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. 2012 Aug;3(8):854-68.
doi: 10.18632/oncotarget.586. Epub 2012 Aug 20.

Regulation of DNA damage following termination of Hedgehog (HH) survival signaling at the level of the GLI genes in human colon cancer

Akwasi Agyeman  1 , Tapati Mazumdar, Janet A Houghton
Affiliations

Regulation of DNA damage following termination of Hedgehog (HH) survival signaling at the level of the GLI genes in human colon cancer

Akwasi Agyeman et al. Oncotarget. 2012 Aug.

Abstract

Transcriptional regulation of the Hedgehog (HH) signaling response is mediated by GLI genes (GLI1, GLI2) downstream of SMO, that are also activated by oncogenic signaling pathways. We have demonstrated the importance of targeting GLI downstream of SMO in the induction of cell death in human colon carcinoma cells. In HT29 cells inhibition of GLI1/GLI2 by the small molecule inhibitor GANT61 induced DNA double strand breaks (DSBs) and activation of ATM, MDC1 and NBS1; γH2AX and MDC1, NBS1 and MDC1 co-localized in nuclear foci. Early activation of ATM was decreased by 24 hr, when p-NBS1(Ser343), activated by ATM, was significantly reduced in cell extracts. Bound γH2AX was detected in isolated chromatin fractions or nuclei during DNA damage but not during DNA repair. MDC1 was tightly bound to chromatin at 32 hr as cells accumulated in early S-phase prior to becoming subG1, and during DNA repair. Limited binding of NBS1 was detected at all times during DNA damage but was strongly bound during DNA repair. Transient overexpression of NBS1 protected HT29 cells from GANT61-induced cell death, while knockdown of H2AX by H2AXshRNA delayed DNA damage signaling. Data demonstrate following GLI1/GLI2 inhibition: 1) induction of DNA damage in cells that are also resistant to SMO inhibitors, 2) dynamic interactions between γH2AX, MDC1 and NBS1 in single cell nuclei and in isolated chromatin fractions, 3) expression and chromatin binding properties of key mediator proteins that mark DNA damage or DNA repair, and 4) the importance of NBS1 in the DNA damage response mechanism.

Keywords: DNA damage; GANT61; GLI1/GLI2 inhibition; Hedgehog; colon cancer.

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Figures

Figure 1
Figure 1. GLI is the critical target in switching off HH survival signaling
A: HT29 cells were treated with equimolar concentrations of GANT61 (20 μM), cyclopamine (20 μM) or GDC-0449 (20 μM) for 72 hr. The extent of cell death was determined at the end of drug exposure. B: HT29 or GC3/c1 cells were selected for resistance to cyclopamine or GDC-0449, respectively, by stepwise selection in increasing drug concentrations from 20 μM to 100 μM. Sensitivity of resistant cells to cyclopamine (100 μM), GDC-0449 (100 μM) or GANT61 (20 μM) was examined after 72 hr drug exposure. C: HT29 cells overexpressing GLI1cDNA or GLI2cDNA were examined for their sensitivity to GANT61 at increasing concentrations of 10 μM, 15 μM and 20 μM for 48 hr. Percent cell death was determined. Inset: Relative expression of GLI1 and GLI2 in HT29 cells overexpressing GLI1 (left) or GLI2 (right). Cell death was determined by Annexin V FITC/PI staining and FACS analysis. Data represent the mean +/− SD, n=2.
Figure 2
Figure 2. Expression of DNA damage-induced mediator proteins at 4 hr and 24 hr in HT29 cells following inhibition of GLI1/GLI2 by GANT61 (20 μM)
A: Localization of γH2AX, MDC1 and NBS1 nuclear foci in single cells and their co-localization was determined after GANT61 administration. After cells were fixed, permeabilized, treated with the respective antibodies, and stained, four-color image acquisition was performed by confocal microscopy and post-processing analysis of the images was as described in Materials and Methods. B: Western analysis of the expression of total and activated forms (γH2AX, p-ATM, p-MDC1, p-NBS1Ser343) of mediator proteins was examined after treatment of HT29 cells with GANT61.
Figure 3
Figure 3. Model of DNA damage and DNA repair in HT29 cells following GLI1/GLI2 inhibition
A,B: DNA damage: HT29 cells treated with GANT61 (20 μM) for 48 hr or 72 hr continuous exposure undergo DNA damage upstream of cell death. A: DNA repair: Cells exposed to GANT61 for 24 hr (which induces DNA damage) followed by placing in drug-free medium for 24 hr or 48 hr can be rescued during which time they repair damaged DNA. B: Cells exposed to GANT61 (20 μM) for 32 hr followed by placing in drug-free medium for a further 16 hr or 40 hr can only be partially rescued from cell death. Cell death was determined by Annexin V FITC/PI staining and FACS analysis. Data represent the mean +/− SD, n=2.
Figure 4
Figure 4. A: The expression of DNA damage-induced mediator proteins was examined following GLI1/GLI2 inhibition for up to 48 hr during continuous exposure to GANT61 (20 μM; DNA damage) or during DNA repair following 24 hr exposure to GANT61 (20 μM) and 16 hr rescue in drug-free medium
A: Western analysis. β-actin was used as the loading control. B: Binding of mediator proteins to chromatin following chromatin extraction and western analysis from GANT61-treated HT29 cells as described in Materials and Methods.
Figure 5
Figure 5. Localization and co-localization of γH2AX and MDC1 nuclear foci during DNA damage or during DNA repair following GLI1/GLI2 inhibition
A: HT29 cells were treated with GANT61 (20 μM) for up to 48 hr (DNA damage), or B: for 24 hr followed by incubation in drug-free medium for 8 hr, 16 hr or 24 hr. γH2AX and MDC1 nuclear foci were examined by confocal microscopy as described in Materials and Methods and in the legend to Figure 2.
Figure 6
Figure 6. Localization and co-localization of MDC1 and NBS1 nuclear foci during DNA damage or during DNA repair following GLI1/GLI2 inhibition
A: HT29 cells were treated with GANT61 (20 μM) for up to 48 hr (DNA damage), or for B: 24 hr followed by incubation in drug-free medium for 8 hr, 16 hr or 24 hr. MDC1 and NBS1 nuclear foci were examined by confocal microscopy as described in Materials and Methods and in the legend to Figure 2.
Figure 7
Figure 7. Effect of modulation of NBS1, nucleosides or H2AX during inhibition of HH signaling at the level of GLI
A. In transient transfections, HT29 cells were transfected with the retroviral vector pQCXIH alone, or pQCXIH-NBS1 for 24 hr prior to treatment for 48 hr with GANT61 at concentrations of 0 μM, 5 μM, 10 μM, or 20 μM. Cell death was determined. Inset: Total NBS1 and p-NBS1Ser343 expression was analyzed by western analysis. B: HT29 cells were treated with 20 μM concentrations of each of adenosine, guanosine, cytidine and thymidine simultaneously with GANT61 (20 μM) for 72 hr, and the extent of cell death determined. C: HT29 cells stably expressing H2AXshRNA or scrambled shRNA were treated with increasing concentrations of GANT61 for 48 hr at which time the extent of cell death was determined. Inset: H2AX knockdown was confirmed by western analysis. D. HT29 cells stably expressing scrambled shRNA or H2AXshRNA were treated with GANT61 (20 μM) for up to 24 hr, and the expression of γH2AX determined by western analysis. β-actin was used as the loading control.
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