Evaluation of the toxic effects evoked by the transient expression of protease genes from human pathogens in HEK293 cells

Shubin, Andrey V.; Lunina, N. A.; Шедова, Е. Н.; Roshina, M. P.; Demidyuk, Ilya V.; Виноградова, Т. В.; Копанцев, Е. П.; Чернов, И. П.; Kostrov, Sergey V.

doi:10.1134/s0003683813090044

Cited by 6 publications

(4 citation statements)

References 28 publications

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“…Thus, 3Cpro and its inactive variant 3Cmut were expressed in several model cell lines. Fully compliant with the previous data [ 10 , 11 ], here, we showed that the observed cytotoxic effect of 3Cpro expression depends on the protease activity, and cell death is accompanied by cytoplasmic vacuolization without caspase activation.…”

Section: Discussionsupporting

confidence: 92%

“…Thus, the effect of 3Cpro on human cells in the pCI-based expression system in vitro is similar to that previously reported by us [10,11].…”

Section: Cpro Expression Induces Caspase-independent Cell Death With Cytoplasmic Vacuolizationsupporting

confidence: 90%

“…Previously, we demonstrated that hepatitis A virus 3C protease (3Cpro) induces cell death that is also independent of caspase activity and features a unique cell morphology, specifically, the accumulation of cytoplasmic vacuoles with previously undocumented features. This suggested that the 3Cpro-induced cell death proceeds by an unknown pathway or is a new variant of a known RCD type [ 10 , 11 , 12 ].…”

Section: Introductionmentioning

confidence: 99%

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Individual Expression of Hepatitis A Virus 3C Protease Induces Ferroptosis in Human Cells In Vitro

Komissarov

Karaseva

Roschina

et al. 2021

IJMS

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Regulated cell death (RCD) is a fundamental process common to nearly all living beings and essential for the development and tissue homeostasis in animals and humans. A wide range of molecules can induce RCD, including a number of viral proteolytic enzymes. To date, numerous data indicate that picornaviral 3C proteases can induce RCD. In most reported cases, these proteases induce classical caspase-dependent apoptosis. In contrast, the human hepatitis A virus 3C protease (3Cpro) has recently been shown to cause caspase-independent cell death accompanied by previously undescribed features. Here, we expressed 3Cpro in HEK293, HeLa, and A549 human cell lines to characterize 3Cpro-induced cell death morphologically and biochemically using flow cytometry and fluorescence microscopy. We found that dead cells demonstrated necrosis-like morphological changes including permeabilization of the plasma membrane, loss of mitochondrial potential, as well as mitochondria and nuclei swelling. Additionally, we showed that 3Cpro-induced cell death was efficiently blocked by ferroptosis inhibitors and was accompanied by intense lipid peroxidation. Taken together, these results indicate that 3Cpro induces ferroptosis upon its individual expression in human cells. This is the first demonstration that a proteolytic enzyme can induce ferroptosis, the recently discovered and actively studied type of RCD.

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Section: Discussionsupporting

confidence: 92%

“…Thus, the effect of 3Cpro on human cells in the pCI-based expression system in vitro is similar to that previously reported by us [10,11].…”

Section: Cpro Expression Induces Caspase-independent Cell Death With Cytoplasmic Vacuolizationsupporting

confidence: 90%

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Individual Expression of Hepatitis A Virus 3C Protease Induces Ferroptosis in Human Cells In Vitro

Komissarov

Karaseva

Roschina

et al. 2021

IJMS

View full text Add to dashboard Cite

show abstract

“…Previously, we demonstrated that hepatitis A virus 3C protease (3Cpro) induces cell death, which is also independent of caspase activity and features a unique cell morphology, specifically, the accumulation of cytoplasmic vacuoles with previously undocumented features. This suggested that the 3Cpro-induced cell death proceeds by an unknown pathway or is a new variant of a known RCD type [10-12].…”

Section: Introductionmentioning

confidence: 99%

Individual expression of hepatitis A virus 3C protease induces ferroptosis in human cellsin vitro

Komissarov

Karaseva

Roschina

et al. 2021

Preprint

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Cytotoxic effect of co-expression of human hepatitis A virus 3C protease and bifunctional suicide protein FCU1 genes in a bicistronic vector

et al. 2017

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Recent reports on various cancer models demonstrate a great potential of cytosine deaminase/5-fluorocytosine suicide system in cancer therapy. However, this approach has limited success and its application to patients has not reached the desirable clinical significance. Accordingly, the improvement of this suicide system is an actively developing trend in gene therapy. The purpose of this study was to explore the cytotoxic effect observed after co-expression of hepatitis A virus 3C protease (3C) and yeast cytosine deaminase/uracil phosphoribosyltransferase fusion protein (FCU1) in a bicistronic vector. A set of mono- and bicistronic plasmid constructs was generated to provide individual or combined expression of 3C and FCU1. The constructs were introduced into HEK293 and HeLa cells, and target protein synthesis as well as the effect of 5-fluorocytosine on cell death and the time course of the cytotoxic effect was studied. The obtained vectors provide for the synthesis of target proteins in human cells. The expression of the genes in a bicistronic construct provide for the cytotoxic effect comparable to that observed after the expression of genes in monocistronic constructs. At the same time, co-expression of FCU1 and 3C recapitulated their cytotoxic effects. The combined effect of the killer and suicide genes was studied for the first time on human cells in vitro. The integration of different gene therapy systems inducing cell death (FCU1 and 3C genes) in a bicistronic construct allowed us to demonstrate that it does not interfere with the cytotoxic effect of each of them. A combination of cytotoxic genes in multicistronic vectors can be used to develop pluripotent gene therapy agents.

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Evaluation of the toxic effects evoked by the transient expression of protease genes from human pathogens in HEK293 cells

Cited by 6 publications

References 28 publications

Individual Expression of Hepatitis A Virus 3C Protease Induces Ferroptosis in Human Cells In Vitro

Individual Expression of Hepatitis A Virus 3C Protease Induces Ferroptosis in Human Cells In Vitro

Individual expression of hepatitis A virus 3C protease induces ferroptosis in human cellsin vitro

Cytotoxic effect of co-expression of human hepatitis A virus 3C protease and bifunctional suicide protein FCU1 genes in a bicistronic vector

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