Article Figures & Data
Figures
- FIGURE 1.
Expression of EFEMP1 in vivo. Immunofluorescence of EFEMP1 expression in vivo in tumors originating from FG (A) and L3.6pl cells (B) 37 d after implantation. Insets, DAPI counterstain. Confocal microscopy of EFEMP1 expression in cultured FG cells (C) and L3.6pl cells (D). EFEMP1 staining (green) is increased in L3.6pl cells and follows a homogeneous perinuclear pattern. Bars, 50 μm.
- FIGURE 2.
Regulation of EFEMP1 expression. Relative expression of EFEMP1 in L3.6pl cells, determined by quantitative RT-PCR. Treatment with IFN-α results in a marked up-regulation of EFEMP1 expression that is counteracted by the NF-κB inhibitor BAY11-7082. Representative data from three independent experiments are shown.
- FIGURE 3.
In vivo growth of EFEMP1-overexpressing tumors. A. Expression levels of EFEMP1 protein in FG, FG-EFEMP1, and L3.6pl cells. B. Orthotopic pancreatic tumor growth after injection of FG, FG-EFEMP1, and L3.6pl cells. FG-EFEMP1 versus FG: §, P < 0.05; *, P < 0.01. C. Tumor weight on necropsy 28 d after tumor cell injection. FG versus FG-EFEMP1, and FG-EFEMP1 versus L3.6pl; *, P < 0.01. D. Macroscopic incidence of hepatic and lymphogenic metastases (n = 10). E to G. H&E staining of FG (E), FG-EFEMP1 (F), and L3.6pl (G) tumors grown orthotopically. Necrosis (black arrows) and fibrotic tissue (white arrows) both are most pronounced in L3.6pl tumors. Bars, 200 μm.
- FIGURE 4.
EFEMP1 induced angiogenesis. A. In FG-EFEMP1 transfectants, a significant increase in VEGF production was observed (*, P < 0.01 in FG vector controls versus FG-EFEMP1). Notably, no further stimulation of VEGF production was seen in EFEMP1-transfected L3.6pl cells compared with L3.6pl vector controls. B. Proliferation of HUVECs induced by VEGF-A (50 ng/mL) and EFEMP1 protein (10 and 100 ng/mL). No significant direct effect of EFEMP1 protein on the proliferation was observed. VEGF-A versus medium control; *, P < 0.01. C. Migration of HUVECs induced by VEGF-A (50 ng/mL) and EFEMP1 protein (10 and 100 ng/mL) in a modified Boyden chamber assay. Again, there was no significant direct effect of EFEMP1 protein. VEGF-A versus medium control; *, P < 0.01. D. Microvascular density is increased in tumors grown from FG-EFEMP1 cells compared with FG (*, P < 0.05). E to G. CD31 staining of tumor specimens grown from FG (E), FG-EFEMP1 (F), and L3.6pl cells (G). Bars, 100 μm.
- FIGURE 5.
Suppression of apoptosis by EFEMP1 expression in vitro and in vivo. Under serum starvation conditions, a FACS scan of FG vector controls (A) shows 83.4% apoptotic tumor cells, whereas the proportion of apoptotic FG-EFEMP1 cells (B) is reduced (48.0%). Data from a typical experiment are shown. C. Proportions of apoptotic (solid bar), G0-G1–phase (dense bands), S-phase (sparse bands), and mitotic cells (white) of FG, FG-EFEMP1, and L3.6pl cells cultivated under serum starvation conditions. D. TUNEL staining of apoptotic tumor cells in xenografts of FG, FG-EFEMP1, and L3.6pl cells. Bar, 100 μm.
- FIGURE 6.
Relative expression of EFEMP1 mRNA in human pancreatic ductal adenocarcinoma specimens. EFEMP1 mRNA expression in the tumor and surrounding normal pancreatic tissue was compared, and individual samples are shown. EFEMP1 mRNA expression was up-regulated in 13 of 15 tumors. Columns, mean relative mRNA expression of quantitative RT-PCR done in triplicate.
Tables
- Table 1.
Differentially Expressed Genes in FG versus L3.6pl Cells In vitro
Item_name Fold change Title Gene symbol 244758_at 1.561 Zinc finger protein 452 ZNF452 223220_s_at 1.665 Poly(ADP-ribose) polymerase family, member 9 PARP9 205483_s_at 1.675 IFN, α-inducible protein (clone IFI-15K) G1P2 216252_x_at 1.702 Fas (tumor necrosis factor receptor superfamily, member 6) FAS 211137_s_at 1.755 ATPase, Ca2+ transporting, type 2C, member 1 ATP2C1 214291_at 1.792 Ribosomal protein L17/similar to dJ612B15.1 [novel protein similar to 60S ribosomal protein L17 (RPL17)] RPL17/dJ612B15.1 218280_x_at 1.816 Histone 2, H2aa HIST2H2AA 1555852_at 1.825 97935_MB_at 1.831 Signal transducer and activator of transcription 1, 91 kDa STAT1 212239_at 1.837 Phosphoinositide-3-kinase, regulatory subunit 1 (p85α) PIK3R1 219234_x_at 1.912 Secernin 3 SCRN3 225669_at 2.005 IFN (α, β, and ω) receptor 1 IFNAR1 227249_at 2.089 Myosin, heavy polypeptide 11, smooth muscle MYH11 241916_at 2.104 Phospholipid scramblase 1 PLSCR1 218736_s_at 2.121 Palmdelphin PALMD 238035_at 2.211 Sp3 transcription factor SP3 212764_at 2.274 209732_at 2.308 C-type lectin domain family 2, member B CLEC2B 221911_at 2.431 ets variant gene 1/hypothetical protein LOC221810 ETV1/LOC221810 202237_at 2.506 Nicotinamide N-methyltransferase NNMT 226603_at 2.596 Chromosome 7 open reading frame 6 C7orf6 204160_s_at 2.662 Ectonucleotide pyrophosphatase/phosphodiesterase 4 (putative function) ENPP4 239294_at 2.684 Transcribed locus 231929_at 2.707 MRNA; cDNA DKFZp586O0724 (from clone DKFZp586O0724) 204326_x_at 2.831 Metallothionein 1X MT1X 229450_at 3.004 IFN-induced protein with tetratricopeptide repeats 3 IFIT3 201667_at 3.270 Gap junction protein, α1, 43 kDa (connexin 43) GJA1 229764_at 3.477 FLJ41238 protein FLJ41238 227997_at 3.530 Interleukin-17 receptor D IL17RD 206632_s_at 4.106 Apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like 3B APOBEC3B 223218_s_at 4.722 Nuclear factor of κ light polypeptide gene enhancer in B cells inhibitor, ζ NFKBIZ 219511_s_at 5.341 Synuclein, α interacting protein (synphilin) SNCAIP 204595_s_at 5.429 Stanniocalcin 1 STC1 201842_s_at 6.449 Epidermal growth factor–containing fibulin-like extracellular matrix protein 1 EFEMP1 208892_s_at 6.504 Dual specificity phosphatase 6 DUSP6 201860_s_at 86.594 Plasminogen activator, tissue PLAT NOTE: A fold change of >1.5 was considered significant.
- Table 2.
Apoptosis of FG, FG-EFEMP1, and L3.6p1 Cells after Treatment with Gemcitabine, 5-Fluorouracil, and Irinotecan
FG (vector) FG-EFEMP1 L3.6pl (vector) No treatment Apoptosis (%) 16.5 18.9 17.4 Fold change 1.00 1.00 1.00 Gemcitabine (50 ng/mL) Apoptosis (%) 44.3 39.9 37.3 Fold change 2.68 2.11 2.14 5-Fluorouracil (10 μg/mL) Apoptosis (%) 75.6 31.8 37.6 Fold change 4.58 1.68 2.16 Irinotecan (470 ng/mL) Apoptosis (%) 76.6 45.8 52.9 Fold change 4.64 2.42 3.04 NOTE: Proportions of apoptotic FG, FG-EFEMP1, and L3.6pl cells as determined by FACS after treatment with gemcitabine, 5-fluorouracil, and irinotecan at their respective IC50 doses (IC50 determination not shown).
Volume 7, Issue 2
