1. Generation of an immortalized human endothelial cell line as a model of neovascular proliferating endothelial cells to assess chemosensitivity to anticancer drugs
- Author
-
Alessia Muzi, Olindo Forini, Grazia Graziani, Lucio Tentori, Lauretta Levati, Matteo Vergati, Pedro Miguel Lacal, Federica Ruffini, and Patrizia Vernole
- Subjects
CD31 ,Male ,virus T antigen ,von Willebrand factor ,Mice ,Cell Movement ,genetics ,endothelium cell ,comparative study ,Inbred BALB C ,CD164L1 protein ,drug effect ,Settore BIO/14 ,Flow Cytometry ,Alkylating ,Neoplasm Proteins ,Endothelial stem cell ,Platelet Endothelial Cell Adhesion Molecule-1 ,Cell Transformation, Neoplastic ,Oncology ,FLT1 protein ,Western ,Endothelium ,Blotting, Western ,Antineoplastic Agents ,Transfection ,reverse transcription polymerase chain reaction ,HT29 Cells ,tumor protein ,Antigens, CD ,Humans ,human ,cell strain HT29 ,xenograft ,Antineoplastic Agents, Alkylating ,mouse ,Vascular Endothelial Growth Factor Receptor-1 ,Vascular Endothelial Growth Factor Receptor-2 ,DNA binding protein ,chemistry ,Transformed ,Immunology ,drug derivative ,genetic transfection ,Cancer Research ,Angiogenesis ,Antigens, Polyomavirus Transforming ,Nude ,Gene Expression ,cell transformation ,temozolomide ,Western blotting ,chemistry.chemical_compound ,Bagg albino mouse ,cell motion ,Neoplasms ,membrane protein ,vasculotropin receptor 2 ,animal ,vasculotropin receptor 1 ,Telomerase ,Tumor Stem Cell Assay ,Cell Line, Transformed ,Mice, Inbred BALB C ,telomere ,Heterologous ,Blotting ,Reverse Transcriptase Polymerase Chain Reaction ,article ,cell line ,Vascular endothelial growth factor ,Dacarbazine ,DNA-Binding Proteins ,medicine.anatomical_structure ,alkylating agent ,CD164L1 protein, human ,CD31 antigen ,dacarbazine ,FLT1 protein, human ,telomerase ,cell proliferation ,cytology ,drug screening ,experimental neoplasm ,flow cytometry ,gene expression ,male ,metabolism ,nude mouse ,pathology ,plasmid ,Animals ,Antigens, CD31 ,Cell Line ,Cell Proliferation ,Endothelial Cells ,Membrane Proteins ,Mice, Nude ,Neoplasms, Experimental ,Plasmids ,Telomere ,Transplantation, Heterologous ,von Willebrand Factor ,Biology ,Experimental ,Antigens, Neoplasm ,medicine ,Antigens ,Neoplastic ,Transplantation ,Kinase insert domain receptor ,Cell culture ,Cancer research ,Polyomavirus Transforming - Abstract
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.