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

Summary

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.

Abstract

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 γ-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 γ-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.

Introduction

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 mi....

Protocol

1. Preparation of cell culture and experimental set-up

1. 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's 5a Medium Modified, supplemented with 10% fetal bovine serum and 1% antibiotic antimycotic solution.
2. Grow cells in a humidified 5% CO₂ environment at 37 °C until 70% of confluency with medium renewal every 2 to 3 days.
3. To gain BNCT beam (e.g., neutron beam plus ¹⁰BPA) and BNCT effect of killing cancer cells as heavy particles and to deposit most of their energy within the boron-....

Results

Firstly, we performed an analysis of a standard marker of detection of DNA-DSBs, γH2AX foci in colon cancer cells, non-radiated, and irradiated with the neutron-mixed beam. γ-H2AX foci appear as distinct fluorescent dots and show the formation of DNA-DSBs (as each γ-H2AX fluorescent dot represents a single DSB) (see Figure 1).

Discussion

The frequencies of foci, immunochemically stained for γ-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 γ-H2AX and 53BP1 is a standard procedure for the detection of DNA-DSBs. Formation of γ-H2AX foci is associated with the recruitment of 53BP1, a regulator of the DNA damage response²³

Materials

List of materials used in this article

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