Endothelial cell growth factor

Renal carcinoma Caki1 and KTCTL-26 cell lines, both derived from a clear cell renal cell carcinoma and von Hippel-Lindau (VHL) positive, were purchased from LGC Promochem (Wesel, Germany). A498 cells with disrupted VHL function were derived from Cell Lines Service (Heidelberg, Germany). The tumor cells were grown and subcultured in RPMI 1640 medium supplemented with 10% fetal calf serum (FCS), 1% Glutamax (all Gibco/Invitrogen, Karlsruhe, Germany), 2% HEPES (2-(4-(2-Hydroxyethyl)-1-piperazinyl)-ethansulfonsäure) buffer and 1% penicillin/streptomycin (both Sigma-Aldrich, München, Germany), at 37 °C in a humidified 5% CO₂ incubator. Subcultures from passages 5–30 were selected for experimental use.
Human umbilical vein endothelial cells (HUVEC), isolated from human umbilical veins, were grown in Medium 199 (M199; Biozol, Munich, Germany), 10% FCS, 10% pooled human serum, 20 μg/mL endothelial cell growth factor (Boehringer, Mannheim, Germany), 0.1% heparin, 100 ng/mL gentamycin and 20 mM HEPES-buffer. Subcultures from passages 2 to 6 were selected for experimental use.

The human prostate tumor cell line PC3 was obtained from DSMZ (Braunschweig, Germany). The tumor cells were grown and subcultured in RPMI 1640 medium (Gibco/Invitrogen, Karlsruhe, Germany) supplemented with 10% fetal calf serum (FCS), 2% HEPES (2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid) buffer (1 M, pH 7.4), 2% glutamine, and 1% penicillin/streptomycin at 37 °C in a humidified, 5% CO₂ incubator. Temsirolimus-resistant sublines were developed over 12 months of continuous exposure to temsirolimus (Torisel^®, LC Laboratories, Woburn, MA, USA), starting at 1 nm/mL and increasing stepwise to 10 µm/mL (PC3^res). Control cells remained untreated (PC3^par). The cell doubling times of 22.07 h (PC3^par) and 24.05 h (PC3^res) were calculated according to the following formula: $\mathrm{Duration}\mathrm{of}\mathrm{culture}=\frac{\ln \left(2\right)}{\ln \left(\mathrm{final}\mathrm{cell}\mathrm{number}\right)-\ln \left(\mathrm{initial}\mathrm{cell}\mathrm{number}\right)}$
Human endothelial cells (human umbilical vein endothelial cells; HUVECs) were isolated from human umbilical veins and harvested by enzymatic treatment with dispase (Gibco/Invitrogen). They were grown in Medium 199 (M199; Biozol, Munich, Germany), supplemented with 10% FCS, 10% pooled human serum, 20 µg/mL endothelial cell growth factor (Boehringer, Mannheim, Germany), 0.1% heparin, 100 ng/ml gentamycin, and 20 mm HEPES buffer (pH 7.4). Subcultures from passages 2–6 were selected for experimental use.

RT112, UMUC-3 (ATCC/LGC Promochem GmbH, Wesel, Germany) and TCCSUP (DSMZ, Braunschweig, Germany) bladder carcinoma cells were grown and subcultured in RPMI 1640, 10% fetal calf serum (FCS), 20 mM HEPES-buffer, 1% glutamax and 1% penicillin/streptomycin (all: Gibco/Invitrogen; Karlsruhe, Germany). Subcultures from passages 7–24 were selected for experimental use. Human endothelial cells (HUVEC) were isolated from human umbilical veins and harvested by enzymatic treatment with dispase (Gibco/Invitrogen). HUVEC were grown in Medium 199 (M199; Biozol, Munich, Germany), supplemented with 10% FCS, 10% pooled human serum, 20 µg/ml endothelial cell growth factor (Boehringer, Mannheim, Germany), 0.1% heparin, 100 ng/ml gentamycin and 20 mM HEPES-buffer (pH 7.4). Subcultures from passages 2–6 were selected for experimental use. HUVEC were used in the study. The institutional ethics committee of the Goethe-University Hospital, Frankfurt, Germany, approved the investigation and waived the need for consent, since HUVEC were used anonymously for in vitro assays with no link to patient data.