Genotoxic stress: novel biomarkers and detection methods : Uncovering RNAs role in epigenetics of carcinogenesis
Sammanfattning: Background: Cancer is a complex disease that is caused by the interplay of multiple genes and diverse environmental factors. As such, there has been continual debate as to which component of this complicated process towards development of neoplasia plays the most important role. The central dogma has, hitherto, been orientated towards DNA damage and its sequel as the major contributor to chemical/environmental carcinogenesis. More recently however, an important role for aberrant RNA structure and function in controlling cellular functions during normal and pathological conditions has emerged. Objectives: The overall goal of the present investigation was to further characterize the molecular mechanisms and effects of different types of genotoxic stress on the biology and function of cells exposed and cultured in vitro. In particular, we were intrigued and interested in studying novel molecular mechanisms underlying the relative carcinogenicity potential of bulky DNA adduct-forming compounds. Methodology: In this multi-dimensional investigation we have applied a broad spectrum of methods within fields of biochemistry, molecular and cellular biology, transcriptomics, biostatistics and bioinformatics. Genotoxic compounds used include pro-oxidants (H2O2, KBrO3, diamide) and diol epoxides (DEs), which are reactive metabolites of PAHs (the bayregion BPDE, the fjord-region DBPDE). Results: Data from experiments where cells were exposed to diamide or H2O2 indicated that levels of induced protein S-glutathionylation [Paper I] correlate well to selective stress gene expression, many of which were related to the induction of nucleic acid damage recognition/repair. Moreover this adaptation was translated to a functional phenotype in form of increased resistance in subsequent exposures to heat shock or oxidative stress. Based on the dramatic alternations (Affymetrix) in steady-state level of mRNAs in cells exposed to the most carcinogenic compound (DBPDE) we hypothesise that RNA and their modifications may function as central components of the epigenome [Paper IV]. A number of novel gene targets were identified, the functions of which have not previously been associated with response to genotoxic stress. These results support the notion that the analysis of alternations in gene expression patterns may provide a useful surrogate biomarker in identification of genotoxic agents with high carcinogenic potential. In parallel, we were interested to examine the relative propensity of RNA and DNA to sustain damage in cells undergoing oxidative stress. In Paper III we clearly show that RNA was far more sensitive in sustaining oxidative damage on guanine, than DNA. Due to the fact that detection of oxidised DNA/RNA in Paper III requires rather complicated and expensive mass spectrometric methods, we have also attempted to develop simpler methodologies for the differential detection/visualisation of modified RNA and DNA pools in intact cells [Paper V]. The data clearly show that combinations of avidin or neutravidin staining, combined with or without RNase or DNase treatments, can be used to visualise oxidative modification to DNA (nuclear and mitochondrial) and RNA in cells undergoing oxidative stress. Small differences in chemical structure of studied DEs have been shown to have pronounced effects on their conformation, target binding preferences and removal efficiency from DNA [Paper II] which, in turn, mark distinctive biochemical and biological effects on cellular biology [Papers II, IV and VI]. DEs and KBrO3 showed distinct, chromatin-based responses in DNA damage signalling pathways, as measure of induction of H2AX or H2B phosphorylation [Paper VI]. Conclusion: The perception of DNA and DNA-driven carcinogenesis must be updated by modern epigenetics, which includes the changes in structure, function and distribution of RNAs. And these re-discovered carriers, executors and directors of genetic information have to be recognised as independent components of epigenome and placed more centrally in efforts to understand mechanisms of chemically-derived and perhaps spontaneously derived carcinogenesis. It is hoped that the potential biomarkers, analytical methods and mechanistic results presented in this thesis provide some stimulus to further study.
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