Long-term survival of functional hepatocytes from adult rat in the presence of phenobarbital in primary culture

Miyazaki, Masahiro; Handa, Yoshihiko; Oda, Munehiro; Yabe, Tamae; Miyano, Keiko; Sato, Jiro

doi:10.1016/s0014-4827(85)80047-1

Cited by 94 publications

(24 citation statements)

References 23 publications

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“…The extent of the EndoG expression was decreased as the incubation time was increased after the transfection of EndoG siRNA (date not shown). Incubation time is known to be an important determinant affecting the outcomes of experiments, especially those using the primary culture of hepatocytes (68,69). In fact, we found that incubation times longer than 14 h before ATZϩMS treatment caused significant differences in some parameters compared with those at 2 h of incubation (date not shown).…”

Section: Discussionmentioning

confidence: 74%

Involvement of Endonuclease G in Nucleosomal DNA Fragmentation under Sustained Endogenous Oxidative Stress

Ishihara

Shimamoto

2006

Journal of Biological Chemistry

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We have previously shown that inhibition of catalase and glutathione peroxidase activities by 3-amino-1,2,4-triazole (ATZ) and mercaptosuccinic acid (MS), respectively, in rat primary hepatocytes caused sustained endogenous oxidative stress and apoptotic cell death without caspase-3 activation. In this study, we investigated the mechanism of this apoptotic cell death in terms of nucleosomal DNA fragmentation. Treatment with ATZ؉MS time-dependently increased the number of deoxynucleotidyl transferase-mediated nick end-labeling (TUNEL)-positive nuclei from 12 h, resulting in clear DNA laddering at 24 h. The deoxyribonuclease (DNase) inhibitor, aurintricarboxylic acid (ATA), completely inhibited nucleosomal DNA fragmentation but the pan-caspase inhibitor, z-VAD-fmk was without effects; furthermore, the cleavage of inhibitor of caspase-activated DNase was not detected, indicating the involvement of DNase(s) other than caspase-activated DNase. Considering that endonuclease G (EndoG) reportedly acts in a caspase-independent manner, we cloned rat EndoG cDNA for the first time. Recombinant EndoG alone digested plasmid DNA and induced nucleosomal DNA fragmentation in isolated hepatocyte nuclei. Recombinant EndoG activity was inhibited by ATA but not by hydrogen peroxide, even at 10 mM. ATZ؉MS stimulation elicited decreases in mitochondrial membrane potential and EndoG translocation from mitochondria to nuclei. By applying RNA interference, the mRNA levels of EndoG were almost completely suppressed and the amount of EndoG protein was decreased to approximately half the level of untreated cells. Under these conditions, decreases in TUNEL-positive nuclei were significantly suppressed. These results indicate that EndoG is responsible, at least in part, for nucleosomal DNA fragmentation under endogenous oxidative stress conditions induced by ATZ؉MS.

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Section: Discussionmentioning

confidence: 74%

Involvement of Endonuclease G in Nucleosomal DNA Fragmentation under Sustained Endogenous Oxidative Stress

Ishihara

Shimamoto

2006

Journal of Biological Chemistry

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“…Maintenance of hepatocytes on liver biomatrix (Reid et al, 1980), basement membrane gels from EHS sarcoma (Bissell et al, 19871, or collagen gels in single (Michalopoulos and Pitot, 1975) and, more recently, in double gel layers (Dunn et al, 1989) has strong effects on cell morphology, longevity, and differentiated phenotype. Similar effects, however, are also seen without any substrate modification but with the addition of compounds such as dimethyl sulfoxide (DMSO) (2%) (Isom et al, 1985) and phenobarbital (3 mM) (Miyazaki et al, 1985). Full expression of markers of differentiation was shown in hepatocytes that had been kept in continuous primary culture for more than 12 months (Isom et al, 1985).…”

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confidence: 80%

Multiple sequential periods of DNA synthesis and quiescence in primary hepatocyte cultures maintained on the DMSO‐EGF on/off protocol

Chan

Kost

Michalopoulos

1989

Journal Cellular Physiology

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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.

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“…The supplementation of culture medium with dimethyl sulfoxide (DMSO) (10, 111, phenobarbital (12) and nicotinamide (131, coculture with nonparenchymal epithelial cells (14) and the use of extracellular matrix components (8, [15][16][17] have been successfully used to maintain the differentiated functions of hepatocytes. Under these experimental conditions, the growth of hepatic parenchymal cells was suppressed.…”

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confidence: 99%

Small Cell Colonies Appear in the Primary Culture of Adult Rat Hepatocytes in the Presence of Nicotinamide and Epidermal Growth Factor

Mikami²,

et al. 1992

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Colonies of small hepatocytes appeared after the culture of primary adult rat hepatocytes for 4 days in serum-free modified Dulbecco's modified Eagle's medium containing 10 mmol/L nicotinamide and 10 ng/ml epidermal growth factor. Each colony consisted of cells that had a single nucleus and a higher nucleus/cytoplasm ratio than surrounding hepatocytes, and immunocytochemically these cells were stained with albumin and transferrin. Ultrastructurally these cells had mitochondria, peroxisomes and desmosomes, indicating that they were derived from hepatocytes. When 6 x 10(5) cells were plated on 35-mm dishes, about 5.5 colonies/mm2 were observed. This result suggested that about 1.5% of adult rat hepatocytes has the potential for multiple replications and of forming a focal colony. These cell populations had higher proliferative activities than surrounding hepatocytes. DNA synthetic activity could not be inhibited by 2% dimethyl sulfoxide. Flow cytometric analysis showed that both 2N and 4N nuclei synthesized their DNA until day 4 but that the number of 2N nuclei rapidly increased at day 5. This result correlated with the observation of the appearance of small cell populations indicating that the cells of these focal colonies were predominantly diploid.

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Long-term survival of functional hepatocytes from adult rat in the presence of phenobarbital in primary culture

Cited by 94 publications

References 23 publications

Involvement of Endonuclease G in Nucleosomal DNA Fragmentation under Sustained Endogenous Oxidative Stress

Involvement of Endonuclease G in Nucleosomal DNA Fragmentation under Sustained Endogenous Oxidative Stress

Multiple sequential periods of DNA synthesis and quiescence in primary hepatocyte cultures maintained on the DMSO‐EGF on/off protocol

Small Cell Colonies Appear in the Primary Culture of Adult Rat Hepatocytes in the Presence of Nicotinamide and Epidermal Growth Factor

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