Andrea Kindler-Röhrborn

In vitro differentiation of neural progenitor cells from prenatal rat brain: Common cell surface glycoprotein on three glial cell subsets

Glial progenitor cell differentiation and cell lineage relationships during brain development are complex hierarchical processes depending on genetic programming, cell‐cell interactions, and microenvironmental factors. The identification of precursor cell‐specific antigens provides a tool for the study of both normal development and deviations from lineage‐specific differentiation associated with malignant transformation. Monoclonal antibody (mAb) RB13‐6 recognizes a 130‐kDa cell surface glycoprotein (gp130RB13‐6) expressed by a subset of 9OAcGD3‐positive glial precursor cells scattered in the rat neuroepithelium on prenatal day (PRD) 13. During prenatal development the fraction of gp130RB13‐6‐positive fetal brain cells (FBC) decreased from about 18% (PRD 14) to about 1.5% (PRD 22), coinciding with increasing fractions of more mature cell types, as indicated by the elevated expression of p24RB21‐15, another cell surface determinant specified by mAb RB21‐15 (Kindler‐Röhrborn et al.; Differentiation 30:53–60, 1985) and other neural cell type‐specific markers. Accordingly, gp130RB13‐6‐positive precursor cells were localized in the ventricular zones throughout brain development. Concomitant with their formation and in the adult rat brain, ependymal layers lining the ventricular surface, choroid plexus, and the leptomeninges were intensely labeled by anti‐gp130RB13‐6 mAb. As visualized by confocal laser scanning microscopy of FBC cultures from PRD 13, gp130RB13‐6 was coexpressed with the RC1 antigen by progenitor cells morphologically resembling radial glia cells. In addition, a very small subpopulation of astrocytes coexpressing gp130RB13‐6 and glial fibrillary acidic protein (GFAP; <5%) occurred 3 days after seeding. Primary FBC cultures from PRD 18 contained an increased subset of astrocytes coexpressing gp130RB13‐6 and GFAP (∼25% of all gp130RB13‐6 expressing cells), apparently generated from gp130RB13‐6‐positive precursors. Corresponding to in vivo conditions, ciliated ependymal cells but also microglial cells/macrophages and leptomeningeal cells showed strong expression of gp130RB13‐6 in culture. We thus present a new glycoprotein on the cell surfaces of a glial progenitor cell subset for further studies of cell lineage relationships between radial glia cells, astrocytes, and ependymal cells. J. Neurosci. Res. 48:95–111, 1997.

Differential modulation of malignant peripheral nerve sheath tumor growth by omega-3 and omega-6 fatty acids

Neurofibromatosis type 1 (NF1) is a common genetic disorder of the nervous system resulting in neurofibromas and malignant peripheral nerve sheath tumors (MPNST). In this study, we report the modulation of murine and human MPNST cell growth by the fatty acids docosahexaenoic acid (DHA) and arachidonic acid (AA). DHA demonstrated a tendency to stimulate cell growth at low doses and induce apoptosis at high doses, paralleled by the activation of ERK and caspase-3. Furthermore, high-dose DHA reversed the stimulation of MPNST cell growth by a number of growth factors suggested to have a pathogenic effect in NF1 and inhibited MPNST growth in vivo. AA was found to have a reciprocal activity in vitro, stimulating MPNST cell growth at comparable concentrations and reducing DHA activation of ERK. These findings introduce fatty acids as a possible regulator of MPNST development in NF1 patients.

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 × 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 × 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.

The circling behavior of the deafblind LEW-ci2 rat is linked to a segment of RNO10 containing Myo15 and Kcnj12.

The LEW/Ztm-ci2 rat is an autosomal recessive mutant that displays circling behavior, deafness, progressive retinopathy, locomotor hyperactivity, ataxia, and opisthotonus. We performed a genome-wide scan of a (LEW/Ztm-ci2 × BN/Ztm) F1 × LEW/Ztm-ci2 backcross population with anonymous microsatellite markers to analyze the genetics of this mutant rat. This linkage analysis demonstrated a very strong association of RNO10 SSLP markers to the phenotype with a core region in the central part of the chromosome. The knowledge of genes mapping to this part of the rat genome and their linkage to SSLP markers is still poor. We developed SSLP markers closely linked to genes, which might be responsible for the mutant phenotype by using the growing amount of rat-specific DNA sequences available at World Wide Web databases. Application of this method facilitated the search for candidate genes for the phenotype of the LEW-ci2 rat. We were able to map Myo15 and its neighboring genes, Znf179 and Aldh3a1, to the region of interest and Myo1c to a more distal location on RNO10. Further rat BAC clones were used to create a physical map of the region of interest. This map revealed the position of further genes. Among those is Kcnj12. Owing to their localization on RNO10 and their involvement in a similar pathology in human and mouse, Myo15 and Kcnj12 can be regarded as candidate genes for the deafblind phenotype of the LEW-ci2 rat.

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 (αNCA1, αNCA2, αNCA3, αNCA4, and αNCA5) 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 αNCA1 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.

In vitro experiments on neuronal and glial cell lineages among the ventricular cells of the mouse neural plate

The proliferative ventricular cells of the early neural plate of the mouse are generally assumed to be pluripotent and equivalent to one another in their developmental capability. Ventricular cells from the rostral parts of the neural plates of mice (Theiler stages 11 and 12, embryonic days 71/2 and 8) were studied in tissue culture with respect to their potential to give rise to neurons or glial cells, or both. Auto‐radiographic and immunohistochemical analyses showed that ventricular cells developing into neuronal phenotypes stopped proliferating immediately upon transfer to cell culture. Using polyclonal anti‐GFAP antibodies, a small proportion of immunoreactive cells could be detected after 4 days of culture. These cells retained their proliferative activity, displayed morphological characteristics of radial glial cells and may have either developed from specific glial progenitor cells or have been induced to proceed along the glial differentiation pathway at the beginning of culture. Therefore, two distinct types of progenitor cells, committed either to neuronal or glial lineages, appear to co‐exist among the cultured neural plate ventricular cells.

Ablation of T cell immunity differentially influences tumor risk in inbred BD rat strains

Inbred rat strains BDIX and BDIV are constitutionally susceptible and resistant, respectively, to the development of malignant peripheral nerve sheath tumors (MPNST) induced by neonatal exposure to N-ethyl-N-nitrosourea (EtNU). They represent a model system for analysis of molecular and cellular processes underlying differential cancer susceptibility. A point mutation in the Neu/ErbB-2 gene is an early marker of Schwann precursor cells at high risk of malignant conversion and is diagnostic of the resulting MPNST predominantly developing in the trigeminal nerves. Initially considerable amounts of Neu/ErbB-2-mutant cells arise in nerve tissue of both rat strains subsequently disappearing in resistant BDIV rats, but persisting and giving rise to MPNST in susceptible BDIX animals. An almost identical cellular immune response—sequentially involving macrophages, T helper- and cytotoxic T lymphocytes—is mounted in the trigeminal nerves of EtNU-treated rats of both strains. In this study, T cell maturation was prevented by neonatal thymectomy following EtNU-exposure. While resistance against MPNST development significantly decreased in BDIV rats MPNST incidence and survival time remained unaltered in thymectomized BDIX rats. Contrary to euthymic animals a number of both thymectomized BDIV and BDIX rats developed MPNST lacking the Neu/ErbB-2-mutation. This suggests that Schwann cells initiated by other genetic alterations can progress to full malignancy in immune-compromised rats only. T cell-dependent resistance against tumorigenesis originating from non-Neu/ErbB-2-mutant Schwann precursors might thus be shared by both strains while BDIV T lymphocytes additionally prevent the development of Neu/ErbB-2-mutant MPNST. Rat strain-specific differences in the interaction of T lymphocytes with (pre)malignant Neu-mutant cells may thus critically contribute to susceptibility and resistance towards EtNU-induced MPNST development.

Sex-biased suppression of chemically induced neural carcinogenesis in congenic BDIX.BDIV-Mss4a rats

We previously mapped several gene loci influencing cancer risk of inbred BDIV and BDIX rats, resistant and susceptible, respectively, to N-ethyl-N-nitrosourea (ENU)-induced malignant peripheral nerve sheath tumors (MPNSTs). On the basis of a genomewide association analysis using a (BDIV × BDIX) F(2) generation the Mss4 locus on rat chromosome 6 was predicted to mediate resistance to MPNST development in the trigeminal nerves, preferentially in females. F(2) females homozygous for D6Mit1 proved almost exclusively resistant to peripheral neurooncogenesis, with no effect detectable in males. To functionally verify Mss4, a congenic BDIX rat strain was generated carrying a corresponding BDIV genomic fragment. On treatment with ENU, congenic BDIX.BDIV-Mss4a rats showed a 2.4-fold lower MPNST rate and a 55-day-longer survival time compared with BDIX animals. The sex-specific effect observed in F(2) rats was less pronounced in BDIX.BDIV-Mss4a congenics, with males, too, being protected against MPNST but to a lesser extent than females. Transcription profiling using trigeminal nerve tissue of BDIX, BDIV, and BDIX.BDIV-Mss4a congenics of both sexes revealed 61 genes located in the Mss4a fragment differentially expressed between BDIV and BDIX rats. In congenic rats each gene either displayed trans-regulated BDIX-like expression strength or cis-regulated BDIV-like transcript levels or intermediate expression without marked sex differences. Genomewide a number of genes exhibiting male-biased expression in the BDIX rat strain displayed a reversal of the sexual dimorphism in congenic rats similar to the BDIV expression pattern, which might be the basis of preferential protection of females against MPNST development.

Allele-specific losses of heterozygosity on chromosomes 1 and 17 revealed by whole genome scan of ethylnitrosourea-induced BDIXBDIV hybrid rat gliomas

The induction of neural tumors by N‐ethyl‐N‐nitrosourea (EtNU) in inbred strains of rats has evolved as a valuable model system of developmental stage– and cell type–dependent oncogenesis. Tumor yield and latency times are strongly influenced by genetic background. Compared with BDIX rats, BDIV rats are relatively resistant to the induction of brain tumors by EtNU, with a lower tumor incidence and latency periods prolonged by a factor of 3. To characterize genetic abnormalities associated with impaired tumor suppressor gene function in neuro‐oncogenesis, losses of heterozygosity (LOHs) and microsatellite instability (MI) were investigated in brain tumors induced by EtNU in (BDIV×BDIX) F1 and F2 rats. The polymerase chain reaction was used to amplify 55 polymorphic microsatellite markers spanning the entire rat genome. The tumors displayed different histologies and grades of malignancy, corresponding to part of the spectrum of human gliomas. MI was not observed in any of the tumors. LOH of rat chromosome 1q was predominantly detected in oligodendrogliomas and mixed gliomas, with a 30% incidence in informative cases. 11p15.5, the human genome region syntenic to the consensus region of LOHs observed on rat chromosome 1, has been shown to be involved in the formation of gliomas in humans. Furthermore, rat brain tumors of different histologies often showed allelic imbalances on chromosome 17p. In both cases of LOH, there was a clear bias in favor of the parental BDIV allele, suggesting the involvement of tumor suppressor genes functionally polymorphic between the two rat strains. Mol. Carcinog. 26:163–171, 1999.