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-dependent Growth Regulatory Genes1
Department of Veterinary Science, Center for Molecular Toxicology and Carcinogenesis, Pennsylvania State University, University Park, Pennsylvania 16802
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. It has become increasingly apparent that the role of the PPs in the development of cancer encompasses many different aspects of cell growth regulation. Immortalized hepatocytes from wild-type (PPAR+/+) and PPAR-/- mice were generated using a temperature-sensitive SV40 virus. Characterization of the murine SV40 hepatocytes (MuSH) generated from both genotypes (MuSH+/+, MuSH-/-) show markers of differentiation such as albumin expression, but is devoid of Kupffer cell contamination. Hallmark PPAR-mediated responses such as induction of acyl-CoA oxidase mRNA by PPs are present in the MuSH+/+ but are absent in MuSH-/- cells. In contrast to most cell culture systems, the wild-type MuSH hepatocytes retain the mitogenic activity of PPs, whereas the MuSH-/- does not respond in this manner, thus making this cell culture system an ideal tool to examine growth regulatory gene expression affected by PPs. Microarray experiments performed on both cell types identified many genes in which regulation is dependent on the presence of PPAR, and these changes were verified with reverse transcriptase-PCR. Genes involved in carcinogenesis and control of the cell cycle that are regulated by PPs in a PPAR-dependent manner include ubiquitin COOH-terminal hydrolase 37 (also known as UCT-L5) and cyclin T1. These results show that MuSH cells reflect the biological properties of both the wild-type and PPAR-null animals and can be used to identify novel PPAR-regulated genes that could be involved in regulation of the cell cycle and carcinogenesis. |
INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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It is now known that PPs exert their effects, both on lipid metabolism and carcinogenesis, through the PPARs. PPARs are members of the NR superfamily and exist as three subtypes designated , ß (or 
), and 
(NR1C1, NR1C2, NR1C3, respectively). The PPARs have differential expression in tissues and apparently have evolved to fulfill different biological roles. The involvement of PPAR in cancer (specifically in liver cancer) has become an area of great interest. Apparently, in addition to having influence on fatty acid metabolism, PPAR serves a biological role in many aspects of the regulation of cell cycle control and apoptosis (5
, 6)
.
Although PPs can bind to and activate all three PPARs to various degrees, research with the PPAR-null mouse has demonstrated that this subtype is predominantly responsible for the peroxisome proliferation and cancer-causing effects of PPs in liver (7)
. For example, the effects of the PP Wy-14,643 in wild-type mice include increased DNA synthesis and increased expression of growth regulatory genes and ultimately hepatocellular carcinoma; these responses are not present in PPAR-null mice (4
, 8
, 9)
.
Although it is clear that PPAR plays a critical role in the ability of PPs to produce a mitogenic responses in rodent liver, some debate continues as to whether this is a direct response of hepatocytes to the chemical or if it requires involvement of other cell types. The controversy stems from the fact that most hepatocyte culture systems do not recapitulate the mitogenic effect seen in vivo (10
, 11)
and because an enhanced hepatocyte proliferative response is observed when Kupffer cells are activated concomitantly with PP administration (reviewed in Ref. 12
). Additional complications on this matter arise from the fact that primary rat hepatocytes are responsive to the mitogenic effect of PPs but require specific culture conditions (13)
. Thus, mechanistic studies on how PPAR affects the cell cycle have been limited to in vivo studies or primary hepatocytes and suffer from numerous confounding factors.
In these studies, immortalized hepatocyte cell lines were generated from both the wild-type and PPAR-null mouse. The wild-type cells retain many of the classic PPAR-mediated responses to PPs such as regulation of gene expression and increases in cell growth and altered cell cycle regulation; these effects are absent in the immortalized hepatocytes derived from the PPAR-null cells. Microarray analysis of the gene expression profile for each of these cell types in response to a known PP has provided a large list of candidate genes that may help explain the carcinogenic potential of PPAR agonists. The creation of these cell lines provides a useful tool for mechanistic examination of the role of PPAR in regulation of cell cycle control and tumorigenesis.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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MEM-4% FBS. Cells were incubated with stock temperature sensitive SV40 virus (14)
at 37°C and agitated every 15 min for 2 h. Media was changed to MEM-8% FBS, and the cells were incubated at 34°C for 7 days. Virus-containing media was removed upon gross cell pathology (nuclear inclusion, cytoplasmic vacuolation, and lysis) and stored in liquid nitrogen. Quantification of virus titer was not performed.
Isolation, Maintenance, and Infection of Primary Hepatocytes.
The isolation and infection of hepatocytes was adapted from a previously described method (14)
. Purebred wild-type and PPAR-null mice on a SV/129 background were used and have been described previously (7
, 15)
. Primary hepatocytes were isolated from two male wild-type mice (10 days old) and two male PPAR-null mice (5 days old). Mice were euthanized by overexposure to carbon dioxide, and whole liver was removed and incubated at 37°C with 1% collagenase in HBSS for 5 min. Hepatocytes were isolated by centrifugation in 10% Percoll (Amersham, Piscataway, NJ) in HBSS at 1000 rpm for 20 min at 4°C. The cells were washed with 10% FBS in MEM, centrifuged, and resuspended in MEM-10% FBS. Each liver was separated into three populations to be infected separately; individual clones were not selected. Cells were cultured with MEM, 4% FBS, dexamethasone and 1% penicillin/streptomycin and allowed to grow to 
75% confluency at 34°C. The cells were then washed with MEM-10% FBS, overlayed with 1 ml of MEM-10% FBS, and infected with 200 µl of stock virus/well. Cells were incubated at 34°C for 2–3 h and gently agitated by hand. Virus-containing media was then aspirated, and the cells were given MEM-4% FBS-0.1 µM dexamethasone. Media were changed twice weekly for 6 weeks. Resultant colonies were isolated as mixed populations (MuSH) or as individual clones and are designated MuSH+/+ (wild-type) or MuSH-/- (PPAR null).
PCR Genotyping of Cell Genomic DNA.
Total genomic DNA was isolated by incubation of cells with lysis buffer [100 mM Tris-HCl, 0.005 M EDTA, 0.2% SDS, 0.02 M NaCl (pH 8.0)] and proteinase K overnight at 55°C with gentle shaking. Cell debris was removed by centrifugation (12,000 rpm for 5 min), and supernatant containing DNA was removed. Genomic DNA was then precipitated using 1 volume isopropanol and resuspended in water. Stock DNA was diluted 1:100 with water, and PCR genotyping was performed using a 60°C annealing temperature for 1 min and 30 cycles using primers for PPAR (F1, 5'-GAGAAGTTGCAGGAGGGGATTGTG-3'; R1, 5'-CCCATTTCGGTAGCAGGTAGTCTT-3') and the NEO cassette (NEOR1, 5'-GCAATCCATCTTGTTCAATGGC-3'). The PPAR forward primer was designed for bases 1191–1215 of mouse PPAR, whereas the PPAR reverse primer was designed for bases 1579–1602. The neomycin reverse primer was designed for the neo cassette, which was inserted between the PPAR primers.
Western Blot Analysis.
Total cell protein was isolated using cell lysis solution [50 mM HEPES (pH 7.4), 250 mM NaCl, 0.1% NP40, 1 mM DTT, 1 mM EDTA, 1 mM NaF, 10 mM ß-glycerophosphate, 0.1% protease inhibitor mixture (Sigma), and 0.1 mM sodium orthovanadate] and lysing on ice for 30 min followed by centrifugation at 12,000 x g for 10 min. The supernatant was removed as total soluble protein. Total soluble protein (150 µg) was separated on a 12% Tris-glycine gel and electrotransferred to a nitrocellulose membrane (Hybond; Amersham). Membranes were washed three times with TBS/0.1% Tween 20 (TBS+), blocked with 5% BSA in TBS+ for 1 h at room temperature, washed three times with TBS+, and incubated while rocking at room temperature for 1 h with primary antibodies. Immunoblotting was performed using a 1:1000 dilution of antimouse albumin antibody raised in rabbit (ICN, Costa Mesa, CA) or a 1:1000 dilution of mouse monoclonal SV40 TAg antibody (Oncogene Research Products, San Diego, CA). The blot was washed three times with TBS+ and incubated at room temperature for 1 h with a horseradish peroxidase-conjugated antirabbit or antimouse antibodies at a 1:10000 dilution (Amersham). The blot was then washed three times, and visualization was performed using enhanced chemiluminescent visualization (Amersham).
Cell Cycle Analysis.
Cell cycle analysis was performed using MuSH hepatocytes that had been incubated at 37°C for 24 h with low serum media (0.1% FBS) and then split to appropriate vessels. Cells were pretreated for 30 min with the G2-M cell cycle inhibitor etoposide (50 ng/ml) and then treated for 24 h with 50 µM Wy-14,643 or DMSO in serum-free media. Cells were harvested, washed with cold PBS, and fixed in cold 75% ethanol overnight at 4°C. Fixed cells were stained with propidium iodide (20 µg/ml in PBS/0.1% Triton X-100) and incubated at room temperature for 30 min before analysis. Flow analysis was performed using a Coulter XL-MCL benchtop cytometer at the Pennsylvania State University Center for Quantitative Cell Analysis. Data analysis was performed using the flow cytometric analysis software package (FlowJo; Treestar, San Carlos, CA).
Determination of Growth Doubling Time.
Doubling times for each cell line at each temperature was determined using trypan blue exclusion. Cells were plated at low density and allowed to recover overnight. Treatment using 50 µM Wy-14,643 or DMSO was started at day 0. Cells were counted at days 0, 2, 4, and 6 in triplicate. Media were replaced with fresh treatment media on every counting day to the remaining wells. Cells were resuspended in PBS, and an aliquot was diluted 1:4 in a 0.4% Trypan Blue solution in PBS, and live cells were counted. Cell counts were normalized to day 0 (100%), and growth curves were plotted, fitted with an exponential growth curve, and doubling time was calculated using GraphPad Prism (San Diego, CA).
Mouse Oligonucleotide Arrays.
The Mouse Genome Oligo Set Version 1 was purchased from Operon (Alameda, CA) and contains 6800 optimized 70-mers plus 24 controls, melting temperature normalized to 78°C. Sequences were optimized by the manufacturer using BLAST against all known mouse genes to minimize cross-hybridization. Oligonucleotides were printed onto glass slides using GeneMachines Omnigrid (San Carlos, California) with additional controls obtained from Stratagene (SpotReport system, La Jolla, CA) at the Penn State University microarray core facility.
Microarray Analysis.
Total RNA was isolated by TriReagent (Sigma) and additionally purified with RNAEasy (Qiagen) according the manufacturers’ instructions. Subsequently, 2.5 µg of oligo(dT) were added to 15 µg of total RNA in a volume of 15.5 µl, and the sample was heated for 10 min at 65°C. While the RNA cooled, 6 µl of 5x first-strand buffer, 3 µl of 0.1 M DTT, and 3 µl of 10x deoxynucleotide triphosphate mix were added and incubated at 42°C. The 10x deoxynucleotide triphosphate mix contained 5 mM each dATP, dCTP, dGTP, 3 mM dTTP, and 2 mM aminoallyl dUTP. The RNA sample was added to the prewarmed reaction mix at 42°C, and 2 µl of 200 units/µl Superscript II and 0.5 µl of RNase inhibitor were added. The reaction (volume is 30 µl) was incubated at 42°C for 1 h. Subsequently, 1.6 µl of 5 M NaOH (for final concentration of 0.25 M) and 20 µl of 0.5 M EDTA were added and incubated for 15 min at 65°C followed by neutralization with 10 µl of 2 M HEPES (pH 8). The reverse transcription reaction was cleaned, and Tris was removed using a Nanosep 30 K concentrator (Pall Filtron) and dried under vacuum. The cDNA pellet was resuspended in 4.5 µl of 0.1 M NaHCO3 (pH 9.0). An aliquot (4.5 µl in DMSO) of NHS-ester Cy3/Cy5 dye (Amersham) was added to the resuspended cDNA pellet and incubated for 1 h at room temperature in the dark. Before combining samples for hybridization, reactions were quenched to prevent cross-coupling. To each reaction, 4.5 µl of 4 M hydroxylamine was added and incubated for 15 min at room temperature in the dark. Reactions were purified on QiaQuick column (Qiagen), dried under vacuum, and resuspended in 15 µl of hybridization buffer (7.5 µl of formamide, 3.75 µl of 20x SSC, 1.5 µl of H2O, 0.75 µl of 2% SDS, and 1.5 µl of 1 mg/ml human Cot1 DNA). Slides were prepared by steaming with hot tap water and quick drying on heat block. Slides were UV cross-linked (1900 µJ x 100) and washed with 0.1% SDS (30 s) and then water (30 s). Slides were boiled in water for 3 min, washed in 70% ethanol for 3 min, spun briefly to remove residual ethanol, and placed into hybridization chamber with prehybridization buffer at 42°C. Labeled cDNA was denatured in a 100°C water bath for 2 min and cooled for 5 min at room temperature. Slides were washed five times in water, then once in isopropanol and allowed to air dry. A lifter slip (rinsed with dH2O and air dried) was placed on the array and denatured probe added by pipetting under the lifter slip. In the present experiments, cohybridization was performed with cDNA from DMSO-treated cells labeled with Cy5 and those from Wy14,643-treated samples labeled with Cy3. Treatment conditions are described above. The comparisons were made within the cell types (MuSH+/+ or MuSH -/-), and no cross-cell-type hybridization was performed in the present experiments. Hybridizing continued at 42°C for 20–24 h. Posthybridization washes included 2x SSC and 0.1% SDS (3 min), 1x SSC (2 min), 0.2x SSC (1 min), and 0.05x SSC (10 s) followed by spin drying and immediate scanning. Arrays were scanned within 1 h after drying. Scanning was performed by GenePix 4000A scanner (Axon Instruments, Inc., Foster City, CA) with dedicated PC running Axon GenePix image acquisition and analysis software. Analysis of gene expression was performed using GeneSpring software (SiliconGenetics, Inc., Redwood City, CA). Supplemental data can be obtained on line.4
Statistical Analysis of Microarray.
Normalization and analysis of the gene expression profiles was performed as follows: some genes were used as negative controls for a background subtraction; their median value was subtracted from the raw values for each gene before anything else was done. Each gene’s measured intensity was divided by its control channel value in each sample. When the control channel value was <10.0, the data point was considered bad. Intensity-dependent normalization was also applied, where the ratio was reduced to the residual of the Lowess fit of the intensity versus ratio curve. The 50th percentile of all measurements was used as a positive control for each sample; each measurement for each gene was divided by this synthetic positive control, assuming that this was at least 0.01. The bottom 10th percentile was used as a test for correct background subtraction. This was never less than the negative of the synthetic positive control. Statistical analysis was performed using a Student t test with a P of 0.05 with the additional criteria of being either 2-fold increased or decreased by Wy14,643 treatment. Differences between wild-type and null cells were judged similarly (P < 0.05 and 2-fold different). Genes that met these parameters were classified by molecular function using annotations from Silicon Genetics.
Reverse Transcriptase-PCR.
Initial verification of altered gene expression observed in the microarray experiments was performed using semiquantitative relative reverse transcriptase-PCR. Total RNA was isolated from cells as above. Relative quantitation was performed using Ambion QuantumRNA ß-actin Internal Standards (Ambion, Austin, TX) according to the manufacturer’s protocol. MuSH+/+ and MuSH-/- cells were incubated at 37°C for 24 h and split to appropriate vessels. Total RNA was diluted to 50 ng/µl, and 100 ng were used in relative reverse transcriptase-PCR. Primers for ACO, cyclin T1, JunB oncogene, UCTH37, and ß-actin mRNA were designed (Table 1)
and optimized. Primer linearity tests and competimer ratio curves were performed for each gene examined individually. The PCR products were separated on a 2% fine resolution agarose gel (NuSieve; FMC, Philadelphia, PA), visualized with ethidium bromide staining, and quantitated (OptiQuant; Packard, Meriden, CT). Data for each gene is expressed relative to the ß-actin amplicon.
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+/+ and MuSH-/- were treated with 50 µM Wy-14,643, 100 µM CLA mix, 10 µM ETYA, 50 µM clofibric acid, and 100 µM ciprofibrate or vehicle (DMSO) for 6 h. Total RNA was isolated using TRI Reagent according to the manufacturer’s protocol. Quantitative reverse transcriptase-PCR was performed, and PCR products were visualized and quantitated as described previously (16)
. Molecules of target gene mRNA/0.1 µg total RNA were determined and expressed relative to molecules ß-actin mRNA in the same sample. Statistical analysis of reverse transcriptase-PCR gene expression data were performed using the corrected values (target gene/ß-actin). One-way ANOVA and Dunnett’s multicomparison tests were performed in Minitab (State College, PA). Means that are statistically different from the DMSO control (P < 0.05) are indicated with an asterisk.
Nonquantitative reverse transcriptase-PCR for CD36 was performed as above without internal standard using primers designed for this gene (17)
. Total RNA was isolated from the RAW264.7, MuSH+/+, MuSH-/-, and FaO cell lines as above.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-null and -wild-type Mouse Hepatocytes.-null mice was immortalized (MuSH-/-). The wild-type population with the highest level of albumin expression (data not shown) was chosen for characterization and is used in all subsequent studies (MuSH+/+). PCR genotyping was performed using primers designed for PPAR and the neomycin cassette. As shown in Fig. 1A
, the expected PCR products were observed from the wild-type and null hepatocytes indicating PPAR+/+ and PPAR-/- genotypes, respectively. To characterize the differentiation status and purity of the MuSH cells, albumin was also examined using Western blot and CD36 examined by reverse transcriptase-PCR. As seen in Fig. 1
, both the MuSH+/+ and MuSH-/- cell lines express albumin at similar levels (Fig. 1C
, bottom gel). CD36 (Fig. 1B)
mRNA, however, is not expressed in these cell lines. Thus, the MuSH cells retain the hepatocyte phenotype and are devoid of detectable Kupffer cell contamination. Because the SV40 TAg has a number of hormone response elements (18
, 19)
, there was a concern that PPs may effect its expression and thus confound interpretation of the cell cycle analysis. Examination of the regulation of SV40 TAg shows that levels of SV40 TAg are not increased by treatment with Wy-14,643 (Fig. 1C
, top gel and graph).
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+/+ and MuSH-/- lines passed for several generations at 34°C, 37°C, or 40°C retain the same phenotypic characteristics as the original passages and express albumin (data not shown). Thus, the MuSH cell lines generated are stable populations of immortalized hepatocytes. Tests to determine whether they are transformed have not been performed and hence are considered to be immortalized.
Loss of Ligand-induced Cell Cycle Progression in PPAR-null Hepatocytes.
A well-documented effect of PP exposure in vivo is the increased proliferation of hepatocytes and increased entry into the cell cycle. To examine if the MuSH cells retain this ability, cell cycle analysis was performed after synchronization with low serum media (0.1%) and etoposide (30-min pretreatment) before PP administration. As seen in Fig. 2
, treatment of the synchronized wild-type population with Wy-14,643 leads to an increase in cells in G2 phase of the cell cycle (10–22.6%). Because of high variability that is inherent in this technique, representative data are shown and represent a consistent pattern seen in three independent experiments. The increase in G2 cells was not seen in the similarly synchronized MuSH-/- cells. A slight increase in cells in S phase can be seen in the MuSH-/- upon treatment with Wy-14,643, but this effect is not consistent and is an artifact of the particular experiment shown. The movement of wild-type cells into the G2 phase of the cell cycle upon treatment with Wy-14,643 is supported by a concomitant decrease in doubling time for cell growth. As shown in Fig. 3
, MuSH+/+ cells grown at 37°C and treated with Wy-14,643 increase in number at a faster rate than untreated cells or those grown at 34°C regardless of treatment. The MuSH-/- cells exhibit no change in growth rate regardless of treatment or temperature. This increase in growth is shown graphically in the form of cell doubling times as shown in Fig. 3B
. The doubling time for the wild-type cells grown at 37°C decreased upon treatment with Wy-14,643 from 2.5 to 1.5 days. This effect is not seen in wild-type cells grown at 34°C or in PPAR-null cells grown at either temperature.
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+/+ and MuSH-/- cells treated with Wy-14,643 (50 µM for 6 h). Quantitation of gene regulation was performed using the GeneSpring software package on normalized data as described in methods. A total of nine slides was analyzed for both MuSH+/+ and MuSH-/- cells. Of the 6800 genes on the array, 4500 gave a detectable signal in at least five of the nine slides for MuSH+/+ cells, whereas 4900 did so in the MuSH-/-. In the MuSH+/+ cells, 1140 genes met the 2-fold change criteria and 560 had a P of <0.05. A smaller subset of genes met both parameters, represented by 110 gene products (Table 2)
. For the MuSH-/- cell lines treated with Wy-14,643, there were 1000 genes that met either criteria with 290 that met both (Table 3)
. A comparison between cell types (MuSH+/+ and MuSH-/-) for selected genes can be found in Table 4
. In this instance, 376 genes were different between the genotypes, with 102 being 2-fold different in response. Regulated genes are grouped by functional classification if known.
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+/+ but not MuSH-/- cells. Several genes of this class were chosen for confirmation using quantitative competitive reverse transcriptase-PCR. Initial screening of the data lead to the selection of two initial candidate genes: JunB and UCTH37. Another other intriguing gene (cyclin T1), the values of which were significant but did not fit the above criteria (<2-fold regulation), was chosen as well.
The initial verification of genes identified through microarray experiments was performed using a relative reverse transcriptase-PCR technique. Three genes chosen for verification were UCTH37 (also known as UCT-L5), cyclin T1, and junB. Primers designed for these genes can be found in Table 1
. As shown in Fig. 4, B–D
, all of the genes chosen were verified as being regulated in accordance with the results from the microarray using relative reverse transcriptase-PCR techniques. Another gene, zinc finger protein 145, was shown to be up-regulated by Wy-14,643 through microarray analysis, but this regulation could not be verified thorough relative reverse transcriptase-PCR (data not shown). The amount of ACO mRNA induction seen in the MuSH+/+ cells is less than what has been reported in other rodent models such as FaO rat hepatocytes (4–6-fold) (16)
but was statistically significant (P < 0.05).
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target genes was performed with multiple known PPAR activators using the standard quantitative competitive reverse transcriptase-PCR technique. The PPs chosen for these experiments were Wy-14,643, CLA mix, ETYA, clofibric acid, and ciprofibrate. MuSH+/+ and MuSH-/- populations were grown at 37°C were treated for 6 h with each of the chemicals listed above. Before these genes could be analyzed further, clonal populations of the MuSH+/+ cells were isolated to eliminate the drift in responsiveness seen within the mixed cell population over the course of passage. Three responsive lines were found, and one was chosen for use in additional gene expression verification (MuSH+/+ CL3). Cells from this clonal line at very low passage were used for all subsequent experiments. An identical clonal expansion was performed with the MuSH-/- cell population, but no clones were found that were responsive to Wy-14,643, therefore, the pooled population was used. As shown in Fig. 5
, the Wy-14,643 effects seen using the relative reverse transcriptase-PCR method are detectable using quantitative reverse transcriptase-PCR, except for the down-regulation of JunB, which shows a small decrease but not statistically significant. In Fig. 5A
, it can be seen that of the chemicals tested, only Wy-14,643 can significantly regulate ACO mRNA in the wild-type cells. No significant regulation was seen in the MuSH-/- cells for any chemical treatment tested. A significant increase in UCTH37 mRNA (Fig. 5B)
was observed with Wy-14,643 as well as clofibric acid. Ciprofibrate, ETYA, and CLA all increased UCTH37 mRNA levels but not to the same extent. JunB up-regulation (Fig. 5C)
was seen in response to CLA, clofibric acid, and ciprofibrate but not in response to ETYA. Finally, cyclin T1 mRNA expression was increased by CLA, ETYA, and ciprofibrate but not by clofibric acid. Interestingly, none of these regulatory trends are seen in the MuSH-/- cells, suggesting a role for PPAR in the regulation of these genes in response to many structurally diverse PPs.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-null and -wild-type hepatocytes using a temperature-sensitive SV40 virus. The cell populations generated are genotypically distinct (MuSH+/+ and MuSH-/-) and are differentiated hepatocytes with no detectable Kupffer cell contamination. Numerous passages have been made with no loss in PP responsiveness or differentiation, although clonal lines have shown a more robust response with some PPAR target genes. As expected, based on in vivo studies (20)
, expression of PPAR-responsive genes and PP-induced cell cycle control was attenuated in the MuSH-/- relative to MuSH+/+. This genotypic difference is seen in cells incubated at 37°C but not at 34°C, indicating that the presence of components of the SV40 virus such as T-antigen, may affect the activity of PPAR. The expression of ACO and the amount of PP induction are lower than seen in other rodent cell lines. Other lipid metabolizing genes such as fatty-acid binding protein and cytochrome P450IVA (CYP4A) show a similar phenomena (data not shown). The reason for this difference in response may be the age of the mice at the time of sacrifice (5–10 days) or the immortalization and cell culture procedure. Although the MuSH cells may not be a good model for the examination of fatty acid-metabolizing enzymes, they are appropriate for the examination of chemically induced mitogenesis. Being able to recapitulate the increase of cell proliferation in an in vitro model system makes these cells amenable to future studies on the mechanism of tumor promotion by PPs.
It has been proposed that Kupffer cells are responsible for the certain effects seen upon exposure to PPs such as increased DNA synthesis in hepatocytes (11)
. For example, ablating the tumor necrosis factor response produced by Kupffer cells decreased PP-induced cell proliferation in the liver (21)
. Support for this hypothesis also comes from evidence that most hepatocyte cell lines or primary cultures of purified hepatocytes are refractory to the mitogenic response of these chemicals. Kupffer cells, however, do not express PPAR, which is required for many of the alterations induced by PPs (22)
. Therefore, it is difficult to conceive of an absolute requirement for this cell type to confer responsiveness to PPs because it is known, through the use of the PPAR-null mouse, that this receptor subtype is required for the chemicals’ carcinogenic responses (4)
. Our results support the hypothesis that the hepatocytes are directly affected by the cell growth response to PPs. This response was PPAR-dependent as shown by an increase in cells entering G2 and a decrease in doubling time in response to Wy-14,643 and is not related to the presence of SV40 TAg in the cells. In addition, these findings are consistent with the lack of inducible DNA replication by Wy-14,643 in the null animal (4)
.
As shown by the cell counting studies, cell growth in response to Wy-14,643 is greatly increased in the MuSH+/+ cells but not in the MuSH-/- cells. An increase in cell number may not be fully explained by the rather modest changes in cell cycle progression. This inconsistency could be attributable to a suppressive effect of Wy-14,643 and PPAR on other aspects of cell survival such as apoptosis. Evidence already exists that PPAR plays a role in the suppression of apoptosis in response to PPs (5)
, and the results shown here are in agreement with such a conclusion.
Microarray analysis performed on both cell types identified a number of genes that were regulated by a known PP (Wy-14,643). A subset of genes was selected for verification by multiple reverse transcriptase-PCR techniques based on the potential role for these genes in cancer and cell cycle regulation. JunB is a gene implicated in the regulation of cell proliferation in tumorigenic cells (3) . High levels of JunB protein exhibit a negative effect on cell proliferation and AP1 activity (23) . A significant down-regulation of JunB mRNA was found in response to Wy-14,643 using microarray and relative reverse transcriptase-PCR techniques. This gene was also seen to be up-regulated with other known PPs such as CLA. The PP CLA has been suggested to have some anticarcinogenic effects and an opposite regulation with respect to Wy-14,643 (a carcinogen) would seem to be in line with the end effects of both chemicals.
UCTH37 (also known as UCT-L5) is a member of a group of proteins that removes the ubiquitin group from polyubiquitinated proteins targeted for degradation (24)
. Ubiquitination is a vital mechanism by which proteins are degraded in the cell. Deubiquitinating enzymes provide a means by which the half-life of proteins within the cell can be prolonged. Increasing the stability of proteins within the cell through deubiquitination suppresses apoptosis, thus leading to prolonged life of the cell (25)
. This mechanism of protein stabilization has become a target of cancer treatment in recent years (26)
. Microarray analysis showed an increase in mRNA for this gene in response to Wy-14,643 that required a functional PPAR. This regulation was tested using both relative and competitive quantitative reverse transcriptase-PCR. Increase UCTH37 mRNA in response to Wy-14,643 was seen with relative reverse transcriptase-PCR but not seen using competitive quantitative reverse transcriptase-PCR techniques. The reason for this difference in methodology is not known. Interestingly, regulation of UCTH37 mRNA was seen in response to other known PPs (specifically ETYA, clofibric acid, and possibly ciprofibrate). In no instance was regulation of UCTH37 mRNA seen in the absence of active PPAR (MuSH-/-). This is the first study that UCTH37 is a PPAR target gene. Additional work is required to determine the role of this change in the underlying mechanism of PP-induced hepatocarcinogenesis. Although not directly verified, other ubiquitin COOH-terminal hydrolases were found to be regulated by Wy-14,643 in a PPAR dependent manner as well, additionally supporting a role for the regulation of protein turnover by PPs.
Cyclin T1, a member of the positive transcription elongation factor b complex, stabilizes the RNA polymerase II complex and suppresses premature termination of mRNA transcripts (27
, 28)
. In addition, cyclin T1 interacts with cdk9 (29)
suggesting that this PPAR-regulated gene could directly modulate the cell cycle. This gene was increased in microarray experiments and verified using both relative and competitive quantitative reverse transcriptase-PCR and required PPAR expression for PP responsiveness. Interestingly, cyclin T1 mRNA was augmented in response to other known PPs such as ETYA and ciprofibrate suggesting a broader role for cyclin T1 in cellular responses to PPs through PPAR.
The genes chosen for additional analysis by reverse transcriptase-PCR represent a small subset of those genes found to be regulated by Wy-14,643 in a PPAR-dependent or -independent manner. The functions of regulated genes spans many aspects of cell growth and homeostasis. Examples of such genes found to be regulated in the MuSH+/+ cells include the RNA polymerase II coactivator, which is thought to help stabilize transcription complexes (30)
and U2 small nuclear ribonucleoprotein, which is involved in the splicing of RNA transcripts in preparation for translation. Members of the fibrinogin/angiopoietin-related protein family were shown to be highly up-regulated by Wy-14,643 in a PPAR-dependent manner through microarray analysis. This family of proteins contains known PPAR-regulated genes (31)
. The function of this family of proteins includes a wide array of effects from response to fasting to suppression of apoptosis (32)
. Certainly, the potential to suppress apoptosis would play a major role in the formation of tumors in response to PPs.
The MuSH cell model has provided insight into the regulation of genes in a PPAR-dependent manner. The MuSH-/- cells, in particular, provide an opportunity to examine genes that are regulated by PPs in a PPAR-independent manner. Array analysis has yielded a wide variety of genes that are regulated in the MuSH-/- cells in response to Wy-14,643. The most interesting of the genes found include the retinoblastoma protein 1 and the retinoblastoma binding protein 6. These genes have been implicated in cell cycle control and their role in cancer has been well documented (reviewed in Ref. 33
). The fact that the PPARß and PPAR are expressed in this model suggest that these genes and others regulated by Wy14,643 may be affected by these other subtypes in MuSH-/-. Alternatively, PPs may affect other signaling cascades irrespective of the PPARs through effects at the cell membrane, stress kinase regulation or potentially other soluble receptors.
The results described herein show that the MuSH cell lines respond similar to the PP response observed in vivo, especially with regard to PPAR-dependent mitogenesis. Immortalized cells, however, are more amenable to a detailed examination of genetic responses and can be manipulated more readily. A large list of candidate genes that potentially can be regulated by PPs has been generated through the use of microarray techniques. Some of these genes have already been verified as PP regulated and thus merit examination in more detail to elicit the role in which they play in tumorigenesis and cancer. These MuSH cells will be useful in the identification of novel PP regulated genes, mechanistic studies of PPAR mode of action, and understanding how these important xenobiotics regulate the cell cycle and ultimately result in cancer.
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ACKNOWLEDGMENTS |
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-null mice, Janice Chou (NIH) for providing the SV40ts virus, Ben Belda and Dan Hannon, Janardan K. Reddy (Northwestern University) for supplying ciprofibrate as well as Dr. Craig A. Praul of the Penn State DNA Microarray Facility. |
FOOTNOTES |
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1 This work was supported by NIH Grant DK49009 (to J. V. H.). Oligonucleotides for the production of the gene expression microarrays were obtained in part as the result of the College of Agricultural Sciences seed grant program (to J. V. H.) and a consortium of users in the Department of Veterinary Science, Penn State University. 
2 To whom requests for reprints should be addressed, at Center for Molecular Toxicology and Carcinogenesis, 226 Fenske Laboratory, University Park, PA 16802. Phone: (814) 863-8532; Fax: (814) 863-1696; E-mail: jpv2{at}psu.edu
3 The abbreviations used are: PP, peroxisome proliferator; PPAR, peroxisome proliferator-activated receptor; NR, nuclear receptor; FBS, fetal bovine serum; MuSH, murine SV40-immortalized hepatocyte; ACO, acyl-CoA oxidase; UCTH37, ubiquitin COOH-terminal hydrolase 37; ETYA, 5,8,11,14-eicosatetraynoic acid; CLA, conjugated linoleic acid.
4 Internet address: http://moltox.cas.psu.edu/Microarray/MuSH.html.
Received 10/ 2/02. Revised 6/25/03. Accepted 7/ 8/03.
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