Aristolochic Acid Activates ras Genes in Rat Tumors at Deoxyadenosine Residues Heinz H. Schmeiser, Johannes W. G. Janssen, John Lyons, et al. Cancer Res 1990;50:5464-5469. Published online September 1, 1990.
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(CANCER RESEARCH 50, 5464-5469, September I, 1990]
Aristolochic Acid Activates ras Genes in Rat Tumors at Deoxyadenosine Residues1 Heinz H. Schmeiser,2 Johannes W. G. Janssen, John Lyons, Hans R. Scherf, Wolfgang Pfau, Albrecht Buchmann, Claus R. Bartram, and Manfred Wiessler InstÃ-lateof Toxicology and Chemotherapy ¡H.H. S., H. R. S., W. P., M. W.J and Institute of Biochemistry [A. B./, German Cancer Research Center, Im Neuenheimer Feld 280, 6900 Heidelberg, and Section of Molecular Biolog)\ Department of Pediatrics II, University of Vim, Prittwit:strasse 43, 7900 UIm, Federal Republic of Germany fj. W. G. J., J. L., C. R. B.J
ABSTRACT
tions found in the tumor with promutagenic DNA adducts formed by the initiator (2, 4). The ras gene family is thought to be involved in the control systems of cell growth and cell differentiation, and activated ras genes have been observed in various human malignancies and at high frequencies in colorectal cancers, pancreatic tumors, and thyroid tumors (5). Some of the most potent carcinogens known, such as aflatoxin, safrole, estragóle, and cycasin, are natural products. In 1982, Mengs and coworkers (6) reported aristolochic acid, a plant extract of Aristolochia species, to be a potent forestomach carcinogen in rats. Prior to 1982, AA3 was used as an antiin-
Aristolochic acid I (AAI), a nitrophenanthrene derivative, is the major component of the carcinogenic plant extract aristolochic acid, which has been used as a medicine since antiquity. Long term oral administration of AAI to male Wistar rats induces multiple tumors, mainly in the forestomach, ear duct, and small intestine. The presence of activated transforming genes was investigated in various tumors of 18 AAI treated rats, namely in 14 squamous cell carcinomas of the forestomach, 7 squamous cell carcinomas of the ear duct, 8 tumors of the small intestine, 3 tumors of the pancreas, 1 adenocarcinoma of the kidney, 1 lymphoma, and 2 métastasesin the lung and the pancreas. By utilizing the tumorigenicity assay and Southern blot analysis, we have detected an activated c-Ha-rai gene in the DNAs of 5 of 5 squamous cell carcinomas of the flammatory agent in several pharmaceutical preparations, forestomach. Direct sequencing of amplified material revealed an AT —» which subsequently were withdrawn from the market. AA is a TA transversion mutation at the second position of codon 61 of the cmixture of structurally related nitrophenanthrene carboxylic Ha-ras gene (CAA to CTA) in all transfectants as well as in the 5 original acids, with aristolochic acid being the major component (Fig. rat tumors. Enzymatic amplification of ras sequences followed by selective 1). oligonucleotide hybridization detected identical mutations in 93% (13 of Studies by our laboratory (7) showed that AAI was a direct 14) of forestomach tumors, in 100% (7 of 7) of ear duct tumors, and in mutagen in the Ames assay in the Salmonella strains TA 100 the lung metastasis. Among those tumors tested, we had 4 cases in which the forestomach tumors and the ear duct tumors originated from the and TA 1537 but not in the nitroreductase deficient strain TA 100NR. Using the 32P postlabeling assay developed by Randersame rat, showing the same mutation in both tissues. Moreover, similar mutations were demonstrated at c-Ki-nu codon 61 ath et al. (8), DNA adducts of AAI were detected in vitro when in 1 of 7 ear duct tumors (CAA to CAT) and in 1 of 8 tumors of the small AAI was incubated with calf thymus DNA and liver homogeintestine (CAA to CTA) as well as at c-N-ras 61 (CAA to CTA) in a nate or an enzymatic nitroreduction system consisting of xanpancreatic metastasis. Additional transfection experiments of some tu thine oxidase and hypoxanthine (9). Similar patterns of DNA mors scoring negative for ras gene mutations in dot blot analyses revealed a CAA to CTA transversion at codon 61 of the c-IIa-rav gene in 1 adducts were observed in vivo in the DNA of target and nontarget organs of Wistar rats that had received five doses of AAI forestomach tumor as well as at codon 61 of the c-N-ras in 1 hyperplasia (10 mg/kg/day) by gavage. of the pancreas and in 1 lymphoma. The apparent selectivity for mutations The purpose of this study was to identify the activated onat adenine residues in AAI induced tumors is consistent with the identi fication of an N6-deoxyadenosine-AAI adduct formed by reaction of AAI cogene(s) in tumors from Wistar rats induced by AAI. Our with DNA in vitro, suggesting that carcinogen-deoxyadenosine adducts results provide insights into the molecular mechanisms involved are the critical lesions in the tumor initiation by aristolochic acid. in tumor initiation by AAI and suggest that carcinogen-deoxy
adenosine adducts may be the critical lesions. INTRODUCTION The initiation of cancer by naturally occurring or synthetic chemicals usually involves the interaction of reactive, electrophilic intermediates (ultimate carcinogens) with DNA (1). This damage may give rise to mutations at critical genomic sites in target tissues. Protooncogenes have been identified as genetic targets that are involved in chemical carcinogenesis (2). Most of the transforming genes identified so far in carcinogen induced animal tumors have been shown to be mutated versions of the ras gene family (3). Cellular ras genes can acquire transforming activity by a single point mutation, resulting in the alteration of amino acid residue 12, 13, 61, or 117 (2, 3). Evidence for the direct involvement of chemical carcinogens in the activation of ras genes has come from studies which used animal tumor systems to correlate the type of ras gene mutaReceived 3/5/90; revised 5/29/90. 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. 1This work was supported in part by a grant from the Deutsche Forschungs gemeinschaft to C. R. B. : To whom requests for reprints should be addressed, at Deutsches Krebsfor schungszentrum, P. 101949, 6900 Heidelberg 1. Federal Republic of Germany.
MATERIALS
AND METHODS
Carcinogen Treatment and Tumor Induction. AAI was isolated from commercially available aristolochic acid (Dr. Madaus & Co., Köln, Federal Republic of Germany) as previously described (10). The purity was greater than 99% based on high pressure liquid chromatography. Male Wistar rats (8 weeks old) were treated, according to the protocol of Mengs et al. (6), 5 times a week for 3 months, with AAI (10 mg/kg/ day) as the sodium salt dissolved in water, or water only. Animals were killed over a 15-week period after treatment. Animals were sacrificed after showing weight loss or symptoms of pain or when tumors were visible or palpable in the peritoneal cavity. Individual tumors were collected, quick frozen in liquid nitrogen, and stored at —80°C until DNA isolation. A representative portion of the induced tumors, when available, was fixed in 10% (v/v) neutral buffered formalin for histological examination. Southern Blot Analysis. Cellular DNAs were digested with restriction endonucleases (Pharmacia) using a 4-fold excess of enzyme. BamHl and/or EcoRl digested DNAs (10 ^g/'ane) from nude mice induced tumors and rat and mouse control DNAs were subjected to electropho3The abbreviations used are: AA, aristolochic acid; AAI, aristolochic acid I; SDS, sodium dodecyl sulfate; SSPE, standard saline phosphate with EDTA (180 HIMNaCI-10 HIMNaH2PO4-1.0 ITIMEDTA, pH 7.4); bp, base pair(s).
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containing 0.5 /
taining 0.3% SDS and 500 Mg/ml of denatured salmon sperm DNA. Hybridization with oligonucleotide probes end labeled (5') with 32Pwas performed overnight at 50°Cin the same solution. Thereafter, the membranes were washed in 2 x SSPE-0.1 % SDS at room temperature, followed by a stringent wash in 5 x SSPE-0.1% SDS for 10 min. After a final wash in 2 x SSPE-0.1% SDS at room temperature, the mem branes were exposed to Kodak X-ray films overnight at —70°C using intensifying screens. Oligonucleotide probes used were either 19-mers or 20-mers. For Table 1, we selected those oligomers that proved to be informative for the present study. Oligomers for hybridization of codons 12 and 61 of the Ki-ras gene were from NEN Division, Dreieich, Federal Republic of Germany; all of the others were prepared using an Applied Biosystems model 380 B DNA synthesizer. A complete list of oligomers used for our analyses of ras gene mutations is available on request. Direct Sequencing. Direct sequencing of polymerase chain reaction amplified DNA fragments was done according to modified protocols described by McMahon et al. (22) and Collins (23). The double strand amplified DNA was electrophoresed on a 2-3% agarose gel. The gel slice containing the desired DNA fragment was cut out of the gel, and DNA was isolated by centrifugation through a Spin-X column (Costar) for 30 min as described by Vogelstein (24). The DNA was treated with phenol, precipitated, and then sequenced directly using the dideoxy termination method and the modified T7 DNA polymerase. Sequenase (United States Biochemical Corp.), and reagents as provided in the Sequenase T7 DNA polymerase kit. Sequencing primers (5 pmol), 2-5 x 10' cpm, end labeled with [7-32P]dATP (>5000 Ci/mmol; Amerslutm). and polynucleotide kinase (Pharmacia) were mixed with ap proximately 100 ng of amplified DNA in 8 n\ H2O, heated for 5-10 min at 95V. and chilled on ice. Subsequently, the following solutions were added: 2 n\ of 5x Sequenase buffer, 1 ^1 0.1 M dithiothreitol, 2.5 ill H.(), and 2 n\ Sequenase (2 units/Ml). After mixing, 3.5 //I of this solution were added to 2.5 n\ of the G, A, T, and C stop mixes. These tubes were incubated for 5 min at 37°C,and the reactions were stopped by the addition of 5 til formamide-dye mix. The samples were heated at 95°Cfor 2 min and subjected to electrophoresis on 12% polyacrylamide-7 M urea gels at 60 W for approximately 2.5 h. Gels were fixed, dried, and exposed to X-ray film for 24 h using two intensifier screens.
RESULTS Tumor Incidence and Distribution. Forty male Wistar rats
incubations were performed without DNA. were treated with AAI (10 mg/kg/day) according to the protocol The sequences used to design the different amplimers and hybridi of Mengs et al. (6). Macroscopically, all animals showed papzation primers were obtained from the published sequence of rat Ha ras (19) and of rat Ki-roj (20). As the sequence of rat N-ros has not illomatosis of the entire forestomach. Forestomach tumors were been available, we have used the mouse N-rav sequence, assuming a observed in 38% (15 of 40) and ear duct tumors in 18% (7 of high conservation of ros genes between these two species (21). 40) of the rats subjected to moribund sacrifice. Both forestom The primers used for amplification of the region around codons 12 ach tumors and ear duct tumors used in this study were classi and 13 of the H-rosgene were 5'-ACCCCTGTAGAAGCGATGAC-3' fied as squamous cell carcinomas (Table 2). Fifty-eight % (23 and 5'-CACAAAATGGTTCTGGATCA-3'. Primers used for the re of 40) of the rats developed adenocarcinomas or sarcomas of gion around codon 61 of the H-ros gene were 5'-TTGATGGGGAGACGTGTTTA-3' and 5'-AGGAAGCCCTCCCCTGTGCG-3'. Am Table Oligonucleotides used for detection of mutations at codon 61 of the plifications yielded a 102- and at 99-bp DNA fragment, respectively. Ha-ras, Ki-ras, and N-ras gene Primers for the region around codons 12 and 13 of the Ki-ros gene were 5'-ATGACTGAGTATAAACTTGTGGT-3' and 5'-TCCACAAAGTGATTCTGAATTAG-3' and for the region around codon No.Ha-61AHa-6IBKÌ-61AKÌ-61BKÌ-61CN-61AN-61BN-61COligonucleotidesSequenceACA temperature64'C62'C63'C6I-C6I'C61-C61-C 61 were 5'-CTCCTACAGGAAACAAGTAGTAA-3' and 5'-TGATTTAACÕ GCA GGT CAA GAA GAG TAGTATTATTTATGGCAA-3'. Amplification yielded a 89- and a TAACA GCA GGT CTA GAA GAG TACAGCA GGT CAA GAG GAG 154-bp fragment, respectively. Primers used for the amplification of TACAGCA GGT CTA GAG GAG the region around codon 61 of the N-ros gene were 5'-GGTGAGACCTACAGCA GGT CAT GAG GAG TGCCTGCTGGA-3' and 5'-ATACACAGAGGAACCCTTCG-3'. TAACA GCT GGA CAA GAG GAG TAACA GCT GGA CTA GAG GAG N-ros product is 103 bp. GCT GGA CAT GAG GAG TADiscrimination Amplified DNA fragments were visualized on 2.5% agarose gels 5465
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Table 2 Characterization ofAAl induced raÃ-tumors ductMutationHa
ForestomachWistar
intestineType of MutationNDM* tumor 'Adenocarcinoma
of of ratno.4789II12131617222425262731363738Type tumorSCC°secsecsecsecsecsecsecsecsecsecsecsecsecMutationHa tumorsecsecsecsecsecsecsecEar CTAHa 61 CTAHa 61 CTAHa 61 CTAHa 61 CTAHa 61
CTAHa 61
of tumor(Pancreas) — NDMHyperplasia
—NDMAdenocarcinoma
CTAHa 61 CTAHa 61 CTAHa 61
—Adenocarcinoma
inthe metastasis CTAN 61 lungLymphoma(Pancreas)
KÕ61CTA'Adenocarcinoma CTAHa 61 CTAHa 61 CTAHa 61 CTAHa 61 CTAHa 61 CTAHa 61 CTA''Ha 61
CTA''Ha 61
thepancreasSCC of
CTAHa 61 —Adenocarcinoma CTAHa 61 61 CTA/KÕ61 CATSmall CTA¿—N 61 NDMSCC inthe metastasis CTA— 61 pancreasAdenocarcinomaof
—SarcomaMiscellaneousType
CTAHa 61 the kidneyMutation—N 61 CTAType a SCC. squamous cell carcinoma. 4 NDM, not determined morphologically.
' Not detected. Mutation found in transformant. ' In duodenum.
the small intestine. Neoplastia changes in the pancreas and kidney were also found. Furthermore, two métastasesof squa mous cell carcinomas in the lung and the pancreas were de tected. Due to the small tumor size, some of the neoplasias of the small intestine and pancreas were not available for histol ogy. No spontaneous tumors were observed in eight rats treated with vehicle. Tumors from 18 AAI treated rats were available for molecular analysis. To determine whether any of these rat tumors contained transforming activity, DNAs isolated from five AAI induced forestomach tumors were tested in the tumorigenicity assay. All five DNAs induced tumor formation in nude mice. Southern blot analysis of these primary transfectants showed the presence of an additional rat c-Ha-ras hybridizing fragment in addition to the normal mouse c-Ha-ras fragment (data not shown). These results suggested the activation of a c-Ha-ras gene in the AAI induced rat tumors. It is known that ras genes can be activated by point mutations at codons 12, 13, 61, or 117. In order to elucidate the molecular mechanisms involved in tumor initiation by AAI, we have investigated these primary transfectants for base pair mutations at the positions around codon 12/13 and 61 of the rat c-Ha-ras alÃ-ele.For that purpose, two sets of primers were used to amplify DNAs of the primary transfectants around codon 12/13 and codon 61 of the rat cHa-ras alÃ-ele.The amplified material was run on an agarose gel, and the amplified rat c-Ha-ras fragment was purified from the gel and sequenced directly using the dideoxy termination method and end labeled primers. Fig. 2, A and C, shows an example of such a sequence analysis. An AT —* TA transversion mutation (boxed T) at the second position of the rat c-Ha-ras gene can be identified. As a consequence, the normal sequence of codon 61 of the rat c-Ha-ras gene (CAA), coding for glutamine, has been changed to CTA, coding for leucine. As ex pected, we could also identify the same mutation in the original rat tumor (Fig. 2, B and D). The normal (CAA) as well as the mutated rat c-Ha-ras (CTA) are present. The weak signal of the mutated alÃ-ele(CTA) in Fig. 2D is
G
A
T
C
199
199
(]
Codon 61
Codoi-161
IE
166
166
G
A
T
C 199
199
Codon 61 [_
Codon 61
— c G
166
166
Fig. 2. Identification of AT —• TA mutations at the second position of codon 61 of the Ha-ras gene by direct sequencing of polymerase chain reaction amplified DNA. A and C, transfectants of AAI induced forestomach tumors; B and D, the original forestomach tumors, respectively.
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possibly due to contaminating normal cells in the analyzed tumor material. The results of all five rat forestomach tumors were identical. These preliminary results encouraged us to analyze the other AAI induced rat tumors for point mutations in the three different ras genes. DNAs from AAI induced rat tumors were amplified for regions around codon 12/13 and 61 of the c-Ha-ras and c-Ki-ras genes and codon 61 of the c-N-ras gene (18), followed by selective oligonucleotide hybridization (25) or direct sequencing. The probes used for hybridization included oligomers specific for every possible amino acid sub stitution at codons 12, 13, 59, and 61 of both Ha-ras and Kiras oncogenes and codon 61 of the N-ras oncogene. An AT —» TA transversion mutation at the second position of codon 61 of the c-Ha-ras gene, resulting in a change of glutamine (codon CAA) with leucine (codon CTA), was de tected in all of the AAI induced squamous cell carcinomas of the ear duct (7 of 7) (Fig. 3; Table 2). The same activating mutation was found in 13 of 14 forestomach tumors (Fig. 3; Table 2). The forestomach tumor induced in rat 36 failed to show a ras point mutation. However, DNA transfection of this tumor scored positive in the tumorigenicity assay. Southern blot analy sis detected a c-Ha-ras gene in the primary transfectant of rat tumor 36, and direct sequencing of this material revealed an identical c-Ha-ras point mutation as in the other forestomach tumors (Table 2). This result supports the current view that DNA transfection analyses may possess more sensitivity in detecting minor ( TA transversion at the second position in the c-Ki-ras gene (Fig. 3; Table 2). Another c-Ki-ras mutation was found in the ear duct tumor of rat 25, characterized by a concurrent c-Ha-ras muta tion (Fig. 3; Table 2). This c-Ki-ras mutation was also an AT — » TA transversion, but this time at the third position of codon 61, resulting in an amino acid change of glutamine with hist i dine. c-N-ras mutations at the middle A of codon 61 were observed in the transformants of neoplasia of the pancreas of two animals (rats 11 and 36) and in one lymphoma (rat 27) but were not detectable in the original tumor DNAs (Table 2; Fig. 4). All other tumors analyzed failed to show mutations in codons 12, 13, 59, and 61 of both the c-Ha-ras and c-Ki-ras genes or in codon 61 of the c-N-ras gene. DISCUSSION
B 61
T
A
wt-CAAmutation-CTA-
Ki
G
wt-CAAmutation-CAT-
Fig. 3. Hybridization of mutation specific oligomers to in vitro amplified DNAs from panel A, a-g, ear duct tumors of animals 7, 8. 9. II, 22, 24, 25; h, normal forestomach; panel B, a-g. tumors of the small intestine of animals 27. 9, 12, 16, 24, 37, 17; h, normal forestomach; and panel C, a-h. as in panel A. Approximately 5 ng of DNAs were spotted onto a nylon membrane and hybridized to oligomers (Table 1) representing wild-type (HT)and mutation specific codon 61 Ha-ras (panel A) or Ki-ra5 sequences (panels B and C). Note that sample g (ear duct tumor of animal 25) in panels A and C contains mutations in the 61st codon of both Ha- and Ki-ros genes.
The tumor induction in the forestomach and the ear duct by AAI was associated with activation of the c-Ha-ras protoonco gene. Since spontaneous tumors of the stomach (26) and ear duct (27) in rats are very rare and were not observed in this study, tumor formation and activating mutations can be re garded as direct results of the carcinogen treatment. In only a single adenocarcinoma of the small intestine an activating mutation of the c-Ki-ras gene was found. However, due to the small size of these tumors, only one other adenocar cinoma could be tested in the tumorigenicity assay, and it failed to show transforming activity. The other tumor DNAs were analyzed by selective oligonucleotide hybridization and, as noted before (28), tumors that consist primarily of nonneoplastic tissue may yield a hybridization signal which is too weak for unambiguous conclusions and may remain undetected. N-ras mutations were observed in a hyperplasia and a metas tasis of the pancreas and in a lymphoma. This observation is in good agreement with the idea of a preferential selection in a particular tissue of activating mutations in a subset of the ras family members. As reported by Leon et al. (29), this prefer ential selection may be related to a more prevalent expression
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of the Ha-, Ki-, or N-ras gene for a given tissue. Previous studies showed that the alkylating agent ./V-methyl/V-nitrosourea consistently activates the rat Ha-ras gene by mutation at guanine in codon 12 (30), whereas the hydrocarbon 7,12-dimethylbenz[a]anthracene activates rat Ha-ras by muta tion at adenine residues in codon 61 in 23% of the mammary carcinomas induced (30). In AAI induced rat tumors of the ton-stomach and ear duct, we found in all tumors analyzed the activating mutation at the middle adenine residue of codon 61 of the Ha-ras gene. The apparent selectivity for mutations at adenine residues found in our study was surprising because we expected by analogy with DNA binding studies of other nitroaromatics (31, 32) that guanine residues would be the major targets for the ultimate carcinogenic species of AAI. However, recent studies in our laboratory showed that an N6-deoxyadenosine-AAI adduct is the major adduci formed by reaction of AAI and DNA after activation with the nitroreductase xanthine oxidase (33). These results support the conclusion that the AAI-DNA adducts give rise to mutations directly. These promutagenic ade nine adducts could lead to AT —» TA transversion mutations by different mechanisms. As discussed by Bigger et al. (34), the postulated mechanisms include conformational changes of a carcinogen modified adenine to allow adenine-adenine mispairing, insertion of adenine opposite a carcinogen induced apurinic site (35), and insertion of adenine opposite a noncoding lesion (36). Since a shift from anti to syn conformation of an N6deoxyadenosine adduct does not prevent the adduci from inter fering with base pairing (37), the adenine mispairing model seems unlikely. The same is true for the formation of apurinic sites, because AAI does not give rise to single strand breaks in DNA in vitro (38). Thus, adenine insertion opposite a noncoding AAI modified adenine is most probably the mechanism that accounts for all AT —» TA transversion mutations observed. The fact that mutations are solely detected at deoxyadenosine residues of either the c-Ha-, c-Ki-, or c-N-ras genes can also be explained by the formation of promutagenic AAI-adenine adducts. Like 7,12-dimethylbenz[fl]anthracene (39) and benzo[c]phenanthrene (40), AAI binds extensively to adenosine residues (33). However, changes of adenosine residues at other positions than codon 61 in the three ras genes result in amino acid changes that are phenotypically silent. It has been demonstrated that ras oncogenes can induce metastatic potential in tumor cells as well as in nontransformed cells (41), although the mechanisms responsible for this induc tion have yet to be elucidated. We have shown that one metas tasis in the lung contained the same Ha-ras mutation found in the primary tumor of the forestomach. In contrast, rat 36 showed a CAA to CTA transversion in the Ha-ras gene of the primary forestomach tumor, whereas transfection analysis of a pancreatic metastasis of this animal surprisingly revealed an identical mutation, albeit in the N-ras gene. Vousden and Marshall (42) reported a lymphoma cell line of which one subclone was metastatic and carried an activated Ki-ras gene unlike the nonmetastatic parental tumor. These observations could be based on the heterogeneity of the primary tumor tissue, which in our case might contain only a subpopulation of N-ras activated cells that remained undetected by dot blot analyses. In summary, induction of rat tumors by AAI appears to be a novel /// vivo model that may be helpful in elucidating the complex interactions between mutagens and ras oncogenes in carcinogenesis.
ACKNOWLEDGMENTS We thank Monika Schmidberger and Marta Scharnbacher for expert technical assistance; E. Kleihauer, B. Kubanek, and H. Seliger for continuous support; and M. Mann for typing the manuscript.
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