Generation of an immortalized human endothelial cell line as a model of neovascular proliferating endothelial cells to assess chemosensitivity to anticancer drugs.
Assessment of chemosensitivity of neovessel endo-thelium associated to tumor mass is hindered by the limited availability of experimental models of actively proliferating endothelial cells. In fact, primary endothelial cells possess a limited lifespan and replicative senescence represents a major limit to their long-term culture. Moreover, non-dividing senescent cells undergo a gradual loss of phenotypic markers and become unable to respond to mitogenic stimuli. We report the generation of an immortalized human endothelial cell line by transfection of human umbilical vein endothelial cells (HUVEC) with both SV40 large/small T antigens and the catalytic subunit of human telomerase. This cell line (HUV-ST) possesses stabilized telomere length and increased proliferation rate with respect to parental cells or to cells transfected with SV40 T antigens only (HUV-S). Nevertheless, even at PD > 100 it is not tumorigenic and displays all major endothelial phenotypic markers, such as von Willebrand factor, CD31, vascular endothelial growth factor (VEGF) receptors (VEGFR1/Flt-1, VEGR2/KDR) and CD105/endoglin. HUV-ST cells are capable of organizing into tubule-like networks with branching morphology in response to appropriate stimuli and migrate upon exposure to VEGF. Interestingly, HUV-ST cells over-express the tumor endothelial marker-1/endosialin which is regarded as the most differentially expressed molecule in tumor-derived endothelium versus normal-derived endothelium. Analysis of chemosensitivity to the wide spectrum methylating agent temozolomide (TMZ), an anticancer drug more effective against actively dividing cells than against resting or slowing proliferating cells, indicated that HUV-ST cells are more susceptible to the drug with respect to HUVEC or HUV-S cells. Abrogation of poly(ADP-ribose) polymerase activity significantly enhances growth inhibition induced by TMZ. In conclusion, the immortalized human endothelial line HUV-ST represents a suitable model for studying the efficacy of anti-neovascular therapy, mimicking proliferating neovascular endothelial cells associated to the tumor mass.
Tentori
Lucio
L
Department of Neuroscience, University of Rome Tor Vergata, I-00133 Rome, Italy.
Vergati
Matteo
M
Muzi
Alessia
A
Levati
Lauretta
L
Ruffini
Federica
F
Forini
Olindo
O
Vernole
Patrizia
P
Lacal
Pedro Miguel
PM
Graziani
Grazia
G
D000818:Animals; D015703:Antigens, CD; D000951:Antigens, Neoplasm; D000952:Antigens, Polyomavirus Transforming / Q000235:genetics; D018906:Antineoplastic Agents, Alkylating / Q000494:pharmacology; D015153:Blotting, Western; D002460:Cell Line; D002461:Cell Line, Transformed; D002465:Cell Movement / Q000235:genetics; D049109:Cell Proliferation / Q000187:drug effects*; D002471:Cell Transformation, Neoplastic / Q000235:genetics; D004268:DNA-Binding Proteins / Q000235:genetics; D003606:Dacarbazine / Q000031:analogs & derivatives* / Q000494:pharmacology; D042783:Endothelial Cells / Q000166:cytology / Q000378:metabolism*; D005434:Flow Cytometry; D015870:Gene Expression; D019073:HT29 Cells; D006801:Humans; D008297:Male; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D051379:Mice; D008807:Mice, Inbred BALB C; D008819:Mice, Nude; D009363:Neoplasm Proteins / Q000235:genetics / Q000378:metabolism; D009374:Neoplasms, Experimental / Q000235:genetics / Q000473:pathology; D010957:Plasmids / Q000235:genetics; D019408:Platelet Endothelial Cell Adhesion Molecule-1 / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D019098:Telomerase / Q000235:genetics; D016615:Telomere / Q000235:genetics / Q000378:metabolism; D000077204:Temozolomide; D014162:Transfection; D014183:Transplantation, Heterologous; D014410:Tumor Stem Cell Assay; D040281:Vascular Endothelial Growth Factor Receptor-1 / Q000235:genetics / Q000378:metabolism; D040301:Vascular Endothelial Growth Factor Receptor-2 / Q000235:genetics / Q000378:metabolism; D014841:von Willebrand Factor / Q000235:genetics / Q000378:metabolism
D015703:Antigens, CD; D000951:Antigens, Neoplasm; D000952:Antigens, Polyomavirus Transforming; D018906:Antineoplastic Agents, Alkylating; C078203:CD248 protein, human; D004268:DNA-Binding Proteins; D008565:Membrane Proteins; D009363:Neoplasm Proteins; D019408:Platelet Endothelial Cell Adhesion Molecule-1; D014841:von Willebrand Factor; D003606:Dacarbazine; C501162:FLT1 protein, human; D040281:Vascular Endothelial Growth Factor Receptor-1; D040301:Vascular Endothelial Growth Factor Receptor-2; D019098:Telomerase; D000077204:Temozolomide