Maintenance of liver function in long term culture of hepatocytes following in vitro or in vivo Ha-rasEJ transfection.
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 micrograms albumin and 0.32 microgram transferrin per 10(6) cells, and 11.06 nmol free and conjugated bile acids (BA) per mg protein. Also, it metabolized 2-acetylaminofluorene (2-AAF) 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-p17hGHneo 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 micrograms albumin and 11.0 micrograms transferrin per 10(6) cells, 38.75 nmol total BA per mg cell protein, and up to 31 ng hGH per 10(6) cells without cloning hGH recombinant cells. A 24 hr control culture of primary hepatocytes isolated from non transfected rats secreted 25.5 micrograms albumin and 11.7 micrograms transferrin per 10(6) cells, and produced 21.64 nmol total BA and 2.13 nmol N-OH-2-AAF per mg cell protein. Hence, Ha-rasEJ transfection of either hepatocytes in vitro or liver cells in vivo, initiated cell cycles leading to presumptive proliferating hepatocytes which express liver function.
Fischbach
M
M
Laboratoire de Biochimie Médicale, Faculté de Médecine, Université de Bourgogne, Dijon, France.
Cao
H W
HW
Diez Ibanez
M
M
Tsaconas
C
C
Alouani
S
S
Montandon
F
F
el Baraka
M
M
Padieu
P
P
Dreano
M
M
Chessebeuf-Padieu
M
M
D015073:2-Acetylaminofluorene / Q000378:metabolism; D000418:Albumins / Q000378:metabolism; D000818:Animals; D001647:Bile Acids and Salts / Q000378:metabolism; D015139:Blotting, Southern; D015153:Blotting, Western; D002455:Cell Division; D002460:Cell Line; D002461:Cell Line, Transformed; D002478:Cells, Cultured; D015870:Gene Expression / Q000235:genetics*; D011905:Genes, ras; D013006:Growth Hormone / Q000235:genetics / Q000378:metabolism; D008099:Liver / Q000166:cytology* / Q000378:metabolism; D008297:Male; D051381:Rats; D011919:Rats, Inbred Strains; D014162:Transfection; D014168:Transferrin / Q000378:metabolism
D000418:Albumins; D001647:Bile Acids and Salts; D014168:Transferrin; D013006:Growth Hormone; D015073:2-Acetylaminofluorene