ATR-Chk1 pathway inhibition promotes apoptosis after UV treatment in primary human keratinocytes: potential basis for the UV protective effects of caffeine

Abstract

New approaches to prevent and reverse UV damage are needed to combat rising sunlight-induced skin cancer rates. Mouse studies have shown that oral or topical caffeine promotes elimination of UV-damaged keratinocytes through apoptosis and markedly inhibits subsequent skin cancer development. This potentially important therapeutic effect has not been studied in human skin cells. Here, we use primary human keratinocytes to examine which of several caffeine effects mediates this process. In these cells, caffeine more than doubled apoptosis after 75 mJ cm(-2) of ultraviolet light B (UVB). Selectively targeting two of caffeine's known effects did not alter UVB-induced apoptosis: inhibition of ataxia-telangiectasia mutated and augmentation of cyclic AMP levels. In contrast, siRNA against ataxia-telangiectasia and Rad3-related (ATR) doubled apoptosis after UV through a p53-independent mechanism. Caffeine did not further augment apoptosis after UVB in cells in which ATR had been specifically depleted, suggesting that a key target of caffeine in this effect is ATR. Inhibition of a central ATR target, checkpoint kinase 1 (Chk1), through siRNA or a new and highly specific inhibitor (PF610666) also augmented UVB-induced apoptosis. These data suggest that a relevant target of caffeine is the ATR-Chk1 pathway and that inhibiting ATR or Chk1 might have promise in preventing or reversing UV damage.

Figure 1. Caffeine augments UVB-induced apoptosis in human keratinocytes

(a–c) Human keratinocytes were treated with vehicle (medium) or 2 mM of caffeine 30 min prior to 75 mJ/cm² of UVB irradiation. (a) Western blots using the indicated antibodies. Cells were harvested 8 h after UVB irradiation. (b) Percentage of sub-2N DNA content measured by flow cytometry. Cells were harvested 24 h after UVB irradiation and stained with propidium iodide. (c) Relative cell death was calculated by comparing percentage of sub-2N DNA content in caffeine/UVB-treated cells with that in medium/UVB-treated cells in each experiment. Average of relative cell death is shown (n=4). Error bar, standard error of the mean. (d) Percentage of sub-2N DNA content measured by flow cytometry. Human keratinocytes were treated with caffeine (Caf) or dibutyryl cyclic AMP (db-cAMP) at the indicated doses 30 min prior to 75 mJ/cm² of UVB irradiation. Cells were harvested 24 h after UVB irradiation and stained with propidium iodide.

Figure 2. ATR siRNA mimics caffeine: inhibition of Chk1 phosphorylation and augmentation of apoptosis after UV

(a) Western blots using the indicated antibodies. Human keratinocytes were treated with vehicle (medium) or 2 mM of caffeine 30 min prior to 75 mJ/cm² of UVB irradiation. Cells were harvested 2 h after UVB irradiation. (b–d) Human keratinocytes were transfected by nucleofection with siRNA against ATR or a non-targeting/scrambled control (scr). Forty-eight hours later, cells were exposed to 75 mJ/cm² of UVB. (b) Western blots using the indicated antibodies. Cells were harvested 1 or 8 h after UVB irradiation as indicated. (c) Percentage of sub-2N DNA content measured by flow cytometry. Cells were treated with vehicle (medium) or 2 mM of caffeine 30 min prior to UVB irradiation. Cells were harvested 24 h after UVB irradiation and stained with propidium iodide. (d) Relative cell death was calculated by comparing percentage of sub-2N DNA content in scr + caffeine/UVB-, ATR + medium/UVB-, or ATR + caffeine/UVB-treated cells with that in scr + medium/UVB-treated cells in each experiment. Average of relative cell death is shown (n=3). Error bar, standard error of the mean.

Figure 3. ATM inhibition does not augment UVB-induced apoptosis

Human keratinocytes were transfected by nucleofection with siRNA against ATR and/or ATM, or a non-targeting/scrambled control (scr). Forty-eight hours later, cells were exposed to 75 mJ/cm² of UVB. (a) Western blots using the indicated antibodies. Cells were harvested 8 h after UVB irradiation. (b) Percentage of sub-2N DNA content measured by flow cytometry. Cells were harvested 24 h after UVB irradiation and stained with propidium iodide. (c) Relative cell death was calculated by comparing percentage of sub-2N DNA content in ATR/UVB-, ATM/UVB-, or ATR+ATM/UVB-treated cells with that in scr/UVB-treated cells in each experiment. Average of relative cell death is shown (n=3). Error bar, standard error of the mean.

Figure 4. Genetic and chemical inhibition of Chk1 augments UVB-induced apoptosis

(a) Human keratinocytes were transfected by nucleofection with siRNA against Chk1 or a non-targeting/scrambled control (scr). Cells were exposed to 75 mJ/cm² of UVB 48 h after transfection. Cells were harvested 24 h after UVB irradiation and stained with propidium iodide. Percentage of sub-2N DNA content was measured by flow cytometry. Relative cell death was calculated by comparing percentage of sub-2N DNA content in Chk1/UVB-treated cells with that in scr/UVB-treated cells in each experiment. Average of relative cell death is shown (n=2). Error bar, standard error of the mean. Western blots using the indicated antibodies are shown in inset. Cells were harvested 48 h after transfection. (b) Front view of the docked pose of PF610666 into Chk1. See text for description of how the compound binds in the kinase catalytic domain. Chemical structure of PF610666 is shown in inset. (c) Western blots using the indicated antibodies. Human keratinocytes were treated with PF610666 at the indicated doses 30 min prior to 75 mJ/cm² of UVB irradiation. Cells were harvested 8 h after UVB irradiation. (d) Relative cell death was calculated by comparing the percentage of cells with sub-2N DNA content in each experiment among cells that received UVB plus no PF610666 to those that received UVB plus 500 or 1000 nM PF610666. Cells were harvested 24 h after UVB irradiation and stained with propidium iodide. Percentage of sub-2N DNA content was measured by flow cytometry. Average of relative cell death is shown (n=3). Error bar, standard error of the mean.

Figure 5. ATR-induced apoptosis is largely p53-independent in human keratinocytes

Human keratinocytes were transfected by nucleofection with siRNA against ATR and/or p53, or a non-targeting/scrambled control (scr). Forty-eight hours later, cells were exposed to 75 mJ/cm² of UVB. (a) Western blots using the indicated antibodies. Cells were harvested 8 h after UVB irradiation. (b) Percentage of sub-2N DNA content measured by flow cytometry. Cells were harvested 24 h after UVB irradiation and stained with propidium iodide. (c) Relative cell death was calculated by comparing percentage of sub-2N DNA content in ATR/UVB-, p53/UVB-, or ATR+p53/UVB-treated cells with that in scr/UVB-treated cells in each experiment. Average of relative cell death is shown (n=3). Error bar, standard error of the mean.