Various chemotherapeutic strategies are designed to induce DNA damage in cancer cells and to affect the DNA synthesis during proliferation.[1] The most toxic DNA lesions caused by genotoxic agents used for cancer treatment are double-strand breaks of DNA (DSBs). DSBs can be formed directly via an anticancer drug. However, DSBs may also arise indirectly as a consequence of other DNA lesions like single-strand breaks (SSBs), base mispairing, or inter-strand crosslinks (ICLs), which compromise the progression of the replication fork.[2, 3] The ability to form these DNA lesions as well as the rate of conversion of these lesions into DSBs resulted in a significant increase in the toxicity of numerous chemotherapeutic drugs.[4]
An example of a chemotherapeutic drug, which activity is based on the induction of DSBs is bleomycin. This anticancer agent is known to cause both DSBs and SSBs. For the activity, the bleomycin requires presence of transition metal ions (e.g., Fe(II) or Cu(I)), one-electron reductant, and oxygen to form a so-called activated bleomycin complex, e.g., bleomycin-Fe(III)-OOH in the presence of Fe ions. After ca. 2 minutes at 4°C, this complex decays to the bleomycin-Fe(III) form.[5, 6] Activated bleomycin removes the hydrogen atom from the deoxyribose moiety in the position C4’ and forms C4’radical. In the presence of oxygen, a series of further chemical rearrangements is followed, which leads directly to the strand break resulting in 3′-phosphoglycolate and 5′-phosphate ends. In the absence of oxygen, the interaction of C4’ radical with an oxidant and water results in 4′-oxidized abasic sites.[7] It was suggested that DSBs are the consequence of randomly and independently arising SSBs. The DSBs:SSBs ratio was reported to be in the range from 1:3 to 1:20.[5] On the other hand, Steighner and Povirk proposed that the double-strand break might be mediated via one bleomycin molecule, which requires reactivation after the initial cut followed by relocation to induce the cleavage of the second strand.[5, 6]
BLM interacts with G-rich segments of DNA via minor groove binding or/and intercalation. The strand breakage results from the attack on the pyrimidine nucleotides (T, C) neighbouring the guanosyl-3-phosphate. Thus, the sequence specificity of bleomycin is referred to 5’-G- pyrimidine (5′-GT and 5′-GC). However, the development of the methodology enabled determining the expanded sequences for cellular, genomic, and purified DNA in which bleomycin cleavage occurs. These sequences vary, which is referred to a different environment that may influence DNA conformation, and thus alter the DNA sequence cleaved by bleomycin [5, 6].
Bleomycin is currently used in chemotherapeutic regimes in combination with other drugs to treat numerous neoplasms. DNA damage caused by bleomycin activity results in extended cell cycle arrest, apoptosis, and mitotic cell death. [5, 6]
Within 2–3 minutes after the DSBs are made, the epigenetic phosphorylation of serine-139 residue in highly conserved motif of histone H2AX, yielding γ-H2AX takes place.[8] The phosphorylation is initiated at the site of the lesion and eventually spreads up to 1–2 Mbp from its origin.[9] This is followed by the recruitment of various proteins (ATM, DNA-PKc, and ATR) responsible for DSB repair. As a result, a megabase chromatin domain is formed. It can be easily visualized under a fluorescence microscope staining with antibody against γ-H2AX.[10] In this way, a γ-H2AX foci consisting of smaller, spatially clustered nano-foci is formed.[11] The number of foci is proportional to the number of DSBs and remains unchanged until the DSB is repaired. Therefore, the evaluation of DSBs location and number may be used as a quantitative biomarker of genomic damage. Indeed, several γ-H2AX-based methods for the quantitative analysis of DSBs including fluorescence microscopy[12, 13] and flow cytometry[14, 15] were successfully developed, also in the research on bleomycin-induced DSBs. For instance, γ-H2AX immunoimaging was applied by Liu and coworkers[16] to investigate the effect of four chemotherapeutic agents, including bleomycin on DNA damage in telomers in the C18-4 spermatogonial cell line. Analysis of fluorescent images revealed that treatment with bleomycin is not associated with telomere shortening. Recently, Bosnjak et al. demonstrated that evaluation of γ-H2AX foci using immunostaining allowed to determine the underlying mechanism of electrochemotherapy combined with olaparib in inhibition of DNA damage in BRCA1 mutated (HCC1937) and non-mutated (HCC1143) cancer cell lines.[12] A procedure for rapid flow cytometry γ-H2AX-based test for screening of genotoxicity of bleomycin against human lymphocytes was also presented.[15] However, despite their huge advantages including its speed (which allows to avoid false-positive results induced by apoptosis), sensitivity, specificity, and accuracy up to 100%[17], suitability for different cell cultures and applicability of automatic methods, the γ-H2AX-based methods still have serious limitations. Most importantly, for a higher number of DSBs, due to the overlapping of fluorescence signal, the counting procedure may lead to significant errors. Furthermore, due to the use of antibody, γ-H2AX fluorescence tests are considered rather expensive, especially compared to the comet assay. Finally, the obtained results are only semiquantitative as the method does not allow to standardize the dose-response curve and its recalibration is often required. All this combined makes γ-H2AX a potential tool for quantification of DNA damage that occurs in response to treatment with various cytotoxic agents. However, for reliable results one should consider the use of complementary methods such as TUNEL or comet assays and/or spectroscopy.
Fourier transform infrared (FTIR) spectroscopy has proved to be a sensitive, non-invasive, and chemically specific tool delivering unique vibrational signature of molecular processes accruing in. In particular, FTIR has been applied in order to distinguish molecular changes associated with DNA damage induced in cells and their nuclei. In addition, the application of FTIR spectroscopy with the use of a synchrotron radiation (SR-FTIR) benefits from high sensitivity, allowing single-cell measurements with good signal-to-noise ratio.[18] Until now, several studies have used FTIR spectroscopy to monitor chemical modifications upon DSBs formation and cellular response to the influence of different damaging factors.[19] The DNA damage induced by fluxes of UVR radiation was investigated in single melanocytes and their extracted nuclei with the use of SR-FTIR and multivariate data analysis. The studies revealed a shift in the asymmetric phosphodiester vibration of DNA backbone from 1236 cm− 1 to 1242 cm− 1 in exposed cells and from 1225 cm− 1 to 1242 cm− 1 in the case of nuclei extracted from irradiated cells. These shifts indicate a partial conformational transition from B-like DNA form to A-like DNA form. Additionally, an increase in the band associated with amide II vibrations and the decrease in the intensity of O-P-O symmetric and asymmetric stretching was observed. These spectral changes suggest arresting cells in G1 phase upon DNA repair mechanisms.[20] Similar changes were observed in SR-FTIR spectra collected from single prostate cells treated with ionizing radiation. The results demonstrate a shift of the asymmetric stretching of the phosphate bands at 1234 cm− 1 toward lower energy, along with the decrease in intensity of the O-P-O symmetric stretching vibrations observed at 1083 cm− 1, indicating chromatin fragmentation due to the relatively high number of DSBs.[21, 22] Dose-dependent spectral changes of bands positions corresponding to DNA phosphate groups and DNA conformation are hallmarks of DNA damage, including SSBs and DSBs formation.[23, 24] The intercellular effects of chemical agents on the phosphate backbone of DNA were also successfully investigated with FTIR spectroscopy. Chan et al. have shown ATR-FTIR spectra collected from breast cancer cells exposed to the intercalating agent, doxorubicin. The significant decrease in O-P-O and C-O motions observed at 1080 cm− 1 and 1050 cm− 1 was linked to DNA disintegration effect.[25] Schirazi et al. discovered that chemotherapeutic drugs such as cisplatin directly interact with the DNA phosphate backbone and phosphodiester groups.[26] The studies presented here clearly indicate that FTIR spectroscopy is an efficient tool in studies of DNA damage and repair mechanisms at the single-cell level.
Despite extensive investigations described in the above overview, both mechanisms of DNA cleavage and the nature of DNA-BLM interactions have not been resolved satisfactorily, requiring further research. In this report, we present a novel approach that combines fluorescence immunostaining of γ-H2AX and SR-FTIR spectroscopy to follow the DSBs lesions induced in malignant cells under the treatment with genotoxic drug, bleomycin. Based on the results of fluorescence immunostaining, we evaluated the number of DSBs in chromatin arisen after the exposure to studied drug in a way allowing for meaningful comparison between different concentrations of the cleaving agent. Moreover, we monitored the molecular changes occurring in BLM treated cells with SR-FTIR spectroscopy at the level of single cell and isolated cellular nuclei. To reveal spectral markers of bleomycin-induced DNA damage, we treated the spectra acquired from individual cells and cellular nuclei with the principal component analysis (PCA) algorithm.