Name: quantifying replication stress in ovarian cancer cells using single-stranded dna immunofluorescence
Uploaded: 2023-02-10T14:15:00
Description: Watch this Scientific Journal Video about Quantifying Replication Stress in Ovarian Cancer Cells Using Single-Stranded DNA Immunofluorescence at JoVE.com

PV is supported by the Inaugural Pedal the Cause Grant by the Alvin J. Siteman Cancer Center through The Foundation for Barnes-Jewish Hospital, Pilot Research Grant from Marsha Rivkin Center for Ovarian Cancer Research, Cancer Research Grant from Mary Kay Ash Foundation and V-Foundation. NR is supported by the NIH Cell and Molecular Biology training T32 grant to Washington University, St. Louis.

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
<ol>
	<li>Make poly-L-lysine coated coverslips.
	<ol>
		<li>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.</...

<ol>
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I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ATR+prohibits+replication+catastrophe+by+preventing+global+exhaustion+of+RPA.">ATR prohibits replication catastrophe by preventing global exhaustion of RPA.</a> Cell. 155 (5), 1088-1103 (2013).</li><li>Brickner, J. R., Garzon, J. L., Cimprich, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Walking+a+tightrope:+The+complex+balancing+act+of+R-loops+in+genome+stability.">Walking a tightrope: The complex balancing act of R-loops in genome stability.</a> Molecular Cell. 82 (12), 2267-2297 (2022).</li><li>Cejka, P., Symington, L. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+end+resection:+Mechanism+and+control.">DNA end resection: Mechanism and control.</a> Annual Review of Genetics. 55, 285-307 (2021).</li><li>Kowalczykowski, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+overview+of+the+molecular+mechanisms+of+recombinational+DNA+repair.">An overview of the molecular mechanisms of recombinational DNA repair.</a> Cold Spring Harbor Perspectives in Biology. 7 (11), 016410 (2015).</li><li>Tirman, S., Cybulla, E., Quinet, A., Meroni, A., Vindigni, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PRIMPOL+ready,+set,+reprime.">PRIMPOL ready, set, reprime.</a> Critical Reviews in Biochemistry and Molecular Biology. 56 (1), 17-30 (2021).</li><li>Taglialatela, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=REV1-Pol&#950;+maintains+the+viability+of+homologous+recombination-deficient+cancer+cells+through+mutagenic+repair+of+PRIMPOL-dependent+ssDNA+gaps.">REV1-Pol&#950; maintains the viability of homologous recombination-deficient cancer cells through mutagenic repair of PRIMPOL-dependent ssDNA gaps.</a> Molecular Cell. 81 (19), 4008-4025 (2021).</li><li>Cimprich, K. A., Cortez, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ATR:+An+essential+regulator+of+genome+integrity.">ATR: An essential regulator of genome integrity.</a> Nature Reviews. Molecular Cell Biology. 9 (8), 616-627 (2008).</li><li>Tirman, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temporally+distinct+post-replicative+repair+mechanisms+fill+PRIMPOL-dependent+ssDNA+gaps+in+human+cells.">Temporally distinct post-replicative repair mechanisms fill PRIMPOL-dependent ssDNA gaps in human cells.</a> Molecular Cell. 81 (19), 4026-4040 (2021).</li><li>Cong, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Replication+gaps+are+a+key+determinant+of+PARP+inhibitor+synthetic+lethality+with+BRCA+deficiency.">Replication gaps are a key determinant of PARP inhibitor synthetic lethality with BRCA deficiency.</a> Molecular Cell. 81 (15), 3128-3144 (2021).</li><li>Xu, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CCNE1+copy+number+is+a+biomarker+for+response+to+combination+WEE1-ATR+inhibition+in+ovarian+and+endometrial+cancer+models.">CCNE1 copy number is a biomarker for response to combination WEE1-ATR inhibition in ovarian and endometrial cancer models.</a> Cell Reports. Medicine. 2 (9), 100394 (2021).</li><li>Konstantinopoulos, P. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Replication+stress+biomarker+is+associated+with+response+to+gemcitabine+versus+combined+gemcitabine+and+ATR+inhibitor+therapy+in+ovarian+cancer.">A Replication stress biomarker is associated with response to gemcitabine versus combined gemcitabine and ATR inhibitor therapy in ovarian cancer.</a> Nature Communications. 12, 5574 (2021).</li><li>Buisson, R., Boisvert, J. L., Benes, C. H., Zou, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+but+concerted+roles+of+ATR,+DNA-PK,+and+Chk1+in+countering+replication+stress+during+S+phase.">Distinct but concerted roles of ATR, DNA-PK, and Chk1 in countering replication stress during S phase.</a> Molecular Cell. 59 (6), 1011-1024 (2015).</li><li>Belan, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=POLQ+seals+post-replicative+ssDNA+gaps+to+maintain+genome+stability+in+BRCA-deficient+cancer+cells.">POLQ seals post-replicative ssDNA gaps to maintain genome stability in BRCA-deficient cancer cells.</a> Molecular Cell. 82 (24), 4664-4680 (2022).</li><li>da Costa, A. A. B. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+replication+stress+in+cancer+therapy.">Targeting replication stress in cancer therapy.</a> Nature Reviews. Drug Discovery. 22 (1), 38-58 (2023).</li><li>Chen, R., Wold, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Replication+protein+A:+Single-stranded+DNA's+first+responder:+Dynamic+DNA-interactions+allow+replication+protein+A+to+direct+single-strand+DNA+intermediates+into+different+pathways+for+synthesis+or+repair.">Replication protein A: Single-stranded DNA's first responder: Dynamic DNA-interactions allow replication protein A to direct single-strand DNA intermediates into different pathways for synthesis or repair.</a> BioEssays. 36 (12), 1156-1161 (2014).</li><li>Bhat, K. P., Cortez, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RPA+and+RAD51:+Fork+reversal,+fork+protection,+and+genome+stability.">RPA and RAD51: Fork reversal, fork protection, and genome stability.</a> Nature Structural &amp; Molecular Biology. 25 (6), 446-453 (2018).</li><li>Mar&#233;chal, A., Zou, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RPA-coated+single-stranded+DNA+as+a+platform+for+post-translational+modifications+in+the+DNA+damage+response.">RPA-coated single-stranded DNA as a platform for post-translational modifications in the DNA damage response.</a> Cell Research. 25 (1), 9-23 (2015).</li></ol>

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...

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 cancers2,3. 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 efficient regulation of cell cycle progression in response to DNA damage. Notably, many ovarian cancers bear functionally inactivating mutations in p53 and, thus, have a defective G1/S checkpoint, leading the cells to initiate DNA replication despite the presence of unrepaired genomic lesions4,5. The degree of DNA damage in ovarian cancers is further compounded by the observation that more than 50% of high-grade serous ovarian carcinoma (HGSOC) have defects in BRCA1- and BRCA2-mediated homologous recombination, the error-free DNA repair pathway, and around 20% have amplification in the gene CCNE1, which prematurely pushes G1 cells into the S-phase6. Together the high frequency of endogenous DNA damage, defective checkpoints, and malfunctioning repair pathways exponentially enhance the accumulation of genomic lesions in ovarian cancers. These lesions can serve as impediments to the progression of critical cellular processes such as DNA replication and transcription. As discussed below, such impediments catalyze the generation of single-stranded DNA (ssDNA) in cells.
The double helix of DNA is critical for safeguarding the genome from multiple mutagenic processes, such as spontaneous depurination and depyrimidination, the activity of cytosine deaminases, and oxidative DNA damage1,7. In contrast, ssDNA is highly vulnerable to these mutational events. Multiple processes in cells can result in the generation of ssDNA (Figure 1). These include the following:
(i) Stalling of the DNA replication machinery: This leads to an uncoupling of the DNA helicase and polymerase, leaving stretches of ssDNA8,9.
(ii) Stalling of the transcription machinery: Persistent stalling of RNA polymerase leads to the generation of three-stranded hybrid DNA/RNA structures called R-loops. R-loop formation exposes the displaced, non-transcribed DNA as a single strand10.
(iii) DNA end-resection: The initiation of homology-directed repair requires the generation of a 3&#39; ssDNA to catalyze the search for a homologous sequence11.
(iv) D-loop: Strand invasion during homologous recombination can result in the displacement of the non-template complementary strand, resulting in ssDNA12.
(v) Replication-coupled gaps: During DNA replication, lagging strand synthesis happens in a discontinuous fashion, whereby Okazaki fragments are first generated and then ligated. A delay or defect in processing the Okazaki fragments can also result in ssDNA formation. Finally, if the replication fork on a leading strand encounters a stalling lesion, DNA polymerase, and primase, PRIMPOL can reprime the synthesis downstream, leaving an ssDNA gap behind13,14.
Evidently, most of these events either happen when the DNA replication machinery faces genomic lesions or during replication-coupled repair, suggesting that higher DNA damage leads to increased levels of ssDNA. As many of these events are replication-associated, the formation of ssDNA is considered the marker of &#34;replication stress&#34; in cells15,16.
Here, we describe an assay that can be used to reliably quantify ssDNA in cells. The simplicity, reproducibility, and cost benefits of this approach make it amenable to be used for assessing the replication-stress response in cells. Emerging studies have revealed that the level of ssDNA can also be a predictor of responses to chemotherapy, such as inhibitors of PARP1/2 enzymes, ATR, and Wee1 kinase17,18,19,20,21. These inhibitors are being pursued in the treatment regimen of several HGSOCs22. Therefore, this assay can also be a useful tool to predict chemotherapeutic responses in ovarian cancer cells.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>3% Paraformaldehyde (PFA)</td>
			<td>Fisher Scientific</td>
			<td>NC0179595</td>
			<td>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 &#176;C</td>
		</tr>
		<tr>
			<td>5-iodo-2&#39;-deoxyuridine (IdU)</td>
			<td>Sigma Aldrich</td>
			<td>I7125-5G</td>
			<td>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 &#176;C</td>
		</tr>
		<tr>
			<td>Anti-BrdU antibody</td>
			<td>BD Biosciences</td>
			<td>347580</td>
			<td>Storage: Store in 4 &#176;C</td>
		</tr>
		<tr>
			<td>Anti-mouse Alexa Fluor Plus 488 secondary antibody</td>
			<td>Thermo Scientific</td>
			<td>A32766</td>
			<td>Light sensitive - keep in dark 
			Storage: Store in 4 &#176;C</td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin (BSA)</td>
			<td>Sigma Aldrich</td>
			<td>A7906-100G</td>
			<td>Made by adding specific mass to volume of PBS 
			Storage: Store in 4 &#176;C</td>
		</tr>
		<tr>
			<td>Circular Cover Glass&#160;</td>
			<td>Electron Microscopy Sciences</td>
			<td>72230-01</td>
			<td></td>
		</tr>
		<tr>
			<td>NIS GA3 Software&#160;</td>
			<td>Nikon&#160;</td>
			<td>77010604</td>
			<td></td>
		</tr>
		<tr>
			<td>OVCAR3</td>
			<td>ATCC</td>
			<td>HTB-161</td>
			<td>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 &#176;C</td>
		</tr>
		<tr>
			<td>Poly-L-Lysine solution</td>
			<td>Sigma Aldrich</td>
			<td>P4832-50ML</td>
			<td>Storage: Store in 4 &#176;C</td>
		</tr>
		<tr>
			<td>ProLong Diamond Antifade Mountant with DAPI</td>
			<td>Thermo Scientific</td>
			<td>P36962</td>
			<td>Storage: Store in 4 &#176;C</td>
		</tr>
		<tr>
			<td>Trypsin-EDTA, 0.25%</td>
			<td>Genesee Scientific</td>
			<td>25-510</td>
			<td>Storage: Store in 4 &#176;C</td>
		</tr>
		<tr>
			<td>Water, sterile-filtered</td>
			<td>Sigma Aldrich</td>
			<td>W3500-6X500ML</td>
			<td>Storage: Store in 4 &#176;C</td>
		</tr>
	</tbody>
</table>

quantifying replication stress in ovarian cancer cells using single-stranded dna immunofluorescence

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.

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.
<img alt="Figure 1" cl...

Watch this Scientific Journal Video about Quantifying Replication Stress in Ovarian Cancer Cells Using Single-Stranded DNA Immunofluorescence at JoVE.com

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

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.

Replication stress is a hallmark of several ovarian cancers. Replication stress can emerge from multiple sources, including double-strand breaks, ...

Our protocol presents a highly efficient and easily reproducible method to quantify single stranded DNA, which is a marker of replication stress in a variety of cell lines. Additionally, its simplicity allows for high throughput applications and screens. In particular, this method allows for rapid quantification of a replication stress, a hallmark of several ovarian cancers.It is also amenable to a host automatic analysis software, further increasing efficiency. Single stranded DNA is now being actively considered as a biomarker for response to chemotherapy targeting DNA repair pathways. Hence, this method is highly applicable in that scenario.Replication stress exists beyond ovarian cancer contexts or BRCA1, BRCA2 deficient cancers. And thus, this method can be applied to a wide array of other cell types or disease contexts. To begin, make polylysine-coated cover slips by adding autoclaved 12 millimeter diameter cover slips and polylysine solution to a 50 milliliter conical tube, and place on a rocker for 15 minutes.Aspirate the solution on a tissue culture hood. Wash the cover slips by adding sterile water, and place the tube containing the cover slips back on the rocker for five minutes. After washing the cover slips three times, aspirate water from the tube and spread the cover slips on a sterile dish.Aspirate the remaining water and let them dry in the tissue culture hood for one hour or until no water droplets remain. Once dry, seal the dish with Parafilm and place it at 4 degrees Celsius. To prevent the detachment of the cells from the cover slips during the pre-extraction step, place one polylysine-coated cover slip in each well of a 24-well plate.Once the OVCAR3 cells reach 70 to 80%confluency, aspirate the medium from the plate and wash the cells with 5 to 7 milliliters of PBS. Aspirate the PBS from the plate. After aspirating the PBS, add 1 milliliter of 0.25%trypsin and place the dish in the incubator at 37 degrees Celsius for 8 to 10 minutes or until cells lift off from the bottom of the plate.Collect the cells with 5 to 10 milliliters of medium and add the cell suspension to a conical tube. Count the cells manually with a hemocytometer using standard procedures. Next, dilute the cell suspension, and obtain a number that will yield 70 to 80%confluency after three populations.Add 1 milliliter of cell suspension on polylysine cover slips placed in the wells of a 24-well plate, and grow the cells in the culture medium under standard conditions. After one population doubling, aspirate the existing media from the plate and pulse the cells with 10 micromolar IdU for the two subsequent population doublings. To harvest the cells, replace the medium with ice cold 0.5%PBSTx on ice for five minutes.For fixation of cells, aspirate PBSTx and incubate the cells for 15 minutes with 3%paraformaldehyde at room temperature. After three to four washes with PBS, keep fixed cells at 4 degrees Celsius until further use. After fixation, permeabilize the cells using 0.5%PBSTx on ice for five minutes, ensuring to cover the entire cover slip.After washing the cells three to four times with 1 milliliter of 0.2%PBST at room temperature, aspirate the PBST and block the samples using 5%BSA made in PBS for 30 minutes at room temperature. For immunostaining, prepare a unified chamber by putting a wet paper towel on a flat bottom Tupperware. Cover the 24-well plate lid with Parafilm.Place it in the humidified chamber and lay down the cover slips on the plate lid. Add 60 microliters of diluted anti-BrdU primary mouse antibody on the top of the cover slip. Alternatively, to reduce the antibody from drying and use less volume, place a drop of diluted antibody on the Parafilm and flip the cover slip onto it.Then incubate for one hour at 37 degrees Celsius. After incubation, aspirate the primary antibody. Return the cover slips back to a 24-well plate and wash them with 0.2%PBST four times.Add diluted secondary antibody on the cover slip as demonstrated earlier, and incubate for one hour in the dark at room temperature. Label a microscope slide and mount the cover slip on the slides with DAPI mounting medium. Store slides in the dark at room temperature for 24 hours if the mounting medium needs to be cured or hardened.The nuclei derived from the untreated and treated cells with 0.5 millimolar hydroxyurea were stained and identifiable in the DAPI and IdU channels. 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.It is important to consider the doubling time of the cell line of choice as the time of IdU pulsing may vary for longer or shorter doubling periods. Alternatively, we can probe cells for replication protein A to confirm the results and assess various replication stress responses in cells. This technique is used for many cell lines in tandem with any DNA damage-inducing agent to examine the accumulation of single-strained DNA.Thus, this method is widely accessible and applicable.

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Visualizing DNA Damage Repair Proteins in Patient-Derived Ovarian Cancer Organoids via Immunofluorescence Assays

Visualizing DNA Damage Repair Proteins in Patient-Derived Ovarian Cancer Organoids via Immunofluorescence Assays

Immunofluorescence is one of the most widely used techniques to visualize target antigens with high sensitivity and specificity, allowing for the accurate ...