Multiple sequential periods of DNA synthesis and quiescence in primary hepatocyte cultures maintained on the DMSO-EGF on/off protocol.
Repeated periods of DNA synthesis activity (each period consisting of two to three cycles) separated by intervals of quiescence in primary rat hepatocytes can be stimulated by sequential addition and removal of 2% dimethyl sulfoxide (DMSO) in the presence of epidermal growth factor (EGF). Hepatocytes can be kept in nonproliferating cultures for 7 days in media supplemented with 2% DMSO and EGF. If DMSO is removed while EGF is maintained, rat and human hepatocytes enter a 3 to 4 day period of DNA synthesis that declines rapidly by days 4 and 5. If DMSO is reintroduced into cultures at that point, kept on for 3 more days and removed again, hepatocytes reenter into proliferation with another self-limited response of 3 to 4 days. Similar phenomena can seen with hepatocytes maintained in the presence of 3 mM phenobarbital. These protocols demonstrate that loss of responsiveness to mitogens in primary hepatocyte cultures is not an irreversible process. They also raise the possibility that signals for termination of DNA synthesis in hepatocytes emanate from hepatocytes themselves. These studies also suggest for the first time the possibility of designing in vitro systems that will allow clonal expansion of differential hepatocytes.
Chan
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K
Department of Pathology, Duke University Medical School, Durham, North Carolina 27710.
Kost
D P
DP
Michalopoulos
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D000818:Animals; D002455:Cell Division / Q000187:drug effects; D002478:Cells, Cultured; D004247:DNA / Q000096:biosynthesis*; D004121:Dimethyl Sulfoxide / Q000494:pharmacology*; D004815:Epidermal Growth Factor / Q000494:pharmacology*; D008099:Liver / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D008297:Male; D051381:Rats; D011916:Rats, Inbred F344; D013997:Time Factors
D004815:Epidermal Growth Factor; D004247:DNA; D004121:Dimethyl Sulfoxide