NG2-expressing glial precursor cells are a new potential oligodendroglioma cell initiating population in N-ethyl-N-nitrosourea-induced gliomagenesis.

Gliomas are the most common primary brain tumor affecting human adults and remain a therapeutic challenge because cells of origin are still unknown. Here, we investigated the cellular origin of low-grade gliomas in a rat model based on transplacental exposure to N-ethyl-N-nitrosourea (ENU). Longitudinal magnetic resonance imaging coupled to immunohistological and immunocytochemical analyses were used to further characterize low-grade rat gliomas at different stages of evolution. We showed that early low-grade gliomas have characteristics of oligodendroglioma-like tumors and exclusively contain NG2-expressing slow dividing precursor cells, which express early markers of oligodendroglial lineage. These tumor-derived precursors failed to fully differentiate into oligodendrocytes and exhibited multipotential abilities in vitro. Moreover, a few glioma NG2+ cells are resistant to radiotherapy and may be responsible for tumor recurrence, frequently observed in humans. Overall, these findings suggest that transformed multipotent NG2 glial precursor cell may be a potential cell of origin in the genesis of rat ENU-induced oligodendroglioma-like tumors. This work may open up new perspectives for understanding biology of human gliomas.

Defective DNA double-strand break repair underlies enhanced tumorigenesis and chromosomal instability in p27-deficient mice with growth factor-induced oligodendrogliomas.

The tumor suppressive activities of the Kip-family of cyclin-dependent kinase (cdk) inhibitors often go beyond their role directly regulating the cell cycle. In this study, we show that p27 enhances Rad51 accumulation during repair of double-strand DNA breaks. Progression of platelet-derived growth factor (PDGF)-induced oligodendrogliomas was accelerated in mice lacking the cyclin-cdk binding activities of p27(kip1). To understand how p27 deficiency contributes, cell lines were developed from RCAS-PDGF infection of nestin-tv-a brain progenitor cells in culture. p27 deficiency did not affect cell proliferation in early passage cell lines; however, the absence of p27 affected chromosomal stability. In p27-deficient cells, the activation of Atm and Chk2 and the accumulation of gamma-H2AX was unaffected when compared with wild-type cells, and the number of phospho-histone H3 staining mitotic cells was decreased, consistent with G2/M checkpoint activation. However, the percentage of Rad51 foci-positive cells was decreased, and the kinase activity that targets the C-terminus of BRCA2, regulating BRCA2/Rad51 interactions, was increased in lysates derived from p27-deficient cells. Increased numbers of chromatid breaks in p27-deficient cells that adapted to the checkpoint were also observed. These findings suggest that Rad51-dependent repair of double-stranded breaks was hindered in p27-deficient cells, leading to chromosomal instability, a hallmark of cancers with poor prognosis.

CPT-11 for recurrent temozolomide-refractory 1p19q co-deleted anaplastic oligodendroglioma.

OBJECTIVE: A Phase II study of CPT-11 in adults with recurrent, temozolomide (TMZ)-refractory, 1p19q co-deleted, anaplastic oligodendroglioma (AO) with a primary objective of determining 6-month progression free survival (PFS). BACKGROUND: There is no standard therapy for alkylator-resistant AO. METHODS: Twenty-two patients (11 men; 11 women) ages 26-65 (median 40), with radiographically recurrent AO were enrolled. All patients had previously been treated with surgery, involved-field radiotherapy, and adjuvant chemotherapy (TMZ in 15; BCNU in 6). Fifteen patients were treated at first recurrence with an alternative chemotherapy. 13 patients underwent repeat surgery. All patients were treated at either first or second recurrence with CPT-11 administered intravenously once every 3 weeks. Neurological and neuroradiographic evaluations were performed every 8-9 weeks. RESULTS: All patients were evaluable for toxicity and response. A total of 141 cycles of CPT-11 (median 3 cycles; range 3-18) were administered. CPT-11 related toxicity included diarrhea (14 patients; 4 grade 3), neutropenia (8; 4 grade 3), fatigue (12; 3 grade 3), and delayed nausea/vomiting (12; 3 grade 3). 5 patients (23%) demonstrated a partial radiographic response, 8 (36%) demonstrated stable disease and 9 (41%) had progressive disease following three cycles of CPT-11. Time to tumor progression ranged from 2 to 13.5 months (median: 4.5 months). Survival ranged from 3 to 21 months (median: 5.5 months). Six-month and 12-month PFS were 33% and 4.5% respectively. CONCLUSIONS: CPT-11 demonstrated modest efficacy (similar to other salvage glioma regimens) with acceptable toxicity in this cohort of adults with recurrent, 1p19q co-deleted AO all of whom had failed prior TMZ chemotherapy.

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

Evaluation of ENU-induced gliomas in rats: nomenclature, immunochemistry, and malignancy.

Rats developed mixed gliomas, oligodendrogliomas, and a few astrocytomas in response to transplacental ethylnitrosourea. The neoplastic cell composition of mixed gliomas must be defined; this study required a 20-80% admixture of neoplastic astrocytes and oligodendroglia for the diagnosis of mixed glioma. A battery of immunoantibodies, including Leu-7, S-100, and vimentin, were helpful in classifying rat gliomas, and the histologic features of each tumor type are described. Other brain tumor characteristics that may decide the outcome of carcinogenicity studies include incidence, multiplicity, latency, fatality, size, and malignancy. The size of tumors was determined by measuring their 3-dimensional volumes. Brain tumor volume was found to be highly correlated with malignancy and fatality. Systematic evaluation of the malignancy of brain tumors is an important but often overlooked adjunct method of measuring the effectiveness of a carcinogen. A system to estimate malignancy, one that grades 9 tumor characteristics and weights, each according to clinical outcome, was developed. It was found that mixed gliomas grew larger, had a shorter latency, and were significantly more malignant than were other gliomas.

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.

Lectin histochemistry of ethylnitrosourea-induced oligodendrogliomas in the rat.

Oligodendrogliomas (n = 26) induced by ethylnitrosourea (ENU) in wistar rats were examined to assess the lectin specificity to oligodendroglial membranes. Two different types of oligodendrogliomas were found in our material: an isomorphous type (n = 12), and a polymorphous type (n = 14). The first one, with two variants according to its size, macro- (n = 9) and microtumors (n = 3), had predominantly a honey-comb pattern with 'clear halos' around the nuclei without anaplasia. The second type, composed mostly by macrotumours, was anaplastic, with high cellular density, necrosis and intratumoral hemorrhages. Peanut agglutinin (PNA) labelled plasma membranes of well-differentiated cellular components of the first group. The tumoral oligodendrocytes lost the property to bind PNA in the second group of tumours, while Concanavalin A (Con A) showed affinity to intracytoplasmic structures of these tumours. PNA is a reliable marker of oligodendroglial plasma membrane of well-differentiated ENU-induced oligodendrogliomas. This experimental model, using PNA and Con A, may have important clinical applications regarding the biological behaviour of this type of neoplasm.

Influence of the postnatal administration of tumor necrosis factor plus interferon-alpha 2b on the development of ethyl-nitrosourea-induced brain tumors in rats.

Using the experimental model of brain tumors induced by ethyl-nitrosourea (ENU), interferon-alpha 2b and human recombinant tumor necrosis factor-alpha (TNF) have been administered to Wistar rats between 100 and 130 days of life (one injection each week, by intraperitoneal route, of 100 micrograms of TNF and 10(4) IU of interferon-alpha 2b, in a total volume of 1 ml per injection). The results obtained suggest, that at this time, this association achieves a reduction in the number of so-called 'malignant schwannomas', but it does not influence the time of appearance nor the number of so-called 'malignant schwannomas', but it does not influence the time of appearance nor the number of so-called 'oligodendroglioma-like tumors'. On the basis of previous observations about cytokine modulation of these ENU-induced neoplasms, a different course of time for obtaining a postnatal biomodulation of both type of tumors is suggested.

Experimental neurocytomas.

Four ethyl-nitrosourea (ENU)-induced oligodendroglioma-like tumors of the rat showing large rosettes on haematoxylin-eosin stain were studied by means of immunohistochemistry and electron microscopy, and their features compared with six human intraventricular neurocytomas. The similarities between the experimental and human tumors studied support the hypothesis that most of the so-called ENU-induced oligodendrogliomas in the rat are primitive neuroectodermal tumors with the tendency to differentiate toward a neuronal phenotype, and also suggest that the ENU-model of neurocarcinogenesis is useful for the induction of experimental neurocytomas.

Expression of neuronal and glial markers in so-called oligodendroglial tumors induced by transplacental administration of ethyl-nitrosourea in the rat.

A series of 18 tumors with histological features of oligodendrogliomas, induced in the rat by means of transplacental ethyl-nitrosourea administration were studied for immunohistochemical demonstration of neuronal (synaptophysin and neurofilament protein) and glial (gliofibrillar acidic protein and vimentin) markers. Most of the tumors showed cells with strong positivity to synaptophysin and to a lesser degree, to neurofilament protein, suggesting the neuronal character of these neoplasms. In 10 tumors, cells with strong positivity to vimentin were found, and in three cases, tumoral cells expressed gliofibrillar acidic protein. The observation that ENU-induced oligodendroglial tumors express neuronal and, to a minor degree, glial markers, suggests their interpretation as primitive neuroectodermal tumors with clear neuronal differentiation.

Influence of recombinant interleukin-2 and tumor necrosis factor-alpha on the development of ethyl nitrosourea-induced brain tumors.

Using the experimental model of brain tumors induced by ethyl-nitrosourea (ENU), human recombinant interleukin-2 (rIL-2) and tumor necrosis factor-alpha (TNF-alpha) were administered to Wistar rats in early stages of carcinogenesis. The results obtained suggest that IL-2 does not influence the development of nervous system tumors. In contrast, TNF-alpha was capable of modulating the development of ENU-induced tumors, producing a reduction in the number of schwannomas and a delay in the appearance of intraparenchymatous brain tumors.

Spasmus nutans--or is it?

At age 3 1/2 years a child developed what appeared to be classic spasmus nutans. Thorough discussions as to the propriety of neuroimaging studies in such patients are presented. With this late age of onset, the presence of a compressive lesion was suspected and neuroimaging demonstrated a sellar-suprasellar mass lesion.

Immunohistochemical recognition of ethylnitrosourea induced rat brain microtumors by anti-Leu 7 monoclonal antibody.

This immunocytochemical study was undertaken to clarify the histogenesis of ethylnitrosourea-induced rat brain tumors. The tumors induced in offspring of Sprague-Dawley rats injected with ethylnitrosourea on day 18 of gestation were used in these experiments. Controls consisted of pregnant Sprague-Dawley rats similarly injected with saline alone. Both microtumors (less than 1 mm) and macrotumors were examined immunocytochemically. The cells present in both macro- and microtumors were reactive with anti-Leu 7, an antibody which recognizes oligodendrocytes. Intermixed with, but distinct from the tumor cells were glial fibrillary acidic protein positive cells morphologically identical to astrocytes found in other areas distant to tumors in the treated animals, and in controls. These data suggest that both early and late tumors are oligodendrogliomas, not astrocytomas or mixed gliomas, and that the cell of origin of the tumor is the oligodendrocyte rather than an uncommitted stem cell as previously suggested.

Immunohistochemical characterization of rat central and peripheral nerve tumors induced by ethylnitrosourea.

Ethylnitrosourea-induced central and peripheral nerve tumors in Sprague-Dawley rats were tested for GFAP (Glial Fibrillary Acidic Protein), S-100 protein, NSE (Neuron Specific Enolase) and Anti-Leu 7 (HNK-1) immunoreactivity utilizing the ABC method (avidin-biotin-complex) for GFAP, S-100 protein and NSE, and the PAP method (peroxidase-antiperoxidase) for Anti-Leu 7. Peripheral nerve neurinomas were consistently positive for S-100 protein and consistently negative for GFAP and Anti-Leu 7. Neurinomas would occasionally exhibit positive staining for NSE (2 of 55 tumors). The staining intensity for S-100 protein varied from strongly positive in differentiated neurinomas to weakly positive in anaplastic tumors. Neoplastic and reactive astrocytes exhibited positive staining for both S-100 protein and GFAP. Variation in the GFAP staining intensity of glial tumors correlated with the degree of differentiation as anaplastic tumors did not stain with the same intensity as their more differentiated counterparts. Oligodendrogliomas exhibited occasional immunoreactivity to S-100 protein (3 of 36 tumors). NSE reactivity in oligodendrogliomas was rarely observed (1 tumor in 36) and immunoreactivity against GFAP or Anti-Leu 7 was consistently absent. Anti-Leu 7 and NSE proved to be of little value in the classification of ENU-induced neural tumors.

Phenobarbital lacks promoting activity for neurogenic tumors in F344 rats transplacentally exposed to ethylnitrosourea.

A chronic rodent study in F344 rats was conducted to investigate the promoting ability of phenobarbital (PB) on neurogenic tumors initiated by transplacental administration of ethylnitrosourea (ENU). Pregnant F344 rats were given a single intravenous dose of 3.5 mg ENU/kg or vehicle on the twentieth day of gestation. A total of 192 male offspring were divided into four groups: ENU-PB, ENU-control, PB-control, and control-control. Rats in ENU-PB and PB-control groups received 0.05% PB in their drinking water from four to 78 weeks of age. Nervous system tumors were induced only in animals exposed to ENU. The difference in the incidence of neuroectodermal tumors in rats that were ENU initiated only (13/37; 35%) compared to the incidence in rats that were initiated and given PB (13/57; 23%) was not statistically significant (p greater than 0.05). ENU-control and ENU-PB treatment groups exhibited no differences in tumor multiplicity or tumor latency. These results demonstrate that PB lacks promoting activity for neurogenic tumors in F344 male rats transplacentally exposed to ENU.

Cell-specific brain tumour induction in neural transplants: evidence for multistep carcinogenesis in the nervous system.

Using neural grafting techniques, an attempt was made to elucidate the histogenesis of gliomas induced transplacentally by N-ethyl-N-nitrosourea (ENU). Pregnant rats received a single intravenous dose of ENU (50 mg/kg body weight) on day 14 of gestation. One day later, suspensions were prepared from the fetal forebrain and stereotactically injected into the caudoputamen of adult rats. These host animals received additional intravenous injections of ENU (50 mg/kg each) eight days and nine weeks after the neural graft. Histopathologically, these neoplasms were classified as oligodendrogliomas, ranging from early neoplastic foci to large, infiltrating malignant tumours. The selective induction of oligodendrogliomas indicates that neoplastic transformation in the nervous system can occur in oligodendrocytes or in precursor cells committed to oligodendrocytic differentiation and that transformation of a pluripotential stem cell is not necessary. Omission of the first (prenatal) dose of ENU led to a much lower tumour incidence, whereas this dose in itself, i.e., without additional postgrafting exposure, did not produce brain tumours in any of the experimental animals. This differential effect of pre- and postgrafting exposure to ENU constitutes the first evidence for a multistep development of brain tumours in vivo.

Selective induction by N-nitrosoethylurea of oligodendrogliomas in fetal forebrain transplants.

Induction of neuroepithelial neoplasms by a single transplacental exposure to N-nitrosoethylurea (NEU) has been widely used as an experimental model for human brain tumors. NEU-induced gliomas are variably composed of neoplastic oligodendrocytes, astrocytes, and ependymal cells. It has remained controversial whether these neoplasms originate from differentiated glia or from pluripotent precursor cells of the subependymal matrix layer. We have taken a novel approach to define the histogenesis of these gliomas based on neural grafting techniques and the extraordinary difference in susceptibility between the fetal and adult brain to neoplastic transformation by alkylnitrosoureas. Pregnant rats received a single i.v. dose of NEU (50 mg/kg) on the 14th day of gestation. One day later, suspensions were prepared from the fetal forebrain and stereotactically injected into the caudoputamen of adult rats. These host animals received additional i.v. injections of NEU (50 mg/kg each) 8 days and 9 weeks posttransplantation. After a mean survival time of 316 days, all animals developed brain tumors within the neural graft. Histopathologically, these neoplasms were classified as oligodendrogliomas, ranging from early neoplastic foci to large, infiltrating malignant tumors. The selective induction of oligodendrogliomas indicates that neoplastic transformation in the nervous system can occur in a differentiated glial cell or in a precursor cell committed to oligodendrocytic differentiation, and that transformation of a pluripotential stem cell is not necessary. Omission of the first (prenatal) dose of NEU led to a much lower tumor incidence, whereas this dose in itself, i.e., without additional postgrafting exposure, did not produce brain tumors in any of the experimental animals. This differential effect of pre- and postgrafting exposure to NEU constitutes the first in vivo evidence of a multistep development of brain tumors.