FTIR Microspectroscopy in Studies of DNA Damage Induced by Proton Microbeam in Single PC-3 Cells

In recent years, the Fourier transformed infrared spectroscopy is often applied in studies of biological materials on cellular level. Undoubted advantage of this method is high sensitivity. In presented research the FTIR microspectroscopy was used to analyse the DNA damage in single PC-3 cells (prostate cancer cell line derived from bone metastases) irradiated by counted number of protons. Focused proton microbeam 2 MeV from the Van de Graaff accelerator at the Institute of Nuclear Physics, Polish Academy of Sciences, was used as an irradiation source. Four groups of single cells were irradiated with 1000, 2000, 4000, and 8000 protons per cell, respectively. Following irradiation cells were fixed in 70% ethanol and then analyzed by IR microspectroscopy. Bond analysis of IR spectra served as a base for result analysis. This research has focused on the detection of changes in DNA backbone spectral range (950-1240 $cm^{-1}$), which could be related to damages such as single and double strand breaks, DNA-DNA, and DNA-protein cross links. Switches and differences in intensity of DNA backbone bands (980-1149 $cm^{-1}$, 1151-1350 $cm^{-1}$ - symmetric and asymmetric $PO^{2-}$ stretching vibrations, as well as in 1110 $cm^{-1}$ - symmetric stretching of P-O-C band) were observed. Experimental spectra of irradiated and control cells were compared with simulated spectra generated by HyperChem software. The multivariate statistical methods of principal component analysis and hierarchical cluster analysis (Ward's method) were also performed and are discussed.