CD4+CD8+ thymocytes are induced to cell death by a small dose of puromycin via ER stress

Oguma, Takemi; Ono, Takeshi; Kajiwara, Toshimitsu; Satō, Masaki; Miyahira, Yasushi; Arino, Hiroshi; Yoshihara, Yasuo; Tadakuma, Takushi

doi:10.1016/j.cellimm.2009.07.002

Cited by 9 publications

(6 citation statements)

References 29 publications

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“…As a translocon opener, puromycin treatment of cells may result in increased ER calcium release and higher rates of apoptosis. For example, Oguma et al reported that puromycin treatment induces GRP78 expression, resulting in caspase 12-mediated cell death in cluster of differentiation (CD)4 + CD8 + thymic lymphoma cells and in normal thymocytes (61). Johnson et al also revealed that malignant glioma cells treated with a low dose of puromycin exhibit increased GRP78 protein and mRNA levels, and enhanced activation of caspase 4.…”

Section: Discussionmentioning

confidence: 99%

The tumorigenic role of DSPP and its potential regulation of the unfolded protein response and ER stress in oral cancer cells

et al. 2018

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Dentin sialophosphoprotein (DSPP) is upregulated in various human cancers, including head and neck squamous cell carcinoma. Cancer cells are commonly found under constant endoplasmic reticulum (ER) stress and exhibit increased levels of misfolded proteins, due to gene mutations and a stressful microenvironment. The present study examined the effects of DSPP silencing on the regulation of ER stress and the unfolded protein response (UPR) in oral cancer cells. A recently established stable DSPP short hairpin (sh)RNA-silenced OSC2 oral cancer cell line was used. The mRNA expression levels of ER stress-associated proteins, including 78 kDa glucose-regulated protein (GRP78), sarcoplasmic/endoplasmic reticulum calcium ATPase 2b (SERCA2b), inositol 1,4,5-trisphosphate receptor (IP3r), protein kinase R-like endoplasmic reticulum kinase (PERK), serine/threonine-protein kinase/endoribonuclease IRE1 (IRE1), activating transcription factor 6 (ATF6) and matrix metalloproteinase 20 (MMP20), were assessed by reverse transcription-quantitative polymerase chain reaction. The expression levels of apoptosis-related [B‑cell lymphoma 2 (Bcl2), Bcl2-associated X protein (Bax) and cytochrome c] and cell proliferation-related [proliferating cell nuclear antigen (PCNA)] proteins were analyzed by western blotting. Cell viability, apoptosis and migration were monitored by MTT assay, Annexin V-fluorescein isothiocyanate flow cytometry and wound-healing assay, respectively. In transiently transfected puromycin‑free OSC2 cells, DSPP silencing markedly downregulated the mRNA expression levels of major ER stress regulators, including GRP78, SERCA2b, PERK, IRE1 and ATF6, as well as MMP20. DSPP silencing also resulted in decreased cell viability and migration, and enhanced apoptosis. Furthermore, PCNA and Bcl2 levels were decreased, whereas Bax and cytochrome c protein levels were increased in DSPP-silenced OSC2 cells. Sustained puromycin treatment partially counteracted the effects of DSPP silencing on the mRNA expression levels of ER stress-related proteins and MMP20, and on the migratory capacity of OSC2 cells. However, following puromycin treatment of DSPP-silenced cells, cell viability was further reduced and apoptosis was enhanced. In conclusion, these data provide evidence to suggest that DSPP may be involved in ER stress mechanisms in oral squamous cell carcinoma, since its downregulation in OSC2 cells led to significant alterations in the levels of major ER stress-associated proteins, and subsequent collapse of the UPR system.

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Section: Discussionmentioning

confidence: 99%

The tumorigenic role of DSPP and its potential regulation of the unfolded protein response and ER stress in oral cancer cells

et al. 2018

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“…However, it is notable that there is a modest increase in mass (19 mg vs. 11.1 mg, p = 0.02) and volume (17.4 cc vs. 9.0 cc, p = 0.01) among sgRNA‐transduced cells versus control. A component of this increased tumor size among transduced cells may be related to the common practice of implantation of tumor soon after puromycin selection, as puromycin has been noted to induce cell stress changes associated with increased GB growth (Liu et al., 2020; Oguma et al., 2009). Ultimately, however, we must stress that CRISPRi screen relies on hundreds of transduced nontargeting sgRNA controls to function as the experimental controls for the candidate lncRNA sgRNAs.…”

Section: Discussionmentioning

confidence: 99%

In vivo CRISPR screening for novel noncoding RNA functional targets in glioblastoma models

Attenello

Tsung

Bishara

et al. 2021

J of Neuroscience Research

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CRISPR (clustered regularly interspaced short palindromic repeat)‐based genetic screens offer unbiased and powerful tools for systematic and specific evaluation of phenotypes associated with specific target genes. CRISPR screens have been utilized heavily in vitro to identify functional coding and noncoding genes in a large number of cell types, including glioblastoma (GB), though no prior study has described the evaluation of CRISPR screening in GB in vivo. Here, we describe a protocol for targeting and transcriptionally repressing GB‐specific long noncoding RNAs (lncRNAs) by CRISPR interference (CRISPRi) system in vivo, with tumor growth in the mouse cerebral cortex. Given the target‐specific parameters of each individual screen, we list general steps involved in transducing guide RNA libraries into GB tumor lines, maintaining sufficient coverage, as well as cortically injecting and subsequently isolating transduced screen tumor cell populations for analysis. Finally, in order to demonstrate the use of this technique to discern an essential lncRNA, HOTAIR, from a nonessential lncRNA, we injected a 1:1 (HOTAIR:control nonessential lncRNA knockdown) mixture of fluorescently tagged U87 GB cells into the cortex of eight mice, evaluating selective depletion of HOTAIR‐tagged cells at 2 weeks of growth. Fluorescently tagged populations were analyzed via flow cytometry for hiBFP (control knockdown) and green fluorescent protein (HOTAIR knockdown), revealing 17% (p = 0.007) decrease in fluorescence associated with HOTAIR knockdown relative to control. The described in vivo CRISPR screening methodology thus appears to be an effective option for identifying noncoding (and coding) genes affecting GB growth within the mouse cortex.

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“…Immune dysfunction has been demonstrated after central nervous system injury in HIV− patients and thus could be one of many confounders in these findings. [16][17][18] Furthermore, steroid therapy-a routine component of deceased donor management-has also been shown to reduce T cell subpopulations in a variety of patient cohorts [19][20][21] ; however, the effect of steroids on CD4 count has never been systematically evaluated in brain-dead patients.…”

Section: Discussionmentioning

confidence: 99%

CD4 Count in HIV− Brain-Dead Donors

et al. 2017

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CD4+CD8+ thymocytes are induced to cell death by a small dose of puromycin via ER stress

Cited by 9 publications

References 29 publications

The tumorigenic role of DSPP and its potential regulation of the unfolded protein response and ER stress in oral cancer cells

The tumorigenic role of DSPP and its potential regulation of the unfolded protein response and ER stress in oral cancer cells

In vivo CRISPR screening for novel noncoding RNA functional targets in glioblastoma models

CD4 Count in HIV− Brain-Dead Donors

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