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Department of Pathology, Medical College of Ohio, Toledo, Ohio 43699
ABSTRACT
Gap junction-mediated intercellular communication in untreated andphenobarbital-treated C57BL/6 x C3H FI mouse hepatocytes was eval
uated by microinjection of fluorescent Lucifer Yellow CH dye. Intercellular communication (dye coupling) was detected in untreated hepatocytesafter 0.5 h in culture, reached a maximum level in 24- and 48-h-old
Cell growth may be regulated by the cell-to-cell exchange ofsmall molecules and ions through gap junctions (i.e., intercellular communication) (1). The loss of gap junctions or a decrease in cell-to-cell communication may predispose cells toenhanced growth (1). Gap junctions are decreased or absent inregenerating liver (2, 3), and intercellular communication canbe decreased by growth factors (4) and tumor promoters (5-8).Several types of neoplastic cells have also been shown to havea reduced or a complete loss of intercellular communication(D-
The loss or inhibition of intercellular communication is alsothought to be a mechanism involved in tumor promotion (8).Tumor promoters often have mitogenic activity in their targettissue, thus permitting the expansion of the initiated cell population (9, 10). This stimulated cell growth may increase thelikelihood of additional genetic events required for completeneoplastic transformation to occur in an initiated cell (10). Themechanism by which tumor promoters exert their mitogeniceffect remains unclear. However, it is now known that nearlyall tumor promoters inhibit intercellular communication bothi/i vivo (11-13) and in vitro (8). This inhibitory effect appearsto be characteristic of tumor promoters, not genotoxic carcinogens or cytotoxic agents (14). Therefore, one possible mechanism by which tumor promoters enhance initiated cell growthmay be through their ability to inhibit intercellular communication.
Work in our laboratory has focused on defining the mechanisms by which tumor promoters inhibit hepatocyte intercellular communication (7, 14-16). We have previously assessedhepatocyte intercellular communication by gap-junctional passage of [3H]uridine nucleotides from prelabeled "donor" hepatocytes to nonlabeled "recipient" hepatocytes by autoradiogra-
Received 9/22/87; revised 1/12/88; accepted 2/8/88.The costs of publication of this article were defrayed in part by the payment
of page charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1To whom requests for reprints should be addressed.
phy (7). With this method, we have shown that several livertumor promoters inhibited hepatocyte intercellular communication, whereas genotoxic carcinogens and hepatocytotoxinsdid not (7, 14). In addition, the inhibitory effect of liver tumorpromoters on rodent hepatocyte intercellular communicationcorrelated with in vivo strain and species sensitivities to thehepatocarcinogenic activity of the promoters (16).
Unfortunately, the [3H]uridine method to detect hepatocyte
intercellular communication is limited in that the kinetics ofthe inhibitory effect of a promoter cannot easily be studied.This is because there is a delay between gap-junctional passageof [3H]uridine nucleotides from donor to recipient cells and
Animals. Male B6C3F,/CNIBR mice, 4-6 months old, were purchased from Charles River Laboratories, Inc. (Wilmington, MA), andused exclusively in this study. Mice were housed in polycarbonate cagesand fed Purina Lab Chow Blox (Ralston Purina Co., St. Louis, MO)and water ad libitum.
Hepatocyte Isolation and Culture. Hepatocytes were isolated by two-stage collagenase perfusion through the portal vein (20) and plated outat 1 x 10* viable cells per 60-mm plastic dish in 3 ml medium. Initial
viability of the isolated cells, determined by trypan blue dye exclusion,was always above 90%. The cells were cultured in Leibovitz's L-15
medium supplemented with glucose (1 mg/ml), dexamethasone (1 MM),gentamicin sulfate (50 Mg/ml), and fetal bovine serum (10%, v/v;Hyclone Laboratories, Logan, UT) at 37'C (21). The cultures were
refed with 3 ml medium/dish after a 2-h attachment period.Detection of Hepatocyte Intercellular Communication by Lucifer Yel
low Dye Injection. Microelectrodes were pulled from 1.5-mm-diametersingle barrel glass Kwik-Fil capillaries (World Precision Instruments,
Inc., New Haven, CT) using a Narishige model PE-2 vertical micro-electrode puller (Narishige Scientific Instrument Laboratory, Tokyo,Japan). Microelectrode tip diameters measured approximately 1 Aim.Microelectrode tips were backfilled with 5% (w/v) Lucifer Yellow CHin 0.1 M LiCl, and microelectrode barrels were backfilled with 0.1 MLiCl. Hepatocyte cultures (2-120 h old) were observed under a NikonOptiphot UFX-II epifluorescence microscope (Nikon, Inc., GardenCity, NJ) at xlOO at room temperature. "Donor" hepatocytes were
impaled with the microelectrode under phase contrast microscopy anddye was iontophoretically ejected using continuous 3 nA current for 1min. Five min after cessation of dye injection, hepatocytes in directcontact with donor hepatocytes (i.e., recipient hepatocytes) were evaluated under epifluorescence for evidence of dye accumulation (dyecoupling). All recipients in contact with injected donors were evaluatedfor evidence of dye coupling. The percentage of dye-coupled recipientswas determined for each treatment and sampling time. Differences inthe number of dye-coupled and non-dye-coupled recipients betweentreatment groups were statistically evaluated by 2 x 2 x2 analysis (22).Dye-coupled cells were photographed by epifluorescence-phase contrastmicroscopy with Tri-Pan film (ASA 400) (Eastman Kodak Co., Rochester, NY).
Occurrence of Dye Coupling in Nontreated Mouse Hepatocytes duringthe First 120 h of Culture. To determine the extent of intercellularcommunication (dye coupling) in nontreated hepatocytes over a prolonged culture period (120 h), nontreated cultures were sampled at 0.5,1, 2, 4, 6, 8, 12, 24, 48, 72, 96, and 120 h after initial plating andevaluated for dye coupling. The cultures were refed just prior to the2-, 24-, 48-, 72-, 96-, and 120-h sampling times.
Fig. 1 depicts phase contrast-fluorescence photomicrographsof dye-coupled mouse hepatocytes after 2 h (Fig. la) or 24 h
2The abbreviation used is: DMSO, dimethyl sulfoxide.
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Fig. 1. Phase contrast-fluorescence photomicrographs of fluorescent dye coupling between donor mouse hepatocytes (D) microinjected with Lucifer YellowCH and adjacent recipient hepatocytes (R) in (a) 2 li old cultures and (/>)24-h-old cultures, x 250.
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CULTURE DURATION (h)
Fig. 2. Dye coupling levels in untreated mouse hepatocytes in primary culture.
(Fig. Ib) in culture. Intercellular communication (dye coupling)in nontreated mouse hepatocyte cultures over the first 0.5-120h of culture is shown in Fig. 2. Dye coupling increased rapidlyduring the first hours of culture to a value of 56.4% dye-coupled
recipient cells at 2 h. This level of dye coupling displayed amore gradual increase during the next 22 h in culture to amaximal level at the 24-h sampling time of 85.2%. Dye couplingremained stable in 48-h cultures but then decreased markedlyover the next 72 h in culture to 39.2% dye-coupled recipients.Refeeding of cultures with fresh medium had no effect on dyecoupling when compared to nonrefed cultures (data not shown).This suggests that factors in the medium did not contribute tothe decline in dye coupling over the 120-h culture period.
Intercellular communication through gap junctions may beone mechanism by which cell growth is regulated (1). Tumorpromoters might enhance tumor formation in vivo by inhibitingintercellular communication (8). In the present study, we evaluated the kinetics of the development of intercellular communication in untreated and phenobarbital-treated culturedB6C3Fi mouse hepatocytes. Previously we have examined intercellular communication between rodent hepatocytes by following the gap junctional passage of [3H]uridine nucleotidesfrom prelabeled donor cells to nonlabeled recipient cells (7, 14-16). The [3H]uridine method cannot be used to evaluate theshort-term effects of tumor promoters on hepatocyte intercellular communication or the kinetics of the inhibitory effect ofthe promoter. Therefore, in the present study, we have utilizedfluorescent dye coupling techniques to study the kinetics of thedevelopment of intercellular communication in nontreated andphenobarbital-treated hepatocyte cultures.
In nontreated, freshly plated mouse hepatocyte cultures, dye
coupling increased during the first 48 h in culture and thendecreased over the next 3 days in culture (Fig. 2). The nonlineardevelopment of intercellular communication suggests that thereis a rate-limiting component or process of hepatocyte gapjunction formation. This may be the availability of gap junctionsubunits [connexons (2)] in the cultured cells, the number ofhepatocyte-to-hepatocyte contacts, and/or de novo synthesis ofconnexon protein subunits. The peak dye coupling level seen inthe nontreated cultures (85.2%) was similar to the level ofintercellular communication detected by the [3H]uridine
method (83.0%) (7). However, intercellular communication wasnot detected with the [3H]uridine assay until 4 h in culture (7),
whereas dye coupling was seen after 0.5 h in culture in thepresent study. This suggests that the sensitivities of the twomethods for detecting peak levels of hepatocyte intercellularcommunication are similar but that the dye injection method isbetter suited to analyzing the rates of development of intercellular communication.
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