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We have previously described a rat monoclonal antibody, RS-11, which recognizes a tumor-associated antigen common to several species. In the present study, we have cloned and characterized the antigen recognized by RS-11. We screened a phage expression library prepared from HeLa cDNA and identified a clone that reacts with RS-11. DNA sequence analysis revealed that this clone contains sequences of keratin 18 (nucleotides 568-1196). We constructed several glutathione S-transferase fusion proteins and synthetic peptides based on this DNA sequence analysis and examined their reactivity with RS-11 to accurately map the RS-11 epitope. We determined that the epitope resides within a region of seven amino acids on the alpha-helix 2B domain of keratin 18 in which two amino acids (Leu(366) and Lys(370)) are completely conserved among intermediate filaments as well as other keratin members that are immunoreactive with RS-11. These two residues are sequentially discontinuous but spatially adjacent. The RS-11 epitope is constitutively present in human primary cultured hepatocytes; however, its immunoreactivity with RS-11 is up-regulated by malignant transformation or stimulation with either epidermal growth factor or transforming growth factor alpha.

The purpose of this study is to indicate that oxidative stress may contribute to occurrence of meningiomas. Recently, it was reported that aside from the neurofibromatosis type 2 (NF2) gene mutations, the calpain-dependent proteolysis of the NF2 gene product, merlin might be closely related to the development of certain NF2-related tumors. Although meningiomas are well known to occur more frequently in aged persons, it still remains unknown why calpain activation occurs predominantly in them. Because the production of free radicals with aging might be one of the causes of calpain activation especially in leptomeningeal cells being devoid of blood supply, the authors examined the relations between mu-calpain activation and merlin proteolysis induced by the oxidative stress.The authors examined 12 patient-derived sporadic meningiomas and their primary cultured cells. Malignant glioma cell line (U-251MG), which had no relation to NF2, was used as a control. They were exposed to hydrogen peroxide (H2O2) for 1 hour. After oxidative stress, they were examined by Western blot and immunofluorescence microscopic analyses.Despite the consistent expressions of activated mu-calpain in 11 of 12 meningioma tissues, this calpain activation completely disappeared after culture; instead the full-length merlin appeared again in 8 of 11 cases. The treatment of cultured cells with hydrogen peroxide induced both mu-calpain-dependent cleavage of merlin and reduction of an intrinsic calpain inhibitor calpastatin. Such proteolysis was significantly blocked by a specific calpain inhibitor, Z-LLal. The full-length merlin was immunocytochemically colocalized with activated mu-calpain at the plasma membrane, and, after mu-calpain activation, the fragment of merlin translocated to the perinuclear cytoplasm or into the nucleus.These findings suggest that oxidative stress-induced activation of mu-calpain causes proteolysis of merlin conceivably to impair cell adhesion and/or contact inhibition of meningioma cells.

Although mutational inactivation and allelic loss in the NF2 gene appear to be causal events in the majority of vestibular schwannomas, involvement of another potentially important mechanism, transcriptional inactivation, has not been investigated.We cloned and functionally characterized the 5'-flanking region of the human NF2 gene and identified the molecular mechanisms that regulate NF2 expression. Luciferase assay and site-directed mutagenesis demonstrated that a 70-base pair (bp) region (-591 to -522 bp from the translation start site) was essential for the basic expression of the NF2 gene. A gel mobility shift assay indicated recognition by nuclear protein of the unusually long ( approximately 66 bp) sequences in this region. Recognition was inhibited by either mutation of the binding core sequence or by methylation of three CpG sites. Point mutations at these CpG sites significantly decreased promoter activity, suggesting the importance of these sites. In 14 of 23 vestibular schwannomas, these three CpG sites were methylated in a site-specific manner and the methylation status was consistent with the expression of NF2 mRNA.Suppressed expression by aberrant methylation or mutation of the promoter elements could be an alternative mechanism for inactivation of the NF2 gene.

Membrane-associated guanylate kinases (MAGUKs) are known to function as scaffolds for forming multiprotein complexes at the synaptic junctions of neuronal cells and at sites of epithelial cell-cell contact. In Drosophila, mutations of the lethal (1)-discs large (dlg) gene, which encodes a MAGUK protein, leads to post-synaptic structure defects in neuronal cells and neoplastic overgrowth of epithelial cells. We previously showed that NE-dlg (neuronal and endocrine dlg), a human homolog of the dlg, plays a crucial role in formation of synaptic structure in human neuronal cells. Here we demonstrate that NE-dlg, similar to Drosophila dlg, is involved in regulation of cell cycle progression and adhesive ability of non-neuronal cells. Overexpression of NE-dlg in proliferating cells including various cancer cell lines induced growth suppression and impairment of cell adhesive ability. Furthermore, NE-dlg overexpression caused the down-regulation of beta-catenin in cancer cells regardless of mutations in the APC (adenomatous polyposis coli) gene. The PDZ domains of NE-dlg were found to be essential for the growth suppression, loss of adhesive property and down-regulation of beta-catenin. We propose that NE-dlg regulates the cell growth and adhesive ability by controlling the level of beta-catenin through an APC-independent pathway. Inactivation of NE-dlg may therefore contribute to development and/or progression of human neoplasms.

Nedd4 is a ubiquitin-protein ligase containing a calcium/lipid-binding domain, multiple WW domains and a C-terminal Hect domain, which is required for both the ubiquitin transfer and the association with E2 ubiquitin-conjugating enzymes. Nedd4 has been reported to be involved in the selective ubiquitination of some regulatory proteins in transcription and membrane transport.Three mRNA species for human Nedd4 were found to be 6.4-, 7.8- and 9.5-kb in size, and their expression patterns varied among normal tissues and cancer cell lines, indicating the tissue- and cell-specificities of Nedd4 expression. The Nedd4 protein, approximately 120 kDa in weight, was found in the cytoplasm, mainly in the perinuclear region and cytoplasmic periphery, of human cultured cells. Neural differentiation induced not only the down-regulation of Nedd4 but also the localization of the protein to both the cytoplasm and neurites. To identify the ubiquitination pathway that is linked to Nedd4, we demonstrated that specific E2 enzymes, including human Ubc4, UbcH5B, UbcH5C, UbcH6 and UbcH7, could transfer ubiquitin molecules to Nedd4 at the active cysteine residue, whereas E6AP accepted ubiquitins from Ubc4, UbcH5B, UbcH5C and UbcH7. Furthermore, nuclear localization of N-terminal deletion mutant Nedd4 enabled us to investigate the interaction between Nedd4 and E2 enzyme (Ubc4 or UbcH7) in the cell. The simultaneous expression of the full-length Nedd4 and E2 enzyme revealed the both proteins mostly colocalized in the cytoplasmic periphery, while the N-terminal deleted Nedd4 induced the nuclear and perinuclear colocalization with E2 enzyme.Our findings suggested that Nedd4 plays an important role in the cell regulation, including neural differentiation through cooperation with specific E2 ubiquitination pathways.

Neurofibromatosis type 2 (NF2) is an autosomal dominantly inherited disorder, strongly associated with development of benign intracranial tumors, including bilateral vestibular schwannomas and meningiomas. The NF2 gene located on the human chromosome 22q12 was recently cloned, and the protein it encodes (termed merlin/schwannomin) was found to be strikingly similar to the moesin-ezrin-radixin (MER) family of cytoskeleton-associated proteins. Mutations of NF2 gene have been found not only in the NF2 patient but also in NF2-related sporadic tumors such as acoustic schwannomas and meningiomas, suggesting that the NF2 gene functionally acts as a tumor suppressor gene. To elucidate the biological function of merlin, we have detected five merlin binding cellular proteins. The N-terminal region of merlin, the entire merlin-ezrin-radixin-moesin (MERM) homology domain, was found to be essential for binding to all five proteins. Since most reported NF2 mutations in the region were determined to be necessary for binding, the mutations probably impair binding. The majority of NF2 mutations are nonsense mutations or frameshifts that resulted in premature termination of merlin. The lack of these interactions caused by such mutations of NF2 gene may affect the cellular signals, resulting in benign intracranial tumors in NF2 patients.

CD44 is a cell surface receptor of hyaluronate that is implicated in the regulation of tumor growth and metastatic potential. Transformation of colon mucosa to carcinoma is associated with overexpression of several CD44 alternative splice variants. The functional roles of CD44 isoforms in colon carcinoma tumor progression remain unclear. CD44H expression is downregulated in colon carcinomas compared to paired normal mucosa. In the present study we demonstrate that reintroduction of CD44H back into colon carcinoma cells by stable transfection reduces their in vitro growth rate and tumorigenicity. Examination of several colon carcinoma cell lines and use of mutant CD44H reveal that this in vitro and in vivo growth reduction requires the ability of CD44H to bind hyaluronate. These observations indicate that the CD44H downregulation associated with transformation of mucosa to colon carcinoma may provide the carcinoma cells with a growth advantage. Furthermore, the reduction in tumor growth rate mediated by reintroduction of CD44H into colon carcinoma cells is dependent on its ability to bind hyaluronate.

To investigate the biological function of p53 in colon carcinoma cells, a wild-type p53 expression plasmid under the control of the human cytomegalovirus promoter was stably transfected into the human colon adenocarcinoma cell line WiDr, which carries a mutation of the p53 gene at codon 273. Exogenous wild-type p53 transcripts were detected at various expression levels in 8 of 117 G418-resistant clones. The growth rates of the wild-type p53+ clones in culture did not change significantly. The efficiency of colony formation in soft agar, however, was completely suppressed in two wild-type p53+ clones. This is the first to demonstrate the feasibility of stable transfection of the wild-type p53 gene under the control of non-inducible promoter in human colon cancer cells. The major alteration found was that wild-type p53+ cells which were incubated with anti-Fas IgM showed marked cytolysis with preferential over-expression of wild-type p53 accompanied by overexpression of a cyclin-dependent kinase inhibitor, WAF1, whereas the endogenous mutant p53 retained its expression level. The findings suggest that a Fas-initiated pathway is incidentally linked to a p53-dependent apoptotic pathway through the reconstituted wild-type p53 gene in WiDr cells. This model should help elucidating the additional role of the p53 tumor suppressor gene and the mechanism of apoptosis in colon carcinoma cells.

The neurofibromatosis type 2 (NF2) gene was recently cloned, and the protein it encodes (merlin) was revealed to belong to a family of proteins that link cytoskeletal components with proteins in the cell membrane. To elucidate the biological function of merlin, we produced a bacterial fusion protein consisting of glutathione S-transferase and merlin and used it to detect five merlin-binding cellular proteins, designated p165, p145, p125, p85 and p70, by a protein-binding assay. p165 and merlin were phosphorylated on serine/threonine residues, and immunoprecipitation showed that p85 bound the native form of merlin. Although the entire merlin-ezrin-radixin-moesin (MERM) homology domain of merlin was found to be essential for binding to all five proteins, the MERM homology domains of ezrin and moesin did not bind to any of the five proteins. Since most reported NF2 mutations are in the region we determined was necessary for binding, the mutations probably impair binding. Therefore, the formation of the protein complex is probably crucial for tumor suppression.

Mutation of the p53 tumor suppressor gene is the most common genetic alteration in human tumors. The altered protein product of the mutant gene is stabilized in tumor cells and can be detected using monoclonal antibody immunohistochemistry. We report a technique for immunostaining of the altered p53 protein in routinely processed paraffin-embedded tissue sections, comparing this method with a frozen-section immunostain method for concordance. Similarly processed tumor cell lines with known mutations or deletions of the p53 gene served as positive or negative controls. The results in 25 human gliomas and 20 colorectal carcinomas showed a 100% correspondence of positive reactivity in the colorectal carcinomas and an 83% correspondence in the gliomas. In no case did the paraffin-embedded sections react in the absence of frozen section reactivity (that is, there were no false positives). Three of 18 gliomas showed reactivity in frozen sections without reactivity in the companion paraffin-embedded sections. This discrepancy could have been caused by technical factors such as length of fixation time. We propose that this new paraffin-embedded section immunostaining method will be of value as a screening technique for the investigation of p53 mutations in archived human tumors. The data thus obtained may then be correlated with clinical information and perhaps be of value in diagnosis or predicting outcome for various human cancers.

Chronic lymphocytic leukemia (CLL) is a usually indolent disease that can assume an aggressive clinical course in some patients. To develop assays that would be predictive of how a particular patient's disease would evolve, we studied the expression of proliferating cell nuclear antigen (PCNA) by Western blotting in 40 patients with CLL. The concentration of PCNA, a cofactor for delta DNA-dependent DNA polymerase, is indicative of the proliferative state of the cell. Significantly lower PCNA levels were observed in earlier stage CLL when compared with more advanced disease. The leukemic cell proliferative rate, assessed by lymphocyte doubling time and flow cytometry, also correlated significantly with the level of PCNA expression. These results suggest that a high level of PCNA in the cells of CLL patients at presentation identifies a subgroup of patients whose CLL cells have a higher proliferative activity and who may, therefore, have a potentially shorter survival.

The effect of herbimycin A, an ansamycin antibiotic which inhibits cellular transformation by retroviral tyrosine kinases, on the monolayer growth of seven colon tumor cell lines and one cell line established from normal colonic mucosa, CCL239, was examined. Each colon tumor cell line tested showed dose-dependent growth inhibition in response to herbimycin A. A 125ng ml-1 dose of the antibiotic caused greater than 40% growth inhibition in all colon tumor cell lines after two cell doublings. In contrast, at similar herbimycin A concentrations only 12% inhibition was observed in 'normal' CCL239 cells. No major morphologic changes were observed at the light microscopic level in any of the tumor cell lines or CCL239 cells in response to treatment with herbimycin A. Studies using the HT29 colon adenocarcinoma cell line showed dose-dependent inactivation of pp60c-src by herbimycin A, resulting in decreased autophosphorylation, enolase phosphorylation and steady-state levels, which correlated with cellular growth inhibition. Herbimycin A-induced reductions in pp60c-src kinase activity preceded changes in pp60c-src steady-state levels. Growth and pp60c-src inhibition were reversible following removal of herbimycin A from cell culture media. Our results suggest that regulation of pp60c-src tyrosine kinase activity may be important in growth control of colon tumor cells.

A 360 residue region encoded by the neurofibromatosis type 1 (NF1) gene shows significant homology to the catalytic domains of both mammalian GTPase-activating proteins (GAP) and yeast IRA proteins. This GAP-related domain of the NF1 gene (NF1-GRD), like the GAP and IRA protein, has been reported to mediate hydrolysis of Ras-bound GTP to GDP, resulting in inactivation of Ras protein. In the present study, we identified two different types of NF1-GRD cDNA. One (type I) is identical to the previously reported sequence, and the other (type II) contained an additional 63 bp insertion that encodes for a region of 21 amino acids in the center of the NF1-GRD molecule. Alternative splicing is the most likely mechanism by which these two types of transcripts arise. Our observations reveal that the type I transcript is predominantly expressed in undifferentiated cells, whereas the type II transcript predominates in differentiated cells. Furthermore, the expression pattern of type I and type II NF1-GRD mRNA immediately changed in SH-SY5Y neuroblastoma cells when neuronal differentiation programs were induced by retinoic acid treatment. We propose that the differential expression of type I and type II NF1-GRD transcripts might be an 'on/off' switch that regulates the catalytic activity of the NF1 gene product, which plays an important role in the regulation of neuronal differentiation.

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Fischer 344 (F344) rats hyperimmunized with syngeneic 9L/R3 glioma cells produced antibody selective to glioma cells. Hybridomas prepared from the spleen cells of the immunized rat were cloned, and we obtained a hybridoma clone which produced monoclonal IgM antibody, termed FR77, that showed selectivity to glioma cells. Immunoperoxidase staining of cultured cells revealed that FR77 was reactive with 3 lines of rat glioma cells but not with normal F344 rat fibroblasts. Immunohistochemical staining of F344 rat tissue sections with biotinylated FR77 demonstrated that FR77 could bind with glioma tissue developed by intracerebral injection of 9L/R3 glioma cells but not to normal parts of the brain tissues and other normal tissues tested. The FR77-defined antigen was observed to be mainly localized in cytoplasm of glioma cells but a small portion of the antigen was also detected on the glioma cell surface.

Cell kinetics of 91 human brain tumors obtained from 88 patients were analyzed with the following two methods, 1) bivariate (two-color) flow cytometric measurement of cellular DNA content and amount of bromodeoxyuridine (BrdU) incorporated into cellular DNA, in 66 specimens, 2) immunohistochemical detection of BrdU incorporated S-phase cells, in 34 specimens. Patients were given an intravenous 1 hour infusion of 200 mg/sq. m. of BrdU 1-2 hours before the surgical removal. The excised tumor specimen was divided into several portions. One was fixed with 70% ethanol and embedded in paraffin, and another was digested mechanically and/or chemically to obtain a single cell suspension, and fixed in 70% ethanol. Paraffin-embedded tissue sections were stained by the peroxidase-antiperoxidase immunohistochemical method using anti-BrdU monoclonal antibody (MoAb). Single cell suspensions were reacted with fluorescein isothiocyanate (FITC) conjugated anti-BrdU MoAb, or anti-BrdU MoAb and FITC-conjugated second antibody successively by the staining with propidium iodide, for flow cytometry (FCM). Rates of S-phase fraction in single cell suspensions calculated by bivariate FCM were correlated well with labeling indexes (LI, i.e. the percentage of BrdU incorporated cells) calculated in tissue sections, but not with the result of analysis of DNA histogram by Dean's method. This discrepancy is probably due to large coefficient value in several samples. Histological malignancy of the tumors was reflected both in the proliferating index (PI, i.e. % S+G2M phase) calculated by bivariate FCM and the LI by immunohistochemical method. PI tended to be high in primitive neuroectodermal tumors and metastatic carcinomas, moderately high in gliomas, and low in benign tumor groups.(ABSTRACT TRUNCATED AT 250 WORDS)

We investigated the effect of retinoic acid (RA) and herbimycin A (herb-A) on cell growth, cell differentiation, and colony formation of human neuroblastoma cell lines. The neuroblastoma line SK-N-SH expressed both neuroblast and nonneuronal phenotypes, whereas its subclone SH-SY5Y and the Kelly cell line were predominantly neuroblastic. Both herb-A and RA, given alone, moderately reduced cell growth and colony formation of the neuroblastic cell lines. Growth curve analyses with SK-N-SH suggested that herb-A greatly reduced the number of initially growing cells, whereas RA slightly enhanced initial cell growth. Morphological changes were determined with the use of rhodaminephalloidin staining of actin. Retinoic acid caused an increase in the fraction of neuroblast cell in SK-N-SH, and conversely of nonneuronal cells in SH-SY5Y and Kelly cell lines. Both drugs also caused partial differentiation towards a neuronal phenotype, and herb-A induced selective lysis of nonneuronal cells of SK-N-SH. Because of their discrepant effects, RA (10 microM) and herb-A (236 nM) were tested in combination at a concentration that had only moderate effects when given alone. The combination further reduced cell growth and colony formation and dramatically enhanced differentiation towards a neuronal morphology. The Kelly cell line with amplified N-myc genome, which correlates with clinical progression of neuroblastoma, was also sensitive to RA plus herb-A. These results recommend the combination of RA and herb-A for differentiation therapy of neuroblastoma.