| 研究実績の概要 |
The chromatin remodeler ATRX is a tumor suppressor gene, and its recurrent somatic mutations associated with stage 4 NB patients. But our knowledge about ATRX mutation in NB tumorigenecity is still limited. Previously, ATRX knockout (KO) clones in NB cells by CRISPR/Cas9 system was established and interestingly, the KO clones proliferate slowly compared to control group and become differentiated.
Microarray based GSEA analysis showed that gene-set related to DNA double-strand break (DSBs) repair, DNA damage response, negative cell cycle regulation, G2M checkpoint and, p53 pathway activation were induced in ATRX KO cells. By in vitro analysis, ATRX loss results in augmentation of gamma-H2AX, a canonical marker for DSBs, indicate the accumulation of endogenous DNA damage in ATRX KO cells. Furthermore, accumulation of DNA damage results in activation of the ATM/Chk2/p53 pathway, leading to cell cycle arrest. Moreover, lentiviral-mediated inactivation of p53 rescued the decreased cell growth levels in ATRX KO cells, suggesting that ATRX plays a role in maintaining DNA integrity and ATM/p53 pathway inactivation is required for NB tumorigenesis. We also studied the correlation between ATRX-mutation and ATM/p53 pathway inactivation by multiomics database analysis, indicating that ATM/p53 pathway inactivation is required for ATRX mutation-related NB tumorigenesis.
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| 現在までの達成度 (区分) |
現在までの達成度 (区分)
2: おおむね順調に進展している
理由
Current year, we have established some other advance techniques for CRISPR/Cas9 system. So, we got the knock out clone in other neuroblastoma (NB) cell lines, that was necessary for our study. We also compared the biological
characteristics of the knock out clones in different NB cells. That means experimental work is going well.
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| 次年度使用額が生じた理由 |
This year we have submitted our abstract in overseas international meeting. Because of novel corona virus, we couldn't present our study. And also some experiment materials, we couldn't buy last fiscal time.
This year, we are going to do some more experiment and will add the remaining
amount in new fiscal year to analyze the telomere maintenance mechanism in ATRX knock out cells. And also attend the national and international conference to present our data.
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