Automated detection of hepatotoxic compounds in human hepatocytes using HepaRG cells and image-based analysis of mitochondrial dysfunction with JC-1 dye.

In this study, our goal was to develop an efficient in situ test adapted to screen hepatotoxicity of various chemicals, a process which remains challenging during the early phase of drug development. The test was based on functional human hepatocytes using the HepaRG cell line, and automation of quantitative fluorescence microscopy coupled with automated imaging analysis. Differentiated HepaRG cells express most of the specific liver functions at levels close to those found in primary human hepatocytes, including detoxifying enzymes and drug transporters. A triparametric analysis was first used to evaluate hepatocyte purity and differentiation status, mainly detoxication capacity of cells before toxicity testing. We demonstrated that culturing HepaRG cells at high density maintained high hepatocyte purity and differentiation level. Moreover, evidence was found that isolating hepatocytes from 2-week-old confluent cultures limited variations associated with an ageing process occurring over time in confluent cells. Then, we designed a toxicity test based on detection of early mitochondrial depolarisation associated with permeability transition (MPT) pore opening, using JC-1 as a metachromatic fluorescent dye. Maximal dye dimerization that would have been strongly hampered by efficient efflux due to the active, multidrug-resistant (MDR) pump was overcome by coupling JC-1 with the MDR inhibitor verapamil. Specificity of this test was demonstrated and its usefulness appeared directly dependent on conditions supporting hepatic cell competence. This new hepatotoxicity test adapted to automated, image-based detection should be useful to evaluate the early MPT event common to cell apoptosis and necrosis and simultaneously to detect involvement of the multidrug resistant pump with target drugs in a human hepatocyte environment.

A plasma membrane protein is involved in cell contact-mediated regulation of tissue-specific genes in adult hepatocytes.

We have identified the liver-regulating protein (LRP), a cell surface protein involved in the maintenance of hepatocyte differentiation when cocultured with rat liver epithelial cells (RLEC). LRP was defined by immunoreactivity to a monoclonal antibody (mAb L8) prepared from RLEC. mAb L8 specifically detected two polypeptides of 85 and 73 kD in immunoprecipitation of both hepatocyte- and RLEC-iodinated plasma membranes. The involvement of these polypeptides, which are integral membrane proteins, in cell interaction-mediated regulation of hepatocytes was assessed by evaluating the perturbing effects of the antibody on cocultures with RLEC. Several parameters characteristic of differentiated hepatocytes were studied, such as liver-specific and house-keeping gene expression, cytoskeletal organization and deposition of extracellular matrix (ECM). An early cytoskeletal disturbance was evidenced and a marked alteration of hepatocyte functional capacity was observed in the presence of the antibody, together with a loss of ECM deposition. By contrast, cell-cell aggregation or cell adhesion to various extracellular matrix components were not affected. These findings suggest that LRP is distinct from an extracellular matrix receptor. The fact that early addition of mAb L8 during cell contact establishment was necessary to be effective may indicate that LRP is a novel plasma membrane protein that plays an early pivotal role in the coordinated metabolic changes which lead to the differentiated phenotype of mature hepatocytes.

The mitogen-activated protein kinase kinase/extracellular signal-regulated kinase cascade activation is a key signalling pathway involved in the regulation of G(1) phase progression in proliferating hepatocytes.

In this study, activation of the mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) signalling pathway was analyzed in proliferating rat hepatocytes both in vivo after partial hepatectomy and in vitro following epidermal growth factor (EGF)-pyruvate stimulation. First, a biphasic MEK/ERK activation was evidenced in G(1) phase of hepatocytes from regenerating liver but not from sham-operated control animals. One occurred in early G(1) (30 min to 4 h), and the other occurred in mid-late G(1), peaking at around 10.5 h. Interestingly, the mid-late G(1) activation peak was located just before cyclin D1 induction in both in vivo and in vitro models. Second, the biological role of the MEK/ERK cascade activation in hepatocyte progression through the G(1)/S transition was assessed by adding a MEK inhibitor (PD 98059) to EGF-pyruvate-stimulated hepatocytes in primary culture. In the presence of MEK inhibitor, cyclin D1 mRNA accumulation was inhibited, DNA replication was totally abolished, and the MEK1 isoform was preferentially targeted by this inhibition. This effect was dose dependent and completely reversed by removing the MEK inhibitor. Furthermore, transient transfection of hepatocytes with activated MEK1 construct resulted in increased cyclin D1 mRNA accumulation. Third, a correlation between the mid-late G(1) MEK/ERK activation in hepatocytes in vivo after partial hepatectomy and the mitogen-independent proliferation capacity of these cells in vitro was established. Among hepatocytes isolated either 5, 7, 9, 12 or 15 h after partial hepatectomy, only those isolated from 12- and 15-h regenerating livers were able to replicate DNA without additional growth stimulation in vitro. In addition, PD 98059 intravenous administration in vivo, before MEK activation, was able to inhibit DNA replication in hepatocytes from regenerating livers. Taken together, these results show that (i) early induction of the MEK/ERK cascade is restricted to hepatocytes from hepatectomized animals, allowing an early distinction of primed hepatocytes from those returning to quiescence, and (ii) mid-late G(1) MEK/ERK activation is mainly associated with cyclin D1 accumulation which leads to mitogen-independent progression of hepatocytes to S phase. These results allow us to point to a growth factor dependency in mid-late G(1) phase of proliferating hepatocytes in vivo as observed in vitro in proliferating hepatocytes and argue for a crucial role of the MEK/ERK cascade signalling pathway.

Mechanism in the sequential control of cell morphology and S phase entry by epidermal growth factor involves distinct MEK/ERK activations.

Cell shape plays a role in cell growth, differentiation, and death. Herein, we used the hepatocyte, a normal, highly differentiated cell characterized by a long G1 phase, to understand the mechanisms that link cell shape to growth. First, evidence was provided that the mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) cascade is a key transduction pathway controlling the hepatocyte morphology. MEK2/ERK2 activation in early G1 phase did not lead to cell proliferation but induced cell shape spreading and demonstration was provided that this MAPK-dependent spreading was required for reaching G1/S transition and DNA replication. Moreover, epidermal growth factor (EGF) was found to control this morphogenic signal in addition to its mitogenic effect. Thus, blockade of cell spreading by cytochalasin D or PD98059 treatment resulted in inhibition of EGF-dependent DNA replication. Our data led us to assess the first third of G1, is exclusively devoted to the growth factor-dependent morphogenic events, whereas the mitogenic signal occurred at only approximately mid-G1 phase. Moreover, these two growth factor-related sequential signaling events involved successively activation of MEK2-ERK2 and then MEK1/2-ERK1/2 isoforms. In addition, we demonstrated that inhibition of extracellular matrix receptor, such as integrin beta1 subunit, leads to cell arrest in G1, whereas EGF was found to up-regulated integrin beta1 and fibronectin in a MEK-ERK-dependent manner. This process in relation to cytoskeletal reorganization could induce hepatocyte spreading, making them permissive for DNA replication. Our results provide new insight into the mechanisms by which a growth factor can temporally control dual morphogenic and mitogenic signals during the G1 phase.

Expression of helix-loop-helix factor Id-1 is dependent on the hepatocyte proliferation and differentiation status in rat liver and in primary culture.

Id proteins are known as negative regulators of differentiation in various cell types. In this report, we show that the Id-1 gene was down-regulated during the development of rat liver. No Id-1 transcripts were detected in terminal differentiated hepatocytes. We have studied Id-1 expression in proliferating hepatocytes using an in vivo model of liver regeneration after partial hepatectomy and an in vitro growth factor-stimulated hepatocyte culture system. Strong activation of Id-1 was observed in mid-late G1 of the hepatocyte cell cycle at a time corresponding to a mitogen restriction point. These observations suggest that Id-1 is involved in the control of proliferation and differentiation in liver cells.

Isolation and characterization of complementary DNA clones for genes overexpressed in chemically induced rat hepatomas.

In order to characterize the genes overexpressed in an hepatoma cell line, the HTC cells, and in diethylnitrosamine induced solid hepatomas, we constructed a complementary DNA library from HTC cells and performed differential screening with probes from HTC cells, from malignant nodules obtained 70 weeks after the carcinogen treatment, and from hepatocytes from normal rat liver. Eight clones corresponding to messenger RNAs (mRNAs) much more expressed in hepatomas than in hepatocytes from normal liver were isolated. Three, clones pHT 71, pHT 13, and pHT 26, were further analyzed by the study of their corresponding transcripts in hepatocytes from regenerating liver and in the hepatocytes from the nontumorous parts of the liver. Clone pHT 71 corresponds to a single 2.3-kilobase mRNA which is present in high levels in carcinoma nodules in hepatoma cell lines, in the nontumorous parts of the liver, and in hepatocytes isolated from regenerating liver 30 h after partial hepatectomy. Clone pHT 13 hybridizes with three distinct transcripts 3.8, 2.6, and 1.6 kilobases long. High levels of the 3.8- and 1.6-kilobase mRNAs are present in carcinoma nodules, in hepatoma cell lines, and in the nontumorous parts of the liver. However, the levels of these RNAs are similar in hepatocytes from regenerating liver and in hepatocytes obtained from normal rat liver. Clone pHT 26 corresponds to a 0.6-kilobase mRNA which exists at a high level only in cancer nodules and in hepatoma cell lines. We were unable to observe any cross-hybridization between these clones and the oncogenes which have been found to be expressed in hepatomas (c-fos, c-Ha-ras, c-Ki-ras, N-ras, and c-myc). The mRNAs corresponding to the three clones have not been detected in various tissues from normal adult rats. Our study shows that a high level of these mRNAs might be associated with rat liver carcinogenesis.

Identification of a novel Skp2-like mammalian protein containing F-box and leucine-rich repeats.

The F-box protein Skp2 is important for S phase entry and binds to Skp1 and the cyclin A-Cdk2 complex. Here we report the cloning, analysis of genomic organization and characterization of a novel gene product related to Skp2 named FBL2. The human FBL2 gene was found to be a highly interrupted gene of at least 126.6 kb located on chromosome 17 in close proximity to the TRAP220 gene in a head-to-tail orientation. The predicted protein contains an F-box and six perfect C-terminal leucine-rich repeats. Similar to Skp2, this protein interacts with Skp1 and deletion of the F-box inhibits this association. However, in contrast to Skp2, FBL2 was detected in non-proliferating hepatocytes and its expression increased in growth-arrested liver epithelial cells. In addition, FBL2 was localized primarily in the cytoplasm concentrated around the nucleus. Overall, our data indicate that although FBL2 shares strong structural homology with Skp2 as well as having a similar ability to associate with Skp1, these proteins likely play distinct roles and target different substrates to the SCF complex.

Skp2 induction and phosphorylation is associated with the late G1 phase of proliferating rat hepatocytes.

The changes in phosphoproteins purified with the affinity peptide p9CKShs1 were analyzed from extracts of regenerating rat livers in order to define some G1 and G1/S regulations characteristic of mature hepatocytes stimulated to proliferate. We observed a 47 kDa phosphoprotein that occurred first at the end of G1 before peaking in the S phase. P47 was also found to be phosphorylated in late G1 in primary hepatocyte cultures stimulated with mitogens. P47 was still phosphorylated in extracts depleted of Cdc2, but to a lesser extent after Cdk2 depletion. This phosphoprotein was identified as Skp2. (i) P47 shared the same electrophoretic mobility than Skp2, a cell cycle protein essential for S phase entry in human fibroblasts; (ii) Skp2, like P47, started to be expressed and was highly phosphorylated during the G1/S transition of hepatocytes stimulated to proliferate in vivo and in vitro; (iii) P47 was specifically immunoprecipitated by an antibody directed against Skp2. In addition, cyclin A/Cdk2 complexes from regenerating liver clearly interacted with Skp2. This is the first demonstration that Skp2 is induced and phosphorylated in the late G1 and S phase of hepatocytes in vivo in regenerating liver as well as in vitro in mitogen-stimulated hepatocytes.

Lysosomal enzyme activities during ageing of adult human liver cell lines.

Ten lysosomal enzyme activities have been compared during the growth and ageing of adult human liver cell lines. Arylsulfatase A, beta-D-galactosidase and beta-D-glucuronidase activities were significantly lower and arylsulfatase B activity was significantly higher in senescent cells than in actively growing cells. Furthermore, hexosaminidase activity was lower and acid phosphatase activity higher in old cells in every cell line tested but the differences were not significant. On the other hand, no change occurred in alpha-L-fucosidase, alpha-D-mannosidase, alpha-D-galactosidase and alpha-D-glucosidase activities. These results demonstrate that the increase in size and number of secondary lysosomes during ageing is accompanied for a few lysosomal enzymes by an increase or a decrease in activity depending on the enzyme.

[A study of lysosomal enzyme activities in serum and leukocytes in chronic hepatic disease (author's transl)]. / Etude des activités enzymatiques lysosomiques sériques et leucocytaires au cours des hepatopathies chroniques.

Five lysosomal enzyme activities (arylsulfatase A, alpha-D-mannosidase, alpha-L-fucosidase, hexosaminidase and beta-D-galactosidase) were determined in serum and leucocytes of 30 controls and 114 patients suffering from various liver diseases, including 30 with idiopathic hemochromatosis and 34 with alcoholic cirrhosis. The results show (1) a decrease of the serum arylsulfatase A activity in idiopathic hemochromatosis, (2) an increase of this activity in cholestatic jaundice, and (3) mainly, a sharp rise of the leucocyte lysosomal enzyme activities in the liver diseases studied (chiefly idiopathic hemochromatosis and alcoholic cirrhosis). The mechanism and the meaning of these disturbances are discussed.

Primary cultures of heart cells from the scallop Pecten maximus (mollusca-bivalvia).

Primary cultures of Pecten maximus heart cells, isolated by an enzymatic procedure, were routinely obtained with a high level of reproducibility in a simple medium based on sterile seawater. Cells attached to the plastic substratum without the need to add a special factor. The number of adhering cells gradually increased with the time of culture. Two types of adhering cells were observed: epitheliallike cells and fibroblastlike cells, which were more numerous. The latter cells were identified as myocytes by electron microscopy and immunofluorescent staining. Results obtained by autoradiography, after incorporation of [14C]leucine, [3H]thymidine, and [14C]acetate, confirmed functional activity of the cells. These cultures were maintained viable in vitro during at least 1 mo.

Long-term maintenance of taurocholate uptake by adult rat hepatocytes co-cultured with a liver epithelial cell line.

Taurocholate (TC) uptake by adult rat hepatocytes co-cultured with other rat liver epithelial cells (RLEC) was studied comparatively to hepatocytes in primary culture. Cells were cultured on Petri dishes for desired times prior to measuring their ability to transport TC. TC uptake was linear for 150 sec in both culture conditions. In hepatocytes cultured alone, the initial rate of TC uptake at an extracellular concentration of 100 microM was 0.19 +/- 0.02 nmole per min per 10(6) cells after 48 hr of culture and decreased by 75% after 4 to 6 days. In hepatocytes co-cultured with RLEC, the rate of uptake at 48 hr (0.31 +/- 0.01 nmole per min per 10(6) cells) was significantly higher than in hepatocytes cultured alone (p less than 0.01); in addition, TC uptake remained stable at an average rate of 0.17 +/- 0.01 nmole per min per 10(6) cells for up to 56 days. No detectable uptake was found in RLEC cultured alone. TC uptake exhibited both saturable (Vmax = 0.30 +/- 0.03 nmole per min per 10(6) cells and Km = 42.6 +/- 4.4 microM) and nonsaturable components. These kinetic parameters were similar to those previously reported in isolated hepatocytes and in short-term cultured hepatocytes. TC uptake exhibited sodium dependence and was significantly reduced when extracellular sodium was replaced by lithium and sucrose, or in the presence of 1 mM ouabain. After 18 days of co-culture, TC uptake had qualitatively the same characteristics as at 48 hr, with a saturable and a nonsaturable component.(ABSTRACT TRUNCATED AT 250 WORDS)

Influence of medium composition and culture conditions on glutathione S-transferase activity in adult rat hepatocytes during culture.

Glutathione S-transferase (GST) activity was measured in adult rat hepatocytes during either pure culture or coculture with another rat liver cell type in various media. Addition of nicotinamide, selenium, or dimethylsulfoxide, deprivation of cyst(e)ine and the use of two complex media were tested. Whatever the conditions used, after a constant decrease during the first 24 h, GST remained active over the whole culture period (1-2 wk). However, various patterns were observed: GST activity either remained relatively stable to approximately 50% of the initial value or showed a moderate or strong increase. The highest values were found in pure hepatocyte cultures maintained in the presence of nicotinamide or dimethylsulfoxide. Similar changes were observed using 1-chloro-2,4-dinitrobenzene or 1,2-dichloro-4-nitrobenzene as substrates for GST. Addition of 10(-4) M indomethacin resulted in 37 to 60% inhibition of enzyme activity. Thus, these results demonstrate that GST remained expressed during culture but its levels markedly varied depending on the medium composition and type and age of culture.

Distinct effects of cell-cell communication and corticosteroids on the synthesis and distribution of cytokeratins in cultured rat hepatocytes.

Cytokeratins CK 8 and CK 18 are the two keratins expressed in the liver. They are known to undergo extensive changes in expression with alteration of the hepatocyte phenotype in vitro. In this study, we have investigated the variation in levels of these two cytokeratins in hepatocytes selected from different situations in vivo. The amounts of corresponding transcripts were compared; cytokeratin 8 and 18 mRNAs were present at similar levels in hepatocytes freshly isolated from adult liver and, unexpectedly, from 17-day-old foetuses and newborn rats, whereas they were markedly higher in regenerating hepatocytes isolated early after partial hepatectomy. In order to investigate whether the different factors that can promote hepatocyte differentiation also produce a similar set of cytoskeletal changes, we have analysed both the expression and the distribution of cytokeratins in hepatocytes under different culture conditions allowing modulation of differentiation. Establishment of cell-cell contacts and addition of glucocorticoids were used as two modulating factors. Coculturing hepatocytes with rat liver epithelial cells (RLEC), which favours active expression of liver-specific genes, resulted in a gradual decline of cytokeratin mRNAs, whereas pure hepatocyte cultures, which exhibit rapid phenotypic changes, expressed increasing levels of CK 8 and CK 18 transcripts. Furthermore, intracellular CK distribution was dramatically modified in parallel: the CK-positive material formed a fine network of fibrils uniformly distributed in the cytoplasm of hepatocytes in pure culture, whereas in cocultured cells CK immunofluorescence appeared principally located at the cellular periphery and it was regularly arranged in long fibrils just beneath the plasma membrane.(ABSTRACT TRUNCATED AT 250 WORDS)

Constitutive expression of functional P-glycoprotein in rat hepatoma cells.

P-glycoprotein is a plasma-membrane glycoprotein involved in multidrug resistance. P-glycoprotein overexpression has been demonstrated to occur in tumor cells after cytotoxic drug exposure, but also in some cancers including hepatocellular carcinomas before any chemotherapeutic treatment. In order to better analyze this constitutive type of tumoral drug resistance, we have investigated P-glycoprotein expression and function in rat liver tumors induced experimentally by administration of diethylnitrosamine and in two cell clones derived from one of these tumors designated as RHC1 and RHC2. High levels of P-glycoprotein mRNAs were found in both liver tumor samples and the two hepatoma cell clones as assessed by Northern blotting; both RHC1 and RHC2 cells displayed altered liver functions commonly observed in rat hepatoma cells, particularly the decreased expression of albumin and overexpression of the fetal glutathione S-transferase 7-7. The use of specific multidrug resistance (mdr) probes revealed a major induction of the mdr1 gene in liver tumor samples while RHC1 and RHC2 cells expressed both mdr1 and mdr3 genes without displaying a major alteration in the number of mdr gene copies as assessed by Southern blotting. High amounts of P-glycoprotein were also demonstrated in RHC1 and RHC2 cells by Western blotting. These cells were strongly resistant to doxorubicin and vinblastine, two anticancer drugs transported by P-glycoprotein. Doxorubicin intracellular retention was low in RHC1 and RHC2 cells, but was strongly enhanced in the presence of verapamil, a known modulator agent of P-glycoprotein; low retention appeared to occur via a drug efflux mechanism, indicating that P-glycoprotein was fully active. These results show that rat hepatoma cells can display elevated levels of functional P-glycoprotein without any prior cytotoxic drug selection and suggest that these cells represent a useful model for analyzing P-glycoprotein regulation in intrinsically clinical drug-resistant cancers.

Perhexiline maleate toxicity on human liver cell lines.

When added to the culture medium of human liver cell lines, perhexiline maleate induced formation of numerous myeloid bodies containing unicentric or multicentric smooth membranes within a few days. The nine lysosomal enzyme activities studied, except for beta-galactosidase which decreased, remained unchanged. These results indicate that on cultured human liver cells perhexiline maleate has an effect similar to that described on hepatocytes of some patients treated with this drug and suggest that myeloid body formation is not due to impairment of lysosomal enzyme activities.

Expression and activation of cdks (1 and 2) and cyclins in the cell cycle progression during liver regeneration.

In normal adult liver, hepatocytes are arrested in G0, and they rapidly respond to a mass loss by a definite number of divisions. Thus, taking advantage of the in vivo regenerative capacity of the liver following partial hepatectomy, we have analyzed both expression and activation of p34cdc2 (= cdk1) and p33cdk2 through the cell cycle, particularly during the long lasting G1 phase and in the G1/S transition. While p33cdk2 is constantly expressed during the cell cycle, p34cdc2 is completely absent in resting hepatocytes and remains unexpressed for up to 20 h after partial hepatectomy, a time period corresponding to the G1 phase and G1/S transition, and then accumulates in the S, G2, and M phases. No histone H1 kinase activity is detected during the G1 phase, while two peaks of p34cdc2 kinase activity are observed during the S and M phases and only one peak of p33cdk2 kinase activity in the S phase. p34cdc2 forms complexes with both cyclins A and B while p33cdk2 is associated with cyclin A only. Surprisingly, cyclins E and D1 are present in resting liver and with modest variation throughout the cell cycle. Taken together, our data provide evidence that the pattern of G1-associated proteins in hepatocytes during liver regeneration is distinct from that described in other cell types.

Growth factor dependence of progression through G1 and S phases of adult rat hepatocytes in vitro. Evidence of a mitogen restriction point in mid-late G1.

Several hepatocyte mitogens have been identified, but the signals triggering the G0/G1 transition and cell cycle progression of hepatocytes remain unknown. Using hepatocyte primary cultures, we investigated the role of epidermal growth factor/pyruvate during the entry into and progression through the G1 phase and analyzed the expression of cell cycle markers. We show that the G0/G1 transition occurs during hepatocyte isolation as evidenced by the expression of early genes such as c-fos, c-jun, and c-myc. In culture, hepatocytes progress through G1 regardless of growth factor stimulation until a restriction point (R point) in mid-late G1 beyond which they cannot complete the cell cycle without mitogenic stimulation. Changes in cell cycle gene expression were associated with progression in G1; the cyclin E mRNA level is low early in G1 but increases at the G1/S boundary, while the protein is constantly detected during cell cycle but undergoes a change of electrophoretic mobility in mid-late G1 after the R point. In addition, a drastic induction of cyclin D1 mRNA and protein, and to a lesser extent of cyclin D2 mRNA, takes place in mitogen-stimulated cells after the R point. In contrast, cyclin D3 mRNA appears early in G1, remains constant in stimulated cells, but accumulates in unstimulated arrested cells, paralleling the cyclin-dependent kinase 4 mRNA expression. These results characterize the different steps of G1 phase in hepatocytes.

Progression through G1 and S phases of adult rat hepatocytes.

Regenerating liver, hepatocyte primary cultures and differentiated hepatoma cell lines are widely used to study the proliferation/differentiation/apoptosis equilibrium in liver. In hepatocytes, priming factors (TNF alpha, IL6) target G0/G1 transition while growth factors (HGF, EGF, TGF alpha) control a mid-late G1 restriction point. A characteristic pattern of cdk/cyclin expression is observed in hepatocytes, presumably related to their ability to proliferate a limited number of times and to undergo a reversible differentiation. Interestingly, cell-cell interactions between hepatocytes and liver biliary cells in co-cultures, result in a cell cycle arrest in mid G1 of hepatocytes which are insensitive to mitogens. Apoptosis exists in hepatocytes but is still poorly documented. However, hepatoma cell lines stimulated by TGF beta undergo cell death in a p53-independent pathway. In conclusion, the interplay of growth and apoptosis regulators and cell-cell interactions control the proliferation/differentiation/apoptosis balance which is a specific feature of hepatocytes.