TY - JOUR
T1 - An estrogen responsive primary amphibian liver cell culture system
AU - Stanchfield, J. E.
AU - Yager, J. D.
N1 - Funding Information:
The authorsw ould like to thank Joseph Miller and Seri Wilpone for technical assistancea nd Dr Robert Allen for use of microscopic equipment. Also the helpful discussionsw ith Dr Oscar Scornik are greatly appreciated. This work was supportedi n part by grant7 5-17 from the Milheim Foundation for Cancer Research, Contract no. NOl-CN-55199a nd Grant CA AM 18353 from the NCI, DHEW.
PY - 1978/10/15
Y1 - 1978/10/15
N2 - Amphibian hepatocytes have been prepared in both high yield and purity using a collagenase perfusion technique. The isolated cells attach efficiently in serum-free medium to collagen-coated culture dishes and subsequently form monolayers. These cultures can be maintained in an appropriate medium for over one week with minimal cell loss. The nuclear labelling index of cells exposed to [3H]thymidine indicates a very low level of cell growth. Twenty-four hour exposure to dexamethasone induces tyrosine aminotransferase activity throughout the culture period. Monolayers incorporate [3H]leucine linearly into acid-insoluble material with approx. 40% of all synthesis devoted to secreted protein. Polyacrylamide gel electrophoresis of proteins in the presence of sodium dodecyl sulfate shows the majority of proteins present in whole serum are synthesized and secreted by the cultured hepatocytes. The absolute rate of protein secretion on the first day of culture is approx. 73 μg/day/mg cell protein which subsequently declines and plateaus at 30% of this level by the 4th-5th day of culture. However, when hepatocytes are cultured in the continued presence of insulin, the drop in protein secretion is completely inhibited. Cultures of hepatocytes isolated from female frogs and subsequently exposed to 17-B estradiol in culture, synthesize and secrete the egg-yolk protein precursor vitellogenin. The protein initially appears as a minor component in the medium 1-2 days after hormone addition. Its rate of synthesis, relative to other secreted proteins, increases with time so that it ultimately constitutes the majority of protein being exported after 6 days of treatment. Parallel with vitellogenin induction is an increase in rate of total protein secretion reaching a 2-fold increase at maximal stimulation. The results show that viable, monolayer cultures of amphibian hepatocytes can be prepared which retain the ability to respond directly to added estrogen by synthesizing vitellogenin.
AB - Amphibian hepatocytes have been prepared in both high yield and purity using a collagenase perfusion technique. The isolated cells attach efficiently in serum-free medium to collagen-coated culture dishes and subsequently form monolayers. These cultures can be maintained in an appropriate medium for over one week with minimal cell loss. The nuclear labelling index of cells exposed to [3H]thymidine indicates a very low level of cell growth. Twenty-four hour exposure to dexamethasone induces tyrosine aminotransferase activity throughout the culture period. Monolayers incorporate [3H]leucine linearly into acid-insoluble material with approx. 40% of all synthesis devoted to secreted protein. Polyacrylamide gel electrophoresis of proteins in the presence of sodium dodecyl sulfate shows the majority of proteins present in whole serum are synthesized and secreted by the cultured hepatocytes. The absolute rate of protein secretion on the first day of culture is approx. 73 μg/day/mg cell protein which subsequently declines and plateaus at 30% of this level by the 4th-5th day of culture. However, when hepatocytes are cultured in the continued presence of insulin, the drop in protein secretion is completely inhibited. Cultures of hepatocytes isolated from female frogs and subsequently exposed to 17-B estradiol in culture, synthesize and secrete the egg-yolk protein precursor vitellogenin. The protein initially appears as a minor component in the medium 1-2 days after hormone addition. Its rate of synthesis, relative to other secreted proteins, increases with time so that it ultimately constitutes the majority of protein being exported after 6 days of treatment. Parallel with vitellogenin induction is an increase in rate of total protein secretion reaching a 2-fold increase at maximal stimulation. The results show that viable, monolayer cultures of amphibian hepatocytes can be prepared which retain the ability to respond directly to added estrogen by synthesizing vitellogenin.
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U2 - 10.1016/0014-4827(78)90445-7
DO - 10.1016/0014-4827(78)90445-7
M3 - Article
C2 - 30641
AN - SCOPUS:0018083101
SN - 0014-4827
VL - 116
SP - 239
EP - 252
JO - Experimental cell research
JF - Experimental cell research
IS - 2
ER -