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First published online 18 January 2005
doi: 10.1242/jcs.01636

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Mechanism of early biphasic activation of poly(ADP-ribose) polymerase-1 in response to ultraviolet B radiation

Momchil D. Vodenicharov^*, Medini M. Ghodgaonkar, Sabina S. Halappanavar, Rashmi G. Shah and Girish M. Shah

Laboratory for Skin Cancer Research, CHUL Research Center (CHUQ), Faculty of Medicine, Laval University, 2705, Laurier Boulevard, Québec, QC, G1V 4G2, Canada

View larger version (52K): [in a new window]   Fig. 1. Biphasic activation of PARP by UVB. (A) Time course of PARP activation in UVB-irradiated cells. Cells were exposed to 1.6 kJ/m2 UVB and pADPr was visualized by indirect immunofluorescence using monoclonal 10H antibody. Data represent one of five experiments with identical results. (B) Quantification of pADPr signal in UVB-exposed nuclei. Cells were exposed to UVB, as described for A and relative fluorescent intensity was measured in at least 150 nuclei per time point from different fields. Data are expressed as mean±s.d. of relative fluorescence units. (C) Polymer immunoblotting of cells exposed to UVB. Extracts of cells treated with 1.6 kJ/m2 UVB were western blotted for pADPr with polyclonal LP96-10 or for actin as a loading control. One of six replicates is shown.

View larger version (67K): [in a new window]   Fig. 2. The UVB-induced second peak of PARP activation. (A) Time course of oxidant formation in UVB-irradiated cells. Cells exposed to 1.6 kJ/m2 UVB were loaded with DCF-DA prior to harvesting to detect the oxidant load in the cytoplasm by immunofluorescence. (B) The quantification of oxidant signal in UVB-exposed cells. The cytoplasmic fluorescence of UVB-exposed cells described above was quantified in 150 cells per time point. Data are expressed as average fluorescence/cell (mean±s.d.). (C) Suppression of only the second peak of PARP activation by antioxidants. Cells were incubated for 1 hour with 5000 Units/ml catalase or 20 mM N-acetylcysteine prior to exposure to 1.6 kJ/m2 UVB. Cells were then harvested at 15 seconds, 5 minutes or 2 hours. As a control, cells were treated with 100 µM H2O2 for 10 minutes. PARP activation was monitored by immunofluorescent detection of pADPr as described for Fig. 1. Data are representative of three experiments with identical results.

View larger version (59K): [in a new window]   Fig. 3. Monophasic activation of PARP by UVC. (A) Time course of PARP activation in UVC-irradiated cells. Cells were exposed to 0.01 kJ/m2 UVC or 100 µM H2O2 for 10 minutes. The pADPr-modified proteins were detected by immunoblotting with polyclonal LP96-10 or monoclonal 10H antibodies. Blots were reprobed with PARP antibody C-2-10. (B) Immediate activation of PARP at the site of local UVC irradiation. Cells were exposed directly or through a polycarbonate filter to 0.1 kJ/m2 UVC. Samples were fixed using the TCA protocol at times ranging from 15 seconds to 2 hours after irradiation. The signals for T-T (FITC green) or pADPr (Texas red) were detected by indirect immunofluorescence; where the signals co-localized, a yellow colour was present in the merged images. Nuclear DNA was stained with DAPI. Images shown are representative of four experiments with identical results.

View larger version (48K): [in a new window]   Fig. 4. EMSA of UVB-irradiated oligo by PARP. (A) Dose dependent binding of PARP to UVB-irradiated oligo. The unirradiated and UVB-irradiated 50 bp oligo were reacted with 0-36 ng of PARP and resolved on neutral 1.5% agarose-TBE gel. Gels were dried and analysed using an instant imager (top panel). The cpm in each band of oligo-PARP complex from four independent experiments were pooled and shown here as mean±s.d. (bottom panel). (B) Specificity of interaction of PARP with UVB-irradiated oligo. Purified PARP (45 ng) was reacted with 5 ng of one of the three oligos: unirradiated control (lanes 1 and 2), 3.2 kJ/m2 UVB-irradiated (lanes 3 and 4) or UVB + T4 EndoV-treated (lanes 5 and 6). Samples were resolved on agarose gel and analysed, as described above. For immune competition study, anti-PARP C-2-10 (1:100) was added prior to reaction with UVB-irradiated oligo (lane 7). Data are from one of three experiments with identical results.

View larger version (64K): [in a new window]   Fig. 5. PARP activation by UVB-irradiated closed circular plasmid DNA. (A) PARP activation in vitro with UVB-irradiated plasmid. Purified PARP was activated with control, UVB (3.2 kJ/m2)- or UVB + T4 EndoV-treated circular plasmid DNA in the presence of 1.2 µM 32P-NAD. Aliquots were resolved on SDS-PAGE prior to autoradiography. Results of one of four experiments with identical results is shown. (B) Quantification of PARP activation in the in vitro assays. PARP was reacted with three plasmids, as described above, along with activated, i.e. extensively nicked DNA. Reaction was terminated at 30 minutes and 32P-pADPr-modified PARP was TCA precipitated and counted. This experiment was carried out four times with each assay conducted in triplicate and data are presented as mean±s.e.m. of 12 observations. (C) In vitro PARP activation assay with non-isotopic NAD. In vitro PARP activation assay with control and UVB-irradiated plasmid DNA was carried out with 200 µM non-isotopic NAD for 60 minutes. The unmodified PARP and pADPr-modified PARP were detected by immunoblotting with antibodies specific for PARP and pADPr. Data represent four experiments with identical results.

View larger version (40K): [in a new window]   Fig. 6. ChIP assay for in vivo interaction between PARP and T-T. (A) Co-immunoprecipitation of DNA containing T-T photolesions with PARP. ChIP input DNA (top two panels): Chromatin extracts of control or UVB (1.6 and 6.4 kJ/m2 for 15 seconds)-irradiated cells were adjusted prior to ChIP to 100 µg input DNA and an equal aliquot from each extract was assayed for DNA content by ethidium bromide staining and for T-T content by immunodot-blot. ChIP eluate using antibodies to PARP (bottom two panels). DNA was extracted from PARP-ChIP eluates of control and UVB-treated cells, and an equal portion of each eluate was assayed for DNA content by ethidium bromide and for T-T content by immunodot-blot. (B) Recovery of PARP cross-linked to T-T. ChIP was carried out for extracts from control and UVB (6.4 kJ/m2)-irradiated cells with anti-T-T, and proteins eluted in SDS-PAGE sample buffer were immunoblotted for PARP. The 25 and 50 kDa bands are subunits of antibodies used in immunoprecipitation. Apoptotic HL-60 cells were loaded for identification of PARP. Data are from one of five experiments with identical results.