Mice harboring targeted mutations of the Dr5
were crossed with mice with targeted Puma
mice were used to generate Dr5−/−
and wild-type (i.e., Dr5+/+
) littermates. Litters were genotyped as previously described in reference 16
Ionizing irradiation treatment.
At 4–6 weeks of age, mice received a single dose of 5 Gy whole-body γ-irradiation from 137Cs source (at a dose rate of 1.4 Gy/min), according to a University of Pennsylvania Institutional Animal Care and Use Committee (IACUC) protocol. At 8 h after the treatment, animals were euthanized. Tissues were harvested and fixed in freshly prepared 4% paraformaldehyde overnight at 4°C. The samples were washed in phosphate-buffered saline and then transferred to 70% ethanol and embedded in paraffin for histology, snapfrozen in liquid nitrogen for protein gel blot, or single-cell suspensions were prepared for flow cytometric analysis.
In vitro cell culture.
The human colorectal carcinoma cell line HCT116, osteosarcoma cell line U2OS, lung cancer cell line H460 and teratocarcinoma cell line PA-1 were purchased from ATCC. P53−/− HCT116 cells were kindly provided by B. Vogelstein (Johns Hopkins University, Baltimore, MD). HCT116 and PA-1 cells were cultured in McCoy's 5A and BME medium supplemented with 10% FCS and antibiotics in humidified 5% CO2 at 37°C, respectively.
Assessment of long-term cell viability.
To assess the long-term effect of chemotherapy, 5 × 104 cells were seeded in a well of 24-well plates in duplicate. Next day, cells were treated with 5FU (0, 0.5, 1.0, 5.0 and 10.0 µg/ml) or with Adriamycin (0, 0.05, 0.1, 0.5 and 1.0 µg/ml). The media containing the drugs were changed every 48 h, and the culture was maintained for 7 d, and then the remaining cells were fixed with 50% methanol and stained with Coomassie brilliant blue (CBB).
After the indicated treatments, the cells were collected and fixed with 70% ethanol at 4°C. The samples were stained with propidium iodide (Sigma) and subjected to flow cytometric analysis using Epics Elite flow cytometer (Beckman-Coulter, Fullerton, CA). In vivo studies, the bone marrow, spleen and thymus were isolated 8 h after γ-irradiation; single cell suspensions were prepared and analyzed for sub-G1 FACS analysis.
Protein gel blot analysis.
Protein gel blotting was performed by standard methods. Apoptotic responses in in vivo studies were performed as described previously in reference 5
. Briefly, snapfrozen tissues were thawed quickly and homogenized in radioimmunoprecipitation assay (RIPA) buffer (1x phosphate-buffered saline, 1% Nonidet P-40 or Igepal CA-630, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate). Homogenates were sonicated and centrifuged at 14,000 rpm for 20 min, and the protein concentration of the supernatants as determined by the Bradford method (Bio-Rad). The samples were separated on a Tris-Glycine gel (Invitrogen) under denaturing conditions. Proteins were electro-blotted onto a polyvinylidene difluoride membrane. After incubation with the indicated antibodies, the proteins were detected using ECL Protein gel blotting Detection kit (GE Healthcare, Piscataway, NJ).
Anti-Bax, anti-Bcl2 and anti-DR4 antibodies were purchased from Becton Dickinson (Franklin Lakes, NJ). Anti-DR5 antibody was purchased from Sigma (St. Louis, MO). Anti-Bad, anti-Bid, anti-survivin, anti-mouse cleaved caspase 9, anti-mouse caspase 8, anti-caspase 6 antibodies were purchased from Cell Signaling (Boston, MA). Anti-p53 (DO1), anti-Fas and anti-Bnip3L antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-Bak, anti-Noxa, anti-WAF1 (p21) and anti-Puma antibodies were purchased from Calbiochem (San Diego, CA). Following primary antibody incubation, membranes were incubated with secondary horseradish peroxidase-conjugated antibodies (1:4,000) (Pierce, Rockford, IL) and subsequently detected by the enhanced chemiluminescence method (Amersham/GE Healthcare Biosciences, Piscataway, NJ).
Cells were washed, collected, and total RNA was prepared using Trizol LS Reagent (Invitrogen) according to the manufacturer's instruction. One µg total of RNA was reverse-transcribed using TaqMan Reverse Transcription Reagents (Applied Biosystems, Foster City, CA) according to the manufacture's instruction. For amplification of p53AIP1 and their splice variants, 5′-ATG GGA TCT TCC TCT GAG GC-3′ was used as a common forward primer. 5′-TCA CTG CAA CCT CAA CGG TG-3′ was used as a reverse primer. 5′-TTA CTG CAC TGT CAG GAT CC-3′ and 5′-TCA GTT CCC AGC TCT GTC CA-3′ were used as reverse primers for the β-isoform and γ-isoform, respectively. PCR reaction consists of 95°C for 30 sec, 56°C for 1 min and 72°C for 1 min and amplified from 25 to 35 cycles. The PCR products were separated by 2% agarose gel every five cycles for smi quantification, and the bands were detected using Molecular Imager Gel Documentation System (Bio-Rad Laboratories, Hercules, CA).
Gene silencing using siRNA.
For transient silencing of the p53 target genes, siRNAs for PUMA, DR5 and FAS as well as non-silencing control were purchased from Santa Cruz Biotechnology. 5′-AGG AUG AGG AUG GUA CGU G-3′ and 5′-AUA UUC AAG AUG GCU GCC C-3′ were used to silence BNIP3L and BAD , respectively. RNA TransPass RI Transfection Reagent (New England BioLabs) was used for the transfection of siRNA according to the manufacturer's instruction.
Measurement of caspase 3 and -7 activities.
7,500 cells HCT116 cells were seeded in a 96-well plates on day 1. siRNA-transfection was performed on day 2. Adriamycin (1 µg/ml) or 5FU (20 µg/ml) treatment started on day 3. Caspase 3/7 activities were measured 24 h after the treatment using Caspase Glo 3/7 Assay (Promega, Madison, WI) according to the manufacture's instructions. The luminescent signal was detected, visualized and measured using Xenogen In vivo Imaging System (Xenogen, Hopkinton, MA). After gene silencing and the drug treatment, all wells were checked under microscopy to ensure that there were no differences in cell numbers between wells.
Immunohistochemistry was performed as previously described in references 5, 19
. Briefly, the bone marrow, spleen, thymus, small intestine and colon were collected and fixed in 4% paraformaldehyde and embedded in paraffin. Tissue-paraffin blocks were cut in 5 µm. Paraffin sections were dewaxed and immunostained with primary antibodies and the secondary antibodies conjugated with Cy3 (Jackson Immunochemicals). Anti-cleaved caspase 9 (asp353, mouse specific, 1:100) antibody was purchased from Cell Signaling. Terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) assays were performed using the Apoptag plus FITC kit according to the manufacturer's instructions (Chemicon International). Sections were counterstained with 4′-6-diamidino-2-phenylindole (DAPI) or propidium iodide (PI) and evaluated under a fluorescence microscope.