Histologically normal breast tissues, obtained from surgically discarded reduction mammoplasty (B1400 and N17) and prophylactic mastectomy (B1389 & B1450) specimens with patients' informed consent and institutional review board approval, were provided by the UCSF Cancer Center and the Cooperative Human Tissue Network. Specimens B1400 and N17 were derived from disease-free women aged 50 and 17 years, respectively. Specimen B1389 was derived from a 53-year old woman with a family history of breast and ovarian cancer, diagnosed with non-proliferative fibrocystic disease. Specimen B1450 was derived from a woman of unspecified age, diagnosed with invasive ductal and lobular carcinoma in the contralateral breast. Tissue samples were minced and enzymatically dissociated using 0.1% w/v collagenase I in Dulbecco's Modified Eagle Medium at 37°C for 12 to 18 hrs. Small tissue fragments (organoids) remaining after digestion, were collected by centrifugation at 100 × g for two minutes. These organoids were stored frozen or seeded directly into Mammary Epithelial Cell Growth Medium (MEGM; Lonza, Walkersville, MD, USA). The resulting HMEC were cultured in serum-free MEGM medium as previously described [16
] and verified to be mycoplasma-free (Bionique Testing Laboratories (Saranac Lake, NY, USA). HMEC were routinely subcultured when 80% confluent and reseeded at a densities of 5 × 103
. Total population doublings were estimated using the equation, Population Doublings (PD) = log (A/B)/log2, where A was the number of collected cells and B was the number of plated cells. The effects of plating efficiencies were not taken into account in these estimates, and the calculated values were therefore lower than actual values. In some experiments, 5-aza-2'-deoxycytidine was added at a concentration of 3.3 μM to the culture medium for 72 to 96 hours prior to harvest.
Passage 4 HMEC were routinely seeded at a density of 0.25 × 106 cells per T-25 flask. The cells were grown for seven to eight days to 60% confluence prior to radiation exposure. Control plates were sham-irradiated. After irradiation, the cells were allowed to recover for 48 hrs, then dissociated with trypsin and replated at densities of 0.25 × 106 cells per 100 mm dish. The cultures were then incubated for four to six weeks. When visual inspection indicated that the largest patches of vHMEC had attained diameters of 1.3 to 2.5 cm, all the cultures were harvested for analysis. To generate growth curves, both irradiated and unirradiated HMEC cultures were maintained in triplicate through Passage 9.
Low linear energy transfer (LET) exposures were conducted using either 160 kVp X-ray or 137Cs γ-irradiation. Cultures were exposed to single doses of 5, 25, 50, 100 or 200 cGy at dose rates of either 22.5 cGy/minute (5, 25, or 50 cGy) or 1 Gy/minute (100 or 200 cGy). High-LET radiation exposures were conducted using a 1 GeV/amu 56Fe ion source at the NASA Space Radiation Laboratory at Brookhaven National Laboratory (Upton, NY, USA).
Senescence associated β-galactosidase (SA-βgal) activity
For visualization, cells were washed, fixed, and incubated overnight at 37°C with X-gal chromogenic substrate at pH 6.0 as described [17
]. The cells were viewed and photographed using a phase contrast Nikon Eclipse TS100 microscope (Nikon Instruments Inc., Melville, NY, USA). For quantitation of SA-βgal (+) and (-) cells by flow cytometry, fluorescein digalactoside (FDG; Molecular Probes, Eugene, OR, USA) was used as a substrate as described [18
]. Cells were dissociated and resuspended in phosphate buffer saline containing 5% fetal bovine serum (PBS/FBS). Diluted FDG solution was mixed with an equal volume of cell suspension, incubated at room temperature for three minutes and quenched by adding 10 volumes of PBS/FBS. Propidium iodide (1.25 μg/ml, Molecular Probes, Eugene, OR, USA) was added to exclude dead cells from analysis. Flow cytometry was performed using a FACScan flow cytometer and Cell Quest Pro software (Becton Dickinson, Franklin Lakes, NJ, USA). Determinations of the relative sensitivities of HMEC and vHMEC to radiation-induced senescence were estimated using the formula:
, in which Ps(0) and Ps(D) were the respective percentages of SA-βgal(+) cells in a population before and after exposure to a radiation dose (D). The formula included the factor 1-Ps(0) to correct for SA-βgal(+) cells pre-existing in the population.
p16 immunohistochemistry and immunofluorescence
Cells were fixed with 4% paraformaldehyde for 30 minutes, washed and permeabilized with 0.1% Triton: 3% H2O2 for five minutes, incubated with a 1:300 dilution of primary antibodies (JC2, Neomarkers, Fremont, CA, USA), and washed three to four times. For visualization, the cells were incubated with VectaStain ABC reagent, followed by DAB substrate (Vector Laboratories, Burlingame, CA, USA). For quantitation, the cells were incubated with Alexa Fluor 488 conjugated goat anti-mouse IgG (1:200) secondary antibodies. Nuclei were counterstained with 0.5 ng/ml 4,6-diamidino-2-phenylindole (DAPI; Molecular Probes, Eugene, OR, USA). Images for individual channels were acquired and quantitated using a Cellomics Array Scan VTI (Thermo Scientific, Pittsburgh, PA, USA), and merged using Adobe Photoshop 6.0 software (Adobe Systems, San Jose, CA, USA).
Cells were incubated for the final 24 hr prior to harvest with 10 μM bromodeoxyuridine (BrdU; BD Biosciences, San Jose, CA, USA), then dissociated, washed with medium containing 2% FCS, and fixed with 95% ethanol. Nuclei were prepared by incubating the fixed cells with 0.8% pepsin for 20 minutes at 37°C, followed by an IFA buffer (10 mM HEPES, 150 mM NaCl, 4% FBS, and 0.1% sodium azide) wash containing 0.5% Tween. The nuclei were then incubated with a 1:5 dilution of fluoroscein-conjugated anti-BrdU antibodies in IFA (BD Biosciences, San Jose, CA, USA) on ice for 30 minutes, followed by treatment with RNase A (5 μg/ml) and propidium iodide (5 μg/ml). Flow cytometry was performed as described above.
Genomic DNA was isolated from cells using the Wizard Genomic DNA Isolation kit (Promega, Madison, WI, USA). A total of 500 to 1,000 ng of DNA was treated with bisulfite using the EZ DNA Methylation-Gold kit (Zymo Research, Orange, CA, USA). Polymerase chain reaction amplification of the p16 gene promoter region was performed using the primer set 5' TTT TTA GAG GAT TTG AGG GAT AGG 3' (-159 to -136) and 5' CTA CCT AAT TCC AAT TCC CCT ACA 3' (+209 to +233) [21
] and the following conditions: 95°C/two minutes × one cycle; 96°C/20 sec, 60°C/20 sec and 72°C/90 sec × 40 cycles; 72°C/five minutes × one cycle. PCR products were purified with the QIAquick gel extraction kit (Qiagen, Valencia, CA, USA) and cloned in the pGEMT vector (Promega, Madison, WI, USA). Individual clones were sequenced with M13 forward and/or reverse primers.
Total cell lysates were prepared in SDS/Tris buffer with protease inhibitors (Complete, Roche Applied Science, Indianapolis, IN, USA) and 20 to 30 μg/lane were separated on gradient SDS-PAGE gels (Invitrogen). After transfer, the separated proteins were incubated with a 1:1000 dilution of p16 antibodies (JC2, Neomarkers), followed by imaging using an Odyssey infrared imaging system (Licor Biosciences, Lincoln, Nebraska, USA).
Graphpad Prism 5 (GraphPad Software, Inc., La Jolla, CA, USA) and JMP IN 3 (JMP, Cary, NC, USA) software were used for all the statistical analyses.
Simulations were performed using Matlab software (The MathWorks, Natick, MA, USA). Agents were dispersed randomly in silico to simulate plating onto a 400 × 400 grid (1.5 mm/pixel) at initial densities of 1,200 agents/grid (20% confluence). The following rules were used for modeling the growth of HMEC: 1) agents could divide as long as there was open space surrounding them; 2) if there was no more open space, agents could still divide but would be compressed until they reached a minimum size; 3) when an agent had reached the maximum number of divisions allowed, it would stop dividing and its type changed permanently to a senescent type; 4) senescent agents could not reattach after detachment during subculture. The program was reinitiated using the resulting agents when the grid reached 80% saturation (approximately 6,000 agents/grid) to simulate re-plating. For each set of experimental conditions, five independent simulations were performed, leading to an average behavior with a relative standard error less than 10%. By representing the grid as a tensor whose vectors are properties of the included agents, the progression of the tensor could be visualized using the advanced imaging platform DIPimage (Image Processing Toolbox for Matlab, Delft University of Technology, Delft, The Netherlands). A growth plateau was defined as the period during which the rate of population doubling deviated significantly from an exponential rate. Plateau width was determined experimentally as the time to 80% confluence of HMEC cultures in which the majority of cells displayed morphological features of senescence immediately after plating.