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

O'Hare MJ,Bond J,Clarke C,Takeuchi Y,Atherton AJ,Berry C,Moody J,Silver AR,Davies DC,Alsop AE,Neville AM,Jat PS

doi

10.1073/pnas.98.2.646

keywords:

subject

Has Abstract

pub_date

2001-01-16 00:00:00

pages

646-51

issue

2

eissn

0027-8424

issn

1091-6490

pii

98/2/646

journal_volume

98

pub_type

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