TEM-8 and tubule formation in endothelial cells, its potential role of its vW/TM domains.
OBJECTIVE
Tumour endothelial marker-8 (TEM-8) has been found to be selectively up regulated in tumour-associated endothelial cells, it is implicated in tumour specific angiogenesis, but its mechanism in angiogenesis is not defined.


METHODS
A ribozyme transgene (TEM-8) was cloned into a suitable mammalian expression vector (pc DNA 3.1-GFP-NT) and transfected into HECV cells. Various domains of TEM-8 were designed and cloned into pEF6/V5-His TOPO TA vector and transfected into Chinese Hamster ovarian cells (CHO), which do not form tubules and do not express TEM-8 in general (CHO(vW), CHO(TM), CHO(vW/TM), CHO(AE), CHO(AC), CHO(IC), and CHO(FL) domains, respectively). The effect of TEM-8 knocked out HECV cells was tested (by angiogenesis and migration assays), and the effect of each cleavage domain of TEM-8 was tested by microtubule formation assay.


RESULTS
TEM-8 stable transfectants (HECV(DeltaTEM8a)) manifested a complete loss of TEM-8 gene expression at mRNA and protein levels. In contrast, control GFP plasmid (HECV(pControl)) and wild-type HECV cells (HECV(WT)) had similar levels of TEM-8 expression. TEM-8 transfected cell (HECV(DeltaTEM8a)) significantly decreased the micro-vessels formation compared with controls (HECV(pControl)) (mean+/-SE, 20.3+/-4.03 microm; p=0.0086 vs. control 39.5+/-10.1 microm), and migration (38.52+/-2.17; p<0.05 vs. control 80.23+/-3.19), and micro-vessel formation of HECV(DeltaTEM8a) cell was also reduced compared with wild-type (HECV(WT)) (mean+/-SE, 20.3+/-4.03 microm; p=0.0078 vs. wild-type 42.5+/-9.1 microm) and migration (38.52+/-2.17microm; p<0.05 vs. wild-type 82.4+/-4.45 microm). vW together with transmembrane domains of TEM-8 (CHO(vW/TM)) and full-length CHO(FL) showed formation of tubule-like structure in CHO cells, whereas the other domains showed no effect.


CONCLUSIONS
Targeting the TEM-8 gene by way of a hammerhead ribozyme knocks out TEM-8 cells, and is an effective way to reduce the micro-vessel formation or migration potential in tumour-associated endothelial cell through its vW domain. These results suggest that the vW domain together with the transmembrane domain of TEM-8 may play an important biological role in TEM-8 related tubule formation.
Rmali
K A
KA
Metastasis and Angiogenesis Research Group, Department of Surgery, Wales College of Medicine, University of Cardiff, Wales, UK. Rmalika@cf.ac.uk
Puntis
M C A
MC
Jiang
W G
WG
D002460:Cell Line; D002465:Cell Movement / Q000502:physiology; D042783:Endothelial Cells / Q000166:cytology* / Q000502:physiology*; D006801:Humans; D008565:Membrane Proteins; D008833:Microcirculation / Q000166:cytology / Q000254:growth & development* / Q000378:metabolism*; D008840:Microfilament Proteins; D008870:Microtubules / Q000378:metabolism / Q000648:ultrastructure; D009363:Neoplasm Proteins; D018919:Neovascularization, Physiologic / Q000502:physiology*; D017434:Protein Structure, Tertiary; D011956:Receptors, Cell Surface / Q000737:chemistry* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013329:Structure-Activity Relationship
C473287:ANTXR1 protein, human; D008565:Membrane Proteins; D008840:Microfilament Proteins; D009363:Neoplasm Proteins; D011956:Receptors, Cell Surface; D011994:Recombinant Proteins