2020 Fiscal Year Research-status Report
Study of ATRX mediated telomere maintenance in neuroblastoma by using genome editing technology (CRISPR/Cas9)
| Project/Area Number |
18K15256
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| Research Institution | Research Institute for Clinical Oncology, Saitama Cancer Center |
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Principal Investigator |
AKTER JESMIN 埼玉県立がんセンター(臨床腫瘍研究所), 臨床腫瘍研究所, 技師 (70795830)
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| Project Period (FY) |
2018-04-01 – 2022-03-31
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| Keywords | Neuroblastoma / ATRX / Replication stress / G-quadruplex / p53 |
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| Outline of Annual Research Achievements |
ATRX is a tumor suppressor gene, and its recurrent somatic mutations relate to older neuroblastoma (NB) patients with advanced stages. Recent reports indicate that ATRX deficiency has been linked to replication stress and DNA damage by way of G-quadruplex (G4) DNA secondary structure. Here we found that NB cells lacking ATRX depends on p53 deficiency to limit replication–fork progression and genomic instability.
Previously, we knocked out (KO) ATRX in MYCN amplified (NGP) and MYCN single copy (SK-N-AS) NB cells with wild type and mutant TP53, respectively, using CRISPR-Cas9 system. By in vitro analysis, ATRX loss results in increased expression of gamma-H2AX, indicating the accumulation of endogenous DNA damage in the ATRX KO NGP cells but not in SK-N-AS clones. Using a monoclonal antibody known to recognize G4 structures (1H6), we found that ATRX loss increased G4 formation only in ATRX KO NGP cells. Furthermore, lentiviral-mediated inactivation of p53 reduced the G4 accumulation in the ATRX KO NGP cells, suggesting that ATRX plays a role in maintaining genome integrity and p53 deficiency helps to release replication stress and DNA damage in NB cells featuring inactivated ATRX.
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| Current Status of Research Progress |
Current Status of Research Progress
2: Research has progressed on the whole more than it was originally planned.
Reason
Current year, we have done several biological analysis by using ATRX knock-out clone in different NB cells. And we got some exciting data for our project. Start to prepare manuscript about ATRX project. That means experimental work is going well.
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| Strategy for Future Research Activity |
1. Writing the manuscript for this project.
2. Submit the article in some reputed journal.
3. Preparation of additional experiment for possible reviewer comments.
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| Causes of Carryover |
Because of novel corona virus, we couldn't present our study in different meetings. And also some experiment materials, we couldn't buy last fiscal time.
This year, we are going to submit our manuscript and add the remaining
amount in new fiscal year for article cost. And try to attend the national conference to present our data.
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