Antonella Calogero

The transcription factor Egr1 is a direct regulator of multiple tumor suppressors including TGFbeta1, PTEN, p53, and fibronectin.

Recent studies are reviewed indicating that the transcription factor early growth response-1 (Egr1) is a direct regulator of multiple tumor suppressors including TGFβ1, PTEN, p53, and fibronectin. The downstream pathways of these factors display multiple nodes of interaction with each other, suggesting the existence of a functional network of suppressor factors that serve to maintain normal growth regulation and resist the emergence of transformed variants. Paradoxically, Egr1 is oncogenic in prostate cancer. In the majority of these cancers, PTEN or p53 is inactive. It is suggested that these defects in the suppressor network allow for the unopposed induction of TGFβ1 and fibronectin, which favor transformation and survival of prostate tumor epithelial cells, and explain the role of Egr1 in prostate cancer. Egr1 is a novel and logical target for intervention by gene therapy methods, and targeting methods are discussed.

Human herpesvirus 6 infection in neoplastic and normal brain tissue

The presence and variant distribution of human herpesvirus 6 (HHV‐6) was investigated by a nested polymerase chain reaction (PCR) in 118 biopsies from patients affected by nervous tissue tumor (115 primary tumors and 3 metastasis) and in 31 autopsy samples from the brain of healthy individuals. HHV‐6 DNA sequences were detected in normal and neoplastic nervous tissue at a frequency of 32% and 37%, respectively. In both tissues, variant A was three times more frequent than the variant B. Peripheral blood lymphocytes (PBLs) derived from seven tumor affected patients contained the same variant as their respective brain sample, as judged by PCR. The expression of HHV‐6 encoded immediate early protein p41 was detected by immunohistochemistry in neoplastic but not in normal brain. This may reflect viral reactivation from latency in immunocompromised patients. The seroepidemiological data indicated a frequency distribution of anti‐HHV‐6 antibodies in patients with brain tumors similar to that found in healthy donors. J. Med. Virol. 63:45–51, 2001.

Inhibition of Egr-1 expression reverses transformation of prostate cancer cells in vitro and in vivo.

Transcription factor early growth response-1 (Egr-1) is a crucial regulator of cell growth, differentiation and survival. Several observations suggest that Egr-1 is growth promoting in prostate cancer cells and that blocking its function may impede cancer progression. To test this hypothesis, we developed phosphorothioate antisense oligonucleotides that efficiently inhibit Egr-1 expression without altering the expression of other family members Egr-2, Egr-3 and Egr-4. In TRAMP mouse-derived prostate cancer cell lines, our optimal antisense oligonucleotide decreased the expression of the Egr-1 target gene transforming growth factor-β1 whereas a control oligonucleotide had no effect, indicating that the antisense blocked Egr-1 function as a transcription factor. The antisense oligonucleotide deregulated cell cycle progression and decreased proliferation of the three TRAMP cell lines by an average of 54±3%. Both colony formation and growth in soft agar were inhibited by the antisense oligonucleotide. When TRAMP mice were treated systemically for 10 weeks, the incidence of palpable tumors at 32 weeks of age in untreated mice or mice injected with the control scramble oligonucleotide was 87%, whereas incidence of tumors in antisense-Egr-1-treated mice was significantly reduced to 37% (P=0.026). Thus, Egr-1 plays a functional role in the transformed phenotype and may represent a valid target for prostate cancer therapy.

The Potential of GMP-Compliant Platelet Lysate to Induce a Permissive State for Cardiovascular Transdifferentiation in Human Mediastinal Adipose Tissue-Derived Mesenchymal Stem Cells

Human adipose tissue-derived mesenchymal stem cells (ADMSCs) are considered eligible candidates for cardiovascular stem cell therapy applications due to their cardiac transdifferentiation potential and immunotolerance. Over the years, the in vitro culture of ADMSCs by platelet lysate (PL), a hemoderivate containing numerous growth factors and cytokines derived from platelet pools, has allowed achieving a safe and reproducible methodology to obtain high cell yield prior to clinical administration. Nevertheless, the biological properties of PL are still to be fully elucidated. In this brief report we show the potential ability of PL to induce a permissive state of cardiac-like transdifferentiation and to cause epigenetic modifications. RTPCR results indicate an upregulation of Cx43, SMA, c-kit, and Thy-1 confirmed by immunofluorescence staining, compared to standard cultures with foetal bovine serum. Moreover, PL-cultured ADMSCs exhibit a remarkable increase of both acetylated histones 3 and 4, with a patient-dependent time trend, and methylation at lysine 9 on histone 3 preceding the acetylation. Expression levels of p300 and SIRT-1, two major regulators of histone 3, are also upregulated after treatment with PL. In conclusion, PL could unravel novel biological properties beyond its routine employment in noncardiac applications, providing new insights into the plasticity of human ADMSCs.

Expression of metabotropic glutamate receptors in murine thymocytes and thymic stromal cells

RT-PCR combined with immunoblotting showed the expression of group-I (mGlu1 and 5) and group-II (mGlu2 and 3) metabotropic glutamate receptors in whole mouse thymus, isolated thymocytes and TC-1S thymic stromal cell line. Cytofluorimetric analysis showed that mGlu-5 receptors were absent in CD4(-)/CD8(-) but present in more mature CD4(+) CD8(+) and CD4(+)CD8(-) thymocytes. mGlu-1a receptors showed an opposite pattern of expression with respect to mGlu5, whereas mGlu2/3 receptor expression did not differ between double negative and double positive cells. mGlu receptors expressed in both thymic cell components were functional, as indicated by measurements of polyphosphoinositide hydrolysis or cAMP formation. These data suggest a possible role for mGlu receptor signalling in the thymus.

Pharmacological blockade of mGlu2/3 metabotropic glutamate receptors reduces cell proliferation in cultured human glioma cells

Glial cell proliferation in culture is under the control of metabotropic glutamate (mGlu) receptors. We have examined whether this control extends to human glioma cells. Primary cultures were prepared from surgically removed human glioblastomas. RT‐PCR combined with western blot analysis showed that most of the cultures (eight out of 11) expressed group‐II mGlu receptors. In two selected cultures (MZC‐12 and FCN‐9), the mGlu2/3 receptor antagonist, LY341495, slowed cell proliferation when applied to the growth medium from the second day after plating. This effect was reversible because linear cell growth was restored after washing out the drug. LY341495 reduced glioma cell proliferation at concentrations lower than 100 nm, which are considered as selective for mGlu2/3 receptors. In addition, its action was mimicked by the putative mGlu2/3 receptor antagonist (2S)‐α‐ethylglutamate. The anti‐proliferative effect of LY341495 was confirmed by measuring [methyl‐3H]‐thymidine incorporation in cultures arrested in G0 phase of the cell cycle and then stimulated to proliferate by the addition of 10% fetal calf serum or 100 ng/mL of epidermal growth factor (EGF). In cultures treated with EGF, LY341495 was also able to reduce the stimulation of the mitogen‐activated protein kinase (MAPK) pathway, as well as the induction of cyclin D1. Both effects, as well as decreased [methyl‐3H]‐thymidine incorporation, were partially reduced by co‐addition of the potent mGlu2/3 receptor agonist, LY379268. We conclude that activation of group‐II mGlu receptors supports the growth of human glioma cells in culture and that antagonists of these receptors should be tested for their ability to reduce tumour growth in vivo.

Inhibition of cell growth by EGR-1 in human primary cultures from malignant glioma.

BackgroundThe aim of this work was to investigate in vitro the putative role of EGR-1 in the growth of glioma cells. EGR-1 expression was examined during the early passages in vitro of 17 primary cell lines grown from 3 grade III and from 14 grade IV malignant astrocytoma explants. The explanted tumors were genetically characterized at the p53, MDM2 and INK4a/ARF loci, and fibronectin expression and growth characteristics were examined. A recombinant adenovirus overexpressing EGR-1 was tested in the primary cell lines.ResultsLow levels of EGR-1 protein were found in all primary cultures examined, with lower values present in grade IV tumors and in cultures carrying wild-type copies of p53 gene. The levels of EGR-1 protein were significantly correlated to the amount of intracellular fibronectin, but only in tumors carrying wild-type copies of the p53 gene (R = 0,78, p = 0.0082). Duplication time, plating efficiency, colony formation in agarose, and contact inhibition were also altered in the p53 mutated tumor cultures compared to those carrying wild-type p53. Growth arrest was achieved in both types of tumor within 1–2 weeks following infection with a recombinant adenovirus overexpressing EGR-1 but not with the control adenovirus.ConclusionsSuppression of EGR-1 is a common event in gliomas and in most cases this is achieved through down-regulation of gene expression. Expression of EGR-1 by recombinant adenovirus infection almost completely abolishes the growth of tumor cells in vitro, regardless of the mutational status of the p53 gene.

Dual-specificity phosphatase DUSP6 has tumor-promoting properties in human glioblastomas

Dual-specificity phosphatase 6 (DUSP6, mitogen-activated protein kinase (MAPK) phosphatase 3 or PYST1) dephosphorylates phosphotyrosine and phosphothreonine residues on extracellular signal-regulated kinase (ERK1/2; MAPK1/2) to inactivate the ERK1/2 kinase. DUSP6 is a critical regulator of the ERK signaling cascade and has been implicated as a tumor suppressor. We report here experimental evidences that DUSP6 is transcriptionally upregulated in primary and long-term cultures of human glioblastoma, as assayed by northern hybridization and real-time quantitative PCR, producing constitutive high level of protein expression. Functional assays were performed with adenovirus-mediated expression of DUSP6 in glioblastoma cultures. Protein overexpression inhibits growth by inducing G1-phase delay and increased mitogenic/anchorage dependence and clonogenic potential in vitro. Changes in cell morphology were associated with an increased tumor growth in vivo. Chemoresistance is a major cause of treatment failure and poor outcome in human glioblastomas. Importantly, DUSP6 overexpression increased resistance to cisplatin-mediated cell death in vitro and in vivo. Antisense-mediated depletion of DUSP6 acted in lowering the threshold to anticancer DNA-damaging drugs. We conclude that upregulation of DUSP6 exerts a tumor-promoting role in human glioblastomas exacerbating the malignant phenotype.

Serum-activated K and Cl currents underlay U87-MG glioblastoma cell migration

Glioblastoma cells in vivo are exposed to a variety of promigratory signals, including undefined serum components that infiltrate into high grade gliomas as result of blood–brain barrier breakdown. Glioblastoma cell migration has been further shown to depend heavily on ion channels activity. We have then investigated the modulatory effects of fetal calf serum (FCS) on ion channels, and their involvement in U87‐MG cells migration. Using the perforated patch‐clamp technique we have found that, in a subpopulation of cells (42%), FCS induced: (1) an oscillatory activity of TRAM‐34 sensitive, intermediate‐conductance calcium‐activated K (IKCa) channels, mediated by calcium oscillations previously shown to be induced by FCS in this cell line; (2) a stable activation of a DIDS‐ and NPPB‐sensitive Cl current displaying an outward rectifying instantaneous current‐voltage relationship and a slow, voltage‐dependent inactivation. By contrast, in another subpopulation of cells (32%) FCS induced a single, transient IKCa current activation, always accompanied by a stable activation of the Cl current. The remaining cells did not respond to FCS. In order to understand whether the FCS‐induced ion channel activities are instrumental to promoting cell migration, we tested the effects of TRAM‐34 and DIDS on the FCS‐induced U87‐MG cell migration using transwell migration assays. We found that these inhibitors were able to markedly reduce U87‐MG cell migration in the presence of FCS, and that their co‐application resulted in an almost complete arrest of migration. It is concluded that the modulation of K and Cl ion fluxes is essential for the FCS‐induced glioblastoma cell migration. J. Cell. Physiol. 226: 1926–1933, 2011.

The Inhibition of KCa3.1 Channels Activity Reduces Cell Motility in Glioblastoma Derived Cancer Stem Cells

In the present study we evaluated the expression of the intermediate conductance calcium-activated potassium (KCa3.1) channel in human glioblastoma stem-like cells (CSCs) and investigated its role in cell motility. While the KCa3.1 channel is not expressed in neuronal- and glial-derived tissues of healthy individuals, both the KCa3.1 mRNA and protein are present in the glioblastoma tumor population, and are significantly enhanced in CSCs derived from both established cell line U87MG and a primary cell line, FCN9. Consistent with these data, voltage-independent and TRAM-34 sensitive potassium currents imputable to the KCa3.1 channel were recorded in the murine GL261 cell line and several primary human glioblastoma cells lines. Moreover, a significantly higher KCa3.1 current was recorded in U87MG-CD133 positive cells as compared to the U87MG-CD133 negative subpopulation. Further, we found that the tumor cell motility is strongly associated with KCa3.1 channel expression. Blockade of the KCa3.1 channel with the specific inhibitor TRAM-34 has in fact a greater impact on the motility of CSCs (reduction of 75%), which express a high level of KCa3.1 channel, than on the FCN9 parental population (reduction of 32%), where the KCa3.1 channel is expressed at lower level. Similar results were also observed with the CSCs derived from U87MG. Because invasion of surrounding tissues is one of the main causes of treatment failure in glioblastoma, these findings can be relevant for future development of novel cancer therapeutic drugs.