Activated Neu Oncogene Research Articles

Abstract B18: Role of the tumor suppressor gene DLC-2 in ERBB2 induced mouse mammary tumorigenesis and metastasis

Abstract Introduction: Overexpression of dominant oncogenes and the loss of tumor suppressor genes are basic genetic events in acquisition of the malignant phenotype. The ERBB-2 proto-oncogene is overexpressed in 20% - 30% of human breast cancers. The tumor suppressor gene Deleted in liver cancer 2 (DLC-2, also known as STARD13) maps to chromosome 13q12.3 and is frequently downregulated by 72% in human breast cancer. It encodes a RhoGAP protein containing a START lipid binding domain. It is not known what role DLC-2 silencing plays in breast carcinogenesis. The purpose of our study is to assess the biological significance of inactivation of DLC-2 in ERBB2 induced mammary tumorigenesis mouse model. For this, we used MMTV-NIC mice, which carry an activated NEU oncogene (ERBB2) under control of the MMTV-LTR promoter that also co-expresses Cre recombinase. These mice were crossed to a conditional DLC-2 knockout mouse (floxed exon 3). Objectives: To determine if heterozygous or homozygous DLC-2 loss plays a role in mouse mammary tumor progression in MMTV-NIC mice. Results: Our preliminary results showed that heterozygous deletion of DLC-2 in the mammary epithelium of the MMTV-NIC mouse model resulted in increased tumor burden with accelerated tumor growth. Metastases to the lungs were quantified by scanning H&E stained step sections (4X150uM). We observed a significant increase in the incidence of lung metastases in heterozygous DLC-2 exon3floxed/+ MMTV-NIC mice. The lung metastases showed a homogenous morphology and histopathologically similar phenotype to the MMTV-NIC strain. DLC-2 heterozygous tumor cells showed increased tumorosphere formation under low-attachment conditions with minimal growth factor supplementation. These tumor cells also showed increased expression of mesenchymal cell markers, as determined by quantitative real time RT-PCR, suggesting an Epithelial to Mesenchymal transition is occurring. Experiments with homozygous floxed DLC-2 mice are ongoing and will be presented. Conclusion: These results indicate that loss of one allele of DLC-2 is sufficient to accelerate NEU oncogene induced tumorigenesis. Citation Format: Pratima Basak, Pratima Basak, Heather Leslie, Heather Leslie, Afshin Raouf, Afshin Raouf, Michael RA Mowat, Michael RA Mowat. Role of the tumor suppressor gene DLC-2 in ERBB2 induced mouse mammary tumorigenesis and metastasis. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Metastasis; 2015 Nov 30-Dec 3; Austin, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(7 Suppl):Abstract nr B18.

Neu-Induced Retroviral Rat Mammary Carcinogenesis: A Novel Chemoprevention Model for Both Hormonally Responsive and Nonresponsive Mammary Carcinomas

Clinically relevant animal models of mammary carcinogenesis are crucial for the development and evaluation of new breast cancer chemopreventive agents. The neu-induced retroviral rat mammary carcinogenesis model is based on the direct in situ transfer of the activated neu oncogene into the mammary epithelium using a replication-defective retroviral vector. The resulting mammary carcinomas in intact Wistar-Furth rats exhibit a mixed hormonal response in the same proportion as has been observed in women. In intact rats, approximately 50% of mammary carcinomas can be prevented by tamoxifen treatment. In ovariectomized animals, the mammary carcinomas are hormonally nonresponsive and cannot be prevented by tamoxifen. We evaluated the efficacy of retinoic X receptor-selective retinoids (rexinoids) in this novel model of mammary carcinogenesis. The rexinoids LG100268 and bexarotene (LG1069, Targretin) were highly efficacious in the prevention of neu-induced mammary carcinomas. Dietary LG100268 at 100 mg/kg diet decreased tumor multiplicity by 32% (P = 0.0114) in intact rats and 50% (P < 0.0001) in ovariectomized rats. Bexarotene treatment at a dose of 250 mg/kg diet was associated with reductions in tumor multiplicity of 84% (P < 0.0001) and 86% (P < 0.0001) in intact and ovariectomized animals, respectively. In addition to tumor multiplicity, proliferation and apoptosis were modulated by bexarotene treatment independently of estrogen signaling. The neu-induced retroviral rat mammary carcinogenesis model represents a valuable addition to existing rodent chemoprevention models. The model is useful for assessing the efficacy of chemopreventive agents, specifically those compounds that target hormonally nonresponsive tumors.

Trastuzumab mimotope vaccination of HER-2/neu transgenic mice results in prolonged tumor-free survival and reduced tumor load

12510 Background: Passive immunotherapy with the humanized monoclonal antibody trastuzumab (Herceptin) to date is the most effective treatment for patients with HER-2/neu overexpressing breast cancer. In previous studies we could show that active immunization with peptide mimotopes, i.e. structural mimics of the epitope recognized by trastuzumab, leads to formation of antibodies again recognizing HER-2/neu in mice. Functional in vitro analyses of the induced antibodies demonstrated “trastuzumab-like” properties, such as receptor internalization, inhibition of signaling, and antibody-mediated cytotoxicity against HER-2/neu overexpressing breast cancer cells. The aim of the present study was to test the effects of trastuzumab mimotope vaccination in vivo, namely in a HER-2/neu transgenic mouse model. Methods: We used BALB-neuT mice, which carry the activated neu oncogene on the BALB/c background. These mice constitute the most aggressive animal model for HER-2/neu driven carcinogenesis, as all females uniformly develop mammary carcinomas at the age of 12 weeks. One group of mice was immunized with the previously described trastuzumab mimotope - KLH conjugate, a control group with the carrier protein KLH alone, and a further control group was left naïve. Mice were palpated weekly to monitor tumor development and size, and blood samples were taken at regular intervals to follow up the induced immune responses. Results: Trastuzumab mimotope immunizations lead to delayed tumor development and thus to an increase in tumor-free survival. When tumors occurred, they were smaller and grew more slowly than in the control mice. In contrast, control KLH immunizations did not affect tumor growth kinetics as compared to the naïve mice. Serum analysis demonstrated that all immunized animals had mounted an anti-KLH immune response, so we accredit the observed tumor-inhibitory effects in the mimotope group to the biologic properties of induced anti-HER-2/neu antibodies. Conclusion: These results indicate that epitope-specific vaccination with mimotopes elicits trastuzumab-like antibodies that are effective in vivo against HER-2/neu overexpressing tumor cells also in HER-2/neu expressing organisms. [Table: see text]

Vaccination by Genetically Modified Dendritic Cells Expressing a TruncatedneuOncogene Prevents Development of Breast Cancer in Transgenic Mice

Dendritic cells (DCs) are powerful antigen-presenting cells that process antigens and present peptide epitopes in the context of the major histocompatibility complex molecules to generate immune responses. DCs are being studied as potential anticancer vaccines because of their ability to present antigens to naive T cells and to stimulate the expansion of antigen-specific T-cell populations. We investigated an antitumor vaccination using DCs modified by transfer of a nonsignaling neu oncogene, a homologue of human HER-2/neu, in a transgenic model of breast cancer. BALB-neuT mice develop breast cancers as a consequence of mammary gland-specific expression of an activated neu oncogene. We vaccinated BALB-neuT mice with bone marrow-derived DCs transduced with Ad.Neu, a recombinant adenovirus expressing a truncated neu oncoprotein. The vaccine stimulated the production of specific anti-neu antibodies, enhanced interferon-gamma expression by T cells, and prevented or delayed the onset of mammary carcinomas in the mice. Over 65% of vaccinated mice remained tumor free at 28 weeks of age, whereas all of the mice in the control groups developed tumors. When challenged with a neu-expressing breast cancer cell line, vaccinated tumor-free animals had delayed tumor growth compared with controls. The antitumor effect of the vaccine was specific for expression of neu. Studies showed that CD4+ T cells were required in order to generate antitumor immunity. Importantly, the effectiveness of the vaccine was not diminished by preexisting immunity to adenovirus, whereas the protection afforded by vaccination that used direct injection of Ad.Neu was markedly reduced in mice with anti-adenovirus antibody titers. DCs modified by recombinant adenoviruses expressing tumor-associated antigens may provide an effective antitumor vaccination strategy.

The κB and V(D)J Recombination Signal Sequence Binding ProteinKRC Regulates Transcription of the Mouse Metastasis-associated Gene S100A4/mts1

A kappaB-like sequence, Sb, is integral to the composite enhancer located in the first intron of the metastasis-associated gene, S100A4/mts1. Oligonucleotides containing this sequence form three specific complexes with nuclear proteins prepared from S100A4/mts1-expressing CSML100 adenocarcinoma cells. Protein studies show the Sb-interacting complexes include NF-kappaB/Rel proteins, p50.p50 and p50.p65 dimers. Additionally, the Sb sequence was bound by an unrelated approximately 200-kDa protein, p200. Site-directed mutagenesis in conjunction with transient transfections indicate that p200, but not the NF-kappaB/Rel proteins, transactivates S100A4/mts1. To identify candidate genes for p200, double-stranded DNA probes containing multiple copies of Sb were used to screen a randomly primed lambdagt11 cDNA expression library made from CSML100 poly(A)(+) RNA. Two clones corresponding to the DNA-binding proteins KRC and Alf1 were identified. KRC encodes a large zinc finger protein that binds to the kappaB motif and to the signal sequences of V(D)J recombination. In vitro DNA binding assays using bacterially expressed KRC fusion proteins, demonstrate specific binding of KRC to the Sb sequence. In addition, introduction of KRC expression vectors into mammalian cells induces expression of S100A4/mts1 and reporter genes driven by S100A4/mts1 gene regulatory sequences. These data indicate that KRC positively regulates transcription of S100A4/mts1.

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

Transplacentally initiated schwannomas in mice and rats arise preferentially in the Gasserian ganglion of the trigeminal nerve and spinal root ganglia, while those of the Syrian golden hamster most commonly occur subcutaneously. Rat and hamster schwannomas almost invariably contain a mutationally activated neu oncogene. In rat schwannomas, the mutant allele predominates, while the relative abundance of mutant alleles is very low in hamster nerve tumors. We investigated whether neu is mutated in mouse schwannomas and whether the pattern and allelic ratio of the mutation resemble those for the hamster or the rat. Pregnant C3H/HeNCr mice received 0.4 micromol N-nitrosoethylurea/g body weight on day 19 of gestation. Ten trigeminal and one peripheral nerve schwannomas developed in 11 of the 201 offspring. Missense T --> A transversion mutations were detected in the neu transmembrane domain in eight of ten schwannomas analyzed, as determined by MnlI digestion of polymerase chain reaction products. The mutant allele was predominantly detected in two tumors and was abundant in six others. Transfection of eight out of ten mouse tumor DNAs into hamster cells yielded transformed foci; seven out of eight contained mutant mouse neu. Mouse schwannomas closely resembled those of rats both in the preferred anatomical site and in the mutant/wild-type neu allele ratios.

Distinct tyrosine autophosphorylation sites negatively and positively modulate neu-mediated transformation.

A number of cytoplasmic signaling molecules are thought to mediate mitogenic signaling from the activated Neu receptor tyrosine kinase through binding specific phosphotyrosine residues located within the intracellular portion of Neu/c-ErbB-2. An activated neu oncogene containing tyrosine-to-phenylalanine substitutions at each of the known autophosphorylation sites was generated and assessed for its specific transforming potential in Rat1 and NIH 3T3 fibroblasts. Mutation of these sites resulted in a dramatic impairment of the transforming potential of neu. To assess the role of these tyrosine phosphorylation sites in cellular transformation, the transforming potential of a series of mutants in which individual tyrosine residues were restored to this transformation-debilitated neu mutant was evaluated. Reversion of any one of four mutated sites to tyrosine residues restored wild-type transforming activity. While each of these transforming mutants displayed Ras-dependent signaling, the transforming activity of two of these mutants was correlated with their ability to bind either the GRB2 or SHC adapter molecules that couple receptor tyrosine kinases to the Ras signaling pathway. By contrast, restoration of a tyrosine residue located at position 1028 completely suppressed the basal transforming activity of this mutated neu molecule or other transforming neu molecules which possessed single tyrosine residues. These data argue that the transforming potential of activated neu is mediated both by positive and negative regulatory tyrosine phosphorylation sites.

The genetic penetrance of the activated neu oncogene for the induction of mammary cancer in vivo.

Carcinogenesis is most often viewed as a multistage disease process. An exception to this was suggested for neu transformation of mammary cells in a transgenic model (Muller et al., 1988); however, this interpretation is controversial (Bouchard et al., 1989). In order to better define neu mammary transformation in vivo, we directly measured the genetic penetrance of the neu oncogene. Mammary cells in situ were infected with replication-defective retroviral vectors carrying the activated neu oncogene (pJRneu). A limiting dilution in vivo transplantation assay was used to measure the percentage of mammary clonogenic (stem-like) cells that stably and functionally integrated this vector. Based on this, the percentage of clonogens integrating and expressing neu that could progress to mammary carcinomas was quantified to estimate the penetrance of this gene in mammary carcinogenesis. The genetic penetrance of neu was 3.6% (95% confidence interval 2.2%-5.8%). This high degree of genetic penetrance is compatible with the observations that certain neu-transgenic mice develop a very great number of mammary carcinomas (Muller et al., 1988). However, whether these data are compatible with a single-step transformation model (100% penetrance) is uncertain and is discussed.

Activated neu Induces Rapid Tumor Progression

Expression of the activated neu oncogene in transgenic mice has been associated with both the synchronous (single-step) and the stochastic (multistep) transformation of the mammary epithelium. To determine the basis for these conflicting observations, additional strains of transgenic mice carrying the activated neu oncogene under the transcriptional control of the mouse mammary tumor virus promoter/enhancer were produced. Activated neu transgene expression, as measured by in situ hybridization and ribonuclease protection assays, resulted in rapid conversion of the normal mammary epithelium to malignant phenotype in three independent strains of mice. Expression of the transgene in male mice led to epithelial hyperplasia of the epididymis and male infertility but not malignancy. These results indicate that tissue context is an important parameter in malignant progression and that expression of appropriate levels of activated neu is sufficient for rapid production of mammary tumors in transgenic mice.

PML suppresses oncogenic transformation of NIH/3T3 cells by activated neu.

The chromosomal translocation t(15;17)(q22;q12) is a consistent feature of acute promyelocytic leukemia (APL) that results in the disruption of genes for the zinc finger transcription factor PML and the retinoic acid receptor alpha (RAR alpha). We have previously shown that PML is a growth suppressor and is able to suppress transformation of NIH/3T3 by activated neu oncogene. In the study presented here, the full-length PML cDNA was transfected into B104-1-1 cells (NIH/3T3 cells transformed by the activated neu oncogene) by retrovirally mediated gene transfer. We found that expression of PML could reverse phenotypes of B104-1-1 including morphology, contact-limiting properties, and growth rate in both transient-expression and stable transfectants. We also demonstrated that PML is able to suppress clonogenicity of B104-1-1 in soft agar assay and tumorigenicity in nude mice. These results strongly support our previous finding that PML is a transformation or growth suppressor. Our results further demonstrate that expression of PML in B104-1-1 cells has little effect on cell cycle distribution. Western blot analysis demonstrated that suppression of neu expression in B104-1-1 by PML was insignificant in the transient transfection experiment but significant in the PML stable transfectants. This study suggests that PML may suppress neu expression and block signaling events associated with activated neu. This study supports our hypothesis that disruption of the normal function of PML, a growth or transformation suppressor, is a critical event in APL leukomogenesis.

PML, a growth suppressor disrupted in acute promyelocytic leukemia.

The nonrandom chromosomal translocation t(15;17)(q22;q21) in acute promyelocytic leukemia (APL) juxtaposes the genes for retinoic acid receptor alpha (RAR alpha) and the putative zinc finger transcription factor PML. The breakpoint site encodes fusion protein PML-RAR alpha, which is able to form a heterodimer with PML. It was hypothesized that PML-RAR alpha is a dominant negative inhibitor of PML. Inactivation of PML function in APL may play a critical role in APL pathogenesis. Our results demonstrated that PML, but not PML-RAR alpha, is a growth suppressor. This is supported by the following findings: (i) PML suppressed anchorage-independent growth of APL-derived NB4 cells on soft agar and tumorigenicity in nude mice, (ii) PML suppressed the oncogenic transformation of rat embryo fibroblasts by cooperative oncogenes, and (iii) PML suppressed transformation of NIH 3T3 cells by the activated neu oncogene. Cotransfection of PML with PML-RAR alpha resulted in a significant reduction in PML's transformation suppressor function in vivo, indicating that the fusion protein can be a dominant negative inhibitor of PML function in APL cells. This observation was further supported by the finding that cotransfection of PML and PML-RAR alpha resulted in altered normal cellular localization of PML. Our results also demonstrated that PML, but not PML-RAR alpha, is a promoter-specific transcription suppressor. Therefore, we hypothesized that disruption of the PML gene, a growth or transformation suppressor, by the t(15;17) translocation in APL is one of the critical events in leukemogenesis.

PML, a growth suppressor disrupted in acute promyelocytic leukemia.

The nonrandom chromosomal translocation t(15;17)(q22;q21) in acute promyelocytic leukemia (APL) juxtaposes the genes for retinoic acid receptor alpha (RAR alpha) and the putative zinc finger transcription factor PML. The breakpoint site encodes fusion protein PML-RAR alpha, which is able to form a heterodimer with PML. It was hypothesized that PML-RAR alpha is a dominant negative inhibitor of PML. Inactivation of PML function in APL may play a critical role in APL pathogenesis. Our results demonstrated that PML, but not PML-RAR alpha, is a growth suppressor. This is supported by the following findings: (i) PML suppressed anchorage-independent growth of APL-derived NB4 cells on soft agar and tumorigenicity in nude mice, (ii) PML suppressed the oncogenic transformation of rat embryo fibroblasts by cooperative oncogenes, and (iii) PML suppressed transformation of NIH 3T3 cells by the activated neu oncogene. Cotransfection of PML with PML-RAR alpha resulted in a significant reduction in PML's transformation suppressor function in vivo, indicating that the fusion protein can be a dominant negative inhibitor of PML function in APL cells. This observation was further supported by the finding that cotransfection of PML and PML-RAR alpha resulted in altered normal cellular localization of PML. Our results also demonstrated that PML, but not PML-RAR alpha, is a promoter-specific transcription suppressor. Therefore, we hypothesized that disruption of the PML gene, a growth or transformation suppressor, by the t(15;17) translocation in APL is one of the critical events in leukemogenesis.

Cellular transformation by a transmembrane peptide: structural requirements for the bovine papillomavirus E5 oncoprotein.

The E5 oncoprotein of bovine papillomavirus, only 44 amino acids long, occurs as a disulfide-bonded transmembrane dimer. This remarkable oncoprotein stimulates signal transduction through activation of the platelet-derived growth factor (PDGF) receptor, and E5 exhibits limited amino acid sequence similarity with PDGF. Results presented here suggest that a key feature of the hydrophobic transmembrane domain is an amino acid side chain that participates in interhelical hydrogen bond formation. These data are reminiscent of the activated neu oncogene, in which a point mutation in the transmembrane domain leads to ligand-independent dimerization and activation of a receptor tyrosine kinase. Significantly, the transmembrane domain of E5 can be largely replaced by the transmembrane domain from the activated neu receptor tyrosine kinase. Extensive mutagenesis defines the minimal structural features required for transformation by the E5 oncoprotein as, first, the ability to dimerize and, second, presentation of a negatively charged residue at the extracellular side of the membrane. The biological activity of E5 mutants that lack most amino acid residues similar to PDGF suggests that E5 and PDGF activate the PDGF receptor by distinct mechanisms.

Expression of the neu oncogene under the transcriptional control of the myelin basic protein gene in transgenic mice: generation of transformed glial cells.

We have taken a transgenic approach in an effort to specifically transform oligodendrocytes, the myelinating glial cells of the central nervous system (CNS). Transgenic mice were generated with a DNA construct that contained the activated neu oncogene under the transcriptional control of the myelin basic protein (MBP) gene. The MBP/c-neu transgenic animals have experienced a low incidence of brain tumors that express molecular markers specific to oligodendrocytes, providing a mouse model to study the formation and progression of oligodendrocyte tumors. A tumor from a transgenic animal has been dispersed in culture, and transformed cells that express properties of oligodendrocytes and astrocytes have been maintained. The degree to which these cells express phenotypic characteristic of oligodendrocytes or astrocytes is influenced by culture conditions. These transformed cells should serve as a valuable resource with which to study various molecular and biochemical aspects of the myelination process, as well as the lineage interrelationship of CNS glial cells.

Amplification of the proto-neu oncogene facilitates oncogenic activation by a single point mutation.

To determine whether the amplification of the proto-neu oncogene (also called c-erbB-2) plays a role in tumorigenicity, we previously generated an NIH 3T3 transfectant (DHFR/G-8) that carried the amplified proto-neu gene. The DHFR/G-8 cells exhibited normal morphology. Their growth curve was similar to that of NIH 3T3 cells but was different from that of the B104-1 cell, and NIH 3T3 transfectant that carries the activated neu oncogene. When injected into nude mice, B104-1 cells produced tumors within 2 weeks, whereas the DHFR/G-8 cells did not produce tumors until 3 months after injection, and the NIH 3T3 cells did not produce any tumors even after 3 months. The tumors produced by the injection of the DHFR/G-8 cells were excised and grown in culture. The cells derived from the tumors were of transformed morphology and highly tumorigenic. The DNAs from the tumor cells were transfected into NIH 3T3 cells. The transfection resulted in foci on the NIH 3T3 monolayer. Southern analysis indicated that the foci derived from the transfection contained the neu gene. Using oligonucleotides as probes, the neu gene in the foci was found to carry a single-point mutation identical to the one previously found in the rat neuroblastoma and glioblastoma induced by the ethylnitrosourea. We conclude that the DNA region encoding the transmembrane domain of neu is a hot spot for converting the proto-neu gene into an activated oncogene and that amplification of the proto-neu gene facilitates mutation of the hot spot.

Increased tyrosine kinase activity associated with the protein encoded by the activated neu oncogene.

A single mutation altering the transmembrane domain of the receptor-like p185 protein encoded by the rat neu gene converts the normal neu gene into a potent oncogene. The biochemical consequences of this mutation were studied by examining phosphorylation of the normal and transforming p185 molecules in membrane preparations. Here we show that the transforming p185 is phosphorylated to a much higher extent in vitro than its normal counterpart. This preferential phosphorylation has the properties that would be expected of p185 autophosphorylation: it takes place on tyrosine and requires intact p185 kinase activity. The normal p185 protein does not demonstrate increased phosphorylation even when it coexists in a transformed cell with the transforming p185 protein. These data show that transforming p185 is specifically associated with an active tyrosine kinase activity and suggest that this activity is intrinsic to the transforming protein. Thus, the transmembrane domain of p185 appears to directly regulate its kinase activity.

Activated neu oncogene sequences in primary tumors of the peripheral nervous system induced in rats by transplacental exposure to ethylnitrosourea.

Neurogenic tumors were selectively induced in high incidence in F344 rats by a single transplacental exposure to the direct-acting alkylating agent N-ethyl-N-nitrosourea (EtNU). We prepared DNA for transfection of NIH 3T3 cells from primary glial tumors of the brain and from schwannomas of the cranial and spinal nerves that developed in the transplacentally exposed offspring between 20 and 40 weeks after birth. DNA preparations from 6 of 13 schwannomas, but not from normal liver, kidney, or intestine of tumor-bearing rats, transformed NIH 3T3 cells. NIH 3T3 clones transformed by schwannoma DNA contained rat repetitive DNA sequences, and all isolates contained rat neu oncogene sequences. One schwannoma yielded a transformant with rat-specific sequences for both neu and N-ras. A point mutation in the transmembrane region of the putative protein product of neu was identified in all six transformants and in the primary tumors from which they were derived as well as in 5 of 6 schwannomas tested that did not transform NIH 3T3 cells. Of 59 gliomas, only one yielded transforming DNA, and an activated N-ras oncogene was identified. The normal cellular neu sequence for the transmembrane region, but not the mutated sequence, was identified in DNA from all 11 gliomas surveyed by oligonucleotide hybridization. Activation of the neu oncogene, originally identified [Schechter, A.L., Stern, D.F., Vaidyanathan, L., Decker, S.J., Drebin, J.A., Greene, M.I. & Weinberg, R.A. (1984) Nature (London) 312, 513-516] in cultured cell lines derived from EtNU-induced neurogenic tumors that by biochemical but not histologic criteria were thought to originate in the central nervous system in BD-IX rats, appears specifically associated with tumors of the peripheral nervous system in the F344 inbred strain.