Two- and three-dimensional models for risk assessment of radiation-enhanced colorectal tumorigenesis

Abstract

Astronauts may be at an increased risk for developing colorectal cancer after a prolonged interplanetary mission given the potential for greater carcinogenic effects of radiation to the colon. In addition, with an increase in age, there is a greater incidence of premalignant colon adenomas with age. In the present study, we have compared the effects of radiation on human colon epithelial cells in two-dimensional (2D) monolayer culture, in three-dimensional (3D) culture, and in intact human colon tissue biopsies. Immortalized colon epithelial cells were irradiated at the NASA Space Radiation Laboratory (NSRL) with either 1 Gy 1 GeV/nucleon (56)Fe particles or 1 Gy 1 GeV/nucleon protons and were stained at various times to assess DNA damage and repair responses. The results show more persisting damage at 24 h with iron-particle radiation compared to protons. Similar results were seen in 3D colon epithelial cell cultures in which (56)Fe-particle-irradiated specimens show more persisting damage at 24 h than those irradiated with low-LET gamma rays. We compared these results to those obtained from human colon tissue biopsies irradiated with 1 Gy gamma rays or 1 Gy 1 GeV (56)Fe particles. Observations of radiation-induced DNA damage and repair in gamma-irradiated specimens revealed more pronounced early DNA damage responses in the epithelial cell compartment compared to the stromal cell compartment. After low-LET irradiation, the damage foci mostly disappeared at 24 h. Antibodies to more than one type of DNA repair factor display this pattern of DNA damage, and staining of nonirradiated cells with nonphosphorylated DNA-PKcs shows a predominance of epithelial staining over stromal cells. Biopsy specimens irradiated with high-LET radiations also show a pattern of predominance of the DNA damage response in the highly proliferative epithelial cell compartment. Persistent unrepaired DNA damage in colon epithelial cells and the differing repair responses between the epithelial and mesenchymal compartments in tissues may enhance tumorigenesis by both stem cell transformation and alterations in the radiation-induced permissive tissue microenvironment that may potentiate cancer progression.

FIG. 1

Panel a: HCEC CT cells in 2D culture irradiated with protons or ⁵⁶Fe particles at the NSRL and stained for DNA damage response proteins. Panel b: More damage persists at 24 h in the form of co-localized DNA-PKcs pT2609 and γ-H2AX (yellow foci) in iron-particle-irradiated specimens compared to those irradiated with protons (panel a). Standard errors were calculated after averaging the percentage of DNA damage foci remaining per nucleus for each time (n > 50 nuclei). Error bars are smaller than the symbols at 24 h.

FIG. 2

Panel a: DNA damage and repair response in CaCo2 cell organotypic cultures after low- or high-LET irradiation. Epithelial cells in these 3D cultures make circular structures (arrows) resembling the crypt openings observed in human colon tissues. 3D cultures irradiated with (panel b) γ rays and (panel c) ⁵⁶Fe particles. Panel d: More damage persists in the form of DNA-PKcs pT2609 foci (green) in specimens exposed to high-LET radiation compared to those exposed to low-LET radiation. Standard errors were calculated after averaging the percentage of DNA damage foci remaining per nucleus for each time (n > 50 nuclei). Error bars are smaller than the symbols at 24 h for the proton-irradiated specimens.

FIG. 3

Panel a: DNA damage and repair response in intact normal colon tissues after irradiation with 1 Gy γ rays. Human colon biopsies have more DNA damage foci per nucleus (in the form of phosphorylated DNA PKcs pT2609) in the epithelial cells (arrow) compared to the stromal cells (arrowhead), as seen in this specimen fixed 30 min after irradiation. Panel b: This pattern is also present in specimens fixed 30 min after irradiation and stained with 53BP1. Panel c: Nonirradiated colon biopsies stained with nonphosporylated DNA-PKcs show predominance of nuclear staining in the crypt epithelial cells (arrow) compared to stromal cells (arrowhead). Green staining outside of the nucleus is a result of autofluorescence of red blood cells and collagen.

FIG. 4

Panels a and b: DNA damage repair in colon biopsies shows that damage is highest at 30 min and decreases at 2 h, with most of the damage disappearing by 24 h. Note that the pT2609 damage foci in the stromal compartment at 30 min is less than half of that observed in the epithelial cell compartment, with the percentage of damage mostly staying stable up to 24 h (panel c). Colon biopsy fixed at 30 min after irradiation with 1 Gy 1 GeV ⁵⁶Fe particles and stained with pT2609 or 53BP1 (panel d). Note that the HZE-particle tracks are present at the crypts containing the epithelial cells but are not contiguous with the adjacent stromal cells. Green staining outside of the nucleus is secondary to autofluorescent red blood cell and collagen.