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.

N-ethyl-N-nitrosourea-induced spinal tumors in an inbred strain of W albino rats.

Exposure to N-ethyl-N-nitrosourea (ENU) either transplacentally via the maternal bloodstream or postnatally by direct injection into the cerebellum or the cisterna magna resulted in a high incidence of spinal tumors in an inbred strain of W albino rats. After prenatal exposure to 60 mg ENU/kg maternal body weight, as many as 92% of the offspring developed 1 or more tumors in the spinal cord, whereas after postnatal exposure to 0.2 mg ENU/animal, 50% of the animals eventually developed spinal tumors. These tumors included relatively pure oligodendrogllomas, astrocytomas, and the usual mixed gllomas. Obvious clinical symptoms of paralysis of the limbs and weight loss accompanying the high incidence of tumors in the spinal cord make this system pertinent to the study of carcinogenesis in the central nervous system as well as to the study of related problems to the incidence of these tumors are discussed.

Oncogenic response of rats with x-ray-induced microcephaly to transplacental ethylnitrosourea.

The relation of congenital malformations to tumor development was examined. Pregnant Sprague-Dawley rats were given 200 rads of X-rays on the 15th or 16th day of gestation and injections of 10 mg ethylnitrosourea (ENU)/kg 1-4 days later, or they were irradiated or injected only. Surviving weanlings that had been irradiated had micrencephaly and other malformations. Offspring exposed to ENU only had no external deformities. By 15 months of age 16.7% of the offspring exposed to X-rays and ENU prenatally had developed neurogenic tumors, whereas 62.2% of those exposed to ENU alone had developed tumors. Those only irradiated had no tumors. Both of the former groups developed oligodendrogliomas, mixed gliomas, ependymomas, and schwannomas, but the first manifestations of tumors occurred later in the group receiving the combined treatment. This delay persisted furing the subsequent period of the study.

[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.

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.

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.

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.

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.

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.

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.

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.

[Deoxyribonucleases in spontaneous and experimental tumors of the nervous system]. / Desoxyribonucleasen in spontanen und experimentellen Tumoren des Nervensystems

DNA- and RNA-concentrations, as well as in vitro activities of DNase I (EC 3.1.4.5), DNase II (EC 3.1.4.6), and DNase I inhibitor, have been determined in 63 spontaneous (man) and 22 experimentally induced (rat) nervous system blastomas of various types and of different degrees of malignancy. Generally, a distinct elevation of DNA concentrations and of the ratio (Q) of DNase II- to DNase I-activities has been observed when compared with control values. A statistically significant relationship could be demonstrated between increase of DNA concentrations and Q in experimentally induced neurinomas of rats as well as in human astrocytomas and glioblastomas. Whereas the increase of Q may be a biochemical expression of elevated DNA synthesis of tumour cells, no conclusions can be drawn as to the role of DNases in the process of malignant transformation.

Glial fibrillary acidic protein (GFAP) in rat brain tumors transplacentally induced by ethylnitrosourea (ENU).

The immunohistochemical distribution of glial fibrillary acidic protein (GFAP) in neoplastic lesions induced in the rat by ENU is reported. GFAP was present in hypertrophic reactive astrocytes, which were numerous in early neoplastic proliferations, in microtumors of the white matter, and in those collected at the periphery of large tumors. They were absent in cortical oligodendroglial foci and microtumors. No GFAP-positive cells were observed in hyperplasias of the white matter: astrocyte-like cells of large tumors were GFAP-negative. The significance of reactive astrocytes and the problem of the astrocytic component in transplacental ENU tumors are discussed.

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.

[Histochemical demonstration of a zinc activated tartrate resistant acid phosphatase in experimentally induced glial microtumors of the rat brain (author's transl)]. / Histochemische Darstellung einer durch Zink aktivierten tartrat-resistenten sauren Phosphatase in experimentell induzierten gliösen Mikrotumoren des Rattengrosshirns

The tartrat resistant zinc activated acid phosphatase (ZT-AP) was investigated in 17 glial microtumours of the rat brain. The tumours were induced by N-ethyl-N-nitrosourea injected to newborn rats subcutaneously (85 mg/kg body weight). The morphology of these microtumours corresponds to the statements in the literature. In regard to their behaviour of the investigated enzyme the microtumours may be divided in 2 groups: 1) Microtumours with an activity of acid phosphatases but without any histochemically demonstrable activity of ZT-AP. Histologically they correspond to oligodendric microgliomas. 2) Microtumours with a high activity of acid phosphatases and a remarkable activity of ZT-AP especially in the perivascular cell proliferations. This type corresponds histologically to the astrocytic and mixed glial proliferations including glioblastomas. According to our present findings the ZT-AP was found in astrocytes and especially in mesenchymal cells. In some cases a focal increased activity of ZT-AP could also be observed before histological alterations appeared. We regard it as an early stage of the development of tumours.

[Cytophotometric determination of DNA concentration in the cells of experimental brain tumors. II. Primary tumors of rat cerebellum induced by 9, 10-dimethyl-1, 2-benzanthracene]. / Tsitofotometricheskoe opredelenie soderzhaniia DNK v kletkakh éksperimental'nykh opukholei mozga. II. Pervichnye opukholi mozzhecka krys, indutsirovannye 9 10-dimetil-1, 2-benzantratsenom

The Feulgen-DNA cytophotometry was applied for studies of 31 rat cerebellum tumors induced by 9, 10-dimetyl-1,2-bensantracene. Most of these gliomas (22) were astrocytomas of different grades of malignancy. The histological diagnosis of other tumors was: glioblastoma -- 4, oligoastrocytoma -- 2, oligodendroglioma -- 1, gliosarcoma 1. The majority cells of 26 tumors had diploid or paradiploid DNA quantity, 4 tumors (1 astrocytoma, 3 dedifferentiated astroyctomas) had triploid modal classes. The tetraploid modal class and a large number of polyploid cells were found only once for glioblastoma multiforme. A supposition was made that drastic changes of ploidy could arise for the second time during the process of tumor evolution. The authors failed to show any exact differences in the ploidy of gliomas in rats with athyreosis or hyperthyreosis, and in the ploidy of somatic cells in control animals.

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.