title: Immortalization of human bronchial epithelial cells in the absence of viral oncoproteins.
abstract: By expressing two genes (hTERT and Cdk4), we have developed a method to reproducibly generate continuously replicating human bronchial epithelial cell (HBEC) lines that provide a novel resource to study the molecular pathogenesis of lung cancer and the differentiation of bronchial epithelial cells. Twelve human bronchial epithelial biopsy specimens obtained from persons with and without lung cancer were placed into short-term culture and serially transfected with retroviral constructs containing cyclin-dependent kinase (Cdk) 4 and human telomerase reverse transcriptase (hTERT), resulting in continuously growing cultures. The order of introduction of Cdk4 and hTERT did not appear to be important; however, transfection of either gene alone did not result in immortalization. Although they could be cloned, the immortalized bronchial cells did not form colonies in soft agar or tumors in nude mice. The immortalized HBECs have epithelial morphology; express epithelial markers cytokeratins 7, 14, 17, and 19, the stem cell marker p63, and high levels of p16(INK4a); and have an intact p53 checkpoint pathway. Cytogenetic analysis and array comparative genomic hybridization profiling show immortalized HBECs to have duplication of parts of chromosomes 5 and 20. Microarray gene expression profiling demonstrates that the Cdk4/hTERT-immortalized bronchial cell lines clustered together and with nonimmortalized bronchial cells, distinct from lung cancer cell lines. We also immortalized several parental cultures with viral oncoproteins human papilloma virus type 16 E6/E7 with and without hTERT, and these cells exhibited loss of the p53 checkpoint and significantly different gene expression profiles compared with Cdk4/hTERT-immortalized HBECs. These HBEC lines are a valuable new tool for studying of the pathogenesis of lung cancer.
authors.affiliation: Hamon Center for Therapeutic Oncology Research and Department of Internal Medicine, The University of Texas Southwestern Medical Center, Dallas, Texas, USA.
authors.forename: Ruben D
authors.initials: RD
authors.lastname: Ramirez
authors.forename: Shelley
authors.initials: S
authors.lastname: Sheridan
authors.forename: Luc
authors.initials: L
authors.lastname: Girard
authors.forename: Mitsuo
authors.initials: M
authors.lastname: Sato
authors.forename: Young
authors.initials: Y
authors.lastname: Kim
authors.forename: Jon
authors.initials: J
authors.lastname: Pollack
authors.forename: Michael
authors.initials: M
authors.lastname: Peyton
authors.forename: Ying
authors.initials: Y
authors.lastname: Zou
authors.forename: Jonathan M
authors.initials: JM
authors.lastname: Kurie
authors.forename: J Michael
authors.initials: JM
authors.lastname: Dimaio
authors.forename: Sara
authors.initials: S
authors.lastname: Milchgrub
authors.forename: Alice L
authors.initials: AL
authors.lastname: Smith
authors.forename: Rhonda F
authors.initials: RF
authors.lastname: Souza
authors.forename: Laura
authors.initials: L
authors.lastname: Gilbey
authors.forename: Xi
authors.initials: X
authors.lastname: Zhang
authors.forename: Kenia
authors.initials: K
authors.lastname: Gandia
authors.forename: Melville B
authors.initials: MB
authors.lastname: Vaughan
authors.forename: Woodring E
authors.initials: WE
authors.lastname: Wright
authors.forename: Adi F
authors.initials: AF
authors.lastname: Gazdar
authors.forename: Jerry W
authors.initials: JW
authors.lastname: Shay
authors.forename: John D
authors.initials: JD
authors.lastname: Minna
mesh_terms: D048708:Cell Growth Processes / Q000502:physiology; D002471:Cell Transformation, Neoplastic / Q000235:genetics / Q000378:metabolism*; D051358:Cyclin-Dependent Kinase 4; D019941:Cyclin-Dependent Kinase Inhibitor p16 / Q000096:biosynthesis / Q000235:genetics; D018844:Cyclin-Dependent Kinases / Q000096:biosynthesis / Q000235:genetics; D004268:DNA-Binding Proteins; D020869:Gene Expression Profiling; D016147:Genes, Tumor Suppressor; D006801:Humans; D015151:Immunoblotting; D007621:Karyotyping; D008168:Lung / Q000166:cytology / Q000378:metabolism* / Q000502:physiology*; D009693:Nucleic Acid Hybridization; D009856:Oncogene Proteins, Viral / Q000096:biosynthesis / Q000235:genetics; D050725:Papillomavirus E7 Proteins; D010750:Phosphoproteins / Q000096:biosynthesis / Q000235:genetics; D011518:Proto-Oncogene Proteins / Q000096:biosynthesis / Q000235:genetics; D012097:Repressor Proteins / Q000096:biosynthesis / Q000235:genetics; D019098:Telomerase / Q000096:biosynthesis / Q000235:genetics; D016615:Telomere / Q000235:genetics; D015534:Trans-Activators / Q000096:biosynthesis / Q000235:genetics; D014157:Transcription Factors; D014162:Transfection; D016159:Tumor Suppressor Protein p53 / Q000096:biosynthesis / Q000235:genetics; D025521:Tumor Suppressor Proteins; D014466:Ultraviolet Rays
chemical_list: D019941:Cyclin-Dependent Kinase Inhibitor p16; D004268:DNA-Binding Proteins; C068651:E6 protein, Human papillomavirus type 16; D009856:Oncogene Proteins, Viral; D050725:Papillomavirus E7 Proteins; D010750:Phosphoproteins; D011518:Proto-Oncogene Proteins; D012097:Repressor Proteins; C115051:TP63 protein, human; D015534:Trans-Activators; D014157:Transcription Factors; D016159:Tumor Suppressor Protein p53; D025521:Tumor Suppressor Proteins; C059731:oncogene protein E7, Human papillomavirus type 16; C495900:CDK4 protein, human; D051358:Cyclin-Dependent Kinase 4; D018844:Cyclin-Dependent Kinases; D019098:Telomerase