DNA Damage Recognition via Activated ATM and p53 Pathway in Nonproliferating Human Prostate Tissue

Abstract

DNA damage response (DDR) pathways have been extensively studied in cancer cell lines and mouse models, but little is known about how DNA damage is recognized by different cell types in nonmalignant, slowly replicating human tissues. Here, we assess, using ex vivo cultures of human prostate tissue, DDR caused by cytotoxic drugs (camptothecin, doxorubicin, etoposide, and cisplatin) and ionizing radiation (IR) in the context of normal tissue architecture. Using specific markers for basal and luminal epithelial cells, we determine and quantify cell compartment-specific damage recognition. IR, doxorubicin, and etoposide induced the phosphorylation of H2A.X on Ser 139 (γH2AX) and DNA damage foci formation. Surprisingly, luminal epithelial cells lack the prominent γH2AX response after IR when compared with basal cells, although ATM phosphorylation on Ser 1981 and 53BP1 foci were clearly detectable in both cell types. The attenuated γH2AX response seems to result from low levels of total H2A.X in the luminal cells. Marked increase in p53, a downstream target of the activated ATM pathway, was detected only in response to camptothecin and doxorubicin. These findings emphasize the diversity of pathways activated by DNA damage in slowly replicating tissues and reveal an unexpected deviation in the prostate luminal compartment that may be relevant in prostate tumorigenesis. Detailed mapping of tissue and cell type differences in DDR will provide an outlook of relevant responses to therapeutic strategies. Cancer Res; 70(21); 8630-41. ©2010 AACR.

Nkx3.1 controls the DNA repair response in the mouse prostate

“…It is noteworthy that basal epithelial cells do not express NKX3.1 or apparently require an accelerated response to DNA repair. Strikingly, Nkx3.1 deletion in murine luminal epithelial cells results in maximal expression of γH2AX at 8 hr after DNA damage, consistent with the observation that in that time frame after DNA damage basal epithelial cells express more γH2AX than do luminal cells 10.…”

“…S1). This evidence of a more robust DNA damage response at 1.5 hr after irradiation is in contrast to the more robust γH2AX staining seen preferentially in basal cells 8–24 hr after induction of DNA damage 10. Intact mice showed a statistically different result from Nkx3 .1 deletion mice, but heterozygous mice did not (Supplementary Fig.…”

Evaluation of surrogate tissues as indicators of drug activity in a melanoma skin model

“…However, we observed different γ H2AX/apoptosis kinetics between fibroblasts, keratinocytes, and melanoma in the MSM that suggests a differential DNA damage response that is cell‐type dependent. Similar findings were reported in prostate tissues after irradiation or CPT treatment with a fast and transient γ H2AX formation in basal cells and slow and moderate γ H2AX response in luminal cells, differences that were linked to constitutively dissimilar responses to DNA damage in the two different cell types 25, 26. It is also known that fibroblasts are more sensitive to UV radiation than keratinocytes, a discrepancy that can be explained by a lower induction of both DNA damage/apoptosis and rapid decrease of p53 in keratinocytes when compared to fibroblasts (fibroblasts showing late and long‐lasting p53 accumulation and were refractory to apoptosis) 27.…”

Novel Assay to Detect RNA Polymerase I Activity In Vivo

“…We then used the 5′ETS/45S CISH assay to detect Pol I activity in human prostate tissues derived from radical prostatectomies cultured ex vivo (33, 34). The prostate tissues were treated with or without BMH-21.…”

“…Radical prostatectomy specimens were cored and sliced at 300 µm as detailed before (33, 34). The tissue slices were cultured with RPMF-4A medium (35) supplemented with growth factors in a humidified tissue culture incubator at 37°C.…”