Human prostate epithelium lacks Wee1A-mediated DNA damage-induced checkpoint enforcement

Abstract

Cellular DNA damage triggers the DNA damage response pathway and leads to enforcement of cell cycle checkpoints, which are essential for the maintenance of genomic integrity and are activated in early stages of tumorigenesis. A special feature of prostate cancer is its high incidence and multifocality. To address the functionality of DNA damage checkpoints in the prostate, we analyzed the responses of human primary prostate epithelial cells (HPECs) and freshly isolated human prostate tissues to gamma-irradiation. We find that gamma-irradiation activates the ataxia telangiectasia mutated-associated DNA damage response pathway in the HPECs but that the clearance of phosphorylated histone H2AX (gammaH2AX) foci is delayed. Surprisingly, gamma-irradiated HPECs were unable to enforce cell cycle checkpoint arrest and had sustained cyclin-dependent kinase 2 (Cdk2)-associated kinase activity because of a lack of inhibitory Cdk phosphorylation by Wee1A tyrosine kinase. We further show that HPECs express low levels of Wee1A and that ectopic Wee1A efficiently rescues the checkpoints. We recapitulate the absence of checkpoint responses in epithelium of ex vivo irradiated human prostate tissue despite robust induction of gammaH2AX. The findings show that prostate epithelium has a surprising inability to control checkpoint arrest, the lack of which may predispose to accrual of DNA lesions.

Fig. 1.

HPECs lack multiple checkpoints after exposure to DNA damage. (A) HPECs and WS1 cells were mock- or IR-treated, and flow cytometric analysis was performed. Results shown are representative of six separate experiments of three HPEC strains. (B) HPECs and WS1 cells were mock- or IR-treated, incubated for 4 h, and pulsed with BrdU for the last 30 min. Cells were stained with an anti-BrdU-antibody (y axis) and propidium iodide (x axis). (C) After mock or IR treatment, the cells were labeled with BrdU, and cells positive for BrdU were analyzed by immunofluorescence. Error bars represent SD of duplicate samples. The incorporation of BrdU in IR-treated relative to mock-treated cells is shown. White bars, HPECs; gray bars, WS1 cells. (D) Cell extracts were analyzed for the presence of Ser¹⁰-phosphorylated histone H3. GAPDH was used as a loading control.

Fig. 2.

Sustained DNA damage foci formation in IR-treated HPEC cells. (A) HPECs were either mock- or IR-treated (1 Gy), incubated for 45 min, and stained with antibodies against γH2AX, Nbs1, and Rad50 as indicated. Nuclei were stained with Hoechst 33342. (Scale bar, 10 μm.) (B) Sustained γH2AX foci in HPECs. HPECs and WS1 cells were either mock- or IR-treated (1 Gy), incubated for the indicated times, and stained for γH2AX. DNA was stained with Hoechst 33342, and the merged images are shown. (Scale bar, 50 μm.) (C) Graphical presentation of data in B. Fold change in the number of cells positive for γH2AX (>4 foci/cell) is shown. Three hundred cells were analyzed for each time point, and the values represent mean of a duplicate experiment.

Fig. 3.

Sustained Cdk2-associated kinase activity in IR-treated HPECs despite functional ATM-Chk2–Cdc25A–pathway. (A) HPECs were either mock- or IR-treated, and the levels of p53, p53-Ser¹⁵, Chk2, and Chk2-Thr⁶⁸ were analyzed by Western blotting. GAPDH and β-tubulin were used as loading controls. (B) HPECs and U2OS cells were mock- or IR-treated and incubated for the times shown. Cell lysates were precipitated (IP) with an anti-Cdc25A antibody followed by immunoblotting for Cdc25A. (C) HPECs and U2OS cells were either mock- or IR-treated, and cellular lysates were precipitated with an anti-Cdk2 antibody followed by immunoblotting analyses for Cdk2-Tyr¹⁵ and Cdk2. (D) Cell lysates from mock-, IR- (10 Gy, 2 h), UVC- (20 J/m², 2 h) and hydroxyurea (HU)- (20 mM, 3 h) treated HPECs and control U2OS cells were precipitated with an anti-cyclin E antibody. The levels of cyclin E-bound Cdk2 and Cdk2-Tyr¹⁵ were evaluated with respective antibodies by Western blotting. (E) HPEC and WS1 cells were treated with IR followed by analysis for the levels and Tyr¹⁵ phosphorylation of cyclin B-associated Cdk1. (F) U2OS and HPECs were mock- or IR-treated, and cellular lysates were precipitated with an antibody against Cdk2. Cdk2 was detected by Western blotting, and Cdk2-associated kinase activity was analyzed by using histone H1 as a substrate. Phosphorylation of histone H1 was quantitated and is normalized to total Cdk2 expression.

Fig. 4.

Ectopic Wee1A kinase restores Cdk-Tyr¹⁵ phosphorylation and cell cycle checkpoints in response to IR. (A) Western blotting analyses of Wee1A levels. (B) Expression of Wee1A, p63, and p27 in prostate tissues and in HPECs. Histologically normal sections of prostate tissue or HPECs were stained for the expression of the indicated proteins. (Scale bar, 50 μm.) (C) HPECs were transfected either alone or in combination with the expression vectors pAMC, Cdk2, and Myc-Wee1A as indicated and incubated for 48 h. The cells were treated with IR, incubated for 2 h, and total cell lysates were prepared for the analysis of indicated proteins or Tyr¹⁵-phosphorylated Cdk2 (asterisk marks a nonspecific band). Cdk2-Tyr¹⁵ phosphorylation was quantitated and normalized to total Cdk2. Cdk2-Tyr¹⁵ present in control-transfected cells is set as 0. (D) HPECs were transfected with Cdk2 and Myc-Wee1A and were mock- (Left) or IR-treated (Right) as in C. The cells were fixed and stained with a Cdk1/2-Tyr¹⁵-specific antibody and subjected to flow cytometry. The cell cycle distribution of Cdk1/2-Tyr¹⁵-positive cells is shown. The threshold for Tyr¹⁵ positivity was set on the basis of signal present in pAMC control-transfected cells. (E) The change in the cell cycle distribution of Cdk2-Tyr¹⁵-positive cells is indicated as follows: IR(S + G₂/M)/Mock(S + G₂/M). The experiment was performed with three different HPEC strains with similar results. An average of two experiments is shown. (F) HPECs and U2OS cells were treated with 10 μM MG132 for 4 h followed by analysis for Wee1A by Western blotting. Normalized Wee1A levels are shown. (G) HPECs were transfected either alone or in combination with the following plasmids: control, Cdk2, wild-type Wee1A (wt), and Wee1A phosphodegron mutant (mut; S53A/S123A). GFP was used as a transfection control. Cells were IR-treated and analyzed for Cdk2-Tyr¹⁵, Cdk2, and Wee1A. Cdk2-Tyr¹⁵ phosphorylation was quantitated and normalized to total Cdk2. Cdk2-Tyr¹⁵ present in control transfected cells is set as 1.

Fig. 5.

Human prostatic epithelium lacks Cdk-Tyr¹⁵ phosphorylation and p53 responses. After prostatectomy, fresh peripheral zone prostate tissue cores were obtained and sliced at 300 μm. Tissue slices were either mock- or IR-treated and incubated for 4 h. Paraffin-embedded sections were stained for Cdk1/2-Tyr¹⁵, γH2AX, p53, p21, and p63, and nuclei were counterstained with Hoechst 33342. Images were captured at magnifications of ×20 and ×40 as indicated and represent consecutive sections. (Scale bars, 50 μm.) Images shown are representative of tissues from two prostates.