Cooperation of bcl-2 and myc in the neoplastic transformation of normal rat liver epithelial cells is related to the down-regulation of gap junction-mediated intercellular communication
Nestor D. DeoCampo,
Melinda R. Wilson and
James E. Trosko1
National Food Safety and Toxicology Center, Department of Pediatrics and Human Development and Genetics Program, East Lansing, MI 48824, USA
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Abstract
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The objectives of this study were to isolate several rat liver epithelial cell clones containing the human bcl-2 and myc/bcl-2 genes in order to study their potential cooperative effect on neoplastic transformation and gap junction-mediated intercellular communication (GJIC) and to test the hypothesis that the loss of GJIC leads to tumorigenesis. Using anchorage-independent growth as a surrogate marker for neoplastic transformation, we transfected both normal rat liver epithelial cells, WB-F344, and a WB-F344 cell line overexpressing v-myc with human bcl-2 cDNA. Those cell lines that only expressed v-myc or human bcl-2 were unable to form colonies in soft agar. However, those cell lines that overexpressed both v-myc and human bcl-2 showed varying ability to form colonies in soft agar, which did not correlate with their human bcl-2 expression level. In order to test if there was a correlation between cell line growth in soft agar and the ability to communicate through gap junctions, we performed scrape load dye transfer and fluorescence recovery after photobleaching assays. Our results show that v-myc and human bcl-2 can cooperate in the transformation of normal cells, but the degree to which the cells are transformed is dependent on the cells' ability to communicate through gap junctions.
Abbreviations: AIG, anchorage-independent growth; FBS, fetal bovine serum; FRAP, fluorescence recovery after photobleaching; GJIC, gap junctionmediated intercellular communication; PBS, phosphate-buffered saline; SL/DT, scrape load dye transfer; TPA, 12-tetradecanoylphorbol-13-acetate.
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Introduction
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If one accepts the stem cell theory of carcinogenesis (1), then the blockage of terminal differentiation (2) might be involved in the `initiation' phase of carcinogenesis, while clonal expansion (mitogenesis), plus inhibition of apoptosis, might contribute to the `promotion' phase (3). Within this conceptual framework, overexpression of various oncogenes, acting together with other genes, might be involved in either the initiation or promotion phases.
The myc oncogene, a transcription factor, has been associated with the cellular functions of proliferation, differentiation and apoptosis (48). Disruption of any of these basic cellular functions could contribute to the multi-step, multi-mechanism process of carcinogenesis. Overexpression of the myc oncogene has been associated with many tumors (9,10).
The ras oncogene, being a member of the G protein family, has been shown to be involved in one of the many signal transduction pathways affecting mitogenesis, differentiation and apoptosis. ras, as with myc, has been shown to be activated in many types of tumors (11,12).
Cooperation of oncogenes is one of the first important molecular concepts of carcinogenesis. One of the first interactions to be reported was cooperation between the myc and ras oncogenes (13). In addition, it was demonstrated that phorbol esters, such as 12-tetradecanoylphorbol-13-acetate (TPA), seemed to act in a manner similar to the ras oncogene, in that it could cooperate with the myc oncogene to induce a neoplastic phenotype (14). TPA, by activating protein kinase C, triggers signal transduction pathways and can act as a modulator of mitogenesis, differentiation and apoptosis (1517).
The ras and myc oncogenes have also been implicated in apoptosis (1820). While myc seems to confer a susceptibility to apoptosis (6,19,21), ras appears to reduce cellular responses to apoptosis (18,20). Therefore, in cells in which both myc and ras are activated, signal transduction cross-talk interacts to block terminal differentiation, triggering cells to proliferate and become resistant to apoptosis. In effect, these unregulated disruptions of interacting signals bring about the appearance of the tumor.
The bcl-2 proto-oncogene has been one of the major genes implicated in the apoptotic process (22). Early experiments in transgenic animals overexpressing bcl-2 under immunoglobulin promoter control showed an increased frequency of follicular hyperplasia and B cell survival, thus suggesting a role of bcl-2 as an anti-apoptotic gene (23). Since this work many have demonstrated that bcl-2 is not only involved in tumors of lymphoid origin but also in many tumors of epithelial origin (2431). bcl-2 has also been demonstrated to synergistically interact with TPA to transform cells (32). Thus, by blocking apoptosis, bcl-2 seems to be acting as a surrogate `ras' gene. However, bcl-2 itself is unable to induce proliferation or neoplastic transformation, which is often associated with the overexpression of other oncogenes, like myc and ras (3335).
Many investigators have studied the co-expression of myc and bcl-2 in a variety of cell types and transgenic models (3639). These studies were able to show that bcl-2 and myc can cooperate and increase or enhance tumor incidence and formation. However, these experiments studying myc and bcl-2 cooperation have focused on cells of lymphoid origin, fibroblasts and a whey acidic protein promoter-controlled bcl-2 vector in the mouse lactating mammary gland, but not in other epithelial systems. The experiments we describe in this study were designed to test whether the bcl-2 oncogene in cooperation with v-myc could function to neoplastically transform the WB-F344 cell line, a normal rat liver epithelial cell line. We have previously reported the establishment of a WB-F344 cell line stably expressing the v-myc oncogene (40). This cell line demonstrates an increase in proliferation and cell saturation density but it did not form colonies in soft agar or tumors in nude mice. This cell line was used as the target for transformation by bcl-2.
Our laboratory and others have shown that while ras and TPA affect signal transduction, they also affect gap junction-mediated intercellular communication (GJIC) (4144). Gap junctions are channels that directly link the interiors of neighboring cells allowing for the free diffusion of small molecular weight molecules (45). Most tumors demonstrate a reduction in GJIC activity, either between themselves (homologous GJIC) or with other cell types (heterologous GJIC) (3). Presumably, down-regulation of GJIC activity would lead to the removal of growth inhibitory signaling, thereby providing a selective advantage. TPA was the first agent shown to reduce GJIC activity (43,44). Rat liver epithelial cells stably transfected with Ha-ras demonstrate a dose-dependent reduction in GJIC with increasing levels of ras T24 protein (41,42,46). Recently our laboratory reported that Ha-ras and v-myc could cooperate to down-regulate GJIC and that this down-regulation correlated with cell malignancy (40). In this same manner we have investigated the effect that bcl-2 alone and in cooperation with v-myc have on GJIC and looked for any correlation with neoplastic transformation.
In this report we demonstrate that bcl-2 can cooperate with v-myc to induce neoplastic transformation of a normal rat liver epithelial cell line. We also demonstrate that the extent of neoplastic transformation is dependent on the cells' ability to communicate through gap junctions.
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Materials and methods
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Cell culture
WB-F344 rat liver epithelial cells, WB.myc and all subsequent bcl-2 transfectants were cultured in D medium (Formula 78-5470EF; Gibco BRL Life Technologies, Gaithersburg, MD), supplemented with 7% fetal bovine serum (FBS) (Gibco BRL Life Technologies). All cells were incubated at 37°C in a humidified atmosphere containing 5% CO2.
Transfection of bcl-2
The human bcl-2 cDNA cloned into the pSFFV vector (kindly provided by Gabrial Nunez, University of Michigan, Ann Arbor, MI) was transfected into cell line WB-F344 and co-transfected with pTK-Hyg (Clontech Laboratories, Palo Alto, CA) into cell line WB.myc using Lipofectin (Roche Molecular Biochemicals, Indianapolis, IN). Briefly, 40 µg of Lipofectin was added to 1.5 ml of D medium (serum-free) and 20 µg of DNA was added to a separate 1.5 ml of D medium. The two samples were mixed and incubated at room temperature for 15 min. This mixture was then added to subconfluent cultures grown in 100 mm dishes that had been rinsed twice with serum-free D medium. The plates were then incubated overnight at 37°C in a humidified chamber. The following day D medium containing 7% FBS was added directly to the plates and incubated for an additional 24 h. Transformed cells were then split and subsequently plated with appropriate drug selection. Individual clones were then isolated using cloning rings and subsequently recloned. Due to the enhanced apoptotic properties of the WB.myc cells, co-transfection with pTK-Hyg was performed as described above with the following modifications: first, the culture was allowed to reach confluency; second, 20 mg of DNA in a ratio of 1:20 pTK-Hyg:pSFFV.bcl-2 was used to ensure that selection using hyromycin B would yield bcl-2-positive cells.
Protein extraction
Proteins were extracted from confluent cell cultures grown in 25 cm2 flasks using 20% SDS containing 2 mM phenylmethylsulfonyl fluoride, 1 µM aprotinin, 1 µM leupeptin (Roche Molecular Biochemicals), 1 µM antipain, 5 mM sodium fluoride (Fluka, Milwukee, WI), 0.1 mM sodium orthovanadate (Aldrich, Milwaukee, WI) and subsequently sonicated three times at 5 s intervals, aliquoted and stored at 20°C. The protein concentration was determined by diluting the extracts 1:5 and assayed using the Bio-Rad DC protein assay (Bio-Rad Laboratories, Hercules, CA).
Western blot analysis
Proteins were loaded in equal amounts (1015 µg) into each well of a 10% SDSpolyacrylamide gel, in accordance with Laemmli (47). The gels were electrophoresed at 200 mV for ~45 min, removed and equilibrated in transfer buffer and transferred to Immobilon-P PVDF membrane (Millipore Corp., Bedford, MA). Human bcl-2 was detected with hamster anti-human bcl-2 monoclonal antibody (Pharmingen, San Diego, CA), endogenous rat bcl-2 was detected with a polyclonal anti-bcl-2 antibody (Santa Cruz Biotechnology, Santa Cruz, CA) and v-myc was detected with anti-v-myc polyclonal antibody (Caltag Laboratories, Burlingame, CA), using the supersignal ultra substrate (Pierce Chemical Co., Rockford, IL) and appropriate peroxidase-labeled secondary antibody.
Anchorage-independent growth (AIG)
To assess AIG cells were grown in soft agarose as previously described (48). Briefly, an initial hard agarose layer (0.5% agarose 6013; Sigma) made in D medium containing 7% FBS was plated in a 60 mm dish. Individual cell lines were then trypsinized, counted and diluted to a final concentration of 100 total cells in D medium containing 0.33% agarose and 7% FBS and were subsequently plated on top of the 0.5% agarose layer. Additional medium was added 3 days later and changed every 3 days for 28 days. Colonies were stained using 1 mg/ml 2-(p-iodophenyl)-3-(nitrophenyl)-5-phenyltetrazolium chloride (Sigma) in 0.9% NaCl and counted.
Cellcell communication assays
Two methods developed in our laboratory were used to assess GJIC: (i) scrape-loading dye transfer (SL/DT) (49); (ii) fluorescence recovery after photobleaching (FRAP) (50). The SL/DT assay utilized confluent cultures grown in 35 mm dishes. The cultures were then rinsed three times with phosphate-buffered saline (PBS) containing Ca2+ and Mg2+ (Ca2+, Mg2+-PBS). An aliquot of 1.5 ml of PBS containing 0.05% Lucifer yellow CH (Molecular Probes, Eugene, OR) was added and several scrapes (cuts) were made on the monolayer using a surgical scalpel. The cultures were incubated for 3 min at room temperature in the dye solution and then rinsed three times with Ca2+, Mg2+-PBS (to remove any background fluorescence). The cultures were then fixed with 1 ml of 4% formalin and visualized using an Ultima laser cytometer (Meridian Instruments, Lansing, MI).
The SL/DT assay results were confirmed using the FRAP assay. Briefly, cultures grown in 35 mm dishes were rinsed three times with Ca2+, Mg2+-PBS and then incubated with Ca2+, Mg2+-PBS containing 7 mg/ml 5,6-carboxyfluorescein diacetate (Molecular Probes) at 37°C in a humidified incubator for 15 min. The cells were then rinsed several times with Ca2+, Mg2+-PBS and analyzed using an Ultima laser cytometer. Cells were randomly selected under a microscope (four cells were selected per field plus one unbleached control, five fields per scan) and photobleached with an argon laser beam. The transfer of fluorescence was then monitored at 4 min intervals for a total of 12 min. The intensity of recovered fluorescence of the individually bleached cells was then quantitated and rates of dye transfer can be measured as the percentage of an unbleached control cell (one per field).
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Results
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Characterization of isolated clones
To examine the interaction between the v-myc and bcl-2 oncogenes in neoplastic transformation and GJIC in rat liver epithelial cells, we generated clonal cell lines, utilizing established normal WB-F344 and WB.v-myc cell lines, overexpressing human bcl-2 alone and co-expressing human bcl-2 and v-myc. The morphological appearances under phase contrast microscopy of the different cell lines are shown in Figure 1
. The WB-F344, WB.v-myc and WB.bcl-2 cells grew in uniform monolayers of polygonal cells and exhibited contact inhibition of growth. The Wb.myc/bcl-2 cell lines demonstrated different morphologies, such as seen between the different clones (uniform polygonal, spindle-shaped and multinucleated).

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Fig. 1. Phase contrast photomicrographs of WB-F344, control and transfected cells. (A) WB-F344; (B) vector control WB.neo; (C) WB.v-myc; (DF) WB.myc/bcl-2 clones WB.mb3, WB.mb19 and WB.mb39, respectively.
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Western blot analysis of bcl-2
The expression of human bcl-2 was assessed using western blot analysis with an anti-bcl-2 monoclonal antibody (Pharmingen, San Diego, CA) specific for human bcl-2 protein. Figure 2a
shows high expression of a 26 kDa band corresponding to transfected human bcl-2 protein, while the control cell lines show no expression. We also verified the presence of v-myc using western blot analysis with an anti-v-myc polyclonal antibody. Figure 2b
shows the presence of a 110 kDa band that corresponds to the gagpolmyc region of v-myc. Its expression is consistent between the controls and transfected clones, suggesting that overexpression of bcl-2 has not effected the expression of v-myc from the parental WB.v-myc cell line.

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Fig. 2. Analysis of expression of human bcl-2 and v-myc proteins. (a) Detection of a 26 kDa protein corresponding to human bcl-2 from extracts of normal WB-F344 and transfected cell lines. (b) Detection of a 110 kDa protein corresponding to v-myc from extracts of normal WB-F344 and transfected cell lines.
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Anchorage-independent growth
AIG, more commonly referred to as growth in soft agar, has often been used as a marker for neoplastic transformation and to identify tumor cells (5154). While this assay does not confirm that cells are indeed neoplastically transformed, it does demonstrate that those cells that are able to grow contain a characteristic shared with all tumors. To assess if the bcl-2 gene was conferring neoplastic characteristics to the normal rat liver epithelial cells, we tested their ability to grow in soft agar. Using a previously described (40) v-myc/Ha-ras tumorigenic WB-F344 cell line (designated MR-42) as a positive control (100% colony forming efficiency), we plated replicate plates with 100 cells/plate in soft agar. Figure 3
demonstrates that the control cell lines, as well as the bcl-2 only clones, were unable to form colonies in soft agar. However, the v-myc/bcl-2 clones were able to form colonies in soft agar and they also demonstrated clonal differences in their colony forming efficiency ranging from 7 to 90%. When we compared the colony forming efficiencies of the v-myc/bcl-2 clones with their respective bcl-2 expression level we found no correlation.

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Fig. 3. Assessment of AIG using growth in soft agar. The data represent the efficiency of colony formation represented as the number of colonies formed per 102 cells plated.
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Gap junction-mediated intercellular communication
In order to explain the differences in growth in soft agar demonstrated by the myc/bcl-2 cell lines we assessed the ability of the individual cell lines to communicate through gap junctions. Figure 4a
demonstrates typical results obtained with the SL/DT technique. The WB-F344 cell line is used as the control for communication, with the dye traveling ~68 cell layers. When WB-F344 cells were compared with the WB.mb19 clone containing v-myc and bcl-2, there was an ~50% decrease in the ability of the cell to transfer dye (34 cell layers). These results were confirmed using FRAP. Figure 4b
shows the GJIC activity (determined by both SL/DT and FRAP) of the various cell lines. This assay allows monitoring of individual cells in the population in assessing activity for the whole population. When compared with colony forming efficiency, the two communication assays appear to be inversely correlated. Those cell lines that showed no significant changes in GJIC demonstrated a marked decrease in colony forming ability in soft agar. However, those cell lines that demonstrated a marked reduction in cellcell communication demonstrated an enhanced or increased ability to form colonies in soft agar.

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Fig. 4. Analysis of GJIC expression using the SL/DT and FRAP techniques. (a) A representation of the SL/DT assay. Note that the normal WB-F344 cell line (A) transfers the dye to ~68 cells, while the myc/bcl-2 clone, MB.19 (B), is only able to transfer the dye to 23 cells on either side of the scrape line. (b) Assessment of GJIC using the FRAP technique. The data represent the percentage fluorescence recovery after a single cell was photobleached amongst a population of cells.
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Discussion
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In this study our objective was to determine if myc and bcl-2 could act cooperatively in the induction of tumorigenicity by inhibition of GJIC. To test this idea, the myc and bcl-2 genes were co-expressed in WB-F344 cells, a normal rat liver epithelial cell line. The parental WB-F344 cells, myc-transformed and bcl-2-transformed cells grew in uniform monolayers of polygonal cells but did not grow in soft agar. The myc/bcl-2-transformed cells demonstrated various phenotypes and the cells were able to form colonies in soft agar. When these cell lines were compared with a cell line with 100% colony forming efficiency they demonstrated varying abilities to form colonies in soft agar. Colony forming ability did not correlate with bcl-2 expression levels of the respective cell lines. We further characterized these myc/bcl-2 cell lines by examining GJIC. We found that there was an inverse correlation between the ability of cells to communicate and their ability to form colonies in soft agar. Those cells lines that demonstrated reduced GJIC showed an increased ability to form colonies in soft agar.
The v-myc/bcl-2-transformed cells did indeed grow in soft agar, while the controls (WB-F344, WB.v-myc and WB.bcl-2) did not. The degree to which the cell lines were able to form colonies varied greatly. If overexpression of bcl-2 was the main factor in neoplastic transformation of the WB.v-myc cell line, we would expect to see a doseresponse correlation between bcl-2 and cell line colony forming efficiency. We did not see any correlation between bcl-2 expression and colony formation, which suggests that another mechanism was responsible for the differences we were observing.
GJIC has been shown, by our group and others, to play an important role in tumor promotion and progression. Presumably, as cells lose GJIC they are effectively removed from intercellular signals that can regulate proliferation, differentiation and apoptosis. In this manner an `initiated' cell could escape growth regulation.
The role of GJIC in the regulation of cell behavior (e.g. growth control, differentiation, apoptosis and adaptive responses of terminally differentiated cells), while still not known in detail, seems to involve the transfer of ions and small molecular weight regulatory molecules through the gap junction channels to act as either a `sink' or `source' (55). GJIC is known to synchronize electronic or metabolic responses between cells (56). Coupling of normal homologous or heterologous cells could alter the behavior of the cells. When it was shown that tumor promoters, such as phorbol esters (44), could reversibly inhibit GJIC, it was hypothesized that an `initiated' cell (a stem-like cell which has been prevented from terminally differentiating or which has been `immortalized') (57) would be growth suppressed by being coupled by gap junctions to surrounding normal cells. The implication is that while the initiated cell is genotypically altered by the `initiator', it could still be `partially blocked from terminally differentiating' (2) and be `contact inhibited' by regulatory signal equilibration from surrounding normal cells. Growth factors, hormones and tumor-promoting chemicals, by triggering signal transduction in the `initiated' and surrounding normal cells, would (i) cause the down-regulation of GJIC (leading to inhibition of contact inhibition) and (ii) induce gene expression related to cell proliferation/differentiation (57). While normal cells would proliferate and differentiate, the `initiated' cell would only proliferate and not die by terminal differentiation or apoptosis, thereby increasing the number of initiated cells. As long as the external exogenous chemical (tumor promoter) is applied, clonal expansion of the initiated cell can occur. If, and when, other genetic changes within the initiated cell occur that can stably down-regulate GJIC, external dependence on the tumor promoter declines and the cell becomes independent of the suppressing effect of surrounding normal cells.
When we examined the myc/bcl-2 cell lines for GJIC activity, we found a striking correlation between GJIC and colony formation. Those cell lines that possessed functional GJIC formed very few colonies in soft agar. However, those cell lines that demonstrated a reduction in GJIC activity showed dramatic increases in their ability to form colonies. This suggests an active role for GJIC in contributing to neoplastic transformation. The use of myc and bcl-2 was not used to imply that initiated or neoplastically transformed cells represent the initiation/promotion/progression model of carcinogenesis, but rather to show the potential importance of GJIC during that process. The myc-transformed cell still has functional GJIC and could be suppressed by surrounding normal cells. In these experiments, the WB.v-myc/bcl-2 cells were only measured against themselves and were found not to have fully functional GJIC. Future studies will need to be conducted to test if co-cultures of these cells with normal rat liver epithelial cells behave differently and if these cells, when placed back into the rat liver, are either suppressed or whether they can grow into tumors.
These new clones demonstrate that bcl-2 can cooperate with myc to neoplastically transform rat liver epithelial cells. However, the degree to which the cells are neoplastically transformed is dependent on their ability to communicate through gap junctions.
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Notes
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1 To whom correspondences should be addressed Email: trosko{at}pilot.msu.edu 
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Acknowledgments
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This reasearch was supported by a National Cancer Institute grant, CA21104, to J.E.T.
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Received December 1, 1999;
revised March 31, 2000;
accepted April 14, 2000.