Article Figures & Data
Figures
- Fig. 1.
Characterization of anti-CITED4 monoclonal antibody HT13. A, Western blot performed using 50 μg of whole cell extracts prepared from U2-OS cells transfected with plasmids expressing HA-CITED1, HA-CITED2, or HA-CITED4 or untransfected cells (Control). Whole cell extracts were prepared as described previously (10) . The extracts were fractionated on a 12% SDS-PAGE gel, transferred to a polyvinylidene difluoride membrane, and probed with a rat anti-HA tag antibody (Roche) used at 1:200 dilution (top panel) or with HT13 anti-CITED4 antibody used at 1:5 dilution (middle panel). The whole cell extracts were also probed using anti-β-tubulin antibody (T-5293, Sigma, Chemical Co., Poole, Dorset, United Kingdom) at dilution 1:1,000 to confirm equal loading. B (top panel), Western blot of 50-μg ECV304 and MDA231 whole cell extracts fractionated on a 12% SDS-PAGE gel, transferred to a polyvinylidene difluoride membrane, and probed using HT13 antibody supernatant diluted 1:5. Bottom panel, Western blot of the same membrane using anti-β-tubulin antibody (Sigma T-5293) at dilution 1:1,000 to confirm equal loading. C, immunofluorescence: U2-OS cells were transfected with plasmids expressing HA-CITED1 (panels d), HA-CITED2 (panels c), HA-CITED4 (panels b), or untransfected (panels a, Control). Forty-eight hours after transfection cells were coimmunostained. Rat anti-HA tag (Roche) was used at 1:200 dilution followed by secondary antibody [rabbit antirat IgG coupled to FITC (Sigma)] at 1:400 dilution. Neat culture supernatant containing the anti-CITED4 monoclonal HT13 was used in combination with rabbit antimouse antibody coupled to rhodamine (Chemicon, Harrow, United Kingdom) at 1:200 dilutions. Nuclei were counterstained with TOPRO (Molecular Probes Ltd., Eugene, OR). Cells were mounted in Vectamount (Vector Laboratories, Peterborough, United Kingdom) and were visualized using a Bio-Rad MRC 1024 confocal microscope. Data from TOPRO (blue), FITC (green), and rhodamine (red) channels were sequentially accumulated. The merged image is shown in the panels on the extreme right.
- Fig. 2.
CITED4 inhibits hypoxia-activated transcription. A, CITED4 inhibits hypoxia-inducible factor 1α transactivation. Hep3B cells were transiently cotransfected with GAL4-hypoxia-inducible factor 1α or GAL4 DNA-binding domain alone (40 ng), 3xGAL4-luc reporter (100 ng), CMV-lacZ (100 ng), and the indicated HA-CITED4 expressing plasmids (40 ng). HA-CITED4Δ plasmid expresses HA-fused to residues 1–137 of CITED4. The vector plasmid served as an additional control. The effect of HA-CITED4 proteins on GAL4-hypoxia-inducible factor 1α transactivation was tested on cells maintained in normoxia (21% O2) or stimulated with hypoxia (1% O2) for 16 hours. Results are presented as relative luciferase units, corrected for lacZ activity, and show the mean of three independent experiments. B, CITED4 blocks the transcriptional activation of a hypoxia response element. Hep3B cells were cotransfected with 3xHRE-luc reporter (40 ng), CMV-lacZ (100 ng), and the indicated CITED4 or CITED4Δ expressing plasmids, or pcDNA3 vector control (40 ng). The effect of CITED4 proteins was tested on cells maintained in normoxia or stimulated with hypoxia for 16 hours. Results are presented as for A. C, CITED4 blocks hypoxia-inducible factor 1α interaction with p300-CH1. Top panels: Autoradiograms of SDS-PAGE gels. Lane 1, 20% of the input of hypoxia-inducible factor 1α generated as an in vitro translated 35S-labeled peptide. The relative amount of the factor bound to bacterially expressed GST (Lane 2) or to GST-p300CH1 (p300 residues 300–528, Lanes 3–5) immobilized on glutathione-Sepharose beads is shown. Binding was tested in the absence (Lane 3) or presence of 20 μg of a synthetic peptide corresponding to the p300-CH1 binding domain of CITED4 (Lane 4, C4C) or in the presence of 20 μg of a control peptide from the NH2 terminus of CITED4 (Lane 5, C4N). Bottom panels, Coomassie-stained gel showing relative amounts of GST and GST-p300CH1 proteins. Bars, ±SD. (RLU, relative luciferase units; HRE, hypoxia response element; HIF, hypoxia-inducible factor)
- Fig. 3.
Immunohistochemistry for CITED4 strong nuclear staining of CITED4 in the nuclei of luminal cells of a duct (A) and in an acinus (B) of a terminal duct lobular unit. CITED4 staining in the nucleus and cytoplasm of the endothelium of a small capillary (C). Heterogeneous nuclear and cytoplasmic CITED4 immunoreactivity within one duct of an intermediate nuclear grade ductal carcinoma in situ; ∗, absence of up-regulation around the area of necrosis (D). Homogeneous strong staining of CITED4 within individual ducts involved by low-grade ductal carcinoma in situ but with low expression of CITED4 in ducts in an adjacent terminal duct lobular unit (arrow; E). Strong CITED4 positivity in the cytoplasmic compartment of malignant glands (black arrow) in contrast to entrapped normal elements, where CITED4 is predominantly nuclear (∗); inset, occasional tumors showed strong nuclear expression (F). Strong cytoplasmic and weak nuclear CITED4 staining of an invasive ductal carcinoma; inset, CITED4 expression in pericytes surrounding a small vessel (G).
- Fig. 4.
Kaplan and Meier relapse-free (top graph) and overall survival (bottom graph) stratified by CITED4 function.
Tables
- Table 1
Clinicopathological data from the series of patients stratified by CITED4 nuclear immunopositivity
CITED4 nuclear-negative (N = 124) CITED4 nuclear-positive (N = 162) P Age (years) <50 38 43 0.45 ≥50 86 119 Nodal status negative 68 88 0.96 positive 56 74 Tumor size ≤2 cm 57 96 0.03∗ >2 cm 67 66 ER negative 47 47 0.11 positive 77 115 EGFR negative 51 60 0.48 positive 70 98 Grade I 16 31 0.0001 II 27 54 III 49 29 CVC <7 12 22 0.04∗ ≥7 19 12 HIF-1α tumor negative 81 123 0.05∗ positive 29 24 VEGF-A negative 58 83 0.50 positive 57 69 -
NOTE: Where n < 286 data unavailable.
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Abbreviations: ER, estrogen receptor; EGFR, epidermal growth factor receptor; CVC, Chalkley vessel count; HIF, hypoxia-inducible factor.
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∗ , significant.
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Volume 64, Issue 17
