Gap junctional intercellular communication and connexin expression in normal and SV 40-transformed human liver cells in vitro.

The gap junction intercellular communication (GJIC) capacity of two normal human liver-derived epithelial cell strains and their SV40 large T oncogene-transformed counterparts was examined. In homologous cultures the GJIC capacity of the transformed cells was considerably less than the parental cells. In heterologous cultures, transformed cells appeared to be able to form GJIC channels with normal cells. Only non-transformed cells expressed connexin (cx) 43 gene and cx 26 or cx 32 transcripts were not detectable in any cell strains tested. When GJIC was assayed in the presence of the phorbol ester tumour promoter 12-O-tetradecanoylphorbol-13- acetate (TPA), all four strains showed a marked sensitivity to TPA in inhibitory activity at 1-10 ng/ml. In contrast to a rat liver epithelial cell line, this effect of TPA did not appear to become refractory even after 24 h exposure. These studies demonstrate that GJIC of human liver cells in culture can be decreased by viral oncogene and tumour promoter action. Such disturbance may be an important component of the carcinogenic activity of these agents.

Evidence for post-transcriptional regulation of cytokeratin gene expression in a rat liver epithelial cell line.

T51B, a cell line of the rat liver nonparenchymal cell compartment, contains a cytokeratin (CK) pair composed of CK8, a CK typical of simple epithelium, and CK14, a CK usually present in proliferative stratified epithelium. T51B-Ni, a subclone selected by prolonged exposure of the parental clone to nickel subsulfide contains CK8 and CK18 (its usual partner in simple epithelium), as well as CK14, at a lower level. The two clones have comparable levels of vimentin. Northern blot analyses of cytoplasmic mRNA demonstrated that the differences in the steady state mRNA levels of each CK paralleled those observed at the protein level, thus showing that the regulatory events occurred prior to translation. The most prominent difference was a 30-fold higher level of CK18 mRNA in T51B-Ni cells. Run-off assays of isolated nuclei revealed that the level of CK8, CK14, and vimentin was regulated primarily at the transcriptional level. However, the large increase in CK18 mRNA levels in T51B-Ni cells did not result from a corresponding increase in the relative level of CK18 gene transcription nor from a change in cytoplasmic CK18 mRNA stability. Comparative Northern blot analyses of nuclear and cytoplasmic mRNAs further suggested that the control of CK18 gene expression in T51B cells is post-transcriptionally mediated by nuclear events.

Recommended protocols based on a survey of current practice in genotoxicity testing laboratories: study design.

The most commonly used genotoxicity assays for cultured mammalian cells are mammalian cell mutagenesis, chromosome aberrations/SCE, hepatocyte UDS, and cell transformation. Since their inception, protocols for these assays have been modified in various laboratories. It has been observed that minor but potentially significant method modifications frequently remain unpublished (Swierenga et al., 1983) but should be considered in the development of recommended protocols. The present study was undertaken to determine the current 'state of the art' for these tests. Detailed questionnaires on culture conditions and testing protocols for both stock and test cell populations were designed with the assistance of an international advisory committee and sent to all research and contract laboratories that could be readily identified in Canada, U.S.A. and Europe. Responses from 425 completed questionnaires were analyzed to determine the most commonly used approach and modifications for each procedural step. As expected, the results show a large degree of interlaboratory variation. Detailed protocols for conducting each assay have been prepared and include: stepwise instructions, precautionary measures and practical solutions to common problems associated with each assay; recipes for media and solutions; formulas for quantifying genotoxic responses; reference lists of related assays; guidelines for interpretation; and discussions of the applications, advantages and disadvantages of each test.

Recommended protocols based on a survey of current practice in genotoxicity testing laboratories: I. Unscheduled DNA synthesis assay in rat hepatocyte cultures.

A protocol based primarily on current laboratory practices in the performance of the unscheduled DNA synthesis (UDS) assay with primary rat hepatocyte cultures has been developed. These guidelines were developed using tabulated responses to a detailed questionnaire completed by North American and European governmental, university and contract laboratories involved with the UDS test. This report identifies those modifications to previously described methodologies which are used on a regular basis and also serves to clarify confusing or inconsistent practices. Although this protocol pertains specifically to the use of primary rat hepatocyte cultures, it can be modified to incorporate other types of cells in which certain aspects remain the same.

Recommended protocols based on a survey of current practice in genotoxicity testing laboratories: II. Mutation in Chinese hamster ovary, V79 Chinese hamster lung and L5178Y mouse lymphoma cells.

Laboratory protocols and guidelines have been developed for the performance of point mutation assays using Chinese hamster ovary (CHO) cells, V79 cells, and L5178Y mouse lymphoma cells. Since only minor differences in the treatment of CHO and V79 cells exist, these two assays could be combined in one procedural guideline. A second protocol was developed for the mouse lymphoma assay in order to incorporate concerns and methods specific to that cell type and genetic locus. The protocols were based primarily on current laboratory practices as determined by responses to a detailed questionnaire completed by North-American and European governmental, university and contract laboratories involved with in vitro mutation testing. This report identifies those modifications to previously described methodologies which are being used on a regular basis, provides recommendations, and also serves to clarify confusing or inconsistent practices.

Recommended protocols based on a survey of current practice in genotoxicity testing laboratories: III. Cell transformation in C3H/10T1/2 mouse embryo cell, BALB/c 3T3 mouse fibroblast and Syrian hamster embryo cell cultures.

A standardized protocol and guidelines for the performance of cell transformation testing in mouse embryo (C3H/10T1/2), mouse fibroblast (BALB/c 3T3) and Syrian hamster embryo (SHE) cells have been developed. The protocol is based primarily on current laboratory practices as determined by responses to a detailed questionnaire completed by North American and European governmental, university and contract laboratories involved with cell transformation experimentation. This report identifies those modifications to previously described methodologies which are being used on a regular basis and also serves to clarify confusing or inconsistent practices.

Recommended protocols based on a survey of current practice in genotoxicity testing laboratories, IV. Chromosome aberration and sister-chromatid exchange in Chinese hamster ovary, V79 Chinese hamster lung and human lymphocyte cultures.

A recommended protocol has been developed for chromosomal aberration and sister-chromatid exchange assays in CHO, V79 and human lymphocyte cultures. The protocol was based on the responses to a detailed questionnaire completed by North-American and European governmental, university, and contract laboratories using these tests. This report identifies those modifications to previously described methods that are used on a regular basis and clarifies confusing or inconsistent practices. These protocols can be modified for use in other types of cells.

Recommended protocols based on a survey of current practice in genotoxicity testing laboratories: study evaluation.

The approach used in the survey of current methodologies used in mammalian cell genotoxicity testing (unscheduled DNA synthesis, mutation, cell transformation and cytogenetics testing) is discussed. The recommended protocols, described in the preceding papers, were developed using responses to detailed questionnaires. This summary outlines general observations related to the survey methodology and to the protocols themselves. Also discussed are the qualities of an effective survey; the evolution of a survey and of a protocol; and the contributions and limitations of this study.

Use of primary keratinocyte cultures from plucked human hairs for analysis of gap junctional intercellular communication.

Hair follicles are a convenient source of human keratinocytes that are easily derived by non-invasive means. In order to test whether such cells could be used for gap junctional intercellular communication (GJIC) studies for the assessment of tumour-promoting activity of environmental chemicals, primary cultures were initiated from plucked hairs of various donors. GJIC measurements were performed when colonies reached a size of 5 mm in diameter, by microinjection of the fluorescent dye Lucifer Yellow CH into single cells at the colony periphery and scoring dye transfer to surrounding cells. The GJIC of cells that had begun to differentiate (appearance of cornified envelopes) can be quantitated more easily by switching cells to a low calcium (0.05-mm) medium. The tumour promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) inhibited GJIC in a concentration- and time-dependent manner, resembling the TPA response of mouse primary epidermal keratinocytes. Thus, hair follicle cultures can be readily used for GJIC studies and should be appropriate for other in vitro assays requiring attached cells.

Effects on intercellular communication in human keratinocytes and liver-derived cells of polychlorinated biphenyl congeners with differing in vivo promotion activities.

Several purified polychlorinated biphenyl (PCB) congeners with differing toxicity/tumor promotional activities in rat liver in vivo were tested for their effects on gap-junctional intercellular communication (GJIC) in cell strains and lines derived from human liver and skin. This in vitro assay is being developed to detect various classes of tumor promoters. The 3-methylcholanthrene (MCA)-type cytochrome P450 inducer and hepatotoxic promoter 3,3',4,4'-tetrachlorobiphenyl was inactive in this assay for all of the cells tested, suggesting this promoter acts by other mechanisms. The phenobarbital-like enzyme inducer and less toxic promoter 2,2',4,4',5,5'-hexachlorobiphenyl inhibited GJIC in both liver and skin cells, whereas the 2,2',5,5'-tetrachlorobiphenyl congener, which does not act as a promoter in rat liver, inhibited GJIC only in the skin cell types and in one of the liver cell strains thought to be of bile duct origin. 2,3,4,4',5-Pentachlorobiphenyl, a mixed (phenobarbital plus MCA) inducer of cytochrome P450, inhibited GJIC in both liver and skin cells, suggesting that it may be a promoter in vivo. The results suggest that GJIC inhibition is a property of PCB congeners with phenobarbital-like enzyme induction capabilities, and that there exist some tissue/cell type differences in sensitivity to these congeners.

Role of cytokeratin intermediate filaments in transhepatic transport and canalicular secretion.

The role of cytokeratin filaments in the function of hepatocytes was investigated using a nickel-treated hepatocyte in vitro model. Cytokeratin intermediate filaments were selectively dissociated from the cell cortex by nickel treatment. Cytokeratins and ubiquitin were observed using immunofluorescence and immunoelectron microscopy. Hepatocytic function was assessed by visualizing uptake, transhepatic transport and secretion of fluorescein diacetate and horseradish peroxidase into the bile canaliculi. In control primary cultures, most of the bile canaliculi were surrounded by an inner layer of actin filaments and an outer pericanalicular sheath of cytokeratin filaments and microtubules. The cytoplasmic distribution of ubiquitin was diffuse and particulate. After treatment with NiCl2 (150 micrograms/ml) for 24 hr, the cytokeratin filaments and desmoplakin became focally detached from the cell cortex and retracted to form an aggregate around the nucleus. These aggregates were associated with intense ubiquitin immunoreactivity. Only a few attachments of the cytokeratin filaments to the cell cortex remained. F-actin remained attached to the cell cortex in the areas where the cytokeratin filaments had become detached. The pericanalicular sheath of cytokeratin filaments and the bile canaliculi disappeared and actin was dispersed over the entire cell periphery. Fluorescein diacetate secretion and horseradish peroxidase uptake were almost completely absent in the hepatocytes treated with nickel. The effects of nickel persisted 24 hr after its removal from the medium. It is concluded that cytokeratin intermediate filaments play a critical role in the formation of the bile canaliculus, secretion of fluorescein diacetate and uptake of horseradish peroxidase. Further, our study indicates that cytokeratin ubiquitination occurs during collapse and aggregation of the cytokeratin filaments. The formation of cytokeratin-ubiquitin conjugates during aggregation suggests a role of ubiquitin in the control of cytokeratin organization in hepatocytes in the response to cell stress.

Altered cytokeratin expression and differentiation induction during neoplastic transformation of cultured rat liver cells by nickel subsulfide.

Rat liver T51B cells were maintained in the presence of low concentrations of Ni(II) derived from alpha Ni3S2 for 3-15 months in culture in order to monitor cytokeratin, differentiation, and transformation patterns. Nickel exposures caused irreversible, heritable juxtanuclear aggregates of cytokeratin CK55, which increased in size and complexity with prolonged nickel exposure, eventually resembling Mallory bodies and expressing glutamyltransferase. Altered cytokeratin expression was accompanied by induction of differentiation, with markers of both bile ductular cells and hepatocytes, such as induction of cytokeratin polypeptides CK39 and CK49, cell morphology, and cytokeratin filament network changes; whereas control cultures similarly maintained for long periods in culture remained unchanged. Altered cytokeratin expression was also accompanied by acquisition of transformation markers--loss of density dependence, progression toward calcium independence, and (benign) growth in nude mice. Observed cytokeratin aberrations may be a factor in nickel carcinogenesis, in view of the known affinity of the metal for cellular structural proteins, especially keratin, which play a role in maintenance of cell behavior.

Interaction of intermediate filaments with nuclear lamina and cell periphery.

Ultrastructural observations of the cytoskeleton suggest that the connection of the intermediate filaments (IFs) to actin microfilaments (MFs) at the plasma membrane and the nuclear lamina inside the nuclear membrane link signals received at the cell periphery to the nucleus. When these connections are viewed in three dimensions using detergent extracted cytoskeletal preparations from tissue cultures or slices made from tissue, the IFs are seen to run without interruption from the cell periphery to the nucleus and back. The IFs form side to side connections with the nuclear lamina and pore complexes. The nucleus and the centrioles are supported and held suspended in these extracted cells where all organelles and cytosol have been removed. The IFs are particularly dense in the ectoplasm where they form a sheet and provide the scaffolding which maintains the shape of the extracted cells. The IFs in the ectoplasm are attached to desmoplakin at cell-cell desmosome adhesions and to MFs where the cells are attached to the fibronectin substratum possibly through integrin linkages at adhesion plaques. This was graphically shown by immunogold labelling of IF cells treated with nickel. In this way, it was possible to visualize the loss of the cell-cell connections at desmosomes and the disruption of the IF-MF connections in the ectoplasm. The MFs after losing their connections with the IFs, redistribute to cover the entire cell periphery. The nickel treatment of primary liver cell cultures lead to the loss of several functions including formation of the bile canaliculus, the ability to secrete fluorescein diacetate and the ability to take up horseradish peroxidase (HRP) by endocytosis. These observations support the conclusion that the IF-MF connections at the cell periphery provide both structural and functional polarity of the liver cells including uptake and secretion and the formation of bile canaliculi.

A simple fixation procedure for immunofluorescent detection of different cytoskeletal components within the same cell.

In recent studies on the cytoskeletal organization of T51B rat liver cells by indirect immunofluorescence microscopy, we have been unable to achieve double-staining of microtubules and intermediate filaments within the same cell. In acetone-fixed cells, microtubules were poorly preserved, and two out of three monoclonal antibodies tested did not stain them properly. In formaldehyde-fixed cells, the monoclonal anti-cytokeratin produced an incomplete staining pattern against a diffuse background. We have now developed a fixation protocol which includes simultaneous fixation and extraction with formaldehyde and nonionic detergent in the present of microtubule stabilization buffer. Although developed for a specific purpose, it is of general application as it yields excellent preservation of all cytoskeletal components tested so far, without masking antigenic determinants. The procedure is both simple and fast and will, therefore, be valuable for efficient processing of samples from large-scale experiments, such as the screening for cytoskeletal changes during longterm treatment of cells with drugs or carcinogens.

Use of cryopreserved hepatocytes for unscheduled DNA synthesis assays.

Hepatocytes were isolated from Fischer rats by perfusion and tested for unscheduled DNA synthesis (UDS) induction or cryopreserved for long-term storage at -196 degrees C. Thawed cells could be recovered at greater than 90% viabilities, and were cultured on fibronectin-coated coverslips. The cells attached and spread, and could be used for UDS assessment. Data were compared for fresh and frozen cells from the same animal. Results obtained for net nuclear grains and dose response were similar for the fresh and frozen cells, in response to the carcinogenic compounds methyl methanesulfonate and 7,12-dimethylbenzanthracene, benzo[a]pyrene, and N-methyl-N'-nitro-N-nitrosoguanidine.

Cultured liver epithelial cells can incorporate monoclonal antibodies directed against intracellular cytokeratin structures without microinjection.

T51B rat liver cells in the exponential phase of growth were arrested in late G1 by medium calcium deprivation, exposed to mouse monoclonal anti-cytokeratin (Mr = 55,000) by addition of the antibody to the medium, induced to enter the S phase by readdition of 1.5 mM calcium, and incubated for a further 18 h. After fixing and staining with FITC-conjugated anti-mouse IgG, the cells exhibited an intact intracellular cytokeratin-staining pattern. No effect of antibody was observed on entry of cells into the S phase. These results show that rat liver-derived epithelial cells can actively take up macromolecules like cytokeratin antibodies without microinjection.

Selective rearrangement of cytokeratin filaments in cultured liver epithelial cells induced by nickel.

Rearrangements of intermediate filaments (IFs) in the liver cells involve cytokeratins, i.e., Mallory body formation. However, rearrangements of IFs observed in cell culture characteristically involve vimentin or both vimentin and cytokeratin. We report here that nickel treatment of a liver epithelial cell line (T51B) in vitro selectively induced cytokeratin filaments to accumulate in a juxtanuclear location as the cells rounded up. After withdrawal of nickel from the culture medium, vimentin filaments remained attached to the cell periphery as the cells spread out again, but the cytokeratin filaments remained aggregated in a perinuclear position without reestablishing all peripheral connections. Thus, the rearrangement of cytokeratin filaments involved partial loss of the peripheral attachments in this model.

Changes in the cytokeratin intermediate filament cytoskeleton associated with Mallory body formation in mouse and human liver.

Mouse and human extracted liver tissue were examined by indirect immunofluorescent staining and transmission electron microscopy in order to study the alteration of cytokeratin intermediate filaments associated with Mallory body formation. Frozen sections of griseofulvin-fed mouse liver and human liver of primary biliary cirrhosis and primary sclerosing cholangitis were extracted by Triton X-100 and nuclease. Indirect immunofluorescent staining was performed by using anticow hoof keratin antibody for mouse liver and anti-human epidermal keratin antibody (AE1 and AE3) for human liver. Transmission electron microscopy was also performed on extracted and critical point-dried samples. The griseofulvin-fed mouse hepatoma cells showed four different types of altered staining pattern based on the indirect immunofluorescent staining of the cytoplasm and Mallory bodies: Type I--cytoplasm(+), Mallory body(-); Type II--cytoplasm(+), Mallory body(+); Type III--cytoplasm(-), Mallory body(+), and Type IV--cytoplasm(-), Mallory body(-). Types I and III were predominant, however, some hepatoma cells which contain Mallory bodies revealed bright cytoplasmic staining (Type II). The nuclear rims were strongly stained. In human liver, AE1 stained Mallory bodies and the bile duct epithelium intensely, but did not stain normal hepatocytes. AE3 mainly stained Mallory bodies and normal hepatocytes, but also stained bile duct epithelium weakly. Indirect immunofluorescent staining for human liver showed the same staining patterns as found in mouse liver, except that Type IV was not observed. Although many hepatocytes which contained Mallory bodies did not react with either of these two antibodies (Type III), some of the hepatocytes were stained, not only with AE3, but also with AE1 (Type II).(ABSTRACT TRUNCATED AT 250 WORDS)

Proliferation of human carcinoma cells in calcium-deficient culture medium may depend on autocrine growth factor(s).

The ability to proliferate in media with low calcium concentrations (less than 0.1 mM) at clonal seeding densities is a characteristic of malignant cells. Cells of the carcinoma line C-4I are an exception, and are unable to proliferate in medium with 0.02 mM calcium (LCM) when seeded at low densities. Conditioned medium, derived from a subline of C-4I cells that were adapted to grow in LCM, contained an acid-stable factor of greater than 8 kilodaltons molecular weight that mediated proliferation and colony formation of unadapted C-4I cells in LCM in a concentration-dependent manner. The effect was not related to enhanced cell attachment or spreading, and was maximal when the unadapted C-4I cells were seeded in aggregates of 2-20 cells rather than singly. Thus, calcium independence, a component of the neoplastic phenotype, may be mediated by autocrine tumor-cell-derived factor(s) and may involve long- and short-range cell interactions.