Response of isolated hepatocytes from carcinogen sensitive (C3H) and insensitive (C57BL) mice to signals inducing replication or apoptosis

Abstract

The mouse strain C3H shows high incidence of liver tumors in carcinogenicity testing, while the strain C57BL exhibits low incidence. The F1 generation hybrids, B6C3F1, which are widely used in long-term carcinogenesis bioassays, are of intermediate sensitivity. We asked whether this strain difference could be due to different susceptibility of the parenchymal cells to signals inducing replication or apoptosis. Hepatocytes were isolated and cultured according to standard protocols. We tested (1) for the induction of DNA synthesis by epidermal growth factor (EGF), (2) for its inhibition by TGF-beta1, and (3) for the induction of apoptosis by TGF-beta1. Basal rates of DNA synthesis in untreated hepatocytes cultured from C3H and B6C3F1 mice were 6.5 and 3.5 times higher, respectively, than in hepatocytes from C57BL on day 3. Moreover, addition of EGF (10 ng/ml) increased DNA synthesis on day 3 in hepatocytes from C3H (4.2-fold) and B6C3F1 (2.7-fold) more strongly than in hepatocytes from C57BL. Treatment with TGF-beta1 inhibited basal and EGF-stimulated DNA synthesis dose-dependently. Inhibition was maximal at 1 ng TGF-beta1/ml in cultures from C57BL mice, and at 0.3 ng/ml in hepatocytes from C3H mice. In untreated hepatocytes from both strains virtually no apoptotic figures (condensed or fragmented nuclei, Hoechst 33285 staining) were found. After treatment with TGF-beta1 the incidence of apoptotic nuclei in hepatocytes from C57BL was higher than in cells from C3H mice (1.7% vs 3% on day 3). Thus it appears that hepatocytes from C57BL mice possess a lower growth potential, as indicated by a low basal rate of DNA synthesis and low inducibility by EGF, but a higher sensitivity to induction of apoptosis by TGF-beta1 than hepatocytes of the C3H strain. These findings may be helpful to explain the different susceptibility to induction of hepatocarcinogenesis in C3H and C57BL mice.

Induction of apoptosis in mouse liver adenoma and carcinoma in vivo by transforming growth factor-?1

“…The number of intact cells was calculated as average cell number/microscopic field·number of microscopic fields (i.e., 2,500-5,000 cells per section) and the incidence of apoptotic bodies as visualized by TUNEL-reaction was expressed as a percentage; 95% CI: confidence limits. Normal liver tissue and HCC of six animals (''NDEA+PB'': 3 mice/10 HCC; ''NDEA+PB+TGF-ß1'': 3 mice/5 HCC) were analysed The total number of lesions was found to be effective Schulte-Hermann et al 2002). The difference in proapoptotic sensitivity of rat and mouse hepatocytes was recently confirmed in a study with primary cultures of hepatocytes from mice (C3H and C57BL, parental strains of B6C3F1 mice) and rats (F344): at any concentration between 0.1 and 100 ng/ml TGF-b1 exerted only a weak proapoptotic effect (less than 1%), whereas rat hepatocytes cultured under the same conditions were readily stimulated to undergo apoptosis at concentrations of 0.3 ng/ml or higher (up to 6%, ED 50 =0.8 ng/ml; Parzefall et al 2002).…”

“…The TGF-b1 lot (11/RP-07) used in the present experiments was as active as the lot used in our previous experiments with rats . Furthermore, in comparative studies with cultured primary mouse hepatocytes we found that recombinant simian TGF-b1 was as active as human TGF-b1 in inhibiting DNA synthesis (Parzefall et al 2002). TGFb1 doses were 56 lg or 200 lg TGF-b1/kg b.w.…”

“…dissolved in 5 mM HCl in 10 mM Tris-HCl (pH 7.4, containing 1 mg BSA/ml). The high dose of TGF-b1 (200 lg/kg) was chosen because previous studies revealed mouse hepatocytes to be more resistant to the mitoinhibitory effect of TGF-b1 than rat and human hepatocytes (Petersen et al 1994;Schulte-Hermann et al 2002;Parzefall et al 2002). TGF-b1 or solvent (6.7 ml/kg b.w.)…”

Distinct Influences of Neonatal Epidermal Growth Factor Challenge on Adult Neurobehavioral Traits in Four Mouse Strains

“…Thus, one of the potential explanations is that the strain-dependent or area-specific sensitivity to EGF might differ among strains and contribute to the above straindifference in EGF responses in learning. The fact that the cell proliferation activity of EGF is significantly different among mouse strains supports this hypothesis (Parzefall et al, 2002). Alternatively, it is possible that EGF permeability into these brain regions might differ considerably among mouse strains, although this assumption also remains to be tested.…”

Proteome analysis of hepatic non-parenchymal cells of immune liver fibrosis rats

“…NPCs were isolated from liver tissue by collagenase type IV (0.05 mg mL 1 ) digestion and density gradient centrifugation using Percoll (Pharmacia, Sweden) as described elsewhere [22,23] with slight modifications. Briefly, liver tissues were perfused for 10 min with pre-perfusion buffer followed by collagenase perfusion until digestion.…”