Sep 20, 1996 - Rumsby,P.C., Evans,J.G., Phillimore,H.E., Carthew,P. and Smith,A.G.. (1992) Search for Ha-ras codon 61 mutations in liver caused by. 236.
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Carcinogenesis vol.18 no.1 pp.233–236, 1997
SHORT COMMUNICATION
Lack of p53 and ras mutations in Helicobacter hepaticus-induced liver tumors in A/JCr mice
Marek A.Sipowicz3, Christopher M.Weghorst, Yih-Horng Shiao, Gregory S.Buzard1, Richard J.Calvert2, Miriam R.Anver1, Lucy M.Anderson and Jerry M.Rice Laboratory of Comparative Carcinogenesis, National Cancer Institute-FCRDC, Frederick, MD 21702, USA, 1Intramural Research Support Program, SAIC Frederick, Frederick, MD, USA and 2 Clinical Research and Review Staff, Office of Special Nutritionals, Food and Drug Administration, Laurel, MD, USA. 3To
whom correspondence should be addressed
Helicobacter hepaticus is a recently discovered bacterium that invades mouse liver causing chronic active hepatitis followed by development of preneoplastic hepatocellular foci, hepatocellular adenomas and carcinomas. This establishes a unique animal model for study of the mechanisms of cancer development due to a chronic bacterial infection. A possible mechanism of bacteriaassociated tumorigenesis is mutation of oncogenes or tumor suppressor genes. Since mutations in ras oncogenes have been widely detected in a variety of chemically induced and spontaneous mouse liver tumors and specific mutations in the p53 tumor suppressor gene have been associated with human bladder cancers attributed to chronic schistosomal infection, we studied exons 1 and 2 of the N-, K- and H-ras genes and exons 5–8 of the p53 gene for the presence of point mutations in 25 liver tumors from 10 naturally infected A/JCr mice, ranging in age from 16 to 24 months. The 20 adenomas and five carcinomas varied in size from 0.1 to 2.3 cm and arose in livers characterized by a wide assortment of pathological profiles, including hepatitis, inflammation, hyperplasia, hypertrophy, leukocyte infiltration, necrosis and focal phenotypic alteration. DNA samples extracted from formalin-fixed paraffin-embedded tissues were screened by PCR/SSCP analysis and showed no mutations in the analyzed genes. Complete absence of mutations in ras genes in 25 mouse liver tumors is unusual. Other genes may be targeted or H.hepaticus infection causes liver cancer through other pathways than direct damage to DNA.
Several Helicobacter species have been isolated from the gastrointestinal tract of a wide variety of mammals, including humans, non-human primates, cheetahs, ferrets, cats, dogs, rats and mice (1). Human infection with Helicobacter pylori causes gastric ulcers and has been associated with a higher incidence of gastritis and gastric adenocarcinoma, the second most frequent cancer worldwide (2). However, investigation of the mechanism of carcinogenesis in association with chronic infection in vivo has been constrained by lack of an animal model. This deficiency has recently been partially corrected by discovery of a Helicobacter species, H.hepaticus, which *Abbreviations: ROS, reactive oxygen species; SSCP, single-strand conformation polymorphism; NDEA, N-nitrosodiethylamine. © Oxford University Press
colonizes the lower intestinal tract of mice and infects the liver of susceptible strains (3). The resulting chronic active hepatitis is followed by development of hepatocellular tumors (4,5). Causation of mouse liver tumors by this novel bacterium establishes a unique animal model for investigation of the mechanisms of cancer development subsequent to chronic infection and inflammation. Since responses to infection often include elevated levels of reactive oxygen species (ROS) and/or nitric oxide, both potentially mutagenic agents (6), a possible mechanism of bacteria-associated tumorigenesis is mutation of oncogenes or tumor suppressor genes. Mutations in the H-, K- and N-ras oncogenes have been detected in a wide spectrum of chemically induced and spontaneous liver tumors in different mouse strains (7–13). Whereas the p53 tumor suppressor gene is often mutated in human hepatocellular carcinomas (14), such mutations have rarely been detected in rodent liver tumors (11,15–17). However, a relatively high incidence of p53 mutations were found in rat liver tumors caused by tamoxifen (18) and in liver carcinomas in rats caused by a cholinedeficient diet (19), which also was found to increase ROS (20). These findings suggest that under some circumstances, including ones involving ROS, p53 mutations may contribute to rodent liver tumorigenesis. Furthermore, a significant increase in p53 mutations at CpG sites in human bladder cancers associated with schistosomiasis suggested an infectionrelated nitric oxide etiology (21). Since Helicobacter infection in A/JCr mouse liver induces oxidative damage (22), it was therefore of interest to determine whether liver tumors caused by this bacterial infection presented ras or p53 mutations. DNA was extracted from formalin-fixed paraffinembedded tissue, as described previously (23), for 25 liver tumors (20 adenomas and five carcinomas) from 10 naturally infected A/JCr male mice, ranging in age from 16 to 24 months, and showing varying degrees of hepatitis and other liver lesions and extent of neoplasia (Table I). Exons 1 and 2 of the ras genes and exons 5–8 of the p53 genes were amplified by PCR. For amplification of ras genes, we used primers previously published by Manam et al. (24) (Table II). p53 was amplified with intron-derived primers (17; Table III). All chosen primers were analyzed with Oligo 5.0 software (National Biosciences, Plymouth, MN) for optimal annealing temperature. Amplification was carried out with the GeneAmp PCR kit (Perkin Elmer, Norwalk, CT). The reaction mixtures contained one tenth of the DNA extract as template, 10 mM Tris, pH 8.3, 50 mM KCl, 125 µM dNTPs, 1.5 mM MgCl2, 0.15–0.2 µM of each upstream and downstream primer and of 1.25 U Taq polymerase in a volume of 50 µl. Two drops of mineral oil were added on top of the mixture and samples were amplified for 40 cycles in a DNA Thermal Cycler (4800 Perkin Elmer). For amplification of the ras genes, the initial denaturation condition was 94°C for 5 min, followed by 39 cycles of annealing at 60°C for 30 s, extension at 72°C for 1 min and denaturation at 94°C for 30 s. A final extension 233
M.A.Sipowicz et al.
Table I. Microscopic characteristic of H.hepaticus-associated changes in liver Animal age
Tumor no.
Liver tumor histology
Tumor size (cm)
Hepatic pathology Helicobacter hepatitis 1111 Inflammation multifocal 111 Hyperplasia multifocal 1111 Leukocytic infiltration multifocal 111 Hypertrophy multifocal 111 Focus of cell alteration Helicobacter hepatitis 111 Necrosis multifocal 1 Hyperplasia multifocal 1111 Hypertrophy multifocal 111 Extramedullary hematopoiesis 11 Focus of cell alteration Helicobacter hepatitis 11 Inflammation multifocal 11 Necrosis multifocal 11 Hyperplasia multifocal 11 Leukocytic infiltration multifocal 111 Helicobacter hepatitis 111 Inflammation multifocal 11 Necrosis multifocal 1 Hyperplasia multifocal 111 Leukocytic infiltration multifocal 111 Hypertrophy multifocal 111 Focus of cell alteration Helicobacter hepatitis 111 Inflammation multifocal 11 Hyperplasia multifocal 111 Leukocytic infiltration multifocal 11 Hyperplasia multifocal 111 Focus of cell alteration Helicobacter hepatitis 1111 Inflammation multifocal 111 Hepatocellular necrosis 11 Hyperplasia multifocal 111 Leukocytic infiltration multifocal 1111 Hypertrophy multifocal 1111 Focus of cell alteration Helicobacter hepatitis 111 Inflammation multifocal 11 Focus of cell alteration Hepatocellular necrosis multifocal 1 Hyperplasia multifocal 111 Hypertrophy multifocal 11 Helicobacter hepatitis 111 Focus of cell alteration multifocal 111 Leukocytic infiltration multifocal 111 Hepatocellular necrosis multifocal 1 Inflammation multifocal 111 Hyperplasia multifocal 111 Hypertrophy multifocal 1 Helicobacter hepatitis 111 Hepatocellular necrosis multifocal 1 Focus of cell alteration multifocal 111 Leukocytic infiltration multifocal 111 Hyperplasia multifocal 111 Hypertrophy multifocal 11 Helicobacter hepatitis 111 Leukocytic infiltration multifocal 111 Hepatocellular necrosis multifocal 1 Inflammation multifocal 111 Hyperplasia multifocal 111 Hypertrophy multifocal 11
22 months
1 2 3 4
Adenoma; clear cell Carcinoma; basophilic Adenoma; clear cell Adenoma; eosinophilic
0.5 1.2 0.3 0.5
22 months
5 6 7 8
Adenoma; eosinophilic Carcinoma; basophilic Carcinoma; basophilic Adenoma; eosinophilic
1.2 2.3 1.2 0.7
22 months
9
Adenoma; basophilic
1.0
24 months
10 11 12 13
Adenoma; basophilic Adenoma; eosinophilic Adenoma; basophilic Carcinoma; basophilic
1.3 1.0 0.8 1.3
16 months
14 15
Adenoma; eosinophilic Adenoma; basophilic
0.3 0.3
18 months
16 17 18 19 20 21
Adenoma; Adenoma; Adenoma; Adenoma; Adenoma; Adenoma;
1.2 1.3 0.5 1.2 0.7 1.2
18 months
22
Adenoma; clear cell
0.7
18 months
23
Adenoma; clear cell
0.5
18 months
24
Carcinoma; clear cell
1.7
18 months
25
Adenoma; clear cell
0.1
eosinophilic basophilic eosinophilic basophilic basophilic eosinophilic
Microscopic changes: 1111, marked; 111, moderate; 11, mild; 1, minimal.
was performed at 72°C for 5 min. Amplification of the p53 and cycling conditions were as published (17). Amplified samples were then screened for mutations with cold single-strand conformation polymorphism (SSCP) 234
analysis as described by Hongyo et al. (25). Constant buffer temperature was maintained at 10°C for analysis of the H- and N-ras genes and 25°C was used for K-ras. SSCP conditions used for analysis of the p53 gene were as previ-
Helicobacter hepaticus-induced liver tumors
Table II. Primers used for amplification of mouse ras gene exons 1 and 2 Region amplified (size) Sequence (59→39) H-ras exon 1 (111 bp) H-ras exon 2 (138 bp) K-ras exon 1 (111 bp) K-ras exon 2 (141 bp) N-ras exon 1 (108 bp) N-ras exon 2 (144 bp)
ATG CTC GAC GGC ATG CTC GAC TAT ATG TAT GAT ATT
ACA TAT TCC AAA ACT TAT TCC GGC ACT GGT TCT GAT
GAA AGT TAC TAC GAG CGT TAC AAA GAG GGG TAC GGC
TAC GGG CGG ACA TAT AGG AGG TAC TAC ATC CGA AAA
Strand AAG ATC AAA GAG AAA GTC AAA ACA AAA ATA AAG TAC
CTT ATA CAG GAA CTT GTA CAA AAG CTG TTC CAA ACA
GTG CTC GTA GCC GTG CTC GTA AAA GTG ATC GTG GAG
GTG GTC GTC CTC GTG ATC GTA GCC GTG CAC GTG GAA
Sense Antisense Sense Antisense Sense Antisense Sense Antisense Sense Antisense Sense Antisense
Table III. Primers used for amplification of mouse p53 gene exons 5–8 Region amplified (size)
Sequence (59→39)
Strand
Exon 5 (232 bp)
TT CCA GTA CTC TCC TCC CCT CAA AGG CTG CCA GTC CTA ACC CCA CAG TCC CGG CTT CTG ACT TAT TC GAC GCA CAA ACC AAA ACA AA GAG GTA GGG AGC GAC TTC AC GCT GGG GAA GAA ACA GGC TAA CCT ACT GCC TTG TGC TGG TCC TTT TCT ACA GGC TCC TCC GCC TCC TTG GTC
Sense Antisense Sense Antisense Sense Antisense Sense Antisense
Exon 6 (172 bp) Exon 7 (198 bp) Exon 8 (208 bp)
ously described (17). Samples with mobility band shifts were re-examined for conformation by repeated PCR and SSCP analysis. DNA sequencing was performed by the dideoxy termination method with [α-35S]dATP incorporation using the CircumVent DNA sequencing kit (New England Biolabs, Beverly, MA). Sequences were then resolved with a 6% polyacrylamide–8 M urea gel and autoradiography was carried out for 3–5 days of X-ray film exposure. None of the liver tumors analyzed showed any mutations in exons 1 or 2 of the three ras oncogenes or in exons 5–8 of the p53 tumor suppressor gene. In other studies, the frequency of H-ras mutations has varied from 12 to 56% in liver tumors of untreated mice, and from 35 to 97% in those of chemically treated animals, with the CAA→AAA transversion in codon 61 being the most frequent (26). Treatment with certain chemicals, e.g. N-hydroxyl-2-acetylaminofluorene in CD-1 and B6C3F1 mice (8,27), 1-hydroxy-2,3-dehydroestragole and vinyl carbamate in B6C3F1 mice (27), 7,12-dimethylbenz[a]anthracene in CD-1 mice (8) and methylene chloride (28), dichloroacetic acid and trichloro- and tetrachlorethylene in B6C3F1 mice (29) resulted in specific patterns of mutations in liver tumors that differed significantly by statistical test from that in spontaneous tumors in the same study. These results have thus provided evidence for a direct role of chemical mutagenesis in mouse liver tumor etiology. The complete absence of ras mutations from a series of mouse liver tumors, especially a series as large as ours (25 tumors), is rare. For some tumor promotion protocols, the incidence of ras mutations was lower in the promoted tumors versus those appearing after initiator treatment only. Experimental situations showing this phenomenon have included
dieldrin and phenobarbital as promoters in C3H/He mice (30), hexachlorobenzene and Aroclor 1254 in C57BL/10ScSn mice (31) and ciprofibrate in B6C3F1 mice (32). These findings suggest that promotion of mouse liver tumors involves a mechanistic pathway not including ras mutations. Thus, H.hepaticus infection may be causing liver tumors by a similar, promotion-like mechanism, although A/J mice, a strain with a low spontaneous liver tumor incidence, have not been studied in this context. Our p53 data are consistent with previous reports of murine liver tumors. No p53 mutations were found in nine spontaneous and 34 chemically induced tumors in CD-1 mice (15), in 22 of 66 randomly selected N-nitrosodiethylamine (NDEA)-induced liver tumors in B6C3F1 mice (11) or in 93 NDEA-induced liver tumors in three different strains (C3H/He, C57BL/6J and B6C3F1) (16). However, in the latter study, four single base substitutions occurred in 12 hepatoma cell lines established from liver tumors. We also observed a consistent band shift in exon 2 of the H-ras gene from a primary tissue culture of an adenoma from a H.hepaticus-infected liver. The mutation was confirmed in a second PCR-SSCP analysis. Sequencing analysis showed silent mutations in codons 50 (ACA→ACG) and 52 (CTA→ TTA) that did not result in amino acid changes. Since such mutations were not detected in the primary tumor, we believe that they arose during cell culture. In sum, we have begun to explore the mechanism by which H.hepaticus causes liver cancer in mice, by analyzing a series of hepatic adenomas and carcinomas for mutational changes in the p53 tumor suppressor gene and H-, K- and N-ras oncogenes. The mutational analysis failed to establish alterations in the p53 or ras genes as possible pathways for H.hepaticus-associated liver tumors in mice. The complete absence of such mutations is consistent with a tumor promotionlike mechanism, although mutational change in other genes of course remains as a possibility. References 1. Cover,T.L. and Blaser,M.J. (1995) Helicobacter pylori: a bacterial cause of gastritis, peptic ulcer disease, and gastric cancer. ASM News, 61, 21–26. 2. Parsonnet,J. (1993) Helicobacter pylori and gastric cancer. Gastroenterol. Clinics N Am., 22, 89–104. 3. Fox,D.G., Dewhirst,F.E., Tully,J.G., Paster,B.J., Yan,L., Taylor,N.S., Collins,M.J.,Jr, Gorelick,P.L. and Ward,J.M. (1994) Helicobacter hepaticus sp. nov., a microaerophilic bacterium isolated from livers and intestinal mucosal scrapings from mice. J. Clin. Microbiol., 32, 1238–1245. 4. Ward,J.M. et al. (1994) Chronic active hepatitis and associated liver tumors in mice caused by a persistent bacterial infection with a novel Helicobacter species. J. Natl Cancer Inst., 86, 1222–1227. 5. Ward,J.M., Anver,M.R., Haines,D.C. and Benveniste,R.E. (1994) Chronic active hepatitis in mice caused by Helicobacter hepaticus. Am. J. Pathol., 145, 959–968. 6. Wink,D.A. et al. (1991) DNA deaminating ability and genotoxicity of nitric oxide and its progenitors. Science, 245, 1001–1003. 7. Stanley,L.A., Devereux,T.R., Foley,J., Lord,P.G., Maronpot,R.R., Orton,T.C. and Anderson,M.W. (1992) Proto-oncogene activation in liver tumors of hepatocarcinogenesis-resistant strains of mice. Carcinogenesis, 13, 2427–2433. 8. Manam,S., Storer,R.D., Prahalada,S., Leander,K.R., Kraynak,A.R., Ledwith,B.J., van Zwieten,M.J., Bradley,M.O. and Nichols,W.W. (1992) Activation of Ha-, Ki-, and N-ras genes in chemically induced liver tumors from CD-1 mice. Cancer Res., 52,1603–1608. 9. Lord,P.G., Hardaker,K.J., Loughlin,J.M., Marsden,A.M. and Orton,T.C. (1992) Point mutation analysis of ras genes in spontaneous and chemically induced C57BL/10J mouse liver tumours. Carcinogenesis, 13, 1383–1387. 10. Wang,Y., Wang,Y., Stoner,G. and You,M. (1993) Ras mutations in 2acetylaminofluorene- induced lung and liver tumors from C3H/HeJ and (C3HXA/J)F1 mice. Cancer Res., 53, 1620–1624.
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M.A.Sipowicz et al. 11. Chen,B., Liu,L., Castonguay,A., Maronpot,R.R., Anderson,M.W. and You,M. (1993) Dose-dependent ras mutation spectra in N-nitrosodiethylamine induced mouse liver tumors and 4-(methylnitrosamino)-1(3-pyridyl)-butanone induced mouse lung tumors. Carcinogenesis, 14, 1603–1608. 12. Enomoto,T., Weghorst,C.M., Ward,J.M., Anderson,L.M., Perantoni,A.O. and Rice,J.M. (1993) Low frequency of H-ras activation in naturally occurring hepatocellular tumors of C3H/HeNCr mice. Carcinogenesis, 14, 1939–1944. 13. Stanley,L.A., Blackburn,D.R., Devereaux,S., Foley,J., Lord,P.G., Maronpot,R.R., Orton,T.C. and Anderson,M.W. (1994) Ras mutations in methylclofenapate-induced B6C3F1 and C57Bl/10J mouse liver tumours. Carcinogenesis, 15, 1125–1131. 14. Tabor,E. (1994) Tumor suppressor genes, growth factor genes, and oncogenes in Hepatitis B Viruses-associated hepatocellular carcinoma. J. Med. Virol., 42, 357–365. 15. Goodrow,T.L., Storer,R.D., Leander,K.R., Prahalada,S.R., van Zwieten,M.J. and Bradley,M.O. (1992) Murine p53 intron sequences 5–8 and their use in polymerase chain reaction/direct sequencing analysis of p53 mutations in CD-1 mouse liver and lung tumors. Mol. Carcinogen., 5, 9–15. 16. Kress,S., Konig,J., Schweizer,J., Lohrke,H., Bauer-Hofmann,R. and Schwarz,M. (1992) p53 mutations are absent from carcinogen-induced mouse liver tumors but occur in cell lines established from these tumors. Mol. Carcinogen., 6, 148–158. 17. Calvert,R.J., Tashiro, Y., Buzard,G.S., Diwan,B.A., and Weghorst,C.M. (1995) Lack of p53 mutations in chemically-induced mouse hepatoblastomas: an end-stage, highly malignant hepatocellular tumor. Cancer Lett., 95, 175–180. 18. Vancutsem,P.M., Lazarus,P. and Williams,G.M. (1994) Frequent and specific mutations of the rat p53 gene in hepatocarcinomas induced by tamoxifen. Cancer Res., 54, 3864–3867 19. Smith,M.L., Yeleswarapu,L., Scalamogna,P., Locker,J. and Lombardi,B. (1993) p53 mutations in hepatocellular carcinomas induced by a cholinedevoid diet in male Fischer 344 rats. Carcinogenesis, 14, 503–510. 20. Nakae,D. et al. (1994) Different roles of 8-hydroxyguanine formation and 2-thiobarbituric acid-reacting substance generation in the early phase of liver carcinogenesis induced by a choline-deficient, L-amino acid-defined diet in rats. Jpn. J. Cancer Res., 85, 499–505. 21. Warren,W., Biggs,P.J., El-Baz,M., Ghoneim,M.A., Stratton,M.R. and Venitt,S. (1995) Mutations in the p53 gene in schistosomal bladder cancer: a study of 92 tumours from Egyptian patients and a comparison between mutational spectra from schistosomal and non-schistosomal urothelial tumours. Carcinogenesis, 16, 1181–1189. 22. Sipowicz,M.A., Diwan,B.A., Anver,M.R., North,S.L., Anderson,L.M., Rice,J.M. and Kasprzak,K.S. (1995) Oxidative DNA damage in liver of Helicobacter hepaticus infected mice. Proc. Am. Ass. Cancer Res., 36, 166. 23. Wright,D.K. and Manos,M.M. (1990) Sample preparation from paraffin embedded tissue. In Innis,M.A. (ed.), PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, CA, pp. 153–158. 24. Manam,S. and Nichols,W.W. (1991) Multiplex polymerase chain reaction amplification and direct sequencing of homologous sequences: point mutation analysis of the ras genes. Anal. Biochem., 199, 106–111. 25. Hongyo,T., Buzard,G.S., Calvert,R.J. and Weghorst,C.M. (1993) ‘Cold SSCP’: a simple, rapid and non-radioactive method for optimized singlestrand conformation polymorphism analyses. Nucleic Acids Res., 21, 3637–3642. 26. Maronpot,R.R., Fox,T., Malarkey,D.E. and Goldsworthy,T.L. (1995) Mutations in the ras proto-oncogene: clues to etiology and molecular pathogenesis of mouse liver tumors. Toxicology, 101, 125–156. 27. Wiseman,R.W., Stowers,S.J., Miller,E.C., Anderson,M.W. and Miller,J.A. (1986) Activating mutations of the c-Ha-ras protooncogene in chemically induced hepatomas of the male B6C3F1 mouse. Proc. Natl Acad. Sci. USA, 83, 5825–5829. 28. Devereux,T.R., Foley,J.F., Maronpot,R.R., Kari,F. and Anderson,M.W. (1993) Ras proto-oncogene activation in liver and lung tumors from B6C3F1 mice exposed chronically to methylene chloride. Carcinogenesis, 14, 795–801. 29. Anna,C.H., Maronpot,R.R., Pereira,M.A., Foley,J.F., Malarkey,D.E. and Anderson,M.W. (1994) ras proto-oncogene activation in dichloroacetic acid-, trichloroethylene- and tetrachloroethylene-induced liver tumors in B6C3F1 mice. Carcinogenesis, 15, 2255–2261. 30. Bauer-Hofmann,R., Buchmann,A., Mahr,J., Kress,S. and Schwartz,M. (1992) The tumour promoters dieldrin and phenobarbital increase the frequency of c-Ha-ras wild-type, but not c- Ha-ras mutated focal liver lesions in male C3H/He mice. Carcinogenesis, 13, 477–481. 31. Rumsby,P.C., Evans,J.G., Phillimore,H.E., Carthew,P. and Smith,A.G. (1992) Search for Ha-ras codon 61 mutations in liver caused by
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haxachlorobenzene and Aroclor 1254 in C57BL/10ScSn mice with iron overload. Carcinogenesis, 13, 1917–1920. 32. Hegi,M.E., Fox,T.R., Belinsky,S.A., Devereux,T.R. and Anderson,M.W. (1993) Analysis of activated protooncogenes in B6C3F1 mouse liver tumors induced by ciprofibrate, a potent peroxisome proliferator. Carcinogenesis, 14, 145–149. Received on July 2, 1996; revised on September 20, 1996; accepted on September 23, 1996
