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Maintenance of liver function in long term culture of hepatocytes following In Vitro or In Vivo Ha-ras EJ transfection

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Collagenase isolated rat hepatocytes were transfected with liposome encapsulated pEJ (LE-pEJ), a plasmid carrying the human cellular activated Ha-rasEJ oncogene. A proliferative cell line was cloned from these cells transfected in vitro. It secreted per day 0.87 µg albumin and 0.32 µg transferrin per 106 cells, and 11.06 nmol free and conjugated bile acids (BA) per mg protein. Also, it metabolized 2-acetylaminoflourene (2-AFAF) into N- and ring-hydroxylated metabolites and 2-aminofluorene at rates of 1.50, 9.73, and 1.98 nmol/mg cell protein/24 hr, respectively. Rats were i.v. injected with both LE-pEJ and LE-p17hGHnneo carrying the hGH cDNA gene, and secreted hGH in the plasma which induced the synthesis of anti-hGH antibodies. A cell line was cloned from cultures of primary hepatocytes isolated from the liver of transfected rats. After 2 to 3 months in culture, this cell line secreted per day 18.9 µg albumin and 11.0 µg transferrin per 106 cells, 38.75 nmol total BA per mg cell protein, and up to 31 ng hGHper 106 cells without cloning hGH recombinant cells. A 24 hr control culture of primary hepatocytes isolated from non transfected rats secreted 25.5 µg albumin and 11.7 µg transferrin per 106 cells, and produced 21.64 nmol total BA and 2.13 nmol N-OH-2-AFAF per mg cell protien. Hence, Ha-ras EJ transfection of either hepatocytes in vitro or liver cells in vivo, initiated cell cycles leading to presumptive proliferating hepatocytes which express liver function.

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Abbreviations

BWE :

basal Williams' medium E

FBS:

fetal bovine serum

F10 or F12 :

basal Ham's F10 or F12 medium

Ha-ras EJ :

EJ allele of the human cellular ras oncogen of Harvey

hGH :

human growth hormone

hsp :

heat shock protein gene

LE-p :

liposome encapsulated plasmid

N-OH-2-AFAF:

N-hydroxy-2-acetylaminofluorene

RLECC:

rat liver epithelial cell

SF:

serum-free

SS:

serum-supplemented

UGG:

serum substitute UGltroser G®

1-OH-, 3-OH-2-AFAFF:

1-hydroxy-, 3-hydroxy-2-acetylaminofluorene

2-AFAF:

2-acetylaminofluorene

2-AFF:

2-aminofluorene

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  1. Laboratoire de Biochimie Médicale, Faculté de Médecine, UGniversité de Bourgogne, 21033, Dijon

    Michel Fischbach, Hanwei Cao, Miguel Diez Ibanez, Christos Tsaconas, Frédéric Montandon, Mohammed El Baraka, Prudent Padieu & Martine Chessebeuf-Padieu

  2. Department of Genetic Engineering, IntraCel SA, CH-1227, Geneva-Carouge

    Sami Alouani & Michel Dreano

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  1. Michel Fischbach
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  2. Hanwei Cao
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Fischbach, M., Cao, H., Ibanez, M.D. et al. Maintenance of liver function in long term culture of hepatocytes following In Vitro or In Vivo Ha-ras EJ transfection. Cell Biol Toxicol 7, 327–345 (1991). https://doi.org/10.1007/BF00124069

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