Research Of Cell Acetylation And Its Effect On Radiation Damage Through FTIR Spectroscopy

The occurrence and development of cancer is closely associated with epigenetics. and histone acetylation is one of important epigenetic modifications. Histone in most of tumor cells shows low acetylation state, however, histone deacetylase inhibitor (HDACi) can correct abnormal acetylation status, induce cell cycle arrest and apoptosis, change the malignant phenotype, and therefore may achieve the purpose of targeted anti-tumor therapy. Trichostatin A(TSA) is one of the representatives of the drugs of histone deacetylase inhibitors, which can inhibit histone deacetylase, increase the acetylation level of histone and nonhistone in cell, induce transformed cell differentiation or apoptosis, and so it can be utilized to study the impact of acetylation on tumor cells.Fourier transform infrared (FTIR) spectroscopy is a powerful analytical tool which can detect nondestructively and quantitatively biological samples without biotagging and biolabeling. Compared with the traditional biological research methods, FTIR spectroscopy technology has multiple advantages, including finger-print characteristics, rapid analysis, high resolution, good repeatability, and nondestructive measurement, therefore, it has been widely used in the research of biological processes. FTIR spectroscopy parameters such as spectral profile, intensity and frequency can be changed accordingly when the compositions and structures of large molecules such as proteins, nucleic acids, lipids and sugars changes, and the changes of these compositions and structures can be probed and analyzed by FTIR spectroscopy.This thesis work applied FTIR spectroscopy to study the changes in cells after treated with TSA, quantitatively characterize the acetylation level and DNA conformation changes in cells, and furthermore predict the radiosensitivity of the cells with different acetylation levels. Hence, this work may shed new light on the histone deacetylase inhibitor research.From the study we have the following major new results and conclusions:1) We employed FTIR spectroscopy to probe the chemical and structural changes of proteins in the TSA treated cells, and with the aid of fluorescent microscopy, we could scrutinize the time-dependent and dose effects on the acetylation level promoted by TSA. Our results showed that TSA caused an elevated level of cellular acetylation and conformational/structural changes of proteins in the cells, and a higher dosage ofTSA caused a higher percent of α-helix structure accompanied by an increment of acetylation level in both histones and cytoskeleton proteins. This work therefore not only validates the usefulness of FTIR spectroscopy in the quantitative assessment of cellular acetylation but also may open an avenue to the in-depth investigation of the effect of HDAC inhibitor drugs such as TSA on cancer cells.2) We applied FTIR spectroscopy to monitor the DNA signals in cells as the histone acetylation was regulated by TSA. Nucleosome is the fundamental unit of chromatin assembly, which is made up of DNA and histones, and acetylation of histones perturbs the interaction of DNA and histones and thus affects the chromatin conformation and function. However, whether or how acetylation induces DNA conformation changes is still elusiveln this work, our results unambiguously demonstrated the significant transformation of B-DNA to Z-DNA upon histone acetylation in the TSA treated HeLa cells. This is the first report providing the explicit experimental evidence for such a B-Z transformation of DNA in the epigenetic states of cells, which adds research content for epigenetics.3) Since FTIR spectroscopy can be employed to characterize the acetylation level of cells and acetylation is closely related to radiosensitivity of tumor cells, we thus furthermore attempted to predict the radiation damage of cells based on FTIR spectroscopy measurement and analysis. For this purpose, we applied both FTIR and fluorescence imaging methods to analyze the acetylated cells. The experiment was carried our with 25 nM and 200 nM TSA treating cells for 24 h, and the FTIR spectroscopy analysis showed that the FTIR intensity ratio of methyl to methylene stretching vibration can be evaluated to characterize the acetylation level changes in cell. According to the results of FTIR spectroscopy characterization, the damage caused by radiation in acetylated cells can indeed be predicted by the ratio of methyl and methylene intensity which is positively correlated with cellular radiosensitivity. Therefore, this work demonstrates that FTIR spectroscopy can be very useful for the study of relationship between epigenetics and radiation bio-effects.