p16INK4a and ß-catenin alterations in rat liver tumors induced by NNK

Leah C. Pulling, Donna M. Klinge and Steven A. Belinsky1

Lovelace Respiratory Research Institute, PO Box 5890, Albuquerque, NM 87185, USA


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Inactivation of the p16INK4a (p16) tumor suppressor gene by promoter hypermethylation and mutation within exon 3 of ß-catenin represent two of the more common gene alterations in human hepatocellular carcinoma (HCC). One exposure implicated in the development of liver cancer is hepatitis B or C viral infection, which causes chronic destruction and regeneration of liver parenchyma. Treatment of rats with high doses of the tobacco-specific nitrosamine 4-methylnitrosamino-1-(3-pyridyl)-1-butanone (NNK) also causes liver toxicity and a high incidence of tumors. The purpose of the current investigation was to define the prevalence of genetic alterations in p16 and ß-catenin in NNK-induced rat liver cancer to determine if the molecular mechanisms seen in human tumors are the same in this animal model. DNA isolated from 15 adenomas and 14 carcinomas was examined for methylation of p16 by methylation-specific PCR. p16 methylation was detected in five of 15 adenomas and eight of 14 carcinomas (45% of all tumors). Methylation of p16 was extensive within the 5'-untranslated region and exon 1{alpha}, areas shown to correlate with loss of gene transcription. Liver tumors were also screened for mutations within exon 3 of ß-catenin. Single strand conformation polymorphism and DNA sequencing revealed five mutations in four of 29 tumors (14%). Mutations were present in three adenomas and one carcinoma and were located within codons 33, 36 or 37. All mutations resulted in amino acid substitutions; three of these mutations occurred at potential serine phosphorylation sites. Our results link two important regulatory pathways altered in human HCC to cancer induced in the rat NNK model. The fact that common genetic alterations are observed between rodent and human HCC suggests that the rat NNK model could be useful for identifying additional genetic alterations critical to the initiation of HCC.

Abbreviations: GGT+, {gamma}-glutamyl transpeptidase-positive; GSK-3ß, glycogen synthase kinase-3ß; HCC, hepatocellular carcinoma; MSP, methylation-specific PCR; NNK, 4-methylnitrosamino-1,3-pyridyl-1-butanone; p16, p16INK4a; RFLP, restriction fragment length polymorphism; SSCP, single strand conformation polymorphism; 5'-UTR, 5'-untranslated region.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Hepatocellular carcinoma (HCC) is one of the most common diseases worldwide. It is endemic in Southeast Asia and sub-Saharan Africa and is rising in incidence in the USA (1). Although HCC is the major malignancy associated with the liver, the molecular mechanisms underlying its development are still being elucidated. Alterations in oncogenes and growth factors such as c-myc and cyclin D1 and inactivation of tumor suppressor genes such as p53 and Rb have been identified in human HCC (25). Alterations recently detected in the p16INK4a (p16) and ß-catenin genes have been substantiated and appear to play an important role in the pathogenesis of human HCC (611).

The p16 tumor suppressor gene is frequently altered in human neoplasms. p16 is an inhibitor of the cyclin-dependent kinases 4 and 6, which bind cyclin D1 and phosphorylate Rb. Thus, p16 inhibits cell cycle progression by maintaining Rb in the unphosphorylated state. This inhibition is lost if either p16 or Rb is inactivated and can result in abnormal cell cycling and growth (12). The predominant mechanism for inactivation of the p16 gene in most human neoplasms is aberrant methylation of a CpG island that extends from the promoter region into exon 1{alpha} of p16 and leads to transcriptional silencing (1316). Inactivation of p16 by hypermethylation is seen in 63–73% of liver tumors (9,11), while mutations are detected in <4% of tumors (17). Mutations or deletions of ß-catenin are localized in exon 3 and are also frequent events in many human cancers. ß-Catenin is a ubiquitous intracellular protein important in both cadherin-mediated cell adhesion and in cell differentiation and proliferation by its association with the Wnt signal transduction pathway. Mutations in exon 3 of ß-catenin have been detected in 12–41% of human liver tumors (68,10).

While the molecular pathways altered in human HCC are still being identified, the causative agents for this cancer are generally considered to involve infection with the hepatitis B or C virus, exposure to aflatoxin B1 and alcoholic cirrhosis of the liver. All of these agents may initiate the oncogenic process, in part through chronic destruction and regeneration of liver cells. The regenerative process, which involves a rapid turnover of cells, may promote genetic instability in hepatocytes and lead to the development of HCC (18). Like viral hepatitis, the tobacco-specific nitrosamine 4-methylnitrosamino-1-(3-pyridyl)-1-butanone (NNK) also causes hepatocellular toxicity, manifested as centrilobular necrosis and a high incidence of liver cancer in rats (1922). The rapid cell proliferation following acute hepatocellular injury from high doses of NNK most likely leads to the accumulation of genetic damage (21). Thus, we hypothesize that the molecular mechanisms underlying NNK-induced liver cancer in rats will parallel those seen in human HCC. This hypothesis was tested by defining the prevalence of alterations in the p16 and ß-catenin genes in NNK-induced HCC.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Tissue samples and nucleic acid isolation
Hepatocellular tumors were induced in male Fischer 344 rats by treatment with NNK three times a week (50 mg/kg i.p.) for 20 weeks (21). DNA was isolated from 29 of these frozen tumors by digestion with 1% pronase, followed by standard phenol/chloroform extraction and ethanol precipitation. Based on previous histological diagnosis (20) 15 and 14 of the tumors were classified as adenomas and carcinomas, respectively.

Single strand conformation polymorphism (SSCP)
Hepatocellular tumors were screened for mutations in exon 3 of the ß-catenin gene by SSCP as described by Orita et al. (23). After an initial 5 min denaturation at 94°C, exon 3 amplification was carried out for 35 cycles, each consisting of denaturation at 94°C for 60 s, annealing at 55°C for 60 s and extension at 72°C for 60 s, to generate a 227 bp product. Primer sequences were as follows: 5'-GCTGACCTGATGGAGTTGGA-3' (sense) and 5'-CTACTTGCTCTTGCGTGAA-3' (antisense). Water blanks, in which no template DNA was added, were included in each experiment, and no PCR product was detected in these controls. A second PCR reaction to incorporate [32P]dCTP was conducted using the same primers and 2 µl of the first stage reaction. Amplification was carried out for 15 cycles using the same program. 32P-labeled amplimers were resolved under two sets of non-denaturing conditions as follows: 6% acrylamide, 5% glycerol and 1x Tris–borate–EDTA buffer, run at 4°C for 6 h at 40 W; 0.5x mutation detection enhancement (a proprietary acrylamide solution for SSCP; FMC Bioproducts, Rockland, ME), run at room temperature for 17 h at 3 W.

Deletion analysis
RNA was isolated from frozen liver tumors using the Stratagene Strataprep Total RNA Miniprep Kit (Stratagene, La Jolla, CA). First strand cDNA was generated at 42°C from 3 µg total RNA using a SuperScript Kit (Life Technologies, Rockville, MD). Amplimers encompassing exons 1–5 of ß-catenin were generated from these cDNAs. After an initial denaturation at 94°C, amplification was carried out for 40 cycles, each consisting of denaturation at 94°C for 30 s, annealing at 50°C for 30 s and extension at 72°C for 30 s, to generate a 320 bp product. Primer sequences were as follows: 5'-TGGACAATGGCTACTCAA-3' (sense) and 5'-TTCCCTGAG ACACTAGAT-3' (antisense). Bands were resolved on 8% non-denaturing acrylamide gels.

Methylation-specific PCR (MSP)
The methylation status of the p16 gene was determined by MSP (24). In MSP genomic DNA is modified by treatment with sodium bisulfite, converting all unmethylated cytosines to uracil, and then to thymine during the subsequent PCR reaction. Two sets of primers were used to amplify the region of interest: one pair recognizes a sequence in which CpG sites are unmethylated (modified to UpG by the bisulfite treatment); the other recognizes a sequence in which CpG sites are methylated (unmodified by the bisulfite treatment). Primers are localized to regions containing frequent cytosines in order to distinguish unmodified from modified DNA. These primers contain CpGs located at the 3'-end to provide maximal discrimination between methylated and unmethylated DNA. Primer sequences and PCR conditions have been described previously (15). Primers were localized to regions in and around the transcription start site of the p16 gene, a region shown to correlate with loss of gene expression (15,16,24). PCR amplification was performed using ~250 ng bisulfite-modified DNA as template. Normal liver DNA was used as a control in all PCRs performed. One common 5' primer was paired with three different 3' primers to examine methylation density throughout exon 1{alpha} of p16 (Figure 1AGo). The products were visualized on 2% agarose gels.




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Fig. 1. (A) The 5'-UTR and exon 1{alpha} of the rat p16 gene. CpG sites are numbered 1–13; the seven CpG sites examined with MSP primers are shown in bold. Location and orientation of the MSP primers are indicated by the arrows under the sequence. Sequencing of bisulfite-modified DNA from tumors assayed for methylation status of CpG sites 4–10. (B) MSP analysis for p16 methylation in rat liver tumors induced by NNK. Bisulfite-modified DNA was amplified with the methylation-specific primer pair that encompassed CpG sites 2, 3, 11 and 12 as shown in (A). Tumors positive for p16 methylation (T7, T13, T24 and T26) are depicted by the presence of a 155 bp product. NL, normal liver; WB, water blank.

 
Sequencing of p16 and ß-catenin
Exon 1{alpha} of p16 from normal liver tissue and from two liver tumors shown to be positive for methylation was amplified by MSP using the outermost 3' primer paired with the common 5' primer. DNA from normal liver was amplified using primers that anneal to unmethylated template, while DNA from the tumor samples was amplified using methylation-specific primers. The 156 bp PCR product was ligated into the PCR II vector using the TA cloning kit (Invitrogen, San Diego, CA). Four clones from each tumor sample and normal liver were sequenced.

For tumors that showed altered electrophoretic mobility of ß-catenin exon 3 by SSCP, the band was excised from the acrylamide gel. DNA was eluted by incubation of the gel slice overnight at 37°C in dH2O and reamplified with ß-catenin exon 3 primers. The PCR product was ligated into the PCR II vector and sequenced. To look for mutations in the remaining tumors and to determine the percent mutant fraction in tumors where mutations were identified by SSCP, ß-catenin was amplified from genomic DNA using exon 3 primers linked to the M13 forward or reverse sequence. The resulting 266 bp DNA product was directly sequenced. All samples were commercially sequenced in duplicate in both directions (Southwest Scientific Resources, Albuquerque, NM).

Restriction fragment length polymorphism (RFLP) analysis of ß-catenin mutations
Mutations identified in exon 3 of ß-catenin resulted in the creation or deletion of restriction enzyme sites in the three adenomas. To confirm the presence of these mutations, PCR products were treated with the restriction enzyme BstN1 or HinfI. All digestion products were resolved on 8% neutral acrylamide gels.


    Results
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 Abstract
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 Materials and methods
 Results
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 References
 
Methylation of p16 in NNK-induced liver lesions
The development of HCC induced by NNK involves the production of multiple histochemically {gamma}-glutamyl transpeptidase-positive (GGT+) foci (Belinsky, unpublished results). It is likely that only a very small subset of these foci then progress to adenoma and ultimately to carcinoma, as seen with other hepatic carcinogens (2527). Equivalent numbers of adenomas and carcinomas were evaluated for aberrant promoter methylation of the p16 gene using MSP. The density of methylation within the 5'-untranslated region (5'-UTR) and exon 1{alpha}, the region previously shown to correlate with complete loss of transcription (1316), was determined through use of multiple MSP primers. Each tumor sample was amplified with a common 5' primer paired with three different 3' primers to assay a total of seven CpG sites (Figure 1AGo). Only unmethylated p16 alleles were detected in liver from sham-exposed rats (not shown). Overall, methylation of the p16 gene was detected in five of 15 adenomas and eight of 14 carcinomas (see Figure 1BGo for representative results). Unmethylated alleles were also simultaneously detected in these samples because the tumors were contaminated with stromal and inflammatory cells (not shown). Four carcinomas were methylated at all seven CpG sites. The remaining tumors were methylated at three to five CpG sites within the island (Table IGo). The density of methylation within adenomas paralleled that seen in carcinomas. One adenoma showed complete methylation and the remaining four lesions contained methylation at three to six CpG sites (Table IGo). Two tumors with complete methylation, as determined by MSP and normal liver were selected for bisulfite sequencing to confirm the MSP data and to assay an additional four CpG sites (nos 4–7) (Figure 1AGo). A portion of the 5'-UTR and exon 1{alpha} was amplified using the MSP primers flanking this portion of the p16 CpG island. The same region was amplified for normal liver using MSP primers specific to unmethylated alleles. All CpG sites analyzed by sequencing were methylated in both tumors; no methylation was detected in normal liver (not shown).


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Table I. Density of p16 methylation in rat liver tumors induced by NNK
 
Mutation of the ß-catenin gene in NNK-induced liver lesions
Mutations in exon 3 of the ß-catenin gene have been detected in human HCC and shown to alter protein function (68,10). SSCP analysis of adenomas and carcinomas detected alterations in electrophoretic mobility in one carcinoma (no. 14) and two adenomas (nos 11 and 16) (Figure 2AGo). Altered bands detected in tumors 11, 14 and 16 were excised from the gel, reamplified, cloned and sequenced. Mutations were identified in codons 36 or 37, or both (Table IIGo). All mutations resulted in amino acid changes: two CAC (histidine)->GAC (aspartate), one TCT (serine)->GCT (alanine) and one TCT (serine)->CCT (proline) (Table IIGo). To establish the mutant fraction within these three tumors and determine if additional mutations were present, all adenomas and carcinomas were evaluated by direct sequencing of the exon 3 PCR product. In addition to confirming the mutations detected by SSCP, a mutation was detected in one additional adenoma (no. 5) (Figure 2BGo and Table IIGo). This mutation was localized to codon 33 and resulted in an amino acid change TCT (serine)->GCT (alanine). Three of the five mutations observed involved loss of serines from GSK-3ß phosphorylation sites (codons 33 and 37). The mutations seen at codon 36 are neighbors to one of these sites. In all tumors with mutations a wild-type base was also detected (Figure 2BGo), consistent with their dominant nature (8,28). The mutant fraction of each sample was estimated by weighing the wild-type and mutant peaks. The fraction of mutant alleles was 53% for the carcinoma and ranged from 36 to 65% for the adenomas (not shown). RFLP was also used to confirm mutations identified by sequencing in the three adenomas (not shown). The T->C mutation in codon 37 (tumor 11) resulted in the creation of a BstN1 restriction enzyme site that was not present in the wild-type. The C->G mutation in codon 36 (tumors 11 and 16) resulted in the formation of one HinfI site in addition to the two already present in the wild-type. The T->G mutation in codon 33 (tumor 5) resulted in the deletion of a HinfI restriction site. Interestingly, none of the tumors with mutations in the ß-catenin gene was positive for p16 methylation.




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Fig. 2. Mutation analysis for exon 3 of ß-catenin. (A) Representative SSCP analysis. A 0.5x mutation detection enhancement gel is shown. DNA was amplified with the primer pair that encompassed exon 3 of ß-catenin. DNA fragments with altered electrophoretic mobility are indicated by arrows. (B) Direct sequencing of ß-catenin exon 3 from wild-type rat liver (WT) and rat liver tumors. Sequencing data for tumors 5 and 14 are shown in the sense direction; data for tumors 11 and 16 are shown in the antisense direction. The mutant bases are displayed in boxes below the wild-type base.

 

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Table II. Summary of ß-catenin exon 3 mutations in liver tumors induced by NNK
 
Deletions of the ß-catenin gene in NNK-induced liver lesions
Recent studies have also detected deletions in murine hepatoblastomas and HCCs induced by environmental carcinogens (28,29) and in human HCCs (7,10). In order to screen for deletions in rat hepatocellular adenomas and carcinomas, exons 1–5 were amplified using cDNA generated from the 29 tumors. However, no deletions were detected in this region (not shown).


    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
This investigation demonstrates that methylation of the p16 tumor suppressor gene is a frequent event in the development of HCC in the rat. The density of p16 methylation was extensive in an area of the gene that correlates with loss of transcription (1316). Mutations in exon 3 of ß-catenin occurred less frequently; however, all mutations identified were localized to codons associated with altered protein function (8,10). Additionally, alterations in both p16 and ß-catenin were early events, evolving at least by the stage of adenoma. Thus, HCCs induced by NNK in the rat arise via molecular mechanisms linked to the development of HCC in humans.

Methylation of the p16 gene was seen in NNK-induced HCCs at a frequency similar to that observed in human tumors (9,11,30) and appeared to increase in frequency during disease progression from adenoma to carcinoma (38 to 62%, respectively). While the timing of inactivation of p16 has not been defined in human HCC, our results in rat tumors parallel findings in other rodent and human tissues. Methylation of p16 is an early event in esophageal and lung cancer (16,31). For example, in rat lung tumors induced by NNK, methylation of p16 was frequently detected in precursor lesions to adenocarcinoma, i.e. adenomas and alveolar hyperplasias. In the rat lung, where the conversion rate of hyperplasia to adenoma and ultimately carcinoma is high, the frequency of p16 methylation was similar between premalignant lesions and adenocarcinomas (16). While inactivation of p16 is also an early event in human squamous cell carcinoma, the frequency of p16 methylation increased during disease progression from basal cell hyperplasia to squamous metaplasia to carcinoma in situ. The lower frequency seen for p16 methylation in basal cell hyperplasia and squamous metaplasia is consistent with the fact that many of these proliferative lesions in the lung arise due to chronic wounding of the bronchial epithelium by the carcinogens within cigarettes, then regress after tobacco cessation (16). This scenario is similar to that seen in the liver, where hundreds of hepatic GGT+ foci are induced following exposure to environmental carcinogens, yet only a few tumors ultimately develop in the exposed liver (2527). In experimental systems, loss of p16 appears to act as a `gate keeper' in permitting cells to pass through early steps of cellular immortalization (16). Thus, our findings in NNK-induced HCCs are consistent with p16 playing a pivotal role in the early stages of this disease.

Loss of control of intracellular ß-catenin levels by mutation has been proposed as an important step in the development of human and murine HCC (68,10,28). ß-Catenin, a key component of the Wnt signaling pathway, is up-regulated in many cancers following mutation of either the APC or ß-catenin genes. This increase in Wnt signaling is associated with cell proliferation and inhibition of apoptosis (32). Normally, in the absence of Wnt signals ß-catenin is rapidly degraded after targeted phosphorylation of highly conserved serine and threonine residues in the N-terminus by GSK-3ß. Mutation at one or more of the phosphorylation sites inhibits degradation of the protein, thus enhancing its availability as a transcription factor (33). All mutations observed in our study were located at either codons containing putative serine GSK-3ß phosphorylation sites (codons 33 and 37) or in a neighboring codon (codon 36). In studies performed by other investigators mutations within these codons were associated with an increase in cytoplasmic and/or nuclear localization of the ß-catenin protein (8,29). Cellular localization was not examined in this study because fixed tumor blocks from the NNK carcinogenicity study corresponding to the frozen tissue used in this study were no longer available (21).

The timing of mutation in ß-catenin during the development of human HCC is not known. The results of our study, which parallel findings in chemically induced murine HCC (28,29), indicate that mutation of ß-catenin may play a role in initiation in some HCCs. We and others have readily detected mutations in adenomas. Moreover, Devereux et al. (28) detected strong positive immunostaining for ß-catenin protein in an altered hepatocellular focus. In murine studies, alterations in ß-catenin in HCCs were largely point mutations; however, some small deletions were identified within exon 3. Only point mutations were seen in our study, a finding similar to that reported by Huang et al. (8) for HCCs associated with hepatitis C virus infection.

A common feature of all studies of ß-catenin is the lack of an association between the mutation and a specific DNA adduct (68,10). In the case of NNK none of the mutations was indicative of the G:C->A:T or G:C->T:A base mispairings associated with the O6-methylguanine and pyridyloxobutyl DNA adducts, respectively (34). The observed transition mutation in codon 37 could have resulted from the T:A->G:C base mispairing associated with the O4-methylthymine adduct, which is also formed during activation of NNK (34). Similarly, the observed mutations in ß-catenin in HCCs induced by aflatoxin B1 do not reflect the G:C->T:A transversion associated with the aflatoxin B1N7-guanine DNA adduct (35,36). The mutations observed in this and other studies may stem from single-stranded breaks and oxidative damage associated with the acute hepatoxicity of carcinogens like NNK (21,22,34,37).

Genetic alterations in both ß-catenin and p16 were not seen in the same tumors, suggesting that HCC associated with exposure to NNK may evolve through different pathways. Similar comparisons within tumors for these alterations have not been made in human HCC. The fact that liver cancer occurs through the alteration of some of the same critical regulatory pathways in both humans and rodents suggests that the rat NNK model could be useful for further characterizing the aberrant pathways responsible for the initiation and progression of HCC.


    Notes
 
1 To whom correspondence should be addressed Email: sbelinsk{at}lrri.org Back


    Acknowledgments
 
This investigation was supported by the Office of Biological and Environmental Research, US Department of Energy, through Cooperative Agreement DE-FC04-96AL76406.


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received September 5, 2000; revised November 8, 2000; accepted November 15, 2000.