Quantifying Replication Stress in Ovarian Cancer Cells Using Single-Stranded DNA Immunofluorescence

Summary

Here, we describe an immunofluorescence-based method to quantify the levels of single-stranded DNA in cells. This efficient and reproducible method can be utilized to examine replication stress, a common feature in several ovarian cancers. Additionally, this assay is compatible with an automated analysis pipeline, which further increases its efficiency.

Abstract

Replication stress is a hallmark of several ovarian cancers. Replication stress can emerge from multiple sources, including double-strand breaks, transcription-replication conflicts, or amplified oncogenes, inevitably resulting in the generation of single-stranded DNA (ssDNA). Quantifying ssDNA, therefore, presents an opportunity to assess the level of replication stress in different cell types and under various DNA-damaging conditions or treatments. Emerging evidence also suggests that ssDNA can be a predictor of responses to chemotherapeutic drugs that target DNA repair. Here, we describe a detailed immunofluorescence-based methodology to quantify ssDNA. This methodology involves labeling the genome with a thymidine analog, followed by the antibody-based detection of the analog at the chromatin under non-denaturing conditions. Stretches of ssDNA can be visualized as foci under a fluorescence microscope. The number and intensity of the foci directly co-relate with the level of ssDNA present in the nucleus. We also describe an automated pipeline to quantify the ssDNA signal. The method is rapid and reproducible. Furthermore, the simplicity of this methodology makes it amenable to high-throughput applications such as drug and genetic screens.

Introduction

Genomic DNA is frequently exposed to multiple assaults from various endogenous and exogenous sources¹. The frequency of endogenous damage directly correlates with the levels of metabolic byproducts, such as reactive oxygen species or aldehydes, which are intrinsically higher in multiple cancer types, including ovarian cancers^2,3. It is imperative that DNA damage is efficiently resolved; otherwise, it can foster genotoxic lesions and, consequently, mutagenesis. The ability of cells to repair genotoxic lesions is reliant on the functionality of error-free DNA repair pathways and the effic....

Protocol

NOTE: The ovarian cancer cell line, OVCAR3, was used in these steps, but this protocol is broadly applicable to multiple other cell lines, including those derived from non-ovarian sources. A schematic of the protocol is shown in Figure 2.

1. Plating the cells

1. Make poly-L-lysine coated coverslips.
   1. Add autoclaved 12 mm diameter coverslips to a 50 mL conical tube with poly-L-lysine solution, and place on a rocker for 15 min.
   2. Aspirate the solution in a tissue culture hood. Wash the coverslips by adding sterile water, and place the tube containing the coverslips ba....

Results

Representative images and the quantification of IdU foci from the nuclei derived from the untreated cells and cells treated with 0.5 mM hydroxyurea for 24 h are shown in Figure 4. Both nuclei are stained and identifiable in the DAPI channel. The analysis of these images consists of quantifying the number of foci in each nucleus. The number of foci is proportional to the degree of replication stress.

Discussion

As was mentioned in the protocol, it is valuable to include a few experimental controls to ensure that the assay is working. These include a no IdU treated sample as well as a no primary antibody treated sample. Both negative controls should yield cells that are stained by DAPI but contain no IdU signal.

Based on the experimental conditions and cell lines used, different antibody dilutions may be needed to obtain the best fluorescent signal. Too much signal may result in an inability to quanti.......

Materials

Name	Company	Catalog Number	Comments
3% Paraformaldehyde (PFA)	Fisher Scientific	NC0179595	10 g sucrose + 100 mL 10X PBS + water to make volume to 925 mL. Add 75 mL 40% Methanol free PFA, mix, and make aliquots of 50 mL before storage Storage: Store in -20 °C
5-iodo-2'-deoxyuridine (IdU)	Sigma Aldrich	I7125-5G	MW = 354.10 g/mol.For 10 mM stock: dissolve 3.541 mg IdU to 1 mL 1 N liquid ammonia Storage: Stored in -20 °C
Anti-BrdU antibody	BD Biosciences	347580	Storage: Store in 4 °C
Anti-mouse Alexa Fluor Plus 488 secondary antibody	Thermo Scientific	A32766	Light sensitive - keep in dark Storage: Store in 4 °C
Bovine Serum Albumin (BSA)	Sigma Aldrich	A7906-100G	Made by adding specific mass to volume of PBS Storage: Store in 4 °C
Circular Cover Glass	Electron Microscopy Sciences	72230-01
NIS GA3 Software	Nikon	77010604
OVCAR3	ATCC	HTB-161	Growth Media: RPMI supplemented with L-glutamine, 0.01 mg/mL bovine insulin; fetal bovine serum to a final concentration of 20% and 1X Pen Strep Storage: Freezing Media: growth media + 5% DMSO and stored in -80 °C
Poly-L-Lysine solution	Sigma Aldrich	P4832-50ML	Storage: Store in 4 °C
ProLong Diamond Antifade Mountant with DAPI	Thermo Scientific	P36962	Storage: Store in 4 °C
Trypsin-EDTA, 0.25%	Genesee Scientific	25-510	Storage: Store in 4 °C
Water, sterile-filtered	Sigma Aldrich	W3500-6X500ML	Storage: Store in 4 °C