UROKINASE PLASMINOGEN ACTIVATOR RECEPTOR AND ITS ROLE IN TUMOR-ASSOCIATED ANGIOGENESIS

Paz J. Luncsford, Patricia A. Watson, and Sandra W. McLeskey, Ph.D.

School of Nursing, University of Maryland at Baltimore, 655 W. Lombard St., Baltimore, MD 21201-1512

Tumor–associated angiogenesis is the formation of blood vessels in tumors. These blood vessels provide tumors with oxygen and nutrients required for their growth. It is believed that tumor growth can be prevented if tumor-associated angiogenesis is stopped. On the molecular level, more must be studied about genes expressed in angiogenesis to develop safe and effective antiangiogenic breast cancer therapies.

Urokinase plasminogen activator receptor (uPAR) is a cell-surface molecule that urokinase plasminogen activator (uPA) binds to. When bound to uPAR, uPA is activated converting plasminogen into plasmin. Plasmin digests fibrin, allowing room for endothelial tube formation. uPAR is a gene expressed in tumor and mammary fat pad endothelial cells of mice.

Using in vitro and in vivo assays, uPAR’s effects on the motility and tube formation capability of endothelial cells can be studied. In one in vitro assay, HMEC-1 cells (immortal human endothelial cells) are placed in fibrin and the movement and tube formation of the cells are observed. In another in vitro assay, mice mammary fat pad blood vessels are inserted in fibrin to observe the formation of endothelial tubes from the explant. In an in vivo assay, tumors are created in the mammary fat pads of mice to observe angiogenesis. In all three assays, uPAR expression can either be shut down or increased using antisense or sense expression vectors for uPAR. This allows uPAR’s effects on angiogenesis to be studied.

Using long distance PCR, correctly sequenced uPAR cDNA has been obtained. The next step in this project is to ligate uPAR into the retroviral vector pLNCX2. uPAR-pLNCX2 can then produce a virus to infect the endothelial cells in the three above assays. Quantitative data from the assays can be used to gain a better understanding of uPAR’s role in tumor-associated angiogenesis.