Braf Mutations Initiate the Development of Rat Gliomas Induced by Postnatal Exposure to N-Ethyl-N-Nitrosourea.

A single dose of N-ethyl-N-nitrosourea (ENU) during late prenatal or early postnatal development induces a high incidence of malignant schwannomas and gliomas in rats. Although T->A mutations in the transmembrane domain of the Neu (c-ErbB-2) gene are the driver mutations in ENU-induced malignant schwannomas, the molecular basis of ENU-induced gliomas remains enigmatic. We performed whole-genome sequencing of gliomas that developed in three BDIV and two BDIX rats exposed to a single dose of 80 mg ENU/kg body weight on postnatal day one. T:A->A:T and T:A->C:G mutations, which are typical for ENU-induced mutagenesis, were predominant (41% to 55% of all somatic single nucleotide mutations). T->A mutations were identified in all five rat gliomas at Braf codon 545 (V545E), which corresponds to the human BRAF V600E. Additional screening revealed that 33 gliomas in BDIV rats and 12 gliomas in BDIX rats all carried a Braf V545E mutation, whereas peritumoral brain tissue of either strain had the wild-type sequence. The gliomas were immunoreactive to BRAF V600E antibody. These results indicate that Braf mutation is a frequent early event in the development of rat gliomas caused by a single dose of ENU.

N-methyl-N-nitrosourea-induced schwannomas in male Sprague-Dawley rats with a literature review of inducible and spontaneous lesions.

N-methyl-N-nitrosourea (MNU) possesses peripheral nervous system carcinogenic activity in rats and induces benign and malignant schwannomas in systemic organs. In this retrospective study, we compared the characteristics of various immunohistochemical markers in MNU-induced schwannomas in male Crj:CD(SD)IGS rats including: vimentin (Vim), S100, p75 nerve growth factor receptor (LNGFR), CD57, pancytokeratin (CK), myoglobin, desmin and α smooth muscle actin (SMA). Single intraperitoneal exposures of 50 or 75mg/kg MNU in male rats at the age of 4 weeks induced schwannomas in 43 surviving and terminated rats up to 30-weeks-old. The incidence rate of neoplastic lesions was 37% (16 of 43 rats). Benign schwannomas (mesentery, pancreas, thymus) and malignant schwannomas (subendocardium, cardiac intramural, thoracic cavity, abdominal cavity, prostate), occurred in nine and seven rats, respectively. All neoplastic lesions were moderately or strongly positive for Vim, S100 and LNGFR proteins. Benign tumors were weakly positive and malignant tumors strongly positive for Ki-67, suggesting a high active proliferation rate of Schwann cell precursors. All lesions were negative for CD57, CK, myoglobin, desmin and SMA. This data may provide useful immunohistochemical information for the investigation of schwannomas in rat chemical carcinogenicity studies.

A liver schwannoma observed in a female Sprague-Dawley rat treated with MNU.

BACKGROUND: Schwannoma is a tumor of the nervous system composed by Schwann cells. It can occur naturally in several tissues of the body in both humans and animals. Diaphragmatic hernia can be congenital or acquired and is defined as a protrusion of abdominal viscera into the thoracic cavity. MATERIALS AND METHODS: The animal was a female rat from an experiment of mammary tumor chemically induced. It was injected with N-methyl-N-nitrosourea (MNU) and died spontaneously at 22 weeks of age. RESULTS: The animal had a diaphragmatic hernia and a hemorrhagic and multicystic mass in the liver herniated lobule. Microscopically the liver displayed a well circumscribed mass that was a tumor with hemorrhagic areas, necrosis and Antoni A and Antoni B patterns. It also displayed occasional positivity to vimentin and diffuse positivity to S-100 and NSE. CONCLUSION: The tumor was a schwannoma with the origin in the Glisson's capsule.

Estudio morfológico de la neurocarcinogénesis experimental inducida por nitroso-ureas / Morphological study of the experimental neurocarcinogenesis induced by nitrosoureas

En el presente trabajo se hace una revisión de la neurocarcinogénesis química,con especial referencia a los tumores del Sistema Nervioso inducidos por etil-nitrosoureaen la rata Wistar. El estudio morfológico de los tumores del Sistema NerviosoCentral y del Sistema Nervioso Periférico inducidos por exposición prenatala este carcinógeno (50 mg/kg día 17 de gestación) muestra la aparición de tumorescompatibles con un diagnóstico histológico de schwannoma maligno o de oligodendroglioma.Sin embargo, los estudios inmunohistoquímicos y ultraestructuralespermiten clasificar a estos tumores, independientemente de su morfología, comotumores malignos, de tipo neuroectodérmico indiferenciado, con posibilidad dediferenciación hacia líneas neuronales y gliales. Sus características biológicas y suposible diagnóstico in vivo, por medio de Resonancia Magnética, hacen que estemodelo de neurocarcinogénesis sea extraordinariamente útil para valorar el efectode nuevos tratamientos farmacológicos, aplicables a tumores cerebrales malignosen el hombre

This work shows a revision of the chemical neurocarcinogenesis, with specialreference to the Nervous System tumors induced by ethyl-nitrosourea in the Wistarrat. Prenatal administration of this carcinogen (50 mg/kg, day 17 of pregnancy)induces Nervous System tumors with histological features of malignantschwannomas or oligodendrogliomas. However, and independently of theirmorphology, the immunohistochemical and ultrastructural studies allow to classifyto these tumors as undifferentiated malignant tumors, with tumor cells showingpossible differentiation toward neuronal and glial phenotypes. Their biologicproperties and their possible diagnosis in vivo, by means of Magnetic Resonance,make this neurocarcinogenesis model to be extraordinarily useful to value theeffect of new drug treatments, applicable to human malignant brain tumors

[Tumour induction by ethylnitrosourea in the central nervous system]. / Inducción de tumores en el sistema nervioso central mediante etilnitrosourea.

INTRODUCTION: Experimental central nervous system (CNS) tumours have been proposed as a useful model for the study of oncogenesis, epiphenomena related to cancer and for the design of new therapeutic strategies. DEVELOPMENT: The administration of chemical substances is one of the most commonly-used methods to induce CNS neoplasms. N-ethyl-N-nitrosourea (ENU) belongs to the nitrosourea family, a wide group of alkylating agents that are able to induce brain tumours in litters after transplacentary administration at the 15th day of pregnancy. This nitrogenous urea compound has a high mutation inducibility affecting the expression of oncogenes such as p53, neu/erbB-2 and Ras. Prenatal exposition of Sprague Dawley rats to ENU induces intra-axial tumours of glial lineage and extra-axial malignant schwannomas. Although the precise mechanism of tumour induction is unclear, it is known to affect cell differentiation of primitive neuroepithelium from the subventricular plate generating oligodendrogliomas, astrocytomas, mixed gliomas or ependimomas. CONCLUSION: The transplacentary administration of ENU induces the development of gliomas and schwannomas that are similar to those found in humans. Animal models are necessary and useful for further studies to get an early diagnosis and to establish correct therapeutic indications.

Inducción de tumores en el sistema nervioso central mediante etilnitrosourea / Tumour induction by ethylnitrosourea in the central nervous system

Introducción. La utilización de modelos experimentalesin vivo de tumores en el sistema nerviosos central (SNC) ha traídoimportantes avances a la neurooncología. Estos modelos animaleshan permitido el estudio de procesos de oncogénesis, de sus epifenómenosy del diseño de nuevas estrategias terapéuticas. Desarrollo.Existen varios métodos de inducción de neoplasias en el SNC,de los cuales la administración de sustancias químicas es una delas modalidades más utilizadas. La N-etil-N-nitrosourea (ENU) esun agente alquilante capaz de inducir tumores cerebrales en la descendenciade ratas gestantes tras su administración transplacentaria.Se tata de un compuesto nitrogenado de la urea con alto podermutagénico que afecta a la expresión de ciertos oncogenes comop53, neu/erbB-2 y Ras. Mediante la exposición prenatal a ratasSprague Dawley del carcinógeno ENU se inducen tumores intraaxialesde estirpe glial y tumores extraaxiales como los schwannomasmalignos. Aunque se desconoce el mecanismo preciso de inducciónde los tumores gliales, se sabe que afecta a la diferenciaciónde las células neuroepiteliales primitivas de la placa subventricular,lo que genera oligodendrogliomas, astrocitomas, gliomasmixtos o ependimomas. Conclusión. La administración transplacentariade ENU permite obtener gliomas y schwannomas malignossimilares a los encontrados en los humanos. Esto puede ayudaral estudio en profundidad de dichos tumores para llegar a realizarun diagnóstico precoz y asentar unas indicaciones terapéuticasprecisas

Introduction. Experimental central nervous system (CNS) tumours have been proposed as a useful model for thestudy of oncogenesis, epiphenomena related to cancer and for the design of new therapeutic strategies. Development. Theadministration of chemical substances is one of the most commonly-used methods to induce CNS neoplasms. N-ethyl-Nnitrosourea(ENU) belongs to the nitrosourea family, a wide group of alkylating agents that are able to induce brain tumoursin litters after transplacentary administration at the 15th day of pregnancy. This nitrogenous urea compound has a highmutation inducibility affecting the expression of oncogenes such as p53, neu/erbB-2 and Ras. Prenatal exposition of SpragueDawley rats to ENU induces intra-axial tumours of glial lineage and extra-axial malignant schwannomas. Although theprecise mechanism of tumour induction is unclear, it is known to affect cell differentiation of primitive neuroepithelium fromthe subventricular plate generating oligodendrogliomas, astrocytomas, mixed gliomas or ependimomas. Conclusion. The transplacentaryadministration of ENU induces the development of gliomas and schwannomas that are similar to those found inhumans. Animal models are necessary and useful for further studies to get an early diagnosis and to establish correcttherapeutic indications

The interaction mode of premalignant Schwann and immune effector cells during chemically induced carcinogenesis in the rat peripheral nervous system is strongly influenced by genetic background.

Contrary to rats of the highly sensitive inbred strain BDIX, BDIV rats are resistant to the induction of malignant schwannomas by N-ethyl-N-nitrosourea, arising predominantly in the trigeminal nerves. A point mutation of the neu/erbB-2 gene diagnostic of N-ethyl-N-nitrosourea-induced rat schwannomas is an early marker of Schwann precursor cells at high risk of subsequent malignant transformation. Neu-mutant cells initially arise at a similar frequency in sensitive and resistant animals. However, these cells disappear from the trigeminal nerves of resistant rats while giving rise to highly malignant schwannomas in susceptible animals. The resistance of BDIV rats obviously includes mechanisms to recognize and eliminate premalignant cells. The involvement of a cellular immune response was investigated in trigeminal nerves of both strains at different times after neonatal carcinogen exposure. An inflammatory reaction involving sequentially CD4(+) macrophages and T helper cells, CD8(+) cytotoxic T cells, and ED1(+) and ED2(+) macrophages was detected as a consequence of N-ethyl-N-nitrosourea treatment as early as postnatal day 40, briefly after the emergence of premalignant neu-mutant Schwann cells. It persisted throughout the observation period (40-250 days). However, there were no gross differences in immune cell counts between tumor-susceptible and tumor-resistant rats, except for a moderate increase of ED2(+) macrophages in N-ethyl-N-nitrosourea-treated BDIX rats only. Differential interactions of immune effector cells with premalignant Schwann cells may thus be involved in genetically determined tumor susceptibility or resistance, which could include functional differences of immune effector cells and/or a differential capability of premalignant Schwann cells to escape or counteract the cellular immune response.

Gender-specific polygenic control of ethylnitrosourea-induced oncogenesis in the rat peripheral nervous system.

The inbred BD rat strains constitute a model system for analysis of the genetic basis of susceptibility or resistance to the development of neural tumors, as they exhibit distinct strain-specific differences regarding the sensitivity to tumor induction by the alkylating carcinogen N-ethyl-N-nitrosourea (EtNU). Among the different BD strains, BDIX and BDIV rats, respectively, are either highly susceptible or entirely resistant to the development of EtNU-induced malignant schwannomas of the peripheral nervous system (PNS), predominantly of the trigeminal nerves. We have previously mapped one locus associated with susceptibility/resistance to schwannoma induction to the telomeric third of chromosome 10 (Mss1) in segregating (BDIX x BDIV) crosses. We report on the genetic mapping of 6 further loci controlling tumor incidence or survival time on chromosomes 1 (Mss2), 3 (Mss3), 6 (Mss4), 13 (Mss5) and 15 (Mss6) as well as on chromosome 10 (Mss7) close to the centromere. Interestingly, most of these loci mediate gender-specific effects of variable strength ranging from minor influences on tumor development to complete tumor resistance. The gender specificity is reflected by the fact that male (BDIX x BDIV) F2 rats exhibit a 2-fold higher incidence of EtNU-induced schwannomas than females as well as a shorter survival time. A number of human nervous system tumors too arise with a marked gender bias. Genes mediating gender-specific predisposition of developing malignant schwannomas in the rat may be relevant for the human individual risk of developing nervous system tumors.

Loss of heterozygosity in malignant rat schwannomas chemically induced in hybrids of inbred rat strains with differential tumor susceptibility.

Rats of the inbred BD strains strongly differ in their susceptibility to the induction of tumors of the central (CNS) and peripheral nervous system (PNS) by N-ethyl-N-nitrosourea (EtNU). Malignant schwannomas induced in (BDIX x BDIV) and (BDIX x BDVI) rat hybrids were analyzed to identify genetic alterations associated with EtNU-induced tumorigenesis in the PNS. EtNU-induced schwannomas exclusively exhibit an A:T T:A transversion mutation of the neu/Erbb-2 gene located on chromosome 10, with subsequent loss of the wild-type neu/Erbb-2 allele at a post-initiation stage. Targeted allelic deletion mapping previously revealed losses of heterozygosity (LOH) at the distal end of chromosome 10 in a large majority of (BDIX x BDIV) schwannomas. The aims of the present study were (i) to scan the whole genome for further LOHs; (ii) to narrow down the consensus regions of frequently occurring allelic deletions using tumors from different crosses of BD rats; and (iii) to determine the sequence of genetic alterations during schwannoma development. A limited number of (BDIX x BDIV) F(1) tumors were initially screened for LOH and microsatellite instability (MI) by amplifying 58 microsatellite markers spanning the whole genome. LOHs on chromosome 5 were detected in 9/17 tumors, with random loss of the parental alleles. Ninety-two schwannomas from different BD rat-crosses were then analyzed to solidify these data and to determine the consensus region of frequent LOHs. The results indicate that LOHs on chromosomes 10 and 5 are required for the development of EtNU-induced malignant schwannomas from immature neu/Erbb-2 mutant glial cells, and that putative tumor suppressor genes are localized on chromosome 10q32.3, corresponding to human chromosome 17q25.3, and the telomeric region of mouse chromosome 11, and on the telomeric quarter of chromosome 5. MI was detected in <0.2% of cases.

MNU induction of neoplasia in a platyfish model.

Interspecific hybrid crosses between members of the fish genus Xiphophorus have been used for over 70 years to study the genetic aspects of melanoma formation. In the well-established "Gordon-Kosswig" cross, the platyfish X. maculatus is outcrossed to the swordtail X. helleri, and the resulting backcross segregants spontaneously develop melanoma. We recently produced a distinct cross between X. maculatus and another platyfish species, X. couchianus. X. maculatus strain Jp 163 A is homozygous for several X-linked pigment pattern genes, including the Spotted dorsal (Sd), Dorsal red (Dr), and Anal fin spot (Af). Af is a sex-limited trait, coding exclusively for melanophores distributed on the modified anal fin or "gonopodium" in the adult male fish. Within F1 and BC1 hybrids (to X. couchianus), the Sd pigment pattern is phenotypically suppressed, whereas Dr and Af are enhanced. We exposed BC1 hybrids to the direct-acting carcinogen N-methyl-N-nitrosourea (MNU). Treatment led to the development of schwannomas, fibrosarcomas, and retinoblastomas. In addition, numerous MNU-treated males that inherited Af developed a pronounced melanotic phenotype, with melanin-containing cells oftentimes totally covering the gonopodium and extending further to grow within the ventral regions of the fish. Genetic linkage analysis of the BC1 hybrids revealed a significant (p < 0.01) association between CDKN2X genotype and the phenotypic degree of melanization. Such an association is consistent with a locus within linkage group V playing a role in the development of melanosis and delineates three genetic preconditions and a carcinogenic scheme resulting in melanosis of the ventral regions of hybrid fish. The overall study further alludes to the potential of using Xiphophorus fish to study carcinogenic mechanisms for tumors other than melanoma (schwannoma, fibrosarcoma, and retinoblastoma) and should enable extensive pathologic and molecular genetic studies of derived neoplastic abnormalities.

The effects of 860 MHz radiofrequency radiation on the induction or promotion of brain tumors and other neoplasms in rats.

Sprague-Dawley rats were irradiated with a continuous- wave (CW) or a pulsed-wave (P) radiofrequency (RF) for 6 h/day, 5 days/week from 2 up to 24 months of age. The RFs emanated from dipole antennas (1 W average output) 2.0 +/- 0.5 cm from the tip of each rat's nose. The RFs had an 860 MHz frequency, and the specific absorption rate was 1.0 W/ kg averaged over the brain. Fifteen groups of 60 rats (900 total) were formed from offspring of females injected i.v. with 0 (groups 1, 2, 9, 10, 13), 2.5 (groups 5, 6, 7, 8, 11, 12, 14) or 10 mg/kg (groups 3, 4, 15) ethylnitrosourea (ENU) to induce brain tumors. Groups 1, 3, 5 and 7 received the PRF, and groups 9 and 11 the CWRF; groups 2, 4, 6, 8, 10 and 12 were sham-irradiated, and groups 13-15 were cage controls. All rats but 2, totaling 898, were necropsied, and major tissues were studied histopathologically. There was no statistically significant evidence that the PRF or CWRF induced neoplasia in any tissues. Additionally, there was no significant evidence of promotion of cranial or spinal nerve or spinal cord tumors. The PRF or CWRF had no statistically significant effect on the number, volume, location, multiplicity, histological type, malignancy or fatality of brain tumors. There was a trend for the group that received a high dose of ENU and was exposed to the PRF to develop fatal brain tumors at a higher rate than its sham group; however, the result was not significant using the log-rank test (P = 0.14, 2-tailed). No statistically significant differences were related to the PRF or CWRF compared to controls in the low- or zero-dose groups regarding tumors of any kind.

Suppression of ethylnitrosourea-induced schwannoma development involves elimination of neu/erbB-2 mutant premalignant cells in the resistant BDIV rat strain.

Contrary to the response of rats of the highly sensitive inbred strain BDIX, BDIV rats are resistant to the induction of malignant schwannomas by exposure to the alkylating N-nitroso carcinogen N-ethyl-N-nitrosourea (EtNU). In BDIX rats, a point mutation at nucleotide 2012 in the transmembrane region of the neu/erbB-2 gene has proved to be a very early marker of initiated Schwann precursor cells with an elevated risk of malignant transformation, and is diagnostic of the resulting schwannomas. To gain insight into the cellular and molecular mechanisms responsible for the resistance of the BDIV strain, comparative quantitative neu mutation analyses combined with histomorphological studies were performed on the trigeminal nerves of EtNU-treated BDIV and BDIX rats as well as on their (BDIX x BDIV) F1 progeny. It was found that neu-mutant Schwann cells are initially present at comparable frequency in the trigeminal nerves of both resistant and sensitive animals. Contrasting with the progressive multiplication of mutant Schwann cells in BDIX trigeminal nerves, however, the numbers of mutant cells began to decrease during the intermediary phase of the carcinogenic process in BDIV animals, and premalignant neu-mutant cells were no longer detectable by the time BDIX rats developed full-blown trigeminal schwannomas. The resistance of BDIV rats thus involves the elimination of initiated neu-mutant Schwann cells during the postinitiation period of EtNU-induced schwannomagenesis via mechanisms that remain to be clarified.

Evaluation of the potential promoting effect of 60 Hz magnetic fields on N-ethyl-N-nitrosourea induced neurogenic tumors in female F344 rats.

The present study investigated the possible effect of 60 Hz magnetic fields (MFs) as promoters of neurogenic tumors initiated transplacentally by a chemical carcinogen, N-ethyl-N-nitrosourea (ENU). In a preliminary study, 5 mg of ENU was shown to induce 30 to 40% neurogenic tumors in F344 rats offspring after 420 days of observation. In the present study, 400 female rats were divided into eight different groups (50 animals/group) and exposed in utero (on day 18 of gestation) to a single intravenous dose of either Saline (Group I), or ENU, 5 mg/kg (Group II to VIII). Dams in group II were given no further treatment while dams in Groups III to VII were exposed to 5 different intensities of MFs forty eight hours later. Animals in group III were sham exposed (<0.02 microT) while groups IV to VII were exposed to 2, 20, 200, and 2000 microT, respectively. Dams in Group VIII were injected intraperitoneally with 12-O-tetradecanoylphrobol-13-acetate (TPA; 10 micrograms/kg) from day 19 until delivery, and then their female offspring continued to be injected every 15 days, starting at day 14 after birth until sacrifice (positive controls). Accordingly, this study included three different types of controls: Internal controls (Groups II and III) and positive control (Group VIII). Body weight, mortality and clinical observations were evaluated in all groups of animals during in-life exposure. Necropsy was performed on all exposed and control animals that died, were found moribund or sacrificed at termination of the study. Histopathological evaluation was done for all brains, spinal cords, cranial nerves, major organs (lungs, liver, spleen, kidneys, pituitary, thyroid and adrenals) and all gross lesions observed during necropsy. All clinical observations and pathological evaluations were conducted under "blinded" conditions. The findings from this ENU/MFs promotion study clearly demonstrate that, under our defined experimental conditions, exposure to 60 Hz linear (single axis) sinusoidal, continuous wave MFs had no effect on the survival of female F344 rats or on the number of animals bearing neurogenic tumors. These results suggest that MFs have no promoting effect on neurogenic tumors in the female F344 rats exposed transplacentally to ENU.

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.

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

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.

Ethylnitrosourea-induced development of malignant schwannomas in the rat: two distinct loci on chromosome of 10 involved in tumor susceptibility and oncogenesis.

Inbred rodent strains with differing sensitivity to experimental tumor induction provide model systems for the detection of genes that either are responsible for cancer predisposition or modify the process of carcinogenesis. Rats of the inbred BD strains differ in their susceptibility to the induction of neural tumors by N-ethyl-N-nitrosourea (EtNU). Newborn BDIX rats that are exposed to EtNU (80 microg/g body weight; injected s.c.) develop malignant schwannomas predominantly of the trigeminal nerves with an incidence >85%, whereas BDIV rats are entirely resistant. A T:A-->A:T transversion mutation at nucleotide 2012 of the neu (erbB-2) gene on chromosome 10, presumably the initial event in EtNU-induced schwannoma development, is later followed by loss of the wild-type neu allele. Genetic crosses between BDIX and BDIV rats served: (a) to investigate the inheritance of susceptibility; (b) to obtain animals informative for the mapping of losses of heterozygosity (LOH) in tumors with polymorphic simple sequence length polymorphisms (SSLPs); and (c) to localize genes associated with schwannoma susceptibility by linkage analysis with SSLPs. Schwannoma development was strongly suppressed in F1 animals (20% incidence). All of the F1 schwannomas displayed LOH on chromosome 10, with a consensus region on the telomeric tip encompassing D10Rat3, D10Mgh16 and D10Rat2 but excluding neu. A strong bias toward losing the BDIV alleles suggests the involvement of a BDIV-specific tumor suppressor gene(s). Targeted linkage analysis with chromosome 10 SSLPs in F2 intercross and backcross animals localized schwannoma susceptibility to a region around D10Wox23, 30 cM centromeric to the tip. Ninety-four % of F1 tumors exhibited additional LOH at this region. Two distinct loci on chromosome 10 may thus be connected with susceptibility to the induction and development of schwannomas in rats exposed to EtNU.

Three-dimensional imaging of nerve tissue by x-ray phase-contrast microtomography.

We show that promising information about the three-dimensional (3D) structure of a peripheral nerve can be obtained by x-ray phase-contrast microtomography (p-microCT; Beckmann, F., U. Bonse, F. Busch, and O. Günnewig, 1997. J. Comp. Assist. Tomogr. 21:539-553). P-microCT measures electronic charge density, which for most substances is proportional to mass density in fairly good approximation. The true point-by-point variation of density is thus determined in 3D at presently 1 mg/cm3 standard error (SE). The intracranial part of the rat trigeminal nerve analyzed for the presence of early schwannoma "microtumors" displayed a detailed density structure on p-microCT density maps. The average density of brain and nerve tissue was measured to range from 0.990 to 0.994 g/cm3 and from 1.020 to 1.035 g/cm3, respectively. The brain-nerve interface was well delineated. Within the nerve tissue, a pattern of nerve fibers could be seen that followed the nerve axis and contrasted against the bulk by 7 to 10 mg/cm3 density modulation. Based on the fact that regions of tumor growth have an increased number density of cell nuclei, and hence of the higher z element phosphorus, it may become possible to detect very early neural "microtumors" through increases of average density on the order of 10 to 15 mg/cm3 by using this method.

Immune-cell system provides the development of extraneural tumors in the ethyl-nitrosourea model of neurocarcinogenesis.

Immunological suppression of the immune-cell system by means of cyclosporin-A was performed at a stage corresponding to microtumor development in the ethyl-nitrosourea (ENU) model of neurocarcinogenesis. The results that we have obtained suggest that this immunological manipulation is related to the appearance of extraneural undifferentiated tumors, suggesting that the immune-cell system is effective in immunocompetent rodents for providing extraneural ENU carcinogenesis.

Schwannoma cells induce a tumor-cell-specific cytotoxic-T-cell response upon transplantation into syngeneic rats but escape elimination through the secretion of immunosuppressive factors.

Tumor cells often express antigens that can be recognized by the immune system. Despite induction of an immune response, the tumor cells escape their elimination. We have studied the mechanisms and factors which mediate these events in a syngeneic tumor model. NV2Cd rat schwannoma cells were transplanted into BDIX rats. After injection of 10(7) to 2 x 10(7) cells, tumors grew very slowly for 10 to 12 days. After that time, rapid growth was observed. The tumors consisted of compact areas of spindle-shaped cells with small cysts, many blood vessels and central necrotic areas. During tumor growth, the number of spleen cells and T lymphocytes increased, and cytotoxic T cells with specificity for the NV2Cd tumor cells were detected. The strong specific cellular immune response did not prevent the increase in tumor volume. We studied the biological activity of the fluid present in the cysts of the tumor. At a concentration of 1 ng to 10 microg protein per ml, the cyst fluid inhibited the proliferation of splenic T lymphocytes and B lymphocytes and of lymphoma cells, but enhanced the proliferation of NV2Cd tumor cells. The cyst fluids contain the immunosuppressive transforming growth factors (TGF)-beta1, -beta2 and -beta3, also the vascular endothelial growth factor (VEGF) and epidermal growth factor (EGF). Antibodies directed against TGF-beta relieved the suppression of T-cell growth by cyst fluid, but did not influence the proliferation of NV2Cd cells. The growth-modulating factors present in the tumor cyst fluid were also detected in conditioned medium from NV2Cd cells cultured in vitro. Our data suggest that tumors can escape the cellular immune response by the production of factors that inhibit lymphocytes. They also enhance their own growth environment by secreted factors.

Characterization of rat NCA/CD9 cell surface antigen and its expression by normal and malignant neural cells.

As part of investigations on ethylnitrosourea (EtNU)-induced neuro-oncogenesis in the rat, we have produced monoclonal antibodies (Mabs) specific for neural cell surface antigens (NCAs) by immunization with cells of the clonal tumorigenic neural rat cell line BT4Ca. Mabs designated as anti-NCA (alpha NCA1, alpha NCA2, alpha NCA3, alpha NCA4, and alpha NCA5) recognize proteins of 25 kDa and 23 kDa, as shown by immunoprecipitation and Western blot. The predominant 25-kDa protein was purified from BT4Ca cells by immunoaffinity chromatography with immobilized Mab alpha NCA1 and identified by N-terminal sequencing as the rat homologue of the CD9 antigen. Identification of proline as N-terminal amino acid of the purified protein suggests post-translational modification of CD9 in the rat central nervous system. The NCA/CD9 protein was localized in distinct regions of fetal and adult rat brain by immunofluorescence staining of frozen sections. Flow cytometric analyses of isolated fetal rat brain cells (FBC) showed that the proportion and number of NCA/CD9-expressing cells increased during prenatal development. Immunoreactivity of approximately 40% of brain cells isolated 13 days post conception (p.c.) indicated that NCA/CD9 is expressed by neuronal precursors at this stage of development. In primary cultures of rat FBC isolated 18 days p.c., the NCA/CD9 antigen was expressed by all premature and mature astrocytes, oligodendrocytes, ependymal cells, and microglial cells, but not by E-N-CAM-expressing neuronal progenitor cells and neurons. Furthermore, eight out of ten EtNU-induced malignant neural rat cell lines as well as EtNU-induced tumors of the central and peripheral nervous system exhibited intermediate or strong immunoreactivity with Mab alpha NCA1. Expression of the NCA/CD9 protein is, therefore, characteristic of both normal glial precursor cells and their malignant counterparts in the rat.