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Transcriptional mutagenesis in mammalian cell systems: p53 signaling as a probe of cellular effects

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Transcriptional mutagenesis in cancer

Chemical and physical agents represent a continuous threat to DNA integrity and cause DNA lesions. Despite the existence of DNA repair systems in cells, some lesions might not be removed and go on to interfere with DNA replication and transcription.

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The process of incorporation of mistakes into the nascent RNA by RNA polymerases during transcription as a result of DNA lesions is referred to as transcriptional mutagenesis (TM). If expression of a mutant protein results in a growth advantage, TM might contribute to various stages of tumour development. The EU-funded project 'Transcriptional mutagenesis in mammalian cell systems: p53 signaling as a probe of cellular effects' (TMP53COMPMIX) studied cellular effects and the biological significance of TM using tumour suppressor protein p53 as a probe. Selection of p53 was based on its role as a guardian of the genome regulating life or death of cells. In this project, the effects of TM on p53 signalling were investigated using a TM probe vector containing a site-specific damaged tumour suppressor gene p53. Researchers developed new expression constructs encoding a mutant p53 protein using the gapped-duplex technique. In addition, the vectors also encoded a green fluorescent protein, acting as an internal control for all experiments. The cellular effects of p53 TM were assessed in human non-small cell lung carcinoma H1299 cells transiently transfected with the probe or the control vectors. The assessment showed that TM of p53 at codon 248 occurred at 14 % in DNA-repair–competent cells and at 54 % in cells with an inhibited DNA repair system. Biological end points were also investigated and it was found that TM of p53 leads to deregulation of its activity as a transcription factor. Importantly, researchers found that p53 TM deregulates further downstream signalling involved in cell cycle control. Project work was intended to shift the emphasis of the effects of mutagenesis from DNA replication in past DNA lesions to the role that DNA damage plays in RNA synthesis. The research resulted in the development of an innovative and relevant assay system to examine the role of TM in human diseases.

Keywords

Transcriptional mutagenesis, cancer, DNA lesions, DNA repair, mutant protein, tumour development, mammalian cell systems, p53, cellular effects, tumour suppressor protein, p53 signalling, cell cycle, RNA synthesis, human diseases

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