Affiliations of authors: F. M. Venanzi, L. Soverchia, P. Felicetti, M. Mennecozzi, A.Concetti, Department of Biology MCA, University of Camerino, Camerino(MC), Italy.
Correspondence to: Franco M. Venanzi, Ph.D., Department of Biology MCA, University of Camerino, via Camerini #2, I-62032 Camerino (MC), Italy (e-mail: franco.venanzi{at}unicam.it).
Almost 16 years ago, the research groups of Robert Weinberg and Kumao Toyoshima simultaneously published the complete cDNA sequences of rat and human epithelial growth factor type-1 receptors (HER-2/neu) (1,2). The cDNA sequences, first presented at the National Center for Biotechnology Information (NCBI) in 1993, have since had no modified versions reported1. We now show that the rat HER-2/neu cDNA sequence (GenBank accession number X03362) may contain incorrect nucleotide assignments.
In the course of developing DNA vaccines for transgenic models of breast cancer, we cloned the second cystein-rich domain (CRD-2) of the extracellular domain of the rat neu gene (X03362; nucleotides 14561987) by polymerase chain reaction (PCR) amplification of PSV2 neuT plasmid (3) (provided by Professor N. Ranzani, University of Pavia, Italy). Surprisingly, the sequence analysis of a number of CRD-2 clones (n = 24 with 1.3 x 104 total nucleotides examined using an ABI Prism model 337 DNA sequencer) showed a deletion of three cytosine bases that were reported in the original HER-2/neu cDNA sequence GenBank (X03362) entry (deletions at positions C1537, C1543, C1552). Translation of the new cDNA sequence predicted that there was one amino acid deletion (L502), one silent mutation (C503), and three missense mutations (G504 V, L508 S, and E509 S). Repeating the cloning procedures with either MMTV-pBR322 rat neu-encoding plasmid (provided by Dr. P. Vezzoni, Consiglio Nazionale delle Ricerche, Milan, Italy) or with different proofreading Taq polymerase, to minimize the possibility of nucleotide misincorporation during PCR cloning, yielded identical results. Thus, the revised CRD-2 cDNA sequence of the extracellular domain of the rat HER-2/neu gene is available at GenBank (accession number AF393158).
Given that this cDNA sequence did have modifications, we wondered if this new entry represented the wild-type sequence or a mutant version of the oncogene. Therefore, additional CRD-2 clones of kidney mRNA extracted from both rat neu transgenic mice (4) and Wistar or Sprague-Dawley rat strains (Charles River, Calco, Italy) were generated by reverse transcription (RT)PCR. Again all of the CRD-2 clones analyzed (n = 15) yielded the AF393158 version of the rat HER-2/neu gene cDNA sequence. In addition, our sequencing results have been confirmed independently by Dr. C. Spangenberg (University of Mainz, Germany) and by Dr. F. Orlandi (Memorial Sloan-Kettering Cancer Center, New York) who are working with unrelated rat neu DNA constructs (personal communications). Taken together, these results strongly argue that the new CRD-2 cDNA sequence of the rat HER-2/neu gene represents the wild-type sequence.
Correcting errors and proofreading are crucial in a genetic age, when DNA sequences have the potential to be part of effective anti-cancer treatments. Indeed, a number of DNA expression vectors encoding the neu extracellular domain have been generated (mostly by PCR amplification) to induce protective immunity against malignancy associated with the HER-2/neu oncogene (57). However, despite the cDNA sequences that the authors of these studies assigned, neither of them have reported the discrepancy in the HER-2/neu gene that we find. In fact, a recent patent application, which discloses an immunogenic neu-rat fusion gene (Genbank accession number AX380924), still contains the 1986 version of CDR-2 cDNA sequence of the rat neu oncogene.
Therefore, in light of these facts, we suggest that the cDNA sequence information of each plasmid construct intended for preclinical or clinical trials should be supported by the original sequence output.
NOTES
1 On September 1, 2002, while drafting this correspondence, Watson et al. submitted a new entry to the National Center for Biotechonology Information for the rat-neu protooncogene cDNA sequence (AY116182), which fully confirms our earlier AF393158 version.
Supported by a grant (7941.MIUR) from INDENA S.p.a., Italian National Plan in Oncology.
References
1 Bargmann CI, Hung MC, Weinberg RA. The neu oncogene encodes an epidermal growth factor receptor-related protein. Nature1986;319:22630.[Medline]
2 Yamamoto T, Ikawa S, Akiyama T, Semba K, Nomura N, Miyajima N, et al. Similiarity of protein encoded by the human c-erb-B-2 gene to epidermal growth factor receptor. Nature 1986;319:2305.[Medline]
3 Bargmann CI, Hung MC, Weinberg RA. Multiple independent activations of the neu oncogene by a point mutation altering the transmembrane domain of p185. Cell 1986;45:64956.[Medline]
4 Guy CT, Webster MA, Schaller M, Parson TJ, Cardiff RD, Muller WJ. Expression of the neu protooncogene in the mammary epithelium of transgenic mice induces metastatic disease. Proc Natl Acad Sci U S A 1992;89:1057882.[Abstract]
5 Chen Y, Hu D, Eling DJ, Robbins J, Kipps TJ. DNA vaccines encoding full-length or truncated Neu induce protective immunity against Neu-expressing mammary tumors. Cancer Res 1998;58:196571.[Abstract]
6 Rovero S, Amici A, Carlo ED, Bei R, Nanni P, Quaglino E, et al. DNA vaccination against rat Her-2/neu p185 more effectively inhibits carcinogenesis than transplantable carcinomas in transgenic BALB/c mice. J Immunol 2000;165:513342.
7 Foy TM, Bannink J, Sutherland RA, McNeill PD, Moulton GG, Smith J, et al. Vaccination with Her-2/neu DNA or protein subunits protects against growth of a Her-2/neu-expressing murine tumor. Vaccine 2001;19:2598606.[Medline]
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