Report on "Radiation Disaster Recovery Studies"
Course: Radiation Disaster Medicine
Name: Nguyen Quang Tam•Regarding "Radiation Disaster Recovery Studies"

Phoenix Leader Education program was initiated by Hiroshima University in 2011 forrenaissance from radiation disaster and supported by MEXT (Ministry of education, Culture,Sports, Science and Technology of Japan), right after the severe earthquake caused thesecond worst radiation accident in the world at Fukushima Dai-ichi nuclear power plant, since
Chernobyl radiation disaster (Soviet Union, 1986). (l)Japan has suffered during several nuclear disasters such as Hiroshima and Nagasaki atomic
bomb (August, 1945) 、 Tokai-mura nuclear accident (1997, 1999) and recently was Fukushima
nuclear accidents (March, 2011). Those accidents gave large number of damage andexperiences for Japanese government and scientists in facing to serious problems of humanhealth, environment and social recovery. After Fukushima accident, Hiroshima University hasrealized that there is needing of a well-organized education program to foster students inmajor fields that can have enough combined knowledge and interdisciplinary skills inradiation disaster science. That gave me a chance to study in Radiation disaster medicine
course cif the program beside my major in oral health sciences.
During the whole program, I have good chances to learn from the basic knowledge such as
history and impact of nuclear accidents on human health in molecular biology and disease
pathology to advanced course in radiation oncology and head and neck cancer treatment. In
addition, combination with radiation environment subjects helped me to understand clearly
about risk of radioactive sources in our disaster area and its long-term exposure. Together
with direct radiation-induced damage in human health, this is the first time I can learn about
physiological effect that evacuees are having, which can indirectly lead to changes in theirglucose metabolism, causing diabetes [Satoh et al. (2015)].
Beside many useful lectures in school from global experts, I have had many opportunities tojoin several common field trips and short field trips in Tokyo, lbaraki (Chiyoda factory),
Fukushima and many symposiums in radiation physics and health management in Nagasaki,Tokyo lnstitu�e of Technology, Fukushima, Seoul (ICRP 2015), United States of America(Colorado State University, 2016) and especially the long-term internship in International
Atomic Energy Agency (IAEA, 2016) to study about clinical applications of mesenchymal stem
cells in treating radiation-induced lesions.

ーRadiation has various negative effects on human health such as cell death in molecular leveland organ damage and mortality risk to human when exposed to high radiation dose. It also
increased the risk of having cancer when individual exposed with radiation over 1Gy whileradiation effect of under 100mGy is uncertain [Kamiya et al. (2015)]. After Fukushima accident,residents and evacuees were at risk at radiation exposure and changing of their life, giving
many challenges to government and scientists to protect their people and to recovery process[Hasegawa et al. {2015); Ohtsuru et al. (2015)].
Being a graduate student in both Phoenix program and Department of Molecular Oral
Medicine and Maxillofacial surgery gave me a fundamental knowledge in oral cancer, effectof radiation and radio-chemotherapy for treating cancer. With lack of knowledge about effectof low dose radiation on human health and also in cancer treatment which I have learned
during four years in Phoenix program gave me an idea to study effect of radiation on
squamous cell carcinoma, especially with low dose radiation.

References:
1. Phoenix Leader Education Program of Hiroshima University website:
http://phoen ixprogram Ip. hiroshima-1.1.ac.jp/en/background/
2. Hasegawa, A., Tanigawa, K., Ohtsuru, A., Yabe, H 零 ，Maeda, M., Shigemura, J., Ohira, T.,
Tominaga, T.、Akashi, M., Hirohashi, N., Ishikawa, T., Kamiya, K., Shibuya, K., Yamashita,S., & Chhem, R. K. (2015). Health effects of radiation and other health problems in theaftermath of nuclear accidents, with an emphasis on Fukushima. Lancet, 386(9992),479-488. doi:10.1016/50140-6736(15)61106-0
3. Kamiya、 K., Ozasa, K., Akiba, S., Niwa, 0., Kodama, K., Takamura, N., Zaharieva, E. K.,
Kimura, Y., & Wakeford, R. (2015). Long-term effects of radiation exposure on health.
Lancet, 386(9992) 、 469-478. doi:10.1016/50140-6736(15)61167-9
4. Ohtsuru, A., Tanigawa, K., Kumagai, A., Niwa, 0., Takamura, N., Midorikawa, S., Nollet,k會，Yamashita, S., Ohto, H., Chhem, R. K., & Clarke、1\/1. (2015). Nuclear disasters andhealth: lessons learned, challenges, and proposals. Lancet, 386(9992), 489-497.
doi:10.1016/S0140-6736(15)60994--1
5. Satoh, H.、 Ohira, T. 、 Hosoya, M亀，Sakai, A., Watanabe, T., Ohtsuru, A., Kawasaki, Y.,
Suzuki, H., Takahashi, A., Kobashi, G., Ozasa, K、, Yasumura, S., Yamashita、 S 零 ，Kamiya,K., & Abe, M. (2015). Evacuation after the Fukushima Daiichi Nuclear Power Plant
Accident Is a Cause of Diabetes: Results from the Fukushima Health Management
Survey. J Diabetes Res, 2015, 627390. doi:10.1155/2015/627390• Title of Doctoral Thesis:
Establishment and characterization of radiation resistant strains from squamous cellcarcinoma cell lines in serum-free defined culture•

Summary of Doctoral Thesis:

Introduction
Squamous cell carcinoma (SCC), including oral squamous cell carcinoma (OSCC), hasbeen increasing in the world and being the most common cancer in South-East Asian
countries where have a betel-quid and areca-nut chewing habit. It has been considered thatcancer cells are functionally heterogeneous that undergo not only proliferation but also2,＇differentiation and maturation to a certain degree and contain a small population of cancer
stem cells (CSCs). It seems logical that cures of cancer can be achieved only if the CSCs
population is eliminated.
On the other hand, there are growing evidences that CSCs are genetically radio-resistant(RR) and might be resistant to other cancer therapies. Therefore, to clarify biological effectsof radiation on SCC cell lines in vitro and in vivo might be worthwhile to circu"'!vent radiation
resistance for oral cancer. Currently, there are no report of establishment of RR-SCC cells inserum-free defined culture, which ensures exact biological mechanisms and prevent
instability of using serum.
In this study, first I have tried to isolate and establish RR-SCC strains from SCC and OSCC
cell lines, and then characterized their cellular and molecular properties in serum-free defined
culture:Materials and methods

Two sec cell lines were used in this study including A431 derived from vulvar sec, andNA/H0-1-N-1 from OSCC. The cells were cultured in serum-free DF6F medium (1:1 mixture ofDMEM and Ham-F12 medium supplemented with insulin {lOµg/ml), transferrin (Sµg/ml), 2aminoethanol (lOµM), sodium selenite (lOnM), 2-�ercaptoethanol {lOµM), and oleic acidconjugated with fatty acid-free bovine serum albumin (9.4µg/ml)).
All cell lines were irradiated weekly at a dose of 2.2Gy/day、4days/week with a low doserate (LDR) irradi戸tion system (RMlOOO, Chugai Technos,」apan), or at 5Gy/5.75min, twice aweek with a high dose rate (HDR) system {Gamma cell 40 Exactor, Best Theratronics, Canada)
in serum-free defined culture. After irradiating 60Gy as a whole dose by LDR and HDR

irradiation system, we have isolated 4 RR sub-strains from 2 SCC cell lines. These radiation
resistant (RR) strains were continuously irradiated every week at a dose of O.SGy frompassage 5. To confirm the radiation resistance of these sub-strains, we have performed aclonogenic survival assay as follow. The parental and RR strains were irradiated at a dose ofOGy, 2Gy, 4Gy, 6Gy, and 8Gy, respectively, and examined radiation resistance by clonogenic
survival assay. After 14 days of culture, the colonies were stained with Giemsa, and counted.
To clarify the biological properties of these cells, several cellular abilities such as growth
in monolayer culture, sphere formation in suspension culture, and migration in Boyden­
chamber method were examined,. For the growth assay, the cells were seeded at 10 4cells/wellin 24-well plate and counted cell numbers every day by the Coulter Counter. For sphere

formation assay, the cells were seeded in 35mm prime surface (low-attachment) dish in DF6F
serum-free medium at 103cells/dish, and then sphere numbers were counted on day 5. For'migration assay, the cells were seeded at Sx104cells/well in 24-well collagen coatedchemotaxicell well in DF medium supplemented with 0.1% BSA and stained with Giemsa forcounting number of migrated cells/mm2 . The ratio of CD133 positive cell in each cell line wasexamined by flowcytometry as cancer stem cell marker.
Total RNAs extracted and purified from all cell lines, were used for Real Time­quantitative PCR (RT-qPCR), and DNA microarray analysis.

3ヽTo study the tumor forming ability in nude mouse, parental and RR cells {0.25 x106 lx106 cells/100µ1 DF} were injected subcutaneously to the dorsal back skin of nude mice
{BALB/c-nu/nu}, and tumor size was measured every week. Then the tumors were excised,weighted, fixed in 4% paraformaldehyde for 24hr, and embedded in paraffin. for H&E andimmuno-histochemical staining.
To investigate which gene has important role in radiation resistance of those cancercells, several high expressed genes in DNA micro array data were picked up for clonogenic
survival assay after cancer cells were transduced with selected siRNA. Relation of these genes
to pluripotent stem cells markers was verified by RT-qPCR after silencing with siRNA.
Results
By using LDR and HDR system, A431-LDR, NA-LDR, A431-HDR, and NA-HDR wereradiation resistance cell lines and successfully isolated in serum-free defined culture. The D31
value of A431-LDR, A431-HDR, parental A431, NA-LDR, NA-HDR, and parental NA was SGy,3.7Gy, 2.3Gy, 7.SGy, 5.SGy and 4.6Gy, respectively. These cells exhibited higher expression ofcancer stem cell marker such as CD133, higher sphere formation and higher migration abilities
compare to those of parental cell lines. Expression of pluripotent stem cell markers such asNanog, Sox2, and Oct4 were not consistent among the RR cells. As a result of DNA microarray
analysis, the RR cell lines showed elevated expression of KRT13 {200 folds}, insulin-growth
factor 2 {IGF2}, chemokines {IL1R2, CCL20} and genes involved in tumor micro environment{SlOOA} and metastasis {MMPs}. In addition, the RR cells exhibited higher tumor forming
ability compared to parental cells in nude mice xenograft. In addition, two genes wereconfirmed to have effects in this cancer cell radiation resistance.
Discussion
Radiation resistant strains from SCC cell lines were successfully isolated in serum-freedefined culture. These cells exhibited CSCs-like properties such as high stem cell marker
expression, sphere forming ability, migration activity in vitro and a high tumor forming ability
in vivo.
These radiation resistant cells might be very useful not only to study cancer stem cells
but also to circumvent radiation resistance for the novel cancer treatment modality.

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