Proto-oncogene Mutation Is Specific for the Neurofibromas in a N-Nitroso-N-ethylurea-induced Hamster Neurofibromatosis Model but not for Hamster Melanomas and Human Schwann Cell Tumors 1

Point mutations of the transmembrane domain coding region of the n e u proto-oncogene in N-nitroso-N-ethylurea-induced hamster neurofibromas were found at high frequency (93%; 14 of 15). They involved codons 659 as well as 658, the latter not having been reported previously in rat tumors. The mutational change was seen even in the early stage neurofibroma. On the other hand, no mutations were detected in melanomas or Wilms' tumors induced in the same N-nitroso-N-ethylurea-treated animals, even when the melanomas demonstrated extensive schwannian differentiation. Moreover, any human Schwann cell tumors including neurofibroma, schwannoma, and malignant schwannoma did not show the mutation of c e r b B 2 gene (0 of 34), which is homologous to the hamster n e u . Since high expression of n e u mRNA is evident in the hamster Schwann cell at the late gestational and neonatal stages, transplacental administration of N-nitroso-N-ethylurea is considered to interact directly to carcinogenesis of the hamster Schwann cell through n e u gene mutation. I N T R O D U C T I O N The Schwann cell and the melanocyte are 2 major components of the neural crest-derived cell system and these 2 cell types are closely related to each another (1). The close relation between these 2 cell lineages has been emphasized by the several facts: (a) schwannian differentiation is frequently observed in pigmented nevus (2); (b) neuroblastoma cell lines are able to differentiate toward both Schwann cell and melanocyte lineages (3); (c) both cell types are affected in some neurocristopathies such as NF13 (4). Transplacental administration of ENU to Syrian golden hamsters induces multiple neurofibromas at high incidence together with melanomas and Wilms tumors (5). From its biological characteristics the ENU-induced hamster tumor system has been considered as an appropriate animal model for human neurocristopathy, especially for NF1 (5, 6). The growth properties and morphological characteristics of hamster neurofibromas are more similar to human neurofibromas than are those of ENU-induced rat peripheral nervous tumors. Moreover, the close relation between the Schwann cell and melanocyte is also observed in the model, that is: (a) frequent and extensive schwannian differentiation of melanoma; and (b) melanocytic proliferation in neurofibroma (5, 7). However, biological behaviors of these 2 tumors are rather different. Neurofibroma grows slowly and shows Received 8/2/93; accepted 12/15/93. 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. lThe research was supported in part by Grants-in-aid for Scientific Research and Cancer Research from the Ministry of Education, Science and Culture, Japan, and from the Smoking Research Foundation, Japan. 2 To whom requests for reprints should be addressed, at Mammalian Genetics Laboratory, ABL Basic Research Program, National Cancer Institute Frederick Cancer Research and Development Center, Box B, Frederick, MD 21702-1201. 3 The abbreviations used are: NF1, neurofibromatosis type l; ENU, N-nitroso-Nethylurea; PCR, polymerase chain reaction; SSC, standard saline-citrate. solid growth often resulting in the large tumor, whereas melanoma shows aggressive growth and distant metastasis that never occurs in neurofibroma. To investigate the difference of the molecular mechanisms of carcinogenesis in these tumors we examined neu gene mutations in the model. The proto-oncogene neu/HER/c-erbB-2 encodes a receptor-type tyrosine kinase that is in part homologous to the epidermal growth factor receptor (8, 9). The human c-erbB-2 gene is frequently amplified in malignant tumors such as breast cancer and a variety of adenocarcinomas, and the amplification is correlated with a poor prognosis (10-12). Activation of the neu gene was earlier observed in ENUinduced rat peripheral nervous tumors. In these tumors mutation from GTG to GAG was found involving codon 664, resulting in an amino acid substitution from valine to glutamic acid in the transmembrane domain (13). This mutation has been considered to increase tyrosine kinase activity (14) and the mutated neu gene exerts transforming activity on the NIH3T3 cell line (15). However, the mutational change of neu has not been demonstrated in human tumors or tumors in any other animals, and in rats the change was restricted to Schwann cell tumors. In the present report, we document the presence of neu gene mutations at the transmembrane domain coding region in hamster neurofibromas, but not melanomas and Wilms' tumors, using the PCR followed by direct sequencing. Moreover, we examined the c-erbB-2 gene change in human Schwann cell tumors to ascertain whether the change is specific only for the rodent Schwann cell or is found in a wide range of species. M A T E R I A L S AND M E T H O D S Extraction of DNA from the Tumors. Hamster neurofibromas, melanomas, and Wilms tumors were induced by the method reported previously (5). Tumors were frozen in liquid nitrogen immediately after resection and stored at -80~ until use. Tissues were mashed and high molecular weight DNA was extracted according to a reported method (16). DNA was also extracted from formaldehyde-fixed paraffin-embedded sections for PCR as described earlier (17). Histopathological diagnoses were made for all tumor samples and schwannian differentiation of melanomas was confirmed by detection for anti-glial fibrillary acidic protein and/or anti-laminin antibodies (7). Thirtyfour human Schwann cell tumors including 15 schwannomas, 17 neurofibromas, and 2 malignant schwannomas were surgically resected at Toranomon Hospital. Six of 17 neurofibromas were resected from the NF1 patients. The pathological diagnosis was made by more than 2 pathologists. Direct DNA Sequencing. Direct DNA sequencing was performed by a reported method (18). Samples of 0.1~d.5 /xg of DNA were amplified by asymmetric PCR with 2.5 units of Taq polymerase (Takara, Japan) using 50 and 5 pmol of paired primers (Fig. 1). Fifty cycles of the reaction at 94 ~ 55 ~ and 72~ for 0.5, 1, and 2 min, respectively, were carried out in a Perkin-Elmer Cetus thermal cycler. Purification of PCR products was made by phenol/ chloroform extraction, ethanol precipitation, and filtration through a polysulfon filter (Ultrafree C3GC; Millipore, Japan). The primer for sequencing (Fig.