neu mutation in schwannomas induced transplacentally in Syrian golden hamsters by N -nitrosoethylurea: high incidence but low allelic representation

Abstract

Peripheral nerve tumors (PNT) and melanomas induced transplacentally on day 14 of gestation in Syrian golden hamsters by N-nitrosoethylurea were analyzed for activated oncogenes by the NIH 3T3 transfection assay, and for mutations in the neu oncogene by direct sequencing, allele-specific oligonucleotide hybridization, MnlI restriction-fragment-length polymorphism, single-strand conformation polymorphism, and mismatch amplification mutation assays. All (67/67) of the PNT, but none of the melanomas, contained a somatic missense T --> A transversion within the neu oncogene transmembrane domain at a site corresponding to that which also occurs in rat schwannomas transplacentally induced by N-nitrosoethylurea. In only 2 of the 67 individual hamster PNT did the majority of tumor cells appear to carry the mutant neu allele, in contrast to comparable rat schwannomas in which it overwhelmingly predominates. The low fraction of hamster tumor cells carrying the mutation was stable through multiple transplantation passages. In the hamster, as in the rat, specific point-mutational activation of the neu oncogene thus constitutes the major pathway for induction of PNT by transplacental exposure to an alkylating agent, but the low allelic representation of mutant neu in hamster PNT suggests a significant difference in mechanism by which the mutant oncogene acts in this species.

Activation of neu by missense point mutation in the transmembrane domain in schwannomas induced in C3H/HeNCr mice by transplacental exposure to N -nitrosoethylurea

“…These mutation-dependent bands are not present in the MnlI digests of PCR products from normal mouse tissue. A similar MnlI restriction-fragment-length polymorphism (RFLP) was previously shown for both rat and hamster neu TMD mutations (Nikitin et al 1991;Buzard et al 1999).…”

“…A few tumors in mice and rats became homozygous for the mutant allele [two out of ten in the mice, and all six lines in the rat (Nikitin et al 1996)]. This is in contrast to hamsters, where loss of heterozygosity not only does not occur, even after repeated in vivo passages, but the wild-type allele is almost always the predominant allele detected in each tumor, as if many of the tumor cells carry only the wild-type allele (Buzard et al 1999). Only one mouse tumor had this characteristic.…”

“…The mouse neu transgene was variably ampli®ed. Stringency conditions favored hybridization of the rat cDNA probe to the more homologous mouse neu sequences, accounting for the relative faintness of the hamster (H) bands tation previously documented in rats and hamsters after perinatal exposure to ENU or to methylnitrosourea (Perantoni et al 1987;Sukumar and Barbacid 1990;Nikitin et al 1991;Nakamura et al 1994a, b;Buzard et al 1999). However, the mouse neu gene found in one SHOK focus did not contain a TMD point mutation.…”

“…An activated neu oncogene has been found in almost all reported rat (80/84) and hamster (81/82) schwannomas that have been transplacentally or perinatally induced by ENU or methylnitrosourea, whereas neu has never been found to be mutated in rat glial tumors (0/87) or hamster melanomas (0/16) (Perantoni et al 1987;Sukumar and Barbacid 1990;Nikitin et al 1991;Nakamura et al 1994b;Buzard et al 1999). We previously found that spontaneously arising schwannomas in adult rats usually lack activated neu, in contrast to perinatally initiated schwannomas (Perantoni et al 1994).…”

“…In contrast to human tumors, the carcinogeninduced oncogenic activation of neu in experimentally induced rat and hamster schwannomas almost invariably occurs by a point mutation rather than by gene overexpression (Perantoni et al 1987;Sukumar and Barbacid 1990;Nikitin et al 1991;Nakamura et al 1994a, b;Buzard et al 1999). This activation in rats and hamsters is by a unique T ® A transversion within the p185 neu receptor transmembrane domain (TMD), which results in a substitution of glutamic acid for valine and a constitutive activation of receptor signaling (Bargmann et al 1986a; Bargmann and Weinberg 1988).…”