Keratinocytes were isolated from newborn BALB/c, p53+/+
, and p53−/−
mice by using established methods (10
) and plated at a density of 3 × 106
cells per 60-mm dish, 7 × 106
cells per 100-mm dish, and 15 × 106
cells per 150-mm dish, respectively. Cultures were maintained in Eagle's minimum essential medium without Ca2+
(Bio-Whittaker) but with 8% Chelex-treated fetal bovine serum (Gemini Bio-Products), 0.05 mM Ca2+
, and 20 U of penicillin-streptomycin (Gibco) ml−1
. Cell line SP1, derived from a chemically induced mouse skin papilloma (52
), was cultured in the medium described above. The nontumorigenic S1 cell line was derived from normal mouse keratinocytes and maintained in the medium described above but supplemented with keratinocyte growth factor. p53-null keratinocytes (line AK1b) (4
) were maintained in medium consisting of 16% fibroblast-conditioned Eagle's minimum essential medium with 8% Chelex-treated serum and 0.05 mM Ca2+
. The p53 Tet-On Saos-2 cell line (45
) and the Bax Tet-On Saos-2 cell line (40
) were generous gifts from Kevin Ryan and Karen Vousden, National Cancer Institute. The cells were maintained in Dulbecco's modified Eagle's medium (Bio-Whittaker) with 8% fetal bovine serum (Gemini) and 20 U of penicillin-streptomycin ml−1
. Induction of the regulated genes was achieved by treating the cells with doxycycline at a final concentration of 800 ng/ml. The Bax sequence is tagged with a p53 epitope recognized by antibody DO-1 (see below). Cells were treated with etoposide or adriamycin (both from Sigma) in culture medium at various concentrations and times.
The cloning of mtCLIC and the construction of the pEGFP-N1 (GFP-mtCLIC) and pEGFP-C1 (mtCLIC-GFP) fusion vectors have been described elsewhere (15
). The mtCLIC open reading frame was also cloned in the pCR3.0 vector (Invitrogen) in the sense and antisense orientations and sequenced. The plasmid expressing the green fluorescent protein (GFP) spectrum was obtained from Clontech, Palo Alto, Calif., and used as a transfection control and cotransfection marker for fluorescence-activated cell sorter (FACS) analysis.
Antibody generation, immunoprecipitation, and Western blot analysis.
Polyclonal antibodies generated against the N-terminal and C-terminal peptides of mtCLIC have been described elsewhere (15
). The polyclonal sera were purified through a protein A column (Pharmacia) following manufacturer specifications and dialyzed in borate buffer. The polyclonal sera were also affinity purified against the immunogenic peptides at the Core Facility of the Frederick Cancer Research and Development Center.
Protein expression was analyzed by Western blotting. Cells were washed and then gently scraped into radioimmunoprecipitation lysis buffer.Thirty micrograms of protein was separated by 10 or 12% polyacrylamide gel electrophoresis-sodium dodecyl sulfate and transferred to nitrocellulose membranes. In some experiments, subcellular fractions were isolated as described previously (15
). Antibodies against the N terminus and the C terminus of mtCLIC were used at 1:1,000 and 1:4,000 dilutions, respectively. A goat anti-rabbit horseradish peroxidase conjugate (Bio-Rad) was used as a secondary antibody. Monoclonal antibodies directed to murine p53 were raised as culture supernatants from the mouse hybridoma cell line PAb122 (19
). Antibody DO-1 to a p53 sequence tag on Bax was from Oncogene, anti-β-actin mouse polyclonal antibody was from Boehringer Mannheim, and anti-cytochrome c
antibody was from BD PharMingen. Blots were developed with enhanced chemiluminescence and SuperSignal chemiluminescence substrates (Pierce).
Immunoprecipitation was performed as follows. Cells were washed with cold phosphate-buffered saline (PBS) and lysed in 50 mM Tris buffer containing 150 mM NaCl, 1.5 mM MgCl2 10% glycerol, 1% Triton X-100, 5 mM EGTA, 20 μM leupeptin, 10 μg of aprotinin/ml, 1 mM phenylmethylsulfonyl fluoride, 200 μM NaVO3, and 10 mM NaF. Lysates were precleared with protein G/A PLUS-agarose beads and incubated with the desired primary antibody at 4°C for 2 h before overnight incubation with protein G/A PLUS-agarose beads at 4°C. Beads were washed in radioimmunoprecipitation buffer, centrifuged, resuspended, and boiled prior to electrophoresis.
S1 cells transfected with mtCLIC-GFP or GFP were fixed with 2.5% glutaraldehyde in 0.1 M sodium phosphate buffer (pH 7.2), postfixed with 1% osmium tetroxide in the same buffer, treated with 2% aqueous uranyl acetate, dehydrated in a graded ethanol series, and embedded in EMbed 812 epoxy resin (Electron Microscopy Sciences). Sections (60 to 80 nm) were stained with uranyl acetate and lead citrate. All grids, including those with the immunolabeled specimens described below, were examined with a Zeiss EM902 microscope at 80 kV.
Cryosections were prepared and labeled by a modification of the Tokuyasu technique (39
). Briefly, HACAT cells were fixed with 2% formaldehyde-0.2% glutaraldehyde, infiltrated with 2.3 M sucrose, and frozen in liquid nitrogen. Frozen sections were cut, mounted on Formvar-carbon-coated grids, blocked on drops of 1% bovine serum albumin in PBS, and then incubated with the antibody against the C-terminal domain of mtCLIC. Labeling was visualized with 6-nm gold particles coupled to protein A (Aurion) diluted to an A520
of 0.05. After extensive washing with PBS, sections were fixed with 1% glutaraldehyde and embedded in 1.8% methylcellulose-0.4% uranyl acetate. Each labeling experiment was accompanied by a control in which an unrelated rabbit polyclonal antibody (51
) was used instead of the anti-mtCLIC antibody. The dilution of the control antibody was such that it produced robust labeling when the corresponding antigen was present.
RNA isolation, Northern blot hybridization, and reverse transcription (RT)-PCR.
Total RNA was isolated from cultured cells by Trizol extraction. RNA was resolved by formaldehyde-agarose gel electrophoresis and blotted as previously described (57
). A 300-bp DNA fragment for mtCLIC was amplified from the pCRII vector by using M13 amplification primers. cDNA probes were radiolabeled with 32
P (Lofstrand) and hybridized to the blots as described previously (57
). Bands were quantified by using a PhosphorImager (Molecular Dynamics). Loading equivalence was assessed on the basis of the 28S band or by reprobing blots for glyceraldehyde-3-phosphate dehydrogenase.
p53 Tet-On Saos-2 cells were grown to mid-log phase, treated with doxycycline (800 ng/ml), and collected at different time points. Approximately 0.5 μg of purified RNA was used to generate first-strand cDNA by RT with Superscript II (Gibco). Aliquots of the synthesized cDNA were used as a template in a PCR with Supermix (Gibco). The mtCLIC-specific primer set (5′-TTCCCCTTCATTTAAACACCTTT-3′ and 5′-TGCTATCTACATGCAACTCTGGA-3′) and the 18S gene-specific and Competimer primer set (Ambion) were mixed at a 2:8 ratio. PCR was performed for 30 cycles, and the PCR-amplified set (18S internal control, 550 bp; mtCLIC, 450 bp) of DNA fragments from each time point was analyzed on 4% agarose gels containing ethidium bromide.
Construction of a SEAP reporter vector containing the human mtCLIC promoter.
The human mtCLIC cDNA sequence was used as a query to search the public human genome database (National Center for Biotechnology Information, Bethesda, Md.), and the 5′ region upstream from the known 5′ untranslated region of mtCLIC was identified in the human contiguous AL445648.10 segment. From the identified sequence, the putative transcription start site was determined by using web-based bioinformation programs (Pedro's Biomolecular Research Tools). Specific primers and purified genomic DNA from cultured human foreskin keratinocytes were used to amplify by PCR a 3.5-kb DNA fragment that corresponded to the promoter sequence upstream from the putative transcription start site. Two separate sets of primers and the 3.5-kb DNA fragment were used to amplify by PCR 1.5-kb (PmtCLIC A/B) and 1-kb (PmtCLIC C) DNA fragments containing the putative p53-binding sites (TF-Bind and TF-Factor). These two fragments were cloned into the pGEM-T Easy vector (Promega, Madison, Wis.) and then further subcloned into the pSEAP-basic reporter vector (Clontech) by ligating the EcoRI-digested DNA fragment from the pGEM-T Easy plasmid to the EcoRI-digested reporter plasmid. The orientation of the promoters was determined by restriction and sequencing analyses. A positive control vector was constructed by ligating a BamHI/SalI-digested secreted embryonic alkaline phosphatase (SEAP) insert from the pSEAP-basic reporter vector to the BamHI/SalI-digested pp53-TA-Luc vector and was confirmed by restriction and sequencing analyses. Reporter vectors were transfected into p53 Tet-On Saos-2 cells, and expression in transfectants was determined in the presence or absence of doxycycline. The expression of SEAP driven by PmtCLIC A/B and PmtCLIC C promoter activity was measured with a Great EscAPe SEAP chemiluminescence detection kit (Clontech) as described by the manufacturer.
Transfection of the GFP-mtCLIC fusion vectors or the pCR3.0 plasmids into primary BALB/c keratinocytes and SP1 and S1 cell lines was performed by using Lipofectamine Plus reagent (Gibco). Briefly, cell lines were plated 2 days before transfection at a density of 3 × 105
cells per 60-mm dish, and primary cells were plated 2 to 3 days before transfection at 3 × 106
cells per 60-mm dish. Cells were transfected with 4 μg of plasmid DNA (per dish) in serum-free medium that was replaced after 3 h with culture medium. Transfected cells were visualized with an inverted fluorescence microscope (Zeiss). With this approach, transfection efficiencies were 60 to 70% in cell lines and 20 to 30% in primary cultures. p53 Tet-On Saos-2 cells were transfected with Lipofectamine Plus reagent by using an empty adenovirus carrier (49
Apoptosis assays and FACS analysis.
Primary keratinocytes and SP1 and S1 cells transfected with the GFP fusion constructs mtCLIC-GFP and GFP-mtCLIC and with GFP plasmids or cotransfected with GFP and mtCLIC plasmids were analyzed by flow cytometry on a FACSCalibur instrument (Becton Dickinson). At various times after transfection, cells were trypsinized and collected in 1 ml of medium. All samples were assayed in the presence or absence of propidium iodide (PI) at a final concentration of 0.5 μg/ml. In a live-cell suspension, only cells with damaged membranes (dying cells) will take up PI. Double-color analysis was carried out on all samples. Similar results were obtained with cotransfection or transfection of fusion plasmids.
Keratinocytes transfected with the fusion proteins described above were treated with Z-VAD-FMK (Enzyme Systems Products) at a concentration of 40 μM. In one group, the caspase inhibitor was added immediately after the transfection medium was removed; in a second group, Z-VAD-FMK was added 24 h after removal of the transfection medium. Cells were analyzed by flow cytometry 48 h after transfection.
Transfected cultures were analyzed for the presence of cell surface annexin V as a measure of apoptotic death. Briefly, cells were trypsinized 24 and 48 h after transfection, centrifuged, and incubated with a biotin-conjugated antibody against annexin V (Genzyme). Cells were then washed and fixed in 10% neutral buffered formalin for 10 min. Fixed cells were incubated with allophycocyanin-streptavidin (Becton Dickinson) diluted in 1× binding buffer and analyzed by flow cytometry.
Mitochondrial membrane potential.
Transfected cells were trypsinized, resuspended in medium, and incubated for 30 min with Mitotracker. Samples were read immediately in the red and green channels in the flow cytometer. Analysis was performed by gating GFP-transfected cells.
Cells transfected with mtCLIC sense and antisense plasmids together with the GFP spectrum plasmid were trypsinized and fixed in cold 70% ethanol. Samples were kept at −20°C for at least 18 h. Cells were then pelleted, and 1 ml of PBS-Tween was added. After a second centrifugation, cells were treated with RNase A for 30 min, and 500 μl of PI (50 μg/ml) was added before flow cytometric analysis. The presence of the sub-G1 peak in gated green cells was used as a measure of apoptosis.
Cell viability was assessed by the methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay (Promega) following manufacturer instructions.