Justyna Miszczyk

Comparative endothelial profiling of doxorubicin and daunorubicin in cultured endothelial cells.

Although anthracycline antibiotics have been successfully used for nearly half a century in the treatment of various malignancies, their use is limited by their cardiac and vascular toxicities, and the mechanisms of these toxicities are still not entirely clear. Herein, we comprehensively characterized cytotoxic effects of two structurally related anthracyclines, doxorubicin and daunorubicin. In nanomolar concentrations, both drugs induced DNA damage and increased nuclear area that were associated with their accumulation in the nucleus (doxorubicin ⩾50 nM and daunorubicin ⩾25 nM) as evidence by Raman microspectroscopy at 3820-4245 cm(-1). At low micromolar concentrations, doxorubicin (⩾5 μM) and daunorubicin (⩾1 μM) increased the generation of reactive oxygen species, decreased intracellular reduced glutathione, induced an alteration in endothelial elasticity and caused a reorganization of the F-actin cytoskeleton. In isolated mouse aortic rings, doxorubicin (⩾50 μM) was less potent than daunorubicin (⩾5 μM) in impairing the endothelium-dependent response. In summary, using a comprehensive endothelial profiling approach, we demonstrated clear-cut differences in the potencies to induce endotheliotoxic responses for two structurally similar chemotherapeutics, at a nuclear, cytosolic and membrane levels. Furthermore, our results suggest that the differences in the endothelial toxicities of doxorubicin and daunorubicin are linked to differences in their nuclear accumulation and the DNA damage-triggered response of the endothelium.

Do protons and X-rays induce cell-killing in human peripheral blood lymphocytes by different mechanisms?

Purpose Significant progress has been made in the technological and physical aspects of dose delivery and distribution in proton therapy. However, mode of cell killing induced by protons is less understood in comparison with X-rays. The purpose of this study is to see if there is any difference in the mode of cell-killing, induced by protons and X-rays in an ex vivo human peripheral blood lymphocyte (HPBL) model. Materials and methods HPBL were irradiated with 60 MeV proton beam or 250-kVp X-rays in the dose range of 0.3–4.0 Gy. Frequency of apoptotic and necrotic cells was determined by the Fluorescein (FITC)-Annexin V labelling procedure, 1 and 4 h after irradiation. Chip-based DNA Ladder Assay was used to confirm radiation-induced apoptosis and necrosis. Chip-based DNA Ladder Assay was used to confirm radiation-induced apoptosis. Results Ex vivo irradiation of HPBL with proton beams of 60 MeV or 250 kVp X-rays resulted in apoptotic as well as necrotic modes of cell-killing, which were evident at both 1 and 4 h after irradiation in the whole dose and time range. Generally, our results indicated that protons cause relatively higher yields of cell death that appears to be necrosis compared to X-rays. The analysis also demonstrates that radiation type and dose play a critical role in mode of cell-killing. Conclusion Obtained results suggest that X-rays and protons induce cell-killing by different modes. Such differences in cell-killing modes may have implications on the potential of a given therapeutic modality to cause immune modulation via programmed cell death (X-rays) or necrotic cell death (proton therapy). These studies point towards exploring for gene expression biomarkers related necrosis or apoptosis to predict immune response after proton therapy.

Response of human lymphocytes to proton radiation of 60 MeV compared to 250 kV X-rays by the cytokinesis-block micronucleus assay

Particle radiotherapy such as protons provides a new promising treatment modality to cancer. However, studies on its efficacy and risks are relatively sparse. Using the cytokinesis-blocked micronucleus assay, we characterized response of human peripheral blood lymphocytes, obtained from health donors irradiated in vitro in the dose range: 0-4. 0 Gy, to therapeutic proton radiation of 60 MeV from AIC-144 isochronous cyclotron, by studying nuclear division index and DNA damage and compared them with X-rays. Peripheral blood lymphocytes show decreased ability to proliferate with increasing radiation doses for both radiation types, however, in contrast to X-rays, irradiation with protons resulted in a higher proliferation index at lower doses of 0.75 and 1.0 Gy. Protons are more effective in producing MN at doses above 1.75 Gy compared to X-rays. Dose-response curves for micronucleus incidence can be best described by a cubic model for protons, while for X-rays the response was linear. The differences in the energy spectrum and intracellular distribution of energy between radiation types are also apparent at the intracellular distribution of cytogenetic damage as seen by the distribution of various numbers of micronuclei in binucleated cells. Our studies, although preliminary, further contribute to the understanding of the mechanistic differences in the response of HPBL in terms of cellular proliferation and cytogenetic damage induced by protons and X-rays as well as intra-cellular distribution of energy and thus radiobiological effectiveness.

Therapeutic proton irradiation results in apoptosis and caspase-3 activation in human peripheral blood lymphocytes

Background: Proton therapy is effective in controlling many cancer types, allowing to spare the normal tissues and limiting the risk of adverse effects. However, cellular and molecular mechanisms by which protons induce cell death are still not fully understood. The purpose of this study was to investigate apoptotic mode of cell killing in human peripheral blood lymphocytes (HPBL) exposed ex vivo to 60 MeV proton beam radiation. Methods: HPBL obtained from 5 healthy donors were irradiated ex vivo with 60 MeV protons in spread out Bragg peak (SOBP), in the dose range 0.3–4.0 Gy. The average proton dose rate was 0.075 Gy/s. After irradiation, HPBL were stained with Annexin V fluorescein isothiocyanate (V-FITC) at different time-points post-radiation exposure: 1, 4 and 24 hours. To assess caspase-3 activation following irradiation with protons, caspase-3 DEVD-R1100 Fluorometric assay was used. Results: The apoptotic cell fraction stained with Annexin V-FITC, analysed after 1 and 4 h post proton-irradiation showed a dose-response increase in cell death. After 24 h post radiation exposure, the apoptotic fraction of cells represented a similar trend as in 1 and 4 h but less pronounced. Caspase-3 activation measured after 6 h of proton irradiation was significantly higher (P Conclusions: The data clearly demonstrates that 60 MeV therapeutic proton beam induced cell-killing in HPBL via apoptotic cell mode of death appears to be mediated by caspase-3 activation.

Biological effects and inter-individual variability in peripheral blood lymphocytes of healthy donors exposed to 60 MeV proton radiotherapeutic beam

Abstract Purpose: The aim of our study was to investigate the amount of initial DNA damage and cellular repair capacity of human peripheral blood lymphocytes exposed to the therapeutic proton beam and compare it to X-rays. Materials and methods: Lymphocytes from 10 healthy donors were irradiated in the Spread Out Bragg Peak of the 60 MeV proton beam or, as a reference, exposed to 250 kV X-rays. DNA damage level was assessed using the alkaline version of the comet assay method. For both sources of radiation, dose–DNA damage response (0–4 Gy) and DNA repair kinetics (0–120 min) were estimated. The observed DNA damage was then used to calculate the relative biological effectiveness (RBE) of the proton beam in comparison to that of X-rays. Results: Dose–response relationships for the DNA damage level showed linear dependence for both proton beam and X-rays (R2 = 0.995 for protons and R2 = 0.993 for X-rays). Within the dose range of 1–4 Gy, protons were significantly more effective in inducing DNA damage than were X-rays (p < .05). The average RBE, calculated from the proton and X-ray doses required for the iso-effective, internally standardized tail DNA parameter (sT-DNA) was 1.28 ± 0.57. Similar half-life time of residual damage and repair efficiency of induced DNA damage for both radiation types were observed. In the X-irradiated group, significant inter-individual differences were observed. Conclusions: Proton therapy was more effective at high radiation doses. However, DNA damage repair mechanism after proton irradiation seems to differ from that following X-rays.

Evaluation of the premature chromosome condensation scoring protocol after proton and X-ray irradiation of human peripheral blood lymphocytes at high doses range

Abstract Purpose of the study: One of the main difficulties in radiation dose assessment is cells inability to reach mitosis after exposure to acute radiation. Premature chromosome condensation (PCC) has become an important method used in biological dosimetry in case of exposure to high doses. Various ways to induce PCC including mitotic cells fusion, chemical stimulation with calyculin A or okadaic acid give wide spectrum of application. The main goal of this study was to evaluate the utility of drug-induced PCC scoring procedure by testing 2 experimental modes where 150 and 75 G2/M-PCC phase cells were analyzed after exposure to high dose proton and X-ray radiation. Another aim is to determine the differences in cellular response induced by proton and photon radiation using a HPBL in vitro model as a further extension of our previous studies involving doses up to 4.0 Gy. Materials and methods: Total body exposure was simulated by irradiating whole blood collected from a healthy donor. Whole blood samples were exposed to two radiation types: 60 MeV protons and 250 kVp X-rays in the dose range of 5.0–20.0 Gy, the dose rate for protons was 0.075 and 0.15 Gy/s for X-rays. Post 48 h of human peripheral blood lymphocytes (HPBL) culture, calyculin A was added. After Giemsa staining, chromosome spreads were photographed and manually analyzed by scorers in the G2/M-PCC phase. In order to check the consistency of obtained results all scorers followed identical scoring criteria. Additionally, PCC index kinetics was evaluated for first 500 cells scored. Conclusions: Here we provide a different method of results analysis. Presented dose-response curves were obtained by calculating the value of counted excess chromosome fragments. The results indicated that obtained dose estimates as adequate in the high dose range till 18.0 Gy for both studied radiation types, giving an opportunity to further improve PCC assay procedure and shorten the analysis time i.e. in case of partial-body exposure. Moreover, the study presents preliminary results of HPBL cellular response after proton irradiation at high doses range showing differences of PCC index kinetics for different cell classes and cell distribution.

Aberrations Involving Chromosome 1 as a Possible Predictor of Odds Ratio for Colon Cancer--Results from the Krakow Case-Control Study.

Background There is still an open question how to predict colorectal cancer risk before any morphological changes appear in the colon. Objective The purpose was to investigate aberrations in chromosomes 1, 2 and 4 in peripheral blood lymphocytes analyzed by fluorescence in situ hybridization technique as a tool to assess the likelihood of colorectal cancer. Methods A hospital-based case-control study included 20 colon cancer patients and 18 hospital-based controls. Information about potential covariates was collected by interview. The frequency of stable and unstable chromosome aberrations in chromosome 1, 2 and 4 was assessed by fluorescence in situ hybridization technique. Results Colorectal cancer patients, as compared to controls, had a relatively higher frequency of chromosome 1 translocations (median: 3.5 versus 1.0 /1000 cells, p = 0.006), stable aberrations (3.8 versus 1.0 /1000 cells, p = 0.007) and total aberrations (p = 0.009). There were no differences observed for chromosomes 2 and 4. Our results showed an increase in the odds of having colon cancer by about 50–80% associated with an increase by 1/1000 cells in the number of chromosome 1 aberrations. Conclusions The results revealed that the frequency of chromosomal aberrations, especially translocations in chromosome 1, seems to be a promising method to show a colon cancer risk. Additionally, our study suggests the reasonableness of use of biomarkers such as chromosome 1 aberrations in peripheral blood lymphocytes in screening prevention programs for individuals at higher colon cancer risk to identify those who are at increased risk and require more frequent investigations, e.g. by sigmoidoscopy.

Application of the micronucleus assay performed by different scorers in case of large-scale radiation accidents

Abstract Mass casualty scenarios of radiation exposure require high throughput biological dosimetry techniques for population triage, in order to rapidly identify individuals, who require clinical treatment. Accurate dose estimates can be made by biological dosimetry, to predict the acute radiation syndrome (ARS) within days after a radiation accident or a malicious act involving radiation. Timely information on dose is important for the medical management of acutely irradiated persons [1]. The aim of the study was to evaluate the usefulness of the micronuclei (MNi) scoring procedure in an experimental mode, where 500 binucleated cells were analyzed in different exposure dose ranges. Whole-body exposure was simulated in an in vitro experiment by irradiating whole blood collected from one healthy donor with 60 MeV protons and 250 keV X-rays, in the dose range of 0.3-4.0 Gy. For achieving meaningful results, sample scoring was performed by three independent persons, who followed guidelines described in detail by Fenech et al. [2, 3]. Compared results revealed no significant differences between scorers, which has important meaning in reducing the analysis time. Moreover, presented data based on 500 cells distribution, show that there are significant differences between MNi yields after 1.0 Gy exposure of blood for both protons and X-rays, implicating this experimental mode as appropriate for the distinction between high and low dose-exposed individuals, which allows early classification of exposed victims into clinically relevant subgroups.