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Cell Growth & Differentiation Vol. 10, 287-294, May 1999
© 1999 American Association for Cancer Research

Nuclear Factor {kappa}B Cooperates with c-Myc in Promoting Murine Hepatocyte Survival in a Manner Independentof p53 Tumor Suppressor Function1

Robert E. Bellas and Gail E. Sonenshein2

Department of Biochemistry, Boston University School of Medicine, Boston, Massachusetts 02118


    Abstract
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
The nuclear factor-{kappa}B (NF-{kappa}B)/Rel family of transcription factors has been implicated in promoting hepatocyte survival during development and liver regeneration following partial hepatectomy. Inhibition of NF-{kappa}B/Rel activity by microinjection of the specific inhibitor I{kappa}B-{alpha} induces apoptosis in a nontransformed normal murine hepatocyte (NMH) cell line. Here, we demonstrate that apoptosis resulting from such inhibition requires down-regulation of the c-Myc proto-oncoprotein and occurs independently of p53 tumor suppressor function. NMH cells plated at low density displayed low sensitivity to I{kappa}B-{alpha}-induced apoptosis and high levels of c-Myc protein expression. Comicroinjection of I{kappa}B-{alpha} with the c-Myc antagonist Mad1-glutathione S-transferase fusion protein greatly enhanced cell death. In addition, transient cotransfection of low-density NMH and AML12 hepatocytes with vectors expressing I{kappa}B-{alpha} and antisense c-myc transcripts promoted cell death. Conversely, ectopic c-myc expression significantly decreased the extent of cell death in NMH cells plated at saturating density, which were characterized by very low levels of c-Myc and high susceptibility to NF-{kappa}B inhibition-induced cell death. Finally, I{kappa}B-{alpha}-induced apoptosis was unaffected in NMH cells expressing a dominant negative p53 protein. Thus, NF-{kappa}B cooperates with c-Myc in promoting murine hepatocyte survival in a manner independent of p53 tumor suppressor activity.


    Introduction
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
NF-{kappa}B3 is a family of dimeric transcription factors with subunits that contain an NH2-terminal stretch of {approx}300 amino acids that share homology with the v-Rel oncoprotein (reviewed in Refs. 1, 2, 3 ). Classical NF-{kappa}B is composed of a p50 and a p65 (RelA) subunit. NF-{kappa}B is ubiquitously expressed in non-B cells in an inactive form sequestered in the cytoplasm with specific inhibitory proteins termed I{kappa}Bs, the paradigm being I{kappa}B-{alpha} (reviewed in Refs. 4 and 5 ). NF-{kappa}B/Rel factors have been implicated in inflammatory responses and synthesis of adhesion molecules (2 , 3) and in control of genes regulating proliferation (6, 7, 8, 9) . More recently, NF-{kappa}B/Rel factors have been found to promote cell survival. In addition, we demonstrated an antiapoptotic function for constitutively expressed NF-{kappa}B/Rel factors in numerous cell types, including B lymphocytes (10 , 11) , hepatocytes (12 , 13) , and breast cancer epithelial cells (14) . Several groups also found that induction of NF-{kappa}B/Rel upon treatment with tumor necrosis factor, radiation, and chemotherapeutic agents can protect cells from apoptosis (15, 16, 17, 18) .

A first indication that NF-{kappa}B/Rel factors might play a significant role in liver biology came from analysis of their expression following PH, when liver regenerates after surgical removal of two-thirds of its mass. Following PH, highly differentiated, largely quiescent liver cells undergo a synchronous proliferative response, which continues until the original liver mass is restored. PH is accompanied by rapid induction of classical NF-{kappa}B p50/p65 heterodimers and p50 homodimers (19 , 20) . This induction appears to be necessary for regeneration in that interference with NF-{kappa}B activation following PH by infection of animals with an adenoviral vector expressing I{kappa}B-{alpha} inhibits regeneration (21) . In addition, NF-{kappa}B is required for normal liver development. Mice null for the relA gene (encoding the p65 subunit) undergo embryonic death at {approx}15–16 days of gestation accompanied by extensive hepatocyte apoptosis (22) , although these experiments did not indicate whether apoptosis was a direct result and not due to indirect endocrine or paracrine effects such as altered cytokine production. We recently demonstrated that apoptosis ensues upon inhibition of NF-{kappa}B in cultured murine hepatocytes either by microinjection of I{kappa}B-{alpha} (12) or upon receptor-mediated down-modulation following TGF-ß1 treatment (13) . These results argue that liver apoptosis in the relA null mice is a direct result of lack of p65 in protein in hepatocytes and suggests that these cells serve as an appropriate model for the study of apoptosis of hepatocytes. Thus, NF-{kappa}B factors play a significant role in the liver during the normal development. Furthermore, the above studies suggest involvement of these factors in pathophysiological liver regeneration, such as those that might occur in response to injury resulting from toxins or viral infection agents.

Whether NF-{kappa}B/Rel antiapoptotic functions are mediated independent of other known apoptosis genes is unknown. Of particular significance to liver biology is the tumor suppressor p53. The p53 protein, originally identified as a cellular nuclear phosphoprotein bound to the large transforming antigen of the SV40 DNA virus (23 , 24) , plays a role in control of cell cycle progression through G1 into S phase, DNA repair, differentiation, tumor formation, and apoptosis (25, 26, 27) . Induction of p53 is often associated with activation of cell death, and ectopic expression of p53 can induce apoptosis (28) . Normal p53 function is required for apoptosis induced via several agents, including {gamma} irradiation and cytotoxic drugs such as doxorubicin (29, 30, 31) . The mechanisms by which p53 exerts these effects are not clear but seem to depend on the ability of p53 protein to act as a transcription factor. A high incidence of p53 mutations has been noted in several human tumors, including hepatocellular carcinoma (32, 33, 34) .

Another gene found to play a role in control of apoptosis is the c-myc oncogene. Overexpression of c-Myc has been found to promote apoptosis upon deprivation of growth factors (35 , 36) . In addition, a drop in c-Myc expression has been found to induce death of many cell types, including T, pre-B, B, melanoma, esophageal, and breast cancer cell lines (reviewed in Ref. 37 ). For example, we demonstrated that, in WEHI 231 B cells, in which c-myc gene transcription is potently regulated by NF-{kappa}B (6) , that inhibition of c-Myc function, either directly or through modulation of Rel, led to apoptosis. Furthermore, ectopic c-Myc expression ameliorated apoptosis of WEHI 231 cells induced by a number of agents that down-regulate NF-{kappa}B (11 , 38) . On the basis of these observations, we have evaluated the involvement of c-Myc and p53 in NF-{kappa}B survival functions in murine hepatocytes. We find that either endogenous or ectopic c-Myc expression is sufficient to protect hepatocytes from cell death induced by NF-{kappa}B inhibition, although, surprisingly, we observe no evidence that NF-{kappa}B regulates c-Myc expression in these cells. Moreover, the survival pathways evinced by NF-{kappa}B appear to be p53 independent. These findings suggest that targeting this pathway may bypass p53-mediated protection of hepatocarcinomas from chemotherapeutic regimens or other apoptotic stimuli.


    Results
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
The Ability of I{kappa}B-{alpha} to Induce Apoptosis Is Density Dependent.
Previously, we demonstrated that microinjection of NMH or AML12 cells with the specific NF-{kappa}B/Rel inhibitor I{kappa}B-{alpha} induces apoptosis with characteristic nuclear condensation (12) . During the course of these studies, we noticed that the susceptibility of these cells to apoptosis appeared to vary with the density of the cells. Specifically, cells in more densely packed areas of the culture dish appeared to undergo cell death with higher frequency than those in sparser regions of the plate. To more carefully document this finding, we plated cells at three different densities: 75, 150, and 270 cells/mm2. Cultures were incubated for 24 h, and then cells were microinjected with either 1 µg/µl I{kappa}B-{alpha}-GST fusion protein or 1 µg/µl GST protein alone, as control. As an additional control for the effects of microinjection, some cultures were not microinjected. After 20 h, cell viability was assessed via trypan blue staining (Fig. 1)Citation . The ability of microinjected I{kappa}B-{alpha}-GST to induce apoptosis at the lower plating densities was markedly reduced relative to its effects at higher densities. At the lowest density, only 9.1 ± 0.3% of I{kappa}B-{alpha}-GST-injected cells stained positive with trypan blue (Fig. 1)Citation . With cultures plated at 150 cells/mm2, 19.3 ± 3.9% cells underwent apoptosis, whereas at the 270 cells/mm2 plating density, 51.8 ± 5.0% of I{kappa}B-{alpha}-GST-microinjected cells died. In contrast, no density-dependent change was observed in the amount of death induced nonspecifically upon microinjection of control GST protein. Thus, some property of the cells that varies with cell density affects NMH susceptibility to I{kappa}B-{alpha}-induced apoptosis.



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Fig. 1. Induction of cell death by I{kappa}B-{alpha}-GST is cell density dependent. NMH cells were plated at the indicated densities. After 24 h, cells were microinjected, in duplicate, with the indicated proteins (1 µg/µl), or they were not microinjected (none). After 20 h, cells were stained with trypan blue, and cell viability was measured by dye exclusion. The total number of cells analyzed per treatment ranged from 143 to 249.

 
Expression of c-Myc Protein Varies Inversely with Cell Density.
Expression of the proto-oncogene c-Myc has been demonstrated to be highly dependent on cell density (39 , 40) . Because c-Myc has also been implicated in apoptosis, we investigated the variation of the expression of this oncoprotein with cell density. Levels of c-Myc protein in nuclear extracts from cells plated at differing densities were analyzed. Twenty-four h after plating at densities of 75, 150, or 270 cells/mm2, nuclear proteins were isolated and subjected to immunoblotting using a affinity-purified, polyclonal anti-Myc antibody. As seen in Fig. 2Citation (inset), c-Myc protein levels dropped significantly as cells were plated at higher densities. Densitometric analysis revealed that cells plated at 270 cells/mm2 expressed only {approx}23% of the amount of c-Myc protein seen in cultures plated at 75 cells/mm2.



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Fig. 2. Expression of c-Myc protein varies with cell density. NMH cells were plated at the indicated densities onto poly-L-lysine-coated coverslips and analyzed after 20 h for c-Myc expression by immunofluorescent staining. After counting 100 cells, in duplicate, the number of cells staining positive for c-Myc was determined (inset). NMH cells were plated at the indicated densities and nuclear protein prepared after 20 h. Samples (50 µg) were subjected to immunoblotting analysis for expression of c-Myc and p65 using affinity-purified polyclonal antibodies.

 
To further confirm that c-Myc protein expression varied with NMH cell density, we determined the percentage of cells expressing c-Myc by immunofluorescent staining. NMH cells were plated at different densities on coverslips. Sixteen h later, cells were fixed and stained, and the percentage of cells staining positive for c-Myc was determined, as described in "Materials and Methods." As seen in Fig. 2Citation , c-Myc expression varied with a strong, inverse relationship to NMH cell density. For example, for cultures plated at 30 cells/mm2, 91.0 ± 9.8% of NMH cells stained positive for c-Myc. In contrast, for cultures plated at 270 cells/mm2, only 19.0 ± 4.2% of NMH cells were c-Myc protein positive. In addition, as seen in Fig. 2Citation (inset), p65 expression varied only modestly with cell density, suggesting that altered susceptibility to apoptosis induction at differing densities was not related to differential expression of NF-{kappa}B. Thus, culturing cells at a density that promotes decreased c-myc expression increases NMH susceptibility to I{kappa}B-{alpha}-induced apoptosis.

Ectopic c-myc Expression Rescues I{kappa}B-{alpha}-induced Apoptosis.
To test the possibility that c-myc expression protected NMH cells from I{kappa}B-{alpha}-induced cell death, we next examined the ability of ectopic c-myc expression to ablate NMH sensitivity to NF-{kappa}B inhibition. NMH cells were plated at a density that would lead to down-regulation of endogenous Myc levels (270 cells/mm2). After 24 h, the cells were microinjected with 0.5 µg/µl I{kappa}B-{alpha} expression plasmid alone or in combination with 0.5 µg/µl c-myc expression vector pm21 and enough Bluescript DNA to bring the final concentration to 1.0 µg/µl. As controls, cells were microinjected with empty expression vector or pm21 alone. As seen previously, microinjection of the I{kappa}B-{alpha} expression vector led to significant induction of cell death (Fig. 3)Citation . In contrast, comicroinjection of I{kappa}B-{alpha} and c-Myc expression vectors resulted in significantly less death. Specifically, comicroinjection of c-Myc expression plasmid reduced the level of death seen upon microinjection of I{kappa}B-{alpha} expression plasmid alone from 23.4 ± 1.7 to 9.6 ± 0.3%. Microinjection of a c-Myc expression vector alone caused only a moderate effect on the extent of dead cells seen in the cultures compared to the control parental vector. Thus, ectopic c-myc expression partially ablates the death of cells in which NF-{kappa}B activity is reduced.



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Fig. 3. Ectopic expression of c-myc protects NMH cells from I{kappa}B-{alpha}-induced apoptosis. NMH cells were plated at high density (270 cells/mm2). After 24 h, cells were microinjected, in duplicate, with either 1 µg/µl empty expression plasmid PMT2T (control) or with 0.5 µg/µl I{kappa}B-{alpha}-expression plasmid PM2T-I{kappa}B-{alpha} (I{kappa}B-{alpha}), 0.5 µg/µl c-Myc expression plasmid pm21 (c-Myc), or 0.5 µg/µl each vector expressing c-Myc and I{kappa}B-{alpha} (I{kappa}B-{alpha}+c-Myc). Where needed, pBluescript vector DNA was added to adjust final DNA concentrations to 1.0 µg/µl. Twenty h after microinjection, cells were analyzed for cell death via trypan blue staining. The total number of cells analyzed per treatment ranged from 152 to 199. These data represent one experiment that is typical of three independent experiments with essentially identical results.

 
To determine whether the above results could be extended to a second hepatocyte cell line, we used an alternative method of gene transfer, using a transient transfection protocol for assessing apoptosis, as has been used extensively by others (15, 16, 17, 18) . Briefly, AML12 and NMH hepatocytes were cotransfected with a ß-gal reporter in the absence or presence of expression plasmids encoding I{kappa}B-{alpha} and c-myc. Analysis of ß-gal staining identifies live, transfected cells. Data on the extent of death can be determined from the percentage of live, ß-gal-staining cells in the experimental samples relative to controls receiving ß-gal expression vector alone. Cells plated at high density (270 cellc/mm2) were transiently transfected via lipofection with the SR-I{kappa}B-{alpha} vector encoding a dominant negative I{kappa}B-{alpha} protein and/or the pm21 c-Myc expression vector in the presence of a SV40ß-gal vector DNA. Twenty-four h after transfection the percentage of live ß-gal-expressing cells was determined. As seen in Fig. 4Citation , in both AML12 and NMH cells, transfection of the SR-I{kappa}B-{alpha} induced significant cell death relative to cells transfected with the ß-gal expression plasmid. SR-I{kappa}B-{alpha} expression decreased the percentage of live ß-gal-expressing cells to 61.5 ± 4.6 in NMH cells and to 48.9 ± 10.9 in AML12 cells. However, coexpression of c-myc and SR-I{kappa}B-{alpha} significantly increased cell survival compared to SR-I{kappa}B-{alpha} expression alone. Thus, in both NMH and AML12 cells plated at high density, ectopic c-myc expression ablates death of cells in which NF-{kappa}B activity is reduced.



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Fig. 4. c-myc expression rescues NMH and AML12 from I{kappa}B-{alpha}-induced death. NMH or AML12 cells were plated at high density (270 cells/mm2) in 96-well plates. After 24 h, cultures were transiently transfected by lipofection with 7.5.ng/well c-Myc expression vector pm21, 7.5 ng/well dominant negative I{kappa}B-{alpha} expression vector SR-I{kappa}B-{alpha}, or 7.5 ng/well each in the presence of 7.5 ng/well ß-gal reporter SV40ß-gal. The final DNA concentration was adjusted to 30 ng/well with pOPRSVICAT DNA. After 24 h, cells were fixed and stained for ß-gal activity and the number of surviving blue cells determined. Columns, percentage of live cells relative to controls, where the number of live blue cells in the control wells was typically 75–150; bars, SD. Data are presented as the mean of duplicates with the sample SD. {square}, AML12 cells; {blacksquare}, NMH cells.

 
Antagonism of Endogenous c-Myc Function Renders Low-Density NMH Susceptible to I{kappa}B-{alpha}.
If ectopic c-Myc expression can protect high-density NMH cells from I{kappa}B-{alpha}-induced apoptosis, then high endogenous levels of c-Myc in cells plated at low density might similarly provide survival signals. Thus, we next tested whether antagonism of c-Myc function in these low-density cells would make them more susceptible to I{kappa}B-{alpha}-induced apoptosis. In our first experiments, we used Mad1, which has been proposed to function via two mechanisms to negatively regulate c-Myc action. First, by binding Max, Mad1 renders Max unavailable for Myc heterodimerization and thereby prevents c-Myc functions that require c-Myc/Max dimers. Furthermore, Mad1/Max binding to an E-box has been found to promote histone deacetylation, thereby repressing transcription (41 , 42) . NMH cells were plated at low density and microinjected with 0.5 µg/µl I{kappa}B-{alpha}-GST in the absence or presence of 0.5 µl/µl Mad1-GST and enough GST protein to bring the final protein concentration to 1.0 µg/µl. As seen in Fig. 5Citation , Mad1 and I{kappa}B-{alpha} act synergistically in inducing apoptosis. Twenty h after microinjection, cultures of control cells that were microinjected with GST alone displayed only 2.5 ± 0.3% dead cells. Similarly, only 5.7 ± 1.0% and 8.3 ± 0.8% death was observed in cells microinjected with Mad1 or I{kappa}B-{alpha}, respectively. However, 46.7 ± 2.7% cells were killed following microinjection with both fusion proteins Mad1 and I{kappa}B-{alpha}. Thus, the block to I{kappa}B-{alpha}-induced apoptosis observed in low density cells can be overcome by Mad1 protein. Furthermore, as seen below, similar data were obtained with a c-myc antisense expression vector in NMH and AML12 cells. Taken together, these data indicate that endogenous c-Myc activity rescues NMH cells from apoptosis induced by I{kappa}B-{alpha}.



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Fig. 5. Mad1 renders low-density NMH cells sensitive to NF-{kappa}B inhibition. NMH cells were plated at low density (75 cells/mm2). After 24 h, cells were microinjected, in duplicate, with 0.5 µg/µl either I{kappa}B-{alpha}-GST or Mad1-GST solution, alone or in combination. GST was added to adjust final protein concentration to 1 µg/µl. Twenty h after microinjection, cells were analyzed for cell death via trypan blue staining. The total number of cells analyzed per treatment ranged from 226 to 274. These data represent one experiment that is typical of two independent experiments with essentially identical results.

 
NF-{kappa}B Survival Functions Are Independent of p53 Status.
The p53 tumor suppressor has been shown to play an important role in many apoptotic pathways (29, 30 , 31) and is frequently mutated in liver tumors (32, 33 , 34) . Recent evidence has suggested that NF-{kappa}B expression protects ras-transformed fibroblasts from undergoing apoptosis in a p53-independent manner (43) . To determine whether p53 status was important in the apoptotic pathways involving c-Myc and NF-{kappa}B in murine hepatocytes, we used a temperature-sensitive p53 expression vector pLTRp53cGVal135 (44) . At the nonpermissive temperature of 32.5°C, a wt p53 is expressed; at 38.5°C, a dominant-negative p53 is made that abrogates endogenous p53 function. Furthermore, because Mad1 may also affect cellular gene expression by alternative means, an antisense c-myc expression vector was used to specifically inhibit c-Myc activity. NMH and AML12 cells, plated at low density, were transiently transfected via lipofection with the SR-I{kappa}B-{alpha} vector encoding a dominant negative I{kappa}B-{alpha} protein and/or an vector expressing antisense myc in the presence of the ts p53 expression vector and SV40ß-gal vector DNA. Twenty-four h after transfection, cells were transferred to either 32.5°C or 38.5°C and further cultured for 24 h, and the percentage of live ß-gal-expressing cells was determined. For both hepatocyte lines, coexpression of I{kappa}B-{alpha} and antisense c-myc resulted in an extensive increase in the number of dead cells in the population (Fig. 6)Citation . This decrease in live cells was observed in the presence of either functional (permissive temperature) p53 or nonfunctional p53 (nonpermissive temperatures). For example, in NMH cells, the combination of SR-I{kappa}B-{alpha} and antisense-myc led to survival of 44.9 ± 13.0% in the presence of functional p53 (wt p53) and to 39.4 ± 1.3% live cells in the presence of the dominant negative p53 (mut p53).



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Fig. 6. Induction of cell death by I{kappa}B-{alpha} at low cell density is promoted by inhibition of c-myc expression and is independent of p53 status. NMH (A) or AML12 (B) cells were plated at low density (75 cells/mm2) in 96-well plates. After 24 h, cultures were transiently transfected by lipofection. DNAs used were the temperature-sensitive p53 expression vector pLTRp53cGVal135, c-Myc expression vector pm21, dominant negative I{kappa}B-{alpha} expression vector SR-I{kappa}B-{alpha}, and ß-gal reporter SV40-ß-gal, all at 7.5 ng/well. pOPRSVICAT DNA was added where needed to adjust the final DNA concentration to 30 ng/well. Cells were cultured at 38.5°C. After 24 h, half of the cultures were switched to an incubation temperature of 32.5°C (wt p53) and the other half maintained at 38.5°C (mut p53). After an additional 24 h, cells were fixed and stained for ß-gal activity and the number of surviving blue cells determined. Columns, percentage of live cells relative to controls, where the control values are typically in the range of 100–150; bars, SD. Data are presented as the mean of duplicates with the sample SD.

 
A similar experiment was performed with NMH cells plated at high density. As seen in Fig. 7Citation , SR-I{kappa}B-{alpha} expression led to survival of 18.4 ± 2.3% of transfected cells in the presence of wt 53 and of 15 ± 1.0% in the presence of mut p53. As an independent test to confirm the temperature sensitive p53 expression vector was effective, cultures were similarly treated with the chemotherapeutic agent doxorubicin, which induces apoptosis that is known to be significantly affected by p53 status (31) . Death due to treatment with doxorubicin was reduced significantly in the presence of mut p53, as expected (Fig. 7)Citation . Expression of c-Myc vector expression vector pm21 significantly rescued I{kappa}B-{alpha}-induced death regardless of p53 status; however, rescue was somewhat more pronounced in the presence of the dominant negative mutant form of p53. This is in contrast to low-density cells, in which p53 status showed no c-Myc effect. Thus, hepatocyte cell death induced upon inhibition of NF-{kappa}B occurs in a p53-independent manner.



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Fig. 7. Induction of cell death by I{kappa}B-{alpha} is p53 independent. NMH cells were plated at high density (270 cells/mm2). After 24 h, cultures were transiently transfected by lipofection as described in "Materials and Methods." DNAs used were the temperature sensitive p53 expression vector pLTRp53cGVal135, c-Myc expression vector pm21, dominant negative I{kappa}B-{alpha} expression vector SR-I{kappa}B-{alpha}, and ß-gal reporter SV40ß-gal. After 24-h culture at 38.5°C, half of the cultures were switched to an incubation temperature of 32.5°C (wt p53) and the other half maintained at 38.5°C (mut p53). At this time, where indicated some cultures were treated with 1 µM doxorubicin (dox). After an additional 24 h, cells were fixed and stained for ß-gal activity, and the number of surviving blue cells was determined. Columns, percentage of live cells relative to controls; bars, SD. The control values are as follows: the number of live ß-gal-expressing cells in the wt p53 was 141 ± 28 and the number of live ß-gal-expressing cells in the mut p53 was 120 ± 28. Data are presented as the mean of duplicates with the sample SD.

 

    Discussion
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Here, we showed that NF-{kappa}B cooperates with c-Myc in promoting murine hepatocyte survival in a manner independent of p53 tumor suppressor function. In NMH cells, in which c-Myc activity was down-modulated, either by cell culture conditions (density) or by introduction of antisense c-myc expression or of the c-Myc antagonist Mad1, direct inhibition of NF-{kappa}B/Rel via microinjection of I{kappa}B-{alpha} induced apoptosis. However, in cells expressing high levels of c-Myc protein (either the endogenous levels seen in low-density cells or the ectopic expression levels in high-density cells), the extent of apoptosis induced by I{kappa}B-{alpha} was greatly reduced. Survival functions of c-Myc appeared to be p53 independent in low density cells but exhibited a p53-dependent component at high cell density. Finally, the induction of cell death by I{kappa}B-{alpha} occurred independently of p53 function, suggesting that NF-{kappa}B survival functions in hepatocytes occur through pathways not involving p53.

Several targets of NF-{kappa}B action have been implicated in cell survival. Baldwin and coworkers (45) recently demonstrated that NF-{kappa}B survival functions involve activation of TRAF1, TRAF2, c-IAP-1, and c-IAP-2 and resultant inhibition of caspase 8 in TNF-{alpha}-treated fibroblasts; however, it is not clear if these targets account for NF-{kappa}B survival functions in all cases. Wu et al. (46) recently reported that NF-{kappa}B cell survival functions in TNF-{alpha}-treated Jurkat T cells are mediated through the immediate early response gene IEX-1L. Zong et al. (47) have implicated the prosurvival Bcl-2 homologue Bfl/A1 as a direct NF-{kappa}B target in protection against TNF-{alpha}-induced apoptosis of HeLa cells. We demonstrated that, in the case of murine B cell lymphomas, NF-{kappa}B transcriptionally activates c-Myc, and ectopic c-Myc can provide significant protection against apoptosis induced by treatments that down-regulate NF-{kappa}B (11 , 38) . However, NF-{kappa}B regulation of c-Myc is cell type specific and apparently does not occur in hepatocytes. In particular, there is good evidence suggesting that, in hepatocytes, c-Myc is not regulated by NF-{kappa}B. First, although both NF-{kappa}B and c-Myc expression are rapidly induced {approx}10-fold following PH, most of the c-Myc response is posttranscriptional (48) . In addition, modulation of NF-{kappa}B activity in cultured nontransformed hepatocyte cell lines via EGF withdrawal has no significant effect on Myc-promoter-CAT activity in transient transfection analysis, although NF-{kappa}B reporters are significantly inhibited (data not shown). Another potential candidate target gene is IL-6; as discussed previously, IL-6 is regulated by NF-{kappa}B in the liver (49 , 50) . Mice null for the TNF-{alpha} receptor type I gene fail to undergo liver regeneration following PH. NF-{kappa}B is not induced, and IL-6 production is inhibited (51) . However, infusion of IL-6 can partially restore the regenerative process (52) , suggesting that IL-6 is a physiologically relevant NF-{kappa}B target in liver regeneration. Whether the NF-{kappa}B survival functions in liver are identical to its functions in regeneration is not clear; however, they seem to correlate. Inhibition of NF-{kappa}B activation following PH inhibits regeneration and leads to hepatocyte apoptosis (21) .

Interestingly, c-Myc exhibits duality with respect to its role in cellular apoptosis. Recently, both unregulated expression and regulated repression of c-myc gene expression have been associated with apoptotic death (reviewed in Ref. 37 ). In a number of cases, overexpression of c-myc under conditions of growth factor deprivation accelerates cell death (35 , 36) . For example, enforced c-myc expression in murine fibroblasts subjected to low serum potentiates or sensitizes these cells to apoptosis (36) . In contrast, there are many experimental systems in which c-myc down-regulation ensues following treatments that cause apoptosis, and ectopic c-myc expression decreases the extent of cellular death (37 , 38 , 53 , 54) . For example, as stated above, ectopic c-Myc expression ablated apoptosis of WEHI 231 B lymphoma cells stimulated to undergo receptor-mediated cell death (38) . In vivo experiments have shown similar protection for activated immature T cells (54) . The true nature of this duality of action of c-Myc with respect to apoptosis is not understood and is likely complex. Different models have been proposed to account for the ability of c-Myc to either sensitize to or protect from apoptosis (37) . One model suggests apoptosis results from a mismatch in go/stop signals for advancing through the cell cycle. Another model argues that c-Myc itself is responsible for specific signals that both advance the cell cycle and initiate apoptotic death; these latter signals must then be countered by the expression of specific antiapoptotic genes. Experimental evidence exists supporting both models, and future studies are necessary to resolve this apparently paradoxical feature of c-Myc function.

Interestingly, the data presented here that NF-{kappa}B can cooperate with c-myc is particularly intriguing in light of the observations that c-myc and TGF-{alpha} synergize in leading to the development of liver tumors in a double transgenic animal model (51) . TGF-{alpha} uses the same receptor as EGF, and EGF deprivation of NMH leads to decreased NF-{kappa}B levels.4 The ability of TGF-{alpha} and c-Myc to synergize in induction of liver tumors suggest that our observations of cooperativity between NF-{kappa}B and c-Myc may be physiologically relevant in vivo.

Finally, high rates of HBV infection in China, Southeast Asia, and Africa make hepatocarcinoma one of the most prevalent cancers in the world, accounting for up to 50% of all cancers in those regions (55) . The HBV-transforming protein pX is a transcriptional activator required for viral infection (56) . pX has been found to induce NF-{kappa}B (56) , which raises the intriguing possibility that NF-{kappa}B survival functions play an important role in HBV-associated hepatocarcinogenesis. Interestingly, p53 mutations have been detected in 20–35% of hepatocarcinomas (32, 33, 34) . Loss of p53 function contributes to lack of tumor responsiveness to chemotherapeutic regimens (31 , 57 , 58) , suggesting its loss is important in tumor development. The data presented here that inhibition of NF-{kappa}B can induce hepatocyte death independently of p53 status may provide new approaches to treatment of hepatocyte tumors harboring p53 mutations.


    Materials and Methods
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Cell Culture Conditions.
NMH cells were maintained in DMEM:Ham’s F-12 medium (Life Technologies, Inc., Gaithersburg, MD) supplemented with 20 ng/ml EGF (Collaborative Research, Bedford, MA), 5 µg/ml insulin, 5 µg/ml transferrin, 5 ng/ml selenium, 120 µg/ml nicotinamide (all from Sigma Chemical Co., St. Louis, MO), 50 units/ml penicillin, and 50 µg/ml streptomycin, as described previously (59) . AML12 cells were maintained in DMEM:Ham’s F-12 medium supplemented 10% fetal bovine serum, 5 µg/ml insulin, 5 µg/ml transferrin, 5 ng/ml selenium, 120 µg/ml nicotinamide, 0 units/ml penicillin, and 50 µg/ml streptomycin, as described previously (59) .

Microinjection Analysis.
Microinjection of exponentially growing NMH cells was performed as reported previously (12) . Purified I{kappa}B-{alpha}-GST fusion protein, kindly provided by U. Siebenlist (National Cancer Institute/NIH, Bethesda, MD); Mad1-GST, kindly provided by R. DePinho (Harvard Medical School, Boston, MA); or GST alone was used at 1 µg/µl unless otherwise noted. Expression plasmids for I{kappa}B-{alpha} and c-Myc were PM2T-I{kappa}B-{alpha} (10) and pm21 (38) ; these were injected at concentrations of 0.5 µg/µl. Bluescript BS-KS+ DNA was used as control and to bring final DNA concentrations to 1.0 µg/µl. All cells in a given field were microinjected, and successful microinjection was estimated to occur >90% of the time. Following microinjection, the culture was washed 10 times with sterile PBS to minimize potential contamination during microinjection. For trypan blue exclusion analysis, cloning rings were placed over the microinjected areas following microinjection. After 20 h, the culture medium inside the ring, including cells that had lost adherence, was transferred to a well in a 96-well culture dish. The adherent cells were removed by trypsinization, and to the same well, trypan blue was added to 0.04%. After 15 min, the percentage of cells excluding trypan blue was determined by examination under phase contrast microscopy (x100).

Immunoblot Analysis.
Cells were washed twice in cold PBS and resuspended in cold 10 mM Tris-HCl (pH 7.6), 10 mM KCl, 5 mM MgCl2, 0.2 mM phenylmethylsulfonyl fluoride, and 10 µg/ml leupeptin. After incubation on ice for 10 min, cells were lysed by addition of NP40 to 0.5%. Nuclei were removed by centrifugation, and nuclear protein was extracted (radioimmunoprecipitation assay extract) as described previously (38) . Proteins were fractionated on a 7.5% polyacrylamide-SDS gel and transferred to polyvinylidene difluoride membrane. Immunoblotting was performed essentially as described previously (38) . Antibodies used were p65 (sc109, Santa Cruz Biotechnology) and c-Myc (affinity-purified rabbit polyclonal antimurine c-Myc protein), kindly provided by Steve Hann (Vanderbilt University, Nashville, TN).

Immunofluorescent Cell Staining.
NMH cells plated on glass coverslips were washed three times in PBS and fixed in 3.7% formaldehyde. Specimens were incubated with 10% normal serum in PBS for 20 min, followed by incubation with 1:100 dilution of a primary polyclonal anti-c-Myc antibody in 1% BSA/PBS for 60 min at room temperature. After three successive 5-min washes in PBS, specimens were incubated with 1:200 FITC-goat antirabbit secondary antibody in PBS/BSA for 60 min. After three PBS washes, the percentage of cells staining positive for nuclear c-Myc was determined by fluorescence microscopy.

ß-gal Expression-based Cell Viability Assay.
Exponentially growing NMH or AML12 cells were transfected using Lipofectamine reagent (Life Technologies, Inc.), according to the manufacturer’s instructions. Cells were plated either in 96-well plates at a density of either 75 or 270 cells/mm2. After 24 h, cells were transfected with DNA (3 µl of Lipofectamine per µg DNA) as indicated in the legends. Expression vectors used include a dominant negative I{kappa}B-{alpha} SR-I{kappa}B-{alpha} (60) , c-Myc expression vector pm21, a temperature-sensitive p53 expression vector pLTRp53cGVal135, a ß-gal expression vector SV-ß-gal, and a vector expressing antisense c-myc. This last vector was prepared by isolating a 1.8-kb insert which contains exons 2 and 3 of murine c-myc from the pRc-CMV-c-myc expression vector using HindIII and EcoRI. The insert was blunt ended and subcloned in the reverse orientation into the NotI sites of pOPRSVICAT replacing the CAT gene. After an additional 16–24 h, live cells expressing ß-gal were determined. For ß-gal staining, cells were washed three times in PBS and fixed in 2% formaldehyde, 0.2% glutaraldehyde, and PBS for 10 min. Cells were then incubated overnight in 5 mM potassium ferricyanide, 5 mM potassium ferrocyanide, 5 mM MgCl2, and 0.1% 5-bromo-4-chloro-3-indolyl-ß-D-galactoside at 37°C. The number of cells staining blue was determined by examination under phase-contrast microscopy.


    Acknowledgments
 
We thank U. Siebenlist, R. DePinho, J. DiDonato, M. Karin, M. Wu, and S. Hann for generously providing purified proteins, expression vectors, and antibody reagents.


    Footnotes
 
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 in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Supported by NIH Grant CA-36355 (to G. E. S.). Back

2 To whom requests for reprints should be addressed, at Boston University School of Medicine, Department of Biochemistry, 715 Albany Street, Boston, MA 02118-2394. Phone: (617) 638-4120; Fax: (617) 638-5339; E-mail: gsonensh{at}bu.edu Back

3 The abbreviations used are: NF-{kappa}B, nuclear factor-{kappa}B; PH, partial hepatectomy; TGF, transforming growth factor; NMH, normal murine hepatocyte; GST, glutathione S-transferase; ß-gal, ß-galactosidase; wt p53, wild-type p53; mut p53, mutant p53; EGF, epidermal growth factor; IL-6, interleukin 6; HBV, hepatitis B virus; CAT, chloramphenicol acetyltransferase. Back

4 R. E. Bellas, G. E. Sonenshein, and N. Fausto, unpublished observations. Back

Received for publication 11/16/98. Revision received 3/16/99. Accepted for publication 3/17/99.


    References
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 

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