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1.
Int J Mol Sci ; 22(11)2021 May 22.
Article in English | MEDLINE | ID: mdl-34067339

ABSTRACT

Dephosphorylation inhibitor calyculin A (cal A) has been reported to inhibit the disappearance of radiation-induced γH2AX DNA repair foci in human lymphocytes. However, other studies reported no change in the kinetics of γH2AX focus induction and loss in irradiated cells. While apoptosis might interplay with the kinetics of focus formation, it was not followed in irradiated cells along with DNA repair foci. Thus, to validate plausible explanations for significant variability in outputs of these studies, we evaluated the effect of cal A (1 and 10 nM) on γH2AX/53BP1 DNA repair foci and apoptosis in irradiated (1, 5, 10, and 100 cGy) human umbilical cord blood lymphocytes (UCBL) using automated fluorescence microscopy and annexin V-FITC/propidium iodide assay/γH2AX pan-staining, respectively. No effect of cal A on γH2AX and colocalized γH2AX/53BP1 foci induced by low doses (≤10 cGy) of γ-rays was observed. Moreover, 10 nM cal A treatment decreased the number of all types of DNA repair foci induced by 100 cGy irradiation. 10 nM cal A treatment induced apoptosis already at 2 h of treatment, independently from the delivered dose. Apoptosis was also detected in UCBL treated with lower cal A concentration, 1 nM, at longer cell incubation, 20 and 44 h. Our data suggest that apoptosis triggered by cal A in UCBL may underlie the failure of cal A to maintain radiation-induced γH2AX foci. All DSB molecular markers used in this study responded linearly to low-dose irradiation. Therefore, their combination may represent a strong biodosimetry tool for estimation of radiation response to low doses. Assessment of colocalized γH2AX/53BP1 improved the threshold of low dose detection.


Subject(s)
Apoptosis/drug effects , Fetal Blood/drug effects , Histones/metabolism , Lymphocytes/drug effects , Marine Toxins/pharmacology , Oxazoles/pharmacology , Tumor Suppressor p53-Binding Protein 1/metabolism , DNA Damage/drug effects , DNA Repair/drug effects , Dose-Response Relationship, Radiation , Fetal Blood/metabolism , Humans , Intracellular Signaling Peptides and Proteins/metabolism , Lymphocytes/metabolism , Microscopy, Fluorescence/methods , Phosphorylation/drug effects
2.
Toxicol In Vitro ; 73: 105127, 2021 Jun.
Article in English | MEDLINE | ID: mdl-33652125

ABSTRACT

While hyperthermia (HT) is a promising modality for cancer treatment, the knowledge on mechanisms of its effect on cells is still limited. We have investigated DNA double-strand break (DSB) and apoptosis induced by HT. Umbilical cord blood lymphocytes (UCBL) were subjected to HT at 43 °C. We have treated cells for 1 h (1 h HT), 2 h (2 h HT) and by combined HT and ice treatment (both lasting 1 h). Enumeration of DSB by 53BP1/γH2AX DNA repair focus formation and early apoptosis by γH2AX pan-staining was conducted by automated fluorescent microscopy. Apoptotic stages and viability were assessed by the annexin/propidium iodide (PI) assay using flow cytometry 0, 18, and 42 h post-treatment. HT induced either immediate (2 h HT) or postponed (1 h HT) DNA damage. The levels of 53BP1 and γH2AX foci differed under the same treatment conditions, suggesting that the ratio of co-localized γH2AX/53BP1 foci to all γH2AX and also to all 53BP1 foci could be a valuable marker. The ratio of co-localized foci increased immediately after 2 h HT regardless the way of assessment. For the first time we show, by both annexin/PI and γH2AX pan-staining assay that apoptosis can be induced during or immediately after the 2 h HT treatment. Our results suggest that HT may induce DSB in dependence on treatment duration and post-treatment time due to inhibition of DNA repair pathways and that HT-induced apoptosis might be dependent or associated with DSB formation in human lymphocytes. Assessment of γH2AX pan-staining in lymphocytes affected by HT may represent a valuable marker of HT treatment side effects.


Subject(s)
DNA Breaks, Double-Stranded , Fetal Blood/cytology , Hot Temperature/adverse effects , Lymphocytes/radiation effects , Apoptosis/radiation effects , DNA Repair , Histones , Humans , Hyperthermia, Induced , Infant, Newborn , Tumor Suppressor p53-Binding Protein 1
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