Intracellular Synthesis of a-Fetoprotein and Fibrinogen without Secretion by Zajdela Rat Ascites Hepatoma Cells1
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چکیده
Hepatoma Cell Protein Synthesis in Vitro. Zajdela ascites hepatoma cells (C strain) (28) were transplanted i.p. into adult male Charles RivenCD rats, and cells were harvested 6 to 7 days later. Hepatoma cells were separated from the ascitic fluid by centnifugation at 50 x g for 15 mm at 5°, then were washed 3 times with 0.9% NaCI solution . For the short term synthesis studies, hepatoma cells (13 x 10@)were incubated in 3.5 ml of RPMI Medium 1640 with 10% fetal calf serum plus antibiotics containing 3 mCi L-[U 14C]leucine (New England Nuclear, Boston, Mass.) at 37° with shaking in air. Puromycin (Calbiochem, San Diego, Calif.) was added to some flasks at zero time minus 15 mm to a final concentration of 100 pg/mI. After incubation for 1 to 3 hn, the total hepatoma cell-incubation medium was frozen and thawed 3 times, brought to a final concentration of 1% sodium deoxycholate to dissolve cell membranes, then dialyzed against frequent changes of 0.9% NaCI solu tion containing 0.1% unlabeled L-Ieucine at 4°to remove free radioactive leucine. The dialyzed samples were centrifuged and the clear su pennatants were reconstituted to their original 3.5 ml vol ume by the addition of 0.9% NaCI solution. Then either 0.5 ml of pooled matamniotic fluid was added to provide suffi cient cannierAFP for immunoprecipitation on 0.5 ml of nor mal adult rat plasma was added to provide cannier fibnino gen. Aliquots of these samples were used for isolation of AFP, fibninogen, and total cell protein. Isolation of AFP, Fibrinogen, and Total Cellular Protein. AFP was isolated immunochemically from a 1.5-mI aliquot of dialyzed total hepatoma cell-incubation medium to which 0.5 ml of pooled matamniotic fluid had been added, by the addition of 2.4 ml of unabsonbed monospecific rabbit anti serum to rat AFP. This amount rabbit antiserum to rat AFP was shown to yield maximum precipitation of AFP in prelim mary titration and resulted in precipitation of 98 to 100% of the total AFP present in these samples, based on observa tions that (a) a 2nd addition of antiserum did not result in visible immune precipitation, and (b) a 2nd addition of mat amniotic fluid, followed by rabbit antiserum, to these sam pIes did not result in the appearance of additional radioac tivity in immune precipitates.
منابع مشابه
Intracellular synthesis of alpha-fetoprotein and fibrinogen without secretion by Zajdela rat ascites hepatoma cells.
Transplabtable Zajdela rat ascites hepatoma cells, previously considered "nonproducers," synthesize detectable amounts of intracellular alpha-fetoprotein (AFP) and fibrinogen, but fail to secret or release these serum proteins. Evidence for defective secretory mechanisms for serum proteins in these hepatoma cells (a) explains the failure to detect AFP in either the serum or ascitic fluid of rat...
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