Biological Implications and Functional Significance of Transglutaminase Type 2 in Nervous System Tumors.

Transglutaminase type 2 (TG2) is the most ubiquitously expressed member of the transglutaminase family. TG2 catalyzes the transamidation reaction leading to several protein post-translational modifications and it is also implicated in signal transduction thanks to its GTP binding/hydrolyzing activity. In the nervous system, TG2 regulates multiple physiological processes, such as development, neuronal cell death and differentiation, and synaptic plasticity. Given its different enzymatic activities, aberrant expression or activity of TG2 can contribute to tumorigenesis, including in peripheral and central nervous system tumors. Indeed, TG2 dysregulation has been reported in meningiomas, medulloblastomas, neuroblastomas, glioblastomas, and other adult-type diffuse gliomas. The aim of this review is to provide an overview of the biological and functional relevance of TG2 in the pathogenesis of nervous system tumors, highlighting its involvement in survival, tumor inflammation, differentiation, and in the resistance to standard therapies.

Role of Glutathione-S-transferases in neurological problems.

INTRODUCTION: Role of Glutathione-S-transferases (GSTs) has been well explored in the cellular detoxification process, regulation of redox homeostasis and S-glutothionylation of target proteins like JNK, ASK1 etc. However, altered levels or functions of this enzyme or their subtypes have emerged in the development of several pathologies diseases such as Alzheimer's disease, Parkinson's disease, cancer and related conditions. Oxidative stress is one of the possible pathological events that contributes significantly to activation of degenerating cascades inside neuronal cells. The central nervous system is highly sensitive to oxidative stress because of low levels or capacities of antioxidant enzymes. The brain is highly metabolic in nature making it susceptible to oxidative stress. Areas covered: The present review provides a comprehensive overview of the multiple connections of GSTs within diverse neurological diseases including cancer. Furthermore, the authors have made significant efforts to discuss the regulation of different GST isoforms that have been associated with various pathological processes such as glioblastoma, Alzheimer's disease, Parkinson's disease, stroke and epilepsy. Expert opinion: Though GSTs have been one of the key areas of scientific research over the last few decades, much remains to be elucidated about their physiological functions as well as pathological involvement of GSTs and their polymorphic variants.

The aspartyl (asparaginyl) beta-hydroxylase in carcinomas.

Aspartyl-(asparaginyl)-ß-hydroxylase (AAH) is a member of the α-ketoglutarate-dependent dioxygenase family that catalyzes the hydroxylation of aspartyl and asparaginyl residues epidermal growth factor (EGF)-like domains of protein. In human tumorous cell lines from main systems of body, including tumor cells of kidney, throat, breast, liver, bladder, cervical and ovary, the AAH can be detected at both the transcriptional level and the translational level, and moreover, the AAH expression is usually increased, which is associated with the development and progression of carcinomas. Thus, AAH may play an important role in different carcinomas and may be a potential hub in carcinogenesis. In this review, we will discuss the role of AAH in carcinomas, focusing on liver cancers and other digestive tumors, lung cancers, and tumors of nervous system.

The dilemma: does tissue expression of cathepsin D reflect tumor malignancy? The question: does the assay truly mirror cathepsin D mis-function in the tumor?

Three molecular forms of the proteolytic enzyme Cathepsin D (CD) are found in the cell: the precursor (proCD), the intermediate single-chain and the mature double-chain. ProCD, which is found in the Golgi Complex, is enzymatically inactive, while the intermediate and the mature forms, respectively found in endosomes and lysosomes, are enzymatically active. The latter are involved in autophagy and apoptosis pathways thus playing a crucial role in the control of cell and tissue homeostasis. ProCD can be secreted in the extracellular space and, by interacting with membrane receptors, can promote cell proliferation. At slightly acid pH, secreted proCD undergoes partial maturation and becomes active. In the extracellular space, CD can degrade the protein components of the matrix and free growth factors therein embedded, thus favoring tumor growth, invasion and angiogenesis. Based on the multiple tasks performed by CD inside and outside the cell, it is not irrational to hypothesize its involvement in cancer development and progression and a strict link between its tissue expression and the clinico-pathological features of the tumor. Thus, not surprisingly, as many as 519 articles are found in the database of pubmed with the keywords 'cathepsin D, cancer and marker'. Disappointingly, however, in spite of, or because of, this large number of studies, the scientific community has not reached a general agreement on the prognostic value of CD in cancer progression. Here, we will briefly review the relevant literature and offer a possible explanation for the conflicting data.

Neuronal apoptosis by prolyl hydroxylation: implication in nervous system tumours and the Warburg conundrum.

Oxygen sensing is mediated partly via prolyl hydroxylation. The EglN prolyl hydroxylases are well characterized in regulating the hypoxia inducible factor alpha (HIF-alpha) hypoxic response, but also are implicated in HIF-independent processes. EglN3 executes apoptosis in neural precursors during development and failure of EglN3 developmental apoptosis can lead to certain forms of sympathetic nervous system tumours. Mutations in metabolic/mitochondrial enzymes (SDH, FH, IDH) impair EglN activity and predisposes to certain cancers. This is because the EglNs not only require molecular oxygen to execute hydroxylation, but also equally require the electron donor alpha-ketoglutarate, a metabolite from the Krebs cycle. Therefore EglN enzymes are considered oxygen, and also, metabolic sensors. alpha-Ketoglutarate is crucial for EglN hydroxylation activity, whereas the metabolites succinate and fumarate are inhibitors of the EglN enzymes. Since EglN activity is dependent upon metabolites that take part in the Krebs cycle, these enzymes are directly tied into the cellular metabolic network. Cancer cells tend to convert most glucose to lactate regardless of whether oxygen is present (aerobic glycolysis), an observation that was first made by Otto Warburg in 1924. Despite the striking difference in ATP production, cancer cells might favour aerobic glycolysis to escape from EglN hydroxylation, resulting in the accumulation of oncogenic HIFalpha and/or resistance to EglN3-mediated apoptosis.

Pten deletion in adult neural stem/progenitor cells enhances constitutive neurogenesis.

Here we show that conditional deletion of Pten in a subpopulation of adult neural stem cells in the subependymal zone (SEZ) leads to persistently enhanced neural stem cell self-renewal without sign of exhaustion. These Pten null SEZ-born neural stem cells and progenies can follow the endogenous migration, differentiation, and integration pathways and contribute to constitutive neurogenesis in the olfactory bulb. As a result, Pten deleted animals have increased olfactory bulb mass and enhanced olfactory function. Pten null cells in the olfactory bulb can establish normal connections with peripheral olfactory epithelium and help olfactory bulb recovery from acute damage. Following a focal stroke, Pten null progenitors give rise to greater numbers of neuroblasts that migrate to peri-infarct cortex. However, in contrast to the olfactory bulb, no significant long-term survival and integration can be observed, indicating that additional factors are necessary for long-term survival of newly born neurons after stroke. These data suggest that manipulating PTEN-controlled signaling pathways may be a useful step in facilitating endogenous neural stem/progenitor expansion for the treatment of disorders or lesions in regions associated with constitutive neurogenesis.

The role of matrix metalloproteinases in the pathophysiology and progression of human nervous system malignancies: a chance for the development of targeted therapeutic approaches?

BACKGROUND: Matrix metalloproteinases (MMPs) are a group of zinc- dependent endopeptidases involved in the degradation of extracellular matrix components. MMPs have been implicated in a wide variety of physiological processes, such as angiogenesis, wound healing and tissue remodeling. However, recent studies have revealed a significant role for MMPs in tumorigenesis pathophysiology and prediction of patients' clinical outcome. Alterations in the regulation of MMP expression are thought to play an important role in the development and progression of central nervous system (CNS) malignancies. OBJECTIVE/METHODS: This study provides an up-to-date review of the literature on the pathophysiologic involvement of MMPs in the development and progression of human CNS malignancies, as well as the potential use of natural and/or synthetic MMP-inhibitors (MMPIs) as a targeted therapeutic approach to this group of neoplasms. RESULTS/CONCLUSIONS: The currently available data provide clear evidence for the involvement of MMPs in the pathophysiology of astrocytomas, glioblastomas, meningiomas, medulloblastomas/primitive neuroectodermal tumors and pituitary tumors. The use of MMPIs in the treatment of CNS malignancies has, until now, reached controversial (but mainly disappointing) results that can nevertheless provide the basis for further investigation. The co-administration of other agents, the use of surgery and/or radiation, and elimination of the MMPIs-induced adverse effects, as well as the use of antisense technology, might be the tools by which the natural and synthetic MMPIs could find their place in everyday clinical practice for the management of CNS malignancies.

Mutations associated with succinate dehydrogenase D-related malignant paragangliomas.

OBJECTIVE: Hereditary paraganglioma (PGL) syndromes result from germline mutations in genes encoding subunits B, C and D of the mitochondrial enzyme succinate dehydrogenase (SDHB, SDHC and SDHD). SDHB-related PGLs are known in particular for their high malignant potential. Recently, however, malignant PGLs were also reported among a small minority of Dutch carriers of the SDHD founder mutation D92Y. The aim of the study was to investigate which SDHD mutations are associated with malignant PGL. DESIGN: Case histories; collaborative study between referral centres in France, the USA, and the Netherlands. PATIENTS: Six unrelated patients with metastatic PGLs of either sympathetic or parasympathetic origin. MEASUREMENTS: Assessment of SDHD mutations underlying malignant PGL. RESULTS: Germline SDHD mutations underlying metastatic PGL were G148D, Y114X, L85X, W43X, D92Y, and IVS2+5G-->A. CONCLUSION: Our findings indicate that malignant SDHD-related PGL is associated with several mutations besides D92Y.

Inhibition of neuropathy target esterase expressing by antisense RNA does not affect neural differentiation in human neuroblastoma (SK-N-SH) cell line.

Neuropathy target esterase (NTE) is phosphorylated and aged by oraganophosphorus compounds (OP) that induce delayed neuropathy in human and some animals. NTE has been proposed to play a role in neurite outgrowth and process elongation during neural differentiation. However, to date, there is no direct evidence of the relevance of NTE in neural differentiation under physiological conditions. In this study we have investigated a possible role for NTE in the all-trans retinoic acid (ATRA)-induced differentiation of neuroblastoma cells by antisense RNA. A NTE antisense RNA construct was generated and then transfected into human neuroblastoma SK-N-SH cells. A positive cell clone that can stably express NTE antisense RNA was obtained by G418 selection and then identified by western blotting. NTE activity was depressed in the transfected cells with only about 50% activity of the enzyme in the control cells. ATRA-induced differentiation of the neuroblastoma cells with lowered NTE activity revealed that inhibition of NTE expression does not affect neural differentiation in SK-N-SH cells. The result suggested that organophosphates may inhibit neural differentiation by initially acting on other targets other than NTE.

Effects of toxic doses of glutamate on Cu-Zn and Mn/superoxide dismutases activities in human glioma cell lines.

Recent research has implicated glutamate in the growth and invasive migration of gliomas. Superoxide dismutase (SOD) is involved in excitotoxicity and may influence cellular proliferative status. Thus, this study investigated the effects of gliotoxic doses of glutamate on Cu-Zn and Mn/SODs activities in human glioma cell lines. To this end, glioma cell lines (U87MG, U138MG and U251MG) were treated with glutamate (5-200 mM) during 48 h. Then, cell viability assays, clonogenic assay and Cu-Zn and Mn/SODs activities of the cell lines were performed. IC50values of glutamate were similar for both U87MG and U138MG cells (56 and 69 mM, respectively), while a higher value was detected for U251MG cells (110 mM). In the long term, 14 days after glutamate was removed from the culture media, cells showed partial or complete recovery. The effects of glutamate treatment on Cu-Zn and Mn/SODs activities varied among the distinct cell lines. While acute treatment with toxic doses of glutamate caused a significant decrease in the Cu-Zn/SOD activity of U138MG and U251MG cells, it did not affect Cu-Zn/SOD activity in U87MG cells. Only in U251MG cells, acute glutamate treatment decreased significantly Mn/SOD activity. In the long term (14 days after the 48 h treatment), glutamate did not affect either Cu-Zn or Mn/SODs activities. Thus, it may be suggested that SOD vulnerability to glutamate-mediated effects may be related to distinct tumoral cell behavior.

When cell biology and neurobiology meet.

A report on the British Society for Cell Biology (BSCB) meeting on 'Cell Biology and Neurobiology: A Meeting for Martin Raff', London, UK, 3-5 July 2002.

Methylation-related chromatin structure is associated with exclusion of transcription factors from and suppressed expression of the O-6-methylguanine DNA methyltransferase gene in human glioma cell lines.

There is considerable interest in identifying factors responsible for expression of the O-6-methylguanine DNA methyltransferase (MGMT) gene, as MGMT is a major determinant in the response of glioma cells to the chemotherapeutic agent 1,3 bis(2-chloroethyl)-1-nitrosourea. Recently we have shown that MGMT expression is correlated in a direct, graded fashion with methylation in the body of the MGMT gene and in an inverse, graded fashion with promoter methylation in human glioma cell lines. To determine if promoter methylation is an important component of MGMT expression, this study addressed the complex interactions between methylation, chromatin structure, and in vivo transcription factor occupancy in the MGMT promoter of glioma cell lines with different levels of MGMT expression. Our results show that the basal promoter in MGMT-expressing glioma cell lines, which is 100% unmethylated, was very accessible to restriction enzymes at all sites tested, suggesting that this region may be nucleosome free. The basal promoter in glioma cells with minimal MGMT expression, however, which is 75% unmethylated, was much less accessible, and the basal promoter in nonexpressing cells, which is 50% unmethylated, was entirely inaccessible to restriction enzymes. Despite the presence of the relevant transcription factors in all cell lines examined, in vivo footprinting showed DNA-protein interactions at six Sp1 binding sites and one novel binding site in MGMT-expressing cell lines but no such interactions in nonexpressors. We conclude that in contrast to findings of previous in vitro studies, Sp1 is an important component of MGMT transcription. These correlations also strongly suggest that methylation and chromatin structure, by determining whether Sp1 and other transcription factors can access the MGMT promoter, set the transcriptional state of the MGMT gene.

Regulation of phospholipase D activity in a human oligodendroglioma cell line (HOG).

Oligodendroglial cells express many specific proteins, such as myelin basic protein (MBP), which are physiologically phosphorylated by protein kinase C (PKC). Diacylglycerols are physiological activators of PKC and can be liberated from phospholipids by the direct receptor-mediated activation of phospholipase C (PL-C) or indirectly via the activation of phospholipase D (PL-D). In a well-characterized human oligodendroglioma (HOG) cell line, PL-C (measured by release of [3H]inositol phosphates) and PL-D (formation of [3H]myristoylated or palmitoylated phosphatidylethanol) were activated by both carbachol (blocked by pirenzepine, suggesting an M1 receptor) and histamine (H1 receptor) but not glutamate, bradykinin, or phenylephrine. PL-C stimulation by carbachol or histamine was completely inhibited by short-term treatment (< 30 min) with phorbol ester (TPA), a PKC activator. In contrast, PL-D activation by either carbachol or histamine was stimulated in additive fashion by TPA, suggesting at least two distinct mechanisms for PL-D activation. Down regulation of PKC by prolonged (24 hr) treatment with TPA reversed the inhibitory effects of TPA on PL-C and the stimulatory effects on PL-D. However, the PKC inhibitors H-7 and galactosylsphingosine did not inhibit the TPA-mediated stimulation of PLD while the less-specific PKC inhibitor, staurosporine, was only partially inhibitory. Preexposure of cells to carbachol, greatly reduced both PL-C and PL-D activation by carbachol, suggesting homologous desensitization. Time-course studies indicated that PL-D activation (10 sec or less) was at least as fast as PL-C activation, and the affinity of carbachol and histamine for the receptor coupled to either phospholipase (EC50 = 5-10 microM) was about the same.(ABSTRACT TRUNCATED AT 250 WORDS)

Serum neurone-specific enolase levels in patients with neuroendocrine and carcinoid tumours.

We have examined concentrations of neurone-specific enolase (NSE) in sera from 18 patients with various neuroendocrine tumours, 26 patients with carcinoid tumours, 21 patients with non-neuroendocrine tumours and 37 control individuals. No statistically significant difference between the concentrations in patients with neuroendocrine tumours and patients with carcinoid tumours was found. However the NSE concentrations in patients with carcinoid and neuroendocrine tumours, when these two groups were combined, were significantly different from the patients with non-neuroendocrine tumours or the control individuals (P < or = 0.01). 38.5% of the patients with carcinoid tumours had raised NSE concentrations in serum; 55.5% of those with non carcinoid neuroendocrine tumours had raised concentrations. There appeared to be no correlation between the NSE concentrations and the extent of metastases.

Proteolytic activity during the growth of C6 astrocytoma in the murine spheroid implantation model.

General protease and collagenase IV activity are involved in the remodelling of the vascular basement membrane that occurs during tumor-induced angiogenesis. This study has assessed the level of these enzymes in tumor, peritumoral or contralateral cerebral cortex tissue during the growth of C6 astrocytoma in the rat spheroid implantation model. General proteolytic activity was increased in tumor tissue beginning on day 8 following spheroid implantation, then increased to a maximum value on day 11 and decreased to control values on day 18. A similar pattern was seen for collagenase IV activity but maximal activity occurred on day 13. The peritumor and tumor patterns of activity were similar. General protease activity was increased in the hemisphere contralateral to the tumor suggesting that the growth of C6 astrocytoma in rat brain was influencing biochemical events distant from the tumor. C6 astrocytoma cells orchestrate a cascade of proteolytic events which may play a crucial role in angiogenesis associated with tumor growth in the model system studied.

Serum neuron-specific enolase in diagnosis and follow-up of gastrointestinal neuroendocrine tumors.

Neuron-specific enolase (NSE), the glycolytic isoenzyme of the enolase gamma-gamma dimer, is a specific marker for the diffuse neuroendocrine system and derivative tumors (NET). Serum levels of NSE were measured in 39 patients with NET of the gastrointestinal tract (including 3 gastric and 13 intestinal carcinoid tumors, 6 gastrinomas, 3 insulinomas, 1 glucagonoma, 2 mixed islet cell tumors, 11 neuroendocrine pancreatic carcinomas), in 15 healthy subjects and in 15 nonendocrine gastric, pancreatic, and intestinal tumors. Thirty-six of the 39 patients had elevated circulating levels of NSE, 2 insulinomas and 1 gastrinoma had values below 12 ng/ml like healthy subjects and nonendocrine tumors. No significant difference of serum NSE was found between 23 'functioning' and 16 'nonfunctioning' NET. Fourteen of the NET were malignant, and NSE circulating values were significantly higher than those of nonmalignant forms. After curative surgery serum NSE decreased significantly. NSE can be considered a reliable marker in the differential diagnosis between endocrine and nonendocrine neoplasms, in the clinical detection of silent endocrine tumors and in the follow-up of NET.

[Arylsulfatase A--physico-chemical properties and the use of enzyme radioimmunoassay in medical diagnosis]. / Arylosulfataza A--wlasciwosci fizyko-chemiczne i zastosowanie w diagnostyce medycznej.

Arylsulfatase A was isolated from urine and human liver. The enzyme was homogeneous with respect to charge and had high specific activity--64 U/mg and 34 U/mg for arylsulfatase A from urine and liver respectively. The enzyme from urine as well as the liver one contained two nonidentical subunits with molecular weights varying about 5 kDa. Treatment of the enzyme from urine, liver and from placenta with endo-beta-N-acetylglucosaminidase F did not remove all carbohydrate from any subunit even in denaturing conditions. Deglycosylation of the enzyme with this one and other glycosidases under various conditions resulted in a decrease in the apparent molecular weights of subunits only by 1-2 kDa. The difference between molecular weights of subunits did not change upon deglycosylation of arylsulfatase A. The results suggest that the presence of two nonidentical subunits is due to presence of different polypeptides rather than various glycosylation of a single polypeptide chain. Arylsulfatase A from urine was inactivated following reaction with diethyl pyrocarbonate at pH 5.5 or at pH 7.0. This confirmed the presence of histidine essential for its catalytic activity. It was also shown that the enzyme was inactivated with ferrate ion, structural analogue of orthophosphate and strong oxidizing agent. The conditions of inhibition of arylsulfatase A carried out with the use of ferrate as well as catalytic and immunochemical properties of the modified enzyme suggest that ferrate reacted with the active site of arylsulfatase A. The results allow to expect that a reactive histidine is present in enzyme's active site and that this aminoacid is modified with ferrate. A simple, sensitive and specific radioimmunoassay was developed for the determination of arylsulfatase A in human serum and urine. The method allows to measure less than nanogram amounts of the enzyme in human body fluids. The test was used to determine arylsulfatase A in serum specimens of 368 patients with histopathologically confirmed cancer of gastrointestinal tract, breast, lung, central nervous system, kidney and woman genital tract. The highest mean concentration of arylsulfatase A in serum and significantly higher than that in the control group of 96 healthy blood donors was found in the case of groups of lung, kidney and central nervous system cancer. The results indicate that the radioimmunoassay determination of serum level of arylsulfatase A might be helpful in diagnosis of lung and central nervous system cancer. Arylsulfatase A serum level cannot be treated as a valuable indicator in the case of cancer of breast and gastrointestinal tract.(ABSTRACT TRUNCATED AT 400 WORDS)

NaF and guanine nucleotides modulate adenylate cyclase activity in NG108-15 cells by interacting with both Gs and Gi.

1. NaF (10 mM) produced a 2-3 fold increase in adenylate cyclase activity in homogenates of NG108-15 cells incubated in the presence of 1 microM GTP. Higher concentrations of NaF suppressed adenylate cyclase activity. 2. In the presence of the adenosine receptor agonist 5'-(N-ethyl)-carboxamidoadenosine (NECA; 100 microM) or the prostacyclin receptor agonist iloprost (10 nM), NaF produced a much smaller increase in adenylate cyclase activity, whereas in the presence of a saturating concentration of iloprost (1 microM), NaF only inhibited adenylate cyclase activity. 3. Similarly, Gpp(NH)p activated basal adenylate cyclase activity, and inhibited 1 microM iloprost-activated enzyme activity. In the presence of 10 microM forskolin, NaF or Gpp(NH)p increased adenylate cyclase activity synergistically. Analysis of concentration-effect curves indicated that NaF (2 mM) or Gpp(NH)p (100 microM) increased the potency with which forskolin activated adenylate cyclase, whilst reducing the maximum activation of adenylate cyclase by iloprost. 4. Opiate receptors mediate inhibition of adenylate cyclase, and the opiate agonist morphine (100 microM) reduced the capacity of NaF or Gpp(NH)p to inhibit iloprost-activated adenylate cyclase. Unexpectedly, pertussis toxin treatment enhanced the ability of NaF or Gpp(NH)p to inhibit iloprost-activated adenylate cyclase. 5. In the absence of GTP, NaF and Gpp(NH)p remained able both to activate basal adenylate cyclase and to be synergistic with forskolin in activating the enzyme. In contrast the ability of NaF and Gpp(NH)p to inhibit iloprost-activated adenylate cyclase was substantially lost in the absence of added GTP. These results suggest that NaF modulates adenylate cyclase activity in NG108-15 cell membranes by interacting with the alpha subunits of both G0 and Gi regulatory proteins. The effects of NaF and Gpp(NH)p are critically dependent on the prior mode and extent of activation or inhibition of this transmembrane signalling pathway. This simple system may be of use in assessing alterations in GSO-O interaction following manipulations such as hormone receptor desensitization.