Generation of a PAX6 knockout glioblastoma cell line with changes in cell cycle distribution and sensitivity to oxidative stress

Abstract

BackgroundThe transcription factor PAX6 is expressed in various cancers. In anaplastic astrocytic glioma, PAX6 expression is inversely related to tumor grade, resulting in low PAX6 expression in Glioblastoma, the highest-grade astrocytic glioma. The aim of the present study was to develop a PAX6 knock out cell line as a tool for molecular studies of the roles PAX6 have in attenuating glioblastoma tumor progression.MethodsThe CRISPR-Cas9 technique was used to knock out PAX6 in U251 N cells. Viral transduction of a doxycycline inducible EGFP-PAX6 expression vector was used to re-introduce (rescue) PAX6 expression in the PAX6 knock out cells. The knock out and rescued cells were rigorously characterized by analyzing morphology, proliferation, colony forming abilities and responses to oxidative stress and chemotherapeutic agents.ResultsThe knock out cells had increased proliferation and colony forming abilities compared to wild type cells, consistent with clinical observations indicating that PAX6 functions as a tumor-suppressor. Cell cycle distribution and sensitivity to H2O2 induced oxidative stress were further studied, as well as the effect of different chemotherapeutic agents. For the PAX6 knock out cells, the percentage of cells in G2/M phase increased compared to PAX6 control cells, indicating that PAX6 keeps U251 N cells in the G1 phase of the cell cycle. Interestingly, PAX6 knock out cells were more resilient to H2O2 induced oxidative stress than wild type cells. Chemotherapy treatment is known to generate oxidative stress, hence the effect of several chemotherapeutic agents were tested. We discovered interesting differences in the sensitivity to chemotherapeutic drugs (Temozolomide, Withaferin A and Sulforaphane) between the PAX6 expressing and non-expressing cells.ConclusionsThe U251 N PAX6 knock out cell lines generated can be used as a tool to study the molecular functions and mechanisms of PAX6 as a tumor suppressor with regard to tumor progression and treatment of glioblastoma.Electronic supplementary materialThe online version of this article (10.1186/s12885-018-4394-6) contains supplementary material, which is available to authorized users.

Tissue-specific actions of Pax6 on proliferation-differentiation balance in the developing forebrain are Foxg1-dependent

“…Moreover, its role in different tumor cell types is contradictory. It has been shown to have both oncogenic and tumor suppressor effect depending on the tissue affected (Hegge et al, 2018;Muratovska et al, 2003;Zhang et al, 2015;Zhou et al, 2005). Here we show one mechanism by which this versatile transcription factor can control the cell cycle in opposite ways, depending on the presence or absence of another important transcription factor.…”

Oncogenic PAX6 elicits CDK4/6 inhibitor resistance by epigenetically inactivating the LATS2‐Hippo signaling pathway

“…The role of PAX6 in the cell cycle is of particular interest. Interestingly, PAX6 could maintain astrocytic glioma cells in the G1 phase of the cell cycle, 54 suggesting that PAX6 promotes cell cycle arrest in glioma. However, based on the tissue context‐dependent role of PAX6 and its relationship with the poor prognosis of patients with GC, we hypothesized that PAX6 could facilitate the cell cycle in GC cells.…”

Streptococcus pneumoniae Cell Wall-Localized Trigger Factor Elicits a Protective Immune Response and Contributes to Bacterial Adhesion to the Host

“…NSC 34 is a mouse neuroblastoma-spinal cord hybrid immortalized cell-line 38 , which has been used to study differentiation and characteristics of motor neuron 56 , 57 . The glioblastoma multiforme derived cell-line U251 39 has been used to study the nature of glioblastoma multiforme and its susceptibility to anti-cancerous drugs 58 , 59 . To our knowledge, the current study is the first to use NCS 34 and U251 cell-lines as targets for S. pneumoniae adhesion.…”