Insulin-like Growth Factor-1-mediated AKT Activation Postpones the Onset of Ultraviolet B-induced Apoptosis, Providing More Time for Cyclobutane Thymine Dimer Removal in Primary Human Keratinocytes*

Insulin-like growth factor-1 (IGF-1) acts as a potent survival factor in numerous cell lines, primarily through activation of the AKT signaling pathway. Although some targets of this pathway have known anti-apoptotic functions, its relationship with the improved survival of cells after exposure to environmental stresses, including UVB, remains largely unclear. We report that in growth factor-deprived keratinocytes, IGF-1 significantly and consistently delayed the onset of UVB-induced apoptosis by >7 h. This delay allowed IGF-1-supplemented keratinocytes to repair significantly more cyclobutane thymine dimers than their growth factor-deprived counterparts. This increase in cyclobutane thymine removal resulted in enhanced survival if the amount of DNA damage was not too high. To increase cell survival after UVB irradiation, IGF-1 supplementation was required only during this initial time period in which extra repair was executed. Finally, we show that IGF-1 mediated this delay in the onset of UVB-induced apoptosis through activation of the AKT signaling pathway. We therefore believe that the AKT signaling pathway increases cell survival after a genotoxic insult such as UVB irradiation not by inhibiting the apoptotic stimulus, but only by postponing the induction of apoptosis, giving the DNA repair mechanism more time to work.

The UVB fraction (290 -320 nm) of terrestrial sunlight is highly carcinogenic (1). UVB photons damage DNA directly, forming specific photolesions, among which cyclobutane pyrimidine dimers and pyrimidine-pyrimidone  photoproducts are most prevalent (2). These lesions give rise to mutations if not efficiently removed by the nucleotide excision repair complex. The response of primary human keratinocytes to UVB irradiation depends highly on the amount of DNA damage. In healthy epidermis, the keratinocyte undergoes cell cycle arrest and repairs its DNA at low UVB doses, whereas high UVB doses lead to apoptosis, which prevents replication of damaged cells and can be seen as the cell's last escape mechanism.
Apoptosis is a tightly regulated energy-dependent process by which the cell triggers its own death. Among the morphological features associated with apoptosis are membrane blebbing, chromatin condensation, DNA fragmentation, and formation of apoptotic bodies (3).
The threshold of UVB irradiation required to induce apoptosis in human keratinocytes is lowered by growth factor deprivation, and this can be prevented by insulin-like growth factor-1 (IGF-1) 1 supplementation of the cell culture medium (4,5). Keratinocytes do not synthesize IGF-1, and the main sources of IGF-1 in human skin are fibroblasts and melanocytes (6). IGF-1 availability to keratinocytes is limited through synthesis of the insulin-like growth factor-binding protein-3 (IGF-BP3), which has been shown to be dependent on p53 transactivation (7). In skin, IGF-BP3 mRNA localizes to the basal proliferative layer of the epidermis (8), further indicating that IGF-1 signaling in skin is a tightly regulated process. Deregulation of this IGF-1 signaling pathway is often a key to the establishment of a transformed phenotype (9). Consistently, constitutive IGF-1 expression in basal keratinocytes of transgenic mice leads to spontaneous tumor promotion (10,11).
As a higher threshold for the induction of apoptosis in the presence of IGF-1 might imply a higher risk to permanent DNA damage, we wished to study the involvement of the IGF-1mediated AKT signaling pathway in the UV response of human keratinocytes. The proto-oncogene c-akt, encoding a 57-kDa serine/threonine protein kinase, is the cellular homolog of the viral oncogene v-akt (12). AKT, also known as protein kinase B, is catalytically activated by phosphorylation at threonine 308 and serine 473 (13). IGF-1, a major survival factor for a variety of cell types, is a potent activator of the AKT signaling pathway (5, 14 -16). Binding of IGF-1 to its receptor triggers activation of phosphatidylinositol 3-kinase (PI3K), enabling PI3K to phosphorylate phosphoinositides at the D3 position of the inositol ring (17). D3 phosphorylated phosphoinositides bind to the pleckstrin homology domains of AKT and PDK1, resulting in their plasma membrane translocation, where PDK1 phosphorylates AKT at Thr 308 (18). The kinase that phosphorylates AKT at Ser 473 , temporarily termed PDK2, has not been iden-tified yet, although possible candidates have been proposed (13, 19 -23). Dually phosphorylated, active AKT is then able to phosphorylate and thereby inactivate the pro-apoptotic protein Bad (24) and the pro-apoptotic forkhead transcription factor FKHRL1 (25) as well as to phosphorylate IB kinase, promoting the expression of anti-apoptotic genes through activation of nuclear factor-B (26,27).
The mechanism by which IGF-1-mediated AKT activation contributes to increased cell survival after exposure to environmental stresses, including UVB, remains largely unclear. In this study, we show that cell survival is enhanced by IGF-1 supplementation only when cells are irradiated with 16 -32 mJ/cm 2 UVB. However, we show that IGF-1-supplemented cells consistently display a prominent delay in the onset of UVB-induced apoptosis over a broader range of UVB doses compared with their growth factor-deprived counterparts. We demonstrate that, although IGF-1 does not directly inhibit the apoptotic stimulus, this postponement of the onset of UVBinduced apoptosis allows more time for DNA repair, weakening the apoptotic stimulus. This results in increased survival of IGF-1-treated cells when they are exposed to a UVB dose of 16 -32 mJ/cm 2 . Finally, we show that IGF-1 mediates this delay in the onset of apoptosis through activation of the AKT signaling pathway.

EXPERIMENTAL PROCEDURES
Cell Culture-Primary human keratinocytes were isolated and pooled from the foreskins of five different donors (Ͻ6 years old) as described (28). Keratinocytes were grown in serum-free medium (keratinocyte serum-free medium; Invitrogen) containing 5 g/ml insulin, 74 ng/ml hydrocortisone, and 6.7 ng/ml triiodo-L-thyronine (T 3 ) supplemented with bovine pituitary extract (BPE; 50 g/ml) and human recombinant epidermal growth factor (EGF; 5 ng/ml). We refer to this medium as "complete medium." Third to fifth passage cells were used in the experiments.
For growth factor-deprived experiments, cells were initially seeded in normal keratinocyte serum-free medium. When they reached 40% confluency, the cells were washed twice with phosphate-buffered saline (PBS) and incubated for 48 h with component-deficient keratinocyte serum-free medium containing no EGF, BPE, insulin, hydrocortisone, or T 3 . This has been shown to suppress the production of autocrine growth factors, although the cells maintain their ability to proliferate, albeit at lower rates (29). Growth factors individually supplied 0.5 h before UVB irradiation included BPE (50 g/ml; Invitrogen), EGF (10 ng/ml; Invitrogen), insulin (10 ng/ml; Sigma), hydrocortisone (74 ng/ml; Sigma), T 3 (6.7 ng/ml; Sigma), and IGF-1 (10 ng/ml; Sigma). Wortmannin was purchased from Sigma, and PD169316 and Z-VADfmk were purchased from Calbiochem.
UVB Irradiation-Prior to UVB irradiation, cells were washed twice with PBS, irradiated through a thin film of PBS, and refed with their own medium. Cells were exposed through the cover of the dish, filtering out residual UVC. The UVB source was a parallel bank of three Philips TL 20W12 tubes with a peak output around 310 nm. Output was measured with an IL700 radiometer (International Light Inc., Newburyport, MA).
Hoechst Staining-Cells were cultivated in four-well Chamberslides. At the appropriate time point, cells were rinsed with ice-cold PBS and fixed with 100% ice-cold methanol for 20 min. Cells were rinsed again with PBS and stained with 3 M Hoechst 33342 (Sigma) for 10 min, rinsed once more, and viewed under a fluorescence microscope.
MTT Survival Assay-Cell viability was assessed using the tetrazolium salt MTT (Sigma). Cells were seeded in 96-well plates. 48 h after UV irradiation, cells were incubated with MTT (1 mg/ml) in PBS for 1 h. Cleavage of MTT by dehydrogenase enzymes of metabolically active cells yields a blue formazan product. The formazan product was dissolved in Me 2 SO, and the absorbance at 550 nm was measured by spectrophotometry.
Western Blotting-At the indicated time points, cells were scraped in lysis buffer (25 mM HEPES, 0.3 mM NaCl, 1.5 mM MgCl 2 , 20 mM ␤-glycerophosphate, 2 mM EDTA, and 2 mM EGTA, pH 7.5) containing 1% Triton X-100, 10% glycerol, 1 mM Na 3 VO 4 , 0.5 mM dithiothreitol, 10 g/ml leupeptin, 10 g/ml aprotinin, and 10 g/ml antipain. Extracts were incubated on ice and spun down at 14,000 rpm for 20 min. Protein concentration was determined using the BCA protein assay reagent (Pierce). 50 -80 g of total protein extract was separated by 10% SDS-PAGE, followed by wet electrotransfer onto Hybond-C Super membrane (Amersham Biosciences). Equal loading of proteins was verified using Ponceau S staining. Membranes were blocked for 1 h at room temperature in Tris-buffered saline containing 0.1% Tween 20 and 5% nonfat dry milk. The membrane was incubated overnight at 4°C with the primary antibody, washed, and incubated for 1 h at room temperature with the peroxidase-conjugated secondary antibody. Protein bands were visualized using enhanced chemiluminescence (ECL, Amersham Biosciences) as recommended by the manufacturer. Monoclonal anti-human poly(ADP-ribose) polymerase (PARP) antibody was purchased from Pharmingen. Polyclonal anti-phospho-AKT(Ser 473 ) and polyclonal anti-AKT antibodies were purchased from Promega. Polyclonal antiphospho-AKT(Thr 308 ), anti-phospho-p38 MAPK(Thr 180 /Tyr 182 ), and anti-p38 MAPK antibodies were purchased from Cell Signaling.
Southwestern Dot-blot Analysis-At the indicated time points, total cellular DNA was extracted using the QIAamp DNA mini-kit (QIAGEN Inc.) as described by the manufacturer. DNA concentration was determined with a spectrophotometer. 300 ng of DNA was loaded onto a nitrocellulose membrane using a dot-blot manifold and fixed for 3 h at 80°C. The membrane was blocked for 0.5 h with Tris-buffered saline containing 0.1% Tween 20 and 5% nonfat dry milk and incubated overnight at 4°C with the primary antibody. The following day, the membrane was rinsed with Tris-buffered saline containing Tween 20, incubated for 90 min with a biotinylated secondary antibody (Amersham Biosciences), rinsed again, and incubated for 1 h with a streptavidin-alkaline phosphatase conjugate (Amersham Biosciences). Following a final rinse, the membrane was incubated with the ECF substrate (Amersham Biosciences), and DNA damage was quantified by blueexcited fluorescence with the STORM imaging system. The primary antibodies used were monoclonal anti-thymine dimer antibody (clone H3; Affitech), directed against cyclobutane thymine dimers, and monoclonal antibody 6-4M2 (a gift from Dr. O. Nikaido), directed against pyrimidine-pyrimidone (6-4) photoproducts.

IGF-1-mediated PI3K Activation Affects Cell Survival
Only at a Limited Dose Range of UVB-In agreement with a previous study (30), we observed that proliferation of keratinocytes is absolutely dependent on growth factors only during the initial spreading and attachment phase. To investigate the effect of IGF-1 on primary human keratinocyte survival, we used the MTT survival assay.
At the UVB dose range of 16 -32 mJ/cm 2 , growth factordeprived cells displayed a significantly lower survival than cells grown in complete medium ( Fig. 1). IGF-1 supplementation 30 min before irradiation with 16 -32 mJ/cm 2 UVB increased survival of growth factor-deprived primary human keratinocytes to the same extent cells grown in complete medium. To examine the involvement of IGF-1-mediated PI3K activation in this process, we used wortmannin, an irreversible inhibitor of the catalytic subunit of PI3K. A 30-min pretreatment with wortmannin (1 M) before IGF-1 supplementation completely blocked the increase in survival by IGF-1 (Fig. 1). No difference in cell survival was observed between growth factor-deprived cells and IGF-1-treated cells or cells grown in complete medium at higher or lower UVB doses. Lower UVB doses (8 mJ/cm 2 ) did not significantly affect cell survival (Fig.  1); and thus, no effect by IGF-1 could be observed. Higher UVB doses (64 mJ/cm 2 ) reduced survival to zero and thus irreversibly led to the death of the entire cell culture whether IGF-1 was added or not ( Fig. 1). The protective effect of IGF-1 supplementation could be observed only at the limited UVB dose range of 16 -32 mJ/cm 2 .
IGF-1-mediated PI3K Activation Postpones the Onset of UVB-induced Apoptosis-To evaluate the effects of IGF-1 supplementation on apoptosis, we first irradiated keratinocytes with a UVB dose of 100 mJ/cm 2 under the different culture conditions. As shown in Fig. 1, IGF-1 was not able to enhance the survival of cells at a UVB dose of 64 mJ/cm 2 and higher. At a UVB dose of 100 mJ/cm 2 , cell survival was reduced to zero, and every primary human keratinocyte eventually underwent apoptosis no matter what culture condition was used. A common feature of apoptosis is caspase activation. Caspase activation leads to cleavage of a number of proteins, among which PARP cleavage by caspase-3 is generally seen as a molecular marker of apoptosis (3). As we wished to investigate why the protective effect of IGF-1 supplementation was lost at this high UVB dose, a time course for PARP cleavage was determined by Western blot analysis. As shown in Fig. 2A, PARP cleavage in IGF-1-treated cells or cells grown in complete medium could first be observed 10 -14 h after UVB irradiation with 100 mJ/ cm 2 . Surprisingly, growth factor-deprived cells already displayed PARP cleavage 3 h after UVB irradiation ( Fig. 2A). The inevitable onset of apoptosis in IGF-1-treated cells or cells grown in complete medium was postponed for Ͼ7 h compared with their growth factor-deprived counterparts.
To investigate whether this discrepancy in time for the onset of apoptosis in IGF-1-supplemented cells versus growth factordeprived cells also plays a role after irradiation with lower UVB doses, we conducted a similar experiment at a UVB dose of 16 mJ/cm 2 , a dose at which we observed a significant difference in cell survival between IGF-1-treated cells and growth factor-deprived cells (Fig. 1). As shown in Fig. 2B, PARP cleavage in growth factor-deprived cells could again first be observed 3-5 h after UVB irradiation, indicating that a portion of the cell culture undergoes apoptosis at this time point. For IGF-1treated cells or cells grown in complete medium, a cleavage product could only be seen 10 -14 h after UVB irradiation (Fig.  2B). Preincubation with IGF-1 thus postpones the induction of apoptosis by Ͼ7 h, independently of the UVB dose used.
Preincubation with 1 M wortmannin prior to IGF-1 supplementation canceled the effect of IGF-1 supplementation and led to an early induction of apoptosis 3 h after irradiation with 100 mJ/cm 2 UVB, similar to growth factor-deprived cells (Fig.  2C). Next, we examined whether the other growth factors present in complete medium (EGF, BPE, T 3 , insulin, and hydrocortisone) were able to delay the onset of UVB-induced PARP cleavage. None of these growth factors delayed the onset of UVB-induced apoptosis (Fig. 2D), demonstrating an IGF-1specific effect.
To further demonstrate the postponement of the onset of apoptosis in IGF-1-treated cells, we looked at a typical morpho-logical marker of the apoptotic process. To monitor chromatin condensation, nuclei were stained with Hoechst fluorescent dye 5 h after irradiation with the lethal UVB dose of 100 mJ/cm 2 . At this time point, chromatin condensation could be observed in growth factor-deprived cultures (Fig. 3b), whereas growing the cells in complete medium or IGF-1 supplementation prior to UVB irradiation clearly rescued the cells from this early induced apoptosis (Fig. 3, a and c). Once again, preincubating the cells with wortmannin prior to IGF-1 supplementation prevented this postponement of the onset of apoptosis (Fig. 3d). None of the other individually supplied growth factors (EGF, BPE, T 3 , insulin, and hydrocortisone) were able to delay the onset of apoptosis (data not shown).
IGF-1-mediated Delay in the Onset of Apoptosis Provides the Cell with Extra Time for Nucleotide Excision Repair-We hypothesize that this postponement of the induction of apoptosis is a key to understanding the protective effect of IGF-1 on the survival of UVB-irradiated keratinocytes. As the ability of the cell to remove DNA damage induced by UVB is known to be the decisive factor for the induction of apoptosis in UVB-irradiated keratinocytes (31), we wished to investigate the effects of IGF-1 on nucleotide excision repair of cyclobutane thymine dimers and (6-4) photoproducts. For this purpose, we used the dot-blot technique to measure cyclobutane thymine dimer and (6-4) photoproduct removal after UVB irradiation. Following UVB irradiation, a dose-dependent increase in the induction of cyclobutane thymine dimers was observed (Fig. 4A). Next, nucleotide excision repair efficiencies were compared for growth factor-deprived cells and cells grown in complete medium by the detection of the appearance and disappearance of cyclobutane thymine dimers under both conditions. Cells were lysed either immediately or 24 h after UVB irradiation, and the remaining amount of cyclobutane thymine dimers was determined and expressed as a percentage of the initial damage. 24 h after UVB irradiation, an increase in the amount of non-repaired cyclobutane thymine dimers was observed with increasing UVB doses for cells grown in complete medium. This correlates with the need to repair growing amounts of damaged DNA when increasing UVB doses are applied. Equally efficient removal of cyclobutane thymine dimers was observed in growth factor-deprived cells and cells grown in complete medium at low UVB doses (4 and 8 mJ/cm 2 ), where no difference in sur- vival after UVB irradiation could be observed (Fig. 4B). However, when irradiated with 16 mJ/cm 2 UVB, a dose at which a significant lower cell survival was observed for growth factordeprived cells, almost no removal of cyclobutane thymine dimers was detected for these cells, in contrast to cells grown in complete medium, which had repaired up to 40% of the cyclobutane thymine dimers (Fig. 4B). Because cyclobutane thymine dimer removal 24 h after irradiation with 16 mJ/cm 2 UVB was significantly lower in growth factor-deprived cells compared with cells grown in complete medium, we examined whether IGF-1 supplementation could prevent this effect. Indeed, cell pretreatment with IGF-1 led to an increase in cyclobutane thymine dimer removal, similar to that in cells grown in complete medium (Fig. 4C). Bearing in mind that induction of apoptosis in growth factor-deprived cells irradiated with 16 mJ/cm 2 UVB could already be observed 3 h after UVB irradiation, we investigated the possibility that the apparent impaired repair observed in these cells might thus be a consequence of the early induction of apoptosis. To determine whether the impaired nucleotide excision repair of cyclobutane thymine dimers is an event upstream or downstream of early induced apoptosis, we incubated growth factor-deprived cells in the presence of the global caspase inhibitor Z-VAD-fmk. Pretreatment with 100 M Z-VAD-fmk 1 h before UVB irradiation inhibited apoptosis completely (data not shown) and led to an increase in cyclobutane thymine dimer removal, again similar to that in cells grown in complete medium (Fig. 4C).
In contrast to cyclobutane thymine dimers, most of the (6-4) photoproducts were removed within the first 3 h after irradiation with 16 mJ/cm 2 UVB (Fig. 4D). In concordance with this fast removal, no significant differences in (6-4) photoproduct removal after irradiation with 16 mJ/cm 2 UVB were observed between growth factor-deprived cells and cells grown in complete medium. IGF-1 Is Required Only during the First 10 h after UVB Irradiation-If IGF-1 enhances survival of cells only by delaying the onset of UVB-induced apoptosis, then IGF-1 should be required only during the first 10 h after UVB irradiation. We therefore conducted another survival experiment in which we varied the time that cells were exposed to IGF-1 after UVB irradiation (Fig. 5). We used a UVB dose of 16 mJ/cm 2 , a dose at which we previously monitored increased survival when cells were treated with IGF-1 for the duration of the assay. Incubating the cells in the presence of IGF-1 until 5 h after UVB irradiation (16 mJ/cm 2 ) led to a minor increase in keratinocyte survival. Incubating the cells in the presence of IGF-1 until 10 h after UVB irradiation (16 mJ/cm 2 ) led to a significant increase in keratinocyte survival, to an extent equal to incubating these cells in the presence of IGF-1 for the whole duration of the assay (48 h) (Fig. 5).
IGF-1-mediated Delay in the Onset of Apoptosis Is Dependent on AKT Activation-One potential downstream effector of PI3K is AKT. A sustained dual phosphorylation, occurring at Thr 308 and Ser 473 , is essential for full activation of AKT (13, 18, 32). The phosphorylation status of this protein was therefore examined by Western blotting. Of the individually supplied growth  A and B, primary human keratinocytes were cultured either in complete medium or in growth factor-deprived medium (ϪGF) with or without IGF-1 supplementation as indicated. The keratinocytes were irradiated with 100 mJ/cm 2 UVB (A) or 16 mJ/cm 2 UVB (B). Cell lysates were collected at the indicated time points. C, primary human keratinocytes were cultured in growth factor-deprived medium. Wortmannin (1 M) and IGF-1 were added 1 h and 30 min, respectively, before irradiation with 100 mJ/cm 2 UVB. Cell lysates were collected at the indicated time points. D, primary human keratinocytes cultured in growth factor-deprived medium were supplemented with the appropriate growth factor 30 min before UVB irradiation (100 mJ/cm 2 ). Cell lysates were collected 5 h after UVB irradiation. The proteins were separated by SDS-PAGE and analyzed by immunoblotting with anti-PARP antiserum. HC, hydrocortisone. factors, only IGF-1 and EGF supplementation led to dual phosphorylation at both Thr 308 and Ser 473 of AKT as shown by Western blot analysis (Fig. 6A). Thr 308 phosphorylation of AKT was sustained for several hours after supplementation of either one of these growth factors (Fig. 6, B and C). However, although EGF-mediated Ser 473 phosphorylation was transient and leveled off within 5-15 min after EGF supplementation, IGF-1-mediated AKT phosphorylation at Ser 473 was sustained for several hours (Fig. 6, B and C). As shown in Fig. 4, phosphorylation at both Thr 308 and Ser 473 was wortmannin-sensitive and therefore PI3K-dependent.
To elucidate a possible involvement of the stress-induced p38 MAPK pathway in the activation of AKT and in the delay of the onset of UVB-induced apoptosis, we incubated our cells with the p38 MAPK inhibitor PD169316 prior to IGF-1 supplementation and UVB irradiation. As shown in Fig. 7A, pretreatment for 30 min with 5 M PD169316 completely abolished the postponement of the onset of UVB-induced apoptosis by IGF-1 supplementation. PD169316 pretreatment of cells grown in complete medium had no effect. Because the structurally re-lated inhibitor SB203580 has been shown to inhibit PDK1 activation and thus AKT activation independently of p38 MAPK at commonly used concentrations (5-10 M) (33), we examined the dose-response effects of PD169316 on the IGF-1mediated delay in the onset of apoptosis and the phosphorylation status of AKT (Fig. 7B). Whereas PD169316 inhibits p38 MAPK when used at nanomolar concentrations (IC 50 ϭ 89 nM) (34), only high concentrations of PD169316 (5-10 M) abrogated the IGF-1-mediated delay in the onset of UVB-induced apoptosis. Consistent with this observation, the prevention of both Ser 473 and Thr 308 phosphorylation of AKT was achieved only using concentrations of the p38 MAPK inhibitor PD169316 higher than 5 M (Fig. 7C). DISCUSSION The aim of this study was to elucidate the mechanism by which IGF-1-mediated AKT activation increases cell survival of keratinocytes after UVB irradiation. It has been shown before that the threshold dose for UVB-induced cell death in human keratinocytes can be increased by IGF-1 supplementation (4).  , n ϭ 3). B, cyclobutane thymine dimer removal 24 h post-irradiation (expressed as a percentage of the initial damage; mean Ϯ S.E., n ϭ 5). C, cyclobutane thymine dimer removal 24 h post-irradiation with 16 mJ/cm 2 UVB. IGF-1 was added 30 min before UVB irradiation (expressed as a percentage of the initial damage; mean Ϯ S.E., n ϭ 3). Growth factor-deprived cells were pretreated with Z-VAD-fmk for 30 min. D, time course of (6-4) photoproduct removal after irradiation with 16 mJ/cm 2 UVB (expressed as a percentage of the initial damage; mean Ϯ S.E., n ϭ 3). ϩGF, cells grown in complete medium; ϪGF, growth factor-deprived cells.
We show that IGF-1 supplementation enhances survival of cells only at a small dose range of UVB (16 -32 mJ/cm 2 ), whereas it offers no protection against irradiation at higher UVB doses. In this study, we clearly demonstrate that IGF-1 signaling does not directly inhibit the apoptotic process, but, no matter what UVB dose used, consistently postpones the induction of apoptosis for Ͼ7 h. We show that the additional cyclobutane thymine dimer removal, executed by the nucleotide excision repair mechanism during this time period, results in the disappearance of the apoptotic stimulus. This results in an increase in cell survival when the DNA damage (and thus the UVB dose) is not too high. This explains why IGF-1-supplemented cells are able to survive somewhat higher UVB doses than growth factor-deprived cells. Finally, we show that this delay in the onset of apoptosis is dependent on the activation of the AKT signaling pathway.
IGF-1 was found to enhance survival of keratinocytes after UVB irradiation only at the limited UVB dose range of 16 -32 mJ/cm 2 . IGF-1 did not significantly affect cell survival when cells were irradiated with lower UVB doses, whereas survival after irradiation at higher UVB doses was reduced to zero no matter what culture condition was used. We report that cells grown in complete medium or pretreated with IGF-1 prior to UVB irradiation and irradiated with a lethal dose of 100 mJ/ cm 2 UVB started to show signs of apoptosis 10 h after UVB irradiation. In contrast to this, growth factor-deprived cells already showed signs of apoptosis 3 h after UVB irradiation. IGF-1 did not protect cells against apoptosis when the cells were irradiated with this lethal UVB dose; nevertheless, a clear postponement of the onset of apoptosis by 7 h was observed in these cells. This difference in time at which apoptosis was induced after UVB irradiation was observed for doses as low as 16 mJ/cm 2 UVB, corresponding to the lowest dose at which we observed a significant increase in cell survival for IGF-1treated cells. This led us to the hypothesis that IGF-1 does not inhibit UVB-induced apoptosis, but merely postpones the onset of UVB-induced apoptosis by Ͼ7 h.
None of the other growth factors present in the complete medium (EGF, BPE, T 3 , insulin, and hydrocortisone) were able to delay the onset of apoptosis in a similar way to IGF-1, demonstrating an IGF-1-specific effect. Only the supraphysiological levels of insulin (5 g/ml), found in keratinocyte complete medium, were also able to delay the onset of UVB-induced apoptosis (data not shown). Insulin not only binds to the insulin receptor, but also has some affinity for the IGF-1 receptor (IGF-1R). These high concentrations of insulin activate the IGF-1R (4). As physiological concentrations of insulin (10 ng/ ml) did not delay UVB-induced apoptosis, the observed delay by supraphysiological levels of insulin is most likely due to IGF-1R activation.
Because the induction of apoptosis after UVB irradiation is highly dependent on the amount of DNA damage induced and the subsequent (in)ability of the cell to repair damaged DNA (31), we investigated whether IGF-1-mediated postponement of the induction of UVB-induced apoptosis affects nucleotide excision repair of UVB-induced lesions. We studied nucleotide excision repair by the detection of UV-induced photolesions, the cyclobutane thymine dimers and (6-4) photoproducts, using the immuno-dot-blot technique with monoclonal antibodies directed against these bulky DNA adducts. When irradiated with UVB doses that cause early induced apoptosis (16 mJ/cm 2 ), hardly any cyclobutane thymine dimer removal was observed in growth factor-deprived cells, in contrast to cells grown in complete medium or cells supplemented with IGF-1 prior to UVB irradiation. However, these growth factor-deprived cells became as proficient as IGF-1-treated cells in nucleotide excision repair of cyclobutane thymine dimers when the apoptotic process itself was inhibited with a global caspase inhibitor (Fig.  4), indicating that a delay in the onset of apoptosis indeed gives the cell more time to repair DNA damage. The observed in- FIG. 6. IGF-1 supplementation leads to sustained dual phosphorylation of AKT. A, primary human keratinocytes cultured in growth factor-deprived medium (ϪGF) were supplemented with the appropriate growth factor for 5 min and then processed as described under "Experimental Procedures." HC, hydrocortisone. B and C, keratinocytes cultured in growth factor-deprived medium were supplemented with IGF-1 or EGF, respectively, and scraped at the appropriate time point. Wortmannin (Wortm.) was added 30 min before growth factor supplementation. The proteins were separated by SDS-PAGE and analyzed by immunoblotting as described under "Experimental Procedures." crease in removal of cyclobutane thymine dimers in IGF-1supplemented cells can be attributed only to the postponement of apoptosis by Ͼ7 h. These data indicate, in contrast to the study of Héron-Milhavet et al. (35), that the intrinsic nucleotide excision repair capacity of the cell is unaffected by IGF-1 supplementation. However, the observed IGF-1-mediated delay in the onset of apoptosis provides the cell with more time to repair cyclobutane thymine dimers. On the contrary, (6-4) photoproducts are almost completely removed within the first hours after UVB irradiation (36). Due to this fast removal, no significant differences in (6-4) photoproduct repair could be observed for growth factor-deprived cells versus cells grown in complete medium.
Lesions are initially removed quickly from the genome, followed by a slower removal of the remaining lesions. This can be attributed to the biphasic nature of nucleotide excision repair (37). Although the global genome has to be screened for the presence of lesions (global genomic repair), actively transcribed genes can be repaired much faster. Lesions present on these genes stall RNA polymerase II (38), leading to a fast recruitment of the nucleotide excision repair complex (39). This has been called transcription-coupled repair or preferential repair. It is the persistence of lesions on these transcribed strands that leads to a high p53 induction and an increase in cells undergoing apoptosis (31,40). Only cyclobutane thymine dimers on transcribed DNA affect survival (41). A delay in the onset of apoptosis signaling provides enough time for the cell to repair cyclobutane thymine dimers on transcribed genes when they are irradiated with UVB doses ranging from 16 to 32 mJ/cm 2 . Although IGF-1 does not contribute to an increased survival at UVB doses higher than 16 -32 mJ/cm 2 , a clear postponement of the onset of apoptosis by Ͼ7 h can readily be observed at these higher UVB doses. This is a strong argument for our hypothesis that not IGF-1 signaling itself, but increased repair of cyclobutane thymine dimers leads to a weakening of the apoptotic stimulus because DNA damage induced by these high UVB doses is too high to be removed efficiently in these extra hours provided to the cell and thus, with or without IGF-1 supplementation, inevitably leads to the induction of apoptosis.
If IGF-1 enhances survival only by providing more time for nucleotide excision repair of cyclobutane thymine dimers, then IGF-1 should be required only during this time period. A survival experiment in which we varied the time that cells were exposed to IGF-1 after UVB irradiation potentiates this hypothesis. In this experiment, we demonstrated that IGF-1 was required only during the first 10 h after UVB irradiation; a longer incubation with IGF-1 did not contribute to an increased survival of keratinocytes after UVB irradiation.
The delay in the onset of UVB-induced apoptosis by IGF-1 was prevented by pretreatment with wortmannin, an irreversible inhibitor of PI3K, implying a role for PI3K. IGF-1 is a potent activator of the AKT pathway through activation of PI3K (13). We subsequently examined the phosphorylation status of AKT after growth factor supplementation. Of all the growth factors tested, only IGF-1 supplementation led to sustained phosphorylation of Thr 308 as well as Ser 473 and therefore to sustained AKT activation (Fig. 6), further supporting the role of AKT in the delay of the onset of UVB-induced apoptosis. On behalf of the AKT phosphorylation at Ser 473 by the hypothetical PDK2 kinase, two different views emerge. One view proposes that by interaction with other enzymes such as protein kinase C-related kinase-2, PDK1 itself could serve as the PDK2 kinase, phosphorylating AKT at Ser 473 (20,23). The second view is supported by p38 MAPK inhibitor studies and suggests that the PDK2 kinase is a member of the p38 MAPK pathway (13,21,22). Both MAPK-activated protein kinase-2 FIG. 7. p38 MAPK inhibitor PD169316 prevents the AKT-mediated delay in UVB-induced apoptosis. A and B, primary human keratinocytes were cultured with or without growth factors as described under "Experimental Procedures," and growth factor-deprived cells were supplemented with IGF-1 30 min before irradiation. Cell lysates were collected 5 h after UVB irradiation (100 mJ/cm 2 ). C, the phosphorylation status of AKT was examined by collecting cell lysates 5 min after IGF-1 supplementation. The proteins were separated by SDS-PAGE and analyzed by immunoblotting as described under "Experimental Procedures." ϩGF, cells grown in complete medium; ϪGF, growth factor-deprived cells.
(MAPKAP2) and mitogen-and stress-activated protein kinase-1 (MSK1), downstream targets of p38 MAPK, are able to phosphorylate AKT at Ser 473 in vitro (13,21). We used the pyridinylimidazole inhibitor PD169316 to examine a possible involvement of p38 MAPK in the activation of AKT and the delay in the onset of UVB-induced apoptosis. PD169316 not only blocked Ser 473 phosphorylation, but prevented AKT phosphorylation at Thr 308 as well when used at high concentrations (5-10 M). Lali et al. (33) reported that the structurally related p38 MAPK inhibitor SB203580 is able to inhibit PDK1-induced AKT activation independently of p38 MAPK activation at a commonly used concentration range (5-10 M), resulting not only in inhibition of Ser 473 phosphorylation, but also in inhibition of Thr 308 phosphorylation of AKT. Therefore, a dose-response experiment was conducted to examine the specificity of the PD169316 inhibitor. Lower concentrations of PD169316, which still inhibit p38 MAPK efficiently (34), had no effect on the phosphorylation status of AKT, suggesting p38 MAPKindependent inhibition of AKT. Strikingly, these conditions also led to a strong inhibition of the IGF-1-mediated delay in the onset of UVB-induced apoptosis (Fig. 7B), supporting the involvement of AKT in the postponement of UVB-induced apoptosis. As shown in Fig. 7A, PD169316 pretreatment of cells grown in complete medium (which contained supraphysiological levels of insulin) did not lead to early induced apoptosis. We hypothesize that this short preincubation with PD169316 is not enough to cancel the effects of prolonged IGF-1R signaling. As a consequence, it is plausible that the UVB response of keratinocytes grown in cell culture medium containing high concentrations of insulin does not accurately reflect the in vivo situation, as prolonged IGF-1R activation might lead to sustained activation of the PI3K/AKT pathway.
The IGF-1R mediates a variety of tumor-promoting effects (10,42), among which delaying the induction of apoptosis probably is the most important. It is evident from our results that when global genomic repair is at stake, sustained activation of the IGF-1/AKT pathway could lead to an increased rate of permanent DNA damage incorporation caused by repeated UVB exposure. In contrast to "in vitro" conditions, where high concentrations of insulin are sometimes used, IGF-1 signaling is restricted in healthy epidermis. The main source of IGF-1 for epidermal keratinocytes (which do not synthesize IGF-1 themselves) is fibroblasts and melanocytes (6). This IGF-1 availability is limited through IGF-BP3 and repressed IGF-1R expression. Both up-regulation of IGF-BP3 and repression of IGF-1R expression are dependent on functional wild-type p53 (7,(43)(44)(45). However, p53 is often mutated in non-melanoma skin cancer. Indeed, Ͼ90% of squamous cell carcinomas and Ͼ50% of basal cell carcinomas contain p53 mutations. Moreover, p53 mutations are also observed in actinic keratoses, indicating that p53 mutation is not only a frequent event, but also an early event, in skin carcinogenesis. The corresponding p53 mutation hot spots in these premalignant and malignant keratinocytes contain predominantly C-T transitions at dipyrimidine sites, a typical signature indicating that UVB light is the mutagen (1,2,46). Wild-type p53 is also required for efficient global genomic repair (41,47,48). We believe that repeated UVB exposure, causing p53 mutation, not only distorts global genomic repair, but also up-regulates IGF-1 signaling in skin because a deregulated IGF-1 signaling pathway is linked to the mutation status of p53 (45,49). Based on our results, this may lead to an increase in the incorporation of permanent DNA damage in the genome. This would at least partially explain why deregulated IGF-1 signaling accelerates tumor promotion. As targeting the IGF-1 signaling pathway might potentially slow down or halt tumor promotion, further research should be conducted on the importance of IGF-1 signaling in healthy and cancerous epidermal keratinocytes.