Date of Award:

5-1998

Document Type:

Dissertation

Degree Name:

Doctor of Philosophy (PhD)

Department:

Animal, Dairy, and Veterinary Sciences

Department name when degree awarded

Toxicology

Committee Chair(s)

Howard M. Deer

Committee

Howard M. Deer

Committee

David B. Drown

Committee

M. Keven Jackson

Committee

Jeffery O. Hall

Committee

Scott A. Ensign

Committee

Robert C. Lamb

Abstract

These studies investigated the effects of retinoic acid (RA) on angiogenesis. Three human, neoplastic cell lines were used to examine angiogenic promotion and/or inhibition. The cell lines, U-373MG glioblastoma, DU-145 prostate carcinoma, and TCCSUP bladder transitional cell carcinoma, were treated with the following: all-trans, 9-cis, or 13-cis RA, at doses from 0.0001 to 100 μM. Hypoxia was used to assist the expression of the angiogenic phenotype. Conditioned media (CM) were prepared by growing the tumor cells in the presence of RA and hypoxia for 24 hours, and then the CM was transferred to bovine, capillary endothelial cells (EC) for 48 hours. The EC were counted for each CM and compared to controls. Multiplex, reverse transcriptase-polymerase chain reaction (MRT-PCR) was used on tumor cell RNA to demonstrate that mRNA expression of several angiogenic growth factors was modified. Angiogenic proteins in the CM were identified by enzyme-linked immunosorbent assay (ELISA).

The CM from the U-373 and DU-145 cells, but not the TCCSUP cells, treated with all-trans or 9-cis RA caused significant increases (Pcis RA did not increase EC proliferation.

MRT-PCR demonstrated that the tumor cells expressed vascular endothelial growth factor (VEGF), midkine, interleukin-8 (IL-8), thrombospondin (TSP), and leukemia inhibitory factor (LIF) mRNAs. Increases VEGF mRNA corresponded to increase EC proliferation. IL-8 mRNA increased at RA concentrations greater than 0.1 μM. Midkine mRNA concentrations were generally unaffected by RA treatments. ELISA demonstrated that VEGF was increased by all-trans RA, to a lesser degree by 9-cis, and even less by 13-cis. IL-8 protein secretion was inhibited by RA. The expression of TSP and LIF mRNAs was almost completely inhibited by hypoxia.

EC were grown with sodium heparin at 0, 10, 100, 500, or 1000 μg/ml in CM produced with DU-145 cells and 0.0001, 0.001, or 1 μM all-trans RA. Heparin increased EC proliferation at 10 μg ml and inhibited proliferation at higher concentrations.

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