Cell culture, transfection, and treatments.
The human small-cell lung carcinoma (SCLC) cell line NCI-H82 (ATCC HTB-175), the human embryonic kidney 293 cell line (ATCC CRL-1573), the human neuroblastoma SH-SY5Y cell line (ATCC CRL-2266), the human cervical carcinoma HeLa S9 cell line (NCCC), and the rat pheochromocytoma PC12 cell line (ATCC CRL-1721) were used in this study. Cells were cultured at 37°C and 5% CO2 in RPMI 1640 medium (H82 and PC12), Dulbecco's modified Eagle's medium (MEM) (HEK-293), MEM mixed with F-12 nutrient mixture (Ham) at ratio of 1:1 (SH-SY5Y), or MEM mixed with 1× MEM nonessential amino acids and 1 mM MEM sodium pyruvate (HeLa), all supplemented with 10% heat-inactivated fetal calf serum, 2 mM l-glutamine, penicillin (100 units/ml), and streptomycin (100 mg/ml). Twenty-four hours after seeding into culture dishes with fresh medium, cells were transfected using Lipofectamine 2000 (Invitrogen) according to the manufacturer's protocol. Twenty-four hours after transfection, cells were treated with 5 μM VP16. The cell density was kept at levels allowing exponential growth.
Reagents and antibodies.
VP16 was obtained from Bristol-Myers, and PKC inhibitor PKC-412 was obtained from Novartis. Hoechst 33342 dye was obtained from Molecular Probes, Geneticin was obtained from Gibco, propidium iodide (PI) was obtained from Sigma, and RNase A was obtained from Boehringer. For Western blot and immunofluorescence experiments, primary mouse monoclonal anti-p73α/β (Ab-4) antibody (NeoMarkers), rabbit polyclonal anti-p73 (Ab-6) antibody (NeoMarkers), rabbit polyclonal anti-Bax antibody (BD Pharmingen), mouse monoclonal anti-Bax (6A7) antibody (BD Pharmingen), mouse monoclonal antihemagglutinin (12CA5) antibody (Roche), rabbit polyclonal anti-Mdm2 (N-20) antibody (Santa Cruz Biotechnology, Inc.), or rabbit polyclonal anti-phosphoserine antibody (Zymed Laboratories) was used. Secondary Alexa Fluor 488- and 594-conjugated goat anti-immunoglobulin G (IgG) (Molecular Probes) was used in immunofluorescence and fluorescence-activated cell sorter (FACS) analysis experiments. For Western blot experiments, rabbit polyclonal anti-glyceraldehyde-3-phosphate dehydrogenase (anti-G3PDH) antibody (Trevigen) and horseradish peroxidase-conjugated goat anti-mouse or goat anti-rabbit IgG (Pierce) were used. For bromodeoxyuridine (BrdU) incorporation studies, BrdU and fluorescein isothiocyanate-conjugated mouse anti-BrdU monoclonal antibody (BD Pharmingen) were used. Predesigned and tested ON-TARGETplus siRNA SMARTpool against human PKCα (GenBank accession number NM_002737) and human PKCβ2 (accession number NM_002738) were obtained from Dharmacon. Nocodazole and hydroxyurea were obtained from Roche.
Human p73 isoforms (α, β, ΔNα, γ, δ, and
) and p73 TA domain-inactive mutants (having an amino acid substitution at position 156) were kind gifts from G. Melino and were described previously (10
). To generate the naturally occurring mutants P405R and P425L, having amino acid substitutions at either position 405 or 425, either full-length p73α p73β or their NH2
-TA-mutated counterparts were used. To generate p73δΔ380 and TAmut p73δΔ380, full-length p73δ and TAmut p73δ were used, and a stop codon was introduced at amino acid 381. To generate PKC phosphorylation site mutant TAmut p73β S388A, a base pair substitution was made at amino acid 388. Mutagenesis was performed according to the manufacturer's protocol (QuikChange site-directed mutagenesis kit; Stratagene) using primers P405R (5′-CAC CTA CAG CCC CGG TCC TAC GGG C-3′ [forward] and 5′-G CCC GTA GGA CCG GGG CTG TAG GTG-3′ [reverse]), P425L (5′-ATG AAC AAG CTG CTC TCC GTC AAC C-3′ [forward] and 5′-G GTT GAC GGA GAG CAG CTT GTT CAT-3′ [reverse]), 380stop (5′-CTG ATG GAG TTG TAG CCG CAG CCA CTG-3′ [forward] and 5′-CAG TGG CTG CGG CTA CAA CTC CAT CAG-3′ [reverse]), and S388A (5′-G CCA CTG GTG GAC GCC TAT CGG CAG CA-3′ [forward] and 5′-TG CTG CCG ATA GGC GTC CAC CAG TGG C-3′ [reverse]). Gal4DBD-p73/N (amino acids 1 to 112) and Gal4DBD-p73/C (amino acids 380 to 513) were kind gifts from K. Somasundaram and were described previously (9
). Plasmids containing the PIG3
promoter-luciferase construct (PIG3
-luc) (containing a 1.4-kb promoter fragment [positions −861 to +546] encompassing the canonical p53 binding site between positions −330 and −309) (28
), the CD95
promoter-luciferase construct (CD95
-luc) (containing bases −1435 to +236 of the human FAS/CD95 gene) (37
), p63α, and p63γ were kind gifts from T. Soussi. T. Perlmann kindly provided us with plasmids encoding β-galactosidase and MH100 Gal4RE-luc. The promoter-luciferase constructs mdm2
-luc (containing the promoter and upstream promoter sequence of human mdm2, including the two p53-responsive elements) (41
-luc (containing the fragment at positions −687 to −318 of the human bax promoter, including one p53-responsive element) (14
-luc (containing 2.4 kb of the human p21 promoter and upstream sequence, including one p53-responsive element) (14
), and cyclin G
-luc (1.48 kb of the cyclin G gene) (14
) were kind gifts from M. Oren. All promoters were cloned into the pGL reporter vector (Promega), except for the CD95 promoter, which was cloned into a pGV-B vector (Wako PicaGene). Both vectors bear a simian virus 40 promoter upstream of the luciferase gene. Enhanced green fluorescent protein (EGFP) plasmid was obtained from Clontech, and pCMV-DsRed-Express (pDsRed) was obtained from BD Biosciences. Plasmid encoding wild-type p53 was kindly provided by B. Vogelstein. PKC and dominant negative PKC (DN-PKC) expression plasmids were kind gifts from Jae-Won Soh. The protein levels of p73, p63, and p53 variants after expression of the various cDNAs were examined by immunoblotting. There was no significant difference in the levels of protein (25
) (see Fig. S3 in the supplemental material).
Immunofluorescence and laser-scanning confocal microscopy.
One day after transfection with p73 plasmids, H82 cells were harvested, and cytospins were prepared. Cells were fixed in 4% paraformaldehyde (PFA) and blocked/permeabilized in phosphate-buffered saline (PBS) with 10 mM HEPES, 0.3% Triton X-100, and 3% bovine serum albumin. Slides were incubated with primary mouse anti-p73α/β antibody or primary rabbit anti-p73 antibody at room temperature for 4 h, followed by secondary Alexa Fluor 488-conjugated antibody (room temperature for 1 h). Subsequently, slides were incubated with primary rabbit anti-Bax antibody, primary mouse anti-Bax (6A7) antibody, or primary rabbit anti-Mdm2 antibody (4°C overnight) and then incubated with secondary Alexa Fluor 594-conjugated antibody (room temperature for 1 h). Nuclei were counterstained with Hoechst dye (1 μg/ml), and slides were mounted in Vectashield H-1000 (Vector Laboratories, Inc.) and analyzed under a Zeiss 510 Meta confocal laser scanning microscope equipped with an inverted Zeiss Axiovert 200m microscope. Mix dyes were acquired by sequential multiple-channel fluorescence scanning to avoid bleedthrough. Three independent experiments were made, and average values are shown. Error bars represent standard deviations (SD).
H82 cells were cotransfected with plasmids encoding EGFP and p73 at a ratio of 1:10. One day after transfections, cells were treated with 5 μM VP16. At 24 h posttreatment, cells were stained with Hoechst dye and scored in a fluorescence microscope as the percentage of EGFP-expressing cells with condensed nuclei.
Flow cytometric analysis of BrdU incorporation.
H82 cells were cotransfected with pCMV-DsRed-Express and p73 plasmids (ratio of 1:10). Forty-eight hours after transfection, cells were incubated with 10 μM BrdU for 15 min, harvested, and prepared according to a protocol for BrdU incorporation (BD Pharmingen). Samples were analyzed using a FACSCalibur flow cytometer using Cell Quest software (BD Biosciences). For each sample, 10,000 cells sorted for red fluorescence were assessed for BrdU incorporation.
Cell cycle analysis.
PC12 cells were cotransfected with EGFP and p73 expression plasmids (ratio of 1:5). Twenty-four hours posttransfection, cells were fixed in 1% PFA and subsequently frozen overnight in 95% ethanol. Thirty minutes prior to analysis, cells were resuspended in PBS with 50 μg/ml PI and 5 μg/ml RNase A. Samples were analyzed using a FACSCalibur flow cytometer using Cell Quest software (BD Biosciences). In each sample, 10,000 cells sorted for green fluorescence were assayed.
Flow cytometric analysis of mdm2 expression.
One day after cotransfection with pDsRed and p73 plasmids (ratio of 1:5), H82 cells were harvested, and cells were fixed in 1% PFA and blocked/permeabilized in PBS with 10 mM HEPES, 0.3% Triton X-100, and 3% bovine serum albumin. Cells were incubated with primary rabbit anti-mdm2 antibody at room temperature for 1 h, followed by secondary Alexa Fluor 488-conjugated antibody (room temperature for 30 min). Subsequently, cells were resuspended in PBS, and samples were analyzed using a FACSCalibur flow cytometer using Cell Quest software (BD Biosciences). For each sample, 10,000 cells sorted for red fluorescence were assessed for intensity of mdm2 staining. Kolmogorov-Smirnov (K-S) statistics (CellQuest Software) were generated to compare the fluorescence intensity distributions between mock-transfected control and p73-transfected cells.
Reporter gene assays.
Transfections were performed in 24-well plates with Lipofectamine 2000 according to the manufacturer's protocol. For promoter-luciferase constructs, each well was transfected with 100 ng of reporter plasmid (mdm2, bax, p21, cyclin G, PIG3, or CD95), 300 ng of p73 expression vector, and 100 ng of pCMX-β-gal reference plasmid containing a bacterial β-galactosidase gene. For Gal4-RE-luciferase constructs, each well was transfected with 100 ng reporter plasmid (MH100 Gal4RE-luc), 200 ng p73 expression vector, and 100 ng pCMX-β-gal reference plasmid. Where indicated, a total of 600 ng of the different PKC or DN-PKC expression vectors or 100 nM PKCα or PKCβ2 small interfering RNA (siRNA) was added to each transfection mixture. The same amount of DNA was added to each well. For PKC-412 treatment, 1 μM PKC-412 was added to cells 6 h after transfection. For VP16 treatment, a final concentration of 5 μM VP16 was added to cells 24 h after transfection and incubated for 6 h before harvesting. Cells were harvested 24 h after transfection and lysed, and extracts were assayed for luciferase and β-galactosidase activities in a microplate luminometer/photometer reader (Orion microplate luminometer; Berthold Detection Systems). Values shown are representative of at least three independent experiments made in triplicate, with error bars representing SD.
Growth rate analysis.
PC12 cells were transfected with mock or p73 vectors and treated with 500 μg/ml Geneticin for 48 h. Transfected/surviving cells were counted and reseeded. Cell counts were then performed at 3, 6, and 9 days, as indicated, using a hemacytometer and/or a CellCoulter apparatus (BD).
Cell cycle synchronization.
Nocodazole (50 ng/ml) or hydroxyurea (2 mM) was added to H82 cells 24 h after transfection, and cells were incubated for 12 or 15 h, respectively. Cells were washed to remove the nocodazole or hydroxyurea and left in culture for the indicated time periods. Cells were subsequently assayed for cell cycle phase using PI or BrdU staining and FACS analysis (as described above). For gene reporter assays, H82 cells were transfected with mdm2-luc, β-gal, and mock plasmid or TA-mutated p73δ. Twenty-four hours after transfections, nocodazole or hydroxyurea was added to cells and incubated for 12 or 15 h, respectively. Cells were washed and left in culture for the indicated time periods before being subjected to gene reporter analysis, as described above.
Immunoprecipitation and immunoblotting.
Twenty-four hours after transfection with plasmids encoding p73β, TAmut p73β, and TAmut p73β S388A, H82 cells were lysed in NP-40 buffer (150 mM sodium chloride, 1% NP-40, 50 mM Tris-HCl [pH 8]). Samples were precleared with protein G-Sepharose beads and then incubated with p73 antibody overnight. Protein G-Sepharose beads were added, and samples were incubated for 1 h. Beads were washed in radioimmunoprecipitation assay lysis buffer (150 mM sodium chloride, 1% NP-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate, 50 mM Tris-HCl [pH 8]), resuspended in Laemmli loading buffer, and boiled for 3 min. Samples were resolved on 10% sodium dodecyl sulfate-polyacrylamide gels and blotted onto nitrocellulose membranes (Amersham Biosciences). Membranes were then probed with rabbit antiphosphoserine antibody and mouse antihemagglutinin antibody. Primary antibody binding was detected using horseradish peroxidase-conjugated goat anti-mouse IgG (Pierce). After repeated washing in Tris-buffered saline, bands were visualized by enhanced chemiluminescence (ECL Plus) according to the manufacturer's instructions (Amersham Biosciences) and analyzed using phosphorimaging (Image Reader LAS-1000 Pro V2.6; Fujifilm).
H82 cells were transfected with plasmids encoding p73β, TAmut p73β, and TAmut p73β S388A. Chromatin immunoprecipitations were carried out using a ChIP assay kit (Upstate, MilliPore) according to the manufacturer's protocol and p73 antibody (Ab-4). PCRs were performed using primers for the p21 promoter (forward primer 5′-GTG GCT CTG ATT GGC TTT CTG-3′ and reverse primer 5′-CTG AAA ACA GGC AGC CCA AG-3′), the distal p21 promoter (2.8 kb upstream of the p53-responsive element) (forward primer 5′-GGA GTC CTG TTT GCT TCT GG-3′ and reverse primer 5′-CTT TGG CCA CAC TGA GGA AT-3′), the bax promoter (forward primer 5′-TAA TCC CAG CGC TTT GGA AG-3′ and reverse primer 5′-TGC AGA GAC CTG GAT CTA GCA-3′), the distal bax promoter (~2.5 kb upstream of the p53-responsive element) (forward primer 5′-GAC CTT GCT TTG CTC TAA GCT ATC-3′ and reverse primer 5′-GAG CCT GTC TCA AAA AGA AAA AAG-3′), the mdm2 promoter (forward primer 5′-GGT TGA CTC AGC TTT TCC TCT TG-3′ and reverse primer 5′-GGA AAA TGC ATG GTT TAA ATA GCC-3′), and the distal mdm2 promoter (~2.5 kb upstream of the p53-responsive element) (forward primer 5′-TGA ATC TAC TCT TGG TGG TCC-3′ and reverse primer 5′-AAG GAA ATT TGG GCT TTC GAC-3′). Quantification of DNA band intensity was made using ImageJ software according to the supplier's instructions.
Statistical analyses were performed using a two-tailed, paired Student's t test and one-way analysis of variance (ANOVA), where P values of <0.01 and P values of <0.05 were considered to be significant.