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ATR–Chk1 Pathway Inhibition Promotes Apoptosis after UV Treatment in Primary Human Keratinocytes: Potential Basis for the UV Protective Effects of Caffeine 

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Presentation on theme: "ATR–Chk1 Pathway Inhibition Promotes Apoptosis after UV Treatment in Primary Human Keratinocytes: Potential Basis for the UV Protective Effects of Caffeine "— Presentation transcript:

1 ATR–Chk1 Pathway Inhibition Promotes Apoptosis after UV Treatment in Primary Human Keratinocytes: Potential Basis for the UV Protective Effects of Caffeine  Timothy P. Heffernan, Masaoki Kawasumi, Alessandra Blasina, Kenna Anderes, Allan H. Conney, Paul Nghiem  Journal of Investigative Dermatology  Volume 129, Issue 7, Pages (July 2009) DOI: /jid Copyright © 2009 The Society for Investigative Dermatology, Inc Terms and Conditions

2 Figure 1 Caffeine augments UVB-induced apoptosis in human keratinocytes (HKC). (a–c) HKC were treated with vehicle (medium) or 2mM of caffeine 30minutes before 75mJcm-2 of UVB irradiation. (a) Western blots using the indicated antibodies. Cells were harvested 8hours after UVB irradiation. (b) Percentage of sub-2N DNA content measured by flow cytometry. Cells were harvested 24hours 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. HKC were treated with caffeine (Caf) or dibutyryl cyclic AMP (db-cAMP) at the indicated doses 30minutes before 75mJcm-2 of UVB irradiation. Cells were harvested 24hours after UVB irradiation and stained with propidium iodide. Journal of Investigative Dermatology  , DOI: ( /jid ) Copyright © 2009 The Society for Investigative Dermatology, Inc Terms and Conditions

3 Figure 2 ATR siRNA mimics caffeine: inhibition of Chk1 phosphorylation and augmentation of apoptosis after UV treatment. (a) Western blots using the indicated antibodies. Human keratinocytes (HKC) were treated with vehicle (medium) or 2mM of caffeine 30minutes before 75mJcm-2 of UVB irradiation. Cells were harvested 2hours after UVB irradiation. (b–d) HKC were electroporated with siRNA against ATR or a nontargeting/scrambled control (scr). Forty-eight hours later, cells were exposed to 75mJcm-2 of UVB. (b) Western blots using the indicated antibodies. Cells were harvested 1 or 8hours after UVB irradiation as indicated. (c) Percentage of sub-2N DNA content measured by flow cytometry. Cells were treated with vehicle (medium) or 2mM of caffeine 30minutes before UVB irradiation. Cells were harvested 24hours 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. Journal of Investigative Dermatology  , DOI: ( /jid ) Copyright © 2009 The Society for Investigative Dermatology, Inc Terms and Conditions

4 Figure 3 ATM inhibition does not augment UVB-induced apoptosis. HKC were electroporated with siRNA against ATR and/or ATM, or a nontargeting/scrambled control (scr). Forty-eight hours later, cells were exposed to 75mJcm-2 of UVB. (a) Western blots using the indicated antibodies. Cells were harvested 8hours after UVB irradiation. (b) Percentage of sub-2N DNA content measured by flow cytometry. Cells were harvested 24hours 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. Journal of Investigative Dermatology  , DOI: ( /jid ) Copyright © 2009 The Society for Investigative Dermatology, Inc Terms and Conditions

5 Figure 4 Genetic and chemical inhibition of Chk1 augments UVB-induced apoptosis. (a) HKC were electroporated with siRNA against Chk1 or a nontargeting/scrambled control (scr). Cells were exposed to 75mJcm-2 of UVB 48hours after transfection. Cells were harvested 24hours 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 48hours after transfection. (b) Front view of the docked pose of PF into Chk1. See text for description of how the compound binds in the kinase catalytic domain. Chemical structure of PF is shown in inset. (c) Western blots using the indicated antibodies. HKC were treated with PF at the indicated doses 30minutes before 75mJcm-2 of UVB irradiation. Cells were harvested 8hours 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 PF with those that received UVB plus 500 or 1,000nM PF Cells were harvested 24hours 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. Journal of Investigative Dermatology  , DOI: ( /jid ) Copyright © 2009 The Society for Investigative Dermatology, Inc Terms and Conditions

6 Figure 5 ATR-induced apoptosis is largely p53 independent in human keratinocytes (HKC). HKC were electroporated with siRNA against ATR and/or p53, or a nontargeting/scrambled control (scr). Forty-eight hours later, cells were exposed to 75mJcm-2 of UVB. (a) Western blots using the indicated antibodies. Cells were harvested 8hours after UVB irradiation. (b) Percentage of sub-2N DNA content measured by flow cytometry. Cells were harvested 24hours 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. Journal of Investigative Dermatology  , DOI: ( /jid ) Copyright © 2009 The Society for Investigative Dermatology, Inc Terms and Conditions

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