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Biomedical subjects

A Calogero

Publications and source records attributed to A Calogero.

At least 19 recordsLinked to original sources
PubMed2006

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 TGFbeta1, 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 TGFbeta1 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.

Early Growth Response Protein 1↗
PubMed2001

Characterization of BIS20x3, a bi-specific antibody activating and retargeting T-cells to CD20-positive B-cells.

This paper describes a bi-specific antibody, which was called BIS20x3. It retargets CD3varepsilon-positive cells (T-cells) to CD20-positive cells and was obtained by hybrid-hybridoma fusion. BIS20x3 could be isolated readily from quadroma culture supernatant and retained all the signalling characteristics associated with both of its chains. Cross-linking of BIS20x3 on Ramos cells leads to DNA fragmentation percentages similar to those obtained after Rituximab-cross-linking. Cross-linking of BIS20x3 on T-cells using cross-linking F(ab')2-fragments induced T-cell activation. Indirect cross-linking of T-cell-bound BIS20x3 via Ramos cells hyper-activated the T-cells. Furthermore, it was demonstrated that BIS20x3 effectively re-targets T-cells to B-cells, leading to high B-cell cytotoxicity. The results presented in this paper show that BIS20x3 is fully functional in retargeting T-cells to B-cells and suggest that B-cell lymphomas may represent ideal targets for T-cell retargeting bi-specific antibodies, because the retargeted T-cell is maximally stimulated in the presence of B-cells. Additionally, since B-cells may up-regulate CD95/ Fas expression upon binding of CD20-directed antibodies, B-cells will become even more sensitive for T-cell mediated killing via CD95L/ Fas L, and therefore supports the intention to use T-cell retargeting bi-specific antibodies recognizing CD20 on B-cell malignancies as a treatment modality for these diseases.

Antibodies, Monoclonal↗
PubMed2001

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.

Antibodies, Viral↗
PubMed2001

The early growth response gene EGR-1 behaves as a suppressor gene that is down-regulated independent of ARF/Mdm2 but not p53 alterations in fresh human gliomas.

PURPOSE: EGR-1 is an immediate early gene with diverse functions that include the suppression of growth. EGR-1 is down-regulated many cancer cell types, suggesting a tumor suppressor role, and may critically involve the p53 pathway. The aim of this work was to measure the expression of EGR-1 and the p16/INK4a/ARF-Mdm2-p53 pathway status in fresh human gliomas. EXPERIMENTAL DESIGN: Thirty-one human gliomas with different grades of malignancy were investigated for Egr-1 mRNA and the protein expression, frequency, and spectrum of p53 gene mutations, mdm2 gene amplification, and p16/INK4a/ARF allele loss. RESULTS: The amplification of Mdm2 and the deletion of the p16/INK4a gene was found in 3 and 5 cases, respectively, whereas mutations of p53, including two novel mutations, were observed in 10 other cases. The three types of changes occurred strictly mutually exclusively, emphasizing that these genes operate in a common pathway critical to glioma progression. EGR-1 mRNA was significantly down-regulated in astrocytomas (14.7 +/- 5.1%) and in glioblastomas (33.6 +/- 10.0%) versus normal brain. Overall, EGR-1 mRNA was strongly suppressed (average, 15.2 +/- 13.9%) in 27 of 31 cases (87%), independent of changes in p16/INK4a/ARF and Mdm2; whereas 4 of 31 cases with residual EGR-1 expression as well as the highest EGR-1 variance segregated with p53 mutations. Immunohistochemical analyses confirmed the suppression of EGR-1 protein. CONCLUSIONS: These results indicate that EGR-1 is commonly suppressed in gliomas independent of p16/INK4a/ARF and Mdm2 and that suppression is less crucial in tumors bearing p53 mutations, and these results implicate an EGR-1 growth regulatory mechanism as a target of inactivation during tumor progression.

Blotting, Northern↗
PubMed2000

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.

Animals↗
PubMed2000

Monitoring of herpes simplex virus thymidine kinase enzyme activity using positron emission tomography.

9-[(1-[18F]Fluoro-3-hydroxy-2-propoxy)methyl]guanine ([18F]FHPG) was evaluated as a tracer for noninvasive positron emission tomography (PET) imaging of herpes simplex virus type 1 thymidine kinase (HSV-tk) gene expression. C6 rat glioma cells with and without the HSV-tk gene were incubated with [18F]FHPG for 2 h. The in vitro tracer uptake in HSV-tk-containing C6tk cells was 35 +/- 5 times higher than that in control cells. In nude rats carrying both a C6 and a C6tk tumor, the average ratio of tracer accumulation between the tumors was 15 +/- 5 at 2 h postinjection. The tracer is rapidly cleared from nontarget tissue into the urine because only the HSV-tk-expressing tumor, kidneys, and bladder remained visible on the late PET images. HPLC analysis revealed that three metabolites, tentatively assigned as FHPG mono-, di-, and triphosphate, were formed in the C6tk tumors only. In conclusion, we have demonstrated that [18F]FHPG is a promising tracer for monitoring HSV-tk enzyme activity in vivo with PET.

Animals↗
PubMed2000

Limitations of the nested reverse transcriptase polymerase chain reaction on tyrosinase for the detection of malignant melanoma micrometastases in lymph nodes.

The specificity and sensitivity of the nested reverse transcriptase polymerase chain reaction (RT-PCR) on tyrosinase was studied, for the detection of micrometastases of malignant melanoma. The specificity was assessed in the blood of six healthy donors, four patients with non-melanoma cancers of which one patient was treated with granulocyte-colony stimulating factor. Lymph nodes of nine patients without malignant melanoma were tested and four cell lines of various other tumours. Six of the nine non-melanoma lymph nodes were positive in this assay. The sensitivity was tested in a spike experiment in vitro, using a melanoma cell line. The detection limit was ten melanoma cells per 10(7) peripheral blood lymphocytes.

Biomarkers, Tumor↗
PubMed2000

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.

B-Lymphocytes↗
PubMed1999

Stage-specific expression of a Schistosoma mansoni polypeptide similar to the vertebrate regulatory protein stathmin.

The ubiquitous vertebrate protein stathmin is expressed and phosphorylated in response to a variety of external and internal signals. Stathmin, in turn, controls cell growth and differentiation through its capacity to regulate microtubule assembly dynamics. This is the first report on the molecular cloning and characterization of a stathmin-like protein (SmSLP) in an invertebrate, the human blood fluke Schistosoma mansoni. SmSLP is first synthesized at high levels in the intermediate molluscan host and completely disappears 48 h after penetration into the mammalian host. The protein is preferentially iodinated in intact immature parasites using the Bolton-Hunter reagent, can be quantitatively extracted in high salt buffers, and remains soluble after boiling. Native SmSLP was partially sequenced, and its complete structure was derived from the cloning and sequencing of its cDNA. The sequence is up to 26% identical to vertebrate stathmin sequences and contains two potential phosphorylation sites. Native SmSLP is indeed phosphorylated because phosphatase digestion shifts its mobility in electrofocusing gels. SmSLP associates with tubulin, as suggested by immune co-precipitation results. In vitro experiments demonstrated that SmSLP inhibits tubulin assembly and causes the depolymerization of preassembled microtubules, thus probably fulfilling regulatory roles in critical steps of schistosome development.

Amino Acid Sequence↗
PubMed1999

An enhanced expression of the immediate early gene, Egr-1, is associated with neuronal apoptosis in culture.

Cultured cerebellar granule cells grown in medium containing 10 mM K+ (K10) underwent apoptosis after four to five days in vitro, unless they were rescued by the addition of insulin-like growth factor-I. The few GABAergic neurons present in the cultures were more resistant to apoptotic degeneration, as indicated by double fluorescent staining with the chromatin dye Hoechst 33258 and with glutamate decarboxylase-67 antibodies. As compared with sister cultures grown in 25 mM K+, K10 cultures showed an increased expression of the Egr-1 protein and a reduced expression of the Fos protein. The increase in Egr-1 was more substantial in granule cells than in GABAergic neurons, and was not observed in K10 cultures chronically exposed to insulin-like growth factor-I. To examine the temporal relationship between the increase in Egr-1 and the development of programmed cell death, we induced apoptosis in K25 cultures at six days in vitro by replacing their medium with serum-free K10 medium. A substantial, but transient, increase in Egr- expression was observed in granule cells 6 h after switching the medium, a time that preceded the appearance of the phenotypical markers of apoptotic death. An early reduction in the Fos protein was observed after switching the medium from K25 into serum-free K10, but also after switching the medium into serum-free K25, a condition which was not associated with the development of apoptosis nor with the increase in Egr-1. We suggest that a transient induction of Egr-1 contributes to the chain of events leading to the execution phase of neuronal apoptosis in culture.

Animals↗
PubMed1998

Upregulation of Epstein-Barr virus-encoded latent membrane protein by human herpesvirus 6 superinfection of EBV-carrying Burkitt lymphoma cells.

The effect of HHV-6 strain A infection on the expression of Epstein-Barr virus- (EBV-) encoded growth transformation-associated genes in two EBV-positive Burkitt lymphoma cell lines, Akata and P3HR-3, was investigated. The results indicate that HHV-6A upregulates the expression of the latent membrane protein LMP-1 in both cell lines. Expression of EBNA-2 was also upregulated in Akata cells following HHV-6A infection. Transfection of reporter constructs carrying the LMP-1 regulatory sequences (LRS; -634/+40) or its 5' deleted derivatives in Akata and in a T-lymphoblastoid cell line, J-Jhan, confirmed the presence of positive and negative regulatory elements responsive to HHV-6A infection in LMP-1 regulatory sequence (LRS). The majority of LRS constructs were under the influence of dominant negative factors. HHV-6A was able to override the effect of such factors acting on reporter plasmids containing the -634/-54, -324/-54, -214/-54, and -106/-54 parts of LRS. The plasmid that carried only the -54/+40 LRS region was constitutively active in both Akata and J-Jhan cells; in Akata, its activity was influenced by HHV-6A. The finding that HHV-6A infection may activate LMP-1 and EBNA-2 expression, which is essential for the immortalization of B-lymphocytes by EBV, shows a novel aspect of the interaction between these two herpesviruses.

Antibodies, Monoclonal↗
PubMed1997

Transcription of latent and replicative Epstein-Barr-virus genes in bone-marrow and peripheral-blood mononuclear cells of healthy donors.

Reverse-transcriptase polymerase chain reaction has been used to analyze the expression of 2 latent genes (EBNA-1 and LMP-1) and one replicative gene (BZLF-1) of Epstein-Barr virus in mononuclear cells from bone marrow and peripheral blood of healthy donors. EBV-gene transcription was detected in 8 out of 15 bone-marrow samples. Among these, 5 allowed the detection of latency-associated transcripts in the absence of BZLF-1 expression. Only one sample showed positivity for expression of both latent and lytic genes. In 2 cases, BZLF-1 was the only transcript detected. In peripheral blood, 4 out of 7 samples showed evidence of EBNA-1 transcription; LMP-1 was expressed in 5 samples, and in 2 cases concomitant expression of EBNA-1 and BZLF-1 was detected. These results provide a direct demonstration by RT-PCR of EBV-gene transcription in bone-marrow-resident viral infected cells and suggest, in contrast to previous studies on peripheral blood, that LMP-1 and BZLF-1 are frequently transcribed also in absence of EBV-related disease. The heterogeneous viral gene expression found makes it difficult to define a pattern of viral latency in vivo which coincides with that described for lymphoblastoid or Burkitt's-lymphoma cell lines at different stages of differentiation.

Animals↗
PubMed1997

Synthetic oligonucleotides: useful molecules? A review.

Specific inhibition of mammalian genes is possible through the use of antisense oligonucleotides (AS ODNs) or ribozymes. These strategies have led to a better understanding of several cellular and molecular mechanisms, among which cancer development. Recently, these strategies have been applied also for therapeutical purposes in diseases such as AIDS and cancer. In some of these therapeutical trials the antisense strategy is combined with gene transfer technology: the AS ODN or the ribozyme are expressed within the cell by the use of adenoviral or retroviral vectors. However, many difficulties have still to be overcome before ODNs and ribozymes can be used routinely in the clinic.

Animals↗
PubMed1996

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.

B-Lymphocytes↗
PubMed1996

EGR-1, the reluctant suppression factor: EGR-1 is known to function in the regulation of growth, differentiation, and also has significant tumor suppressor activity and a mechanism involving the induction of TGF-beta1 is postulated to account for this suppressor activity.

The transcription factor EGR-1 is a potential regulator of over 30 genes and plays a role in growth, development, and differentiation and, in addition, has significant transformation suppression activity. The regulatory properties are reviewed and a hypothesis for the transformation suppression activity is proposed. EGR-1 contains three "zinc-finger" motifs in the C-terminal portion of the molecule that constitute the DNA-binding domain and interact with the promoters by virtue of two classes of GC-rich elements: single GC-elements (GCEs) with the consensus 5'-T-G-C-G-T/g-G/A-G-G-C/a/t-G-G/T-3' and overlapping sites consisting of an Sp-1 binding site and the GCE consensus or close homolog of these sequences. The Wilm's tumor suppressor gene product WT1 interacts with the same GCE and, owing in part to four alternate splice products, interacts with a broader range of GC-rich elements with the consensus 5'-GNGNGGGNG-3' and 5'-TCCTCCTCCTCCTC-3'. WT1 commonly but not invariably acts as repressor of transcription, whereas EGR-1, in the absence of overlapping Sp-1 binding sequences, is often an activator. The well-known rapid response of the EGR-1 gene following mitogenic stimulation together with the occurrence of GCEs in the promoters of many growth factors and protooncogenes suggests a role of EGR-1 in growth. Moreover, EGR-1 is constitutively expressed in several viral-transformed systems. On the other hand, studies of model and human tumor lines reveal that EGR-1 has significant growth and transformation suppression roles. Recent studies show that this effect can be accounted for by the ability of EGR-1 to induce the expression and secretion of TGF-beta1, a potent growth suppressor of many cell types, by binding to a single GCE of the TGF-beta1 promoter. Although the effects of EGR- at overlapping Sp1/EGR-1 DNA binding sites are not predictable, known cases fall into two loose groups. Sp1 is usually activating and increasing concentrations of EGR-1 lead to displacement that results in either inhibition of transactivation or EGR-1-dependent transactivation. Moreover, recent studies suggest that displaced Sp1 binds to and activates the endogenous Egr-1 gene, thereby leading to "facilitated inhibition" of Sp1 function by the resulting increased EGR-1. This effect may augment the growth suppressive function of EGR-1 based on induction of TGF-beta1.

Animals↗
PubMed1995

Human herpesvirus 6 variant A, but not variant B, infects EBV-positive B lymphoid cells, activating the latent EBV genome through a BZLF-1-dependent mechanism.

Human herpesvirus 6, a predominantly T lymphotropic virus, has been recently shown to infect some EBV-positive B cell lines, and to induce in them the activation of the EBV lytic cycle. Here we have confirmed and extended such observations, showing that (1) this phenomenon is restricted to the variant A of HHV-6: in fact two isolates belonging to the HHV-6 variant B (BA92 and Z29) were neither able to infect any B cell line, independently of the EBV status, nor to induce the EBV genome expression. The only exception is represented by the P3HR1 cells, in which, however, the infection by the variant B does not determine induction of EBV antigens; (2) the presence of the EBV genome contributes to the susceptibility of the B cell lines to HHV-6 infection, increasing the binding sites and the percentage of infectable cells, as detected by immunoelectron microscopy; and (3) HHV-6 infected T cells, transfected with plasmids bearing the promoter regions of the EBV early genes BZLF1 and BMRF1, show a strong transactivation of these promoters.

B-Lymphocytes↗
PubMed1995

Cerebrospinal fluid and plasma concentrations of SRIH, beta-endorphin, CRH, NPY and GHRH in obese and normal weight subjects.

Numerous hypothalamic peptides are involved in the control of eating behaviour. We assessed plasma and cerebrospinal fluid (CSF) levels of SRIH, beta-endorphin (beta-EP), CRH, NPY and GHRH in a group of massively obese patients and in normal weight subjects. In the obese patients, CSF SRIH and beta-EP levels were significantly reduced and increased, respectively, compared with controls (20.6 +/- 2.62, mean +/- s.e.m., vs 34.5 +/- 2.14 pg/ml, P < 0.05, for SRIH and 111.2 +/- 5.00 vs 80.4 +/- 5.32 pg/ml, P < 0.001, for beta-EP). Considering the data of obese and control subjects altogether, SRIH and beta-EP concentrations correlated negatively and positively, respectively, with BMI values (r = -0.641, P < 0.005 and r = 0.518, P < 0.05). No significant differences were observed in CSF levels of CRH, NPY and GHRH between obese and normal weight subjects, though GHRH levels were close to the assay sensitivity. CSF concentrations of CRH were positively correlated with those of SRIH in obese patients (r = 0.60, P < 0.05) and with those of NPY both in obese (r = 0.69, P < 0.02) and in control subjects (r = 0.83, P < 0.005). Plasma levels of SRIH, beta-EP, NPY and GHRH did not differ significantly in the two groups of subjects; plasma CRH was undetectable. Our results argue against the hypothesis of an enhanced SRIH tone as the cause of impaired GH secretion in obese patients, a primary defect in GHRH or GH release seems more likely. Moreover, they emphasise the importance of an increased tone of endogenous opioids in the pathophysiology of human obesity.

Adult↗