TITLE:
X-Ray Induced Mutation Frequency at the Hypoxanthine Phosphoribosyltransferase Locus in Clinically Relevant Radioresistant Cells
AUTHORS:
Yoshikazu Kuwahara, Mehryar Habibi Roudkenar, Yusuke Urushihara, Yohei Saito, Kazuo Tomita, Amaneh Mohammadi Roushandeh, Tomoaki Sato, Akihiro Kurimasa, Manabu Fukumoto
KEYWORDS:
Clinically Relevant Radioresistant (CRR) Cell, Hypoxanthine Phosphoribosyltransferase (HPRT), Mutation Frequency, Phosphorylated Histone H2AX (γH2AX), X-Rays
JOURNAL NAME:
International Journal of Medical Physics, Clinical Engineering and Radiation Oncology,
Vol.6 No.4,
November
3,
2017
ABSTRACT: To elucidate the molecular
mechanisms underlying cellular radioresistance, clinically relevant
radioresistant cell lines were established via long-term exposure to X-rays
with stepwise dose escalation. Established cells continue to proliferate
despite exposure to 2 Gy X-rays/day for more than 30 days, a standard protocol
in cancer radiotherapy. DNA repair fidelity in radioresistant and the parental
cells by evaluating the mutation frequency at the hypoxanthine phosphoribosyltransferase (HPRT) locus after exposure to X-rays was determined. Mutation spectrum at the HPRT locus
was examined by multiplex polymerase chain reaction. Rejoining kinetics of
X-ray-induced DNA double strand breaks (dsbs) was evaluated by the detection of
phosphorylated histone H2AX (γH2AX)
after X-irradiation. The fold increase in the HPRT mutation frequency due to acute radiation
was similar between radioresistant and the parental cell lines. However, fractionated radiation (FR) consisting of 2
Gy X-rays/day increased the mutation frequency at the HPRT locus
in parental but not in radioresistant cells. Analysis of the FR-induced
mutations at the HPRT locus
revealed a high frequency of deletion mutations (>70%) in parental but not
in radioresistant cells. As assessed by γH2AX
immunostaining, DNA dsbs induced by acute exposure to 10 Gy of X-rays were
repaired to the control level within 7 days in radioresistant but not in the
parental cells. Moreover, 2 Gy × 5 FR increased the number of γH2AX-positive
cells in parental cultures but not in radioresistant cultures. DNA dsbs induced
by 2 Gy/day FR are repaired with fidelity in radioresistant but not in parental
cells.