DNA damage response following X-irradiation in oral cancer cell lines HSC3 and HSC4

Sirimanas Jiaranuchart; Atsushi Kaida; Yusuke Onozato; Hiroyuki Harada; Masahiko Miura

doi:10.1016/j.archoralbio.2018.02.016

DNA damage response following X-irradiation in oral cancer cell lines HSC3 and HSC4

Arch Oral Biol. 2018 Jun:90:1-8. doi: 10.1016/j.archoralbio.2018.02.016. Epub 2018 Mar 6.

Authors

Sirimanas Jiaranuchart¹, Atsushi Kaida², Yusuke Onozato³, Hiroyuki Harada³, Masahiko Miura⁴

Affiliations

¹ Department of Oral Radiation Oncology, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8549, Japan; Department of Oral and Maxillofacial Surgery, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo, 113-8549, Japan.
² Department of Oral Radiation Oncology, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8549, Japan.
³ Department of Oral and Maxillofacial Surgery, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo, 113-8549, Japan.
⁴ Department of Oral Radiation Oncology, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8549, Japan. Electronic address: masa.mdth@tmd.ac.jp.

PMID: 29522917
DOI: 10.1016/j.archoralbio.2018.02.016

Abstract

Objective: The objective of this study was to characterize the DNA damage response in two human oral cancer cell lines following X-irradiation.

Design: To visualize radiation-induced cell cycle alterations, two human oral cancer cell lines, HSC3 and HSC4, expressing fluorescent ubiquitination-based cell cycle indicator (Fucci) were established in this study. G2 arrest kinetics following irradiation were obtained from two-color flow cytometric analysis and pedigrees of Fucci fluorescence. DNA double strand break repair kinetics were obtained from immunofluorescence staining for phosphorylated histone H2AX, p53-binding protein 1, phosphorylated DNA-dependent protein kinase catalytic subunit, and breast cancer susceptibility gene 1.

Results: Both cell lines showed apparent G2 arrest after 10 Gy of irradiation, but it was more enhanced in the HSC3-Fucci cells. Radiosensitivity was higher in the HSC3-Fucci cells than in HSC4-Fucci cells. Pedigree analysis of Fucci fluorescence revealed that the HSC3-Fucci cells exhibited a significantly longer green phase (normally indicating S/G2/M phases, but here reflective of G2 arrest) when irradiated in the red phase (G1 phase) than HSC4-Fucci cells irradiated in either red or green phases. Non-homologous end joining was marginally suppressed during the G1 phase and markedly more likely to be impaired during the S/G2 phases in HSC3-Fucci cells. When G2 arrest was abrogated by checkpoint kinase 1 or Wee1 inhibitors, only HSC4-Fucci cells exhibited radiosensitization.

Conclusions: We characterized DNA damage response in HSC3-Fucci and HSC4-Fucci cells following irradiation and the former demonstrated inefficient non-homologous end joining, especially during the S/G2 phases, resulting in enhanced G2 arrest. These findings may have clinical implications for oral cancer.

Keywords: DNA double strand break repair; Fluorescent ubiquitination-based cell cycle indicator; G2 arrest; Non-homologous end joining; Oral cancer.

MeSH terms

Carcinoma, Squamous Cell / radiotherapy
Cell Cycle / physiology*
Cell Cycle / radiation effects*
Cell Cycle Checkpoints / radiation effects
Cell Cycle Proteins / metabolism
Cell Division / radiation effects
Cell Line, Tumor / physiology
Cell Line, Tumor / radiation effects
Checkpoint Kinase 1 / metabolism
DNA Breaks, Double-Stranded / radiation effects
DNA Damage / radiation effects*
DNA-Activated Protein Kinase
Dose-Response Relationship, Radiation
G2 Phase Cell Cycle Checkpoints / radiation effects
Histones / metabolism
Histones / radiation effects
Humans
Kinetics
Microscopy, Fluorescence
Mouth Neoplasms / radiotherapy*
Nuclear Proteins / metabolism
Protein-Tyrosine Kinases / metabolism
Proteins / metabolism
Proteins / radiation effects
Tumor Suppressor p53-Binding Protein 1
Ubiquitination
X-Rays / adverse effects*

Substances

Cell Cycle Proteins
H2AX protein, human
Histones
Nuclear Proteins
Proteins
TP53BP1 protein, human
Tumor Suppressor p53-Binding Protein 1
Protein-Tyrosine Kinases
WEE1 protein, human
CHEK1 protein, human
Checkpoint Kinase 1
DNA-Activated Protein Kinase
PRKDC protein, human