Immortalized differentiated hepatocyte lines derived from transgenic mice harboring SV40 T-antigen genes

doi:10.1016/0014-4827(88)90199-1

Experimental Cell Research

Volume 175, Issue 2, April 1988, Pages 354-362

https://doi.org/10.1016/0014-4827(88)90199-1 Get rights and content

Abstract

Hepatocytes of transgenic mouse fetuses harboring SV40 virus transforming gene sequences in the SVΔe-MGH fusion gene construct 202 driven by the mouse metallothionein (MT-I) enhancer [R. D. Palmiter, H. Y. Chen, A. Messing, and R. L. Brinster (1985) Nature (London) 316, 457–460] were cultured at Day 19 of gestation and established as a differentiated line expressing albumin and α-fetoprotein (AFP) mRNAs. Hepatocyte line FMH-202 contains integrated SV40 sequences, expresses SV40 T-antigen genes, and exhibits unlimited growth potential because it has been cultured 18 months without apparent decrease in cell viability or in growth rate that could suggest the occurrence of a crisis period. Immortalized cells multiply in chemically defined medium deficient in arginine with transferrin plus insulin, whereas EGF, insulin, and transferrin are obligatory requirements for fetal or newborn mouse hepatocyte multiplication in primary cultures. Cells did not grow in agar and were not tumorigenic in nude mice. Their immortalized. nonmalignant phenotype was further documented by low saturation densities of confluent monolayers showing no overgrowth, and by growth arrest in the absence of insulin with subsequent induction of DNA synthesis and resumption of cell growth in response to insulin. Thus, it appears that immortalized SV40 T-antigen-expressing hepatocytes are present in the liver of the transgenic mice. However, at later points in liver development the transforming activity of T-antigen becomes apparent and leads to hepatocellular carcinoma formation in vivo.

References (42)

I. Georgoff et al.
J. Biol. Chem
(1984)
T.A. Stewart et al.
Cell
(1984)
R.L. Brinster et al.
Cell
(1984)
M.R.D. Scott et al.
Cell
(1983)
E.H. Postel et al.
Virology
(1976)
M.A. Innis et al.
Arch. Biochem. Biophys
(1979)
R. Risser et al.
Virology
(1974)
D.F. Clayton et al.
Mol. Cell. Biol
(1983)
R. Etat et al.
D.M. Jefferson et al.
Mol. Cell. Biol
(1984)

B.B. Knowles et al.

G.I. Darlington et al.

J. Natl. Cancer Inst

(1980)

H.P. Morris et al.

Methods Cancer Res

(1968)

F. Pinet et al.

S. Yasumoto

Mol. Cell. Biol

(1984)

S.E. Schlegel-Haueter et al.

M. Höhne et al.

Oncogene

(1987)

R.D. Palmiter et al.

Nature (London)

(1985)

J.M. Adams et al.

Nature (London)

(1985)

D. Hanahan

Nature (London)

(1985)

I.A. Small et al.

Mol. Cell. Biol

(1985)

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^☆: This work was supported by grants from the Deutsche Forschungsgemeinschaft, the Bundesminister für Forschung und Technologie (Bonn), and the Forschungsrat Rauchen und Gesundheit (Hamburg).

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Immortalized differentiated hepatocyte lines derived from transgenic mice harboring SV40 T-antigen genes☆

Abstract

J. Biol. Chem

Cell

Cell

Cell

Virology

Arch. Biochem. Biophys

Virology

Mol. Cell. Biol

Mol. Cell. Biol

J. Natl. Cancer Inst

Methods Cancer Res

Mol. Cell. Biol

Oncogene

Nature (London)

Nature (London)

Nature (London)

Mol. Cell. Biol