The DNA damage/repair cascade in glioblastoma cell lines after chemotherapeutic agent treatment.

Therapeutic resistance in glioblastoma multiforme (GBM) has been linked to a subpopulation of cells with stem cell-like properties, the glioma stem cells (GSCs), responsible for cancer progression and recurrence. This study investigated the in vitro cytotoxicity of three chemotherapeutics, temozolomide (TMZ), doxorubicin (Dox) and paclitaxel (PTX) on glioma cell lines, by analyzing the molecular mechanisms leading to DNA repair and cell resistance, or to cell death. The drugs were tested on 16 GBM cell lines, grown as neurospheres (NS) or adherent cells (AC), by studying DNA damage occurrence by Comet assay, the expression by immunofluorescence and western blotting of checkpoint/repair molecules and apoptosis. The three drugs were able to provoke a genotoxic injury and to inhibit dose- and time-dependently cell proliferation, more evidently in AC than in NS. The first cell response to DNA damage was the activation of the damage sensors (p-ATM, p-53BP1, γ-H2AX), followed by repair effectors; the expression of checkpoint/repair molecules appeared higher in NS than in AC. The non-homologous repair pathway (NHEJ) seemed more involved than the homologous one (HR). Apoptosis occurred after long treatment times, but only a small percentage of cells in NS underwent death, even at high drug concentration, whereas most cells survived in a quiescent state and resumed proliferation after drug removal. In tumor specimens, checkpoint/repair proteins were constitutively expressed in GBMs, but not in low-grade gliomas.

Positive-charged solid lipid nanoparticles as paclitaxel drug delivery system in glioblastoma treatment.

Paclitaxel loaded solid lipid nanoparticles (SLN) of behenic acid were prepared with the coacervation technique. Generally, spherical shaped SLN with mean diameters in the range 300­600 nm were obtained. The introduction of charged molecules, such as stearylamine and glycol chitosan into the formulation allowed to obtain positive SLN with Zeta potential in the 8-20 mV range and encapsulation efficiency in the 25­90% range.Blood­brain barrier (BBB) permeability, tested in vitro through hCMEC/D3 cells monolayer, showed a significantly increase in the permeation of Coumarin-6, used as model drug, when vehicled in SLN. Positive-charged SLN do not seem to enhance permeation although stearylamine-positive SLN resulted the best permeable formulation after 24 h.Cytotoxicity studies on NO3 glioblastoma cell line demonstrated the maintenance of cytotoxic activity of all paclitaxel-loaded SLN that was always unmodified or greater compared with free drug. No difference in cytotoxicity was noted between neutral and charged SLN.Co-culture experiments with hCMEC/D3 and different glioblastoma cells evidenced that, when delivered in SLN, paclitaxel increased its cytotoxicity towards glioblastoma cells.

Stem cell niches in glioblastoma: a neuropathological view.

Glioblastoma (GBM) stem cells (GSCs), responsible for tumor growth, recurrence, and resistance to therapies, are considered the real therapeutic target, if they had no molecular mechanisms of resistance, in comparison with the mass of more differentiated cells which are insensitive to therapies just because of being differentiated and nonproliferating. GSCs occur in tumor niches where both stemness status and angiogenesis are conditioned by the microenvironment. In both perivascular and perinecrotic niches, hypoxia plays a fundamental role. Fifteen glioblastomas have been studied by immunohistochemistry and immunofluorescence for stemness and differentiation antigens. It has been found that circumscribed necroses develop inside hyperproliferating areas that are characterized by high expression of stemness antigens. Necrosis developed inside them because of the imbalance between the proliferation of tumor cells and endothelial cells; it reduces the number of GSCs to a thin ring around the former hyperproliferating area. The perinecrotic GSCs are nothing else that the survivors remnants of those populating hyperproliferating areas. In the tumor, GSCs coincide with malignant areas so that the need to detect where they are located is not so urgent.

Solid lipid nanoparticles for potential doxorubicin delivery in glioblastoma treatment: preliminary in vitro studies.

The major obstacle to glioblastoma pharmacological therapy is the overcoming of the blood-brain barrier (BBB). In literature, several strategies have been proposed to overcome the BBB: in this experimental work, solid lipid nanoparticles (SLN), prepared according to fatty acid coacervation technique, are proposed as the vehicle for doxorubicin (Dox), to enhance its permeation through an artificial model of BBB. The in vitro cytotoxicity of Dox-loaded SLN has been measured on three different commercial and patient-derived glioma cell lines. Dox was entrapped within SLN thanks to hydrophobic ion pairing with negatively charged surfactants, used as counterions. Results indicate that Dox entrapped in SLN maintains its cytotoxic activity toward glioma cell lines; moreover, its permeation through hCMEC/D3 cell monolayer, assumed as a model of the BBB, was increased when the drug was entrapped in SLN. In conclusion, SLN proved to be a promising vehicle for the delivery of Dox to the brain in glioblastoma treatment.

Repressor element-1 silencing transcription factor (REST) is present in human control and Huntington's disease neurones.

AIMS: The repressor element-1 silencing transcription factor/neurone-restrictive silencer factor (REST/NRSF) is a master regulator of neuronal gene expression. REST/NRSF functions by recruiting other cofactors to genomic loci that contain the repressor element 1/neurone restrictive silencer element (RE1/NRSE) binding motif. In brain, demonstration of REST protein presence in neurones has remained controversial. However, RE1/NRSE containing neuronal genes are actively modulated and REST dysregulation is implicated in Huntington's disease (HD). We aimed to investigate REST distribution in autopsy brain from control and HD patients. METHODS: Brain tissues from six controls and six HD cases (Vonsattel grade 3 and 4) were investigated using immunohistochemical analysis. RESULTS: REST was present in neurones and glial cells of the cortex, caudate nucleus, hippocampus and cerebellum. REST labelling was mainly cytoplasmic in neurones while preferential nuclear staining of REST was found in glial cells. We also found that REST and huntingtin (HTT) colocalize in human neurones. Low levels of cytoplasmic REST were detected in neurones of the HD cortex and caudate but no direct relationship between decreased neuronal REST expression and disease grade was observed. CONCLUSIONS: These data support the notion of REST presence in human brain neurones and glial cells and indicate the importance of developing compounds able to restore REST-regulated transcription of neuronal genes in HD.

Temozolomide down-regulates P-glycoprotein in human blood-brain barrier cells by disrupting Wnt3 signaling.

Low delivery of many anticancer drugs across the blood-brain barrier (BBB) is a limitation to the success of chemotherapy in glioblastoma. This is because of the high levels of ATP-binding cassette transporters like P-glycoprotein (Pgp/ABCB1), which effluxes drugs back to the bloodstream. Temozolomide is one of the few agents able to cross the BBB; its effects on BBB cells permeability and Pgp activity are not known. We found that temozolomide, at therapeutic concentration, increased the transport of Pgp substrates across human brain microvascular endothelial cells and decreased the expression of Pgp. By methylating the promoter of Wnt3 gene, temozolomide lowers the endogenous synthesis of Wnt3 in BBB cells, disrupts the Wnt3/glycogen synthase kinase 3/ß-catenin signaling, and reduces the binding of ß-catenin on the promoter of mdr1 gene, which encodes for Pgp. In co-culture models of BBB cells and human glioblastoma cells, pre-treatment with temozolomide increases the delivery, cytotoxicity, and antiproliferative effects of doxorubicin, vinblastine, and topotecan, three substrates of Pgp that are usually poorly delivered across BBB. Our work suggests that temozolomide increases the BBB permeability of drugs that are normally effluxed by Pgp back to the bloodstream. These findings may pave the way to new combinatorial chemotherapy schemes in glioblastoma.

Down-modulation of SEL1L, an unfolded protein response and endoplasmic reticulum-associated degradation protein, sensitizes glioma stem cells to the cytotoxic effect of valproic acid.

Valproic acid (VPA), an histone deacetylase inhibitor, is emerging as a promising therapeutic agent for the treatments of gliomas by virtue of its ability to reactivate the expression of epigenetically silenced genes. VPA induces the unfolded protein response (UPR), an adaptive pathway displaying a dichotomic yin yang characteristic; it initially contributes in safeguarding the malignant cell survival, whereas long-lasting activation favors a proapoptotic response. By triggering UPR, VPA might tip the balance between cellular adaptation and programmed cell death via the deregulation of protein homeostasis and induction of proteotoxicity. Here we aimed to investigate the impact of proteostasis on glioma stem cells (GSC) using VPA treatment combined with subversion of SEL1L, a crucial protein involved in homeostatic pathways, cancer aggressiveness, and stem cell state maintenance. We investigated the global expression of GSC lines untreated and treated with VPA, SEL1L interference, and GSC line response to VPA treatment by analyzing cell viability via MTT assay, neurosphere formation, and endoplasmic reticulum stress/UPR-responsive proteins. Moreover, SEL1L immunohistochemistry was performed on primary glial tumors. The results show that (i) VPA affects GSC lines viability and anchorage-dependent growth by inducing differentiative programs and cell cycle progression, (ii) SEL1L down-modulation synergy enhances VPA cytotoxic effects by influencing GSCs proliferation and self-renewal properties, and (iii) SEL1L expression is indicative of glioma proliferation rate, malignancy, and endoplasmic reticulum stress statuses. Targeting the proteostasis network in association to VPA treatment may provide an alternative approach to deplete GSC and improve glioma treatments.

Promoter hypermethylation of the EMP3 gene in a series of 229 human gliomas.

The epithelial membrane protein 3 (EMP3) is a candidate tumor suppressor gene in the critical region 19q13.3 for several solid tumors, including tumors of the nervous systems. The aim of this study was to investigate the EMP3 promoter hypermethylation status in a series of 229 astrocytic and oligodendroglial tumors and in 16 GBM cell lines. The analysis was performed by methylation-specific PCR and capillary electrophoresis. Furthermore, the EMP3 expression at protein level was evaluated by immunohistochemistry and Western blotting analysis. Associations of EMP3 hypermethylation with total 1p/19q codeletion, MGMT promoter hypermethylation, IDH1/IDH2 and TP53 mutations, and EGFR amplification were studied, as well as its prognostic significance. The EMP3 promoter hypermethylation has been found in 39.5% of gliomas. It prevailed in low-grade tumors, especially in gliomas with an oligodendroglial component, and in sGBMs upon pGBMs. In oligodendroglial tumors, it was strongly associated with both IDH1/IDH2 mutations and total 1p/19q codeletion and inversely with EGFR gene amplification. No association was found with MGMT hypermethylation and TP53 mutations. In the whole series, the EMP3 hypermethylation status correlated with 19q13.3 loss and lack of EMP3 expression at protein level. A favorable prognostic significance on overall survival of the EMP3 promoter hypermethylation was found in patients with oligodendroglial tumors.

Temozolomide downregulates P-glycoprotein expression in glioblastoma stem cells by interfering with the Wnt3a/glycogen synthase-3 kinase/ß-catenin pathway.

BACKGROUND: Glioblastoma multiforme stem cells display a highly chemoresistant phenotype, whose molecular basis is poorly known. We aim to clarify this issue and to investigate the effects of temozolomide on chemoresistant stem cells. METHODS: A panel of human glioblastoma cultures, grown as stem cells (neurospheres) and adherent cells, was used. RESULTS: Neurospheres had a multidrug resistant phenotype compared with adherent cells. Such chemoresistance was overcome by apparently noncytotoxic doses of temozolomide, which chemosensitized glioblastoma cells to doxorubicin, vinblastine, and etoposide. This effect was selective for P-glycoprotein (Pgp) substrates and for stem cells, leading to an investigation of whether there was a correlation between the expression of Pgp and the activity of typical stemness pathways. We found that Wnt3a and ABCB1, which encodes for Pgp, were both highly expressed in glioblastoma stem cells and reduced by temozolomide. Temozolomide-treated cells had increased methylation of the cytosine-phosphate-guanine islands in the Wnt3a gene promoter, decreased expression of Wnt3a, disrupted glycogen synthase-3 kinase/ß-catenin axis, reduced transcriptional activation of ABCB1, and a lower amount and activity of Pgp. Wnt3a overexpression was sufficient to transform adherent cells into neurospheres and to simultaneously increase proliferation and ABCB1 expression. On the contrary, glioblastoma stem cells silenced for Wnt3a lost the ability to form neurospheres and reduced at the same time the proliferation rate and ABCB1 levels. CONCLUSIONS: Our work suggests that Wnt3a is an autocrine mediator of stemness, proliferation, and chemoresistance in human glioblastoma and that temozolomide may chemosensitize the stem cell population by downregulating Wnt3a signaling.

MGMT hypermethylation and MDR system in glioblastoma cancer stem cells.

BACKGROUND: Cancer stem cells (CSCs) in gliomas are associated with resistance to radio- and chemotherapy, based on O(6)-methylguanine-DNA methyltransferase (MGMT) hypermethylation and the Multidrug resistance (MDR) system activation. MATERIALS AND METHODS: Samples from 21 glioblastomas (GBMs) were put in culture with growth factors or serum in order to obtain neurospheres or adherent cells. Both were genetically and immunohistochemically characterized for ATP-binding cassette, sub-family B (MDR/TAP), member 1 (ABCB1), ATP-binding cassette, sub-family C (CFTR/MRP), member 1 (ABCC1) and MGMT expression together with primary tumors. RESULTS: ABCB1 expression was positive in endothelial cells of primary tumors. ABCC1 expression was variably positive in tumor cells and positive in neurospheres, and less expressed in adherent cells. MGMT was methylated and unmethylated in primary tumors and in neurospheres, respectively, and unmethylated in adherent cells. CONCLUSION: Methylation is an epigenetic event affecting progenitors before the separation of the two glia lineages and maximally the future initiating cells. ABCB1 expression is limited to endothelial cells, whereas ABCC1 expression could mark a minority of tumor cells approaching a stem-like status.

REST controls self-renewal and tumorigenic competence of human glioblastoma cells.

The Repressor Element 1 Silencing Transcription factor (REST/NRSF) is a master repressor of neuronal programs in non-neuronal lineages shown to function as a central regulator of developmental programs and stem cell physiology. Aberrant REST function has been associated with a number of pathological conditions. In cancer biology, REST has been shown to play a tumor suppressor activity in epithelial cancers but an oncogenic role in brain childhood malignancies such as neuroblastoma and medulloblastoma. Here we examined REST expression in human glioblastoma multiforme (GBM) specimens and its role in GBM cells carrying self-renewal and tumorigenic competence. We found REST to be expressed in GBM specimens, its presence being particularly enriched in tumor cells in the perivascular compartment. Significantly, REST is highly expressed in self-renewing tumorigenic-competent GBM cells and its knock down strongly reduces their self-renewal in vitro and tumor-initiating capacity in vivo and affects levels of miR-124 and its downstream targets. These results indicate that REST contributes to GBM maintenance by affecting its self-renewing and tumorigenic cellular component and that, hence, a better understanding of these circuitries in these cells might lead to new exploitable therapeutic targets.

MGMT promoter hypermethylation and its associations with genetic alterations in a series of 350 brain tumors.

MGMT (O6-methylguanine-DNA methyltransferase) promoter hypermethylation is a helpful prognostic marker for chemotherapy of gliomas, although with some controversy for low-grade tumors. The objective of this study was to retrospectively investigate MGMT promoter hypermethylation status for a series of 350 human brain tumors, including 275 gliomas of different malignancy grade, 21 glioblastoma multiforme (GBM) cell lines, and 75 non-glial tumors. The analysis was performed by methylation-specific PCR and capillary electrophoresis. MGMT expression at the protein level was also evaluated by both immunohistochemistry (IHC) and western blotting analysis. Associations of MGMT hypermethylation with IDH1/IDH2 mutations, EGFR amplification, TP53 mutations, and 1p/19q co-deletion, and the prognostic significance of these, were investigated for the gliomas. MGMT promoter hypermethylation was identified in 37.8% of gliomas, but was not present in non-glial tumors, with the exception of one primitive neuroectodermal tumor (PNET). The frequency was similar for all the astrocytic gliomas, with no correlation with histological grade. Significantly higher values were obtained for oligodendrogliomas. MGMT promoter hypermethylation was significantly associated with IDH1/IDH2 mutations (P = 0.0207) in grade II­III tumors, whereas it had a borderline association with 1p deletion (P = 0.0538) in oligodendrogliomas. No other association was found. Significant correlation of MGMT hypermethylation with MGMT protein expression was identified by IHC in GBMs and oligodendrogliomas (P = 0.0001), but not by western blotting. A positive correlation between MGMT protein expression, as detected by either IHC or western blotting, was also observed. The latter was consistent with MGMT promoter hypermethylation status in GBM cell lines. In low-grade gliomas, MGMT hypermethylation, but not MGMT protein expression, was associated with a trend, only, toward better survival, in contrast with GBMs, for which it had favorable prognostic significance.

Antigenic and Genotypic Similarity between Primary Glioblastomas and Their Derived Neurospheres.

Formation of neurospheres (NS) in cultures of glioblastomas (GBMs), with self-renewal, clonogenic capacities, and tumorigenicity following transplantation into immunodeficient mice, may denounce the existence of brain tumor stem cells (BTSCs) in vivo. In sixteen cell lines from resected primary glioblastomas, NS showed the same genetic alterations as primary tumors and the expression of stemness antigens. Adherent cells (AC), after adding 10% of fetal bovine serum (FBS) to the culture, were genetically different from NS and prevailingly expressed differentiation antigens. NS developed from a highly malignant tumor phenotype with proliferation, circumscribed necrosis, and high vessel density. Beside originating from transformed neural stem cells (NSCs), BTSCs may be contained within or correspond to dedifferentiated cells after mutation accumulation, which reacquire the expression of stemness antigens.

IDH1 and IDH2 mutations, immunohistochemistry and associations in a series of brain tumors.

A total of 343 brain tumors were studied for IDH1 and IDH2 mutations by direct sequencing and for protein expression by immunohistochemistry with mIDH1(R132H) antibody. Of these, 287 were gliomas (17 pilocytic astrocytomas, 13 grade II and 5 grade III astrocytomas, 167 primary (pGBMs) and 19 secondary (sGBMs) glioblastomas, 36 grade II and 26 grade III oligodendrogliomas and 4 grade II-III oligoastrocytomas). In gliomas, IDH1 mutations at codon R132 were identified in 22.3%, of which 93.7% were c.395G>A (p.R132H). Mutations were more frequent in oligodendrogliomas (53.2%) than in astrocytic tumors (22.8%) and in sGBMs (84.2%) upon pGBMs (1.8%). There was a statistically significant correlation between mIDH1(R132H) antibody immunostaining and the relevant mutation c.395G>A (p.R132H) (P = 0.0001). No mutations were identified in non-glial tumors which were also negative to immunohistochemistry, with the exception of one PNET. A c.515G>T (p.R172M) mutation of the IDH2 gene was only identified in a grade II oligodendroglioma patient which was wild-type for IDH1. A direct correlation with MGMT promoter hypermethylation status and an inverse correlation with EGFR amplification was found, whereas the relationships with 1p/19q co-deletion and TP53 mutations only showed a trend toward correlation. In all gliomas, a positive correlation was found between IDH1 mutations and a young age (P = 0.0001). In contrast, a correlation with overall survival could only be obtained in low-grade gliomas. Immunohistochemistry appeared to be useful in differential diagnoses, especially toward non-tumor pathologic nervous tissue, and in recognizing infiltrating glioma cells. The mIDH1(R132H) antibody positivity was complementary with Cyclin D1 expression.

SOX2 expression and amplification in gliomas and glioma cell lines.

BACKGROUND: The transcription factor SOX2 controls gene expression during development and has roles in both neurogenesis and gliogenesis. MATERIALS AND METHODS: The current study utilized immunohistochemistry, immunofluorescence, molecular genetics (gene copy number analysis) and Western blotting to examine SOX2 expression in surgical samples from 133 brain gliomas of different grades of malignancy, and in cell lines from 16 glioblastomas. RESULTS: Our results reveal a positive correlation between SOX2 expression and malignancy grade in gliomas and identify the hypercellular and hyperproliferative areas of glioblastomas as the areas with the highest SOX2 expression. SOX2 gene amplification was found in neurospheres and 14.4% and 11.1% of glioblastomas and anaplastic oligodendrogliomas, respectively. In contrast, SOX2 expression was not observed in neuronal tumors and varied in medulloblastomas depending upon their neuronal differentiation status. CONCLUSION: It is concluded that SOX2 is a marker for undifferentiated and proliferating cells and that its expression is up-regulated in the most anaplastic areas of glioblastomas and oligodendrogliomas. The hypothesis is that antigenic and genetic analogy exist between these areas and neurospheres and that glioblastoma stem cells may actually be dedifferentiated tumor cells that re-acquire a stem cell-like status.

On the origin and growth of gliomas.

This review begins with the description of the nervous cytogenesis, proliferation of primitive cells, migration and differentiation with antigen expression and regulation through factors. Emphasis is given to neural stem cells, progenitors and to radial glia as belonging to early stages of gliogenesis. Experimental production of brain tumors in the rat by transplacental administration of ethylnitrosourea and systemic administration of methylnitrosourea effective through neural stem cells and progenitors is briefly described as a preamble to the recent conception of gliomas as originating from stem cells or brain tumor stem cells (BTSCs). The nature and origin of BTSCs, their molecular regulation and their recognition in vivo are discussed. In the growth of tumors, the role of nestin, migration of neural stem cells toward the tumor from the subventricular zone or from the tumor to the brain and the migration from bone marrow of mesenchymal stem cells for angiogenesis of the tumors are considered. Special mention is made of the relationship between glio-vascular niches in the subventricular zone and the neo-niches around the tumors with their importance in the tumor growth as points of joint between cytogenesis and angiogenesis. Finally, besides the importance of hypoxia, cell traffic in the brain adjacent to tumor is emphasized in relation to the growth of gliomas.

MGMT promoter hypermethylation in a series of 104 glioblastomas.

AIM: To evaluate MGMT promoter hypermethylation as prognostic factor in a retrospective study of 104 cases of glioblastoma multiforme (GBM). MATERIALS AND METHODS: The O(6)-methylguanine-DNA methyltransferase (MGMT) status was evaluated by methylation-specific PCR (MSP), immunohistochemistry and Western blotting analysis in formalin-fixed paraffin-embedded surgical samples. RESULTS: The MGMT gene was found to be methylated in 29 of 101 tumors (28.7%) by MSP, according to the evaluation methods employed. By immunohistochemistry, different categories were identified on the basis of reaction intensity, percentage of positive cells and homogeneous or heterogeneous distribution. MSP did not correlate with immunohistochemistry, with the exception of the category with the highest percentage of positive cells and homogeneity of immunostaining. Western blotting analysis correlated with immunohistochemical findings (Pearson's correlation coefficient r=0.268, p=0.0211), but not with MSP. By Kaplan-Meier survival analysis, radiotherapy was a significant prognostic factor (p=0.0001). When uncensored patients alone were considered, MGMT methylation status showed a significant correlation with survival (p=0.026). Temozolomide therapy correlated with survival (p=0.022), but not with MGMT methylation. After multivariate Cox regression analysis, only radiotherapy remained as an independent prognostic factor (p=0.0001). CONCLUSION: Correlation was inconclusive among MSP, immunohistochemistry and Western blotting analysis, despite the sophisticated score system for the immunohistochemical quantitative evaluation. MGMT expression is a complex event in which many factors beside epigenetic silencing are implicated.

mTOR, S6 and AKT expression in relation to proliferation and apoptosis/autophagy in glioma.

BACKGROUND: The mammalian target of rapamycin (mTOR) controls cell growth through protein synthesis regulation. It can be activated by protein kinase B (AKT) or through ribosomal S6 kinase (S6K1). In malignant glioma, mTOR inhibitors have antiproliferative and proapoptotic effects and mTOR has been suggested as a target of therapies, thus it is worthwhile to verify its relations with the phosphatidylinositol-3-kinase (PI3)/AKT cascade, proliferation and apoptosis in human gliomas. MATERIALS AND METHODS: In a series of 64 gliomas, including high- and low-grade tumors, AKT, mTOR, S6, caspase-3, poly(ADP-ribose) polymerase 1 (PARP1) and cleaved PARP1, Ki-67/MIB.1 and beclin 1 were studied by molecular biology techniques, quantitative immunohistochemistry and Western blotting. RESULTS: mTOR (phospho-mTOR), S6 (phospho-S6), AKT (phospho-AKT) levels and Ki-67/MIB.1 labelling index (LI) increased with increasing grade of malignancy. Epithelial growth factor receptor (EGFR) amplification correlated with EGFRwt and EGFRvIII immunohistochemistry, and with AKT expression; the latter correlated with mTOR expression, whereas S6 expression correlated with Ki-67/MIB.1 LI. Within the category of glioblastoma, S6 but not mTOR correlated with proliferation. mTOR did not show correlation with apoptosis, whereas it was inversely correlated with beclin 1, in line with the observation that autophagy is not activated in many malignancies. CONCLUSION: The relationship of S6 with the proliferation markers emphasizes the importance of the position of S6K1 downstream AKT in the PI3/AKT pathway.

Survivin expression in glioblastomas correlates with proliferation, but not with apoptosis.

BACKGROUND: Survivin is expressed in proliferating tissues and in tumors. It is a member of the inhibitory apoptosis protein (IAP) family known to regulate mitosis and to inhibit apoptosis. It has therefore been regarded as a target for therapies. In malignant gliomas it increases with malignancy, even though in glioblastomas it does not seem to correlate with outcome. MATERIALS AND METHODS: Survivin was immunohistochemically studied in 39 selected viable glioblastoma areas belonging to 20 cases which were assayed for apoptosis, using a TUNEL assay, caspase-3, poly(ADP-ribose)polymerase 1 (PARP-1), Bid (BH3-interacting domain death agonist) and with the proliferation index Ki-67/MIB-1 and mitotic index (MI). RESULTS: A positive linear correlation was found between the survivin labelling index (LI) and the Ki-67/MIB-1 LI and MI. No inverse correlation was found with apoptosis. CONCLUSION: This double behavior can be attributed to mechanisms mediating survivin activity, either as a mitosis regulator and apoptosis inhibitor, and should be taken into account in therapeutic strategies using survivin.

Stat3 expression and its correlation with proliferation and apoptosis/autophagy in gliomas.

Signal transducer and activator of transcription-3 (Stat3) was studied along with several steps of the PI3/Akt pathway in a series of 64 gliomas that included both malignant and low-grade tumors, using quantitative immunohistochemistry, Western blotting, and molecular biology techniques. The goal of the study was to investigate whether activated Stat3 (phospho-Stat3) levels correlated with cell proliferation, apoptosis, and autophagy. Stat3 and activated Akt (phospho-Akt) expression increased with malignancy grade, but did not correlate with proliferation and survival within the category of glioblastomas. A correlation of Stat3 with Akt was found, indicating a regulation of the former by the PI3/Akt pathway, which, in turn, was in relation with EGFR amplification. Stat3 and Akt did not show any correlation with apoptosis, whereas they showed an inverse correlation with Beclin 1, a stimulator of autophagy, which was rarely positive in glioblastomas. Autophagy seems then to be inactivated in malignant gliomas.