Type-3 metabotropic glutamate receptors regulate chemoresistance in glioma stem cells, and their levels are inversely related to survival in patients with malignant gliomas.

Drug treatment of malignant gliomas is limited by the intrinsic resistance of glioma stem cells (GSCs) to chemotherapy. GSCs isolated from human glioblastoma multiforme (GBM) expressed metabotropic glutamate receptors (mGlu3 receptors). The DNA-alkylating agent, temozolomide, killed GSCs only if mGlu3 receptors were knocked down or pharmacologically inhibited. In contrast, mGlu3 receptor blockade did not affect the action of paclitaxel, etoposide, cis-platinum, and irinotecan. mGlu3 receptor blockade enabled temozolomide toxicity by inhibiting a phosphatidylinositol-3-kinase/nuclear factor-κB pathway that supports the expression of O(6)-methylguanine-DNA methyltransferase (MGMT), an enzyme that confers resistance against DNA-alkylating agents. In mice implanted with GSCs into the brain, temozolomide combined with mGlu3 receptor blockade substantially reduced tumor growth. Finally, 87 patients with GBM undergoing surgery followed by adjuvant chemotherapy with temozolomide survived for longer time if tumor cells expressed low levels of mGlu3 receptors. In addition, the methylation state of the MGMT gene promoter in tumor extracts influenced survival only in those patients with low expression of mGlu3 receptors in the tumor. These data encourage the use of mGlu3 receptor antagonists as add-on drugs in the treatment of GBM, and suggest that the transcript of mGlu3 receptors should be measured in tumor specimens for a correct prediction of patients' survival in response to temozolomide treatment.

Expression of the Wnt inhibitor Dickkopf-1 is required for the induction of neural markers in mouse embryonic stem cells differentiating in response to retinoic acid.

Cultured mouse D3 embryonic stem (ES) cells differentiating into embryoid bodies (EBs) expressed several Wnt isoforms, nearly all isotypes of the Wnt receptor Frizzled and the Wnt/Dickkopf (Dkk) co-receptor low-density lipoprotein receptor-related protein (LRP) type 5. A 4-day treatment with retinoic acid (RA), which promoted neural differentiation of EBs, substantially increased the expression of the Wnt antagonist Dkk-1, and induced the synthesis of the Wnt/Dkk-1 co-receptor LRP6. Recombinant Dkk-1 applied to EBs behaved like RA in inducing the expression of the neural markers nestin and distal-less homeobox gene (Dlx-2). Recombinant Dkk-1 was able to inhibit the Wnt pathway, as shown by a reduction in nuclear beta-catenin levels. Remarkably, the antisense- or small interfering RNA-induced knockdown of Dkk-1 largely reduced the expression of Dlx-2, and the neuronal marker beta-III tubulin in EBs exposed to RA. These data suggest that induction of Dkk-1 and the ensuing inhibition of the canonical Wnt pathway is required for neural differentiation of ES cells.

Interactions between ephrin-B and metabotropic glutamate 1 receptors in brain tissue and cultured neurons.

We examined the interaction between ephrins and metabotropic glutamate (mGlu) receptors in the developing brain and cultured neurons. EphrinB2 coimmunoprecipitated with mGlu1a receptors, in all of the brain regions examined, and with mGlu5 receptors in the corpus striatum. In striatal slices, activation of ephrinB2 by a clustered form of its target receptor, EphB1, amplified the mGlu receptor-mediated stimulation of polyphosphoinositide (PI) hydrolysis. This effect was abolished in slices treated with mGlu1 or NMDA receptor antagonists but was not affected by pharmacological blockade of mGlu5 receptors. An interaction among ephrinB2, mGlu1 receptor, and NMDA was supported by the following observations: (1) the NR1 subunit of NMDA receptors coimmunoprecipitated with mGlu1a receptors and ephrinB2 in striatal lysates; (2) clustered EphB1 amplified excitatory amino acid-stimulated PI hydrolysis in cultured granule cells grown under conditions that favored the expression of mGlu1a receptors; and (3) clustered EphB1 amplified the enhancing effect of mGlu receptor agonists on NMDA toxicity in cortical cultures, and its action was sensitive to mGlu1 receptor antagonists. Finally, fluorescence resonance energy transfer and coclustering analysis in human embryonic kidney 293 cells excluded a physical interaction between ephrinB2 and mGlu1a (or mGlu5 receptors). A functional interaction between ephrinB and mGlu1 receptors, which likely involves adaptor or scaffolding proteins, might have an important role in the regulation of developmental plasticity.

Activation of Fas receptor is required for the increased formation of the disialoganglioside GD3 in cultured cerebellar granule cells committed to apoptotic death.

Apoptosis was induced in cultured cerebellar granule cells by lowering extracellular K+ concentrations (usually from 25 to 10 mM). The apoptotic phenotype was preceded by an early and transient increase in the intracellular levels of the disialoganglioside, GD3, which behaves as a putative pro-apoptotic factor. We examined whether activation of Fas receptor mediates the increase in GD3 formation in granule cells committed to die. Degenerating granule cells showed increased expression of both Fas receptor and its ligand (Fas-L), at times that coincided with the increase in GD3 levels and the induction of GD3 synthase mRNA. Addition of neutralizing anti-Fas-L antibodies reduced the extent of 'low-K+'-induced apoptosis and abolished the increase in GD3 levels and GD3 synthase mRNA. Similar reductions were observed in cultures prepared from gld or lpr mice, which harbor loss-of-function mutations of Fas-L and Fas receptor, respectively. In addition, exogenous application of soluble Fas-L further enhanced both the increase in GD3 formation and cell death in cultured granule cells switched from 25 into 10 mM K+. We conclude that activation of Fas receptor is entirely responsible for the increase in GD3 levels and contributes to the development of apoptosis by trophic deprivation in cultured cerebellar granule cells.

Growth inhibition and differentiation induction in murine erythroleukemia cells by 4-hydroxynonenal.

4-Hydroxynonenal (HNE) is one of the major end products of lipid peroxidation. Here we show that the exposure of murine erythroleukemia (MEL) cells to 1 microM HNE, for 10.5 h over 2 days, induces a differentiation comparable with that observed in cells exposed to DMSO for the whole experiment (7 days). The exposure of MEL cells for the same length of time demonstrates a higher degree of differentiation in HNE-treated than in DMSO-treated MEL cells. The protooncogene c-myc is down-modulated early, in HNE-induced MEL cells as well as in DMSO-treated cells. However, ornithine decarboxylase gene expression first increases and then decreases, during the lowering of the proliferation rate. These findings indicate that HNE, at a concentration physiologically found in many normal tissues and in the plasma, induces MEL cell differentiation by modulation of specific gene expression.

Differential regulation of Epstein-Barr virus (EBV) latent gene expression in Burkitt lymphoma cells infected with a recombinant EBV strain.

Epstein-Barr virus (EBV)-negative Burkitt lymphomas (BLs) can be infected in vitro with prototype EBV strains to study how the virus may affect the phenotype of tumor cells. Studies thus far have concentrated on the use of transforming B95-8 and nontransforming P3HR1 strains. Immunological and phenotypic differences between the sublines infected with these two strains were reported. The majority of these differences, if not all, can be attributed to the lack of EBNA-2 coding sequences in the P3HR1 strain. The recent development of a selectable Akata strain has opened up new possibilities for infecting epithelial and T cells as well. We infected five EBV-negative BL lines with the recombinant Akata virus. Our results indicate that the infected cell lines BL28, Ramos, and DG75 express EBNA-1, EBNA-2, and LMP1, the viral proteins associated with type III latency, and use both YUK and QUK splices. In contrast, two EBV-negative variants of Akata and Mutu when reinfected displayed restricted type I latency and expressed only EBNA-1. All clones of infected Mutu cells used the QUK splice exclusively. The usage of Qp was observed in a majority of Akata clones. Some Akata clones, however, were found to have double promoter usage (Qp and C/Wp) but at 4 months after infection did not express EBNA-2. The results demonstrate differential regulation of EBV latency in BLs with the same recombinant viral strain and suggest that the choice of latency type may be cell dependent. The restricted latency observed for infected Akata and Mutu cells indicates that a BL may opt for type I latency in the absence of immune pressure as well.

Wilms' tumor gene expression by normal and malignant human B lymphocytes.

Very little is known about Wilms' tumor gene (WT1) expression in B cells and its importance for growth regulation and differentiation. We have investigated WT1 expression in fresh B lymphocytes and in a panel of B-cell lines of normal and malignant origin, including both Epstein-Barr virus (EBV) genome negative and EBV carrying cell lines. WT1 is constitutively activated in all lymphoblastoid cell lines (LCL) derived from EBV immortalization of lymphocytes from normal donors in vitro. These cell lines are distinguished for the presence of activated B-cell markers and an unrestricted expression of viral latent genes. In contrast, WT1 expression is abrogated in normal B lymphocytes and in all Burkitt tumor derived cell lines, irrespective of the EBV genome carrying status and their phenotype pattern. A single step RT-PCR for simultaneous detection of the four spliced transcript isoforms has been applied to confirm their expression. Analysis of variant relative proportions suggested the maintenance of a balanced expression of the isoforms in LCL, as reported in non tumorous tissues. These data, together with the evidence that the replication in vitro of lymphoblastoid cells is not affected by WT1 activation following viral immortalization, support the hypothesis that gene inactivation, in addition to disrupted alternate splicing, may play a role in growth control derangements.

Expression of neurotrophin receptors in normal and malignant B lymphocytes.

In order to define a cellular model suitable for studying, in vitro, the molecular properties and functions of neurotrophin receptors in human lymphocytes, TrkA, TrkB, TrkC and p75(NTR) expression was investigated in a panel of EBV immortalized lymphoblastoid (LCL) and Burkitt lymphoma-derived cell lines (BLs) compared to primary B lymphocytes by RT-PCR and flow cytometric analysis. Our data show that trkA and trkB are transcribed in most B cell lines of normal and malignant origin. For several of them, we also gained first evidence of trkC expression in B cells. All cell lines and primary B cells lack p75(NTR) expression. These data suggest that neurotrophin receptors expression in the B cell lines correlates to some extent with the phenotypic maturation stage and endogenous viral activity levels. Our data suggest that TrkA and TrkB, once activated, provide a partial rescue from apoptosis, whereas TrkC stimulates the progression through the cell cycle without affecting cell survival. Finally, the identification of a number of cell lines showing single expression of one of the Trk receptors has disclosed the availability of a cellular tool for further studies on their function, and mechanisms of signal transduction in the B cell moiety in the absence of p75(NTR).

4-Hydroxynonenal-induced MEL cell differentiation involves PKC activity translocation.

4-Hydroxynonenal (HNE) is a highly reactive aldehyde, produced by cellular lipid peroxidation, able to inhibit proliferation and to induce differentiation in MEL cells at concentrations similar to those detected in several normal tissues. Inducer-mediated differentiation of murine erythroleukemia (MEL) cells is a multiple step process characterized by modulation of several genes as well as by a transient increase in the amount of membrane-associated protein kinase C (PKC) activity. Here we demonstrate that a rapid translocation of PKC activity from cytosol to the membranes occurs during the differentiation induced by HNE. When PKC is completely translocated by phorbol-12-myristate-13-acetate (TPA), the degree of HNE-induced MEL cells differentiation is highly decreased. However, if TPA is washed out from the culture medium before the exposition to the aldehyde, HNE gradually resumes its differentiative ability. The incubation of cells with a selective inhibitor of PKC activity, bisindolylmaleimide GF 109203X, partially prevents the HNE-induced differentiation in MEL cells. In conclusion, our results demonstrate that HNE-induced MEL cell differentiation is preceded by a rapid translocation of PKC activity, and that the inhibition of this phenomenon prevents the onset of terminal differentiation.

Expression of Egr-1 correlates with the transformed phenotype and the type of viral latency in EBV genome positive lymphoid cell lines.

In this paper we have investigated the role of Egr-1 in B cell growth regulation by examining the gene expression in a panel of B cell lines, including both EBV genome negative and EBV carrying cell lines. Egr-1 expression correlates with the cellular phenotype and the specific pattern of viral latency established within the individual cell lines. Thus, constitutive activation of Egr-1 gene is invariably associated with unrestricted expression of viral latent genes in all group III EBV genome carrying cell lines. In contrast, Egr-1 expression is abrogated in group I Burkitt tumor cells, irrespective of the EBV genome carrying status. Activated viral gene expression associated with phenotypic conversion of group I cell lines in to group II or III restores the Egr-1 gene expression. Several forms of EGR-1 protein are found within the different groups of cell lines, and the binding activity to DNA consensus sequences was investigated. Finally, time course analysis of Egr-1 expression during the early steps of EBV infection in vitro demonstrated that Egr-1 is upregulated within minutes from the initial interaction with the B lymphocyte.

Identification of a 66 kD heat shock protein (HSP) induced in M-14 human melanoma cells by severe hyperthermic treatment.

Following severe hyperthermic treatment M-14 cells synthesize at high rate a new protein of about 66 kD, in addition to the three well known major HSPs (HSP 28, HSP 70 and HSP 90). This 66 kD protein is constitutively expressed at low levels and its rate of synthesis is not enhanced by mild hyperthermic exposures (40 degrees C for 2-4 h; 42 degrees C for 1-3 h), sufficient to induce the three major HSPs. The 66 kD protein is induced whenever the thermal dose administered to cells attains a threshold, roughly corresponding to a 50% reduction in survival. The 66 kDa protein is not induced by a variety of compounds (disulfiram, arsenate, cadmium) able to elicit a stress response in M-14 cells, as indicated by enhanced synthesis of the three major HSPs. Once induced by a treatment at 45 degrees C for 15 min, the rate of synthesis of the 66 kD protein remains above the control level for 16-20 h during recovery from the stress, while the synthesis of HSP 70 is shut off between 8 and 12 h. Immunoblotting and immunoprecipitation studies showed that the 66 kD protein shares immunological determinant(s) with HSP 70. Pulse-chase experiments demonstrated that the 66 kD protein is not a degradation product or a late post-transcriptional modification of HSP 70. It is proposed that the 66 kD protein is a previously unrecognized heat shock protein (HSP 66), characterized by an unusually high threshold for its induction.