Profiling the DNA damage response dynamics at sub-lethal and lethal temozolomide doses

Visser, Hartwig

Abstract

Confronted with DNA damage, cells can undergo cell cycle arrest and DNA repair, or cell death. However, communal DNA damage response (DDR) proteins involved in each of these cell fates, has confounded attempts to unravel the molecular nuances that drive cell fate determinations.

Aim: This study sought to identify whether distinct molecular signatures in the form of DDR protein and miRNA expression exist to underpin cell fate decisions.

Methods: Human lymphoblastoid (TK6) cells were exposed to a temozolomide (TMZ) dose range to identify sub-lethal and lethal doses. These doses (30 μM and 900 μM, respectively) were used for all subsequent experiments. TMZ-induced effects were determined based on DDR protein levels (immunoblotting), DNA damage accumulation (immunofluorescence), cell fate (flow cytometry), and miRNA expression (RNA sequencing and qRT-PCR). Mimics of identified miRNAs were over-expressed in TK6 cells and functional analysis performed with flow cytometry and immunoblotting.

Results: TK6 cells exposed to 30 μM TMZ (30TMZ) preferentially underwent G2 arrest whereas 900 μM (900TMZ) caused apoptosis, identifying these as sub-lethal and lethal doses respectively. ATR-γH2Ax-Chk1-p53 pathway was significantly upregulated among 900TMZ-treated cells when compared to 30TMZ. Collectively, 31 miRNAs were differentially expressed when comparing 30TMZ-, and 900TMZ-treated cells to control-treated cells. Among these, miR-29b-3p, miR-363-5p, and miR-485-3p were determined to be significantly downregulated in response to 900TMZ-treated cells when compared to 30TMZ-treated cells. Moreover, p53 and miRNAs (miR-29b-3p, miR-363-5p, and miR-485-3p) were regulated in opposing directions following TMZ exposure, alluding to a possible miRNA-regulatory role for p53 in this context. Over-expression of miR-29b-3p or miR-363-5p individually or in combination did not affect cell fate.

Conclusion: TK6 cells exposed to a lethal dose of TMZ expresses a unique molecular profile in the form of significantly upregulated ATR-γH2Ax-Chk1-p53 signalling, along with significant downregulation of miR-29b-3p, miR-363-5p, and miR-485-3p when compared to sub-lethally-treated TK6 cells. This suggests these miRNAs to be DDR-responsive and thus, provides a link between DDR proteins and miRNA regulation. This is the first time these miRNAs have been reported in TK6 cells in the context of the DDR and these, along with the other differentially expressed miRNAs identified here, may prove useful as diagnostic biomarkers, druggable targets in future therapies, or become therapies in itself.

• Thesis Type: Thesis
• Deposit Date: Sep 30, 2022
• Publicly Available Date: May 5, 2023
• Public URL: https://uwe-repository.worktribe.com/output/10014381
• Award Date: May 5, 2023