Original Articles

By Mrs. Michelle Moron , Prof. Joao Arruda , Dr. Helena Segreto , Dr. Durvanei Maria , Dr. Luiz Batista , Dr. Godofredo Genofre
Corresponding Author Prof. Joao Arruda
Physics Institute-University of Sao Paulo, Rua do Matao - Travessa R. 187, Cidade Universitaria, Sao Paulo, SP, Brazil - Brazil São Paulo, SP, Brazil
Submitting Author Mrs. Michelle M Moron
Other Authors Mrs. Michelle Moron
Physics Institute - University of Sao Paulo, - Brazil

Dr. Helena Segreto
Department of Clinical and Experimental Oncology, Division of Radiotherapy, Federal University of Sa, - Brazil

Dr. Durvanei Maria
Biomedical and Biophysical Laboratories, Butantan Institute, - Brazil

Dr. Luiz Batista
DNA Repair Laboratory, Institute of Biomedical Sciences, - Brazil

Dr. Godofredo Genofre
CENEPES, Grajau General Hospital, - Brazil


Cancer cells, Electric field, S-phase arrest, Radiation sensitizer, Gamma radiation, DNA repair

Moron M, Arruda J, Segreto H, Maria D, Batista L, Genofre G. Cancer Cells Jointly Exposed To Gamma-radiation And Electric Field Develop S-phase Arrest. WebmedCentral BIOLOGY 2012;3(3):WMC003143
doi: 10.9754/journal.wmc.2012.003143

This is an open-access article distributed under the terms of the Creative Commons Attribution License(CC-BY), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
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Submitted on: 06 Mar 2012 12:25:01 PM GMT
Published on: 12 Mar 2012 04:53:34 PM GMT


It has been shown recently that death of cells simultaneously exposed to ionizing radiation and static electric fields is substantially higher than in cells only irradiated, suggesting that a static electric field interferes with cell repair mechanisms. In the present work, T47D breast cancer cells were firstly irradiated with 1 and 2 Gy of gamma radiation, and then exposed to a SEF of 1250 V/cm by 24 hours. After these treatments cell cycle distributions were evaluated by flow cytometry. It was found that treatment with irradiation plus static electric fields exposure causes a higher accumulation of cells at the S phase, and a corresponding reduction at G1, while the population in G2/M was nearly unchanged. To our knowledge, this is the first time that a static electric field is shown to interfere with the progression trough S-phase in irradiated cells, most likely due to an interference with DNA repair mechanisms. We conclude that this physical agent works as an additional factor accentuating the deleterious effects of DNA damage and therefore, it is a radiation sensitizer for possible clinical application.


Effects on cells simultaneously exposed to ionizing radiation and static electric fields (SEF) have been recently reported [1]. The results showed that cultures of the prokaryote Microcystis panniformis (Cyanobacteria) and of the eukaryote Candida albicans irradiated with gammas (0-5 KGy range) and immediately exposed to a static electric field (from 20 to 200 V/cm), presented substantial increase in cell death in comparison with samples only irradiated. It was suggested that static electric fields could hamper DNA repair processes. In accordance with a biophysical model, an exogenous electric field polarizes the displacement of repair proteins, impairing their harboring to DNA damage sites.
DNA repair performance in irradiated human cells exposed to exogenous static electric fields was shown to be considerably less efficient than in cells only irradiated. This was verified from the quantification of γ-H2AX foci by measurement of fluorescence intensities, as recently discussed elsewhere [1]. It is important to point out that this phosphorylated protein is the first step in recruiting and localizing DNA repair proteins to damage sites [2].
These findings motivated us to investigate the cell cycle distribution in cancer cells submitted to radiation treatments associated with exposure to static electric fields.  It is already known that the cell cycle progression of cells exposed to radiation is altered, mainly due to the accumulation of cells in the G2/M phase [3,4].
Since a SEF hampers DNA repair, we hypothesized that it could lead to changes in the cell cycle distribution, delaying and/or impeding the cell cycle progression.
In this paper we report on results for breast cancer cells cycle (T47D line), obtained after irradiation with gammas and exposure to SEF. This experiment is part of a research program on new radiotherapy procedures and strategies, in progress at this Laboratory.


Cell Culture and Treatments
The cell line T47D, epithelial-like human breast ductal carcinoma (ATCC HTB-133) was obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). Monolayer cell cultures were grown in RPMI 1640 (Gibco Laboratories, Grand Island, NY, USA) supplemented with 10% fetal calf serum (FCS; Cultilab, Campinas, SP, Brazil) and penicillin-streptomycin (100 units/mL and 100 mg/mL, respectively). Cell culture was maintained at 37 ºC in a humidified 5% CO2 atmosphere.
Cells grown on flasks were irradiated with doses of 0 (control), 1 and 2 Gy of gamma from a 60Co source ALCION II (CGR-MeV-Varian) at a dose rate of 1.27 Gy/min at room temperature. Flasks with cell cultures were placed between a capacitor plates, and a static electric field of 1250 V/cm was applied during 24 h. This set was accommodated inside a cell incubator and maintained at 37 ºC.
Cell Cycle Distribution by Flow Cytometry
After the treatments with radiation and SEF, the T47D cells cycle distributions were evaluated by flow cytometry. To this purpose, approximately 1.0 × 105 cells were plated in 25 cm2 plastic flasks 24 h before gamma irradiation. The cells were tripsinized and washed in cold solution of phosphate-buffered-saline (PBS), fixed in 70% cold ethanol and stored at –20ºC. Before analysis, cells were washed in PBS and treated with 50 mg/mL RNase A, incubated with 50 mg/mL propidium iodide (PI) for at least 1h in the dark at 4ºC. Approximately 20.000 cells were analyzed and PI fluorescence was measured by flow cytometer (FACScalibur, Becton Dickinson, San Jose, CA, USA). The data was processed with the software WinMDI version 2.8 (available at http://facs.scripps.edu/software.html. June 21, 2010).


Results are shown in Figure 1, where the number of events is expressed as function of the DNA content.
Relative quantities for the three cycle phases are in Table 1, where we have calculated the number of events-(γ + SEF) divided by the number of events-γ. By events-(γ + SEF) we mean the percentage of cells found in each of the cell phases, after treatments with radiation (γ) and static electric field (SEF). Thus, events-γ were obtained with radiation only. For the sake of simplicity, we define the following expression,
σ = [events-(γ + SEF)] / [events-γ].
In this sense, results in Table 1 are interpreted as:
1. For σ > 1, the number of events in a given phase is higher for the (γ + SEF) treatment,
2. For σ < 1, the number of events in a given phase is smaller for the (γ + SEF) treatment, and
3. For σ = 1, the number of events in a given phase is equal for the two treatments.
The results in Table 1 could be summarized as follows,
(a) G0/G1 phase– the SEF moderately reduced the number of cells (σ < 1) by 10% approximately for the treatments with 1 and 2 Gy.
(b) S phase– the action of the SEF substantially increased the number of cells, particularly for the 1 Gy dose (over 30%).
(c) G2/M phase– the results are statistically compatible with σ ≈ 1 within the experimental errors.


It is clear that the (γ + SEF) treatment causes a higher accumulation of cells at the S phase, and a corresponding reduction at G1, while the population in G2/M was nearly unchanged. Although a delay in G2 phase is the most evident, significant delays also occur in G1, as well as throughout the S phase [5]. Therefore, cells reach S phase but the SEF delays their normal progression through the transition  S → G2/M.
Inhibition at the beginning of the replication phase (as shown by σ > 1 at S) could be, according to Friedberg and collaborators [6], the prevailing response to damages caused by ionizing radiation. Additionally, as pointed out elsewhere [5,7], cells entering S phase with unrepaired DNA lesions may activate S phase checkpoints to provide more time for the DNA repair machinery to remove lesions in the double-helix [8,9,10].
We note that DNA damage can perturb the cellular steady-state quasi-equilibrium and activate or amplify certain biochemical pathways that regulate cell growth and division and pathways that help to coordinate DNA replication, damage and removal. In this regard, it is valid concluding that a SEF negatively interferes in the repairing process  and, thus, this physical agent both contributes to the cell killing and works as an additional factor accentuating inhibition at the replication phase due to the presence of unrepaired DNA lesions in the cell genome. DNA damage checkpoints ensure the fidelity of genetic information both by arresting cell cycle progression and facilitating DNA repair pathways. In the absence of DNA replication checkpoint, stalled forks are thought to collapse, creating strand breaks that threaten genome stability and cell viability [11]. Since DNA repair efficiency mediates the response of cancer cells to treatment, as pointed out elsewhere [12], our present data suggest SEF as a radiation sensitizer for future clinical application.
This is the first study showing that a SEF interferes with the progression trough S-phase in irradiated cells, probably due to the interference of this exogenous physical agent with DNA repair mechanisms as recently demonstrated by us elsewhere [13].


SEF: static electric fields
PBS: phosphate-buffered-saline
PI: propidium iodide


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6. Friedberg EC, Walker GC, Siede W, Wood RD, Schultz RA, Ellenberger T. DNA Repair and Mutagenesis. 2nd ed. Washington: ASM Press, 2006.
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11. McGlynn P, Lloyd RG. Recombinational repair and restart of damaged replication forks. Nat Rev Mol Cell Biol 2002; 3 (11): 859-70.
12. Batista LFZ, Roos WP, Christmann M, Menck CFM, Kaina B. Differential sensitivity of malignant glioma cells to methylating and chloroethylating anticancer drugs: p53 determines the switch by regulating xpc, ddb2, and DNA double-strand breaks. Cancer Res 2007; 67 (24): 11886-95.
13. Arruda-Neto JDT, Friedberg EC, Bittencourt-Oliveira MC, Segreto HRC, Moron MM, Maria DA, Batista LF, Schenberg AC. The role played by endogenous and exogenous electric fields in DNA signaling and repair. DNA Repair 2010; 9 (4): 356-57.

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