Our Research 2019 (92) ～
(92) DNA Damage Response After Ionizing Radiation Exposure in Skin Keratinocytes Derived from Human-Induced Pluripotent Stem Cells
Aug 10, 2019
Mikio SHIMADA
Tokyo Institute of Technology
Epidermal cells are positioned on the body surface and thus risk being exposed9o genotoxic stress, including ionizing radiation (IR), ultraviolet rays, and chemical compounds. The biological effect of IR on the skin tissue is a significant problem for medical applications such as radiation therapy. Keratinocyte stem cells and progenitors are at risk for IR-dependent tumorigenesis during radiation therapy for cancer treatment. To elucidate the molecular mechanism of genome stability in epidermal cells, we derived skin keratinocytes from human-induced pluripotent stem cells (iPSCs) and analyzed their DNA damage response (DDR).
Skin keratinocytes were derived from iPSCs and designated as first- (P1), second- (P2), and third- (P3) passage cells to compare the differentiation states of DDR. After 2 Gy gamma-ray exposure, cells were immunostained with DNA double-strand break markers γ-H2AX/53BP1 and cell senescence markers p16/p21 for DDR analysis. DDR protein expression level, cell survival, and apoptosis were analyzed by western blotting, WST-8 assay and TUNEL assay, respectively. DDR of constructed 3D organoid modeling was also analyzed.
P1, P2, and P3 keratinocytes were characterized with keratinocyte markers keratin 14 and p63 using immunofluorescence, and all cells were positive to both markers. Derived keratinocytes showed high expression of integrin α6 and CD71 (real-time (qRT)-PCR ratio: iPSCs: integrin α6: 1.12, CD71: 1.25, P1: integrin α6: 7.80, CD71: 0.43, P2: integrin α6: 5.53, CD71: 0.48), suggesting that P1 and P2 keratinocytes have potential as keratinocyte progenitors. Meanwhile, P3 keratinocytes showed low expression of integrin α6 and CD71 (qRT-PCR ratio: P3: integrin α6: 0.55, CD71: 0.10), suggesting differentiated keratinocytes. After IR exposure, the P1 and P2 keratinocytes showed an increase in DNA repair activity by a γ-H2AX/53BP1 focus assay (P1: γ-H2AX: 28.0%, 53BP1: 17.0%, P2: γ-H2AX: 37.7%, 53BP1: 28.3%) but not in P3 keratinocytes (P3: γ-H2AX: 74.7%, 53BP1: 63.7%) compared with iPSCs (γ-H2AX: 57.0%, 53BP1: 55.0%). Furthermore, in derived keratinocytes, expression of the cellular senescence markers p16 and p21 were increased compared with iPSCs (P16: non irradiated, iPSCs: 0%, P1: 12.5%, P2: 14.5%, P3: 29.7%, IR, iPSCs: 0%, P1: 19.5%, P2: 34.8%, P3: 64.5%). DDR protein expression, cellular sensitivity, and apoptosis activity decreased in derived keratinocytes compared with iPSCs.
We have demonstrated the derivation of keratinocytes from iPSCs and their characterization of differentiated states and DDR. Derived keratinocytes showed progenitors like character as a result of DDR. These results suggest that derived keratinocytes are useful tools for analyzing the effects of IR, such as DDR on the skin tissue from radiation therapy for cancer.
Reference:
Miyake T, Shimada M(corresponding author), Matsumoto Y, Okino A.
DNA Damage Response After Ionizing Radiation Exposure in Skin Keratinocytes Derived from Human-Induced Pluripotent Stem Cells.
Int J Radiat Oncol Biol Phys. 2019 May 11. pii: S0360-3016(19)30706-0. doi: 10.1016/j.ijrobp.2019.05.006. [Epub ahead of print]
(93) Neutron-Induced Rat Mammary Carcinomas Are Mainly of Luminal Subtype and Have Multiple Copy Number Aberrations
Aug 15, 2019
Hitomi Moriyama
(Graduate School of Human Health Sciences, Tokyo Metropolitan University / QST, NIRS)
Neutrons are a type of high linear energy transfer (LET) radiation and they have stronger carcinogenic effects than low LET radiation. As it is not known whether neutrons induce cancers with special characteristics, we sought to clarify the features of rat mammary carcinomas for which the incidence increases after rats were exposed to neutron radiation. We obtained mammary carcinomas from female Sprague-Dawley rats irradiated at 7 weeks of age with ~0.5 Gy neutron beams or γ rays and compared them with carcinomas of non-irradiated rats. Tumors were classified into luminal and non-luminal subtypes (which are thought to have different cells of origin) based on immunohistochemistry, while their DNA copy number aberrations were determined using microarrays. As a result, neutrons and γ rays significantly increased the incidence of luminal carcinomas but not non-luminal carcinomas. The carcinomas in the irradiated and non-irradiated groups contained multiple DNA copy number aberrations affecting 46 genes for which mutations have been reported in human breast cancer. Thus, neutrons and γ rays increase the incidence of luminal mammary carcinoma in rats, probably via genetic aberrations similar to those found in human breast cancer patients, indicating that neutrons induce cancers without special characteristics.
日本語解説
中性子は高線エネルギー付与（LET）放射線の一種であり、低LET放射線と比較して強い発癌作用を有する。乳腺は放射線による発がん感受性が高い。中性子線によって誘発されるがんが特殊なタイプなのかどうかはわかっていないため、我々は、中性子線照射により発生した乳がんの特徴を明らかにすることを目的として、次の研究を行った。すなわち、約0.5 Gyの中性子線またはγ線を7週齢のSprague-Dawley雌ラットに照射し、発生した乳がんを非照射ラットの乳がんと比較した。腫瘍を免疫組織化学染色によりルミナル型と非ルミナル型（これらは起源細胞が異なると考えられている）に分類し、そのコピー数異常についてマイクロアレイを用いて解析した。その結果、中性子線とγ線はルミナル型の発生を有意に増加させた。これらの乳がんと非照射群の乳がんは、ヒト乳がんで変異が報告されている46の遺伝子のDNAコピー数異常を含んでいた。このように、中性子線とγ線は、ヒト乳がん患者で見られる遺伝子異常と同様の遺伝的異常を介して、ラットのルミナル型乳がんの発生率を増加させていることが推察され、特殊なタイプのがんを誘発するのではないことがわかった。
Reference:
Moriyama H, Daino K, Imaoka T, Nishimura M, Nishimura Y, Takabatake M, Morioka T, Fukushi M, Shimada Y, Kakinuma S.
Neutron-Induced Rat Mammary Carcinomas Are Mainly of Luminal Subtype and Have Multiple Copy Number Aberrations
Anticancer Res. 2019 Mar;39(3):1135-1142. doi: 10.21873/anticanres.13222. PMID: 30842142