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

Final Report Summary - TMP53COMPMIX (Transcriptional mutagenesis in mammalian cell systems: p53 signaling as a probe of cellular effects)

DNA is exposed to several chemical and physical agents that represent a continuous threat to its integrity and cause DNA lesions. Despite the existence and efficiency of DNA repair systems in cells, some lesions may not be removed, and interfere not only with DNA replication but also with DNA transcription. In fact, RNA polymerase can bypass the site of damage and, due to base misincorporation, lead to the production of mutant mRNA through a process termed transcriptional mutagenesis (TM). If expression of mutant protein through TM results in changes in the cellular phenotype, for example growth advantage, TM could have a role in the etiology of human cancers, contributing to various stages of tumor development. The objectives of the project include (i) how defective transcripts originating from TM alter protein function, (ii) what effects expression of these altered proteins has on cells, and (iii) how the cellular signaling events in response to cellular stress are affected in the presence of TM proteins. In this study, the effects of TM on p53 signaling are investigated using a TM probe vector containing a site-specifically damaged tumor suppressor gene p53. The DNA damage is O6-methylguanine (O6-meG) caused by several exogenous and endogenous compounds and previously shown to cause TM. O6-meG damage was placed in codon 248 of p53 which in case of TM would lead to expression of the dominant negative mutation R248W.

We have developed new expression constructs using gapped-duplex technique containing site-specific O6-meG DNA damage yielding transcripts encoding mutant p53 protein. In addition, the vectors also encode a green fluorescent protein (GFP), acting as an internal control for all experiments. The p53 and GFP genes are each independently controlled by one of two identical tetracycline-responsive minimal CMV promoters arranged in tandem allowing for tight control of expression. The use of a set of co-regulated genes functioning as probes and control marker affords a unique approach for this work. As controls for the cellular response to expression of p53, two vectors encoding wt p53 and mutant R248W has been constructed.

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. To characterize the system transfection efficiency was checked using the co-expressed GFP and the results showed similar level of expression with all three vectors and was stable for up to 72 hours. The extent of TM was determined using a restriction enzyme assay and showed that in transfected H1299 cells, O6-meG caused 14% TM of p53 at codon 248 in DNA repair competent cells and 54% in cells with inhibited DNA repair system. DNA repair was blocked by pre-incubating the cells with the specific methyl transferase inhibitor O6-benzylguanine. Biological endpoints were also investigated and it was found that TM of p53 lead to deregulation of its activity as a transcription factor. This was shown by reduced induction at level of mRNA and/or protein of several p53 regulated genes involved in the regulation of cell cycle progress and apoptosis. Importantly we show that p53 TM deregulates further downstream signaling important for maintain proper cell cycle control. In conclusion, these results show that TM can alter the protein function of p53 and that this can lead to effects on cellular signaling crucial for regulating cell cycle progress and/or apoptosis.

This research proposes to shift the long-standing emphasis from the effects of mutagenesis from DNA replication past DNA lesions, to the role DNA damage plays in RNA synthesis. A very important issue is not only to study the effects of expression of TM proteins but also put this into the context of effects on cellular signaling. With these studies we intend to show that TM not only can have impact on cellular phenotypes by itself but also a significant effect on cellular signaling in a stress situation such as genotoxicity. Furthermore we will characterize the detrimental contribution of TM to cancer and tumor progression. Together the proposed research project constitutes a very innovative and relevant assay system to examine the role of TM in human disease development.