title: Conditional immortalization of freshly isolated human mammary fibroblasts and endothelial cells.
abstract: Reports differ as to whether reconstitution of telomerase activity alone is sufficient for immortalization of different types of human somatic cells or whether additional activities encoded by other "immortalizing" genes are also required. Here we show that ectopic expression of either the catalytic subunit of human telomerase (hTERT) or a temperature-sensitive mutant (U19tsA58) of simian virus 40 large-tumor antigen alone was not sufficient for immortalization of freshly isolated normal adult human mammary fibroblasts and endothelial cells. However, a combination of both genes resulted in the efficient generation of immortal cell lines irrespective of the order in which they were introduced or whether they were introduced early or late in the normal proliferative lifespan of the cultures. The order and timing of transduction, however, did influence genomic stability. Karyotype analysis indicated that introduction of both transgenes at early passage, with hTERT first, yielded diploid cell lines. Temperature-shift experiments revealed that maintenance of the immortalized state depended on continued expression of functional U19tsA58 large-tumor antigen, with hTERT alone unable to maintain growth at nonpermissive temperatures for U19tsA58 large-tumor antigen. Such conditional diploid lines may provide a useful resource for both cell engineering and for studies on immortalization and in vitro transformation.
authors.affiliation: Ludwig Institute for Cancer Research-University College London Breast Cancer Laboratory, Department of Surgery, Royal Free and University College School of Medicine, 67-73 Riding House Street, London W1W 7EJ, United Kingdom.
authors.forename: M J
authors.initials: MJ
authors.lastname: O'Hare
authors.forename: J
authors.initials: J
authors.lastname: Bond
authors.forename: C
authors.initials: C
authors.lastname: Clarke
authors.forename: Y
authors.initials: Y
authors.lastname: Takeuchi
authors.forename: A J
authors.initials: AJ
authors.lastname: Atherton
authors.forename: C
authors.initials: C
authors.lastname: Berry
authors.forename: J
authors.initials: J
authors.lastname: Moody
authors.forename: A R
authors.initials: AR
authors.lastname: Silver
authors.forename: D C
authors.initials: DC
authors.lastname: Davies
authors.forename: A E
authors.initials: AE
authors.lastname: Alsop
authors.forename: A M
authors.initials: AM
authors.lastname: Neville
authors.forename: P S
authors.initials: PS
authors.lastname: Jat
mesh_terms: D000328:Adult; D000952:Antigens, Polyomavirus Transforming / Q000235:genetics / Q000502:physiology*; D001940:Breast / Q000166:cytology*; D020134:Catalytic Domain; D002455:Cell Division; D002461:Cell Line, Transformed; D016922:Cellular Senescence; D004261:DNA Replication; D004268:DNA-Binding Proteins; D004727:Endothelium / Q000166:cytology*; D005260:Female; D005347:Fibroblasts / Q000166:cytology*; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D007621:Karyotyping; D012313:RNA; D012190:Retroviridae / Q000235:genetics; D013539:Simian virus 40 / Q000235:genetics; D019098:Telomerase / Q000235:genetics / Q000502:physiology*; D013696:Temperature; D013997:Time Factors; D014162:Transfection; D019076:Transgenes
chemical_list: D000952:Antigens, Polyomavirus Transforming; D004268:DNA-Binding Proteins; C422952:telomerase RNA; D012313:RNA; D019098:Telomerase