Vincenza Barresi

Activation of metabotropic glutamate receptors prevents neuronal apoptosis in culture

Abstract: Cultured granule cells grown in serum‐containing medium with a “low K+” concentration (10 mM) underwent apoptosis after maturation for 5 days in vitro (5 DIV), a time that coincides with the developmental decline in the activity of metabotropic glutamate receptors (mGluRs) coupled to polyphosphoinositide hydrolysis. The mGluR agonist (1S,3R)‐1‐aminocyclopentane‐1,3‐dicarboxylic acid (1S,3R‐ACPD) prevented the development of low K+‐induced apoptosis and the presence of the drug was critical at 6 and 7 DIV, i.e., after the drop of mGluR activity. The neuroprotective action of 1S,3R‐ACPD was prevented by the mGluR antagonist (RS)‐α‐methyl‐4‐carboxyphenylglycine (MCPG) and was mimicked by N‐methyl‐d‐aspartate or carbamylcholine but not by agonists of the mGluR subtypes negatively linked to adenylyl cyclase. In cultures treated either with Li+—which reduced polyphosphoinositide response to concentrations of glutamate (5 µM) that approximate those physiologically present in the incubation medium—or MCPG, the development of low K+‐induced apoptosis already occurred at 4 DIV. Thus, the activation of mGluRs coupled to polyphosphoinositide hydrolysis by endogenous glutamate could contribute to protect cultured granule cells against apoptosis during early stages of maturation.

Ciliary Neurotrophic Factor Activates JAK/Stat Signal Transduction Cascade and Induces Transcriptional Expression of Glial Fibrillary Acidic Protein in Glial Cells

Abstract: In recent reports, ciliary neurotrophic factor (CNTF) has been implicated as an injury factor involved in regulating astrogliosis in the CNS. In this study, we used a rat oligodendroglial progenitor cell line that is highly responsive to CNTF to examine CNTF‐induced alterations that may play a role in activation of the glial fibrillary acidic protein (GFAP) gene. We determined that CNTF induces the transient translocation of Stat1α/p91 to the nucleus. This nuclear translocation was followed by GFAP promoter activation and an up‐regulation of GFAP mRNA and protein. Levels of CNTF‐α receptor mRNA, however, were unaffected by addition of the ligand. Transfection studies using an upstream 5′‐flanking, 1.9‐kb rat GFAP promoter linked to a luciferase reporter gene revealed CNTF‐induced transcriptional activation within 1 h of ligand exposure. Moreover, serial‐deleted constructs identified a distal (−1,857 to −1,546 bp) and a proximal (−384 to −106 bp) region as being important for CNTF‐induced GFAP promoter activation. These two regions showed a strong degree of overlap for CNTF‐ and serum‐induced activation of the GFAP gene. Analysis of the two regions revealed several cis‐elements that are thought to be involved in GFAP regulation and/or the regulation of other genes by members of the interleukin‐6 family of cytokines. Moreover, we are the first to report the presence of several putative CNTF‐responsive elements within our identified distal and proximal regions in the GFAP gene promoter.

Transplantation of prodrug-converting neural progenitor cells for brain tumor therapy

Since neural progenitor cells can engraft stably into brain tumors and differentiate along the neuronal and glial line, we tested the hypothesis that transplanted cytosine deaminase (CD)-expressing ST14A cells (an immortalized neural progenitor cell line) can convert locally 5-fluorocytosine (5-FC) into 5-fluorouracil (5-FU) and produce a regression of glioma tumors. ST14A, retrovirally transduced with the E. coli CD gene, showed a strong bystander effect on glioma cells as assessed by in vitro assay. Intracerebral injection of C6 glioma cells generated a rapidly growing tumoral mass. DiI prelabeled ST14A, coinjected into the rat brain with C6 glioma cells, survived in the tumoral mass up to 10 days and their number was not affected by in vivo 5-FC treatment. In contrast, a significant decrease of the glioma tumoral mass (−50%) was observed in 5-FC-treated rats. 5-FC had no effect on the tumor in the absence of CD-expressing ST14A cells. Our results support the feasibility of systems based on intratumoral transplantation of prodrug-converting cells for brain tumor therapy.

A bioinformatic approach to the identification of candidate genes for the development of new cancer diagnostics.

Abstract A multivariate analysis of the National Cancer Institute gene expression database is reported here. The soft independent modelling of a class analogy approach achieved cell line classification according to histological origin. With the PCA method, based on the expression of 9605 genes and ESTs, classification of colon, leukaemia, renal, melanoma and CNS cells could be performed, but not of lung, breast and ovarian cells. Another multivariate procedure, called partial least squares discriminant analysis (PLS-DA), provides bioinformatic clues for the selection of a limited number of gene transcripts most effective in discriminating different tumoral histotypes. Among them it is possible to identify candidates in the development of new diagnostic tests for cancer detection and unknown genes deserving high priority in further studies. In particular, melan-A, acid phosphatase 5, dopachrome tautomerase, S100-β and acid ceramidase were found to be among the most important genes for melanoma. The potential of the present bioinformatic approach is exemplified by its ability to identify differentiation and diagnostic markers already in use in clinical settings, such as protein S-100, a prognostic parameter in patients with metastatic melanoma and a screening marker for melanoma metastasis.

Design and synthesis of trans 2-(furan-2-yl)vinyl heteroaromatic iodides with antitumour activity

A new molecular modelling approach based on physico-chemical and pharmacokinetic properties, called Volsurf plus, was used to design new heterocyclic compounds with antiproliferative activity. The synthesis and in vitro antitumour tests on a breast carcinoma cell line (MCF7) confirmed VOLSURF predicted activity values.

Structural features of the rat GFAP gene and identification of a novel alternative transcript.

The glial fibrillary acidic protein (GFAP) is expressed in a cell‐specific manner and represents the major subunit of intermediate filaments of astroglial cells. The knowledge of the gene structure is an important step for further understanding the mechanisms of cell‐specific expression. In the present study, we report the complete sequence of the rat GFAP gene and provide evidence for the existence, in the rat brain, of a novel alternative transcript. Since three different transcripts, indicated as GFAPα, β, and γ, have been previously reported (Feinstein et al. [1992] J. Neurosci. Res. 32:1–14; Zelenika et al. [1995] Mol. Brain Res. 30:251–258), we called this novel mRNA isoform GFAPδ. It is generated by the alternative splicing of a novel exon located in the classic seventh intron. This alternative exon (called VII+) contains a 101‐bp coding sequence in frame with exon VII and interrupted by a stop codon TAA at position +5451. Therefore, the novel GFAPδ transcript encodes for an hypothetical GFAP where the forty‐two carboxy‐terminal amino acids encoded by exon VIII and IX are replaced by thirty‐three amino acids encoded by exon VII+. Northern blot analysis with a specific probe for exon VII+ revealed a 4.2‐kb mRNA, expressed in several brain areas, but absent in extracerebral tissues (lung, heart, kidney, liver, spleen). The previously discovered GFAP isoforms (α, β, and γ) produce hypothetical translation products differing in the aminoterminal Head domain. The present data suggest, for the first time, the possible existence of GFAP isoforms differing in the carboxy‐terminal Tail domain. J. Neurosci. Res. 56:219–228, 1999.

Anticonvulsant effects of carbenoxolone in genetically epilepsy prone rats (GEPRs).

Carbenoxolone (CBX), the succinyl ester of glycyrrhetinic acid, is an inhibitor of gap junctional intercellular communication. Systemic administration of CBX was able to decrease the seizure severity score and to increase the latency time of seizure onset in genetically epilepsy prone rats (GEPRs). In particular, intravenous or intraperitoneal administration of carbenoxolone (5-30 mg/kg) produced a dose-dependent and significant reduction in the clonic and tonic phases of the audiogenic seizures in GEPRs. The anticonvulsant doses were not associated with an impairment of motor coordination. The bilateral microinjection of CBX (0.001-0.50 microg/0.5 microl) into the inferior colliculi, the substantia nigra (pars reticulata or compacta) and the inferior olivary complex was able to reduce the seizure severity score in a dose-dependent manner. The anticonvulsant effects were longer lasting after focal microinjection than after systemic administration. No anticonvulsant effects were observed following focal bilateral microinjections of glycyrrhizin into the same brain areas where CBX was shown to be effective.

Antiabsence effects of carbenoxolone in two genetic animal models of absence epilepsy (WAG/Rij rats and lh/lh mice)

Abstract Carbenoxolone (CBX), the succinyl ester of glycyrrhetinic acid, is an inhibitor of gap junctional intercellular communication. We have tested its possible effects upon two genetic animal models of epilepsy (WAG/Rij rats and lethargic (lh/lh) mice). Systemic administration of CBX was unable to significantly affect the occurrence of absence seizures in WAG/Rij rats. In particular, intravenous (5–40 mg/kg) or intraperitoneal (i.p.; 10–80 mg/kg) administration of CBX was unable to significantly modify the number and duration of spike-wave discharges (SWDs) in WAG/Rij rats, whereas the bilateral microinjection (0.05, 0.1, 0.5 and 1 μg/0.5 μl) of CBX into nucleus reticularis thalami (NRT) and nucleus ventralis posterolateralis (VPL) thalami produced a decrease in the duration and the number of SWDs. Bilateral microinjection of CBX into nucleus ventroposteromedial (VPM) thalami did not produce any significant decrease in the number and duration of SWDs. On the contrary, i.p. (5–40 mg/kg) or intracerebroventricular (0.5, 1, 2 and 4 μg/2 μl) administration of CBX in lh/lh mice induced a marked decrease in the number and duration of SWDs in a dose-dependent manner. At the doses used no movement disorders, or other behavioural changes, were recorded in both WAG/Rij rats and lh/lh mice. No effects were observed in both animal models following systemic or focal administration of glycyrrhizin into the same brain areas where CBX was shown to be effective.

Clonal selection of 11q CN-LOH and CBL gene mutation in a serially studied patient during MDS progression to AML

By conventional metaphase and SNP array cytogenetics we serially studied a patient affected by high-risk myelodysplastic syndrome (MDS), documenting the conversion from partial trisomy 8q to trisomy 8 and partial tetrasomy 8q during progression to acute myeloid leukemia (AML). Moreover, the serial application of high resolution genomic array analysis at different disease stages allowed the description of cryptic abnormalities and the demonstration of their enrichment in the AML phase. In particular the detection and quantification of a copy-neutral loss of heterozygosity region located in chromosome 11q guided the search for point mutations in the CBL gene, thus allowing the escription of the novel missense mutation K382E and the demonstration of its selection during progression to secondary AML.

Broad copy neutral‐loss of heterozygosity regions and rare recurring copy number abnormalities in normal karyotype‐acute myeloid leukemia genomes

We analyzed, by the latest high‐resolution SNP arrays, 19 Normal Karyotype (NK)‐AML patients at diagnosis (Dx) and remission (R) phases, to determine the number of tumor‐associated copy number abnormalities (CNAs) and copy neutral‐loss of heterozygosity (CN‐LOH) regions per patient and to identify possible recurring genomic abnormalities. The number of tumor‐associated CNAs was determined after comparison of matched Dx/R samples using stringent conditions able to reduce the number of false positive CNAs. With the exception of a single outlier case, a low number of CNAs per patient was detected (median value of 1 somatic loss or gain per patient). However, a high prevalence of CNAs (60–70% of the patients showed at least one tumor‐associated gain or loss) and few recurring CNAs were observed, thus providing new hints towards identification of cooperating mutations. An extensive search of all tumor‐associated CN‐LOH regions >1 Mb revealed only three broad regions (terminal 12Mb of 22q, terminal 27Mb of 1p and the whole chromosome 21) in three patients out of 19 (16%). CN‐LOH of the whole chromosome 21 was responsible for homozygosity of a missense mutation (R80C) of RUNX1/AML1. Our study suggests that a relative submicroscopic copy number stability NK‐AML genomes is associated with low recurrence of specific CNAs and CN‐LOH in NK‐AML patient population. Sequencing of candidate genes in the identified CNAs and CN‐LOH regions should be considered a priority in the search of novel driver mutations of AML.