RESUMEN
One of the most frequently detected changes in human solid tumors is the mutation of the ras oncogene, which has been associated with production of angiogenic growth factors such as vascular endothelial growth factor/vascular permeability factor (VEGF/VPF). Using the v-Ha-ras Tg-AC transgenic mice and the background FVB/N strain of inbred mice, the pattern of expression of specific VEGF/VPF transcripts was characterized in major organs and in skin, papillomas, and carcinomas during multi-stage skin carcinogenesis. Three VEGF/VPF transcripts were found to be constitutively expressed in skin as well as the major organs in both mouse strains, which corresponded in size and sequence to previously reported murine VEGF120 with a bp size of 331, VEGF164 with a bp size of 333, and VEGF188 with a bp size of 407. A previously unreported fourth murine transcript was also detected in skin and major tissues from both mouse strains which corresponded to rat VEGF144, with a bp size of 404. In addition, a unique 425 bp VEGF transcript which corresponded to human VEGF205 was present in highly vascularized tissues including heart, lung, liver, kidney, brain, as well in papillomas and carcinomas isolated from v-Ha-ras Tg.AC mice. In contrast, VEGF205 was present only in carcinomas derived from FVB/N mice. An antibody generated from a peptide sequence designed to detect each of the five VEGF/VPF peptides defined by RT-PCR analysis confirmed the existence of these five peptides and confirmed that the murine VEGF205 peptide was selectively expressed in papillomas and carcinomas derived from v-Ha-ras Tg.AC mice. These results demonstrate that there is significant alternative splicing of the murine VEGF/VPF gene during multi-stage carcinogenesis, which results in four commonly expressed VEGF transcripts. In addition, these studies identified a fifth VEGF transcript and peptide at the later stages of tumor promotion and in progression which appears to be linked to the presence of v-Ha-ras.
Asunto(s)
Factores de Crecimiento Endotelial/genética , Regulación Neoplásica de la Expresión Génica/genética , Genes ras/genética , Linfocinas/genética , Neoplasias Cutáneas/genética , Empalme Alternativo/genética , Animales , Ratones , Ratones Endogámicos , Ratones Transgénicos , Proteínas de Neoplasias/química , Neovascularización Patológica/fisiopatología , Papiloma/genética , Reacción en Cadena de la Polimerasa , ARN Mensajero/genética , Neoplasias Cutáneas/patología , Transcripción Genética/genética , Factor A de Crecimiento Endotelial Vascular , Factores de Crecimiento Endotelial VascularRESUMEN
The beta2 integrin (CD 18/CD 11 a, b, c) family of proteins mediate adherence of leukocytes to vascular endothelium and the associated ligand, intercellular adhesion molecule-1 (ICAM-1; CD 54), interacts with beta2 integrin proteins to allow transendothelial migration of leukocytes into sites of inflammation. The present study examines the function of these proteins in a murine model of acute cutaneous inflammation induced following topical application of 12-O-tetradecanoylphorbol-13-acetate (TPA) to the dorsal epidermis of SENCAR mice and in a model of skin multistage carcinogenesis. At 24 h following topical application of TPA to the dorsal epidermis of mice, dermal leukocytes expressed higher levels of beta2 integrin protein compared with the lower levels of beta2 integrin protein expression by peripheral blood leukocytes. ICAM-1 protein was localized to epidermal keratinocytes and vascular endothelium in TPA-treated skin and to proliferating papilloma cells. Intravenous (i.v.) injection of either 50 microg anti-beta2 integrin antibody alone or in combination with anti-ICAM-1 antibody significantly inhibited both TPA-stimulated neutrophil infiltration into the dermis (P < 0.001) and myeloperoxidase (MPO) activity (P < 0.03 anti-beta2 integrin antibody; P < 0.01 anti-beta2 integrin + ICAM-1 adhesion molecule antibodies), but had no effect on TPA-induced epidermal hyperplasia. In addition, injection of either anti-ICAM-1 adhesion molecule antibody alone (P < 0.004) or in combination with anti-beta2 integrin antibody (P < 0.001) significantly inhibited TPA-induced production of 7,8-dihydroxy-2'-deoxyguanosine (8-OHdG) immunoreactive proteins by epidermal keratinocytes. Beta2 integrin/ICAM-1 adhesion molecules work in concert to regulate migration, retention and functional activation of leukocytes within the dermis during TPA-induced skin inflammation and within stromal tissue of papillomas that form during multi-stage carcinogenesis. Agents that inhibit these receptor/ligand interactions may be useful in defining the roles of specific cell populations in cutaneous inflammation and multistage carcinogenesis and may also have potential as anti-promoting and anti-progression agents.