CHD4 as a Potential Biomarker in Differentiating Between Cellular Schwannoma and Malignant Peripheral Nerve Sheath Tumor.

Cellular schwannoma is an uncommon variant of benign peripheral nerve sheath tumors, but is commonly misdiagnosed as malignant peripheral sheath tumor (MPNST). Conventional methods that are used to distinguish cellular schwannoma from MPNST include immunohistochemistry (IHC) staining. However, most markers cannot precisely differentiate these 2 tumor types, and thus identification of a better marker is needed to improve the accuracy of diagnosis. Here, we evaluate the use of chromodomain helicase DNA-binding protein 4 (CHD4) as a specific marker for cellular schwannoma by comparing CHD4 and S-100 IHC staining in 14 cellular schwannoma and 17 MPNST tissue samples. Our results indicated that nuclear CHD4 stains were in moderate-to-high in 94% MPNST (16 cases) and 93% cellular schwannoma (13 cases). However, cytoplasmic CHD4 stains were moderate-to-high in 93% cellular schwannoma (13 cases) but negative-to-weak in 100% MPNST (17 cases). In contrast, the S-100 stains were moderate-to-high in 86% of the cellular schwannoma (12 cases) and in 35% of the MPNST (6 cases). Taken together, the results indicated that different location of CHD4 staining is a potential biomarker to differentiate cellular schwannoma from MPNST.

HDAC8, A Potential Therapeutic Target for the Treatment of Malignant Peripheral Nerve Sheath Tumors (MPNST).

INTRODUCTION: HDAC isoform-specific inhibitors may improve the therapeutic window while limiting toxicities. Developing inhibitors against class I isoforms poses difficulties as they share high homology among their catalytic sites; however, HDAC8 is structurally unique compared to other class I isoforms. HDAC8 inhibitors are novel compounds and have affinity for class I HDAC isoforms demonstrating anti-cancer effects; little is known about their activity in malignant peripheral nerve sheath tumors (MPNST). Recently, we demonstrated anti-MPNST efficacy of HDAC8i in human and murine-derived MPNST pre-clinical models; we now seek to consider the potential therapeutic inhibition of HDAC8 in MPNST. METHODS: Four Human MPNST cell lines, a murine-derived MPNST cell line, and two HDAC8 inhibitors (PCI-34051, PCI-48012; Pharmacyclics, Inc. Sunnyvale, CA) were studied. Proliferation was determined using MTS and clonogenic assays. Effects on cell cycle were determined via PI FACS analysis; effects on apoptosis were determined using Annexin V-PI FACS analysis and cleaved caspase 3 expression. In vivo growth effects of HDAC8i were evaluated using MPNST xenograft models. 2D gel electrophoresis and mass spectrometry were used to identify potential HDAC8 deacetylation substrates. RESULTS: HDAC8i induced cell growth inhibition and marked S-phase cell cycle arrest in human and murine-derived MPNST cells. Relative to control, HDAC8i induced apoptosis in both human and murine-derived MPNST cells. HDAC8i exhibited significant effects on MPNST xenograft growth (p=0.001) and tumor weight (p=0.02). Four potential HDAC8 substrate targets were identified using a proteomic approach: PARK7, HMGB1, PGAM1, PRDX6. CONCLUSIONS: MPNST is an aggressive sarcoma that is notoriously therapy-resistant, hence the urgent need for improved anti-MPNST therapies. HDAC8 inhibition may be useful for MPNST by improving efficacy while limiting toxicities as compared to pan-HDACis.

FRAX597, a small molecule inhibitor of the p21-activated kinases, inhibits tumorigenesis of neurofibromatosis type 2 (NF2)-associated Schwannomas.

The p21-activated kinases (PAKs) are immediate downstream effectors of the Rac/Cdc42 small G-proteins and implicated in promoting tumorigenesis in various types of cancer including breast and lung carcinomas. Recent studies have established a requirement for the PAKs in the pathogenesis of Neurofibromatosis type 2 (NF2), a dominantly inherited cancer disorder caused by mutations at the NF2 gene locus. Merlin, the protein product of the NF2 gene, has been shown to negatively regulate signaling through the PAKs and the tumor suppressive functions of Merlin are mediated, at least in part, through inhibition of the PAKs. Knockdown of PAK1 and PAK2 expression, through RNAi-based approaches, impairs the proliferation of NF2-null schwannoma cells in culture and inhibits their ability to form tumors in vivo. These data implicate the PAKs as potential therapeutic targets. High-throughput screening of a library of small molecules combined with a structure-activity relationship approach resulted in the identification of FRAX597, a small-molecule pyridopyrimidinone, as a potent inhibitor of the group I PAKs. Crystallographic characterization of the FRAX597/PAK1 complex identifies a phenyl ring that traverses the gatekeeper residue and positions the thiazole in the back cavity of the ATP binding site, a site rarely targeted by kinase inhibitors. FRAX597 inhibits the proliferation of NF2-deficient schwannoma cells in culture and displayed potent anti-tumor activity in vivo, impairing schwannoma development in an orthotopic model of NF2. These studies identify a novel class of orally available ATP-competitive Group I PAK inhibitors with significant potential for the treatment of NF2 and other cancers.

Expression profiling of 519 kinase genes in matched malignant peripheral nerve sheath tumor/plexiform neurofibroma samples is discriminatory and identifies mitotic regulators BUB1B, PBK and NEK2 as overexpressed with transformation.

About 50% of all malignant peripheral nerve sheath tumors (MPNSTs) arise as neurofibromatosis type 1 associated lesions. In those patients malignant peripheral nerve sheath tumors are thought to arise through malignant transformation of a preexisting plexiform neurofibroma. The molecular changes associated with this transformation are still poorly understood. We sought to test the hypothesis that dysregulation of expression of kinases contributes to this malignant transformation. We analyzed expression of all 519 kinase genes in the human genome using the nanostring nCounter system. Twelve cases of malignant peripheral nerve sheath tumor arising in a background of preexisting plexiform neurofibroma were included. Both components were separately sampled. Statistical analysis compared global changes in expression levels as well as changes observed in the pairwise comparison of samples taken from the same surgical specimen. Immunohistochemical studies were performed on tissue array slides to confirm expression of selected proteins. The expression pattern of kinase genes can separate malignant peripheral nerve sheath tumors and preexisting plexiform neurofibromas. The majority of kinase genes is downregulated rather than overexpressed with malignant transformation. The patterns of expression changes are complex without simple recurring alteration. Pathway analysis demonstrates that differentially expressed kinases are enriched for kinases involved in the direct regulation of mitosis, and several of these show increased expression in malignant peripheral nerve sheath tumors. Immunohistochemical studies for the mitotic regulators BUB1B, PBK and NEK2 confirm higher expression levels at the protein level. These results suggest that the malignant transformation of plexiform neurofibroma is associated with distinct changes in the expression of kinase genes. The patterns of these changes are complex and heterogeneous. There is no single unifying alteration. Kinases involved in mitotic regulation are particularly enriched in the pool of differentially expressed kinases. Some of these are overexpressed and are therefore possible targets for kinase inhibitors.

Differential expression of type 2 3α/type 5 17ß-hydroxysteroid dehydrogenase (AKR1C3) in tumors of the central nervous system.

Human aldo-keto reductase (AKR) 1C3, type 2 3α-hydroxysteroid dehydrogenase (HSC)/ type 5 17ß-HSD, is known to be involved in steroids, prostaglandins, and lipid aldehydes metabolism. The expression of AKR1C3 has been demonstrated in hormone-dependent normal tissues such as breast, endometrium, prostate, and testis; and de -regulated AKR1C3 expression has been shown in breast carcinoma, endometrial hyperplasia, endometrial carcinoma, and prostate carcinoma. AKR1C3 expression has also been demonstrated in hormone-independent normal tissues (renal tubules and urothelium) and neoplastic tissues (renal cell carcinoma, Wilm's tumor, and urothelial cell carcinoma). Extensive expression of AKR1C3 in normal and neoplastic as well as hormone-dependent and hormone-independent tissues indicates that AKR1C3 may have functions beyond steroid hormone metabolism. In this report, we describe a widespread expression of AKR1C3 in glial neoplasms and meningiomas, with limited expression in medulloblastoma and no expression in Schwannoma. These tumors, except meningioma, are not classically considered to be sex hormone-dependent or related brain tumors. The current results corroborate our earlier observations that AKR1C3 is expressed in both sex hormone-dependent and hormone-independent malignancies. Similar to AKR1C3 distribution in Wilm's tumor, we also demonstrate that expression of AKR1C3 is reduced in tumors with embryonic phenotypes.

Clinical and molecular genetics of Carney complex.

Carney complex (CNC) is a rare multiple familial neoplasia syndrome that is characterized by multiple types of skin tumors and pigmented lesions, endocrine neoplasms, myxomas and schwannomas and is inherited in an autosomal dominant manner. Clinical and pathologic diagnostic criteria are well established. Over 100 pathogenic variants in the regulatory subunit type 1A (RI-A) of the cAMP-dependent protein kinase (PRKAR1A) have been detected in approximately 60% of CNC patients, most leading to R1A haploinsufficiency. Other CNC-causing genes remain to be identified. Recent studies provided some genotype-phenotype correlations in CNC patients carrying PRKAR1A-inactivating mutations, which provide useful information for genetic counseling and/or prognosis; however, CNC remains a disease with significant clinical heterogeneity. Recent mouse and in vitro studies have shed light into how R1A haploinsufficiency causes tumors. PRKAR1A defects appear to be weak tumorigenic signals for most tissues; Wnt signaling activation and cell cycle dysregulation appear to be important mediators of the tumorigenic effect of a defective R1A.

PAK kinase regulates Rac GTPase and is a potential target in human schwannomas.

Merlin loss causes benign tumours of the nervous system, mainly schwannomas and meningiomas. Schwannomas show enhanced Rac1 and Cdc42 activity, the p21-activated kinase 2 (PAK2) activation and increased ruffling and cell adhesion. PAK regulates activation of merlin. PAK has been proposed as a potential therapeutic target in schwannomas. However where PAK stands in the Rac pathway is insufficiently characterised. We used a novel small-molecule PAK inhibitor, IPA-3, to investigate the role of PAK activation on Rac1/Cdc42 activity, cell spreading and adhesion in human primary schwannoma and Schwann cells. We show that IPA-3 blocks activation of PAK2 at Ser192/197 that antagonises PAK's interaction with Pix. Accordingly, Pix-mediated Rac1 activation is decreased in IPA-3 treated schwannoma cells, indicating that PAK acts upstream of Rac. We show that this Rac activation at the level of focal adhesions in schwannoma cells is essential for cell spreading and adhesion in Schwann and schwannoma cells.

Fatty acid 2-hydroxylase regulates cAMP-induced cell cycle exit in D6P2T schwannoma cells.

Sphingolipids are ubiquitous components of eukaryotic cells that regulate various cellular functions. In many cell types, a fraction of sphingolipids contain 2-hydroxy fatty acids, produced by fatty acid 2-hydroxylase (FA2H), as the N-acyl chain of ceramide [hydroxyl fatty acid (hFA)-sphingolipids]. FA2H is highly expressed in myelin-forming cells of the nervous system and in epidermal keratinocytes. While hFA-sphingolipids are thought to enhance the physical stability of specialized membranes produced by these cells, physiological significance of hFA-sphingolipids in many other cell types is unknown. In this study, we report novel roles for FA2H and hFA-sphingolipids in the regulation of the cell cycle. Treatment of D6P2T Schwannoma cells with dibutyryl-cAMP (db-cAMP) induced exit from the cell cycle with concomitant upregulation of FA2H. Partial silencing of FA2H in D6P2T cells resulted in 60-70% reduction of hFA-dihydroceramide and hFA-ceramide, with no effect on nonhydroxy dihydroceramide and ceramide. Under these conditions, db-cAMP no longer induced cell cycle exit, and cells continued to grow and divide. Immunoblot analyses revealed that FA2H silencing prevented db-cAMP-induced upregulation of cyclin-dependent kinase inhibitors p21 and p27. These results provide evidence that FA2H is a negative regulator of the cell cycle and facilitates db-cAMP-induced cell cycle exit in D6P2T cells.

Validation of the p21-activated kinases as targets for inhibition in neurofibromatosis type 2.

Neurofibromatosis type 2 (NF2) is a dominantly inherited cancer disorder caused by mutations at the NF2 gene locus. Merlin, the protein product of the NF2 gene, has been shown to negatively regulate Rac1 signaling by inhibiting its downstream effector kinases, the p21-activated kinases (Pak). Given the implication of Paks in tumorigenesis, it is plausible that merlin's tumor suppressive function might be mediated, at least in part, via inhibition of the Paks. We present data indicating this is indeed the case. First, analysis of primary schwannoma samples derived from NF2 patients showed that in a significant fraction of the tumors, the activity of Pak1 was highly elevated. Second, we used shRNAs to knockdown Pak1, 2, and 3 in NIH3T3 cells expressing a dominant-negative form of merlin, NF2(BBA) (NIH3T3/NF2(BBA)), and find that simultaneous knockdown of Pak1-3 in these cells significantly reduced their growth rates in vitro and inhibited their ability to form tumors in vivo. Finally, while attempting to silence Pak1 in rat schwannoma cells, we found that these cells were unable to tolerate long-term Pak1 inhibition and rapidly moved to restore Pak1 levels by shutting down Pak1 shRNA expression through a methylation-dependent mechanism. These data suggest that inhibiting Pak could be a beneficial approach for the development of therapeutics toward NF2. In addition, the finding that the shRNA-mediated Pak1 suppression was silenced rapidly by methylation raises questions about the future application of such technologies for the treatment of diseases such as cancer.

Dissecting and targeting the growth factor-dependent and growth factor-independent extracellular signal-regulated kinase pathway in human schwannoma.

Schwannomas are tumors of the nervous system that occur sporadically and in patients with the cancer predisposition syndrome neurofibromatosis type 2 (NF2). Schwannomas and all NF2-related tumors are caused by loss of the tumor suppressor merlin. Using our human in vitro model for schwannoma, we analyzed extracellular signal-regulated kinase 1/2 (ERK1/2) and AKT signaling pathways, their upstream growth factor receptors, and their role in schwannoma cell proliferation and adhesion to find new systemic therapies for these tumors that, to date, are very difficult to treat. We show here that human primary schwannoma cells show an enhanced basal Raf/mitogen-activated protein/ERK kinase/ERK1/2 pathway activity compared with healthy Schwann cells. Due to a strong and prolonged activation of platelet-derived growth factor receptor beta (PDGFRbeta), which is highly overexpressed, ERK1/2 and AKT activation was further increased in schwannoma, leading to increased proliferation. Using specific inhibitors, we discovered that ERK1/2 activation involves the integrin/focal adhesion kinase/Src/Ras signaling cascades and PDGFRbeta-mediated ERK1/2 activation is triggered through the phosphatidylinositol 3-kinase/protein kinase C/Src/c-Raf pathway. Due to the complexity of signals leading to schwannoma cell proliferation, potential new therapeutic agents should target several signaling pathways. The PDGFR and c-Raf inhibitor sorafenib (BAY 43-9006; Bayer Pharmaceuticals), currently approved for treatment of advanced renal cell cancer, inhibits both basal and PDGFRbeta-mediated ERK1/2 and AKT activity and decreases cell proliferation in human schwannoma cells, suggesting that this drug constitutes a promising tool to treat schwannomas. We conclude that our schwannoma in vitro model can be used to screen for new therapeutic targets in general and that sorafenib is possible candidate for future clinical trials.

Expression of emmprin and matrix metalloproteinases (MMPs) in peripheral nerve sheath tumors: emmprin and membrane-type (MT)1-MMP expressions are associated with malignant potential.

BACKGROUND: Matrix metalloproteinases (MMPs), including membrane-type (MT)-MMPs, correlate with biological aggressiveness in many carcinomas. However, their roles in peripheral nerve sheath tumors (PNSTs) have rarely been investigated. MATERIALS AND METHODS: In this study, the immunohistochemical expression of 6 MMPs, their 3 inhibitors and emmprin, an MMP inducer, was examined in 14 schwannomas, 14 neurofibromas and 12 malignant peripheral nerve sheath tumors (MPNSTs) in relation to malignant potentials. RESULTS: Higher expression levels (>3+) of emmprin and MT1-MMP were noted in 83.3% and 16.7% of MPNSTs, respectively, versus none in schwannomas and neurofibromas (p<0.0001). The overall expression rate (1-4+) of MT1-MMP was 58.3% in MPNSTs versus 7.1% in both schwannomas and neurofibromas (p=0.0093). Gelatinase A (MMP-2) showed higher expression levels (>3+) in all the tumors without significant differencies. Moreover, the expression patterns of MMP-1 and gelatinase B (MMP-9) could divide PNSTs into two groups: schwannoma versus neurofibroma/MPNST. Higher expression levels (>3+) of MMP-9 were observed in 50% of schwannomas versus none in neurofibromas and MPNSTs, while those of MMP-1 were found in 35.7% of neurofibromas and 66.7% of MPNSTs versus none in schwannomas. RECK was the main inhibitor expressed in these 3 tumors, with no significant differences. CONCLUSION: These results suggest that emmprin and MT1-MMP may be malignant potential-related proteins in PNSTs, and that MMP-1 and 9 may help differentiation between schwannoma and neurofibroma, especially in their plexiform types.

A mouse model for the Carney complex tumor syndrome develops neoplasia in cyclic AMP-responsive tissues.

Carney complex is an autosomal dominant neoplasia syndrome characterized by spotty skin pigmentation, myxomatosis, endocrine tumors, and schwannomas. This condition may be caused by inactivating mutations in PRKAR1A, the gene encoding the type 1A regulatory subunit of protein kinase A. To better understand the mechanism by which PRKAR1A mutations cause disease, we have developed conventional and conditional null alleles for Prkar1a in the mouse. Prkar1a(+/-) mice developed nonpigmented schwannomas and fibro-osseous bone lesions beginning at approximately 6 months of age. Although genotype-specific cardiac and adrenal lesions were not seen, benign and malignant thyroid neoplasias were observed in older mice. This spectrum of tumors overlaps that seen in Carney complex patients, confirming the validity of this mouse model. Genetic analysis indicated that allelic loss occurred in a subset of tumor cells, suggesting that complete loss of Prkar1a plays a key role in tumorigenesis. Similarly, tissue-specific ablation of Prkar1a from a subset of facial neural crest cells caused the formation of schwannomas with divergent differentiation. These observations confirm the identity of PRKAR1A as a tumor suppressor gene with specific importance to cyclic AMP-responsive tissues and suggest that these mice may be valuable tools not only for understanding endocrine tumorigenesis but also for understanding inherited predispositions for schwannoma formation.

Implication of telomere length as a proliferation-associated marker in schwannomas.

BACKGROUND AND OBJECTIVES: Some schwannomas in the central nervous system may demonstrate relatively aggressive behavior in pathological findings and clinical course. We evaluate the diagnostic values of telomerase activity and telomere length in the clinicopathological behavior of schwannomas. METHODS: Thirty surgical specimens from intracranial and intraspinal schwannomas were analyzed by polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA) for telomerase activity and terminal restriction fragments (TRFs) using Southern blot for telomere length. Proliferative indices were also studied. RESULTS: Telomerase activity could not be detected in all schwannomas. Elongated telomere length (mean 17,101 +/- 259 bp) was found in four specimens (13.3%). Three of these four were found to have mitotic figures, high vascularity, cellularity, and pleomorphism in the pathological findings. The proliferative indices (35) showed correlative high values. One patient died of this disease, and one was found to have recurrence at follow-up evaluation. Those that displayed benign histopathological pictures showed relatively short telomere length (8,866 +/- 271 base pairs) and low proliferative indices (21). These is a significant difference between these two groups (P = 0.001). CONCLUSIONS: Elongation of telomere length in schwannomas appears to predict aggressive clinicopathological behavior.

Mutations of the gene encoding the protein kinase A type I-alpha regulatory subunit (PRKAR1A) in patients with the "complex of spotty skin pigmentation, myxomas, endocrine overactivity, and schwannomas" (Carney complex).

Carney complex (CNC) is a familial multiple neoplasia syndrome associated with abnormal skin and mucosal pigmentation. The complex has features overlapping those of McCune-Albright syndrome (MAS) and the other multiple endocrine neoplasias (MENs). CNC is inherited as an autosomal dominant trait, and the responsible genes have been mapped by linkage analysis to loci at 2p16 and 17q22-24. Because of its unusual biochemical features (e.g., paradoxical responses to various endocrine signals) and its clinical similarities to MAS, genes implicated in cyclic nucleotide-dependent signaling, including GNAS1 (which is responsible for MAS), had been considered likely candidates for causing CNC. The gene encoding the protein kinase A (PKA) type I-alpha regulatory subunit (RI alpha), PRKAR1A, had been mapped to 17q22-24; loss-of-heterozygosity (LOH) analysis using polymorphic markers from this region revealed consistent changes in tumors from patients with CNC, including those from one family previously mapped to 17q22-24. Investigation of a polymorphic site within the 5' of the PRKAR1A gene showed segregation with the disease and retention of the allele bearing the disease gene in CNC tumors. Mutations of the PRKAR1A gene were also found to have occurred de novo in sporadic cases of CNC; no mutations were found in kindreds mapping to 2p16. Thus, genetic heterogeneity in CNC was confirmed; in total, 41% of all patients with CNC had mutations in the PRKAR1A gene. All mutations were frameshifts, insertions, and deletions that led to nonsense mRNA and premature termination of the predicted peptide product. Functional studies in CNC tumors suggested that inactivating mutations of the PRKAR1A gene led to nonsense mRNA decay (the mutant peptide product was not present) and were associated with dysregulated PKA activity, increased responsiveness to cAMP, and excess of type-II PKA activity. We conclude that the PRKAR1A gene, coding for the RIalpha subunit of PKA, a critical cellular component of a number of cyclic nucleotide-dependent signaling pathways, is mutated in a subset of patients with CNC. In their tumors, there is LOH of the normal allele, suggesting that normal RI-alpha may have tumor suppression function in the tissues affected by CNC. An excess of type-II PKA activity was present in affected tissues, which may be responsible for the apparent tumorigenicity of PRKAR1A mutations in endocrine tissues.

Correlation of clinical features and telomerase activity in human gliomas.

Telomerase is a ribonucleoprotein containing an RNA template that synthesizes telomeric DNA. The expression of telomerase activity is concomitant with the attainment of immortality in tumor tissues and cells. In this report, we analyzed telomerase activity in 39 human gliomas with different histological, and in 10 meningiomas, 3 neurinomas, and 2 normal brain tissues by using a polymerase chain reaction (PCR)-based telomeric repeat amplification protocol (TRAP) assay. Telomerase activity was detectable in almost all of the gliomas (36 of 39), but not in any of the meningiomas, neurinomas, or normal brain tissues. In addition, we also analyzed the level of telomerase activity in the 36 gliomas with positive telomerase activity. The relative telomerase activity of the glioma showed a clear association with the pathological grade of glioma; i.e., most of the tumors with high telomerase activity were pathologically of high grade. And also the relative level of telomerase activity could be correlated with the survival time of the patients. These results suggest that the level of telomerase activity in brain tumors is a diagnostic marker indicating the prognosis of the patient as well as the malignant potential of the tumor.

The expression of Gal beta 1,4GlcNAc alpha 2,6 sialyltransferase and alpha 2,6-linked sialoglycoconjugates in human brain tumors.

CMP-NeuAc: Gal beta 1,4GlcNAc alpha 2,6 sialyltransferase (alpha 2,6-ST) [EC 2.4.99.1] is developmentally regulated, shows a high degree of tissue specificity, and appears to play a role in oncogenic transformation and metastasis. In the present study, we have performed the first detailed analysis of the expression of alpha 2,6-ST and alpha 2,6-linked sialoglycoconjugates in human brain tumors. We used a polyclonal, monospecific anti-rat alpha 2,6-ST antibody and the alpha 2,6-linked sialic acid-specific lectin, Sambucus nigra agglutinin (SNA) for histochemical studies, and a human alpha 2,6-ST-specific cDNA probe for Northern analysis. Meningiomas, chordomas and craniopharyngiomas frequently expressed alpha 2,6-ST and alpha 2,6-linked sialoglycoconjugates. Among the different meningioma subtypes, meningothelial meningiomas stained more strongly with both anti-alpha 2,6-ST antibody and SNA than the fibroblastic and anaplastic meningiomas. On the other hand, all tumors of glial origin and medulloblastomas were virtually devoid of either alpha 2,6-ST or alpha 2,6-linked sialoglycoconjugate expression. Moreover, very weak to negligible expression of both alpha 2,6-ST and alpha 2,6-linked sialoglycoconjugates was observed in brain metastases. In conclusion, alpha 2,6-ST and alpha 2,6-linked sialoglycoconjugate expression is associated with non-neuroectodermal epithelial-like tumors.

Tissue-specific expression and methylation of the human CYP2E1 gene.

The level and number of CYP2E1 gene transcripts were investigated by northern blot analysis in various human adult tissues including liver, lung, placenta, skin and neurinoma. Three transcripts of 1.8, 2.6 and 4 Kb were expressed in a tissue-specific manner. The origin of the various transcripts was studied and showed that both 4 and 2.6 Kb mRNAs contained sequences from the 3' non-translated region of the gene and that the 4 Kb also contained region localized in the 5' non-translated region. Furthermore, it clearly appeared that a catalytically active CYP2E1 enzyme (as proved by NDMA demethylase activity) was only detected in tissues expressing the 1.8 Kb. The human CYP2E1 was also identified through immunohistochemical techniques. Finally, we observed a relation between the hypomethylation of the human CYP2E1 gene and the hypoexpression of the corresponding protein.

Production of matrix metalloproteinases and tissue inhibitor of metalloproteinases-1 by human brain tumors.

The role of matrix metalloproteinases (MMP's) and their inhibitor, tissue inhibitor of metalloproteinases-1 (TIMP-1), in human brain tumor invasion was investigated. Gelatinolytic activity was assayed via gelatin zymography, and four MMP's (MMP-1, MMP-2, MMP-3, and MMP-9) and TIMP-1 were immunolocalized in human brain tumors and in normal brain tissues using monoclonal antibodies. The tissue was surgically removed from 44 patients: glioblastoma (five cases), anaplastic astrocytoma (six cases), astrocytoma (four cases), metastatic tumor (six cases), neurinoma (10 cases), meningioma (10 cases), and normal brain tissue (three cases). Glioblastomas, anaplastic astrocytomas, and metastatic tumors showed high gelatinolytic activity and positive immunostaining for MMP's; TIMP-1 was also expressed in these tumors, but some tumor cells were negative for the antibody. Astrocytomas had low gelatinolytic activity and the tumor cells showed no immunoreactivity for MMP's and TIMP-1. Although neurinomas and meningiomas had only moderate proteinase activity and exhibited positive immunoreactivity for MMP-9, intense expression of TIMP-1 was simultaneously observed in these tumor cells. These findings suggest that MMP's play an important role in human brain tumor invasion, probably due to an imbalance between the production of MMP's and TIMP-1 by the tumor cells.

Changes in lactate dehydrogenase isoenzyme pattern in patients with tumors of the central nervous system.

Lactate dehydrogenase (LDH) isoenzymes were studied in biopsy samples obtained from 100 benign and malignant brain tumors. Diagnosis was confirmed by histopathology. It is observed that all tumors investigated had elevated LDH activity and showed a LDH isoenzyme pattern which is different from that of normal brain. A pronounced cathodal shift was seen in malignant tumors like medulloblastoma, grade 3-4 astrocytomas and neuroblastomas, whereas anodal pattern was seen in benign tumors like grade 1-2 astrocytomas and oligodendrogliomas. Some tumors like meningiomas showed a midzone pattern like increased LDH3. It was possible to differentiate certain tumors on the basis of LDH isoenzyme pattern like medulloblastomas into differentiated and undifferentiated; craniopharyngiomas into recurring and non-recurring ones. LDH1/LDH5 ratio was low (< 1.0) in malignant tumors and high (5.0-14.0) in benign tumors and it was useful in differentiating tumors according to the degree of malignancy and biological behavior. It is observed that both LDH isoenzyme pattern and LDH1/LDH5 ratio could be used as an adjuvant to histopathological grading of brain tumors.