The role of hypoxia and p53 in the regulation of angiogenesis in bladder cancer.
OBJECTIVE
Our previous studies defined thrombospondin-1 (TSP-1) and vascular endothelial growth factor (VEGF) as the primary mediators of angiogenesis in the bladder and the loss of inhibitory TSP-1 as a key event in the transition to an angiogenic phenotype during bladder cancer development. We evaluated the role of p53, which is commonly inactivated in bladder cancer, and hypoxia in the regulation of angiogenesis in the bladder.


METHODS
The p53 status was modulated in normal urothelial and bladder cancer cells, and conditioned media was collected under normal oxygen or hypoxic (0.5% O2) conditions. Angiogenic activity was evaluated with the endothelial cell migration assay, and the levels of secreted TSP-1 and VEGF were determined by Western blot analysis and enzyme-linked immunosorbent assay, respectively.


RESULTS
Retroviral mediated expression of the E6 oncoprotein reduced wild-type p53 levels in normal urothelial cells by greater than 90% but did not significantly alter TSP-1 or VEGF levels, while total inductive and inhibitory activities remained unchanged. Adenoviral mediated expression of wild-type p53 was confirmed in 4 bladder cancer cell lines by Western blot analysis for p53 and its downstream effector protein p21 (2.5 to 5.0-fold increase). TSP-1 levels remained unchanged but the levels of secreted VEGF in the high grade UMUC-3 and 253J cell lines were significantly decreased 5 to 50-fold and a corresponding decrease in net angiogenic activity was observed. However, (increased expression) of p53 had no effect on the angiogenic activity of the low grade RT4 or high grade HT1376 bladder cancer cells. Hypoxia converted normal urothelial cell derived conditioned media from anti-angiogenic to angiogenic and increased the angiogenic activity of bladder cancer cell derived conditioned media. This change was due to 2.5 to 6-fold hypoxic up-regulation of VEGF because the expression of inhibitory TSP-1 was not significantly altered.


CONCLUSIONS
Our results suggest that p53 does not regulate angiogenesis in the bladder in the setting of an otherwise normal genome and gene therapy with wild-type p53, which is currently being studied for this cancer, may have only limited effects on angiogenesis. In contrast, hypoxia regulates angiogenesis in this system, primarily through its effects on VEGF.
Reiher
F K
FK
Department of Urology, R. H. Lurie Comprehensive Cancer Center, Northwestern University Medical School, Chicago, Illinois, USA.
Ivanovich
M
M
Huang
H
H
Smith
N D
ND
Bouck
N P
NP
Campbell
S C
SC
D015153:Blotting, Western; D002295:Carcinoma, Transitional Cell / Q000503:physiopathology* / Q000628:therapy; D015687:Cell Hypoxia / Q000502:physiology; D015536:Down-Regulation; D016228:Endothelial Growth Factors / Q000378:metabolism / Q000502:physiology; D005786:Gene Expression Regulation; D016158:Genes, p53 / Q000502:physiology; D015316:Genetic Therapy; D006801:Humans; D008222:Lymphokines / Q000378:metabolism / Q000502:physiology; D009389:Neovascularization, Pathologic / Q000503:physiopathology*; D019700:Thrombospondin 1 / Q000378:metabolism; D014407:Tumor Cells, Cultured; D016159:Tumor Suppressor Protein p53 / Q000502:physiology*; D015854:Up-Regulation; D001749:Urinary Bladder Neoplasms / Q000503:physiopathology* / Q000628:therapy; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors
D016228:Endothelial Growth Factors; D008222:Lymphokines; D019700:Thrombospondin 1; D016159:Tumor Suppressor Protein p53; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors