Linda M. Parysek

A type III intermediate filament gene is expressed in mature neurons

A cDNA (199E) specific for the 57 kd neural IF protein has been isolated from a PC12 cell lambda gt11 library. Antibody eluted from the fusion protein produced by 199E recognizes the 57 kd protein on immunoblots and, in PC12 cells, labels a pattern of fibrillar structures identical to that seen with 57 kd antiserum. In situ hybridization using antisense RNA transcripts labels areas of the nervous system known to contain the 57 kd protein. 199E hybridizes with a single mRNA species of approximately 2.0 kb from PC12 cells. A 199E-reactive message can be detected as early as E10 in rat embryos. Southern analyses suggest that there is only one gene for this protein. Amino acid sequence predicted from 199E indicates that the 57 kd protein is a type III IF protein like vimentin and desmin. Thus, expression of IF structural genes in neurons is not limited to the type IV neuronal IF triplet proteins.

Identification of ASF/SF2 as a Critical, Allele-Specific Effector of the Cyclin D1b Oncogene

The cyclin D1b oncogene arises from alternative splicing of the CCND1 transcript, and harbors markedly enhanced oncogenic functions not shared by full-length cyclin D1 (cyclin D1a). Recent studies showed that cyclin D1b is selectively induced in a subset of tissues as a function of tumorigenesis; however, the underlying mechanism(s) that control tumor-specific cyclin D1b induction remain unsolved. Here, we identify the RNA-binding protein ASF/SF2 as a critical, allele-specific, disease-relevant effector of cyclin D1b production. Initially, it was observed that SF2 associates with cyclin D1b mRNA (transcript-b) in minigene analyses and with endogenous transcript in prostate cancer (PCa) cells. SF2 association was altered by the CCND1 G/A870 polymorphism, which resides in the splice donor site controlling transcript-b production. This finding was significant, as the A870 allele promotes cyclin D1b in benign prostate tissue, but in primary PCa, cyclin D1b production is independent of A870 status. Data herein provide a basis for this disparity, as tumor-associated induction of SF2 predominantly results in binding to and accumulation of G870-derived transcript-b. Finally, the relevance of SF2 function was established, as SF2 strongly correlated with cyclin D1b (but not cyclin D1a) in human PCa. Together, these studies identify a novel mechanism by which cyclin D1b is induced in cancer, and reveal significant evidence of a factor that cooperates with a risk-associated polymorphism to alter cyclin D1 isoform production. Identification of SF2 as a disease-relevant effector of cyclin D1b provides a basis for future studies designed to suppress the oncogenic alternative splicing event.

Brain lipid binding protein in axon-Schwann cell interactions and peripheral nerve tumorigenesis.

ABSTRACT Loss of axonal contact characterizes Schwann cells in benign and malignant peripheral nerve sheath tumors (MPNST) from neurofibromatosis type 1 (NF1) patients. Tumor Schwann cells demonstrate NF1 mutations, elevated Ras activity, and aberrant epidermal growth factor receptor (EGFR) expression. Using cDNA microarrays, we found that brain lipid binding protein (BLBP) is elevated in an EGFR-positive subpopulation of Nf1 mutant mouse Schwann cells (Nf1 −/− TXF) that grows away from axons; BLBP expression was not affected by farnesyltransferase inhibitor, an inhibitor of H-Ras. BLBP was also detected in EGFR-positive cell lines derived from Nf1:p53 double mutant mice and human MPNST. BLBP expression was induced in normal Schwann cells following transfection with EGFR but not H-Ras12V. Furthermore, EGFR-mediated BLBP expression was not inhibited by dominant-negative H-Ras, indicating that BLBP expression is downstream of Ras-independent EGFR signaling. BLBP-blocking antibodies enabled process outgrowth from Nf1 −/− TXF cells and restored interaction with axons, without affecting cell proliferation or migration. Following injury, BLBP expression was induced in normal sciatic nerves when nonmyelinating Schwann cells remodeled their processes. These data suggest that BLBP, stimulated by Ras-independent pathways, regulates Schwann cell-axon interactions in normal peripheral nerve and peripheral nerve tumors.

Distribution of MAP-4 in cells and in adult and developing mouse tissues.

Over the last decade, a number of proteins have been identified that coassemble with microtubules in vitro. As outlined in this volume and elsewhere,I4 these include low molecular weight species from brain (tau), and high molecular weight proteins from brain ((microtubule-associated protein) MAP1, MAP-2) and cultured cells (210 kD HeLa MAP, MAP-4). Antisera against all of these MAPS stain interphase arrays in cultured cells, and most also react with mitotic spindles. Although studies on the distribution of these proteins in tissues are being initiated, the function of any of these MAPS in vivo is still largely unknown. We have been investigating the occurrence of MAP-43 in a variety of systems. This protein was originally identified as a 215 kD MAP in neuroblastoma cells, the synthesis of which appeared to be induced upon neurite differentiation.2s5 The distribution of MAP-4 in extracts3 and semi-thin sections6 of mouse tissues has recently been described. This paper outlines the species distribution of MAP-4, further analyses on the complexity of this MAP, and the occurrence of MAP-4 during brain development and in early mouse embryos.

Novel protein isoforms of the APC tumor suppressor in neural tissue

The conventional protein isoform of the APC tumor suppressor is 310 kD and is encoded by exons 1 – 15 of the APC gene. Other RNAs are expressed from the APC gene and include one form that contains an exon upstream of exon 1, designated BS, but this transcript does not include exon 1. This transcript recently has been shown to be enriched in non-dividing, terminally-differentiated cells (Santoro and Groden, 1997). To determine if the BS-containing transcript encoded an alternate APC protein isoform, we generated and affinity-purified a polyclonal antibody directed to protein sequence predicted by exon BS. The BS antibody labeled a band of ∼300 kD on immunoblots of cerebral and cerebellar tissue from adult human, baboon, rat and mouse. These same tissue lysates also contained prominent BS-reactive proteins of 290 kD, 200 kD and 150 kD. Lysates from mitotically active cells did not contain these APC isoforms. To verify that BS-reactive proteins were APC isoforms, BS-immunoprecipitates were blotted and labeled with commercially available APC antibodies. All four high molecular weight BS-antibody-precipitated proteins were recognized by antibodies directed against epitopes encoded by APC exons 2 and 15. BS isoforms were not, however, labeled with antibodies to an epitope encoded by APC exon 1, consistent with the prediction that BS – APC isoforms lack the domain encoded by these sequences. Like conventional APC, at least one of the four BS – APC protein isoforms also interacts with β-catenin. BS – APC isoforms that lack exon 1-encoded sequences are incapable of dimerization with the conventional form of APC, yet retain the ability to bind β-catenin. Such isoforms are likely to be functionally distinct from the conventional APC protein.

A syngeneic mouse glioma model for study of glioblastoma therapy

Animal models of human tumors serve a vital role in the development and testing of new anticancer therapies. Since the immune system is likely to play an essential role in tumor eradication, there is a particular need for modeling human disease in immunocompetent hosts. Few models of glioma have been developed in immunocompetent mice that are commercially available and none of these tumors have histological and antigenic characteristics of human gliomas. We have used a cell line, 4C8, derived from a spontaneous glioma-like tumor that arose in a transgenic mouse to develop a new glioma model. The intracranial injection of 4C8 cells into immunocompetent syngeneic B6D2F1 mice resulted in tumors that were densely cellular, developed a pseudopallisading pattern of necrosis, and expressed GFAP; all important features of human malignant gliomas. The average neurological endpoint was 51 days after intracranial injection. The 4C8 cells also grew rapidly in the flank, retaining histologic features seen in intracranial tumors. Flank tumors reached an average volume of 100 mm3, a volume ideal for therapy testing, by 34 days postinjection. These results suggest that the 4C8 mouse glioma model is an excellent system in which to test new antiglioma therapies for use in humans.

Complementary immunohistochemical distribution of the neurofilament triplet and novel intermediate filament proteins in the autonomic and sensory nervous system of the guinea-pig.

We have previously established that immunoreactivity for the triplet of polypeptides that comprise the class IV intermediate filament proteins (NFP-triplet) is localized in specific subpopulations of neurons in guinea-pig sensory and autonomic ganglia. Antibodies to novel neurofilament proteins, including a polyclonal antibody to a 57 kDa neuronal intermediate filament polypeptide (NIF57kD) and a monoclonal antibody (CH1) to a 150 kDa intermediate filament, or associated, protein were used in combination with antibodies to the NFP-triplet for double-labelling immunohistochemistry. The results show that different subpopulations of neurons in the guinea-pig dorsal root ganglia, coeliac ganglion and enteric ganglia can be distinguished by their complementary immunoreactivity for these proteins. In dorsal root ganglia, larger neurons are intensely immunoreactive for the NFP-triplet while immunoreactivity with CH1 and NIF57kD antibodies is restricted to the small to medium-sized neurons. In the coeliac ganglion, two regionally defined subpopulations of neurons can be distinguished by their immunoreactivity for either the NFP-triplet or NIF57kD, whereas CH1 labels all neurons with equal intensity. Three classes of morphologically distinct myenteric neuron subpopulations are also distinguished by their immunoreactivity for either the NFP-triplet, NIF57kD or CH1 antibodies. Two classes of submucous neurons are labelled both with CH1 and NIF57kD antibodies but show faint or no immunoreactivity for the NFP-triplet. It is concluded that intermediate filament protein immunoreactivity marks different subpopulations of neurons, which suggests that these proteins may have specific roles in neuronal function.

The NF2 tumor suppressor regulates microtubule-based vesicle trafficking via a novel Rac, MLK and p38(SAPK) pathway.

Neurofibromatosis type 2 patients develop schwannomas, meningiomas and ependymomas resulting from mutations in the tumor suppressor gene, NF2, encoding a membrane-cytoskeleton adapter protein called merlin. Merlin regulates contact inhibition of growth and controls the availability of growth factor receptors at the cell surface. We tested if microtubule-based vesicular trafficking might be a mechanism by which merlin acts. We found that schwannoma cells, containing merlin mutations and constitutive activation of the Rho/Rac family of GTPases, had decreased intracellular vesicular trafficking relative to normal human Schwann cells. In Nf2−/− mouse Schwann (SC4) cells, re-expression of merlin as well as inhibition of Rac or its effector kinases, MLK and p38SAPK, each increased the velocity of Rab6 positive exocytic vesicles. Conversely, an activated Rac mutant decreased Rab6 vesicle velocity. Vesicle motility assays in isolated squid axoplasm further demonstrated that both mutant merlin and active Rac specifically reduce anterograde microtubule-based transport of vesicles dependent upon the activity of p38SAPK kinase. Taken together, our data suggest loss of merlin results in the Rac-dependent decrease of anterograde trafficking of exocytic vesicles, representing a possible mechanism controlling the concentration of growth factor receptors at the cell surface.

Vimentin filaments in spreading, randomly locomoting, and f-met-leu-phe-treated neutrophils.

SummaryHuman neutrophils contain intermediate filaments of the vimentin type. A cytoskeletal preparation, produced by high-salt and Triton X-100 extraction of human neutrophils, reveals a major band at 57000 Mr that comigrates with 3T3 cell vimentin on one-dimensional gels. Two-dimensional gel electrophoresis of whole neutrophils illustrates the presence of vimentin but not desminor keratin-filament subunits. The presence of vimentin in neutrophils is also shown by its specific staining with avian vimentin antiserum by two-dimensional gel immunoautoradiography. Indirect immunofluorescence studies show that vimentin antiserum labels an area on one side of the nucleus in spreading neutrophils. This bright area appears as a loose knot of vimentin filaments; a few filaments may radiate from the knot. In contrast to spreading neutrophils, those undergoing random locomotion contain a fine network of filaments that are located in the cytoplasm between the nucleus and the trailing end of the cell. Similarly, in chemoattractant-treated neutrophils, vimentin filaments are bundled in the uropod. Transmission electron microscopy of human neutrophil monolayers confirms the intracellular distribution of intermediate filaments as shown by immunofluorescence in spreading and randomly locomoting cells.

Peripherin gene is linked to keratin 18 gene on human chromosome 12

Peripherin is a neuron-specific intermediate filament (IF) protein, found primarily in phylogenetically old regions of the nervous system. Whereas other neuronal IF genes have only two to three introns and are scattered in the genome, the peripherin gene (PRPH) has a complex intron-exon structure like nonneuronal IF genes that are clustered in tandem arrays, e.g., those encoding the keratins. We used a cosmid containing the human peripherin gene (PRPH) to determine its chromosomal location in relationship to nonneuronal IF genes. Using a rodenthuman mapping panel, we localized thePRPH gene to human chromosome 12. Since a cluster of keratin genes maps to 12q12–13, polymorphic markers were developed forPRPH and for one of the keratin genes presumed to be in the cluster, keratin 18 (KRT18). Both markers were typed in CEPH reference families. Pairwise and multipoint analyses of the CEPH data revealed thatKRT17 is tightly linked to DNA markersD12S4, D12S22, D12S90, D12S96 andD12S103, which lie betweenD12S18 andD12S8, with odds greater than 1000∶1. These markers are physically located at 12q11–13, thus supporting the fine localization ofKRT18 in or near the group of type II keratins in this region. Furthermore, linkage analysis showed that the peripherin gene (PRPH) is tightly linked toKRT18 (Ž=15.73, θ=0.013), and therefore appears to be in close proximity to the cluster.