Cell Lines and Plasmids
Human non-small lung carcinoma cell line H1299 and human osteosarcoma cell line SaoS2 were obtained from ATCC and were maintained in Dulbecco's modified Eagle's medium with 10% fetal bovine calf serum, 250 units of penicillin, and 250 μ
g of streptomycin. Expression plasmids encoding TAp63γ
, and TAp73α
were constructed in cytomegalovirus-driven pcDNA3.1A vector as described earlier (28
). VDR full-length and minimal promoter luciferase constructs were constructed as described previously (29
). p53 expression plasmid was a generous gift from Dr. Steven Berberich (Wright State University). VDRE-luciferase (VDRE-Luc) reporter was a generous gift from Dr. Alberto Munoz (University of Madrid). Etoposide, doxorubicin, and 1α
were purchased from Sigma. For constructing VDR short hairpin RNA expression vector, shVDR target sequence was selected using algorithms obtained from Dharmacon, Inc. (Chicago, IL) and Genscript Corp. (Piscataway, NJ). Subsequently, the target sequence to VDR was cloned into the PstI site in the pSilencer 3.1 vector obtained from Ambion Inc. (Austin, TX). Target sequence used for shVDR was as follows: shVDR-sense, 5′-GATCCGCAGCGCATTGCCATATTCTGCAGATATGGCAATGATGCGCTGCTGTTTTTTGGAAA-3′; shVDR-antisense, 5′-AGCTTTTCCAAAAAACAGCAGCGCATCATTGCCATATCTGCAGAATATGGCAATGATGCGCTGCG-3′.
Cells were seeded onto 6-well plates prior to the day of transfection. At around 80% confluence, transient transfections were performed using Lipofectamine2000 reagent (Invitrogen) with the desired plasmids in serum-free Dulbecco's modified Eagle's medium. For all of the studies, a total of 3 μg of expression plasmids, encoding TAp63γ, TAp73β, ΔNp73β, p53, and TAp73α, were used. For p53 dose-dependent studies, 1, 2, and 3 μg of p53 plasmid was used. After a 5-h incubation, the medium was replaced with Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine calf serum and 1% PS.
p73 Knock-down by siRNA
Cells were seeded in 6-well plates prior to the day of transfection. At around 30- 40% confluence, cells were subjected to calcium phosphate-mediated transfection. Briefly, 1 h before transfection, medium was changed, and the cells were kept in an incubator with 3% CO2. For each transfection reaction, siRNA (Qiagen) was mixed with CaCl2 (125 mm final concentration) and 2× BBS (50 mm BES, 280 mm NaCl, 1.5 mm Na2HPO4). The transfection mixture was incubated for 3 min at room temperature and then added to the respective wells. As control, an AllStars negative control nonsilencing RNA (Qiagen) was used. Six hours post-transfection, medium was changed, and cells were transferred to an incubator with 5% CO2. At 24 h, another round of transfection was performed as described above. After an additional 48 h, total RNA was harvested using the RNAeasy kit as per the manufacturer's protocol (Qiagen, Valencia, CA). Sequences used for p73 siRNA were as follows: sense, r(CGGGAUGCUCAACAACCAU)dTdT; antisense, r(AUGGUUGUUGAGCAUCCC G)dGdG.
g of total RNA was used to synthesize cDNA by using the TaqMan reverse transcription kit (Applied Biosystems, Foster City, CA). Quantitative real time PCR analysis was performed in a 96-well microtiter plate format on an ABI Prism7900HT sequence detection system using TaqMan Universal master mix and Assays on Demand specific for VDR (Hs_0017213_m1), p21 (Hs_00355782_m1), p73 (Hs_00232088_m1), and CYP27B1 ((Hs_00168017_m1) (PerkinElmer Life Sciences). Relative mRNA quantitation was performed by using the comparative ΔΔCt
method as described earlier (28
Protein and Immunoblot Studies
At 24 h after transfection or treatments, total protein was extracted from cells using radioimmune precipitation buffer (0.5% sodium deoxycholate, 1% Nonidet P-40, 0.1% SDS, phosphate-buffered saline, pH 7.4). Protein extracts were run on 10% SDS-PAGE and transferred onto polyvinylidene difluoride membrane and blocked with 5% blocking milk solution. Membranes were subsequently immunoblotted with antibodies to detect specific proteins. Monoclonal anti-VDR, rabbit polyclonal anti-p21, rabbit polyclonal anti-p53 FL-193, monoclonal anti-p63 4A4 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), and monoclonal anti-β-actin (Sigma) antibodies were used to detect VDR, p21, p53, p63, and β-actin expression, respectively. Monoclonal Ab-6 (Calbiochem) was used to detect p53. Appropriate horseradish peroxidase-conjugated antibodies (Promega, Madison, WI) were used as secondary antibodies, and the Supersignal West-pico Chemiluminescent Substrate kit (Pierce) was used to detect the chemiluminescence signal.
Immunoprecipitation for Endogenous p73α
After 24 h of doxorubicin and etoposide treatments, cells were harvested for total protein using radioimmune precipitation buffer, and 1 mg of total protein was used for immunoprecipitation. Briefly, total protein was precleared with 20 μl of rec-protein G-Sepharose beads (Invitrogen) for 1 h at 4 °C. After preclearing, beads were removed, and total protein was incubated with rotation overnight at 4 °C with 1 μg of monoclonal anti-p73 antibody (Ab-4 Lab Vision Corp., Fremont, CA). The next day, immunoprecipitated samples were incubated with rec-protein G-Sepharose beads for 1 h, followed by four washes with radioimmune precipitation buffer to remove the unbound proteins. Immunoprecipitated samples with beads were run on 10% SDS gel and immunoblotted with rabbit polyclonal anti-p73 antibody (Bethyl Laboratories Inc., Montgomery, TX).
To measure the VDR-Luc and VDR(min)-Luc reporter activities, cells were seeded in 6-well plates at 2.25 × 105 cells/well. At 24 h after seeding, cells were transfected with 100 ng of reporter constructs along with the desired plasmids as indicated. After 24 h of post transfection, cells were washed once with phosphate-buffered saline and harvested in passive lysis buffer (Promega, Madison, WI). Dual luciferase assays were performed to detect both firefly and Renilla luciferase activity using the Dual-Luciferase Reporter 1000 Assay System as per the manufacturer's protocol (Promega, Madison, WI). The relative luciferase activity was measured by calculating the ratio of Renilla luciferase activity to firefly luciferase activity. To measure the vitamin D3-mediated transcriptional activity of VDR, H1299 cells were seeded in 12-well plates at 6 × 104 cells/well and transfected with 50 ng of VDRE-Luc reporter along with the desired plasmids as indicated. The next day, cells were treated with 8 μm etoposide for 12 h, and then medium was replaced with fresh medium containing 1α,25-dihydroxyvitamin D3 (VD3). After 24 h of post VD3 treatments, cells were subjected to a dual luciferase assay as mentioned earlier.
Chromatin Immunoprecipitation Assay
Chromatin immunoprecipitation analysis was performed by using a chromatin immunoprecipitation kit (Upstate Cell Signaling Solutions, Waltham, MA) according to manufacturer's protocol. Briefly, H1299 cells were seeded onto three 15-cm dishes, and after 24 h, cells were fixed on a plate by using formaldehyde for 10 min. After fixation, cells were homogenized, and nuclei were subjected to sonication to shear the DNA. Fragmented chromatin was precleared using protein G beads for 1.5 h. Precleared chromatin was subjected to overnight immunoprecipitation with Mouse monoclonal anti-p73 Ab-4 (Lab Vision Corp., Fremont, CA) or normal rabbit IgG antibodies (Santa Cruz Biotechnology). Part of the precleared chromatin that was not subjected to immunoprecipitation was used as input DNA. After immunoprecipitation, both input DNA and immunoprecipitated chromatin were reverse cross-linked, and DNA was eluted. The eluted DNA was subjected to PCR amplification using GoTaq Green PCR master mix as per the manufacturer's protocol (Promega, Madison, WI) using primers specific for VDR (forward, 5′-CCCAAGCTTGATGATTATAGGTGCGGATACCCG-3′; reverse, 5′-CGGGGTACCCAGTAACAGGTTGCGACGGAG-3′) and p21 (forward, 5′-GGTACCGGCACTCTTGTTCCCCCAGGCTG-3′; reverse, 5′-CTCGAGACCATCCCCTTCCTCACCTGAAAA-3′). PCR conditions used for both VDR and p21 were as follows. A total of 40 cycles were performed, each consisting of 30 s at 94 °C, 30 s at 60 °C, and 45 s at 68 °C.