Genomic Adaption and Mutational Patterns in a HaCaT Subline Resistant to Alkylating Agents and Ionizing Radiation.

Reinhard Ullmann,Simone Rothmiller,Richard Obermair,Horst Thiermann,Jessica Müller,Julia Jakobi,Hanns Leonhard Kaatsch,Benjamin Valentin Becker,Matthias Port,Annette Schmidt,Gerrit Schrock,Harry Scherthan

doi:10.3390/ijms22031146

Abstract

Sulfur mustard (SM) is a chemical warfare agent that can damage DNA via alkylation and oxidative stress. Because of its genotoxicity, SM is cancerogenic and the progenitor of many chemotherapeutics. Previously, we developed an SM-resistant cell line via chronic exposure of the popular keratinocyte cell line HaCaT to increasing doses of SM over a period of 40 months. In this study, we compared the genomic landscape of the SM-resistant cell line HaCaT/SM to its sensitive parental line HaCaT in order to gain insights into genetic changes associated with continuous alkylation and oxidative stress. We established chromosome numbers by cytogenetics, analyzed DNA copy number changes by means of array Comparative Genomic Hybridization (array CGH), employed the genome-wide chromosome conformation capture technique Hi-C to detect chromosomal translocations, and derived mutational signatures by whole-genome sequencing. We observed that chronic SM exposure eliminated the initially prevailing hypotetraploid cell population in favor of a hyperdiploid one, which contrasts with previous observations that link polyploidization to increased tolerance and adaptability toward genotoxic stress. Furthermore, we observed an accumulation of chromosomal translocations, frequently flanked by DNA copy number changes, which indicates a high rate of DNA double-strand breaks and their misrepair. HaCaT/SM-specific single-nucleotide variants showed enrichment of C > A and T > A transversions and a lower rate of deaminated cytosines in the CpG dinucleotide context. Given the frequent use of HaCaT in toxicology, this study provides a valuable data source with respect to the original genotype of HaCaT and the mutational signatures associated with chronic alkylation and oxidative stress.

Highlights

Sulfur mustard (SM) is infamous for its use as a chemical warfare agent on the battlefield of Ypres during the First World War in 1917 and in several other more recent military conflicts
We identified genetic changes associated with chronic exposure to SM, which may impact resistance toward various alkylating agents and ionizing irradiation
We observed that chronic exposure of the parental HaCaT cell line to SM induced the expansion of a hyperdiploid subclone at the cost of the originally prevailing hypotetraploid cell population

Summary

Introduction

Sulfur mustard (SM) is infamous for its use as a chemical warfare agent on the battlefield of Ypres during the First World War in 1917 and in several other more recent military conflicts. SM poses a continuing threat, as it is probably the most widely distributed chemical weapon to date [1,2,3]. DNA can be damaged by oxidative stress due to SM-induced depletion of endogenous antioxidants [12,13,14,15]. One outcome of SM-induced DNA damage can be an increase in PARP activity resulting in NAD+ depletion, decreased glycolysis, and subsequent protease release, leading to reduced cellular fitness [16]

Methods

Results

Conclusion