Title:
Regulation of angiogenic factors in human malignant melanoma in vitro and in vivo
Project management at the University of Würzburg:
Participating scientists:
Abstract:
Several growth factors with angiogenetic potential were studied in human melanocytes as well as melanoma cells in vitro, and it was shown that besides vascular endothelial growth factor (VEGV), which was assumed to be the only hypoxia-inducible angiogenetic factor up to now, two other angiogenic factors are also hypoxia-inducible: Angiogenin (ANG) and interleukin-8 (IL-8). By Northern blot analysis it was shown that baseline as well as induction levels of ANG and IL-8 in different human melanoma cell lines were correlated to the metastatic potential of the cell lines, whereas normal melanocytes revealed no upregulation. Immunohistochemical studies showed strong expression of ANG and IL-8 protein in primary melanoma and melanoma metastases particularly near small vessels in close vicinity of necrotic/ hypoxic areas, whereas benign nevi were all negative. The upregulation of ANG and IL-8 mRNA in melanoma and metastases was confirmed by RT-PCR and in situ hybridization, respectively.
By IL-8 promoter-dependent reporter gene analysis and mRNA stability assays it was shown that induction of IL-8 mRNA was due to both enhanced transcriptional activation and enhanced IL-8 mRNA stability. Interestingly, low aggressive cell lines with low transcriptional activation of IL-8 revealed point mutations in the AP-1 or the NF-kappaB-like binding sites in the IL-8 promoter. On the other hand, high induced AP-1 and NF-kappaB binding activity was found in highly aggressive melanoma cell lines under hypoxic conditions, indicating that both sites are critical for regulation of IL-8 under hypoxia.
To study the regulation of ANG in melanoma- and endothelial cells at a molecular level, three different regions of the ANG gene (promoter, c-DNA, 3'UTR) were isolated and expressed in firefly luciferase vectors. Melanoma cell lines and endothelial cells (HUVEC, HMEC-1) were transfected with the appropriate plasmid DNA. It was shown that elevated ANG mRNA levels under hypoxic conditions were due mostly to enhanced ANG mRNA stability than to transcriptional activation.
Key words:
angiogenesis
VEGF
angiogenin
growth factors
hypoxia
Projekt period: from 1.1996 to 12.2000
Preceding project:
NN
Publications:
Links:
PubMed