Chronic UVA Irradiation of Human Hacat Keratinocytes Induces Malignant Transformation Associated with Acquired Apoptotic Resistance

Oncogene (2006) 25, 3680–3688 & 2006 Nature Publishing Group All rights reserved 0950-9232/06 $30.00 www.nature.com/onc ORIGINAL ARTICLE Chronic UVA irradiation of human HaCaT keratinocytes induces malignant transformation associated with acquired apoptotic resistance

Y-Y He1,JPi2, J-L Huang1, BA Diwan3, MP Waalkes2 and CF Chignell1

1Laboratory of Pharmacology and Chemistry, National Institute of Environmental Health Sciences, Research Triangle Park, NC, USA; 2Inorganic Carcinogenesis Section, Laboratory of Comparative Carcinogenesis, National Cancer Institute, National Institute of Environmental Health Sciences, Research Triangle Park, NC, USA and 3Basic Research Program, Science Applications International Corporation-Frederick, National Cancer Institute at Frederick, Frederick, MA, USA

Ultraviolet A (UVA, 315–400 nm), constituting about Keywords: UVA; transformation; carcinogenesis; AKT; 95% of ultraviolet irradiation in natural sunlight, keratinocyte; PTEN represents a major environmental challenge to the skin and is clearly associated with human skin cancer. It has proven difficult to showdirect actions of UVA as a carcinogen in human cells. Here, we demonstrate that chronic UVA exposures at environmentally relevant doses Introduction in vitro can induce malignant transformation of human keratinocytes associated with acquired apoptotic resis- Ultraviolet (UV) radiation in sunlight is clearly an tance. As evidence of carcinogenic transformation, UVA- important environmental factor in human skin carcino- long-treated (24 J/cm2 once/week for 18 weeks) HaCaT genesis. Each year approximately one million new cases (ULTH) cells showed increased secretion of matrix of skin cancer are diagnosed in the United States alone, metalloproteinase (MMP-9), overexpression of keratin making it the most common type of cancer in this 13, altered morphology and anchorage-independent country. In animal models, UV radiation is a complete growth. Malignant transformation was established by carcinogen which can initiate and promote skin carci- the production of aggressive squamous cell carcinomas nogenesis resulting in squamous cell carcinoma (SCC), after inoculation of ULTH cells into nude mice (NCr-nu). basal cell carcinoma (BCC) and melanoma (de Gruijl ULTH cells were resistant to apoptosis induced not only et al., 1993; Setlow et al., 1993; Noonan et al., 2001). by UVA but also by UVB and arsenite, two other human UV radiation in sunlight is composed of Ultraviolet B skin carcinogens. ULTH cells also became resistant to (UVB 280–315 nm) and Ultraviolet A (UVA 315– apoptosis induced by etoposide, staurosporine and doxor- 400 nm). UVA has been considered far less carcinogenic ubicin hydrochloride. Elevated phosphorylation of protein based on limited direct damage to DNA (Setlow, 1974). kinase B (PKB, also called AKT) and reduced expression However, UVA is approximately 20-fold more abun- of phosphatase and tensin homologue deleted on chromo- dant than UVB in the sunlight and much more UVA some 10 (PTEN) were detected in ULTH cells. The penetrates the epidermis and reaches the basal germi- resistance of ULTH cells to UVA-induced apoptosis was native layers (Bruls et al., 1984). Recently, UVA was reversed by either inhibition of phosphatidylinositol shown to induce mutations in the basal layer of the skin 3-kinase (PI-3K) or adenovirus expression of PTEN or and UVA signature mutations have been detected in dominant negative AKT. These data indicate that UVA SCC and solar keratosis (Agar et al., 2004). Although has carcinogenic potential in human keratinocytes and UVA does induce mutations, mechanisms different from that the increased AKT signaling and decreased PTEN UVB may be involved (Dahle and Kvam, 2003). expression may contribute to this malignant transforma- Therefore, it is possible that UVA exposure may play tion. Further comparisons between the transformed a greater role in the development of human skin cancers ULTH and control cells should lead to a better under- than is generally assumed. It has been, however, difficult standing of the mechanism of UVA carcinogenesis and to convincingly show direct carcinogenic effects of UVA may help identify biomarkers for UVA-induced skin in vivo in human cells. malignancies. Apoptosis is a genetically programmed, morphologi- Oncogene (2006) 25, 3680–3688. doi:10.1038/sj.onc.1209384; cally distinct form of cell death that can be triggered by published online 8 May 2006 a variety of physiological and pathological stimuli. Apoptosis is characterized by cell rounding, membrane blebbing, cytoskeletal collapse, nuclear pyknosis, chro- Correspondence: Dr Y-Y He, LPC, NIEHS/NIH, 111 TW Alexander matin condensation/fragmentation and the formation Dr, POB 12233, MD F0-06, Research Triangle Park, NC 27709, USA. E-mail: [email protected] of membrane-bound apoptotic bodies (Kerr et al., 1972; Received 4 November 2005; revised 7 December 2005; accepted 7 Wyllie et al., 1980; Clarke, 1990). These bodies are December 2005; published online 8 May 2006 rapidly phagocytosed and digested by macrophages or Chronic UVA irradiation induces malignant transformation Y-Y He et al 3681 neighboring cells. Apoptotic cell death plays a key role a Control ULTH in eliminating damaged and/or potentially transformed cells. Disruption of apoptosis clearly contributes to the pathogenesis of cancer, and is a contributing factor in chemotherapeutic resistance observed in many human tumor cells (Thompson, 1995). Indeed, suppression of apoptosis is a consistent characteristic of tumors and malignantly transformed cells (Johnstone et al., 2002). In order to better define the role of UVA in human skin carcinogenesis, we studied the transforming poten- Control ULTH tial of chronic UVA exposure in HaCaT cells, a human b keratinocyte line. Accordingly, these UVA-long treated HaCaT (ULTH) cells showed clear signs of malignant transformation, including formation of aggressive SCC upon inoculation into nude mice. The ULTH cells became highly resistant to apoptosis induced not only by UVA, and but also by UVB, arsenite, etoposide, staurosporine and doxorubicin. Additional studies indicated that reduced phosphatase and tensin homologue deleted on chromosome 10 (PTEN) levels and increased protein kinase B (PKB also called AKT) signaling may be key events that mediate both c Control ULTH transformation and acquired apoptotic resistance. These data clearly demonstrate the carcinogenic potential of MMP-9 UVA in human skin cells.

d 5 Control ULTH * Results 4

Chronic UVA treatment-induced malignant 3 transformation of normal human HaCaT keratinocytes In an attempt to achieve carcinogenic transformation, 2

normal HaCaT (control) cells were chronically exposed Relative Fold to UVA (24 J/cm2 once a week for 18 weeks). The UVA 1 treated cells, designated ULTH, were subsequently compared to passage-matched control HaCaT cells. 0 MMP-9 Anchorage-independent growth is common in tumor cells (Cox and Der, 1994). To determine if ULTH cells Figure 1 Chronic UVA exposure induced malignant transformation of human HaCaT cells. (a) Anchorage-independent growth had acquired the ability for such growth, cells were assay. Control HaCaT (control) and ULTH cells were seeded at a seeded in soft agar, and colony formation was deter- density of 2 Â 104 in a 0.33% soft agar over a 0.5% agar bottom mined. The colony formation was common with ULTH layer. Colony was observed at 21 days. (b) Plasma membrane and cells (Figure 1a) and more than 800 colonies were nuclei of control and ULTH cells were stained with Image-iTt 4 LIVE Plasma Membrane and Nuclear Labeling Kit. Morphology formed from the initial 2 Â 10 ULTH cells. Thus of the cells was observed by using confocal microscopy. chronic UVA treatment induced anchorage-independent (c) Analysis of MMP-9 activity in control and ULTH in cell growth in ULTH cells. Control cells did not form conditioned medium by zymogram gel showing increased MMP-9 colonies. activity in ULTH conditioned medium. (d) Quantitative analysis of Morphological differences were observed between the zymography results. Data expressed as fold-control activity and are represented as means (n ¼ 3); bars, s.e.m.; *signiﬁcant differences UVA-exposed and passage-matched control cells. Con- from the activity in control cells (Po0.05). trol cells exhibited epithelial-like morphology, with uniformity of cell size and shape (Figure 1b). However, in contrast, cells treated chronically with UVA showed a heterogeneous morphology characterized by the pre- control cells (Figure 1c). The activity of MMP-9 in sence of giant cells with multiple nuclei (Figure 1b), ULTH-conditioned medium was 4.0-fold higher than in possibly indicating genomic instability in ULTH cells. control cell medium (Figure 1d). The hypersecretion of MMPs, secreted enzymes that degrade the extra- this MMP is consistent with the aggressive nature of the cellular matrix, have been implicated in tumor cell inva- tumors derived from these cells (see below). MMP-9 sion and are commonly upregulated in cancer cells activation is commonly observed in SCC from various (Bernhard et al., 1990). Zymographic analysis revealed locations (Juarez et al., 1993; Borchers et al., 1997) and marked increases in secretion of active matrix metallo- indicates a potential role of UVA in both tumor proteinase-9 (MMP-9) from ULTH cells compared to promotion and progression.

Oncogene Chronic UVA irradiation induces malignant transformation Y-Y He et al 3682 a Control ULTH C ULTH

Keratin 13

Figure 3 Increased expression of keratin 13 in ULTH. (a) Control and ULTH cell lysates were analysed by immunoblotting with antibody speciﬁc to keratin 13. (b) Expression of keratin 13 (red) was detected immunoﬂuorescence in control (c) and ULTH cells. Blue represented nuclei stained with Hoechst 33342.

ULTH cells acquire resistances to apoptosis concurrently b with malignant transformation To determine whether the transformed ULTH cells acquired resistance to apoptosis induced by further UVA or UVB radiation, control and ULTH cells were exposed to a single dose of UVA (24 J/cm2) or UVB (20 mJ/cm2) radiation after overnight serum starvation. As measured by phosphatidylserine translocation, a hallmark of apoptosis, 52.8% of control cells became apoptotic compared to only 23.7% of the ULTH cells after UVA exposure (Figure 4a). UVB induced 72.8% apoptosis in control cells while only 27.2% ULTH cells underwent apoptosis (Figure 4a). Similar resistance to cell death was also observed with different doses of UVA and UVB (Figure 4b). After UVA and UVB exposure caspase-3 and -7 were activated and PARP and FAK were cleaved in control cells (Figure 5a). In contrast, these reactions were Figure 2 A squamous cell carcinoma arising from inoculation of dramatically reduced in ULTH cells. A similar effect ULTH cells invading the surrounding renal cell parenchyma. One million cells were injected under the kidney capsule of a female was observed with DNA fragmentation (Figure 5b). nu/nu mouse. The mouse was euthanized 110 days after the Both caspase-8 and -9 were activated in control cells injection; Â 200. (b) A higher magnification of a showing a highly after exposure to either UVA or UVB (Figure 5c). invasive squamous cell carcinoma with multiple mitotic figures However, much less activation of these upstream indicated by arrows, Â 400. caspases in apoptosis was observed in ULTH cells after either UVA or UVB exposure. These data indicate that Malignant transformation of ULTH cells was esta- ULTH cells acquired resistance to UV-induced apop- blished by the formation of malignant SCC after tosis in both the caspase-8-and -9-related pathways. inoculation under the renal capsule of nude mice. Of 11 mice inoculated with ULTH cells seven (64%) showed formation of SCC, while tumors were not ULTH cells acquired generalized resistance to observed after similar inoculation with passage-matched apoptogenic agents control cells (0 tumors/11 mice). Tumors formed by In addition to UVA and UVB radiation, a number of ULTH cell inoculation were highly malignant with agents known to induce apoptosis at different doses numerous mitotic figures, and aggressively invaded into were also used to treat control and ULTH cells, the surrounding renal parenchyma (Figure 2). including arsenite, a known human skin carcinogen, etoposide, staurosporine and doxorubicin. All of these treatments were found to induce only apoptosis but not ULTH cells exhibit increased keratin 13 expression necrosis at their highest concentration used in this study The overexpression of keratin 13 is thought to be a good (data not shown). Arsenite was much more toxic in marker for skin tumor progression (Warren et al., 1993; control cells than ULTH cells, suggesting that chronic Slaga et al., 1995). The expression of keratin 13 was UVA exposure induced resistance to arsenite much higher in ULTH cells than in control cells (Figure (Figure 6a). Similarly ULTH cells were resistant to 3a and b). These findings suggest a potential role of cytolethality induced by apoptogenic agents including UVA exposure in both skin tumor initiation and etoposide, staurosporine and doxorubicin at different progression. concentrations (Figure 6b–d). As these agents are all

Oncogene Chronic UVA irradiation induces malignant transformation Y-Y He et al 3683 a Dark UVA VBU

Control

5.8 52.8 72.8

ULTH

5.7 23.7 27.2

b UVA 120 UVB 120 ControlNH 100 100 ULTH 80 80 60 60

40 40 ControlNH Cell Viability (%) Cell Viability (%) 20 20 ULTH

0 0 020400102030 UVA (J/cm2) UVB (mJ/cm2) Figure 4 ULTH resisted to apoptosis induced by UVA and UVB. (a) At 18 h after UVA (24 J/cm2) or UVB (20 mJ/cm2) exposure, control and ULTH cells were analysed by flow cytometry after annexin-V and propidium iodide staining. The number in the lower right quadrant represented the corresponding percentage of total apoptosis (%). (b) At 24 h after cells were exposed to different doses of UVA and UVB, cell viability was analysed by MTS assay, bars, s.e.m. Data are expressed as percentage (%)-control (c) and are represented as means (n ¼ 8). known to kill cells by apoptosis it appears ULTH cells conditions (data not shown). It seemed that the control had acquired a generalized resistance to apoptosis. cells were less sensitive to these similar treatments with either wortmannin or LY, or exogenous expression of PTEN or DN-AKT, as compared with ULTH cells. Increased AKT signaling and decreased PTEN expression These results confirm that either increased AKT in ULTH cells signaling or decreased PTEN level is crucial for the As compared with control cells, increased AKT acquired increased survival of ULTH cells after UVA phosphorylation at Ser473 was observed in ULTH cells, treatment. Thus chronic UVA exposure allowed ULTH while no difference was detected in total AKT levels cells to acquire increased AKT signaling and decreased (Figure 7a), indicating that chronic UVA exposure PTEN expression thereby conferring resistance to induced enhanced AKT signaling. Interestingly, a apoptosis. decreased PTEN level was observed in ULTH cells (Figure 7a). Pretreatment with either of two PI3-Kinase/ AKT inhibitors, wortmannin (WMT; 100 and 500 nM) Discussion or LY-294002 (LY; 10 and 20 mM), clearly made ULTH cells sensitive to UVA-induced apoptosis (Figure 7b and UV irradiation is an unequivocal human skin carcino- c). In comparison, wortmannin (100 and 500 nM) and gen. SCC and BCC are the most common forms of LY (10 mM) had no significant effect on the control cells human skin cancer. Although UVA, the major compo- (data not shown), while LY (20 mM) increased apoptosis nent of UV in sunlight, can induce melanoma in fish of the control cells to about 72% after UVA exposure. (Setlow et al., 1993), it is unknown whether UVA When ULTH cells were infected with dominant-negative induces human keratinocyte-based skin cancers such as AKT (DN-AKT) (Fujio et al., 1999) or wild-type PTEN SCC and BCC in human cells. Our study provides clear (Huang and Kontos, 2002) adenovirus vectors, more evidence that UVA, at environmentally relevant doses, ULTH cells underwent apoptosis upon UVA exposure can directly transform human keratinocytes such that than did cells infected with the empty vector (EV) they acquire a highly malignant phenotype. UVA, (Figure 7d and e). In contrast, no significant effect was although the much less energetic form of UV radiation, observed with the control cells under the similar is the dominant form of UV in sunlight. UVA clearly

Oncogene Chronic UVA irradiation induces malignant transformation Y-Y He et al 3684 abDark UVA UVB Dark UVA UVB exposed in the present study was chosen to be 2 C U C U C U C U C U C U environmentally relevant. In this regard, 24 J/cm of UVA equates to about 1 h and 12 min in the midday sun during the summer at latitude 481N (Jeanmougin and Caspase 3 Civatte, 1987). In our laboratory, to obtain 24 J/cm2 of UVA irradiation required approximately 1 h and 20 min. Chronic exposure of keratinocytes to UVA from sunlight is likely a common scenario in humans due to Caspase 7 summertime recreational sun exposure (Autier et al., 1999) or fashionable use of tanning beds (Geller et al., PARP 2002). Excessive exposure to UVA may also occur as the consequence of selection of a sunscreen without UVA FAK protection or improper application during a stay in the sun (Gasparro, 2000; Cokkinides et al., 2001; Geller β-Actin et al., 2002). Thus, the levels of exposure used in the present study to malignantly transform human kerati- c 6 Caspase-8 nocytes equate to plausible UVA doses that could occur

5 in vivo in humans. Caspase-9 Chronic UVA exposure suppressed PTEN and 4 upregulated AKT phosphorylation in ULTH cells. As 3 AKT activation provides a key survival signal for cells ** ** 2 to evade apoptosis, the enhanced AKT activation may play a crucial role in acquired apoptotic resistance and 1

Relative activity (a.u.) perhaps malignant transformation induced by chronic 0 UVA exposure. In the two-stage, chemical models of mouse skin carcinogenesis, one of the key elements is thought to be increased AKT signaling (Segrelles et al., C-UVA C-UVB C-DARK 2002). Specifically, the overexpression of AKT both ULTH-Dark ULTH-UVA ULTH-UVB accelerates tumor formation and contributes to tumor Figure 5 Decreased caspase activation and DNA fragmentation progression (Segrelles et al., 2002). PTEN is one of the in ULTH (U) cells after UVA and UVB exposure as compared with that in control cells (c). (a) At 6h after UVA (24 J/cm 2)or tumor suppressor genes commonly mutated in various UVB (20 mJ/cm2) exposure, cell lysates were analysed by immuno- advanced human cancers including glioblastomas, as blotting. (b) Same as in (a) except that DNA was extracted 18 h well as prostate, breast, endometrial and renal cancers after exposure and electrophoresed on agarose gel. (c) Same as in (Stambolic et al., 1998). Skin-specific PTEN knockout (a) except that caspase-8/9 activity was determined. Data are expressed as fold-control (c) activity and are represented as means mice develop spontaneous skin tumors and show earlier (n ¼ 3); bars, s.e.m.; *significant differences from the activity in onset of chemically induced skin carcinogenesis (Suzuki control cells (Po0.05). et al., 2003; Backman et al., 2004). Adenoviral delivery of PTEN suppresses the malignant phenotype of prostate and ovarian cancer cells (Minaguchi et al., transformed normal human HaCaT cells as evidenced 1999; Davies et al., 2002). Decreased PTEN levels and by anchorage-independent growth, hypersecretion of enhanced AKT signaling may be crucial alterations that MMP’s, altered morphology and overexpression of lead to the malignant transformation of human keratino- keratin 13 and acquired apoptotic resistance. The cytes seen in the present work after chronic UVA strongest evidence that UVA can directly induce exposures and the concurrent resistance to apoptosis. malignant transformation in this human keratinocyte More studies are needed to understand the underlying line is the formation of aggressive SCC upon ULTH cell molecular mechanisms for the roles and alterations of inoculation in the nude mouse. Therefore, the present PTEN and AKT activation. Our results suggest that study strongly indicates that UVA is a potential human addition of AKT inhibitors or PTEN inducers to carcinogen, as it provides direct evidence of UVA- sunscreens could potentially impact skin cancer deve- induced carcinogenic transformation leading to the lopment. capacity for SCC formation in human keratinocytes. Interestingly, our ULTH cells showed cross-resistance There are two major implications of this work. Firstly, to apoptosis induced by UVB and arsenite, two known the malignant transformation of normal human kerati- human skin carcinogens. This UVA-induced resistance nocytes provides compelling evidence that UVA can may enhance skin tumor development when the skin is directly target human keratinocytes, altering them in a concomitantly exposed to sunlight (UVA combined with fashion such that they produce a common type of skin UVB) and arsenic. UVB-induced genotoxicity in apop- cancer (i.e. SCC). Secondly, this work provides a model totic resistant cells may allow damaged cells to survive with clear human relevance to help elucidate the and clonally expand with the potential to form high- molecular events involved in UVA-induced skin tumors. grade malignancies. HaCaT cells chronically treated For transformation, the dose of UVA radiation (24 J/ with arsenite also showed resistance to UVA-induced cm2) to which the human keratinocytes were chronically apoptosis (Pi et al., 2005), implying that UVA and

Oncogene Chronic UVA irradiation induces malignant transformation Y-Y He et al 3685 a 200 b 120 NHControl 160 ULTH 90 120 NHControl ULTH 80 60 Cell Viability (%)

Cell Viability (%) 40 30 0 0 50 100 0 50 100 Arsenite (microM) Etoposide (microM)

c 120 d 120 Control Control 100 100 ULTH ULTH 80 80 60 60

40 40 Cell Viability (%) Cell Viability (%) 20 20

0 0 0 0.5 1 0 2.5 5 Staurosporine (microM) Doxorubicin (microg/ml) Figure 6 ULTH resisted to cell death induced by a number of other treatments. (a) Control and ULTH cells were treated with different concentration of arsenite in serum-free medium. At 24 h after treatment, cell viability was analysed by MTS assay. (b) Same as in (a) except that cells were treated with different concentrations of etoposide. (c) Same as in (a) except that cells were treated with different concentrations of staurosporine. (d) Same as in (a) except that cells were treated with different concentrations of doxorubicin, bars, s.e.m. Data are expressed as percentage (%)-control (c) and are represented as means (n ¼ 8). arsenite may synergize skin tumorigenesis. Indeed, the In summary, chronic UVA exposure induced malig- work of Rossman et al. (2001) shows oral arsenite nant transformation of human HaCaT keratinocytes exposure and UV irradiation act together as cocarcino- in vitro, producing tumors upon inoculation into nude gens in mouse skin. Arsenic is a potent human skin mice (NCr-nu), and generalized resistance to apoptosis. carcinogen and common environmental pollutant, and This is the first report of UVA-induced malignant synergy between UV and arsenic could have important transformation of human cells and is particularly consequences for development of human skin cancers. significant because transformation occurred in a cell In the present work, the response to chronic UVA line analogous to a potential in vivo target site of UVA irradiation was investigated using a human keratinocyte carcinogenesis. In addition, this study provides compel- cell line, HaCaT. In rodents, primary cells are efficiently ling evidence that UVA alone, at environmentally converted into tumorigenic cells by the coexpression of relevant doses, has the potential to be a human skin cooperating oncogenes. However, similar experiments carcinogen. The acquisition of apoptotic resistance in with human cells have consistently failed to yield UVA-transformed keratinocytes may be an important tumorigenic transformants, indicating a fundamental factor in oncogenesis and may allow UVA to synergize difference in the biology of human and rodent cells. with other skin carcinogens. Further comparison Although HaCaT cells are spontaneously immortalized between the transformed and control cells should lead through p53 mutation, evidence and our study suggest to a better understanding of the molecular mechanisms this cell line is not tumorigenic and closely approximates involved in UVA carcinogenesis and, perhaps, mole- normal human keratinocytes in terms of differentiation cular markers for identification of UVA-induced skin (Boukamp et al., 1988). HaCaT cells have provided a malignancies. Studies of the progression of UVA- valuable model system for the study of the molecular induced neoplastic transformation in vitro may also events associated with the malignant transformation of provide significant insights for the development of human epithelial cells (Boukamp et al., 1988) and have chemopreventive strategies for skin cancer. been used extensively as an in vitro model of human epidermal skin to investigate the effects of UVB and UVA (Catani et al., 2001; Phillipson et al., 2002). In Materials and methods addition, UVA has clearly been shown to induce p53 Cell culture mutations in human skin (Agar et al., 2004). Therefore, Human HaCaT keratinocytes, obtained from Professor N the HaCaT cells used in this study provided a model to Fusenig, German Cancer Research Center, Heidelberg, Germany investigate UVA-induced tumor promotion after initial (Boukamp et al., 1988), were maintained in a monolayer p53 mutation by UVA exposure. culture in 95% air/5% CO2 at 371C in Dulbecco modified

Oncogene Chronic UVA irradiation induces malignant transformation Y-Y He et al 3686 a C ULTH b Vehicle WMT 100nM WMT 500nM LY 10 µM LY 20 µM p-AKT473

Dark AKT1

PTEN

β-Actin UVA

c 70 d Control ULTH 60 Vector DN-AKT PTEN

50 Dark 40

Apoptotic Cells (%) 20

10 UVA

0 UVA - + - + - + - + - + - + WMT WMT LY LY 100 500 10 20

e 70 ULTH Control 60

20 Apoptotic Cells (%)

0 UVA - + - + - + - + EV DN- PTEN AKT Figure 7 ULTH cells showed increased AKT signaling. (a) Control cells and ULTH cells were lysed and cell lysates were analysed by immunoblotting with antibodies specific to phosphor-AKT (Ser473), AKT1 and PTEN. (b) ULTH cells were infected with vector, wild-type PTEN or dominant negative AKT (DN-AKT) and the cell lysates were subjected to analysis as in (a, c), ULTH cells were pretreated with or without wortmannin (WMT; 100, 500 nM) or LY-294002 (LY; 10 and 20 mM) and then exposed to UVA (24 J/cm2). Apoptosis was analysed by flow cytometry following staining with Annexin-V-FITC and PI. (d) Cell apoptosis was quantified from (b) and compared with that of control cells. (e) ULTH cells were infected with empty vector, dominant-negative AKT or wild-type PTEN and then exposed to UVA (24 J/cm2). Apoptosis was analysed as in (b). (f) Cell apoptosis was quantified from (d) and compared with that of control cells, bars, s.e.m. Data are expressed as percentage (%) of total cell number and are represented as means (n ¼ 3). Eagle medium (DMEM) supplemented with 10% fetal bovine dark under the same conditions. The temperature was serum (FBS), 31 mg/ml penicillin and 50 mg/ml streptomycin. controlled by a fan to avoid heat development during exposure. After treatment, fresh medium was added and the UVA treatment cells were incubated at 371C. At 3 days after exposure, the cells Cells were exposed to UVA as described previously (He et al., were subcultured. Cells were exposed to UVA (24 J/cm2) once 2003a, b, 2004). Briefly, the medium was removed and cells at a week for 18 weeks. The resulting cells, designated as UVA- Goldilux UV meter equipped with a UVA detector (Oriel long-term-treated-HaCaT (ULTH) cells, were compared to the Instruments, Stratford, CT). Control samples were kept in the parental HaCaT cell line.

Oncogene Chronic UVA irradiation induces malignant transformation Y-Y He et al 3687 Adenovirus infection Anchorage-independent growth assay The next day after the cells were seeded, empty-vector, wild- Colony formation in soft agar was assayed as described type PTEN (kind gifts from Christopher Kontos, Duke previously (Cox and Der, 1994). In a 60-mm tissue culture University Medical Center) or dominant-negative AKT (a dish, 2 Â 104 cells were resuspended in 0.33% Noble agar kind gift from Kenneth Walsh, Boston University) adenovirus (Difco, Kansas City, MO) in Eagle’s MEM with 10% FBS and vectors were added into the cells in a multiplicity of infection layered over 5 ml 0.5% agar in Eagle’s MEM with 10% FBS. (MOI) of 50. After 14 h infection by of incubation with the Cells were grown at 371C in a 5% CO2 atmosphere, and virus, cells were washed and fed with fresh medium. At 24 h colonies with more than eight cells were counted 21 days after after the initiation of infection, cells were irradiated with UVA. seeding.

Tumor formation after inoculation into nude mice Determination of apoptosis To test for malignant transformation, separate groups (n 11) Apoptosis was determined by Annexin-V/propidium iodide ¼ of nude (NCr-nu) mice (NCI-Frederick, Design Center Animal (PI) staining followed by flow cytometry, as described Production Area, Frederick, MD) were inoculated under the previously (He et al., 2003b). DNA fragmentation was renal capsule with 1 106 control or ULTH cells after surgical determined as described previously (He et al., 2003a) as was Â exposure under anesthesia. Tumor formation was assessed caspase activity (He et al., 2003b). weekly. The first tumor was detected after 8 weeks in the ULTH cell-injected mice and the average sacrifice time for this Determination of cell viability group was 133 days. Controls were killed when the last ULTH At 24 h after treatment with UVA, UVB, arsenite (Sigma), injected mouse was sacrificed. Kidneys and any resulting etoposide, staurosporine and doxorubicine (EMD Bios- tumors were embedded in paraffin, sectioned, stained with ciences), cell viability was measured using the MTS assay H&E and analysed by light microscopy. (CellTiter 96Aqueous Proliferation Assay Promega) and monitored at 492 nm using a Spectrafluor plate reader Statistical analyses according to the manufacturer’s instructions. Zymography data were expressed as the mean7s.e. of three independent experiments and were analysed by Student’s t-test. Incidence data (tumorigenicity studies) were analysed Western blotting by Fisher’s exact test. A two-sided value of Po0.05 was After treatment, cells were harvested and lysed with M-PER considered significant in all cases. Mammalian Protein Extraction Reagent (Pierce, Rockford, CA) supplemented with Protease Inhibitor Cocktail (Calbio- chem) and Phosphatase Inhibitor Cocktail (Sigma). Western blotting was performed as described previously (He et al., Abbreviations 2003a). Antibodies used were as follows: caspase-3 and Keratin 13 (Santa Cruz), caspase-7 and PARP (Cell Signaling), AKT, Also called PKB^Protein kinase B; BCC, Basal cell FAK, p-AKT (Ser473), AKT1 and PTEN (Santa Cruz) and carcinoma; DN-AKT, Dominant AKT; EV, Empty vector; b-actin (Sigma). Control (C), Control normal HaCaT cells; MMP-9, Matrix metalloproteinase-9; PI-3K, Phosphatidylinositol 3-kinase; PTEN, Phosphatase and tensin homologue deleted on Confocal microscopy chromosome 10; SCC, Squamous cell carcinoma; UVA, Cells were seeded into 35-mm dishes containing a glass Ultraviolet A (315–400 nm); UVB, Ultraviolet B (280– coverslip-covered 15-mm cutout (MatTek, Ashland, MA) for 315 nm); ULTH, UVA-long-term treated HaCaT cells. live/fixed cell microscopy measurement. ULTH cells containing multiple nuclei were visualized by using an Image-iTt LIVE Plasma Membrane and Nuclear Labeling Kit (Mole- Acknowledgements cular Probes/Invitrogen) according to the manufacturer’s instructions. For fixed cells, the cells were stained with anti- This research was supported by the Intramural Research keratin 13 (Santa Cruz) antibody followed by Alexa Fluor 568 Program of the NIH, NIEHS and NCI. The research has been and Hoechst 33342 dye (He et al., 2004). Cell fluorescence was funded in part with Federal funds from the National Cancer monitored using a Zeiss 510Meta confocal microscope. Institute, National Institutes of Health, under contract # NO1- CO-12400. The content of this publication does not necessarily reflect the views of the Department of Health and Human Zymographic analysis of MMP activity Services, nor does mention of trade names, commercial Cultures at 70–80% confluence were washed twice with PBS, products, or organizations imply endorsement by the US and the medium was changed to serum-free DMEM medium. Government. We thank Dr Jerrold M Ward for assistance with After 48 h, the conditioned medium was collected and the microphotography. The authors also wish to thank Drs Carol MMP activity was analysed as described previously (Achanzar Trempus, Jie Liu and Ann Motten for critical reading of the et al., 2001). manuscript.

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