Abstract
The GLI genes are transcription factors and in cancers are oncogenes, aberrantly and constitutively activated. GANT61, a specific GLI inhibitor, has induced extensive cytotoxicity in human models of colon cancer. The FOXM1 promoter was determined to be a transcriptional target of GLI1. In HT29 cells, inhibition of GLI1 binding at the GLI consensus sequence by GANT61 led to inhibited binding of Pol II, the pause-release factors DSIF, NELF and p-TEFb. The formation of R-loops (RNA:DNA hybrids, ssDNA), were reduced by GANT61 at the FOXM1 promoter. Pretreatment of HT29 cells with α-amanitin reduced GANT61-induced γH2AX foci. Co-localization of GLI1 and BrdU foci, inhibited by GANT61, indicated GLI1 and DNA replication to be linked. By co-immunoprecipitation and confocal microscopy, GLI1 co-localized with the DNA licensing factors ORC4, CDT1, and MCM2. Significant co-localization of GLI1 and ORC4 was inhibited by GANT61, and enrichment of ORC4 occurred at the GLI binding site in the FOXM1 promoter. CDT1 was found to be a transcription target of GLI1. Overexpression of CDT1 in HT29 and SW480 cells reduced GANT61-induced cell death, gH2AX foci, and cleavage of caspase-3. Data demonstrate involvement of transcription and of DNA replication licensing factors by non-transcriptional and transcriptional mechanisms in the GLI-dependent mechanism of action of GANT61.
Keywords:
DNA licensing; GANT61; GLI; transcription.
MeSH terms
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Antineoplastic Agents / pharmacology*
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Binding Sites
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Cell Cycle Proteins / genetics
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Cell Cycle Proteins / metabolism
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Cell Death / drug effects
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Colonic Neoplasms / drug therapy*
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Colonic Neoplasms / genetics
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Colonic Neoplasms / metabolism
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Colonic Neoplasms / pathology
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DNA Replication / drug effects*
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Dose-Response Relationship, Drug
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Forkhead Box Protein M1 / genetics
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Forkhead Box Protein M1 / metabolism
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Gene Expression Regulation, Neoplastic / drug effects*
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HT29 Cells
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Histones / metabolism
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Humans
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Minichromosome Maintenance Complex Component 2 / genetics
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Minichromosome Maintenance Complex Component 2 / metabolism
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Nuclear Proteins / genetics
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Nuclear Proteins / metabolism
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Origin Recognition Complex / genetics
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Origin Recognition Complex / metabolism
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Promoter Regions, Genetic
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Pyridines / pharmacology*
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Pyrimidines / pharmacology*
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RNA Polymerase II / genetics
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RNA Polymerase II / metabolism
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Transcription Factors / genetics
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Transcription Factors / metabolism
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Transcription, Genetic / drug effects*
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Transcriptional Elongation Factors / genetics
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Transcriptional Elongation Factors / metabolism
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Transfection
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Zinc Finger Protein GLI1 / antagonists & inhibitors*
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Zinc Finger Protein GLI1 / genetics
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Zinc Finger Protein GLI1 / metabolism
Substances
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Antineoplastic Agents
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CDT1 protein, human
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Cell Cycle Proteins
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FOXM1 protein, human
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Forkhead Box Protein M1
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GANT 61
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GLI1 protein, human
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H2AX protein, human
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Histones
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NSMF protein, human
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Nuclear Proteins
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ORC4 protein, human
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Origin Recognition Complex
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Pyridines
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Pyrimidines
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SUPT5H protein, human
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Transcription Factors
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Transcriptional Elongation Factors
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Zinc Finger Protein GLI1
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RNA Polymerase II
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MCM2 protein, human
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Minichromosome Maintenance Complex Component 2