Oncogene (2001) 20, 6103 ± 6110 ã 2001 Nature Publishing Group All rights reserved 0950 ± 9232/01 $15.00 www.nature.com/onc ORIGINAL PAPERS G2 phase cell cycle arrest in human skin following UV irradiation

Sandra Pavey1, Terry Russell1 and Brian Gabrielli*,1

1Joint Experimental Oncology Program, Department of Pathology, University of Queensland, Brisbane, Australia

The contribution of the short wavelength ultraviolet (UV) equivalent to a minimal erythemal dose (1MED), can component of sunlight to the aetiology of skin cancer has produce DNA damage and stimulate unscheduled been widely acknowledged, although its direct contribu- DNA synthesis (i.e. excision repair mechanisms) in tion to tumour initiation or progression is still poorly basal and suprabasal layer cells in the skin (Freeman et understood. The loss of normal cell cycle controls, al., 1987; Honigsmann et al., 1987). Studies using particularly checkpoint controls, are a common feature cultured cells have reported both G1 and G2 phase cell of cancer. UV radiation causes both G1 and G2 phase cycle arrests as responses to DNA damage induced by checkpoint arrest in vitro cultured cells. In this study we UVR. The G1 arrest appears to be through a p53- have investigated the cell cycle responses to suberythemal dependent mechanism (Lu and Lane, 1993; Herzinger doses of UV on skin. We have utilized short-term whole et al., 1995), whereas the G2 arrest is independent of organ skin cultures, and multi parameter immunohisto- p53, and involves a block in the cdc25-dependent chemical and biochemical analysis to demonstrate that activation of the mitotic cyclin dependent kinase basal and suprabasal layer melanocytes and keratino- (CDK) complexes (Herzinger et al., 1995; Gabrielli et cytes undergo a G2 phase cell cycle arrest for up to 48 h al., 1997b). following irradiation. The arrest is associated with A number of human genetic mutations are asso- increased p16 expression but no apparent p53 involve- ciated with melanoma. Of these, p16/CDKN2A, which ment. This type of organ culture provides a very useful is functionally inactivated in a high proportion of model system, combining the ease of in vitro manipula- melanoma cell lines and in 10 ± 30% of tumours tion with the ability to perform detailed molecular (Castellano and Parmiani, 1999), is a direct e€ector analysis in a normal tissue environment. Oncogene of cell cycle progression. Mutations of p16/CDKN2A (2001) 20, 6103 ± 6110. have also been identi®ed in non-melanoma skin cancers (Sou®r et al., 1999). Ectopic expression of p16 in Keywords: p16; UV; skin; G2 checkpoint di€erent tumour lines has been shown to inhibit cell growth via a Rb-dependent G1 phase arrest, due to the ability of p16 to speci®cally bind and inhibit CDK4 Introduction and CDK6 (Ruas and Peters, 1998). The physiological roles for p16 are still poorly de®ned, but it has been The role of ultraviolet radiation (UVR) as one implicated in replicative senescence (Haddad et al., aetiological factor in the development of skin cancer, 1999; Dickson et al., 2000), and a G1 phase checkpoint including melanoma, is well established (Elder, 1989), response to DNA damage (Shapiro et al., 1998). We with UVR acting as both an initiator and promoter in have previously reported increased p16 levels in the course of multi-step carcinogenesis (Wikonkal and response to suberythemal doses of short wavelength Brash, 1999). UVR has a range of e€ects on UVR in cultures of melanocyte and keratinocyte- epidermally-derived cells in vitro and in vivo. It directly derived cells (Wang et al., 1996; Milligan et al., increases the pigment production and the dendricity of 1998), which correlates with a G2 phase delay. Loss melanocytes (Friedmann and Gilchrest, 1987), and may of the UV-induced G2 checkpoint arrest corresponded also act as an independent mitogen for these cells to an increase in the proportion of cells with fractured (Libow et al., 1988). Studies have also demonstrated nuclei, a result of cells undergoing aberrant mitosis that UVR induces the production of keratinocyte- (Milligan et al., 1998), demonstrating the importance derived factors that regulate keratinocyte and melano- of this checkpoint for the maintenance of genomic cyte growth in an autocrine/paracrine manner (Ley and integrity. We have also reported a strong increase in Ellem, 1992; Im et al., 1998; Thody and Graham, p16 expression in response to suberythemal doses of 1998). Exposure to simulated solar radiation at doses UVR in human skin (Pavey et al., 1999), suggesting that the elevated p16 levels may be involved in the normal cellular responses to sunlight exposure, possibly in a cell cycle response. *Correspondence: B Gabrielli, Department of Pathology, University Much work on UV responses, particularly cell cycle of Queensland Medical School, Herston Road, Brisbane, Queensland responses, has used cultured cell lines, many of which 4006, Australia; E-mail: [email protected] Received 5 March 2001; revised 31 May 2001; accepted 8 June are non-epidermal in origin, e.g. ®broblasts. The 2001 anatomical organization of skin and penetration UV responses in skin S Pavey et al 6104 characteristics of di€erent wavelength UVR mean that form of cdc2 (PY15) in irradiated compared with conclusions from in vitro studies may not be directly control samples (Figure 3a). PY15 was also detected in transferable to human skin. This is in part because the basal and suprabasal layer cells at 24 h following mutagenic potential of a particular wavelength in vivo irradiation by immunohistochemistry and localized to will be a function of both the dose received by the stem both cytoplasm and nucleus, similar to the localization cells in the basal layer and the e€ectiveness of the of cyclin B1 (data not shown). Although there is an radiation in inducing DNA damage (Campbell et al., absolute requirement for cyclin association with cdc2 1993; Pavey et al., 1999). Studies examining the e€ects to permit Tyr15 phosphorylation, the direct association of UVR on primary cultures of melanocytes and of the increased cyclin B1 and PY15 staining detected keratinocytes, which are physiological targets for the by immunohistochemistry and immunoblotting was mutagenic UVB component of solar radiation, have con®rmed by analysis of cyclin B1 immunoprecipitates overlooked the potential paracrine e€ects on the from similar tissue extracts. This revealed a strong biological and physiological responses of these cells. accumulation of the inactive phosphotyrosine 15 form In this study we have utilized a short-term whole skin of cdc2 associated with cyclin B1 in UV irradiated organ culture model to examine whether the G2 phase samples over controls similar to that detected by cell cycle arrest we have observed after low dose UVR immunoblotting of the tissue extracts (Figure 3a,b). in cultured cell lines was also a response of basal layer The accumulation of inactive phosphotyrosine 15 cells in the skin. We have used multi-parameter cdc2/cyclin B1 in the basal layer cells of irradiated skin analyses of a number of markers of cell cycle stage, was suggestive of a G2 phase cell cycle arrest in these and biochemical analysis to demonstrate a G2 phase cells. The delay of up to 16 h before signi®cant cell cycle arrest in the basal and suprabasal layer accumulation of cyclin B1 was observed would predict keratinocytes and melanocytes 24 ± 48 h after exposure that these cells had progressed through S phase during to suberythemal doses of UVB. The G2 arrest is the ®rst 24 h after irradiation, and if arrested, would associated with elevated levels of p16, but surprisingly not enter S phase during the next 24 h. To examine little or no increase in p53 or its downstream e€ectors this, skin samples were incubated with BrdU from 0 ± of cell cycle arrest, p21/WAF1 or GADD45, were 24 h or 24 ± 48 h following UV irradiation. Parallel detected. The cell cycle responses observed are di€erent BrdU labelling experiments of unirradiated controls from that observed with in vitro cultured primary were also performed. Few cells in the control skin were melanocytes and keratinocytes, suggesting that the BrdU positive, and there was a 4sevenfold increase in tissue environment in¯uences cell cycle responses of irradiated skin incubated with BrdU during the initial the basal layer cells. 24 h after irradiation (Figures 1c,d and 4). Skin samples labelled with BrdU from 24 ± 48 h after irradiation revealed a fourfold decrease in the number Results of BrdU positive cells compared to the 0 ± 24 h BrdU labelling experiments (Figure 4), suggestive of a The expression and localization of a number of decrease in S phase cells from 24 ± 48 h after irradia- markers of cell cycle stage were examined by tion. There were no signi®cant di€erences in control immunohistochemical and biochemical analysis of levels of incorporation during the two time periods. unirradiated control skin and after irradiation with The intensity and uniformity of BrdU staining suberythemal doses of UVB (250 ± 875 Jm72). Cyclin throughout the nucleus indicated that cells had under- B1 was examined as marker of G2 phase. Its gone S phase rather than DNA repair, which is marked expression increases from late S phase, and in UVR by intense patches of nucleotide incorporation (Lu and treated, G2 arrested cells, it is expressed at normal G2 Lane, 1993). phase levels and complexed with the Tyr15 phosphory- As a separate measure of S phase, immunohisto- lated, inactive form of cdc2 (Gabrielli et al., 1997b). chemical staining for PCNA, a co-factor for DNA When the levels of cyclin B1 expressed in skin were polymerase delta, was performed. In control unirra- examined, it was found to be low to undetectable in diated samples, PCNA staining was uniform through- unirradiated, control skin, but increased in both out the basal and suprabasal layer cells and exclusively number of positive staining cells and intensity of nuclear (Figure 1e). By 24 h after UV irradiation, there staining at 24 h after UVR treatment (Figure 1a,b). was a dramatic change in the localization of PCNA, Cyclin B1 staining in the irradiated skin was both with it relocating from the nucleus to the cytoplasm at nuclear and cytoplasmic, and con®ned to basal and this time (Figure 1f). Strong cytoplasmic staining was suprabasal layer melanocytes and keratinocytes (Figure detected at 16 h following UV irradiation, and by 24 h 1b). The proportion of epidermal cells immunolabelled 97% of cells displayed strong cytoplasmic staining with cyclin B1 in skin samples increased from control (Figure 5). There was a corresponding loss of nuclear levels at 8 h to a peak at 24 h, which was maintained PCNA, with only 10% of cells displaying weak nuclear at 48 h and decreased by 72 h following UV irradiation staining. By 48 h, 26% of cells had nuclear PCNA (Figure 2). Immunoblotting of tissue extracts of similar staining, and by 72 h the majority of PCNA staining control and UV irradiated samples harvested at 24 h was con®ned to the nucleus, with the intensity of the after irradiation revealed strong increase in both cyclin remaining cytoplasmic staining observed reduced B1 and cdc2 levels, and the level of phosphotyrosine 15 compared to 24 h. Immunoblotting of tissue extracts

Oncogene UV responses in skin S Pavey et al 6105

Figure 1 Mock irradiated (a,c,e) and irradiated with 250 Jm72 UVB (b,d,f) skin samples were harvested after 24 h and immunostained with antibodies to cyclin B1 with melanocytes indicated by arrows (a,b), labelled with BrdU during the initial 24 h after irradiation (c,d), or stained for PCNA (e,f), Panels a,b are at magni®cation of 10006and panels c,d,e,f,g at 4006

of the basal layer cells provided by UVR. However, the persistence of the elevated cyclin B1 staining at 48 h and relative reduction in S phase cells from 24 ± 48 h as compared to the initial 24 h after irradiation, as measured by both the BrdU incorporation and loss of nuclear PCNA, indicated that it was likely to be a G2 phase arrest. Further evidence to support this was found with immunoblotting of extracts of skin harvested at 48 h after irradiation. Elevated levels of cyclin B1 and corresponding increased PY15 were found to persist in the irradiated compared to control skin (Figure 6). Ca€eine is a potent inhibitor of the ATM-related ATR (Sarkaria et al., 1999) which is Figure 2 Skin samples were harvested at the indicated times implicated in the UV-induced G2 arrest (Cliby et al., after irradiation and immunostained for cyclin B1. The propor- 1998), and addition of ca€eine abrogates the UV- tion of cyclin B1 positive cells were quanti®ed as described in induced G2 phase arrest in HeLa cells (Gabrielli et al., Materials and methods 1999). When ca€eine was added to irradiated skin samples at 24 h after irradiation then harvested at 48 h, there was a loss of the cyclin B1 accumulation revealed no changes in PCNA levels following and PY15 band detected in the 48 h irradiated samples irradiation (Figure 2a). indicative of exit from the G2 phase arrest (Figure 6). These data were suggestive of either a G2 phase We have previously demonstrated that p16 is arrest or a wave of cells passing through G2/M at 24 h induced in human skin following low dose UV after irradiation due to quasi synchronous stimulation radiation (Pavey et al., 1999) (Figure 7a,b). By using

Oncogene UV responses in skin S Pavey et al 6106

Figure 5 Tissue sections from similar time course to that in Figure 2 were immunostained for PCNA and the proportion of cells with cytoplasmic and/or nuclear staining quanti®ed

Figure 3 (a) Tissue extracts from three separate control and irradiated skin samples, 24 h after irradiation, were immuno- blotted for the indicated cell cycle regulatory proteins. Tubulin levels were used as a loading control. (b) Cyclin B1 immunopre- cipitations from similar tissue extracts as used in (a) were immunoblotted for cyclin B1, cdc2 and phosphotyrosine 15 form Figure 6 Tissue extracts from either unirradiated control skin of cdc2 (PY15) samples (con), irradiated with 250 Jm72 UVB (UV) or irradiated and addition of 50 mM ca€eine at 24 h after irradiation (UV+ca€.) then harvested at 48 h after irradiation. Samples were immunoblotted for cyclin B1 and PY15 as in Figure 3. The empty arrowhead indicates a non-speci®c band and the ®lled arrowhead the cyclin B1 band

expression were detected (Figure 3a), con®rming our earlier immunohistochemical observations (Pavey et al., 1999). We have also examined the levels of transcrip- tional targets of p53, p21/WAF1 and GADD45, to assess whether p53 was transcriptionally active after UVR exposure. Control unirradiated skin contained detectable levels of p21/WAF1 within the suprabasal, spinous and granular cell layers (Figure 7g), with approximately 10% of epidermal cells expressing low levels of p21/WAF1 (Figure 8). The proportion of cells Figure 4 Control and irradiated skin samples were incubated with BrdU from either 0 ± 24 h or 24 ± 48 h after irradiation, then expressing p21/WAF1 increased twofold by 16 h after stained for BrdU incorporation and the proportion of BrdU irradiation, then dropped dramatically by 24 h to positive cells quanti®ed approximately 2%, and remained at these levels through to 72 h post-UV (Figures 7h and 8). The increase in p21/WAF1 staining was restricted to the double immunohistochemical staining techniques, cy- spinous and granular cell layers. Only very low levels clin B1 was found to co-localize with p16 in 480% of of GADD45 were detected and these increased p16 positive cells (Figure 7c,d). Strong p16 staining 5twofold following UV exposure (Figure 3). The was also found in BrdU positive cells from the initial small changes in GADD45 levels detected by immuno- 24 h labelling experiments (Figures 6e,f), with 67% of blotting of the tissue extracts were not associated with cells staining for p16 also labelled with BrdU. p16 any detectable change in GADD45 staining in the staining also corresponded to the relocation of PCNA basal layer compartment, where only weak staining from the nucleus to the cytoplasm in irradiated cells was observed. GADD45 has been demonstrated to act with 480% of p16 positive cells displaying predomi- as direct binding inhibitor of the cyclin B/cdc2 complex nantly cytoplasmic staining for PCNA at 24 h after (Zhan et al., 1999). However, we were unable to detect irradiation. any GADD45 associated with cyclin B1 immunopreci- The level of p53 was also examined by immunoblot- pitates from either control or irradiated samples (data ting of the tissue extracts. No changes in p53 not shown). These data demonstrate that p21/WAF1

Oncogene UV responses in skin S Pavey et al 6107

Figure 7 Control (a,g), and UV radiated skin (b ± f,h) were immunostained for (a,b) p16; (c) p16; (d) same section as in (c) doubled-stained for p16 (red) and cyclin B1 (purple/black); (e) BrdU; (f) same section as in (e) doubled-stained for BrdU (red) and p16 (purple/black); p21 (g,h). Panels a,b,e,f,g,h are at a magni®cation of 4006and panels c,d at 10006

and GADD45 were unlikely to contribute to the G2 The relocation of PCNA from the nucleus to the arrest observed in the basal layer cells. cytoplasm at this time was unexpected, although this has been reported previously in UV-irradiated mela- nocytes associated with a G2 arrest (Iyengar, 1994). Discussion The relocation to the cytoplasm would act to reinforce a block on S phase entry, suggesting the cells undergo This is the ®rst detailed analysis of the cell cycle a cell cycle arrest either in G2 phase immediately after responses of melanocytes and keratinocytes in human the ®rst round of DNA replication, or in the skin to UVR. We have observed increased BrdU subsequent G1. The elevated levels of cyclin B1, incorporation during the ®rst 24 h following irradia- accumulation of inactive phosphotyrosine 15 form of tion, suggesting that UVR is acting as a mitogen, cdc2 complexed with cyclin B1, a marker of G2 arrest stimulating a rapid round of DNA synthesis. The (Gabrielli et al., 1997b), and the lack of mitotic ®gures reduced BrdU labelling from 24 ± 48 h demonstrated (unpublished observations), all point to a cell cycle that cells did not re-enter S phase during this period. arrest in G2 phase from 24 ± 48 h. The ability of

Oncogene UV responses in skin S Pavey et al 6108 the e€ects of the interactions between cells in tissue, e.g. cell ± cell contacts and paracrine interactions. These cultures are also typically homogeneous cell popula- tions, whereas the epidermal layer of the skin is an interdependent mix of predominantly keratinocytes, interspersed with the occasional melanocyte which are connected with up to 36 of these keratinocytes by dendritic projections (Quevedo, 1969). After UV exposure keratinocytes produce a range of cytokines and growth factors, and these may in¯uence the short- term cell cycle responses and longer-term growth and melanizing responses of melanocytes (reviewed in Thody and Graham, 1998). The keratinocyte derived Figure 8 Sections from a similar time course to that used in factors, melanotropin and endothelin has been demon- Figure 2 was immunostained for p21 and the proportion of p21 strated to overcome the UVB-induced G1 arrest in positive cells quanti®ed cultured melanocytes (Im et al., 1998; Tada et al., 1998). These paracrine interactions, and possibly other cell ± cell interactions, can in¯uence cell responses to ca€eine to abrogate this G2-like state is further UVR, and are poorly mimicked by the tissue culture evidence that the growth stimulated cells arrest in G2 systems currently used. phase. Initiation of the G2 phase arrest in the basal layer This G2 phase arrest appears similar to the p53- cells may facilitate more than a protective checkpoint independent G2 arrest following low dose UVR response to cellular damage. Another skin response to previously described in in vitro cell culture systems, UVR is an increase in melanin content to provide a with the accumulation of high levels of Tyr15 further level of protection against UV-induced DNA phosphorylated, inactive cdc2/cyclin B1 (Gabrielli et damage and possibly oxidative damage (Thody and al., 1997a). The lack of p53 involvement in either the Graham, 1998). aMSH expression increases after UVR, UV response or cell cycle arrest characterized here is and the binding of aMSH and tyrosinase activity are surprising, considering that earlier studies have demon- increased in normal G2 phase and G2 phase arrested strated a p53-dependent G1 arrest in cell lines and melanoma cells, these e€ects co-operate to increase the primary cultures of epidermal melanocytes and sensitivity of irradiated cells to aMSH and stimulate keratinocytes (Medrano et al., 1995; Petrocelli et al., melanin synthesis (McLane and Pawelek, 1988; Watters 1996). The di€erences between the observations et al., 1998; Chakraborty et al., 1999). The G2 arrest in reported here and the work using cultured primary the surrounding basal layer keratinocytes may also be epidermal cells are likely to be due to a number of a response to increased aMSH binding to these cells factors. The low levels of UVB used in the experiments (Chakraborty and Pawelek, 1993) and contribute to the reported here are not likely to produce DNA strand increased melanization of the basal layer. breaks in the basal and suprabasal layer cells due to The strong increase in p16 expression in the basal the shielding e€ects of the upper epidermal layers. and suprabasal layer cells following exposure to Although p53 can be activated by a single DNA strand suberythemal doses of UVB previously reported (Pavey break (Huang et al., 1996), the lack of any p21/WAF1 et al., 1999), is tightly correlated with all the markers or GADD45 induction in the basal layer cells is further of cell cycle arrest examined here. The p16 staining evidence of the absence of strand breaks in these cells. cells also stained for BrdU and cyclin B1, and The role of p53 in the response to high doses of UVR displayed cytoplasmic relocation of PCNA, indicating has been implicated in an apoptotic rather cell cycle that they had transited through S phase and were in response (Ziegler et al., 1994; Wikonkal and Brash, G2 phase at 24 h after irradiation. These ®ndings point 1999). The lack of p53 induction in response to low to the cells containing high levels of p16 being arrest in doses of UVR used in the present study may simply G2 phase, similar to the correlation between elevated re¯ect this, and no apoptosis was detected under the p16 and G2 arrest we have reported with in vitro conditions used in the present studies (Pavey et al., cultured cells (Wang et al., 1996; Milligan et al., 1998). 1999). P16 is also been reported to increase in cells stimulated Another factor that may contribute to the di€erence to re-enter the cell cycle (Tam et al., 1994). Although between UV responses of cultured cell lines and whole the strong p16 expression reported here was found in skin is the tissue environment. Basal layer cells have growth stimulated cells, it is unlikely to be simply a low mitotic activity in unirradiated skin, although they consequence of re-entry into the cell cycle. The small are competent to proliferate (indicated by the uniform number of BrdU positive cells found in control skin staining of basal and suprabasal cell nuclei with did not have detectable p16 staining, and treatment of PCNA), and are readily stimulated to proliferate by skin with retinoic acid which stimulated proliferation in UVR. By contrast, in vitro cultured cells are constantly the basal layer compartment, did not increase p16 stimulated to proliferate with exogenously supplied expression (Pavey and Gabrielli, manuscript in pre- growth factors. In vitro culture also fails to account for paration). The contribution of the elevated levels p16

Oncogene UV responses in skin S Pavey et al 6109 to the G2 phase arrest is still unclear, but we have described (Pavey et al., 1999). For BrdU staining of formalin- previously demonstrated in in vitro cultured cells that ®xed tissues, sections were incubated in 4 M HCl for 30 min p16 inhibits the activation of a cdk4/cyclin D activity at room temperature, followed by digestion with trypsin at in late S/early G2 phase that appears to be required for 1mgml71 in PBS for 10 min at 378C. The sections were then G2 phase progression (Gabrielli et al., 1999). Alter- blocked and stained as for other antigens. natively, the p16 may have a role in establishing a G1 phase arrest subsequent to the G2 arrest (Shapiro et Tissue extracts al., 1998), or may possibly contribute to both. Tissue samples were removed from the media and snap In summary, we have shown that in response to frozen and stored at 7808C until analysis. Tissue was cut suberythemal dose of UVR, basal and suprabasal layer into small pieces and placed in 1.2 ml extraction bu€er cells in human skin arrest in G2 phase, and this is containing 300 mM NETN/NaCl (300 mM NaCl, 1 mM associated with increased levels of p16. De®ning the EDTA, 0.5% Nonidet P-40, and 20 mM Tris, 8 pH) normal cell cycle checkpoint responses to UVR supplemented with 5 mg/ml aprotinin, 5 mg/ml leupeptin, provides new insights into how loss of these check- 5 mg/ml pepstatin, 10 mM NaF, 0.1 mM sodium orthovana- points may contribute to UVR induced skin cancer. date, and 0.5 mM phenylmethylsulphonyl ¯uoride. Tissue was homogenized on ice using a Polytron Homogeniser with a This work also demonstrates that although cultured 7 mm aggregate (Kinematic AG, Switzerland), using 3 ± 4 cells o€er an excellent model system for de®ning pulses at 5000 r.p.m. for a duration of 5 s/pulse. Extracts molecular mechanisms, tissue environment is an were then ultracentrifuged in a TL-100 ultracentrifuge important in¯uence on cellular responses. The devel- (Beckman), at 135 000 g for 1 h at 48C. Samples were opment of whole organ models such as one reported equilibrated on the basis of the level of tubulin expression here will be essential to help de®ne the normal by SDS ± Page and immunoblotting with a tubulin antibody physiological roles of cellular regulators. 1 : 500 (Amersham Pharmacia Biotech, Sweden).

Immunoprecipitation and immunoblotting The tissue extracts were precleared with 50 ml of a 50% suspension of Protein A-Sepharose (CL-4B Amersham Materials and methods Pharmacia Biotech, Sweden) for 1 h, and the cleared extracts were incubated with 0.5 mg of anti-cyclin B1 antibody pre- Organ cultures bound to 30 ml of a 50% solution of Protein-A-Sepharose Organ cultures were established and maintained as previously overnight at 48C. The supernatant was then removed and described (Pavey et al., 1999). Brie¯y, neonatal foreskins were precipitates were washed four times with the extraction obtained after circumcision, and cut into two, and irradiated bu€er. SDS sample bu€er was then added to the precipitates with 250 ± 875 Jm72 UVB (875 Jm72 is equivalent to one and supernatants and boiled for 20 min and 3 min respec- minimal erythemal dose, 1 MED) or used as a control, tively. Immunoprecipitates were resolved on a 10% SDS- maintained in specialized media supplemented with anti- polyacrylamide gel, transferred to PVDF membrane (NEN biotics and incubated at 378C in a humidi®ed incubator with Life Science Products), and immunoblotted for associated the epidermis exposed as previously described (Pavey et al., proteins using the indicated antibody at 48C overnight. 1999). Protein-A conjugated-HRP (ICN), secondary antibody was used for detection using chemiluminescence with a Western blot Chemiluminescence Reagent Plus Kit (NEN Life Science Bromodeoxyuridine labelling Products). Tissue samples were incubated in base medium supplemented with 25 m 5-bromo-2'-deoxyuridine (BrdU), with the M Evaluation of expression epidermis submerged enabling maximal absorption and incorporation. BrdU labelling was undertaken 0 ± 24 h There were a total of 88 samples collected and analysed, incubation, or 24 ± 48 h incubation following irradiation, consisting of: (a) 16 unirradiated controls; (b) 16 irradiated and harvested at 24 or 48 h as indicated. with 250 Jm72 UVB; (c) 16 unirradiated controls and 16 UVB irradiated samples used for BrdU incorporations studies; (d) 12 unirradiated controls and 12 UVB irradiated Immunohistochemistry samples used for preparation of tissue extracts. A time course Immunohistochemistry was performed as described pre- was undertaken on 16 of the control and 16 of the UV viously (21), using a Zymed Histostain-Plus kit (Zymed, irradiated samples, and were harvested at 0, 8, 16, 24, 48 and CA, USA) with the substrate AEC (Zymed) or Vector 72 h in organ culture for both irradiated and unirradiated NovaRed (Vector laboratories) to produce a red end product control samples. A cell was considered to be immunolabelled for single antigen detection, or a Zymed Histostain-DS kit and positive for the cell cycle markers used, if there was a (Zymed), for detection of two di€erent antigens within the visibly detectable signal within the nucleus, cytoplasm, or same sample (AEC ± red; NBT/BCIP ± black). Antibodies both nucleus and cytoplasm of a cell. A ®eld on each slide used were as follows: p16 1 : 300 (Wang et al., 1996), PCNA representative of the entire sample was selected and the 1 : 200 (NCL-PCNA, Novocastra), cyclin B1 1 : 4500 (Gab- number of cells expressing the protein of interest were rielli et al., 1996), BrdU 1 : 400 (Ab-2, NeoMarkers, measured and reported as a percentage of the total number LabVision), p21/WAF1 1 : 300 (Calbiochem), phospho-cdc2 of cells counted in each ®eld as previously described (Pavey et (PY15) 1 : 20 (New England Biolabs), and GADD45 1 : 200 al., 1999). All nucleated cells within the epidermal layers were (Santa Cruz). Antigen retrieval was performed for p16, included in the analyses. From the ®elds analysed, there was PCNA, cyclin B1, BrdU, p21/WAF1 and PY15 as previously an average of 168 cells counted per ®eld.

Oncogene UV responses in skin S Pavey et al 6110 Acknowledgements funded by the National Health and Medical Research We would like to thank Dr Nicholas Hayward for his Council of Australia. B Gabrielli was supported by an continued support and Drs Hayward and Nick Saunders Australian Research Council Fellowship. for critical reading of the manuscript. This project was

References

Campbell C, Quinn AG, Angus B, Farr PM and Rees JL. Lu X and Lane DP. (1993). Cell, 75, 765 ± 778. (1993). Cancer Res., 53, 2697 ± 2699. McLane JA and Pawelek JM. (1988). Biochemistry, 27, Castellano M and Parmiani G. (1999). Melanoma Res., 9, 3743 ± 3747. 421 ± 432. Medrano EE, Im S, Yang F and Abdel-Malek ZA. (1995). Chakraborty A and Pawelek J. (1993). J. Cell. Physiol., 157, Cancer Res., 55, 4047 ± 4052. 344 ± 350. Milligan A, Gabrielli BG, Clark JM, Hayward NK and Chakraborty AK, Funasaka Y, Slominski A, Bolognia J, Ellem KA. (1998). Mutat. Res., 422, 43 ± 53. Sodi S, Ichihashi M and Pawelek JM. (1999). Ann. N.Y. Pavey S, Conroy S, Russell T and Gabrielli B. (1999). Cancer Acad. Sci., 885, 100 ± 116. Res., 59, 4185 ± 4189. Cliby WA, Roberts CJ, Cimprich KA, Stringer CM, Lamb Petrocelli T, Poon R, Drucker DJ, Slingerland JM and JR, Schreiber SL and Friend SH. (1998). EMBO J., 17, Rosen CF. (1996). Oncogene, 12, 1387 ± 1396. 159 ± 169. Quevedo Jr WC. (1969). Am. Zool., 9, 531 ± 540. Dickson MA, Hahn WC, Ino Y, Ronfard V, Wu JY, Ruas M and Peters G. (1998). Biochim. Biophys. Acta, 1378, Weinberg RA, Louis DN, Li FP and Rheinwald JG. F115 ± F177. (2000). Mol. Cell. Biol., 20, 1436 ± 1447. Sarkaria JN, Busby EC, Tibbetts RS, Roos P, Taya Y, Elder DE. (1989). J. Invest. Dermatol., 92, 297S ± 303S. Karnitz LM and Abraham RT. (1999). Cancer Res., 59, Freeman SE, Gange RW, Sutherland JC, Matzinger EA and 4375 ± 4382. Sutherland BM. (1987). J. Invest. Dermatol., 88, 430 ± 433. Shapiro GI, Edwards CD, Ewen ME and Rollins BJ. (1998). Friedmann PS and Gilchrest BA. (1987). J. Cell. Physiol., Mol. Cell. Biol., 18, 378 ± 387. 133, 88 ± 94. Sou®r N, Moles JP, Vilmer C, Moch C, Verola O, Rivet J, Gabrielli BG, Clark JM, McCormack AK and Ellem KA. Tesniere A, Dubertret L and Basset-Seguin N. (1999). (1997a). J. Biol. Chem., 272, 28607 ± 28614. Oncogene, 18, 5477 ± 5481. Gabrielli BG, Clark JM, McCormack AK and Ellem KA. Tada A, Suzuki I, Im S, Davis MB, Cornelius J, Babcock G, (1997b). Oncogene, 15, 749 ± 758. Nordlund JJ and Abdel-Malek ZA. (1998). Cell Growth GabrielliBG,DeSouzaCP,TonksID,ClarkJM,Hayward Di€er., 9, 575 ± 584. NK and Ellem KA. (1996). J. Cell Sci., 109, 1081 ± 1093. Tam SW, Shay JW and Pagano M. (1994). Cancer Res., 54, Gabrielli BG, Sarcevic B, Sinnamon J, Walker G, Castellano 5816 ± 5820. M, Wang XQ and Ellem KA. (1999). J. Biol. Chem., 274, Thody AJ and Graham A. (1998). Pigment Cell Res., 11, 13961 ± 13969. 265 ± 274. Haddad MM, Xu W, Schwahn DJ, Liao F and Medrano EE. Wang XQ, Gabrielli BG, Milligan A, Dickinson JL, Antalis (1999). Exp. Cell Res., 253, 561 ± 572. TM and Ellem KA. (1996). Cancer Res., 56, 2510 ± 2514. Herzinger T, Funk JO, Hillmer K, Eick D, Wolf DA and Watters D, Garrone B, Coomer J, Johnson WE, Brown G Kind P. (1995). Oncogene, 11, 2151 ± 2156. and Parsons P. (1998). Biochem. Pharmacol., 55, 1691 ± Honigsmann H, Brenner W, Tanew A and Ortel B. (1987). 1699. J. Photochem. Photobiol. B, 1, 33 ± 43. Wikonkal NM and Brash DE. (1999). J. Investig. Dermatol. Huang LC, Clarkin KC and Wahl GM. (1996). Proc. Natl. Symp. Proc., 4, 6±10. Acad. Sci. USA, 93, 4827 ± 4832. Zhan Q, Antinore MJ, Wang XW, Carrier F, Smith ML, Im S, Moro O, Peng F, Medrano EE, Cornelius J, Babcock Harris CC and Fornace Jr AJ. (1999). Oncogene, 18, G, Nordlund JJ and Abdel-Malek ZA. (1998). Cancer 2892 ± 2900. Res., 58, 47 ± 54. Ziegler A, Jonason AS, Le€ell DJ, Simon JA, Sharma HW, Iyengar B. (1994). Melanoma Res., 4, 293 ± 295. Kimmelman J, Remington L, Jacks T and Brash DE. Ley KD and Ellem KA. (1992). Carcinogenesis, 13, 183 ± 187. (1994). Nature, 372, 773 ± 776. Libow LF, Scheide S and DeLeo VA. (1988). Pigment Cell Res., 1, 397 ± 401.

Oncogene