Schwann cell tumors express characteristic patterns of CD44 splice variants

Members of the CD44 family of cell surface hyaluronate-binding proteins have been implicated in cell migration, cell-matrix interactions and tumor progression. To determine whether these proteins might play a role in the normal functions of Schwann cells and in their tumorigenesis, we examined the patterns of CD44 expression in Schwann cells from rat peripheral nerve, rat Schwann cell tumor lines, and human schwannomas. Normal rat spinal nerves and primary Schwann cell cultures expressed standard CD44 (CD44s) but not alternatively spliced variant isoforms. In contrast, rat Schwann cell tumor lines expressed both CD44s and a number of variants, including proteins containing sequences encoded by exon v6. Furthermore, we found that these cell lines bind hyaluronate, and that their cell surface hyaluronate binding correlates with CD44 expression. All of the human schwannomas also expressed CD44 variants, especially epitopes encoded by exon v5, the border between v7 and v8, and v9-10. These data indicate that Schwann cells normally express CD44s, that Schwann cell tumors express both CD44s and particular variants of CD44, and that CD44s and possibly variants of CD44 are involved in hyaluronate recognition by Schwann cell tumors.     

Effects of bicuculline and baclofen on paired-pulse depression in the dentate gyrus of epileptic patients

Neurofibromatosis 2 (NF2) is an uncommon, autosomal dominant disorder in which patients are predisposed to neoplastic and dysplastic lesions of Schwann cells (schwannomas and schwannosis), meningeal cells (meningiomas and meningioangiomatosis) and glial cells (gliomas and glial hamartomas). Clinical and genetic criteria that distinguish NF2 from neurofibromatosis 1 have allowed more accurate assignment of specific pathological features to NF2. The NF2 tumor suppressor gene on chromosome 22q12 encodes a widely expressed protein, named merlin, which may link the cytoskeleton and cell membrane. Germline NF2 mutations in NF2 patients and somatic NF2 mutations in sporadic schwannomas and meningiomas have different mutational spectra, but most NF2 alterations result in a truncated, inactivated merlin protein. In NF2 patients, specific mutations do not necessarily correlate with phenotypic severity, although grossly truncating alterations may result in a more severe phenotype. In schwannomas, NF2 mutations are common and may be necessary for tumorigenesis. In meningiomas, NF2 mutations occur more commonly in fibroblastic than meningothelial subtypes, and may cluster in the first half of the gene. In addition, in meningiomas, a second, non-NF2 meningioma locus is probably also involved. Future efforts in NF2 research will be directed toward elucidating the role of merlin in the normal cell and the sequelae of its inactivation in human tumors.     

Ruffling membrane, stress fiber, cell spreading and proliferation abnormalities in human Schwannoma cells

Schwannomas are peripheral nerve tumors that typically have mutations in the NF2 tumor suppressor gene. We compared cultured schwannoma cells with Schwann cells from normal human peripheral nerves (NHSC). Both cell types expressed specific antigenic markers, interacted with neurons, and proliferated in response to glial growth factor, confirming their identity as Schwann cells. Schwannoma cells frequently had elevated basal proliferation compared to NHSC. Schwannoma cells also showed spread areas 5-7-fold greater than NHSC, aberrant membrane ruffling and numerous, frequently disorganized stress fibers. Dominant negative Rac inhibited schwannoma cell ruffling but had no apparent effect on NHSC. Schwannoma cell stress fibers were inhibited by C3 transferase, tyrphostin A25, or dominant negative RhoA. These data suggest that the Rho and Rac pathways are abnormally activated in schwannoma cells. Levels of ezrin and moesin, proteins related to the NF2 gene product, merlin, were unchanged in schwannoma cells compared to NHSC. Our findings demonstrate for the first time that cell proliferation and actin organization are aberrant in schwannoma cells. Because NF2 is mutant in most or all human schwannomas, we postulate that loss of NF2 contributes to the cell growth and cytoskeletal dysfunction reported here.     

Alterations of microsatellites in neurofibromas of von Recklinghausen's disease

von Recklinghausen's disease, or type I neurofibromatosis, a common familial tumor syndrome, is characterized by the occurrence of multiple benign neoplasms of nerve sheath cells. The disease is caused by germ-line mutations of the NF1 gene, which encodes a member of the GTPase-activating superfamily of Ras regulatory proteins. We analyzed 5 dinucleotide repeat loci in DNAs from neurofibromas and matched normal skin from 16 NF1 patients. Eight cases (50%) manifested microsatellite alterations. Expansions or compressions of dinucleotide repeats were observed at one locus in four cases and at two loci in one case. Banding patterns compatible with the loss of a microsatellite allele were observed in four cases, including one that also presented microsatellite instability. The surprisingly high frequency of microsatellite alterations suggests that the NF1 gene or another gene(s) contributing to the pathogenesis of neurofibromas might be directly or indirectly implicated in the control of genomic integrity.     

Expression of the ERBB2/neu and neurofibromatosis type 1 gene products in reactive and neoplastic schwann cell proliferation

In the present study we investigated the expression of the ERBB2 protein and neurofibromin in human benign and malignant Schwann cell tumors, traumatic neuromas and peripheral nerves without pathological findings. By immunohistochemistry and Western analysis ERBB2 expression was not detectable in normal nerves but in proliferating Schwann cells of traumatic neuromas. While none of the malignant schwannomas exhibited ERBB2 expression, weak expression was seen in a small proportion of the benign schwannomas. Low levels of neurofibromin were detectable in normal nerves but not in traumatic neuromas or tumors. These data indicate an inverse expression pattern of ERBB2 and neurofibromin in human non-neoplastic Schwann cells, but not in neurinoma cells.     

Expression of Kit in neurofibromin-deficient human Schwann cells: role in Schwann cell hyperplasia associated with type 1 neurofibromatosis

Type 1 Neurofibromatosis (NF1) is characterized by the formation of neurofibromas, benign tumors composed mainly of Schwann cells, which can turn malignant to form neurofibrosarcomas. Neurofibromin, the protein product of the Nf1 gene, is believed to act as a tumor suppressor, accelerating the conversion of the oncogene Ras to its inactive form. The absence of neurofibromin could therefore lead to higher Ras activity in Schwann cells, resulting in uncontrolled growth through a cascade of events not yet elucidated. We describe the abnormal expression of high levels of the Kit tyrosine kinase receptor in both NF1-derived Schwann cell lines and tissue, as compared to primary Schwann cells or schwannoma-derived cells. High levels of Kit expression in the neurofibrosarcoma-derived Schwann cells correlate with a decrease in neurofibromin expression. Using inhibitors of tyrosine kinase receptors, we found that proliferation of the neurofibrosarcoma-derived cells is dependent upon activation of a subclass of tyrosine-kinase receptors. The proliferation of these cells is not dependent upon an autocrine loop involving typical Schwann cell mitogens. Finally, the proliferation of the neurofibrosarcoma-derived Schwann cells can be increased by stimulation with Kit ligand. These data implicate Kit as one of the components leading to the Schwann cell hyperplasia observed in NF1.     

Comparative immunoreactivity of CD-68 and HMB-45 in malignant melanoma, neural tumors and nevi

The monoclonal antibody CD 68 (KP 1) reacts with fibrohistiocytic and some epithelial neoplasms; its reactivity compared with that of HMB 45 in malignant melanoma (MM) and neural tumors needs further elucidation. Using a streptavidin-biotin-immunoperoxidase procedure, we examined the reactivity of 65 MM (46 conventional, 1 polypoid, 6 desmoplastic [DMM], and 12 metastatic), 21 neurofibromas, 1 neurofibrosarcoma, 10 schwannomas, 1 perineurioma, 2 neurothekeomas, and 14 blue and 26 other nevi for CD-68, HMB-45-defined antigen, S 100 and neurofilament protein. A positive staining for CD 68 was observed in 38 of 42 primary, 5 of 6 DMM, and 11 of 12 metastatic melanomas; 6 of 10 schwannomas; 5 of 10 nevi with junctional component and all 14 blue nevi. All 21 neurofibromas, 1 each neurofibrosarcoma and perineurioma, both neurothekeomas, and all 12 nevi with dermal component were CD 68-negative. HBM 45 was expressed by all 44 primary, none of 6 DMM, and 7 of 12 metastatic melanomas; by none of 10 schwannomas, 6 neurofibromas, 1 neurofibrosarcoma, 1 perineurioma and 2 neurothekeomas. Both junctional nevi, 8 of 10 nevi with junctional components, 1 of 10 dermal components of junctional nevi, and 11 of 13 blue nevi were also HMB 45 positive. Except for 1 perineurioma, S 100 decorated all tumors examined. NF was immunoreactive in 1 of 45 conventional melanomas, 2 of 21 neurofibromas, 2 of 10 schwannomas, and 3 of 10 blue nevi; it was non-reactive in all polypoid, desmoplastic and metastatic melanomas; neurofibrosarcoma, perineurioma, neurothekeoma and other nevi. We conclude that the CD-68-reactivity in primary melanomas, neurofibromas, neurofibrosarcomas, perineuriomas, and nevi was similar to that of HMB 45. The significantly higher CD 68-positivity than of HMB 45 in metastatic and desmoplastic melanomas and schwannomas may be of diagnostic value.     

Preparing physicians for indicatory physical examination of the nervous system

Recently, splice variants of CD44 have been described that confer metastatic potential to non-metastasizing rat pancreatic carcinoma and sarcoma cell lines. Using antibodies against variant CD44 (CD44v) sequences, we have examined the expression of variant CD44 glycoproteins on human lymphoid cells and tissues and in colorectal neoplasia. Lymphohematopoietic cells express low levels of CD44v glycoproteins. During the process of lymphocyte activation in vitro and in vivo, expression of CD44v glycoproteins is transiently upregulated. The reaction pattern of various antibodies indicates that these CD44 variants contain the domain encoded by exon v6, which is part of the variant that confers metastatic capability. In human colorectal neoplasia we observed overexpression of CD44 splice variants in all invasive carcinomas. Already at early stages of colorectal tumor progression exon v5 epitopes were overexpressed. Tumor progression was strongly related to expression of CD44 isoforms containing exon v6 encoded domains. The findings establish CD44 variants as tumor progression markers in colorectal cancer.     

The splice-pattern of CD44 is altered in chronic pancreatitis exhibiting dysplastic changes

Recent studies have shown that several splice variants of CD44, might be involved in tumor progression. Since chronic pancreatitis is suggested to be a risk factor for pancreatic cancer we investigated the splice pattern of CD44 in chronic pancreatitis to elucidate the role of CD44 in pancreas tumorigenesis. The expression of CD44-isoforms was examined in 40 specimens of chronic pancreatitis and 12 specimens of normal pancreas by immunohistochemistry, Westernblotting and exon specific RT-PCR. Pancreatic cancer tissue from two patients who developed pancreatic cancer 2 and 3 years following surgery for chronic pancreatitis were analyzed. Strong expression of CD44s was found in all cells, whereas the expression of CD44v6 was restricted to ductal cells. Westernblotting revealed an overexpression of CD44v6 in chronic pancreatitis as compared to normal pancreas. Exon specific analysis revealed an altered splice pattern of CD44, similar to that in pancreatic cancer, in 12.5% of the chronic pancreatitis specimens. Both patients who developed pancreatic cancer after chronic pancreatitis exhibited this altered splice pattern in both, chronic pancreatitis and pancreatic cancer. These results suggest that variant forms of CD44-mRNA might be expressed in early dysplastic alterations in chronic pancreatitis.     

Complex CD44 splicing combinations in synovial fibroblasts from arthritic joints

CD44 is a broadly expressed cell surface glycoprotein which is the major cell surface receptor for the glycosaminoglycan, hyaluronan. In humans, alternative splicing of up to 9 variant exons (v2-v10) into CD44 mRNA, together with post-translational modification via glycosylation and chondroitin sulfate attachment has the potential of generating a large number of CD44 isoforms. Insertion of these various exons has the potential to change the functional capacities of the molecule and has implications in disease. We have analyzed CD44 splice variant expression in cultured VCAM-1-positive synovial fibroblasts isolated from patients with osteo- or rheumatoid arthritis and from normal synovium. Rheumatoid and osteoarthritic tissue express CD44 splice variants at the cell surface level. At the mRNA level exons v3, v6, v7, v8, v9 and v10 were detected in different splicing combinations. Rheumatoid tissue showed high expression, osteoarthritic tissues showed great variation. In contrast, non-inflamed tissue showed no splicing events. Our results indicate that the nature of CD44 splice variant expression may be linked to the inflammatory state of the synovial joint.     

CD44H expression by human neuroblastoma cells: relation to MYCN amplification and lineage differentiation

The human CD44 cell surface glycoprotein has been involved in a variety of functions including lymphocyte homing, extracellular cell matrix attachment, and tumor metastasis. Due to the alternative splicing of the single gene, a large family of different variants or isoforms is generated. Several reports have indicated an up-regulation of CD44 variant (v) isoforms in malignant process, conferring metastatic potential to non-metastatic cells. Neuroblastoma is a tumor characterized by an aggressive and metastatic behavior in advanced stages with amplification of the MYCN protooncogene. In this report we show that the CD44 standard molecule is highly expressed in 100% of stage I-III, IVs neuroblastomas and ganglioneuromas but only in a subset of stage IV tumors. In contrast, no expression of CD44 was detected on MYCN amplified stage IV tumors, thus demonstrating a highly significant negative relationship between MYCN amplification and CD44 expression in neuroblastoma. The expression of CD44 on neuroblastoma cultured cell lines was not shown to be related to MYCN amplification but rather linked to the S-type, schwann/glial differentiation lineage. Immunochemical analysis of tumor samples with anti-CD44v3 and -v6 antibodies and Northern blot analysis of mRNA from cell lines with probes spanning exons 4-10 did not reveal any expression of splice variants on neuroblastomas of all stages and cell lines, thus ruling out a major role of these isoforms in neuroblastoma progression and metastasis.     

Hepatocyte growth factor and c-MET in benign and malignant peripheral nerve sheath tumors

Hepatocyte growth factor (HGF), secreted by mesenchymal cells, has pleiotropic biological activities on several cell types. HGF and its receptor, the c-met proto-oncogene product (c-MET) have been implicated in the genesis and progression of several carcinomas and sarcomas. It has been suggested that MET/HGF autocrine signaling may contribute to tumorigenesis in sarcomas. HGF has been recently found to be a mitogen for rat Schwann cells and to be present in neurofibromas in NF1 patients. In this investigation, we assessed the immunoreactive patterns of HGF and MET in benign and malignant peripheral nerve sheath tumors (PNST) using archival formalin-fixed tissue. The standard avidin-biotin-peroxidase method was used. All benign tumors were negative with HGF. Eight cases of MPNST were positive with both HGF and MET. In some malignant PNST, positivity with both ligand and the receptor may be indicative of an autocrine mediated signal transduction and may implicate HGF/MET in tumor progression. Immunoreactivity with MET was strikingly greater in MPNST in contrast to benign PNST; this finding may prove to be helpful in distinguishing some histologically low-grade MPNST from cellular and atypical benign PNST.     

NF1 mutation analysis using a combined heteroduplex/SSCP approach

Neurofibromatosis type 1 (NF1) is a common autosomal dominant disorder characterized predominantly by neurofibromas, café-au-lait spots, and Lisch nodules. The disease is caused by disruptive mutations of the large NF1 gene, with half of cases caused by new mutation. Less than 100 constitutional mutations have thus far been published, ranging from very large deletions to point mutations. We have pursued NF1 mutation analysis by heteroduplex analysis (HDA) and single-strand conformational polymorphism analysis (SSCP) of individual exons. We streamlined these techniques to eliminate the use of radioactivity, to apply both methods to the same PCR product, and to multiplex samples in gels. Applied simultaneously to a set of 67 unrelated NF1 patients, HDA and SSCP have thus far identified 26 mutations and/or variants in 45 of the 59 exons tested. Disease-causing mutations were found in 19% (13/67) of cases studied. Both techniques detected a variety of mutations including splice mutations, insertions, deletions, and point changes, with some overlap in the ability of each method to detect variants.     

Heterogeneity of CD44 expression among human B-cell subpopulations

CD44 is a widely distributed cell surface glycoprotein that participates in a number of cellular adhesion and signal transduction processes. Germinal center B cells express very low levels of CD44, whereas their precursors and differentiation products express much higher levels. In immunofluorescence studies comparing 20 antibodies classified as being against the hematopoietic isoform of CD44, one antibody, A1G3, was unreactive with germinal center B cells, whereas the other antibodies showed low intensity but definite reactivity. Western blotting and sequential immunoprecipitation studies of lysates from density-separated lymphocyte fractions showed two bands that were differentially expressed and reacted differently with A1G3 compared with the other CD44 antibodies. These results suggest that germinal center B cells and non-germinal center B cells express different forms of CD44. Of 21 malignant B-cell populations examined, 5 showed reactivity with a "standard" CD44 reagent and significantly reduced reactivity with A1G3, while one sample showed the opposite pattern and the remainder were positive for both reagents. Of 10 cell lines studied, 5 were differentially stained by A1G3 and a standard CD44 antibody. PCR amplification of reverse transcribed mRNA from sorted human tonsil B-cell subpopulations and Southern blotting showed that B cells express a number of splice isoforms of CD44. These results demonstrate that B cells express multiple forms of CD44; both splice insert isoforms and variants distinguished by A1G3; the form of CD44 expressed depends on B-cell differentiation state.     

In vivo chiro-inositol metabolism in the rat: a defect in chiro-inositol synthesis from myo-inositol and an increased incorporation of chiro-[3H]inositol into phospholipid in the Goto-Kakizaki (G.K) rat

BACKGROUND: CD44 is a cell adhesion molecule often expressed in the form of various splice variants. The role of standard CD44 isoform (CD44s) and its variants in colorectal carcinogenesis is partly conflicting. Therefore, we compared the expression of CD44s (hermes-3) and its splice variants (v3 and v6) with traditional prognostic factors in 194 colorectal cancer patients treated at Kuopio University Hospital and followed up for a mean of 14 years. METHODS: Formalin-fixed, paraffin-embedded tissue sections from 194 patients with colorectal carcinoma were examined immunohistochemically to detect the expression of different forms of CD44. The hypothesis that CD44s, CD44v3, and CD44v6 expression intensities and distribution in cancer cells correlated with survival was tested with the log-rank test, hazard ratios, and their confidence intervals. RESULTS: In high-grade tumours CD44s and CD44v6 expression intensities and CD44s percentages were stronger than in low-grade tumours. CD44v6, CD44v3, and CD44s expression intensities in tumour epithelium were also stronger in Dukes C and D tumours than in A and B tumours. In the univariate survival analysis patients with strong CD44s, CD44v3, and CD44v6 expression intensities in tumour epithelium had lower cancer-related survival than the patients who had weak CD44s, CD44v3, and CD44v6 expression intensities. Recurrence-free survival was also shorter in patients with intense signals for CD44v3 and CD44v6 in tumour epithelium. In the multivariate analysis the CD44v6 expression intensity in tumour epithelium predicted independently both cancer-related and recurrence-free survival in T1-4N0-3M0 and T1-3N0M0 cases. In addition, the CD44v3 expression intensity in tumour epithelium was a significant predictor of RFS in T1-3N0M0 cases. CONCLUSIONS: These results strongly suggest that the CD44 splice variants v6 and v3 have prognostic significance in colorectal cancer.     

Expression of CD44 variant isoforms in malignant melanoma

In a variety of human tumors, expression of splice variants of the adhesion molecule CD44 (CD44v) has been described as correlating with tumor progression. Here, we report on the expression of CD44v in melanocytes, nevi, primary melanomas, and cutaneous and lymph node metastases. Thirteen nevi, 65 primary melanomas of varying thickness, 39 cutaneous and 15 lymph node metastases, and melanocytes and a panel of melanoma lines were tested for surface expression of the standard form of CD44 and the variant exons v5, v6, v7, v7-v8, and v10 by immunohistology or fluorescence-activated cell sorting. Melanocytes did not express any variant isoform of CD44. However, nevi, as well as primary melanoma and melanoma metastases, stained to a varying degree with anti-CD44v5, anti-CD44v7-v8, and anti-CD44v10. Exons v6 and v7 were not detected on any of these tissue specimens. Compared with nevi, expression of exon v10 was up-regulated in thick primary tumors and skin metastases. Lymph node metastases displayed elevated levels of exon v5. Expression of CD44v in melanoma lines (n = 20) differed, inasmuch as many lines did not express variant isoforms; in particular, exon v10. Interestingly, however, the few CD44v5-positive melanoma lines metastasized in the nu/nu mouse. Because benign as well as malignant growth of melanocytes was accompanied by expression of CD44 variant isoforms, a linkage between expression of CD44 variant isoforms and malignant transformation or tumor progression was excluded. Considering the function of distinct isoforms, one might speculate that expression of exon CD44v5, which was up-regulated in lymph node metastases compared with nevi and primary melanoma, provided a growth stimulus. Exon v10 is present at high density in epidermal cells. The de novo expression of this exon in nevi and the increased expression in thick melanoma and skin metastases would be in line with the assumption of an anchoring advantage in the surrounding epidermal tissue.