Hostname: page-component-78c5997874-4rdpn Total loading time: 0 Render date: 2024-11-06T11:12:28.853Z Has data issue: false hasContentIssue false
  • English
  • Français

Quantification of DNA double-strand break induced by radiation in cervix cancer cells: in vitro study

Published online by Cambridge University Press:  10 October 2018

Sothing Vashum ,
Rabi Raja Singh I ,
Saikat Das ,
Mohammed Azharuddin KO  and
Prabhakaran Vasudevan
Sothing Vashum*
Affiliation:
Department of Radiation Therapy, Christian Medical College and Hospital, Vellore, Tamil Nadu, India
Rabi Raja Singh I
Affiliation:
Department of Radiation Therapy, Christian Medical College and Hospital, Vellore, Tamil Nadu, India
Saikat Das
Affiliation:
Department of Radiation Therapy, Christian Medical College and Hospital, Vellore, Tamil Nadu, India
Mohammed Azharuddin KO
Affiliation:
Department of Neurosciences, Christian Medical College and Hospital, Vellore, Tamil Nadu, India
Prabhakaran Vasudevan
Affiliation:
Department of Neurosciences, Christian Medical College and Hospital, Vellore, Tamil Nadu, India
*
Authors for correspondence: Sothing Vashum, Department of Radiation Therapy, Christian Medical College and Hospital, Ida Scudder Road, Vellore – 632004, Tamil Nadu, India. Tel: +91-8754867454. E-mail: [email protected] Prabhakaran Vasudevan, Department of Neurosciences, Christian Medical College and Hospital, Ida Scudder Road, Vellore – 632004, Tamil Nadu, India. Tel: +91-4162282701. E-mail: [email protected]
Get access Rights & Permissions [Opens in a new window]

Abstract

Aim

DNA double-strand break (DSB) results in the phosphorylation of the protein, H.2AX histone. In this study, the effect of radiotherapy and chemotherapy on DNA DSB in cervical cancer cells is analysed by the phosphorylation of the protein.

Methods

The cervical cancer cells (HeLa cells) were cultured and exposed to ionising radiation. Radiation sensitivity was measured by clonogenic survival fraction after exposing to ionising radiation. Since the phosphorylation of H.2AX declines with time, the DNA damage was quantified at different time points: 1 hour, 3 hours and 1 week after exposed to the radiation. The analysis of γ-H.2AX was done by Western-blot technique. The protein expression was observed at different dose of radiation and combination of both radiation and paclitaxel.

Results

Low-dose hypersensitivity was observed. By 1 week after radiation at 0·5, 0·8 and 2 Gy, there was no expression of phosphorylated H.2AX. Previous experiments on the expression of phosphorylated H.2AX (γ-H.2AX) in terms of foci analysis was found to peak at 1 hour and subsequently decline with time. In cells treated with the DNA damaging agents, the expression of phosphorylated H.2AX decreases in a dose-dependent manner when treated with radiation alone. However, when combined with paclitaxel, at 0·5 Gy, the expression peaked and reduces at 0·8 Gy and slightly elevated at 2 Gy.

Findings

In this study, the peak phosphorylation was observed at 3 hour post irradiation indicating that DSBs are still left unrepaired.

Keywords

chemotherapyDNA double-strand breakphosphorylated H.2AXRadiationWestern blotting
Type
Original Article
Information
Journal of Radiotherapy in Practice , Volume 18 , Issue 1 , March 2019 , pp. 52 - 54
Copyright
© Cambridge University Press 2018 

Access options

Get access to the full version of this content by using one of the access options below. (Log in options will check for institutional or personal access. Content may require purchase if you do not have access.)

Footnotes

Cite this article: Vashum S, Singh I RR, Das S, Azharuddin KO M and Vasudevan P. (2019) Quantification of DNA double-strand break induced by radiation in cervix cancer cells: in vitro study. Journal of Radiotherapy in Practice18: 52–54. doi: 10.1017/S1460396918000456

References

1. Kuo, LJ, Yang, L. “Gamma-H2AX - a novel biomarker for DNA double-strand breaks.” In vivo 22 3 (2008): 305-9. Radiobiology Laboratory, California Pacific Medical Center Research Institute, San Francisco, CA 94118; 2St. Mary’s Medical Center, San Francisco, CA 94117, U.S.A.Google Scholar
2. Rogakuo, EP, Pilch, DR, Orr, AH, Ivanova, VS, Boriner, WM. DNA double strand breaks induce histone H2AX phosphorylation on serine 139. J Biol Chem 1998; 273 (10): 5858–5868.Google Scholar
3. Paull, TT, Rogakou, EP, Yamazaki, V, Kirchgessner, CU, Gellert, M, Bonner, WM. A critical role for histone H2AX in recruitment of repair factors to nuclear foci after DNA damage. Curr Biol 2000; 10 (15): 886–895.Google Scholar
4. Fuhrman, CB, Kilgore, J, LaCoursiere, YD et al. Radiosensitization of cervical cancer cells via double-strand DNA break repair inhibition. Gynecol Oncol 2008; 110 (1): 93–98.Google Scholar
5. MacPhail, SH, Banáth, JP, Yu, TY, Chu, EH, Lambur, H, Olive, PL. Expression of phosphorylated histone H2AX in cultured cell lines following exposure to X-rays. Int J Radiat Biol 2003; 79 (5): 351–358.Google Scholar
6. Goutham, HV, Mumbrekar, KD, Vadhiraja, BM et al. DNA double-strand break analysis by γ-H2AX foci: a useful method for determining the overreactors to radiation-induced acute reactions among head-and-neck cancer patients. Int J Radiat Oncol Biol Phys 2012; 84 (5): e607–e612. Epub Jul 24, 2012 .Google Scholar
7. Lowry, OH, Rosebrough, NJ, Farr, A, Randall, RJ. Protein measurement with the Folin phenol reagent. J Biol Chem 1951; 193 (1): 265–275.Google Scholar
8. ImageJ Software National Institutes of Health, Bethesda, Maryland, USA.Google Scholar
9. Das, S, Singh, R, George, D, Vijaykumar, TS, John, S. Radiobiological response of cervical cancer cell line in low dose region: evidence of low dose hypersensitivity (HRS) and induced radioresistance (IRR). https://doi.org/10.7860/JCDR/2015/14120.6074 Google Scholar