Cyclin-dependent kinase inhibitor 1 plays a more prominent role than activating transcription factor 4 or the p53 tumour suppressor in thapsigargin-induced G1 arrest

Erin van Zyl; Claire Peneycad; Evan Perehiniak; Bruce C McKay

doi:10.7717/peerj.16683

Cyclin-dependent kinase inhibitor 1 plays a more prominent role than activating transcription factor 4 or the p53 tumour suppressor in thapsigargin-induced G1 arrest

PeerJ. 2023 Dec 18:11:e16683. doi: 10.7717/peerj.16683. eCollection 2023.

Authors

Erin van Zyl¹, Claire Peneycad¹, Evan Perehiniak¹, Bruce C McKay^{1

2}

Affiliations

¹ Department of Biology, Carleton University, Ottawa, ON, Canada.
² Institute of Biochemistry, Carleton University, Ottawa, ON, Canada.

Abstract

Background: Thapsigargin (Tg) is a compound that inhibits the SERCA calcium transporter leading to decreased endoplasmic reticulum (ER) Ca2+ levels. Many ER chaperones are required for proper folding of membrane-associated and secreted proteins, and they are Ca2+ dependent. Therefore, Tg leads to the accumulation of misfolded proteins in the ER, activating the unfolded protein response (UPR) to help restore homeostasis. Tg reportedly induces cell cycle arrest and apoptosis in many cell types but how these changes are linked to the UPR remains unclear. The activating transcription factor 4 (ATF4) plays a key role in regulating ER stress-induced gene expression so we sought to determine if ATF4 is required for Tg-induced cell cycle arrest and apoptosis using ATF4-deficient cells.

Methods: Two-parameter flow cytometric analysis of DNA replication and DNA content was used to assess the effects of Tg on cell cycle distribution in isogenic HCT116-derived cell lines either expressing or lacking ATF4. For comparison, we similarly assessed the Tg response in isogenic cell lines deleted of the p53 tumour suppressor and the p53-regulated p21^WAF1 cyclin-dependent kinase inhibitor important in G₁ and G₂ arrests induced by DNA damage.

Results: Tg led to a large depletion of the S phase population with a prominent increase in the proportion of HCT116 cells in the G₁ phase of the cell cycle. Importantly, this effect was largely independent of ATF4. We found that loss of p21^WAF1 but not p53 permitted Tg treated cells to enter S phase and synthesize DNA. Therefore, p21^WAF1plays an important role in these Tg-induced cell cycle alterations while ATF4 and p53 do not. Remarkably, the ATF4-, p53-and p21^WAF1-deficient cell lines were all more sensitive to Tg-induced apoptosis. Taken together, p21^WAF1 plays a larger role in regulating Tg-induced G₁ and G₂ arrests than ATF4 or p53 but these proteins similarly contribute to protection from Tg-induced apoptosis. This work highlights the complex network of stress responses that are activated in response to ER stress.

Keywords: ATF4; CDKN1A; Cell cycle; ER stress; TP53; Thapsigargin.

MeSH terms

Activating Transcription Factor 4* / genetics
Cell Line, Tumor
Cyclin-Dependent Kinases / metabolism
DNA
Humans
Thapsigargin / pharmacology
Tumor Suppressor Protein p53* / genetics

Substances

Tumor Suppressor Protein p53
Thapsigargin
Activating Transcription Factor 4
DNA
Cyclin-Dependent Kinases

Grants and funding

This work was supported by the Natural Sciences and Engineering Research Council of Canada (RGPIN-2019-06146). Erin van Zyl was supported with a Vanier Canada Graduate Scholarship through the Natural Sciences and Engineering Research Council of Canada. Erin van Zyl and Claire Peneycad received studentship support through the Ontario Graduate Scholarship program from the Ontario Ministry of Colleges and Universities. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.