 Gy) did not result in any p16 staining (data not shown).

The staining for p16 at 24 h after exposure to UVB radiation was found to be positive in the epidermis and absent in the dermis (Fig. 1,E). The p16 staining in the epidermal layers was patchy in appearance with staining confined to specific areas in basal and suprabasal nests or clusters of cells (Fig. 1,E). These were often located deep in the rete pegs (Fig. 1,E). In the epidermis, staining was observed in keratinocytes of the basal layer and in those in the spinous layer immediately suprabasally. Staining was also present in cells identified as melanocytes (Fig. 1, F and G) by simultaneous immunostaining with the melanocyte-specific antibody B8G3 (see “Materials and Methods”) and was absent in cells identified as Langerhan’s cells by CD1a antibody stain (results not shown). Both cytoplasmic and nuclear staining were evident in most cells, with cytoplasmic staining predominating and varying in intensity (Fig. 1, F and G). The general staining pattern was similar in an adult foreskin irradiated with UVB (data not shown).

UVC radiation also produced patchy p16 staining localized to the epidermis. This was evident predominantly within granular layer cells and throughout the anuclear deepest cells of the cornified layer (Fig. 1 B). The shorter wavelength UVC was the only treatment to induce high levels of p16 staining confined to cells predominantly within the granular layer, which suggests that cells in the different epidermal layers may respond to UV radiation and induce p16 expression in a wavelength-dependent manner.

The low levels of staining detected in some of the unirradiated samples was often found toward the edges of the samples and seemed to be associated with physical trauma to the skin, for example, pinching and cutting, although this was confined to keratinocytes of the basal layer and the spinous layer immediately suprabasally.

For each sample, the number of cells with positive staining within the epidermis was recorded. The increase in the number of cells staining for p16 postirradiation was quantitated and expressed as a percentage of the total number of cells in a given field (Fig. 2). All of the doses of UV resulted in a dramatic increase in p16 levels within the epidermis. The response observed post-UVB irradiation was independent of the dose, increasing from 3% in controls to 15–20% in irradiated samples (P = 0.012). The response observed after UVC irradiation suggests that there may be a dose-dependent effect on p16 induction, increasing from 3% in controls to 13% for 10 Jm− 2 (P = 0.003), and to 32% for 20 Jm− 2 (P = 0.012), although this was only a small sample (n = 4).

The induction of p16 was not evident until 12 h postirradiation and peaked at 24 h, with the levels maintained until 48 h with all of the doses of UVB used (Fig. 3). The level of p16 was reduced again at 72 h postirradiation. There was a shift in the epidermal distribution of cells expressing p16 during the time course after UVB irradiation. P16-staining cells moved from predominantly within the basal and suprabasal spinous layer at 24 h to higher levels in the spinous layer and to the granular layer at 48 and 72 h postirradiation (Fig. 1 H). The staining was confined to keratinocytes in these layers.

We have demonstrated for the first time that exposure of human skin to UV radiation induces the accumulation of p16 protein. The observed differences between the UVB- and UVC-induced p16 expression can be explained by the penetrative ability of the different wavelengths through the epidermal layers of the skin. The shorter wavelength UVC penetrates only to the granular and cornified layers. The longer wavelength UVB induced p16 predominantly within the basal layer cells and cells immediately suprabasally, parallelling the findings for UV-induced p53 accumulation in human skin (17). The time course of p16 induction was somewhat delayed compared with p53, with little p16 detected before 12 h. p53 was detectable by 4 h postirradiation and peaked between 24 to 48 h, similar to p16, then resolved by 5–15 days postirradiation (22, 23, 24). Whereas the localization of p16 expression can in part be attributed to the penetrative ability of the UV wavelengths, this does not explain why the responses observed with UVC are not detected with irradiation using the longer wavelength UVB. This may point to some differences in the responses of cells to the different wavelengths used. We have also observed differences in the induction of p16 by UVB and UVC in cultured cell lines (15).

The observation of p16 accumulation in the cornified layer after UVC radiation is itself interesting and prompts the question as to whether the p16 response is due to UVC-induced DNA damage as might be expected, particularly as DNA is likely to be the primary cellular target for short wavelength UV radiation (25). This p16 response seems to be specific for UV-induced damage, however, because no p16 staining was detected in skin after exposure to ionizing radiation. This same UV-specific p16 response has also been observed with similar studies in cell lines.4

The consequences of p16 induction are unclear. The demonstrated role for p16 is to block G1 phase progression by inhibiting cdk4- and cdk6-dependent phosphorylation of Rb (5, 6, 7), and the induction of p16 in senescence is likely to use this mechanism (8, 26, 27). We have previously reported that p16 protein accumulates in response to low doses of UVB and UVC in cultured cell lines, correlated with a G2 phase cell cycle delay, and the loss of the p16 function correlated with the loss of the UV-induced G2 arrest (14, 15). Recently, we have demonstrated that UV-induced p16 inhibits a cdk4/cyclin D kinase activity that has a role in G2-M progression, strongly supporting the hypothesis that p16 is involved in a UV-induced G2 checkpoint response (28). Loss of p16 function may not only provide a growth advantage by disabling the senescence mechanism, thus extending the proliferative capacity of the cells, but may also result in cells undergoing mitosis with an increased burden of UV-induced DNA mutations, the result of an inability to delay in G2 phase to permit complete repair of the UV-induced lesions. The time course of p16 induction in skin closely resembles that observed in the cell lines and may also be associated with a G2 phase delay similar to that observed in the cultured cells. The cell cycle effects of the increased p16 expression will require further study with markers specific for different cell cycle stages.

The loss of two potential growth-limiting mechanisms—senescence and a UV-induced cell cycle checkpoint—is reminiscent of the dual roles of the tumor suppressor, p53, in damage-induced cell cycle arrest and apoptosis. p53 has also been implicated in UV-induced skin cancers, with the loss of p53 function associated with squamous and basal cell carcinomas ( see Ref. 29 and references therein), and p53 is strongly induced by UV radiation in skin keratinocytes (22, 23, 24, 30). In chronically sun-exposed skin, clonal populations of keratinocytes express high levels of p53 that are in many cases mutant (31); this provides evidence for the potential contribution of p53 mutations to the transformation of these cells in skin. Interestingly, there is no evidence of p53 induction in skin melanocytes from any of the reported studies; the lack of this induction is highlighted in one report (23). Immunohistochemical staining of serial sections of a number of the skin samples used in the present study revealed only low levels of p53 staining because of the low fluences of UV used, although the p53 expression pattern was essentially the same as reported by others. In contrast, the p16 response was heterogenous, and the suberythemal doses of UV used in this experiment were not sufficient to induce apoptosis, as confirmed by TUNEL staining. UVB-irradiation of cultured melanocytes does induce p53 and is associated with a G1 arrest (32); thus, the absence of this effect in skin melanocytes is currently unexplained. There are relatively few mutations of p53 in melanomas (33), although a high proportion of melanomas and melanoma cell lines express high levels of p53, which suggests that it may be functionally compromised in some way (33, 34). It may, however, indicate that melanocytes in the skin do not normally use a p53-dependent response pathway either to elicit a cell cycle arrest or to initiate apoptosis, as seems to be the case for skin keratinocytes (29, 35). Thus, the induction of p16 may be the primary response to UV-induced damage in melanocytes.

In summary, we have demonstrated that p16 is induced in cells of the basal layer in human skin, including melanocytes. A better understanding of the role of p16 in the UV-induced response may provide new insight into how the loss of p16 function contributes to melanoma development.

Fig. 1.

p16 is induced in human skin after UV exposure. p16 immunostaining in human skin after 24 h in culture: A, unirradiated control; B, post-10 Jm −2 UVC irradiation; C, post-250 Jm− 2 UVB irradiation; D, peptide-blocked control; E, the distribution of p16 staining throughout the epidermis was patchy. Both cytoplasmic and nuclear p16 staining was evident in most stained cells within the epidermis, with cytoplasmic p16 predominating (F and G); arrows, melanocytes (m). There was a shift in cells containing p16 staining upwards through the epidermis at 48 h post-UVB irradiation (H).

Fig. 1.

p16 is induced in human skin after UV exposure. p16 immunostaining in human skin after 24 h in culture: A, unirradiated control; B, post-10 Jm −2 UVC irradiation; C, post-250 Jm− 2 UVB irradiation; D, peptide-blocked control; E, the distribution of p16 staining throughout the epidermis was patchy. Both cytoplasmic and nuclear p16 staining was evident in most stained cells within the epidermis, with cytoplasmic p16 predominating (F and G); arrows, melanocytes (m). There was a shift in cells containing p16 staining upwards through the epidermis at 48 h post-UVB irradiation (H).

Close modal
Fig. 2.

Increase in p16 expression in human skin in relation to dose at 24 h postirradiation. The percentage of cells per field expressing p16 for control and after UVB and UVC irradiation with indicated dose.

Fig. 2.

Increase in p16 expression in human skin in relation to dose at 24 h postirradiation. The percentage of cells per field expressing p16 for control and after UVB and UVC irradiation with indicated dose.

Close modal
Fig. 3.

p16 expression is a delayed response, peaking at 24 h after UV exposure. The proportion of cells expressing p16 dramatically increased at 16 h postirradiation with 250 Jm−2 UVB, peaked at 24 h, and substantially diminished by 72 h postirradiation. Similar data were obtained with 150 and 875 Jm−2 UVB.

Fig. 3.

p16 expression is a delayed response, peaking at 24 h after UV exposure. The proportion of cells expressing p16 dramatically increased at 16 h postirradiation with 250 Jm−2 UVB, peaked at 24 h, and substantially diminished by 72 h postirradiation. Similar data were obtained with 150 and 875 Jm−2 UVB.

Close modal

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

1

This work was supported by a grant from the Queensland Cancer Fund. B. G. is the recipient of an Australian Research Fellowship.

3

The abbreviations used are: cdk, cyclin-dependent kinase; UVB, ultraviolet-B; UVC, ultraviolet-C; TUNEL, terminal deoxynucleotidyl transferase-mediated nick end labeling.

4

S. Goldstone, J. Sinnamon, S. Pavey, and B. Gabrielli, manuscript in preparation.

We thank Dr. Nicholas Hayward for support and encouragement during this work, Professor Adele Green and Dr. Peter Parsons for review of the manuscript, and Dr. Margaret Cummings for assistance with histology.

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