Conditioned medium from tumor cells induces Flt-1, but not VWF, mRNA and promoter activity.

(A) HUVECs were serum-starved and then incubated with or without tumor cell–conditioned medium. In RNase protection assays, an α-[³²P] UTP-labeled 413-bp human Flt-1 antisense riboprobe was incubated with 10 μg yeast RNA (lane 1) or total RNA from untreated HUVECs (lane 2), HUVECs treated with conditioned medium from Lewis lung carcinoma (LLC) cells (lane 3), or HUVECs treated with conditioned medium from B16-F1 melanoma cells (lane 4). An α-[³²P] UTP-labeled 392 bp human VWF antisense riboprobe was incubated with 10 μg yeast RNA (lane 5) or total RNA from untreated HUVECs (lane 6), HUVECs treated with conditioned medium from Lewis lung carcinoma (LLC) cells (lane 7), or HUVECs treated with conditioned medium from B16-F1 melanoma cells (lane 8). The protected fragments (316-bp hFlt-1 and 288-bp hVWF) represent the human Flt-1 and VWF transcripts, respectively. The upper band in the VWF gel represents undigested probe. An α-[³²P] UTP-labeled human GAPDH antisense riboprobe was hybridized with total RNA as an internal control. The results are representative of 2 independent experiments. (B) HUVECs were transiently transfected with Flt-1-luc and VWF-luc-2 then incubated for 24 hours with conditioned medium from Lewis lung carcinoma, B16-F1 melanoma, NHEK-neos, or RPTECs. The results show the means and standard deviations of luciferase light units (relative to untreated cells) obtained in triplicate from 3 independent experiments.