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.
MeSH terms
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Adaptor Proteins, Signal Transducing
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Apoptosis
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Ataxia Telangiectasia Mutated Proteins
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Cell Cycle Proteins / metabolism
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Cell Line, Tumor
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Chromatin / metabolism
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Colonic Neoplasms / genetics*
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Colonic Neoplasms / metabolism*
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DNA Breaks, Double-Stranded / drug effects
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DNA Damage*
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DNA Repair
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DNA-Binding Proteins / metabolism
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Hedgehog Proteins / antagonists & inhibitors
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Hedgehog Proteins / metabolism*
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Histones / genetics
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Histones / metabolism
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Humans
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Kruppel-Like Transcription Factors / antagonists & inhibitors*
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Kruppel-Like Transcription Factors / genetics
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Kruppel-Like Transcription Factors / metabolism
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Nuclear Proteins / antagonists & inhibitors*
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Nuclear Proteins / genetics
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Nuclear Proteins / metabolism
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Protein Serine-Threonine Kinases / metabolism
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Pyridines / pharmacology
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Pyrimidines / pharmacology
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RNA Interference
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RNA, Small Cytoplasmic
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Receptors, G-Protein-Coupled / antagonists & inhibitors
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Receptors, G-Protein-Coupled / genetics
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Receptors, G-Protein-Coupled / metabolism
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Signal Transduction
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Smoothened Receptor
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Trans-Activators / metabolism
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Transcription Factors / antagonists & inhibitors*
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Transcription Factors / genetics
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Transcription Factors / metabolism
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Transcription, Genetic
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Tumor Suppressor Proteins / metabolism
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Zinc Finger Protein GLI1
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Zinc Finger Protein Gli2
Substances
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Adaptor Proteins, Signal Transducing
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Cell Cycle Proteins
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Chromatin
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DNA-Binding Proteins
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GANT 61
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GLI1 protein, human
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GLI2 protein, human
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H2AX protein, human
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Hedgehog Proteins
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Histones
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Kruppel-Like Transcription Factors
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MDC1 protein, human
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NBN protein, human
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Nuclear Proteins
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Pyridines
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Pyrimidines
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RNA, Small Cytoplasmic
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Receptors, G-Protein-Coupled
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SMO protein, human
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Smoothened Receptor
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Trans-Activators
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Transcription Factors
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Tumor Suppressor Proteins
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Zinc Finger Protein GLI1
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Zinc Finger Protein Gli2
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ATM protein, human
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Ataxia Telangiectasia Mutated Proteins
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Protein Serine-Threonine Kinases