Evidence for DNA damage checkpoint activation in barrett esophagus

Abstract

Barrett esophagus is an epithelial metaplasia that predisposes to adenocarcinoma. Better markers of cancer risk are urgently needed to identify those patients who are likely to benefit most from emerging methods of endoscopic ablation. Disease progression is associated with genomic DNA changes (segmental gains, losses, or loss of heterozygosity). Although these changes are not easily assayed directly, we hypothesized that the underlying DNA damage should activate a DNA damage response (DDR), detectable by immunohistochemical (IHC) assays of checkpoint proteins and the resulting replicative phase cell cycle delays. Surgical specimens and endoscopic biopsies (N = 28) were subjected to IHC for the cell cycle markers cyclin A and phosphorylated histone H3 (P-H3), the DDR markers gammaH2AX and phosphorylated ATM/ATR substrates (P-ATM/ATRsub), and the DNA damage-responsive tumor suppressors p16 and p53. Correlations were made with histologic diagnoses. The fractions of cells that stained for cyclin A, P-H3, and gammaH2AX increased in parallel in dysplastic tissue, consistent with checkpoint-mediated cell cycle delays. Foci of nuclear gammaH2AX and P-ATM/ATRsub were demonstrated by standard and confocal immunofluorescence. Staining for p16 was more prevalent in early-stage disease with lower staining for gammaH2AX and P-H3. Staining for p53 was moderately increased in some early-stage disease and strongly increased in some advanced disease, consistent with checkpoint-mediated induction and mutational inactivation of p53, respectively. We suggest that IHC for DDR-associated markers may help stratify risk of disease progression in Barrett.

Figure 1

Validation of IHC staining. We exposed U2-OS cell clones with inducible expression of p16 to conditions predicted to yield low and high expression, respectively, of each target antigen (see Materials and Methods for additional details). Cells were processed in parallel for IHC (left, center) or immunoblot analysis (right (C, F, I, L, O)). The conditions were as follows. Cyclin A: low (A), synchrony in G₁ using a mitotic arrest in nocodazole (Noc) followed by release for 4 hours; high (B), G₁/S arrest mediated by hydroxyurea (HU). P-H3: low (D), G₁/S arrest in HU; high (E), mitotic arrest in Noc. γH2AX: low (G), standard culture; high (H), induction of DNA double-strand breaks by etoposide. p16: low (J), standard culture (in tetracycline); high (K), withdrawal of tetracycline. p53: low (M), standard culture conditions; high (N), etoposide. Immunoblot analysis for tubulin (tub) served as a loading control. In immunoblots (I) and (O), intervening lanes (from an intermediate induction condition not shown) were removed digitally.

Figure 2

Increased cyclin A, P-H3, and γH2AX staining in Barrett-associated dysplasia within surgical resection specimens. Esophageal resection specimens from patients with HGD or EAC were subjected to IHC staining (brown) and counterstained with hematoxylin (blue). (A, B) Tissue with no dysplasia (ND). Cyclin A staining (A) is confined to nuclei of cells in basal regions of intestinal-like crypts. Note the goblet cells of Barrett. P-H3 staining (B) is rare. (C, D) LGD: Cyclin A and P-H3 staining are more prevalent. (E, F) EAC: Cyclin A and P-H3 staining are prevalent. (G) γH2AX staining is higher in EAC (left) than neighboring Barrett tissue without dysplasia (ND, right). (H) LGD: Scattered cells display distinct nuclear γH2AX staining.

Figure 3

Quantitation of increased cyclin A and P-H3 staining in Barrett surgical specimens and endoscopic biopsy samples with dysplasia. (A) Surgical specimens. IHC was performed on formalin-fixed specimens (N = 6). Approximately 267 to 938 epithelial cells were counted per stained tissue sample (from three to four 10x microscope fields per specimen), and the percentage of (+) cells were determined (16,979 cells were counted in total). The results are graphed in “box and whisker” plots of the mean, upper and lower quartiles (boxes), and SDs (thin lines). Tissue with no dysplasia (ND) was compared with tissue with dysplasia (LGD, HGD, and EAC). Cyclin A and P-H3 staining was significantly higher in the dysplastic tissues (P < .02 and P < .003, respectively). (B) Endoscopic biopsy samples. Cyclin A and P-H3 staining was scored in 20x microscope fields from endoscopic biopsies with no dysplasia (left) or indefinite for dysplasia (IND)/LGD/HGD (right) and graphed as in (A). A total of 8763 cells from tissues with ND (N = 8) and 9247 cells from tissues with IND/LGD/HGD (N = 7), respectively, were scored for cyclin A staining. A total of 8799 cells from tissues with ND and 10,121 cells from tissues with IND/LGD/HGD, respectively, were scored for P-H3 staining. Cyclin A and P-H3 staining were significantly higher in the dysplastic biopsies (P < .03 and P < .01, respectively).

Figure 4

Increased staining for replicative cell cycle phases, DDR, p16, and p53 in Barrett endoscopic biopsy samples. Nearby formalin-fixed sections were stained from samples with no dysplasia and no history ofHGDor EAC (left) or no dysplasia but with a history of HGD (right). Sections were stained for (A, B) cyclin A, (C, D) P-H3, (E, F) γH2AX, (G, H) pATM/ATRsub, (I, J) p16, and (K, L) p53. p16 staining was more prominent in the specimen with Barrett alone (I), whereas pATM/ATRsub (H) and p53 (L) staining were more prominent in the sample with a history of HGD.

Figure 5

Two patterns of p53 staining in Barrett tissue. Endoscopic biopsies were subjected to IHC for p53. (A) LGD showing moderate p53 staining in nuclei of scattered cells. Note the diversity of cell types in the epithelium, indicating that this tissue is not neoplastic. (B) No p53 staining in a sample with no dysplasia (ND). (C) Uniformly high p53 staining in a sample with uniform cell morphology indicative of HGD or EAC.

Figure 6

Immunofluorescence staining of nuclear DNA damage foci in Barrett tissue. (A–D) An endoscopic biopsy sample that showed no dysplasia but displayed IHC staining for γH2AX and P-H3 and was from a patient with a history of intramucosal carcinoma was subjected to coimmunofluorescence staining for γH2AX (A), P-ATM/ATRsub (B), and DAPI staining for nuclear DNA (C). The combined image is shown in (D). Arrows: nuclear foci costaining for γH2AX and P-H3. Control stains without primary Abs showed no nuclear foci (not shown). (E–H) Confocal microscopy confirmed the nuclear location of the costained foci (arrows). Magnification denoted by scale bars in (D) and (H).