Thyroid epithelial cell transformation by a retroviral vector expressing SV40 large T

Burns, Jorge S.; Lemoine, Louise; Nr, Lemoine; Ed, Williams; Wynford-Thomas, David

doi:10.1038/bjc.1989.158

Cited by 19 publications

(10 citation statements)

References 16 publications

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“…The reduced level of iodide-trapping activity after extended passage of these lines is similar to that which we have observed in SV40-transformed rat thyroid cells (Burns et al, 1989). Other human epithelial cell types transformed by SV40 show either 20* v-a partial loss (such as keratinocytes; Banks-Schlegel & Howley, 1983) or complete loss (such as urothelial cells; Christian et al, 1987) of their normal differentiated phenotype.…”

Section: Discussionsupporting

confidence: 64%

Characterisation of human thyroid epithelial cells immortalised in vitro by simian virus 40 DNA transfection

Es²,

Jones³

et al. 1989

Br J Cancer

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159

105

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Section: Discussionsupporting

confidence: 64%

Characterisation of human thyroid epithelial cells immortalised in vitro by simian virus 40 DNA transfection

Es²,

Jones³

et al. 1989

Br J Cancer

Self Cite

159

105

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“…Primary cells and their derivatives were routinely cultured in Coon's modified F-12 medium [9] containing 5% calf serum (CS) (GIBCOIBRL, Paisley, UK) supplemented with six growth factors (6H) (all from Calbiochem/Novobiochem, Nottingham, UK): 5 mU/mL TSH, 10 pg/mL insulin, M hydrocortisone, 10 ng/mL glycyl-histidyl-lysine acetate, 10 ng/mL somatostatin, and 5 pg/mL transferrin, as used previously for the immortal rat thyroid line FRTL-5 [9,10].…”

Section: Primary Thyroid Cell Culturesmentioning

confidence: 99%

Stepwise transformation of primary thyroid epithelial cells by a mutant Ha‐ras oncogene: An in vitro model of tumor progression

Burns

Blaydes

Wright

et al. 1992

Molecular Carcinogenesis

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Activating mutations of the ras oncogene family occur at high frequency in all stages of thyroid tumorigenesis, both human and experimental. To test the causal nature of this association, and to investigate the biological role of ras mutation, we introduced a mutant c-Ha-ras gene into normal rat thyroid follicular cells using an ecotropic retroviral vector. The major immediate effect was to greatly extend the proliferative lifespan of these cells in culture from less than 3 to more than 15 doublings, without any observable loss of growth-factor dependence or differentiated functions. This in vitro phenotype strongly supports an initiating role for ras mutation in the genesis of benign thyroid tumors (adenomas) in vivo. Spontaneous transformation was observed at low frequency on continuous culture of mutant ras-expressing cells, giving rise to fully immortalized, growth factor-independent, highly tumorigenic lines. Transformation was associated with (i) loss of responsiveness to the growth inhibitor TGF-beta 1, and (ii) greatly increased nuclear levels of p53 protein, which unexpectedly was not due to point mutation in the conserved regions of the p53-coding sequence. We postulate that these two phenomena are causally related to each other and to the transformed phenotype.

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“…For gene transfer, cells were plated at 2 × 10 5 per 60-mm dish and infected with undiluted viral supernatant (containing 8 µg ml Ð1 polybrene, Aldrich, Gillingham, UK) from amphotropic producer cell lines as described previously (Burns et al, 1989). Two days later, cultures were passaged into G418 (400 µg ml Ð1 ) and colonies subsequently selected.…”

Section: Retroviral Gene Transfermentioning

confidence: 99%

Human thyroid cancer cells as a source of iso-genic, iso-phenotypic cell lines with or without functional p53

et al. 1999

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Summary Differentiated thyroid carcinomas (in contrast to the rarer anaplastic form) are unusual among human cancers in displaying a remarkably low frequency of p53 mutation and appear to retain wild-type (wt) p53 function as assessed by the response of derived cell lines to DNA damage. Using one such cell line, K1, we have tested the effect of experimental abrogation of p53 function by generating matched subclones stably expressing either a neo control gene, a dominant-negative mutant p53 (143 ala ) or human papilloma virus protein HPV16 E6. Loss of p53 function in the latter two groups was confirmed by abolition of p53-dependent 'stress' responses including induction of the cyclin/CDK inhibitor p21WAF1 and G1/S arrest following DNA-damage. In contrast, no change was detected in the phenotype of 'unstressed' clones, with respect to any of the following parameters: proliferation rate in monolayer, serum-dependence for proliferation or survival, tumorigenicity, cellular morphology, or tissue-specific differentiation markers. The K1 line therefore represents a 'neutral' background with respect to p53 function, permitting the derivation of functionally p53 + or -clones which are not only iso-genic but also iso-phenotypic. Such a panel should be an ideal tool with which to test the p53-dependence of cellular stress responses, particularly the sensitivity to potential therapeutic agents, free from the confounding additional phenotypic differences which usually accompany loss of p53 function. The results also further support the hypothesis that p53 mutation alone is not sufficient to drive progression of thyroid cancer to the aggressive anaplastic form.Keywords: p53; differentiation; iso-genic; tumour progression; thyroid 1111British Journal of Cancer (1999) 79(7/8), 1111-1120 © 1999 Cancer Research Campaign Article no. bjoc.1998 Received 27 April 1998 Revised 8 July 1998 Accepted 13 July 1998Correspondence to: D Wynford-Thomas from differentiated to anaplastic thyroid cancer requires events additional to loss of p53 function. Furthermore, we reasoned that if K1 cells were indeed indifferent to the presence or absence of wt p53 function, they would represent an ideal, ÔneutralÕ, background on which to derive matched functionally p53 + or Ð lines for use as a tool to investigate p53-dependency of therapeutic agents.Our earlier study examined only a few phenotypic features, however, and employed just a single p53 mutant which has subsequently been found to be an inefficient dominant-negative in some contexts (Williams et al, 1995). We have therefore now re-examined this model in more detail, analysing multiple clones, multiple parameters of proliferation and differentiation state, and using not only mutant p53 but also the human papilloma virus protein HPV16 E6 as a means of abrogating p53 function. MATERIALS AND METHODS Cell cultureThe K1 and FTC human thyroid cancer cell lines were kindly provided by Prof. M Schlumberger (Villejuif, France) and Dr P Goretski (Dusseldorf, Germany), respectively. Both lines and their...

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Thyroid epithelial cell transformation by a retroviral vector expressing SV40 large T

Cited by 19 publications

References 16 publications

Characterisation of human thyroid epithelial cells immortalised in vitro by simian virus 40 DNA transfection

Characterisation of human thyroid epithelial cells immortalised in vitro by simian virus 40 DNA transfection

Stepwise transformation of primary thyroid epithelial cells by a mutant Ha‐ras oncogene: An in vitro model of tumor progression

Human thyroid cancer cells as a source of iso-genic, iso-phenotypic cell lines with or without functional p53

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