Biology subjects

Nakanishi, M.

Publications and source records attributed to Nakanishi, M..

23 records · Page 2Linked to original sources

bioRxiv2020

ATR inhibition enhances 5-fluorouracil sensitivity independent of non-homologous end-joining and homologous recombination repair pathway

The anticancer agent, 5-fluorouracil (5-FU), is typically applied in the treatment of various types of cancers because of its properties. Thought to be an inhibitor of the enzyme thymidylate synthase which plays a role in nucleotide synthesis, 5-FU has been found to induce single- and double-strand DNA breaks. The activation of ATR occurs as a reaction to UV- and chemotherapeutic drug-induced replication stress. In this study, we examined the effect of ATR inhibition on 5-FU sensitivity. Using western blotting, we found that 5-FU treatment led to the phosphorylation of ATR. Surviving fractions were remarkably decreased in 5-FU with ATR inhibitor (ATRi) compared to 5-FU with other major DNA repair kinases inhibitors. ATR inhibition enhanced induction of DNA double-strand breaks and apoptosis in 5-FU-treated cells. Using gene expression analysis, we found that 5-FU could induce the activation of intra-S checkpoint. Surprisingly, BRCA2-deficient cells were sensitive to 5-FU in the presence of ATRi. In addition, ATR inhibition enhanced the efficacy of 5-FU treatment, independent of non-homologous end-joining and homologous recombination repair pathways. Findings from the present study suggest ATR as a potential therapeutic target for 5-FU chemotherapy.

cancer biology↗

bioRxiv2019

Human URAT1/SLC22A12 gene promoter is regulated by 27-hydroxycholesterol through estrogen response elements

Elevated levels of uric acid, a metabolite of purine in humans, is related to various diseases, such as gout, atherosclerosis and renal dysfunction. The excretion and reabsorption of uric acid to/from urine is tightly regulated by uric acid transporters. The amino acid sequences of uric acid reabsorption transporters, URAT1/SLC22A12, OAT4/SLC22A11, and OAT10/SLC22A13, share closer phylogenic relationship, whereas the gene promoter sequences are distant phylogenic relationship. Through the single-cell RNA-sequencing analysis of an adult human kidney, we found that only a small number of cells express these transporters, despite their role in the regulation of serum uric acid levels. Transcriptional motif analysis on these transporter genes, revealed that the URAT1/SLC22A12 gene promoter displayed the most conserved estrogen response elements (EREs) among the three transporters. The endogenous selective estrogen receptor modulator (SERM) 27-hydroxycholesterol (27HC) had positive effects on the transcriptional activity of URAT1/SLC22A12. We also found that 27HC increased the protein and gene expression of URAT1/SLC22A12 in mouse kidneys and human kidney organoids, respectively. These results strongly suggest the role of 27HC for URAT1/SLC22A12 expression in renal proximal tubules and upregulation of serum uric acid levels and also show the relationship between cholesterol metabolism and serum uric acid regulation. Significance StatementThe elevated levels of serum uric acid cause various diseases, and the excretion/reabsorption of uric acid to/from urine is tightly regulated by the uric acid transporters. We found that despite the role in serum uric acid regulation, only a small number of cells express URAT1/SLC22A12. We also found that URAT1/SLC22A12 gene promoter region has effective estrogen response elements, and endogenous selective estrogen receptor (ER) modulator 27-hydroxycholesterol (27HC) increased URAT1/SLC22A12 expression in the mice kidneys and human kidney organoids. These suggest that 27HC increases URAT1/SLC22A12 expression and upregulate serum uric acid levels. Since 27HC connects cholesterol metabolism, our study indicates the important link between cholesterol metabolism and serum uric acid regulation, and also provides a novel therapeutic approach to hyperuricemia.

biochemistry↗

bioRxiv2019

Brainstem organoids from human pluripotent stem cells contain neural crest population

The brainstem controls heartbeat, blood pressure and respiration, which are life-sustaining functions, therefore, disorders of the brainstem can be lethal. Brain organoids derived from human pluripotent stem cells recapitulate the course of human brain development and are expected to be useful for medical research on central nervous system disorders. However, existing organoid models have limitations, hampering the elucidation of diseases affecting specific components of the brain. Here, we developed a method to generate human brainstem organoids (hBSOs), containing neural crest stem cells as well as midbrain/hindbrain progenitors, noradrenergic and cholinergic neurons, and dopaminergic neurons, demonstrated by specific electrophysiological signatures. Single-cell RNA sequence analysis, together with proteomics and electrophysiology, revealed that the cellular population in these organoids was similar to that of the human brainstem and neural crest, which raises the possibility of making use of hBSOs in grafting for transplantation, efficient drug screenings and modeling the neural crest diseases.

developmental biology↗

bioRxiv2019

Toxic PR poly-dipeptides encoded by the C9orf72 repeat expansion target Kapβ2 and dysregulate phase separation of low-complexity domains

Low-complexity (LC) domains of proteins are found in about one fifth of human proteome, and a group of LC-domains form labile cross-{beta} polymers and liquid-like droplets. Polymers and droplets formed from LC-domains are dynamically regulated by posttranslational modifications and molecular chaperones including nuclear transport receptors. Repeat expansion in the first intron of a gene designated C9orf72, which is the most prevalent form of familial amyotrophic lateral sclerosis (ALS), causes nucleocytoplasmic transport deficit, however, the detailed mechanism remains unsolved. Here we show that the proline:arginine (PR) poly-dipeptides encoded by the C9orf72 repeat expansion bound nuclear transport receptor Kap{beta}2 through its nuclear localization signal (NLS) recognition motif, and inhibited the ability of Kap{beta}2 to melt fused in sarcoma (FUS) droplets by competing interaction with FUS. The findings in this study offer mechanistic insights as to how the C9orf72 repeat expansion disables nucleocytoplasmic transport and causes neurodegenerative diseases.

molecular biology↗

bioRxiv2018

Epigenetic-scale comparison of human iPSCs generated by retrovirus, Sendai virus or episomal vectors

Human induced pluripotent stem cells (iPSCs) are established by introducing several reprogramming factors, such as OCT3/4, SOX2, KLF4, c-MYC. Because of their pluripotency and immortality, iPSCs are considered to be a powerful tool for regenerative medicine. To date, iPSCs have been established all over the world by various gene delivery methods. All methods induced high-quality iPSCs, but epigenetic analysis of abnormalities derived from differences in the gene delivery methods has not yet been performed. Here, we generated genetically matched human iPSCs from menstrual blood cells by using three kinds of vectors, i.e., retrovirus, Sendai virus, and episomal vectors, and compared genome-wide DNA methylation profiles among them. Although comparison of aberrant methylation revealed that iPSCs generated by Sendai virus vector have lowest number of aberrant methylation sites among the three vectors, the iPSCs generated by non-integrating methods did not show vector-specific aberrant methylation. However, the differences between the iPSC lines were determined to be the number of random aberrant hyper-methylated regions compared with embryonic stem cells. These random aberrant hyper-methylations might be a cause of the differences in the properties of each of the iPSC lines.

cell biology↗