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- Bajak, Edyta Zofia
(författare)
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Genotoxic stress : novel biomarkers and detection methods : uncovering RNAs role in epigenetics of carcinogenesis
- 2005
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- 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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| 2. |
- Edholm, Dan, et al.
(författare)
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Adenovirus vector designed for expression of toxic proteins
- 2001
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Ingår i: Journal of Virology. - 0022-538X .- 1098-5514. ; 75:20, s. 9579-9584
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Tidskriftsartikel (refereegranskat)abstract
- To construct recombinant adenoviruses expressing biologically active proteins may be impossible, or result in a significant reduction in virus yield, if the protein expressed has an inhibitory effect on virus replication or cellular growth. To overcome this problem, we previously designed adenovirus vectors expressing foreign proteins from inducible promoters. However, during our work with a replication-deficient virus expressing the ASF/SF2 splicing factor from a progesterone antagonist-inducible gene cassette, we discovered that ASF/SF2 was expressed at a significant level in the 293 producer cell line, even in the absence of inducer. 293 cells code for adenovirus E1A and E1B proteins and thus support the growth of E1-deficient adenoviruses. Here we show that this background ASF/SF2 expression results from a low level of E1A-mediated transactivation of the basal promoter driving transgene expression. To overcome the problem of leaky expression, we reconstructed a novel gene cassette that combines an inducible promoter and a Lac repressor protein-based block to reduce transcriptional elongation. We show that this novel vector system dramatically reduced background transgene expression and therefore should be useful for the rescue and propagation of high-titer stocks of recombinant adenoviruses expressing toxic proteins.
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| 3. |
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| 4. |
- Mattsson, Ase, et al.
(författare)
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H2AX phosphorylation in A549 cells induced by the bulky and stable DNA adducts of benzo[a]pyrene and dibenzo[a,l]pyrene diol epoxides
- 2009
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Ingår i: Chemico-Biological Interactions. - : Elsevier BV. - 0009-2797 .- 1872-7786. ; 177:1, s. 40-47
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Tidskriftsartikel (refereegranskat)abstract
- Early events in the cellular response to DNA damage, such as double strand breaks, rely on lesion recognition and activation of proteins involved in maintenance of genomic stability. One important component of this process is the phosphorylation of the histone variant H2AX. To investigate factors explaining the variation in carcinogenic potency between different categories of polycyclic aromatic hydrocarbons (PAHs). we have studied the phosphorylation of H2AX (H2AX gamma). A549 cells were exposed to benzo[a]pyrene diol epoxide [(+)-anti-BPDE] (a bay-region PAH) and dibenzo[a,l]pyrene diol epoxide [(-)-anti-DBPDE] (a fjord-region PAH) and H2AX gamma was studied using immunocytochemistry and Western blot. Hydrogen peroxide (H2O2) was used to induce oxidative DNA damage and strand breaks. As showed with single cell gel electrophoresis, neither of the diol epoxides resulted in DNA strand breaks relative to H2O2. Visualisation of H(2)AX gamma formation demonstrated that the proportion of cells exhibiting H2AX gamma staining at 1 h differed between BPDE, 40% followed by a decline, and DBPDE, <10% followed by an increase. With H2O2 treatment, almost all cells demonstrated H(2)AX gamma at 1 h. Western blot analysis of the H2AX gamma formation also showed concentration and time-dependent response patterns. The kinetics of H2AX gamma formation correlated with the previously observed kinetics of elimination of BPDE and DBPDE adducts. Thus, the extent of H2AX gamma formation and persistence was related to both the number of adducts and their structural features.
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