{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,4,30]],"date-time":"2026-04-30T23:08:50Z","timestamp":1777590530415,"version":"3.51.4"},"reference-count":48,"publisher":"Proceedings of the National Academy of Sciences","issue":"2","content-domain":{"domain":["www.pnas.org"],"crossmark-restriction":true},"short-container-title":["Proc. Natl. Acad. Sci. U.S.A."],"published-print":{"date-parts":[[2001,1,16]]},"abstract":"<jats:p>\n            Reports differ as to whether reconstitution of telomerase\n activity alone is sufficient for immortalization of different types of\n human somatic cells or whether additional activities encoded by other\n \u201cimmortalizing\u201d genes are also required. Here we show that\n ectopic expression of either the catalytic subunit of human telomerase\n (hTERT) or a temperature-sensitive mutant (U19tsA58) of simian virus 40\n large-tumor antigen alone was not sufficient for immortalization of\n freshly isolated normal adult human mammary fibroblasts and endothelial\n cells. However, a combination of both genes resulted in the efficient\n generation of immortal cell lines irrespective of the order in which\n they were introduced or whether they were introduced early or late in\n the normal proliferative lifespan of the cultures. The order and timing\n of transduction, however, did influence genomic stability. Karyotype\n analysis indicated that introduction of both transgenes at early\n passage, with hTERT first, yielded diploid cell lines.\n Temperature-shift experiments revealed that maintenance of the\n immortalized state depended on continued expression of functional\n U19tsA58 large-tumor antigen, with hTERT alone unable to maintain\n growth at nonpermissive temperatures for U19tsA58 large-tumor\n antigen. Such conditional diploid lines may provide a useful\n resource for both cell engineering and for studies on immortalization\n and\n            <jats:italic>in vitro<\/jats:italic>\n            transformation.\n          <\/jats:p>","DOI":"10.1073\/pnas.98.2.646","type":"journal-article","created":{"date-parts":[[2002,7,26]],"date-time":"2002-07-26T14:45:09Z","timestamp":1027694709000},"page":"646-651","update-policy":"https:\/\/doi.org\/10.1073\/pnas.cm10313","source":"Crossref","is-referenced-by-count":182,"title":["Conditional immortalization of freshly isolated human mammary fibroblasts and endothelial cells"],"prefix":"10.1073","volume":"98","author":[{"given":"Michael J.","family":"O'Hare","sequence":"first","affiliation":[{"name":"Ludwig Institute for Cancer Research\u2013University College London\r Breast Cancer Laboratory, Department of Surgery, Royal Free and\r University College School of Medicine, 67-73 Riding House Street,\r London W1W 7EJ, United Kingdom; Ludwig Institute for\r Cancer Research, Royal Free and University College School of\r Medicine, Courtauld Building, 91 Riding House Street, London W1W 7BS,\r United Kingdom; Wohl Virion Centre, Windeyer\r Institute, Royal Free and University College School of Medicine, 46\r..."}]},{"given":"Jacquelyn","family":"Bond","sequence":"additional","affiliation":[{"name":"Ludwig Institute for Cancer Research\u2013University College London\r Breast Cancer Laboratory, Department of Surgery, Royal Free and\r University College School of Medicine, 67-73 Riding House Street,\r London W1W 7EJ, United Kingdom; Ludwig Institute for\r Cancer Research, Royal Free and University College School of\r Medicine, Courtauld Building, 91 Riding House Street, London W1W 7BS,\r United Kingdom; Wohl Virion Centre, Windeyer\r Institute, Royal Free and University College School of Medicine, 46\r..."}]},{"given":"Catherine","family":"Clarke","sequence":"additional","affiliation":[{"name":"Ludwig Institute for Cancer Research\u2013University College London\r Breast Cancer Laboratory, Department of Surgery, Royal Free and\r University College School of Medicine, 67-73 Riding House Street,\r London W1W 7EJ, United Kingdom; Ludwig Institute for\r Cancer Research, Royal Free and University College School of\r Medicine, Courtauld Building, 91 Riding House Street, London W1W 7BS,\r United Kingdom; Wohl Virion Centre, Windeyer\r Institute, Royal Free and University College School of Medicine, 46\r..."}]},{"given":"Yasuhiro","family":"Takeuchi","sequence":"additional","affiliation":[{"name":"Ludwig Institute for Cancer Research\u2013University College London\r Breast Cancer Laboratory, Department of Surgery, Royal Free and\r University College School of Medicine, 67-73 Riding House Street,\r London W1W 7EJ, United Kingdom; Ludwig Institute for\r Cancer Research, Royal Free and University College School of\r Medicine, Courtauld Building, 91 Riding House Street, London W1W 7BS,\r United Kingdom; Wohl Virion Centre, Windeyer\r Institute, Royal Free and University College School of Medicine, 46\r..."}]},{"given":"Amanda J.","family":"Atherton","sequence":"additional","affiliation":[{"name":"Ludwig Institute for Cancer Research\u2013University College London\r Breast Cancer Laboratory, Department of Surgery, Royal Free and\r University College School of Medicine, 67-73 Riding House Street,\r London W1W 7EJ, United Kingdom; Ludwig Institute for\r Cancer Research, Royal Free and University College School of\r Medicine, Courtauld Building, 91 Riding House Street, London W1W 7BS,\r United Kingdom; Wohl Virion Centre, Windeyer\r Institute, Royal Free and University College School of Medicine, 46\r..."}]},{"given":"Catherine","family":"Berry","sequence":"additional","affiliation":[{"name":"Ludwig Institute for Cancer Research\u2013University College London\r Breast Cancer Laboratory, Department of Surgery, Royal Free and\r University College School of Medicine, 67-73 Riding House Street,\r London W1W 7EJ, United Kingdom; Ludwig Institute for\r Cancer Research, Royal Free and University College School of\r Medicine, Courtauld Building, 91 Riding House Street, London W1W 7BS,\r United Kingdom; Wohl Virion Centre, Windeyer\r Institute, Royal Free and University College School of Medicine, 46\r..."}]},{"given":"John","family":"Moody","sequence":"additional","affiliation":[{"name":"Ludwig Institute for Cancer Research\u2013University College London\r Breast Cancer Laboratory, Department of Surgery, Royal Free and\r University College School of Medicine, 67-73 Riding House Street,\r London W1W 7EJ, United Kingdom; Ludwig Institute for\r Cancer Research, Royal Free and University College School of\r Medicine, Courtauld Building, 91 Riding House Street, London W1W 7BS,\r United Kingdom; Wohl Virion Centre, Windeyer\r Institute, Royal Free and University College School of Medicine, 46\r..."}]},{"given":"Andrew R. J.","family":"Silver","sequence":"additional","affiliation":[{"name":"Ludwig Institute for Cancer Research\u2013University College London\r Breast Cancer Laboratory, Department of Surgery, Royal Free and\r University College School of Medicine, 67-73 Riding House Street,\r London W1W 7EJ, United Kingdom; Ludwig Institute for\r Cancer Research, Royal Free and University College School of\r Medicine, Courtauld Building, 91 Riding House Street, London W1W 7BS,\r United Kingdom; Wohl Virion Centre, Windeyer\r Institute, Royal Free and University College School of Medicine, 46\r..."}]},{"given":"Derek C.","family":"Davies","sequence":"additional","affiliation":[{"name":"Ludwig Institute for Cancer Research\u2013University College London\r Breast Cancer Laboratory, Department of Surgery, Royal Free and\r University College School of Medicine, 67-73 Riding House Street,\r London W1W 7EJ, United Kingdom; Ludwig Institute for\r Cancer Research, Royal Free and University College School of\r Medicine, Courtauld Building, 91 Riding House Street, London W1W 7BS,\r United Kingdom; Wohl Virion Centre, Windeyer\r Institute, Royal Free and University College School of Medicine, 46\r..."}]},{"given":"Amber E.","family":"Alsop","sequence":"additional","affiliation":[{"name":"Ludwig Institute for Cancer Research\u2013University College London\r Breast Cancer Laboratory, Department of Surgery, Royal Free and\r University College School of Medicine, 67-73 Riding House Street,\r London W1W 7EJ, United Kingdom; Ludwig Institute for\r Cancer Research, Royal Free and University College School of\r Medicine, Courtauld Building, 91 Riding House Street, London W1W 7BS,\r United Kingdom; Wohl Virion Centre, Windeyer\r Institute, Royal Free and University College School of Medicine, 46\r..."}]},{"given":"A. Munro","family":"Neville","sequence":"additional","affiliation":[{"name":"Ludwig Institute for Cancer Research\u2013University College London\r Breast Cancer Laboratory, Department of Surgery, Royal Free and\r University College School of Medicine, 67-73 Riding House Street,\r London W1W 7EJ, United Kingdom; Ludwig Institute for\r Cancer Research, Royal Free and University College School of\r Medicine, Courtauld Building, 91 Riding House Street, London W1W 7BS,\r United Kingdom; Wohl Virion Centre, Windeyer\r Institute, Royal Free and University College School of Medicine, 46\r..."}]},{"given":"Parmjit S.","family":"Jat","sequence":"additional","affiliation":[{"name":"Ludwig Institute for Cancer Research\u2013University College London\r Breast Cancer Laboratory, Department of Surgery, Royal Free and\r University College School of Medicine, 67-73 Riding House Street,\r London W1W 7EJ, United Kingdom; Ludwig Institute for\r Cancer Research, Royal Free and University College School of\r Medicine, Courtauld Building, 91 Riding House Street, London W1W 7BS,\r United Kingdom; Wohl Virion Centre, Windeyer\r Institute, Royal Free and University College School of Medicine, 46\r..."}]}],"member":"341","published-online":{"date-parts":[[2001,1,16]]},"reference":[{"key":"e_1_3_3_1_2","doi-asserted-by":"publisher","DOI":"10.1016\/0014-4827(61)90192-6"},{"key":"e_1_3_3_2_2","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.95.18.10614"},{"key":"e_1_3_3_3_2","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.85.14.5112"},{"key":"e_1_3_3_4_2","first-page":"1","volume":"1072","author":"Shay J W","year":"1991","unstructured":"J W Shay, W E Wright, H Werbin Biochim Biophys Acta 1072, 1\u20137 (1991).","journal-title":"Biochim Biophys 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