2015IMP&HIRFL Annual Report·175·
3-71MSH2Mediates Sensitization of Liver Cancer Cell
to Heavy Ions∗
Miao Guoying and Zhang Hong
The DNA mismatch repair(MMR)system,composed of several proteins such as MLH1,MSH2,MSH6,MSH3, and PMS2,eliminates replication errors and maintains genomic stability.MutSα,an MSH2/MSH6heterodimer, recognizes single base mismatches,whereas MutSβ,an MSH2/MSH3heterodimer,primarily recognizes2∼4bp insertiondeletion loops[1,2].The MutL complex,mainly MutLα,an MLH1/PMS2heterodimer,forms a ternary complex with a MutS heterodimer that binds to DNA mismatches during replication and leads to recruitment of other proteins to complete the process of DNA MMR.Germ line mutations in MMR genes result in Lynch syndrome, which is characterized by hereditary predisposition to cancers with microsatellite instability(MSI)in the colon, endometrium,ovaries,and urinary tract[3,4].The MMR system also participates in repairing certain DNA damage. MutSαrecognizes these mispairs and recruits MutLαfor the subsequent repair reactions.These two complexes are also linked to a signal transduction pathway that leads to cell growt
h arrest or cell death.Heavy ion radiotherapy like carbon ion has showed a strong role for killing malignancies especially for radio resistant malignancies that are insensitive to conventional radiotherapy,such as X-ray.Carbon ion radiotherapy produces the higher relative biological effectiveness(RBE)and increased linear energy transfer(LET)than conventional radiotherapy,there by carbon ion radiotherapy results in the more DNA damages of cancer cells.Considering that the MutSαand MutSβcomplex plays a role in repairing DNA damage,especially double strand break(DSB)repair.We hypothesized that MSH2deficiency may halt the repair of DNA damage induced by radiotherapy,resulting in enhanced cytotoxicity of radiotherapy in cancer patients.In addition,MSH2protein also can modulate the homologous recombination that is a prominent double-strand break repair pathway.MSH2-deficient cells are profoundly sensitive to methotrexate in vitro culture,intuitively,if MSH2deficiency dictates the toxicity of radiotherapy in a clinical setting,MSH2 status can be used as a predictive marker for the radio therapeutic outcome in patients with MSH2-deficient cancers. However,the precise role of MSH2in mediating the cytotoxic effects of radiotherapy remains poorly understood. In this study,wefirst examined the effects of MSH2deficiency on cytotoxicity caused by Heavy ion radiotherapy. To explore this possibility,using liver cancer cell line in which MSH2protein expression can be regulated thorough siRNA expression,we investigated the effect of MSH2deficiency on the cellular sensitivity to carbon ion.Using liver cancer HepG2cell in whi
modulate
ch MSH2expression is controlled by siRNA expression,we discovered that MSH2 deficiency sensitized cells to carbon ion radiotherapy.Interestingly,Furthermore,MSH2-deficient cells maintained higher levels of phosphorylated histone H2AX after carbon ion treatment in comparison with MSH2-proficient cells, suggesting that MSH2plays an important role in repairing DNA double strand breaks(DSBs).This role of MSH2 was further supported by ourfindings that MSH2-deficient cells were sensitive to a poly polymerase(PARP-1) inhibitor.Moreover,the combination of carbon ion and PARP-1inhibitor exhibited a synergistic effect compared with either treatment individually.Collectively,our results provide novel evidence that MSH2deficiency contributes
Fig.1(color online)Knockdown MSH2of liver cancer HepG2cell，The cells appeared G2/M phase arrest After12h by carbon ion treatment,increased from0.6%in the control group to59%in the siRNA group,and the difference between two group was significant(P<0.05).It is exhibited MSH2may be involved in the regulation of cell cycle.
∗Foundation item:Key Program of National Natural Science Foundation of China(U1432248),National Natural Science Foundation of China(11305226)
·176·IMP
&HIRFL Annual Report 2015to the cytotoxicity of carbon ion treatment through deficient DSB repair shown in Fig.1.These data lay the foundation for the development of effective prediction and treatments for cancers with MSH2deficiency.
References
[1]T.A.Kunkel,D.A.Erie.,Annu.Rev.Biochem,74(2005)681.
[2]J.Jiricny.Nat.Rev.Mol.Cell Biol,7(2006)335.
[3]Y.M.Hendriks,A.E.de Jong,H.Morreau,et al.,Cancer J.Clin.,56(2006)213.
[4]  C.R.Boland,A.Goel.,Gastroenterology,138(2010)2073.
3-72Early Embryonic Exposure of Ionizing Radiation
Disrupts Zebrafish Pigmentation ∗
Wang Yupei,Zhou Xin and Zhang Hong
Mitochondrial F1F0-ATPase has been shown to modulate pigmentation negatively in zebrafish [1,2].Both of these works hypothesize that the alkalization of cellular endomembrane system,as induced by inhibition of mitochondrial ATPase-mediated transport of H +ions,facilitates trafficking of Tyr to the melanosomes that enhance zebrafish pig-mentation.Wang [3]et al.further identified that Beta adrenergic receptors (β-ARs)regulate zebrafish pigmentation partly
by modulating F1F0-ATPase.These studies suggest the important role of mitochondrial F1F0-ATPase in zebrafish pigmentation,but whether IR could affect F1F0-ATPase function has not been studied.
Fig.1The morphological alterations of melanocyte melanization defects caused by IRof half lethal expo-
sure dose in zebrafish embryos or larvae.Zebrafish embryos were exposed to 2Gy proton beams at 3hpf and imaged on different developmental stages(24,48and 72hpf).Representative images were shown.
Here we employed two kinds of IR,including X-rays
and proton radiations to study the effects of IR on ze-
brafish pigmentation.We show that early embryonic
exposure of ionizing radiation leads to significant dis-
turbance of zebrafish pigmentation(Fig.1).We find IR
induces markedly increase of ATP5α1expression,sug-
gesting the important role of F1F0ATPase/ATP syn-
thase in zebrafish pigmentationing(Fig.2).By using
specific ATPase/ATP synthase inhibitors,we identify
that the activity of ATP synthase contributes to ze-
brafish pigmentation after IR exposure.ATP synthase
inhibitor Oligomycin administration partially restored pigmentation in irradiated zebrafish embryos,but AT-Pase inhibitor BTB06584has no effect.Taken together,we here show that IR exposure down-regulated zebrafish pigmentation though ATP synthase regulation.Fig.2IR induced gene expression alterations in atp5a α1and abrb2a.3hpf zebrafish embryos were exposed or not to
IR.20h post-irradiation,ATP5α1and abrb2a mRNA was quantified by qRTPCR.The expression of target gene was ana-
lyzed to normalize actin mRNA levels.All data was then compared to unirradiated data,which was adjusted to a value of one.
(a)ATP5α1expression after 1and 2Gy proton beam radiation.(b)abrb2a expression after 1and 2Gy proton beam
radiation.Results showed represent mean ±SE (N =3),*P <0.05,**P <0.01.

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