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

Kiviharju-af Haalström 10.1073/pnas.0609299104pnas.XXYYYYY103.

Supporting Information

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SI Figure 6
SI Figure 7
SI Figure 8
SI Figure 9
SI Figure 10
SI Figure 11
SI Methods




SI Figure 6

Fig. 6. Altered p53 responses in ionizing radiation (IR)-treated human prostate epithelial cells (HPECs). (A) Levels of p53 and its target proteins were analyzed in mock- or IR-treated HPEC cells by Western blotting. GAPDH was used as a loading control. (B) p53 reporter assay. HPECs were transfected with p53-GFP-13XRE reporter construct, and the cells were treated with IR as indicated. Reporter activity (GFP levels) and p53 levels were analyzed by Western blotting. Loading was controlled by b-tubulin. The functionality of the reporter assay was verified by cotransfecting the cells with ectopic wild-type p53 and p53-GFP-13XRE reporter (lane 3). Ectopic expression of eGFP was used as a positive control (lane 2).





SI Figure 7

Fig. 7. Restoration of p53 response does not increase Wee1A or Cdk2-Tyr15 levels after irradiation. (A) HPECs were either treated with a vehicle or Mdm2 inhibitor Nutlin-3 (8 mM) for 12 h followed by either mock or IR treatment. Samples were collected 2 h after IR treatment and immunoprecipitated with an anti-Cdk2 antibody followed by immunoblot analyses for Cdk2-Tyr15 and Cdk2. Total cell lysates were analyzed for p53, p21, Wee1A, and GAPDH by Western blotting. Results shown are representative of two separate experiments. (B) HPECs treated as in A were pulse-labeled for 30 min with BrdU. Cells were harvested, stained with an anti-BrdU antibody and propidium iodide, and analyzed by multiparameter flow cytometry. Percentages of cells in the different phases of the cell cycle are indicated.





SI Figure 8

Fig. 8. Tyr15 phosphorylation in checkpoint-proficient Mv1Lu cells. Mv1Lu cells were transfected either alone or in combination with pAMC control vector, Cdk2, and Myc-Wee1A as indicated and incubated for 48 h. The cells were either mock- or IR-treated, incubated for 2 h, and total cell lysates were prepared followed by Western blot analyses.





SI Figure 9

Fig. 9. Ectopic expression of Wee1A in HPECs does not lead to a p53 response. HPECs were transfected either alone or in combination with pAMC, Cdk2, and Myc-Wee1A as indicated and incubated for 48 h. The cells were treated with IR and incubated for 2 h, and total cell lysates were prepared for the analysis of p53. GAPDH was used as a loading control.





SI Figure 10

Fig. 10. Immunohistological staining controls for p53, p21, and Tyr15. Paraffin-embedded tissue sections were stained for Cdk1/2-Tyr15 (A), p53 (B), and p21 (C), and nuclei were counterstained with Hoechst 33342. IR-treated prostate tissues represent ex vivo tissue slices. Colon adenoma and carcinomas are from routine histological specimens. [Magnification, ´20 (A and B); ´40 (C, and D).] Several exposures were obtained representing the high- and low-expression levels of the antigens present in the indicated tissues. (D) Conjugate controls. (Scale bar, 50 mm.)





SI Figure 11

Fig. 11. Induction of p53 in prostatic tissues by Nutlin-3a and leptomycin B. The ex vivo prostatic tissues were treated with 18 mM Nutlin-3a or 10 ng/ml leptomycin B for 18 h followed by immunohistochemical staining for p53 with anti-p53 antibody DO-7. (Magnification, ´40.)





SI Methods

Plasmids, Transfections, and p53 Reporter Assay. HPECs were transfected by lipofection [FuGENE 6 transfection reagent (Roche Diagnostics, Indianapolis, IN) or Lipofectamine 2000 (Invitrogen, Carlsbad, CA], and Mv1Lu cells by electroporation [Gene Pulser II (Bio-Rad, Hercules, CA)]. The following plasmids were used: pAMC, pCDNA3, eGFP (Clontech, Mountain View, CA), Cdk2, Myc-Wee1A (1), wt Wee1A and Wee1A (S53A/S123A) (2), wild-type p53, and p53-GFP-13XRE reporter (3).

DNA Damage Foci. Cells grown on collagen-coated coverslips were either mock- or IR-treated and fixed with 3.5% paraformaldehyde in PBS. Cells were washed, permeabilized with ice-cold 70% (vol/vol) ethanol (-20°C) for 5 min, and blocked with 8% BSA in Tris-buffered saline for 1 h. Cells were stained with the following antibodies: gH2AX (obtained from W. Bonner, National Institutes of Health, Bethesda, MD) or purchased from Upstate Biotechnology (Lake Placid, NY) (clone JBW301), Nbs1 and anti-Rad50 (Novus Biologicals, Littleton, CO). The primary antibodies were detected with Alexa 594-conjugated secondary antibodies (Molecular Probes, Eugene, OR), and DNA was stained with Hoechst 33342 (Sigma-Aldrich, St. Louis, MO). The stainings were analyzed with an Axioplan 2 Imaging epifluorescence microscope equipped with a ´63 Plan-Neofluar objective (NA 1.25) (Zeiss, Thornwood, NY). Images were captured with AxioCam HRc CCD-camera using Axiovision 4.4 software (Zeiss).

Flow Cytometry. Semiconfluent cell cultures were either mock- or IR-treated and processed for flow cytometry as described before (3). For analyzes of cell replication, the cells were pulse-labeled with 50 mM BrdU for the last 30 min of incubation. Cells were washed, fixed with ice-cold 70% (vol/vol) ethanol, treated with 0.1 N HCl, and heated for 10 min at 90°C to denature DNA. Cells were stained with an anti-BrdU-antibody (Amersham Pharmacia, Piscataway, NJ), followed by incubation with Alexa 488-conjugated secondary antibody (Molecular Probes), RNase A, and propidium iodide. For detection of Tyr15-phosphorylated Cdk2, fixed cells were stained with anti-Cdk2-Tyr15 antibody (Cell Signaling Technology, Beverly, MA), secondary antibody, RNase A, and propidium iodide. All analyzes were performed with an LSR flow cytometer (BD Biosciences, San Jose, CA), and spectral overlap was corrected by routine compensation. Aggregates were excluded from the analyzes. DNA analysis was performed by ModFit LT vs. 3.1 (Verity Software House, Topsham, ME).

BrdU Incorporation Assay. Cells were incubated in the presence of 50 mM BrdU followed by fixing of the cells with 3.5% paraformaldehyde. Cells were permeabilized with cold 70% (vol/vol) ethanol (-20°C) for 10 min, and DNA was denatured with 1.5 M HCl. Immunostaining was performed with an anti-BrdU antibody (Amersham Pharmacia) followed by detection with Alexa 594-conjugated secondary antibody (Molecular Probes) and counterstaining of the nuclei with Hoechst 33342. The number of BrdU-positive cells was compared with the number of all nuclei in the same field. More than 300 nuclei were analyzed per condition.

Western Blotting. Total cell lysates were prepared by resuspending cell pellets in urea/Tris buffer (UTB) (9 M urea/75 mM Tris[chemp]HCl, pH 7.5/0.15 M 2-mercaptoethanol) and sonicating briefly. Alternatively, cells were lysed in Nonidet P-40/lysis buffer containing 50 mM Tris[chemp]HCl, pH 7.5/150 mM NaCl/1% Nonidet P-40/50 mM NaF/1 mM Na3VO4/1 mM PMSF/1 mM DTT/0.1 mg/ml E64/0.1 mg/ml leupeptin/0.1 mg/ml soybean trypsin inhibitor. The protein concentrations of UTB samples were determined by Bio-Rad assay and Nonidet P-40-lysed samples by Bio-Rad DC protein assay. Proteins (50 mg for UTB lysates or 20-25 mg for Nonidet P-40 lysates) were separated by SDS/PAGE, transferred to polyvinylidene fluoride membranes (Immobilon P; Millipore Corporation, Billerica, MA), and blocked in PBS/5% nonfat milk. The following antibodies were used: p53, DO-1; Cdc25A, F-6; Cdk2, M2; Wee1A, H-300 (Santa Cruz Biotechnology, Santa Cruz, CA); p21WAF1/CIP1, 6B6; b-tubulin (BD Biosciences); Chk2, DCS-270 (obtained from Jiri Lukas, Danish Cancer Society); Chk2-Thr68, p53-Ser15, Cdk1/2-Tyr15 (Cell Signaling Technology); Ser10 histone H3 (Upstate Biotechnology); GFP (BD Biosciences, Clontech), and GAPDH (Europa Bioproducts, Cambridge, U.K.). Hdm2 was detected with a mix of three antibodies [SMP14 (Santa Cruz Biotechnology) and 2A10 and IF2 (Calbiochem)]. Anti-species horseradish peroxidase-conjugated secondary antibodies were obtained from Dako (DakoCytomation) and detected by using enhanced chemiluminescence (ECL, Amersham Pharmacia). MultiAnalyst version 1.0.2 was used for signal quantitation (Bio-Rad).

Immunoprecipitation. Cell lysates were prepared according to Nonidet P-40 lysis protocol, and protein levels were adjusted and precipitated with anti-Cdc25A (F-6), anti-Cdk2 (M2), and anti-cyclin E (HE111) antibodies (Santa Cruz Biotechnology). The immunocomplexes were collected with GammaBind G-Sepharose (Amersham Pharmacia) and washed extensively. Laemmli sample buffer (1´) was added, and the precipitates were boiled for 5 min. The supernatants were loaded onto SDS/PAGE and separated followed by immunoblotting analyses as described above.

Kinase Assay. Cells were lysed with Nonidet P-40 lysis buffer, protein concentrations were determined, and samples were precleared with GammaBind G-Sepharose and subjected to immunoprecipitation with an antibody against Cdk2 (M2) (Santa Cruz Biotechnology). The kinase reaction was carried out in kinase buffer (50 mM Hepes[chemp]KOH, pH 7.5/10 mM MgCl2/1 mM DTT/2.5 mM EGTA/0.1 mM sodium orthovanadate/1 mM NaF/20 mM ATP/10 mCi of [g-32P]ATP (3,000 Ci/mmol, Amersham Pharmacia) by using 5 mg of histone H1 (Boehringer Mannheim) as a substrate, and incubated for 30 min at 30°C with occasional mixing. Phosphorylated products were resolved on SDS/PAGE, dried, and exposed to radiographic film (Fujifilm).

Imaging Equipment and Settings. The microscope was an Axioplan 2 Imaging e (Zeiss). The objective lenses were Zeiss Plan-Neofluar, ´20, NA 0.5 (model 1004-989); Zeiss Plan-Neofluar, ´40, NA 0.75 (model 440351); and Zeiss Plan-Neofluar, ´63, NA 1.25 (model 440460). Filter sets were: DAPI, ex filter spectrum D360/40 (ex filter catalog no. 39869), em filter spectrum D460/50 (em filter catalog no. 39194); FITC, ex filter spectrum HQ480/40 (ex filter catalog no. 39552), em filter spectrum HQ535/50 (em filter catalog no. 38074); Texas red, ex filter spectrum HQ560/55 (ex filter catalog no. 36363), em filter spectrum HQ645/75 (em filter catalog no. 35279). The camera was an AxioCam HRc, color 3 ´ 14 bit; acquisition software was Zeiss Axiovision version 4.4.

Ex Vivo Prostate Tissue Images (Fig. 5). Scale factor for x and y = 0.521375 mm per pixel. (Magnification, ´20.) Scale factor for x and y = 0.267737 mm per pixel. (Magnification, ´40.) Pixel type, 48-bit RGB color; acquisition bit depth, 42; camera resolution, 1,300 ´ 1,030 standard color; Channel, Alexa 594 excitation wavelength and 590 emission wavelength; 617-ms exposure time with ´20 magnification (both mock and IR-treated): p53, 1678; gH2AX, 282; p63, 500; Tyr15, 2,360; p21, 1,550; conjugate controls: Alexa 594-conjugated goat anti-rabbit, 2,360; Alexa 594-conjugated goat anti-mouse, 1,500.

Exposure time (in milliseconds; both mock and IR-treated): p53, 1,114; gH2AX, 756; p63, 373; Tyr15, 2,360; p21, 950. Conjugate controls, Alexa 594-conjugated goat anti-rabbit, 950; Alexa 594-conjugated goat anti-mouse, 950. (Magnification, ´40.)

1. Rothblum-Oviatt CJ, Ryan CE, Piwnica-Worms H (2001) Cell Growth Differ 12:581-589.

2. Watanabe N, Arai H, Iwasaki J, Shiina M, Ogata K, Hunter T, Osada H (2005) Proc Natl Acad Sci USA 102:11663-11668.

3. Peltonen K, Kiviharju TM, Jarvinen PM, Ra R, Laiho M (2005) Pigment Cell Res 18:196-202.

This Article

  1. Published online before print April 12, 2007, doi: 10.1073/pnas.0609299104 PNAS April 24, 2007 vol. 104 no. 17 7211-7216
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