Name: immunofluorescence imaging of dna damage and repair foci in human colon cancer cells
Uploaded: 2020-06-09T15:00:00
Description: Watch this Scientific Journal Video about Immunofluorescence Imaging of DNA Damage and Repair Foci in Human Colon Cancer Cells at JoVE.com

Neutron-mixed beam composed of neutron/gamma radiation was accessed from the Maria research reactor in the National Centre for Nuclear Research in Poland. K.M.O. was supported by the National Science Centre, Poland (Miniatura 2) grant no. #2018/02/X/NZ5/02849.

1. Preparation of cell culture and experimental set-up
<ol>
	<li>Maintain human colon cancer cells, HCT-116 as recommended by the supplier, in monolayers in 75 cm2 culture flasks containing 10 mL of formulated McCoy&#39;s 5a Medium Modified, supplemented with 10% fetal bovine serum and 1% antibiotic antimycotic solution.</li>
	<li>Grow cells in a humidified 5% CO2 environment at 37 &#176;C until 70% of confluency with medium renewal every 2 to 3 days.</li>
	<li>To gain BNCT beam (e.g., neutron bea...

<ol>
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J., 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=Complex+DNA+Damage+Induced+by+High+Linear+Energy+Transfer+Alpha-Particles+and+Protons+Triggers+a+Specific+Cellular+DNA+Damage+Response.">Complex DNA Damage Induced by High Linear Energy Transfer Alpha-Particles and Protons Triggers a Specific Cellular DNA Damage Response.</a> International Journal of Radiation Oncology, Biology, Physics. 100 (3), 776-784 (2018).</li><li>Kondo, N., 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=DNA+damage+induced+by+boron+neutron+capture+therapy+is+partially+repaired+by+DNA+ligase+IV.">DNA damage induced by boron neutron capture therapy is partially repaired by DNA ligase IV.</a> Radiation Environmental Biophysics. 55 (1), 89-94 (2016).</li><li>Sakai, W., Sugasawa, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+Damage+Recognition+and+Repair+in+Mammalian+Global+Genome+Nucleotide+Excision+Repair.">DNA Damage Recognition and Repair in Mammalian Global Genome Nucleotide Excision Repair.</a> DNA Replication, Recombination, and Repair: Molecular Mechanisms and Pathology. , 155-174 (2016).</li><li>Rodriguez, C., 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=In+vitro+studies+of+DNA+damage+and+repair+mechanisms+induced+by+BNCT+in+a+poorly+differentiated+thyroid+carcinoma+cell+line.">In vitro studies of DNA damage and repair mechanisms induced by BNCT in a poorly differentiated thyroid carcinoma cell line.</a> Radiation Environmental Biophysics. 57 (2), 143-152 (2018).</li><li>Sollazzo, 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=Live+Dynamics+of+53BP1+Foci+Following+Simultaneous+Induction+of+Clustered+and+Dispersed+DNA+Damage+in+U2OS+Cells.">Live Dynamics of 53BP1 Foci Following Simultaneous Induction of Clustered and Dispersed DNA Damage in U2OS Cells.</a> International Journal of Molecular Sciences. 19 (2), (2018).</li><li>Jeggo, P., L&#246;brich, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Radiation-induced+DNA+damage+responses.">Radiation-induced DNA damage responses.</a> Radiation Protection Dosimetry. 122 (1-4), 124-127 (2006).</li><li>Sigurdsson, S., Van Komen, S., Petukhova, G., Sung, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Homologous+DNA+pairing+by+human+recombination+factors+Rad51+and+Rad54.">Homologous DNA pairing by human recombination factors Rad51 and Rad54.</a> The Journal of Biological Chemistry. 277 (45), 42790-42794 (2002).</li><li>Choi, E. H., Yoon, S., Hahn, Y., Kim, K. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+Dynamics+of+Rad51+and+Rad54+in+Response+to+Postreplicative+Stress+and+DNA+Damage+in+HeLa+Cells.">Cellular Dynamics of Rad51 and Rad54 in Response to Postreplicative Stress and DNA Damage in HeLa Cells.</a> Molecules and Cells. 40 (2), 143-150 (2017).</li><li>Burma, S., Chen, B. P., Murphy, M., Kurimasa, A., Chen, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ATM+phosphorylates+histone+H2AX+in+response+to+DNA+double-strand+breaks.">ATM phosphorylates histone H2AX in response to DNA double-strand breaks.</a> The Journal of Biological Chemistry. 276 (45), 42462-42467 (2001).</li><li>Nakamura, T. M., Du, L. L., Redon, C., Russell, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histone+H2A+phosphorylation+controls+Crb2+recruitment+at+DNA+breaks,+maintains+checkpoint+arrest,+and+influences+DNA+repair+in+fission+yeast.">Histone H2A phosphorylation controls Crb2 recruitment at DNA breaks, maintains checkpoint arrest, and influences DNA repair in fission yeast.</a> Molecular and Cellular Biology. 24 (14), 6215-6230 (2004).</li><li>Kuo, L. J., Yang, L. X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gamma-H2AX+-+a+novel+biomarker+for+DNA+double-strand+breaks.">Gamma-H2AX - a novel biomarker for DNA double-strand breaks.</a> In Vivo. 22 (3), 305-309 (2008).</li><li>Sage, E., Shikazono, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Radiation-induced+clustered+DNA+lesions:+Repair+and+mutagenesis.">Radiation-induced clustered DNA lesions: Repair and mutagenesis.</a> Free Radical Biology and Medicine. 107, 125-135 (2017).</li><li>Boguszewska, K., Szewczuk, M., Urbaniak, S., Karwowski, B. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Review:+immunoassays+in+DNA+damage+and+instability+detection.">Review: immunoassays in DNA damage and instability detection.</a> Cellular and Molecular Life Sciences. 76 (23), 4689-4704 (2019).</li><li>M&#248;ller, P., 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=Potassium+bromate+as+positive+assay+control+for+the+Fpg-modified+comet+assay.">Potassium bromate as positive assay control for the Fpg-modified comet assay.</a> Mutagenesis. , (2020).</li><li>Sommer, S., Buraczewska, I., Kruszewski, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Micronucleus+Assay:+The+State+of+Art,+and+Future+Directions.">Micronucleus Assay: The State of Art, and Future Directions.</a> International Journal of Molecular Sciences. 21 (4), 1534 (2020).</li><li>Bennett, B. T., Bewersdorf, J., Knight, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunofluorescence+imaging+of+DNA+damage+response+proteins:+optimizing+protocols+for+super-resolution+microscopy.">Immunofluorescence imaging of DNA damage response proteins: optimizing protocols for super-resolution microscopy.</a> Methods. 48 (1), 63-71 (2009).</li><li>Cheng, L., 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=Simultaneous+induction+of+dispersed+and+clustered+DNA+lesions+compromises+DNA+damage+response+in+human+peripheral+blood+lymphocytes.">Simultaneous induction of dispersed and clustered DNA lesions compromises DNA damage response in human peripheral blood lymphocytes.</a> PLoS One. 13 (10), 0204068 (2018).</li><li>Paull, T. T., 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+critical+role+for+histone+H2AX+in+recruitment+of+repair+factors+to+nuclear+foci+after+DNA+damage.">A critical role for histone H2AX in recruitment of repair factors to nuclear foci after DNA damage.</a> Current Biology. 10 (15), 886-895 (2000).</li><li>Okumura, 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=Relative+biological+effects+of+neutron+mixed-beam+irradiation+for+boron+neutron+capture+therapy+on+cell+survival+and+DNA+double-strand+breaks+in+cultured+mammalian+cells.">Relative biological effects of neutron mixed-beam irradiation for boron neutron capture therapy on cell survival and DNA double-strand breaks in cultured mammalian cells.</a> Journal of Radiation Research. 54 (1), 70-75 (2013).</li><li>Dagrosa, M. 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=First+evaluation+of+the+biologic+effectiveness+factors+of+boron+neutron+capture+therapy+(BNCT)+in+a+human+colon+carcinoma+cell+line.">First evaluation of the biologic effectiveness factors of boron neutron capture therapy (BNCT) in a human colon carcinoma cell line.</a> International Journal of Radiation Oncology, Biology, Physics. 79 (1), 262-268 (2011).</li><li>Dagrosa, 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=Studies+for+the+application+of+boron+neutron+capture+therapy+to+the+treatment+of+differentiated+thyroid+cancer.">Studies for the application of boron neutron capture therapy to the treatment of differentiated thyroid cancer.</a> Applied Radiation and Isotopes. 69 (12), 1752-1755 (2011).</li><li>Reynolds, P., 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=The+dynamics+of+Ku70/80+and+DNA-PKcs+at+DSBs+induced+by+ionizing+radiation+is+dependent+on+the+complexity+of+damage.">The dynamics of Ku70/80 and DNA-PKcs at DSBs induced by ionizing radiation is dependent on the complexity of damage.</a> Nucleic Acids Research. 40 (21), 10821-10831 (2012).</li><li>Rasche, L., 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=Analysis+of+Lymphocytic+DNA+Damage+in+Early+Multiple+Sclerosis+by+Automated+Gamma-H2AX+and+53BP1+Foci+Detection:+A+Case+Control+Study.">Analysis of Lymphocytic DNA Damage in Early Multiple Sclerosis by Automated Gamma-H2AX and 53BP1 Foci Detection: A Case Control Study.</a> PLoS One. 11 (1), 0147968 (2016).</li><li>Ban&#225;th, J. P., Macphail, S. H., Olive, P. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Radiation+sensitivity,+H2AX+phosphorylation,+and+kinetics+of+repair+of+DNA+strand+breaks+in+irradiated+cervical+cancer+cell+lines.">Radiation sensitivity, H2AX phosphorylation, and kinetics of repair of DNA strand breaks in irradiated cervical cancer cell lines.</a> Cancer Research. 64 (19), 7144-7149 (2004).</li><li>Barth, R. F., 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=Current+status+of+boron+neutron+capture+therapy+of+high+grade+gliomas+and+recurrent+head+and+neck+cancer.">Current status of boron neutron capture therapy of high grade gliomas and recurrent head and neck cancer.</a> Radiatiation Oncology. 7, 146 (2012).</li><li>Mirzaei, H. 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The frequencies of foci, immunochemically stained for &#947;-H2AX and 53BP1 are commonly used in radiobiology and are associated with DNA-DSB number and are considered as efficient and sensitive markers for monitoring the induction and repair of DNA-DSBs19. The co-staining procedure of &#947;-H2AX and 53BP1 is a standard procedure for the detection of DNA-DSBs. Formation of &#947;-H2AX foci is associated with the recruitment of 53BP1, a regulator of the DNA damage response23</sup...

Radiation-induced DNA damage response activated by neutron mixed-beam used in boron neutron capture therapy (BNCT) has not been fully determined. This protocol provides a step-by-step procedure to perform the detection of radiation-induced foci (RIF) of repair proteins by immunofluorescence staining e.g., in human colon cancer cell line after irradiation with the neutron-mixed beam.
Ionizing radiation (IR) induces a multitude of different types of DNA damage (DNA-DSBs, DNA-SSBs, DNA base damage) out of which DNA double-strand breaks are the most genotoxic DNA lesions1. Unrepaired breaks can cause cell death, while misrepaired breaks raises the probability of chromosome rearrangement, mutagenesis, and loss of decisive genetic information. The damage response pathways responding to DSBs include pathways of DNA repair like non-homologous end joining (NHEJ), a mechanism that requires Ku 70/80, DNA&#8211;PKcs, Xrcc4, and DNA ligase IV as key factors2,3,4,5,6. In mammals, the second main DNA repair pathway is homologous recombination repair (HRR) pathway which requires key components - the Rad52 epistasis gene family&#8211; Rad51, Rad52, Rad54, Rad55, Rad57 and Rad587. Rad51 and Rad54 are key human recombination factors involved in repair mechanisms related to the replication stress and DNA breaks in eukaryotes. Interestingly, it was observed that the downregulation of the HRR pathway enhances the NHEJ pathway which is error-prone pointing at HR pathway relevance for the genome stability8.
The first step of DSBs formation is the phosphorylation of the &#947;-H2AX histone at Ser-139 by Ataxia telangiectasia mutated kinase (ATM) from the PI3 kinase family7,8. Interestingly, H2AX phosphorylation can be visualized easily by immunofluorescence technique as foci (&#947;-H2AX foci) using antibodies specific for phosphorylated H2AX6. There is a 1:1 relationship between the number of DSBs and &#947;-H2AX foci, therefore, DSB marker, &#947;-H2AX, is studied extensively through the characterization of foci formation, size, and quantity6,7,8,9,10,11. Formation of &#947;-H2AX foci leads to the recruitment and accumulation of DNA damage response (DDR) proteins and chromatin-modifying factors, such as 53BP1 (p53 binding protein 1), MDC1 (mediator of DNA damage checkpoint), BRCA1, Mre11/Rad50/Nbs1, PARP-1, and many others repair factors thus forming radiation-induced foci (RIF). All these proteins co-localize with &#947;-H2AX through direct or indirect binding1,11,12.
It is important to detect DNA damage and repair with a proper sensitive test, therefore, more attention is paid to the development of highly precise techniques13. In the context of DNA damage and repair studies, the methodology is crucial for sensitive detection of genome DNA damage, description of damage category, and quantification of DNA damage and repair mechanisms13. To detect single cells with damaged DNA, comet assay is used commonly in radiobiological studies14. Other available cytogenetic methods recognize chromosomal aberrations, including dicentrics, translocations, acentric fragments, rings, and chromatid type aberrations, and micronucleus chromosomal damage (Mn). The most frequently used method in radiobiology, especially in biological dosimetry, is the dicentric chromosome assay due to its high specificity for radiation15. For example, PCR, a classic molecular method, cannot recognize the type of detected DNA damage. In this case, the immunometric methods pass the sensitivity level because reactions are specific between the antigen and the antibody. Immunofluorescence imaging provides visual evidence for the appearance of different proteins in distinct foci in response to DNA damaging agents like ionizing radiation16. However, the activation levels of damage and repair proteins&#8217; mRNA levels are easily detectable by real-time PCR which is a proper quantitative method for further molecular studies in the context of DNA damage response17.
Taking into account that &#947;-H2AX foci attract repair factors18, to monitor DNA damage and repair at the cellular level, we have developed a reliable immunofluorescence staining procedure, based on the analysis of the representative repair protein foci from the NHEJ pathway (DNA-PKcs) and Rad52 from the HRR pathway.
Here, we propose the use of immunodetection for these proteins as the efficient and sensitive procedure for monitoring DNA-DSB induction and repair. Up till now, there have been no available data on DNA-DSBs based on foci of repair proteins at the cellular level after the neutron mixed-beam irradiation for BNCT, except &#947;-H2AX and 53BP1 markers19. We suggest the adaptation of the HCT-116 colon cancer cell line, as it is rich itself in DSBs foci, as a standard cell line for DNA damage analysis, because RIFs are easily detectable. This adherent cell line is easy to maintain and proper for irradiation procedures. The proposed procedure is based on a huge amount of previous studies related to the general immunofluorescence procedure of &#947;-H2AX staining. However, it includes all details regarding the selection of appropriate antibodies with tested dilutions for each representative protein belonging to each repair pathway. Moreover, it describes the utilization of a unique neutron-mixed beam used in BNCT therapy. However, we recommend to extend the studies with both methods, immunofluorescence staining firstly and then, with high-cost molecular analysis as performed previously4,17.

<table>
	<tbody>
		<tr>
			<td>12 mm Coverslips</td>
			<td>VWR</td>
			<td>89015-725</td>
			<td></td>
		</tr>
		<tr>
			<td>35 mm Petri dishes</td>
			<td>Sarstedt</td>
			<td>7.183.390.000</td>
			<td></td>
		</tr>
		<tr>
			<td>4-Borono-L-phenylalanine</td>
			<td>SIGMA-ALDRICH</td>
			<td>17755</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibiotic-Antimycotic (100X)</td>
			<td>Gibco</td>
			<td>15240062</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-DNA PKcs (phospho S2056) antibody - ChIP Grad</td>
			<td>Abcam</td>
			<td>AB18192</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Mouse IgG (whole molecule)&#8211;FITC antibody produced in goat</td>
			<td>SIGMA-ALDRICH</td>
			<td>F0257</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-phospho-Histone H2A.X (Ser139) Antibody, clone JBW301</td>
			<td>MerckMillipore</td>
			<td>05-636</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-RAD52 antibody</td>
			<td>Abcam</td>
			<td>AB117097</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin Fraction V (BSA)</td>
			<td>Roche</td>
			<td>BSAV-RO</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI (4&#39;,6-Diamidino-2-Phenylindole, Dihydrochloride)</td>
			<td>ThermoFisher SCIENTIFIC</td>
			<td>D1306</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (Heat Inactivated)</td>
			<td>SIGMA-ALDRICH</td>
			<td>F9665</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat Anti-Rabbit IgG H&#38;L (Alexa Fluor 488)</td>
			<td>Abcam</td>
			<td>AB150077</td>
			<td></td>
		</tr>
		<tr>
			<td>HCT-116 cell line</td>
			<td>ATCC</td>
			<td>CCL-247&#8482;</td>
			<td></td>
		</tr>
		<tr>
			<td>ImageJ</td>
			<td>National Institute of Health (NIH)</td>
			<td>https://imagej.nih.gov/ij/</td>
			<td></td>
		</tr>
		<tr>
			<td>Image Pro</td>
			<td>Media cybernetics</td>
			<td>http://www.mediacy.com/imagepro</td>
			<td></td>
		</tr>
		<tr>
			<td>LUNA II Automated Cell Counter</td>
			<td>Logos Biosystems</td>
			<td>L40002</td>
			<td></td>
		</tr>
		<tr>
			<td>McCoy&#8217;s 5A Medium (Modified, with L-glutamine and sodium bicarbonate)</td>
			<td>SIGMA-ALDRICH</td>
			<td>M9309</td>
			<td></td>
		</tr>
		<tr>
			<td>microscope slides</td>
			<td>ThermoFisher SCIENTIFIC</td>
			<td>B-1198</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline (PBS)</td>
			<td>Hirszfeld Institute of Immunology and Experimental Therapy, PAS</td>
			<td>20.59.52.0</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>SIGMA-ALDRICH</td>
			<td>X100</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan Blue Stain, 0.4%</td>
			<td>Logos Biosystems</td>
			<td>T13001</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA solution 0.25%</td>
			<td>SIGMA-ALDRICH</td>
			<td>T4049</td>
			<td></td>
		</tr>
	</tbody>
</table>

immunofluorescence imaging of dna damage and repair foci in human colon cancer cells

The purpose of the manuscript is to provide a step-by-step protocol for performing immunofluorescence microscopy to study the radiation-induced DNA damage response induced by neutron-gamma mixed-beam used in boron neutron capture therapy (BNCT). Specifically, the proposed methodology is applied for the detection of repair proteins activation which can be visualized as foci using antibodies specific to DNA double-strand breaks (DNA-DSBs). DNA repair foci were assessed by immunofluorescence in colon cancer cells (HCT-116) after irradiation with the neutron-mixed beam. DNA-DSBs are the most genotoxic lesions and are repaired in mammalian cells by two major pathways: non-homologous end-joining pathway (NHEJ) and homologous recombination repair (HRR). The frequencies of foci, immunochemically stained, for commonly used markers in radiobiology like &#947;-H2AX, 53BP1 are associated with DNA-DSB number and are considered as efficient and sensitive markers for monitoring the induction and repair of DNA-DSBs. It was established that &#947;-H2AX foci attract repair proteins, leading to a higher concentration of repair factors near a DSB. To monitor DNA damage at the cellular level, immunofluorescence analysis for the presence of DNA-PKcs representative repair protein foci from the NHEJ pathway and Rad52 from the HRR pathway was planned. We have developed and introduced a reliable immunofluorescence staining protocol for the detection of radiation-induced DNA damage response with antibodies specific for repair factors from NHEJ and HRR pathways and observed radiation-induced foci (RIF). The proposed methodology can be used for investigating repair protein that is highly activated in the case of neutron-mixed beam radiation, thereby indicating the dominance of the repair pathway.

Firstly, we performed an analysis of a standard marker of detection of DNA-DSBs, &#947;H2AX foci in colon cancer cells, non-radiated, and irradiated with the neutron-mixed beam. &#947;-H2AX foci appear as distinct fluorescent dots and show the formation of DNA-DSBs (as each &#947;-H2AX fluorescent dot represents a single DSB) (see Figure 1).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61399/61399fig01.jpg" /> 
<...

Watch this Scientific Journal Video about Immunofluorescence Imaging of DNA Damage and Repair Foci in Human Colon Cancer Cells at JoVE.com

Immunofluorescence Imaging of DNA Damage and Repair Foci in Human Colon Cancer Cells

Radiation-induced DNA damage response activated by neutron mixed-beam used in boron neutron capture therapy (BNCT) has not been fully established. This protocol provides a step-by-step procedure to detect radiation-induced foci (RIF) of repair proteins by immunofluorescence staining in human colon cancer cell lines after irradiation with the neutron-mixed beam.

The purpose of the manuscript is to provide a step-by-step protocol for performing immunofluorescence microscopy to study the radiation-induced DNA damage ...

This protocol provides a step-by-step procedure to detect radiation-induced foci of repair proteins by immunofluorescence staining in human colon cancer cell lines after irradiation with the neutron-mixed beam. Immunofluorescence imaging is a sensitive method and provides visual evidence for the appearance of repair pathway proteins in foci in response to DNA damaging-agents, like ionizing radiation. Neutron-mixed beam is used in boron neutron capture therapy, BNCT, and the radiation-induced DNA damage response has not been fully established.Our protocol can also be useful for analysis of biological effects at the cellular level due to radiation by other high-LET beams, like protons using proton beam therapy or carbon ions using hadron therapy. After irradiation, remove the medium from the attached cells, and wash them once with 2.5 milliliters of PBS. Then fix the cells with one milliliter of 70%ethanol for 10 minutes.To permeabilize the cells, remove the ethanol, and wash them with 2.5 milliliters of PBS. Gently add one milliliter of 2%Triton X-100 in PBS to cover the coverslips, and incubate the cells for five minutes at room temperature. Wash the cells three times with PBS, and block the permeabilization with one milliliter of 2%BSA diluted in PBS.Then incubate the cells for a minimum of 30 minutes with the 2%BSA. To stain the cells, add the proper amount of primary antibody diluted in PBS with BSA, as recommended in the text manuscript. Then cover the Petri dish, and place it in a plastic box with moisturized lignin to maintain humidity.Incubate it for 30 minutes at 37 degrees Celsius. After the incubation, perform three washes with PBS, and add the proper amount of secondary antibody. Return the dish to the plastic box with moisturized lignin, and incubate it for at least 30 minutes at 37 degrees Celsius.Then repeat the washes with PBS, and add 100 microliters of DAPI diluted to a final concentration of one microgram per milliliter to counterstain the nuclei. Incubate the cells for a maximum of two minutes at room temperature, and repeat the washes with PBS. After the last wash, remove the PBS, and gently put a coverslip on top of the mounting medium, avoiding the formation of air bubbles.Seal the edges of the coverslip with nail polish, and allow the mounting medium to harden before performing fluorescence microscopy. Gamma-H2AX foci, which are standard markers for DNA double-stranded breaks, were detected in neutron-mixed, beam-irradiated, and non-irradiated colon cancer cells. The foci appear as distinct fluorescent dots.A higher diameter of gamma-H2AX foci was observed in the irradiated cells. Moreover, a single track of a high-LET alpha particle was detected crossing the nuclei. Immunofluorescence microscopy was used to detect representative repair proteins Rad52 and DNA-dependent protein kinase.A high mean value of DNA-dependent protein kinase foci diameter was measured at DNA breaks after irradiation in comparison with control cells. Clustered DNA double-stranded breaks were observed in irradiated cells as more complex, larger, and clustered foci with higher intensity. The most important stage of immunofluorescence staining is fixation of cells and permeabilization of the cell membrane.These steps are required to allow antibodies to easily penetrate both into those cells and intracellular repair proteins. This procedure gives information about the activation of each repair pathway at a cellular level. The results should then be confirmed at a molecular level using an assay, such as real-time PCR.This technique makes it possible for researchers to explore radiation-induced DNA damage response, which has not been fully determined in the case of different beams using anticancer therapies.

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JoVE Journal

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Cell Biology

Cancer Research

Unit 1

DNA Replication and Repair

SCIENTISTS IN ACTION

Evaluating In Vitro DNA Damage Using Comet Assay

DNA damage is a common phenomenon for each cell during its lifespan, and is defined as an alteration of the chemical structure of genomic DNA. Cancer therapies, such as radio- and chemotherapy, introduce enormous amount of additional DNA damage, leading to cell cycle arrest and apoptosis to limit cancer progression. Quantitative assessment of DNA damage during experimental cancer therapy is a key step to justify the effectiveness of a genotoxic agent. In this study, we focus on a single cell electrophoresis assay, also known as the comet assay, which can quantify single and double-strand DNA breaks in vitro. The comet assay is a DNA damage quantification method that is efficient and easy to perform, and has low time/budget demands and high reproducibility. Here, we highlight the utility of the comet assay for a preclinical study by evaluating the genotoxic effect of olaparib/temozolomide combination therapy to U251 glioma cells.

Evaluating In Vitro DNA Damage Using Comet Assay

The comet assay is an efficient method to detect DNA damage including single and double-stranded DNA breaks. We describe alkaline and neutral comet assays to measure DNA damage in cancer cells to evaluate the therapeutic effect of chemotherapy.

DNA damage is a common phenomenon for each cell during its lifespan, and is defined as an alteration of the chemical structure of genomic DNA. Cancer ...

Immunofluorescence Microscopy of &#947;H2AX and 53BP1 for Analyzing the Formation and Repair of DNA Double-strand Breaks

DNA double-strand breaks (DSB) are serious DNA lesions. Analysis of the formation and repair of DSB is relevant in a broad spectrum of research areas including genome integrity, genotoxicity, radiation biology, aging, cancer, and drug development. In response to DSB, the histone H2AX is phosphorylated at Serine 139 in a region of several megabase pairs forming discrete nuclear foci detectable by immunofluorescence microscopy. In addition, 53BP1 (p53 binding protein 1) is another important DSB-responsive protein promoting repair of DSB by nonhomologous end-joining while preventing homologous recombination. According to the specific functions of &#947;H2AX and 53BP1, the combined analysis of &#947;H2AX and 53BP1 by immunofluorescence microscopy may be a reasonable approach for a detailed analysis of DSB. This manuscript provides a step-by-step protocol supplemented with methodical notes for performing the technique. Specifically, the influence of the cell cycle on &#947;H2AX foci patterns is demonstrated in normal fibroblasts of the cell line NHDF. Further, the value of the &#947;H2AX foci as a biomarker is depicted in x-ray irradiated lymphocytes of a healthy individual. Finally, genetic instability is investigated in CD34+ cells of a patient with acute myeloid leukemia by immunofluorescence microscopy of &#947;H2AX and 53BP1.

Immunofluorescence Microscopy of &amp;#947;H2AX and 53BP1 for Analyzing the Formation and Repair of DNA Double-strand Breaks

This manuscript provides a protocol for the analysis of DNA double-strand breaks by immunofluorescence microscopy of &#947;H2AX and 53BP1.

DNA double-strand breaks (DSB) are serious DNA lesions. Analysis of the formation and repair of DSB is relevant in a broad spectrum of research areas ...

Characterization of Tumor Cells Using a Medical Wire for Capturing Circulating Tumor Cells: A 3D Approach Based on Immunofluorescence and DNA FISH

Circulating tumor cells (CTCs) are associated with poor survival in metastatic cancer. Their identification, phenotyping, and genotyping could lead to a better understanding of tumor heterogeneity and thus facilitate the selection of patients for personalized treatment. However, this is hampered because of the rarity of CTCs. We present an innovative approach for sampling a high volume of the patient blood and obtaining information about presence, phenotype, and gene translocation of CTCs. The method combines immunofluorescence staining and DNA fluorescent-in-situ-hybridization (DNA FISH) and is based on a functionalized medical wire. This wire is an innovative device that permits the in vivo isolation of CTCs from a large volume of peripheral blood. The blood volume screened by a 30-min administration of the wire is approximately 1.5-3 L. To demonstrate the feasibility of this approach, epithelial cell adhesion molecule (EpCAM) expression and the chromosomal translocation of the ALK gene were determined in non-small-cell lung cancer (NSCLC) cell lines captured by the functionalized wire and stained with an immuno-DNA FISH approach. Our main challenge was to perform the assay on a 3D structure, the functionalized wire, and to determine immuno-phenotype and FISH signals on this support using a conventional fluorescence microscope. The results obtained indicate that catching CTCs and analyzing their phenotype and chromosomal rearrangement could potentially represent a new companion diagnostic approach and provide an innovative strategy for improving personalized cancer treatments.

We present a new approach to characterize tumor cells. We combined immunofluorescence with DNA fluorescent-in-situ-hybridization to evaluate cells captured by a functionalized medical wire capable of in vivo enriching CTCs directly from patient blood.

Circulating tumor cells (CTCs) are associated with poor survival in metastatic cancer. Their identification, phenotyping, and genotyping could lead to a ...

Isolation of Human Endothelial Cells from Normal Colon and Colorectal Carcinoma - An Improved Protocol

Primary cells isolated from human carcinomas are valuable tools to identify pathogenic mechanisms contributing to disease development and progression. In particular, endothelial cells (EC) constituting the inner surface of vessels, directly participate in oxygen delivery, nutrient supply, and removal of waste products to and from tumors, and are thereby prominently involved in the constitution of the tumor microenvironment (TME). Tumor endothelial cells (TECs) can be used as cellular biosensors of the intratumoral microenvironment established by communication between tumor and stromal cells. TECs also serve as targets of therapy. Accordingly, in culture these cells allow studies on mechanisms of response or resistance to anti-angiogenic treatment. Recently, it was found that TECs isolated from human colorectal carcinoma (CRC) exhibit memory-like effects based on the specific TME they were derived from. Moreover, these TECs actively contribute to the establishment of a specific TME by the secretion of different factors. For example, TECs in a prognostically favorable Th1-TME secrete the anti-angiogenic tumor-suppressive factor secreted protein, acidic and rich in cysteine-like 1 (SPARCL1). SPARCL1 regulates vessel homeostasis and inhibits tumor cell proliferation and migration. Hence, cultures of pure, viable TECs isolated from human solid tumors are a valuable tool for functional studies on the role of the vascular system in tumorigenesis. Here, a new up-to-date protocol for the isolation of primary EC from the normal colon as well as CRC is described. The technique is based on mechanical and enzymatic tissue digestion, immunolabeling, and fluorescence activated cell sorting (FACS)-sorting of triple-positive cells (CD31, VE-cadherin, CD105). With this protocol, viable TEC or normal endothelial cell (NEC) cultures could be isolated from colon tissues with a success rate of 62.12% when subjected to FACS-sorting (41 pure EC cultures from 66 tissue samples). Accordingly, this protocol provides a robust approach to isolate human EC cultures from normal colon and CRC.

Tumor endothelial cells are important determinants of the tumor microenvironment and the course of the disease. Here, a protocol for the isolation of pure and viable endothelial cells from human colorectal carcinoma and normal colon to be used in drug testing and pathogenesis research is described.

Primary cells isolated from human carcinomas are valuable tools to identify pathogenic mechanisms contributing to disease development and progression. In ...

Characterizing DNA Repair Processes at Transient and Long-lasting Double-strand DNA Breaks by Immunofluorescence Microscopy

The repair of double-stranded breaks (DSBs) in DNA is a highly coordinated process, necessitating the formation and resolution of multi-protein repair complexes. This process is regulated by a myriad of proteins that promote the association and disassociation of proteins to these lesions. Thanks in large part to the ability to perform functional screens of a vast library of proteins, there is a greater appreciation of the genes necessary for the double-strand DNA break repair. Often knockout or chemical inhibitor screens identify proteins involved in repair processes by using increased toxicity as a marker for a protein that is required for DSB repair. Although useful for identifying novel cellular proteins involved in maintaining genome fidelity, functional analysis requires the determination of whether the protein of interest promotes localization, formation, or resolution of repair complexes. 
The accumulation of repair proteins can be readily detected as distinct nuclear foci by immunofluorescence microscopy. Thus, association and disassociation of these proteins at sites of DNA damage can be accessed by observing these nuclear foci at representative intervals after the induction of double-strand DNA breaks. This approach can also identify mis-localized repair factor proteins, if repair defects do not simultaneously occur with incomplete delays in repair. In this scenario, long-lasting double-strand DNA breaks can be engineered by expressing a rare cutting endonuclease (e.g., I-SceI) in cells where the recognition site for the said enzyme has been integrated into the cellular genome. The resulting lesion is particularly hard to resolve as faithful repair will reintroduce the enzyme's recognition site, prompting another round of cleavage. As a result, differences in the kinetics of repair are eliminated. If repair complexes are not formed, localization has been impeded. This protocol describes the methodology necessary to identify changes in repair kinetics as well as repair protein localization.

Repair of double-strand DNA breaks is a dynamic process, requiring not only formation of repair complexes at the breaks, but also their resolution after the lesion is addressed. Here, we use immunofluorescence microscopy for transient and long-lasting double-stranded breaks as a tool to dissect this genome maintenance mechanism.

The repair of double-stranded breaks (DSBs) in DNA is a highly coordinated process, necessitating the formation and resolution of multi-protein repair ...

In Vivo Immunofluorescence Localization for Assessment of Therapeutic and Diagnostic Antibody Biodistribution in Cancer Research

Monoclonal antibodies (mAbs) are important tools in cancer detection, diagnosis, and treatment. They are used to unravel the role of proteins in tumorigenesis, can be directed to cancer biomarkers enabling tumor detection and characterization, and can be used for cancer therapy as mAbs or antibody-drug conjugates to activate immune effector cells, to inhibit signaling pathways, or directly kill cells carrying the specific antigen. Despite clinical advancements in the development and production of novel and highly specific mAbs, diagnostic and therapeutic applications can be impaired by the complexity and heterogeneity of the tumor microenvironment. Thus, for the development of efficient antibody-based therapies and diagnostics, it is crucial to assess the biodistribution and interaction of the antibody-based conjugate with the living tumor microenvironment. Here, we describe In Vivo Immunofluorescence Localization (IVIL) as a new approach to study interactions of antibody-based therapeutics and diagnostics in the in vivo physiological and pathological conditions. In this technique, a therapeutic or diagnostic antigen-specific antibody is intravenously injected in&#160;vivo and localized ex vivo with a secondary antibody in isolated tumors. IVIL, therefore, reflects the in vivo biodistribution of antibody-based drugs and targeting agents. Two IVIL applications are described assessing the biodistribution and accessibility of antibody-based contrast agents for molecular imaging of breast cancer. This protocol will allow future users to adapt the IVIL method for their own antibody-based research applications.

The in vivo immunofluorescence localization (IVIL) method can be used to examine in vivo biodistribution of antibodies and antibody conjugates for oncological purposes in living organisms using a combination of in vivo tumor targeting and ex vivo immunostaining methods.

Monoclonal antibodies (mAbs) are important tools in cancer detection, diagnosis, and treatment. They are used to unravel the role of proteins in ...

In Vitro Evaluation of Oncogenic Transformation in Human Mammary Epithelial Cells

Tumorigenesis is a multi-step process in which cells acquire capabilities&#160;that allow their growth, survival, and dissemination under hostile conditions. Different tests seek to identify and quantify these hallmarks of cancerous cells; however, they often focus on a single aspect of cellular transformation and, in fact, multiple tests are required for their proper characterization. The purpose of this work is to provide researchers with a set of tools to assess cellular transformation in vitro from a broad perspective, thereby making it possible to draw sound conclusions.
A sustained proliferative signaling activation is the major feature of tumoral tissues and can be easily monitored under in vitro conditions by calculating the number of population doublings achieved over time. Besides, the growth of cells in 3D cultures allows their interaction with surrounding cells, resembling what occurs in vivo. This enables the evaluation of cellular aggregation and, together with immunofluorescent labeling of distinctive cellular markers, to obtain information on another relevant feature of tumoral transformation: the loss of proper organization. Another remarkable characteristic of transformed cells is their capacity to grow without attachment to other cells and to the extracellular matrix, which can be evaluated with the anchorage assay.
Detailed experimental procedures to evaluate cell growth rate, to perform immunofluorescent labeling of cell lineage markers in 3D cultures, and to test anchorage-independent cell growth in soft agar are provided. These methodologies are optimized for Breast Primary Epithelial Cells (BPEC) due to its relevance in breast cancer; however, procedures can be applied to other cell types after some adjustments.

This protocol provides experimental in vitro tools to evaluate the transformation of human mammary cells. Detailed steps to follow-up cell proliferation rate, anchorage-independent growth capacity, and distribution of cell lineages in 3D cultures with basement membrane matrix are described.

Tumorigenesis is a multi-step process in which cells acquire capabilities&#160;that allow their growth, survival, and dissemination under hostile ...

An Automated Microscopic Scoring Method for the &#947;-H2AX Foci Assay in Human Peripheral Blood Lymphocytes

Ionizing radiation is a potent inducer of DNA damage and a well-documented carcinogen. Biological dosimetry comprises the detection of biological effects induced by exposure to ionizing radiation to make an individual dose assessment. This is pertinent in the framework of radiation emergencies, where health assessments and planning of clinical treatment for exposed victims are critical. Since DNA double strand breaks (DSB) are considered to be the most lethal form of radiation-induced DNA damage, this protocol presents a method to detect DNA DSB in blood samples. The methodology is based on the detection of a fluorescent labelled phosphorylated DNA repair protein, namely, &#947;-H2AX. The use of an automated microscopy platform to score the number of &#947;-H2AX foci per cell allows a standardized analysis with a significant decrease in the turn-around time. Therefore, the &#947;-H2AX assay has the potential to be one of the fastest and sensitive assays for biological dosimetry. In this protocol, whole blood samples from healthy adult volunteers will be processed and irradiated in vitro in order to illustrate the usage of the automated and sensitive &#947;-H2AX foci assay for biodosimetry applications. An automated slide scanning system and analysis platform with an integrated fluorescence microscope is used, which allows the fast, automatic scoring of DNA DSB with a reduced degree of bias.

An Automated Microscopic Scoring Method for the &amp;#947;-H2AX Foci Assay in Human Peripheral Blood Lymphocytes

This protocol presents the slide preparation and automated scoring of the &#947;-H2AX foci assay on peripheral blood lymphocytes. To illustrate the method and sensitivity of the assay, isolated lymphocytes were irradiated in vitro. This automated method of DNA DSB detection is useful for fast and high-throughput biological dosimetry applications.

Ionizing radiation is a potent inducer of DNA damage and a well-documented carcinogen. Biological dosimetry comprises the detection of biological effects ...

Modeling Brain Metastasis by Internal Carotid Artery Injection of Cancer Cells

Brain metastasis is a cause of severe morbidity and mortality in cancer patients. Critical aspects of metastatic diseases, such as the complex neural microenvironment and stromal cell interaction, cannot be entirely replicated with in vitro assays; thus, animal models are critical for investigating and understanding the effects of therapeutic intervention. However, most brain tumor xenografting methods do not produce brain metastases consistently in terms of the time frame and tumor burden. Brain metastasis models generated by intracardiac injection of cancer cells can result in unintended extracranial tumor burden and lead to non-brain metastatic morbidity and mortality. Although intracranial injection of cancer cells can limit extracranial tumor formation, it has several caveats, such as the injected cells frequently form a singular tumor mass at the injection site, high leptomeningeal involvement, and damage to brain vasculature during needle penetration. This protocol describes a mouse model of brain metastasis generated by internal carotid artery injection. This method produces intracranial tumors consistently without the involvement of other organs, enabling the evaluation of therapeutic agents for brain metastasis.

Brain metastasis is a cause of severe morbidity and mortality in cancer patients. Most brain metastasis mouse models are complicated by systemic metastases confounding analysis of mortality and therapeutic intervention outcomes. Presented here is a protocol for internal carotid injection of cancer cells that produces consistent intracranial tumors with minimal systemic tumors.

Brain metastasis is a cause of severe morbidity and mortality in cancer patients. Critical aspects of metastatic diseases, such as the complex neural ...

Orthotopic Implantation of Patient-Derived Cancer Cells in Mice Recapitulates Advanced Colorectal Cancer

Over the last decade, more sophisticated preclinical colorectal cancer (CRC) models have been established using patient-derived cancer cells and 3D tumoroids. Since patient derived tumor organoids can retain the characteristics of the original tumor, these reliable preclinical models enable cancer drug screening and the study of drug resistance mechanisms. However, CRC related death in patients is mostly associated with the presence of metastatic disease. It is therefore essential to evaluate the efficacy of anti-cancer therapies in relevant in vivo models that truly recapitulate the key molecular features of human cancer metastasis. We have established an orthotopic model based on the injection of CRC patient-derived cancer cells directly into the cecum wall of mice. These tumor cells develop primary tumors in the cecum that metastasize to the liver and lungs, which is frequently observed in patients with advanced CRC. This CRC mouse model can be used to evaluate drug responses monitored by microcomputed tomography (&#181;CT), a clinically relevant small-scale imaging method that can easily identify primary tumors or metastases in patients. Here, we describe the surgical procedure and the required methodology to implant patient-derived cancer cells in the cecum wall of immunodeficient mice.

This protocol describes the orthotopic implantation of patient-derived cancer cells in the cecum wall of immunodeficient mice. The model recapitulates advanced colorectal cancer metastatic disease and allows for the evaluation of new therapeutic drugs in a clinically relevant scenario of lung and liver metastases.

Over the last decade, more sophisticated preclinical colorectal cancer (CRC) models have been established using patient-derived cancer cells and 3D ...