Induction of tubulogenesis in telomerase-immortalized human microvascular endothelial cells by glioblastoma cells.
To facilitate the study of human endothelial cells we have used a replication defective retrovirus encoding the catalytic subunit of telomerase (hTERT) to derive populations of telomerase-immortalized human microvascular endothelial (TIME) cells. Whereas parental HMVECs became senescent on average within 35-45 population doublings (PDs), TIME cells have continued to proliferate for at least 200 PDs. TIME cells express readily detectable telomerase activity but display only a modest increase in telomere length. Karyotypic analysis reveals the cells to have a normal complement of human chromosomes with no evidence of gross genetic abnormalities. Furthermore, TIME cells retain many of the characteristics of the primary endothelial cells from which they were derived. For example, they express a panel of characteristic endothelial cell surface marker proteins such as CD31/PECAM-1 and alpha(v)beta3-integrin. In addition, TIME cells express receptors for low-density lipoprotein (LDL) receptor as they are competent for receptor-mediated endocytosis of fluorescent acetylated LDL. Importantly, when plated on matrigel, TIME cells undergo tubule formation. Moreover, when cocultured in the presence of human glioma cells, but not primary human astrocytes, TIME cells are induced to form stable tubules. Detachment of TIME cells from extracellular matrix leads to a form of programmed cell death known as anoikis. Conditional activation of the protein kinase Akt (Akt:ER*) significantly inhibited the onset of TIME cell anoikis under these conditions. We believe that the ability of hTERT to immortalize primary human endothelial cells, and the fact that such cells retain the endothelial characteristics of the cells from which they were derived, will greatly facilitate the analysis of human endothelial cell biology in vitro.
Venetsanakos
Eleni
E
Department of Neurological Surgery, Cancer Research Institute, San Francisco, California 94143, USA.
Mirza
Amer
A
Fanton
Christie
C
Romanov
Serguei R
SR
Tlsty
Thea
T
McMahon
Martin
M
D017209:Apoptosis; D015153:Blotting, Western; D001932:Brain Neoplasms / Q000098:blood supply* / Q000473:pathology; D002478:Cells, Cultured; D018920:Coculture Techniques; D004268:DNA-Binding Proteins; D004730:Endothelium, Vascular / Q000201:enzymology / Q000473:pathology*; D005909:Glioblastoma / Q000098:blood supply* / Q000473:pathology; D006801:Humans; D017404:In Situ Hybridization, Fluorescence; D007621:Karyotyping; D008077:Lipoproteins, LDL; D009389:Neovascularization, Pathologic / Q000378:metabolism / Q000473:pathology*; D019408:Platelet Endothelial Cell Adhesion Molecule-1 / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt; D012333:RNA, Messenger / Q000378:metabolism; D011973:Receptors, LDL / Q000378:metabolism; D019030:Receptors, Vitronectin / Q000378:metabolism; D012190:Retroviridae / Q000235:genetics; D019098:Telomerase / Q000235:genetics / Q000378:metabolism*; D016615:Telomere / Q000378:metabolism
D004268:DNA-Binding Proteins; D008077:Lipoproteins, LDL; D019408:Platelet Endothelial Cell Adhesion Molecule-1; D011518:Proto-Oncogene Proteins; D012333:RNA, Messenger; D011973:Receptors, LDL; D019030:Receptors, Vitronectin; C494918:AKT1 protein, human; D017346:Protein Serine-Threonine Kinases; D051057:Proto-Oncogene Proteins c-akt; D019098:Telomerase