Article Figures & Data
Figures
- FIG. 1.
The E1A-12 tumorigenic spacer does not mediate MHC class I downregulation. (A) Schematic representations of E1A-5, E1A-12, and E1A-12sm. The E1A proteins contain four conserved regions, CR1 to CR4. A salient feature of E1A-12 is the unique 20-amino-acid spacer region between CR2 and CR3 that is essential for tumorigenesis. E1A-12sm is the same as E1A-12 except for a single amino acid substitution (alanine to proline) at position 138 in the spacer region. (B) Northern blot analysis of MHC class I. Total RNAs isolated from cell lines DP5-2, 12-1, and 12-1sm, which were transformed by Ad5, Ad12, and spacer point mutant Ad12, respectively, were analyzed by Northern blotting using 32P-labeled MHC class I DNA as the probe. As a quantitative control, the same blot was rehybridized with a 32P-labeled RNase P probe.
- FIG. 2.
Microarray analysis of differential gene expression between tumorigenic and nontumorigenic Ad12-transformed cells. Total RNAs isolated from tumorigenic 12-1 cells and nontumorigenic 12-1sm cells were used for microarray analysis on an Affymetrix rat genome U34A microarray. The number of genes displaying a threefold or greater change in expression between the two cell lines is indicated.
- FIG. 3.
Neuronal and tumor-related genes are expressed in Ad12 tumorigenic cells. Neuronal and tumor-related genes identified by microarray analyses (upper portion) or not reported in the microarray (bottom portion) were analyzed by Northern blotting. Total RNAs isolated from different E1A-expressing cell lines were hybridized with a 32P-labeled DNA probe derived from the indicated gene. As a quantitative control, RNA levels of RNase P were compared in each cell line. DP5-2 is a cell line transformed by Ad5, and 12-1, 12-1sm, and 12-1sd are cell lines transformed by wild-type, spacer point mutant, and spacer deletion mutant Ad12, respectively. MT3-MMP, membrane-type matrix metalloproteinase.
- FIG. 4.
Stable transfection of E1A-12 into Ad5 nontumorigenic cells induces neuronal and tumor-related gene expression. (A) Northern blot analyses of representative neuronal and tumor-related genes. Ad5-transformed cells (DP5-2) were transfected with 13S E1A-12-expressing plasmids to generate a stable cell line (DP5-2/E1A-12) that coexpresses E1A-5 and E1A-12. Total RNAs prepared from normal DP5-2 cells, Ad12-transformed cells (12-1), vector-transfected cells (DP5-2/vector), and stable DP5-2/E1A-12 cells were used for Northern blot hybridization. All stably transfected cell lines were independently derived. (B) Western blot analyses of E1A-12 expression. Whole-cell extracts from the cells shown in panel A were immunoblotted using an antibody against E1A-12. As a quantitative control, the same blot was reprobed with β-actin antibody. The number assigned to each lane corresponds with that in panel A.
- FIG. 5.
Northern blot profiles of representative neuronal genes from different Ad5- and Ad12-transformed cells. Total RNAs isolated from Ad5- and Ad12-transformed cells and from normal mouse brain and kidney cells (serving as controls) were hybridized with 32P-labeled DNA probes derived from neuronal genes. As a quantitative control, RNA levels of RNase P are also shown.
- FIG. 6.
NRSF is retained in the cytoplasm of Ad5- and Ad12-transformed cells. (A) Western blot analyses of NRSF expression. Protein extracts of whole cells (WE), cytoplasm (CE), and nuclei (NE) from DP5-2 (Ad5) and 12-1 (Ad12) cells were analyzed by Western blotting using an anti-NRSF antibody. As a quantitative control, the same blots were reprobed with an antibody against β-actin. (B) Confocal immunofluorescent microscopy. Indirect immunofluorescence was performed for staining NRSF (green). Nuclei were stained with DAPI (blue). (C) EMSA. Equal amounts (15 μg) of CE and NE from DP5-2 and 12-1 cells were analyzed using a 32P-labeled double-stranded DNA probe that contains an RE1 sequence strongly recognized by NRSF. Positions of free and NRSF-bound RE1 probe are indicated.
- FIG. 7.
Promoter activation of α-internexin in Ad12 cells requires E-box elements. (A) Schematic representations of firefly luciferase reporter driven by the rat α-internexin promoter. The α-internexin promoter (Intx, −1100 to +73) is denoted by a thin line and the luciferase gene by a black bar. The sequences and locations of two E-box elements in the promoter relative to the transcription initiation site (+1, indicated by an arrow) are given. Conserved E-box sequences are shown in bold, and mutations in the E-box elements are underlined. Names of each reporter are indicated on the left side. (B) Luciferase activity assay. DP5-2 and 12-1 cells were transfected with plasmids of firefly luciferase reporter shown in panel A together with Renilla luciferase reporter plasmids (internal control). At 24 h posttransfection, the cells were lysed and luciferase activities were measured. Data represent averages and standard deviations from three independent experiments. The control is firefly luciferase reporter lacking a promoter. (C) EMSA. EMSA was conducted by incubating nuclear extracts (10 μg) of DP5-2 and 12-1 cells with 32P-labeled E-box1 double-stranded oligonucleotide (only sense sequences shown) derived from the α-internexin promoter. For DNA binding competition assay, the nuclear extract of 12-1 cells was preincubated with 1-, 5-, and 10-fold unradiolabeled wild-type (wt) or mutant (mt) E-box1 double-stranded oligonucleotides, followed by EMSA as described above. The E-box 1 conserved sequences are shown in bold, and mutations in E-box 1 are underlined.
Tables
- TABLE 1.
Genes differentially expressed in tumorigenic and nontumorigenic Ad12-transformed cells as determined by microarray analysis
Categorya Ratiob GenBank accession no. Gene product name Neuronal 55.7 U02096 Fatty acid binding protein (brain type) 24.3 M63656 Aldolase C (brain type) 24.3 X73411 snRNP-associated polypeptide N (brain type) 13.0 M29293 Neural snRNP 10.6 X52817 Neural C1-13 9.2 U17604 rS-Rex-b (brain type) 4.9 AF041082 Transmembrane receptor Robo1 4.0 U08290 Neuronatin alpha 3.5 U09357 Neural receptor-type protein tyrosine phosphatase zeta/beta 3.5 AF004431 Homeodomain protein Nkx6.1 3.2 X06984 Aldolase C (brain type) 3.2 X55812 SKR6, a CB1 cannabinoid receptor −3.2 AF023621 Sortilin Cell cycle 10.6 D14014 Cyclin D1 4.6 X75207 Cyclin D1 Transcription 6.1 Y00396 c-MYC oncogene and flanking regions 3.0 U56242 Transcription factor Maf2 −3.2 L03556 Hox1.3 protein (hox1.3) −3.2 X60769 Silencer factor B −6.5 L23148 Inhibitor of DNA binding, splice variant Id1.25 Tumor invasiveness 4.0 D10926 Tissue factor pathway inhibitor 3.2 D63886 Membrane-type matrix metalloproteinase 3.2 M23697 Tissue-type plasminogen activator Cell structure 12.1 AJ224879 Collagen alpha 1 type II −3.0 AF097887 Chp mRNA −3.7 X81448 Keratin 18 −6.5 S76054 Cytokeratin-8 Immune response 3.7 U77777 Gamma interferon-inducing factor isoform alpha precursor −7.0 U17035 Mob-1 Apoptosis −3.0 X96437 PRG1 Miscellaneous 10.8 M81681 Biliverdin reductase −4.0 X06483 Ribosomal protein L32 −4.9 L16764 HSP70 −10.6 Z27118 HSP70 −12.1 X62951 mRNA (pBUS19) with repetitive elements −21.1 AJ001044 EGP-314 protein homologue ESTs/unknown 9.2 AI230247 9.2 AI179399 9.2 AI105137 8.0 AA817854 6.1 AA893743 5.7 AA945737 4.3 RX013713 4.3 AA900476 4.0 AA899106 3.7 AI232379 3.5 AA893082 3.5 AA800503 3.5 AA800708 3.2 AI231292 3.2 AA800908 3.2 AAA799328 3.0 AA894305 3.0 AA891204 −3.0 AI070295 −3.2 AI177161 −3.7 AA894130 −5.3 RX049893 −6.1 H31287 −13.9 AA818604 −19.7 AA892468 −21.1 AI072634 −59.7 AA848563
