Proc. NatI. Acad. Sci. USA Vol. 86, pp. 2545-2548, April 1989 Amplification of the proto-neu facilitates oncogenic activation by a single point mutation (gene amplification/multiple-step ) MIEN-CHIE HUNG, DUEN-HWA YAN, AND XIAOYAN ZHAO Department of Tumor , The University of Texas M. D. Anderson Center, 1515 Holcombe Boulevard, Houston, TX 77030 Communicated by Robert A. Weinberg, December 13, 1988

ABSTRACT To determine whether the amplification of encoded p185 protein (13, 14) but exhibit normal morphology the proto-neu oncogene (also called c-erbB-2) plays a role in and form monolayers when growing to confluency (15). tumorigenicity, we previously generated an NIH 3T3 transfec- We studied the tumorigenicity of the DHFR/G-8 cells and tant (DHFR/G-8) that carried the amplified proto-neu gene. found that amplification of the proto-neu gene in DHFR/G-8 The DHFR/G-8 cells exhibited normal morphology. Their cells facilitated a single point mutation that rendered the cells growth curve was similar to that of NIH 3T3 cells but was tumorigenic. The results have important implications in the different from that of the B104-1 cell, an NIH 3T3 transfectant pathogenesis of human tumors-namely, the amplified pro- that carries the activated neu oncogene. When injected into tooncogene in human tumors could be activated by convert- nude mice, B104-1 cells produced tumors within 2 weeks, ing one (or a small number) of the amplified protooncogenes whereas the DHFR/G-8 cells did not produce tumors until 3 into an activated oncogene in addition to producing a high months after injection, and the NIH 3T3 cells did not produce level of protooncogene protein product. any tumors even after 3 months. The tumors produced by the injection of the DHFR/G-8 cells were excised and grown in MATERIALS AND METHODS culture. The cells derived from the tumors were oftransformed morphology and highly tumorigenic. The DNAs from the DNA Transfection. DNA transfection procedures were tumor cells were transfected into NIH 3T3 cells. The transfec- performed by the calcium phosphate precipitation technique tion resulted in foci on the NIH 3T3 monolayer. Southern of Graham and Van der Eb as modified by Anderson et al. the foci derived from the transfection (16). In each transfection, 75 ,g ofgenomic DNA was applied analysis indicated that to 8 x 105 NIH 3T3 cells (2 x 10 cm dishes). Cells were split contained the neu gene. Using oligonucleotides as probes, the in a ratio of 1:6 after 24 hr. Two weeks later, foci of neu gene in the foci was found to carry a single-point mutation morphologically transformed cells were scored and analyzed. identical to the one previously found in the rat neuroblastoma Cell Culture. NIH 3T3 cells and transfectant clones derived and glioblastoma induced by the ethylnitrosourea. We con- from NIH 3T3 cells were grown in Dulbecco's modified clude that the DNA region encoding the transmembrane Eagle's medium (DMEM) supplemented with 10% calf se- domain of neu is a hot spot for converting the proto-neu gene rum. The methotrexate-resistant or -sensitive properties into an activated oncogene and that amplification of the were analyzed by plating 1 x 105 cells in a 10-cm plate dish proto-neu gene facilitates mutation of the hot spot. with 10 ml of DMEM containing 10% calf serum and 0.6 ,uM methotrexate as described (15). After 2 weeks, the sensitive Rat neuro/glioblastomas induced by transplacental injection cells were completely killed, and the resistant cells were ofethylnitrosourea frequently carry an oncogene, termed neu allowed to grow to confluency. (also called murine c-erbB-2), that is detectable by transfec- Southern Analysis. Southern blot analysis was performed tion into mouse NIH 3T3 cells (1, 2). The neu oncogene was essentially by published techniques (17) as described (18). shown to be homologous to but distinct from the gene The nitrocellulose filters were hybridized at high stringency encoding epidermal growth factor receptor (EGFR) (3-5). (50% formamide/0.75 M NaCl/75 mM sodium citrate, 420C) The mechanism that induces activation of the neu oncogene for 36 hr and then were washed twice at room temperature for was found to be a single-point mutation converting adenine to 5 min in 0.3 M NaCI/30 mM sodium citrate and at 650C for thymidine. The activation mutation results in a change of 1 hr in 15 mM NaCI/1.5 mM sodium citrate/0.1% NaDodSO4. valine to glutamic acid in the transmembrane domain of the Oligonucleotide Hybridization. The oligonucleotides ini- neu-encoded protein, p185 (6). tially were provided by Cornelia Bargmann at MIT and later Many human tumor DNAs contain the amplified protoon- synthesized on the Applied Biosystems 380A DNA synthe- cogene, suggesting its amplification may play an important sizer at M. D. Anderson Cancer Center. Full-length oligo- role in tumorigenicity (7-12). The human homologue of the nucleotides were purified by polyacrylamide gel electropho- murine neu gene (human gene symbol NGL for neuro/ resis, and 100 ng ofeach oligonucleotide was phosphorylated glioblastoma-derived; has been called ERBB2, HER2, human by polynucleotide kinase by using the published technique c-erbB-2, or TKRI) has also been shown to be amplified in (17). Unincorporated nucleotide was removed by chroma- many human tumors (10-12). To study the possible role of tography over a Sephadex G-25 column. The oligonucleotide amplified neu in tumorigenicity, we have obtained a cell line, hybridization was carried out essentially by the procedures DHFR/G-8, that is an NIH 3T3 transfectant containing described by Bargmann et al. (6). approximately 100 copies of the proto-neu gene, the normal allele of the activated neu oncogene, by coamplification with the dihydrofolate reductase gene (DHFR) after methotrexate RESULTS treatment. The DHFR/G-8 cells produce a high level of neu- We had previously generated an NIH 3T3 transfectant, the DHFR/G-8 cell line, containing approximately 100 copies of The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" Abbreviations: DHFR, dihydrofolate reductase; EGFR, epidermal in accordance with 18 U.S.C. §1734 solely to indicate this fact. growth factor receptor. 2545 Downloaded by guest on October 1, 2021 2546 Biochemistry: Hung et al. Proc. Natl. Acad. Sci. USA 86 (1989) the rat genomic proto-neu gene-a normal and unmutated 1 2 3 4 5 6 7 allele of the rat neu oncogene-by cotransfection with 23.1- pSV2-DHFR plasmid and selection with methotrexate (15). 94- The DHFR/G-8 cell line expresses high levels of the neu- 6-5- _ encoded p185 protein but exhibits normal morphology and 4.4- I__ibm-4Ep forms monolayers in culture. To further investigate if the 2.3. overexpression of proto-neu gene could play a role in 2.0 tumorigenicity, the DHFR/G-8 cells were injected into nude .- mice. The DHFR/G-8 cells produced tumors only after a long 0.5- lag period of 3 months; the control cell line, B104-1, an NIH 3T3 transfectant containing the mutation-activated neu on- cogene, produced tumors rapidly within 2 weeks after injec- tion, and the NIH 3T3 cells did not produce any tumor even 3 months after injection (Table 1). FIG. 1. Southern blot analysis of DNA isolated from the When two independent tumors induced by injection of the indicated sources. Ten micrograms of DNA was digested with DHFR/G-8 cells were excised and grown in culture, they BamHI and hybridized with a 0.4-kilobase (kb) BamHI fragment of rat neu cDNA (5). Lanes: 1, NIH 3T3 cells; 2-5, PNT-2.1, PNT-2.2, (PNT-1 and PNT-2) exhibited typical transformed morphology PNT-1.1, and PNT-1.2 cells, respectively (four independent trans- similar to that of B104-1 cells. The PNT-1 and PNT-2 cells fectant cell lines derived from PNT-2 and PNT-1 by NIH 3T3 produced tumors within 2 weeks after injection (Table 1). focus-forming assay); 6 and 7, PNT-1 and PNT-2 cells (lines derived Since the morphology and tumorigenicity ofPNT-1 and PNT-2 from tumors that were excised from the tumor-bearing mice 3 months cells were different from those ofparental DHFR/G-8 cells, it after injection of DHFR/G-8 cells). Phage A HindIII size markers in is conceivable that a second hit involving genetic alteration in kb are indicated to the left. the cell DHFR/G-8 occurred during the 3-month lag period. potential mutation in the same region that contains the We reasoned that the genetic alteration by the second hit mutation in rat neuro/glioblastomas. might activate some protooncogene and then convert the Oligonucleotides corresponding in sequence to the wild type DHFR/G-8 cells into the transformed PNT-1 and PNT-2 or the adenosine-to-thymidine mutant version ofthe neu gene cells. To test this possibility, we carried out the NIH 3T3 were as described (6). These two 20-mers were hybridized focus-forming assay. The DNAs prepared from PNT-1, under stringent conditions to dried agarose gels containing PNT-2, and DHFR/G-8 cells were transfected into NIH 3T3 DNAs that had been digested with appropriate restriction cells. Both PNT-1 and PNT-2 DNA produced foci and endonucleases. The hybridization condition was shown to be DHFR/G-8 DNA did not. Four independent foci (PNT-1.1, able to distinguish the wild-type and the mutant version ofneu PNT-1.2, PNT-2.1, and PNT-2.2) were picked, and they all gene from each other (6). The 20-mer corresponding to the exhibited transformed morphology (data not shown). All four wild-type sequence hybridized with DNA from DHFR/G-8 foci were further shown to be tumorigenic two weeks after but not with DNA from B104-1 or any ofthe four independent injection. The results suggest that PNT-1 and PNT-2 indeed foci (Fig. 2). When the gel was stripped of probe and rehy- contained activated oncogene. bridized with the 20-mer corresponding to the mutant se- Since the DHFR/G-8 cell line is an NIH 3T3 transfectant quences, positive signals were observed for DNAs from containing 100 copies ofthe rat proto-neu gene and since only B104-1 and the four independent foci but not for DNA from injection ofDHFR/G-8 cells but not NIH 3T3 cells produced DHFR/G-8. Since it is known that transfectants usually tumors in the nude mice, we suspected that the target of the contain multiple copies of the transfected gene, the different second hit might reside in the amplified rat proto-neu gene. intensities shown by the four independent foci (Fig. 2b, lanes If so, we expected the foci generated by PNT-1 and PNT-2 3-6) are due to different copy numbers of the transfected neu would contain the rat neu gene. The DNAs from the four gene in the foci. From these results we conclude that the independent foci were analyzed by Southern blot with a rat second hit of DHFR/G-8 cells is a single point mutation that neu-specific probe (Fig. 1). All four foci exhibited the rat is identical to the one previously found in the rat neuro/ neu-specific restriction pattern, indicating that the target of glioblastoma (6). We also tried to determine how many copies the second hit was the amplified rat proto-neu gene in the of the proto-neu gene were mutated to become activated neu DHFR/G-8 cells. oncogene in the PNT-1 and PNT-2 cells. The majority of the It was previously shown that a single point mutation at the neu genes in PNT-1 and PNT-2 cells were normal versions of DNA region encoding the transmembrane domain is suffi- the neu gene, since the hybridization signals with the wild-type cient to convert the proto-neu gene into an activated neu 20-mer were similar to that of DHFR/G-8. When the mutant oncogene ih rat neuro/glioblastomas (6). Since the PNT-1 20-mer was hybridized with DNA from PNT-1 and PNT-2, we and PNT-2 cells carry transfected neu genes, it was ofinterest detected only a very weak signal after a long exposure ofx-ray to determine whether the second hit in the DHFR/G-8 cells film. However, a signal with similar intensity was also pro- contained the same mutation. To answer this question, we duced by cross-hybridization of the mutant 20-mer to the 100 used synthetic oligonucleotides as probes to search for the copies ofproto-neu gene in DHFR/G-8 cells (19/20-nucleotide match) under the same conditions (data not shown). Since it is Table 1. Tumorigenicity assay difficult to distinguish the hybridization signal of the mutant No. of tumors/ Time to 20-mer to a single copy of mutated neu oncogene (20/20- Cell line no. of injections* develop tumorst nucleotide match) from the cross-hybridization signal to 100 copies of the proto-neu gene (19/20-nucleotide match) in the B104-1 6/6 2 weeks PNT-1 and PNT-2 cells (see below), we could not accurately DHFR/G-8 6/6 3 months estimate exactly how many copies ofthe proto-neu gene were NIH 3T3 0/6 3 months mutated. However, the results suggest that at most only a very PNT-1 6/6 2 weeks few copies of proto-neu in PNT-1 and PNT-2 cells were PNT-2 6/6 2 weeks mutated to become activated neu oncogene. *Each mouse was injected s.c. with 106 cells in 0.2 ml of phosphate- Since the mutations of the neu gene in these four indepen- buffered saline. dent foci were identical to that in B104-1, it might be possible tThe tumors were at least 1 cm in diameter. that the DHFR/G-8 cells used for injection were contam- Downloaded by guest on October 1, 2021 Biochemistry: Hung et al. Proc. Natl. Acad. Sci. USA 86 (1989) 2547

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FIG. 3. Southern blot analysis of DNA isolated from the indicated sources. Ten micrograms of DNA was digested with EcoRI and hybridized with either the 0.4-kb BamHI fragment of rat neu cDNA (a) or a DHFR DNA probe derived from pSV2-DHFR (b). FIG. 2. Oligonucleotide hybridization with tumor cell lines and Lanes: 1, DHFR/G-8 cells; 2, PNT-1 cells; 3, PNT-2 cells; 4, B104-1 neu transfectants. DNA was digested with BgI II and hybridized cells; 5, NIH 3T3 cells; 6, BDIX rat liver cells; 7, Rat-1 cells; 8, phage either with wild-type oligonucleotide (a) or mutant oligonucleotide A HindIll size markers in kb. (6) (b). The arrows to the right indicate the hybridization signals. Lanes: 1, DHFR/G-8; 2, B104-1; 3-6, PNT-1.1, PNT-1.2, PNT-2.1, In the present study, the mutation responsible for activa- and PNT-2.2, respectively; M, overexposed phage A HindIII size tion is a single point mutation. However, it is conceivable that markers that are indicated to the left. amplification could facilitate rearrangement or deletion, too. The recent results reported by E. Helseth (personal commu- inated with a small fraction of B104-1 cells, and the PNT-1 nication) and colleagues support the concept. They found and PNT-2 cells were derived from the contaminating B104-1 EGFR (formerly called ERBBJ; also called human c-erbB-J) cells. To rule out this possibility, the PNT-1 and PNT-2 cells was amplified and overexpressed in a human carcinoma cell were shown to be of DHFR/G-8 origin. Like DHFR/G-8, line, T-CAR-1, derived from the adrenal cortex. In addition both PNT-1 and PNT-2 were methotrexate-resistant and to the high level of normal EGFR (170 kDa), they detected a contained highly amplified pSV2-DHFR DNA and the neu 50-kDa protein that interacted with antibody against the gene (Fig. 3); B104-1 did not contain pSV2-DHFR and was C-terminal domain of EGFR. Their results suggest that the methotrexate-sensitive. When compared to the BDIX rat 50-kDa protein may be a mutated form of EGFR. From our liver and Ratl cells that presumably contained two copies of model system in the present study, we would suggest that the neu gene (Fig. 3), the copy number of the neu gene in EGFR was amplified in the carcinoma, and later one of the B104-1 was approximately 20, which was much lower than amplified EGFR genes was mutated to produce the 50-kDa those seen in PNT-1, PNT-2, and DHFR/G-8, which con- protein. Since the virus-associated oncogene v-erbB is tained approximately 100 copies. known to encode a truncated form of EGFR, the 50-kDa Since both PNT-1 and PNT-2 cells were derived from protein may be activated in a similar way to the v-erbB DHFR/G-8 cells and since all four foci derived from PNT-1 protein. and PNT-2 cells contain the same mutation-activated neu Several protooncogenes have been shown to be amplified oncogene, we conclude that amplification of the proto-neu in many human tumors (7-12, 19, 20). Our study raises an gene facilitated oncogenic activation by a single point muta- interesting question: "could some of the amplified protoon- tion and that this point mutation, which is identical to the one cogenes bear activating mutations?" If the mutated alleles found in the rat neuro/glioblastomas, is a "hot spot" for can be frequently found in those human tumor DNAs, activating the proto-neu gene. containing amplified neu (ERBB2; NGL) or EGFR (ERBBI ), our present model may be appropriate for human tumors- i.e., amplification of the protooncogene may facilitate addi- DISCUSSION tional oncogenic mutation, and amplification and mutation We have shown that amplification of the proto-neu gene can may form a two-step mechanism in multistepped carcinogen- facilitate oncogenic activation by a single point mutation in esis. some of the amplified proto-neu genes. Our results indicate In vitro mutagenesis studies have shown that mutations that one way to convert a protooncogene into an activated that convert valine to glutamine or aspartic acid can also oncogene is through a two-step mechanism-namely, ampli- activate the proto-neu gene into an activated neu oncogene fication and then mutation. (21). However, the point mutations induced by ethylni- Downloaded by guest on October 1, 2021 2548 Biochemistry: Hung et al. Proc. Natl. Acad. Sci. USA 86 (1989) trosourea and spontaneous mutation in the present study are 5. Bargmann, C. I., Hung, M.-C. & Weinberg, R. A. (1986) all thymidine-to-adenosine transversions that convert valine Nature (London) 319, 226-230. (6), 6. Bargmann, C. I., Hung, M.-C. & Weinberg, R. A. (1986) Cell to glutamic acid. The codon for the specific valine is GTG 45, 649-657. which thus requires only one thymidine-to-adenosine muta- 7. Libermann, T. A., Nusbaum, H. R., Razon, N., Kris, R., Lax, tion to mutate valine to glutamic acid. However, double I., Soreq, H., Whittle, N., Waterfield, M. D., Ullrich, A. & mutations are required to convert the specific valine to Schlessinger, J. (1985) Nature (London) 313, 144-147. glutamine or aspartic acid. This difference probably explains 8. Semba, K., Kamata, N., Toyoshima, K. & Yamamoto, T. why all ofthe oncogenic mutations of neu detected in vivo so (1985) Proc. Natl. Acad. Sci. USA 82, 6497-6501. 9. Xu, Y. H., Richert, N., Ito, S., Merlino, G. T. & Pastan, I. far are valine-to-glutamic acid mutations. (1984) Proc. Natl. Acad. Sci. USA 81, 7308-7312. Recently, overexpression of the human homologue of the 10. Slamon, D. J., Clark, G. M., Wong, S. G., Levin, W. J., neu gene has been shown to transform NIH 3T3 cells (22, 23). Ullrich, A. & McGuire, W. L. (1987) Science 235, 177-182. It is not yet clear why overexpression of the rat proto-neu 11. Kraus, M. H., Popescu, N. C., Amsbaugh, S. C. & King, gene in DHFR/G-8 cells does not result in a transforming C. R. (1987) EMBO J. 6, 605-610. phenotype. It could be possible that the rat proto-neu gene is 12. Vijver, M., Bersselaar, R., Devilee, P., Cornelisse, C., Peterse, less potent than the human counterpart or that DHFR/G-8 J. & Nusse, R. (1987) Mol. Cell. Biol. 7, 2019-2023. 13. Stern, D. F., Heffernan, P. A. & Weinberg, R. A. (1986) Mol. cells do not produce enough normal P185 to transform NIH Cell. Biol. 6, 1729-1740. 3T3 cells. 14. Padhy, L., Shih, C., Cowing, D., Finkelstein, R. & Weinberg, R. A. (1982) Cell 28, 865-871. These studies were initially conducted in the Whitehead Institute 15. Hung, M.-C., Schechter, A. L., Chevray, P.-Y. M., Stern, laboratory of Dr. Robert Weinberg, whom we thank for enthusiastic D. F. & Weinberg, R. A. (1986) Proc. Natl. Acad. Sci. USA 83, advice and support. We also thank Ms. Carolyn Davis and Ms. Jean 261-264. Eakins for typing this manuscript. This work was supported in part 16. Anderson, P., Goldfarb, M. P. & Weinberg, R. A. (1979) Cell by Grant CA-45265 of the National Institutes of Health, Grant 16, 63-75. CD-360 of The American Cancer Society, and Award 5-621 of the 17. Maniatis, T., Fritsch, E. & Sambrook, J. 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H., Kraus, M. H., Segatto, 0. S., 4. Schechter, A. L., Hung, M.-C., Vaidyanathan, J., Weinberg, King, R. & Aaronson, S. A. (1987) Science 237, 178-182. R. A., Yang-Feng, T., Franke, U., Ullrich, A. & Coussens, L. 23. Hudziak, R. M., Schlessinger, J. & Ullrich, A. (1987) Proc. (1985) Science 229, 976-978. Natl. Acad. Sci. USA 84, 7159-7163. Downloaded by guest on October 1, 2021