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G Raschellà

Publications and source records attributed to G Raschellà.

At least 19 recordsLinked to original sources
PubMed2005

Silencing of endogenous IGFBP-5 by micro RNA interference affects proliferation, apoptosis and differentiation of neuroblastoma cells.

Signal transduction through the IGF axis is implicated in proliferation, differentiation and survival during development and adult life. The IGF axis includes the IGF binding proteins (IGFBPs) that bind IGFs with high affinity and modulate their activity. In neuroblastoma (NB), a malignant childhood tumor, we found that IGFBP-5 is frequently expressed. Since NB is an IGF2-sensitive tumor, we investigated the relevance and the function of endogenous IGFBP-5 in LAN-5 and in SY5Y(N) cell lines transfected with micro and small interfering RNAs directed to IGFBP-5 mRNA. Cells in which IGFBP-5 expression was suppressed were growth-inhibited and more prone to apoptosis than the parental cell line and controls. Apoptosis was further enhanced by X-ray irradiation. The ability of these cells to undergo neuronal differentiation was impaired after IGFBP-5 inhibition but the effect was reversed by exposure to recombinant IGFBP-5. Together, these data demonstrate the importance of IGFBP-5 for NB cell functions and suggest that IGFBP-5 might serve as a novel therapeutic target in NB.

Apoptosis↗
PubMed2002

Cyclin D1-dependent regulation of B-myb activity in early stages of neuroblastoma differentiation.

Levels of the transcription factor B-myb must be down-regulated to allow terminal differentiation of neuroectodermal cells and yet its constitutive expression induces early markers of neural differentiation. Thus, we investigated potential mechanisms of enhanced B-myb activity in early stages of neural differentiation. We report here that B-myb expression does not decrease, cyclin A and Sp1 levels remain constant while p21 levels increase continuously upon retinoic acid-induced differentiation of the LAN-5 neuroblastoma cell line. In contrast, cyclin D1 expression is down-regulated at the onset of the differentiative process by protein destabilization. Luciferase assays of promoter activity indicate that B-myb-dependent transactivation is enhanced in LAN-5 cells treated with retinoic acid (RA) for 24 h. The enhancement is independent from cyclin A but is suppressed by a degradation-resistant mutant form of cyclin D1. The importance of cyclin D1 in controlling B-myb activity is further suggested by co-immunoprecipitation experiments, showing that the amount of cyclin D1 co-immunoprecipitated with B-myb decreased after RA treatment. Thus, B-myb may play an active role in the early stages of differentiation when its transactivation activity is enhanced as a consequence of cyclin D1 down-modulation.

Antineoplastic Agents↗
PubMed2001

Retinoblastoma family proteins induce differentiation and regulate B-myb expression in neuroblastoma cells.

BACKGROUND: The expression of several genes is modulated during neuroblastoma differentiation. The retinoblastoma family proteins, pRb, p107 and pRb2/p130, act in the repression of proliferation genes, interacting mainly with the E2F transcription factors. PROCEDURE AND RESULTS: In this study, we found that, in neuroblastoma cell lines, pRb and p107 proteins decreased, undergoing progressive dephosphorylation, whereas pRb2/p130 increased at late stages of differentiation. B-myb expression was down-regulated in association with the up-regulation of pRb2/p130, the major partner of E2F on the E2F site of the B-myb promoter in differentiated cells. Transfection of each of the retinoblastoma family genes in neuroblastoma cells was able to induce neural differentiation, to inhibit 3H-thymidine incorporation, and to down-regulate B-myb promoter activity. CONCLUSIONS: In conclusion, our data suggest a major contribution of retinoblastoma proteins, and especially of pRb2/p130, in B-myb promoter regulation and demonstrate the induction of neural differentiation by p107 and pRb2/p130, suggesting a role of these proteins in triggering differentiation-specific genes.

Animals↗
PubMed2001

DR-nm23 expression affects neuroblastoma cell differentiation, integrin expression, and adhesion characteristics.

BACKGROUND AND PROCEDURE: Nm23 gene family has been associated with metastasis suppression and differentiation. We studied DR-nm23 during neuroblastoma cells differentiation. DR-nm23 expression increased after retinoic acid induction of differentiation in human cell lines SK-N-SH and LAN-5. RESULTS: In several cell lines, overexpression of DR-nm23 was associated with more differentiated phenotypes. SK-N-SH cells increased vimentin expression, increased deposition of collagen type IV, modulated integrin expression, and underwent growth arrest; the murine neuroblastoma cell line N1E-115 showed neurite outgrowth and a striking enhancement of beta1 integrin expression. Up-regulation of beta1 integrin was specifically responsible for the increase in the adhesion to collagen type I-coated plates. Finally, cells overexpressing DR-nm23 were unable to growth in soft agar. CONCLUSIONS: In conclusion, DR-nm23 expression is directly involved in differentiation of neuroblastoma cells, and its ability to affects the adhesion to extracellular substrates and to inhibit growth in soft agar suggests an involvement in the metastatic potential of neuroblastoma.

Agar↗
PubMed2001

Structural and catalytic properties and homology modelling of the human nucleoside diphosphate kinase C, product of the DRnm23 gene.

The human DRnm23 gene was identified by differential screening of a cDNA library obtained from chronic myeloid leukaemia-blast crisis primary cells. The over-expression of this gene inhibits differentiation and induces the apoptosis of myeloid precursor cell lines. We overproduced in bacteria a truncated form of the encoded protein lacking the first 17 N-terminal amino acids. This truncated protein was called nucleoside diphosphate (NDP) kinase CDelta. NDP kinase CDelta had similar kinetic properties to the major human NDP kinases A and B, but was significantly more stable to denaturation by urea and heat. Analysis of denaturation by urea, using size exclusion chromatography, indicated unfolding without the dissociation of subunits, whereas renaturation occurred via a folded monomer. The stability of the protein depended primarily on subunit interactions. Homology modelling of the structure of NDP kinase CDelta, based on the crystal structure of NDP kinase B, indicated that NDP kinase CDelta had several additional stabilizing interactions. The overall structure of the two enzymes appears to be identical because NDP kinase CDelta readily formed mixed hexamers with NDP kinase A. It is possible that mixed hexamers can be observed in vivo.

Amino Acid Sequence↗
PubMed2000

Neuroblastoma specific effects of DR-nm23 and its mutant forms on differentiation and apoptosis.

DR-nm23 belongs to a gene family which includes nm23-H1, originally identified as a candidate metastasis suppressor gene. Nm23 genes are expressed in different tumor types where their levels have been alternatively associated with reduced or increased metastatic potential. Nm23-H1, -H2, DR-nm23 and nm23-H4 all possess NDP kinase activity. Overexpression of DR-nm23 inhibits differentiation and promotes apoptosis in hematopoietic cells. By contrast, it induces morphological and biochemical changes associated with neural differentiation in neuroblastoma cells. In this study, we show that mutations in the catalytic domain and in the serine 61 phosphorylation site, possibly required for protein-protein interactions, impair the ability of DR-nm23 to induce neural differentiation. Moreover, neuroblastoma cells overexpressing wild-type or mutant DR-nm23 are less sensitive to apoptosis triggered by serum withdrawal. By subcellular fractionation, wild-type and mutant DR-nm23 localize in the cytoplasm and prevalently in the mitochondrial fraction. In co-immunoprecipitation experiments, wild-type DR-nm23 binds other members of nm23 family, but mutations in the catalytic and in the RGD domains and in serine 61 inhibit the formation of hetero-multimers. Thus, the integrity of the NDP kinase activity and the presence of a serine residue in position 61 seem essential for the ability of DR-nm23 to trigger differentiation and to bind other Nm23 proteins, but not for the anti-apoptotic effect in neuroblastoma cells. These studies underline the tissue specificity of the biological effects induced by DR-nm23 expression.

Animals↗
PubMed1999

Expression of B-myb in neuroblastoma tumors is a poor prognostic factor independent from MYCN amplification.

The transcription factors of the Myb family are expressed in several tissues and play an important role in cell proliferation, differentiation, and survival In this study, the expression of A-myb, B-myb, and c-myb was investigated in a group of 64 neuroblastomas at different dinical stages by a sensitive reverse transcription-PCR tchnique and correlated with patients' survival. All of the myb genes were frequently expressed in neuroblastoma tumors. Interestingly, the expression of B-myb, which was detected in 33 cases, was associated with an increased risk of death (P = 0.027 in a univariate analysis), whereas there was no correlation with A-myb and c-myb expression. In addition, in a multivariate Cox regression analysis that included myb gene expression, MYCN status, age at diagnosis, and tumor staging, MYCN amplification and B-myb expression were independently associated to an increased risk (P < 0.01 and P = 0.015, respectively). In overall survival curves obtained by stratifying the neuroblastoma cases on the basis of MYCN status and B-myb expression, the group of patients without MYCN amplification and positive for B-myb expression had worse survival probability than that without MYCN amplification and nonexpressing B-myb (P < 0.01). In summary, these findings provide the first demonstration that B-myb expression can be a useful prognostic marker in human neuroblastoma. Moreover, B-myb expression has a prognostic value complementary to MYCN amplification and can identify a group of high-risk patients that would not be predicted on the basis of the MYCN status only.

Cell Cycle Proteins↗
PubMed1998

The RB-related gene Rb2/p130 in neuroblastoma differentiation and in B-myb promoter down-regulation.

The retinoblastoma family of nuclear factors is composed of RB, the prototype of the tumour suppressor genes and of the strictly related genes p107 and Rb2/p130. The three genes code for proteins, namely pRb, p107 and pRb2/p130, that share similar structures and functions. These proteins are expressed, often simultaneously, in many cell types and are involved in the regulation of proliferation and differentiation. We determined the expression and the phosphorylation of the RB family gene products during the DMSO-induced differentiation of the N1E-115 murine neuroblastoma cells. In this system, pRb2/p130 was strongly up-regulated during mid-late differentiation stages, while, on the contrary, pRb and p107 resulted markedly decreased at late stages. Differentiating N1E-115 cells also showed a progressive decrease in B-myb levels, a proliferation-related protein whose constitutive expression inhibits neuronal differentiation. Transfection of each of the RB family genes in these cells was able, at different degrees, to induce neuronal differentiation, to inhibit [3H]thymidine incorporation and to down-regulate the activity of the B-myb promoter.

Animals↗
PubMed1997

Retinoblastoma-related protein pRb2/p130 and its binding to the B-myb promoter increase during human neuroblastoma differentiation.

Neuroblastoma cells can undergo neural differentiation upon treatment with a variety of chemical inducers and growth factors. During this process, many cell cycle-related genes are downregulated while differentiation-specific genes are triggered. The retinoblastoma family proteins, pRb, p107, and pRb2/p130, are involved in transcriptional repression of proliferation genes, mainly through their interaction with the E2F transcription factors. We report that pRb2/p130 expression levels increased during differentiation of neuroblastoma cell line LAN-5. On the other hand, both pRb and p107 decreased and underwent progressive dephosphorylation at late differentiation times. The expression of B-myb and c-myb, two targets of the retinoblastoma family proteins, were downregulated in association with the increase of pRb2/p130, which was detected as the major component of the complex with E2F on the E2F site of the B-myb promoter in differentiated cells. Interestingly, E2F4, a preferential partner of p107 and pRb2/p130, was upregulated and underwent changes in cellular localization during differentiation. In conclusion, our data suggest a major role of pRb2/p130 in the regulation of B-myb promoter during neural differentiation despite the importance of cofactors in modulating the function of the retinoblastoma family proteins.

Carrier Proteins↗
PubMed1997

DR-nm23 gene expression in neuroblastoma cells: relationship to integrin expression, adhesion characteristics, and differentiation.

BACKGROUND: Neuroblastoma, a childhood tumor originating from cells of the embryonic neural crest, retains the ability to differentiate, yielding cells with epithelial-Schwann-like, neuronal, or melanocytic characteristics. Since nm23 gene family members have been proposed to play a role in cellular differentiation, as well as in metastasis suppression, we investigated whether and how DR-nm23, a recently identified third member of the human nm23 gene family, might be involved in neuroblastoma differentiation. METHODS: Three neuroblastoma cell lines (human LAN-5, human SK-N-SH, and murine N1E-115) were used in these experiments; cells from two of the lines (SK-N-SH and N1E-115) were also studied after being stably transfected with a plasmid containing a full-length DR-nm23 complementary DNA. Cellular expression of specific messenger RNAs and proteins was assessed by use of standard techniques. Cellular adhesion to a variety of protein substrates was also evaluated. RESULTS: DR-nm23 messenger RNA levels in nontransfected LAN-5 and SK-N-SH cells generally increased with time after exposure to differentiation-inducing conditions; levels of the other two human nm23 messenger RNAs (nm23-H1 and nm23-H2) remained essentially constant. Transfected SK-N-SH cells overexpressing DR-nm23 exhibited some characteristics of differentiated cells (increased vimentin and collagen type IV expression) even in the absence of differentiation-inducing conditions. Compared with control cells, DR-nm23-transfected cells exposed to differentiation-inducing conditions showed a greater degree of growth arrest (SK-N-SH cells) and greater increases in integrin protein expression, especially of integrin beta1 (N1E-115 cells). DR-nm23-transfected N1E-115 cells also showed a marked increase in adhesion to collagen type I-coated tissue culture plates that was inhibited by preincubation with an anti-integrin beta1 antibody. CONCLUSIONS: DR-nm23 gene expression appears to be associated with differentiation in neuroblastoma cells and may affect cellular adhesion through regulation of integrin protein expression.

Animals↗
PubMed1997

Phenotypic and morphological characterization of neuroblastoma cells constitutively expressing B-myb.

B-myb gene is expressed in neuroblastoma cells and down-regulated during differentiation. We used B-myb-transfected LAN-5 cells, which constitutively express high level of B-myb, to detect changes at phenotypic and morphological levels in basal and differentiation conditions. Our results demonstrate that the overexpression of B-myb markedly affects the cytoskeletal composition, the pattern of neurotransmitter enzymes and the extracellular matrix expression. In general, B-myb transfected neuroblastoma cells show a broad potentiality without a direction toward a specific neuroectodermal differentiation pathway. On the other hand, we confirm inhibition of the neuronal differentiation upon retinoic acid (RA) treatment of B-myb transfected cells. Furthermore, the ultrastructural analyses are supportive of a change in the metabolism in B-myb transfected cell treated with RA. Our data suggest that B-myb expression is compatible with an early phase of differentiation of neuroectodermal cells, but must be down-regulated for the completion of the differentiative programme.

Cell Cycle Proteins↗
PubMed1996

B-myb transcriptional regulation and mRNA stability during differentiation of neuroblastoma cells.

B-myb and c-myb expression is high in neuroblastoma cells and declines during retinoic acid-induced differentiation. We show here that B-myb down-regulation during retinoic acid-induced differentiation of LAN-5 neuroblastoma cells occurs at the transcriptional level. In addition, we measured B-myb and c-myb messenger RNA half-lives, and found that, unlike c-myb, B-myb messenger RNA was remarkably stable (> 10 h). Inhibition of protein synthesis by treatment with cycloheximide increased B-myb messenger RNA levels, suggesting that one or more labile proteins act as repressors of B-myb transcription. In the same cell line, blocking protein synthesis decreased the level of c-myb mRNA under both normal and differentiative conditions. Thus, B-myb and c-myb undergo similar transcriptional regulation, but there are specific differences in the stability of their messenger RNAs and in the mechanisms through which their transcription is controlled. These differences could reflect different functional roles played by c-myb and B-myb in neuroblastoma cells.

Cell Cycle Proteins↗
PubMed1995

Requirement of b-myb function for survival and differentiative potential of human neuroblastoma cells.

The B-myb gene belongs to a family of transcription factors that also includes A-myb and c-myb. B-myb is expressed in many cell types including human neuroblastoma cells. Here we demonstrate that B-myb expression is down-regulated during retinoic acid-induced neural and glial differentiation of neuroblastoma cells. This modulation is an early event, is maintained at late times of induction, and is in part regulated at the transcriptional level. Constitutive expression of B-myb prevents retinoic acid-induced neural differentiation as reflected by morphological features and the expression of (or lack of) biochemical markers associated with the undifferentiated phenotype. Furthermore, the expression of antisense B-myb transcripts does not allow the rescue of viable cells, suggesting an important role for B-myb in the survival of neuroblastoma cells. These results indicate that B-myb plays a functional role in the differentiative potential of neuroblastoma cells, raising the possibility that this gene is one of the nuclear regulators in the cascade of events leading to cellular differentiation.

Base Sequence↗
PubMed1992

Inhibition of proliferation by c-myb antisense RNA and oligodeoxynucleotides in transformed neuroectodermal cell lines.

Transfection of a neuroblastoma cell line with expression vectors containing two different segments of human c-myb complementary DNA in antisense orientation yielded far fewer transfectant clones than did the transfection with the identical segments in sense orientation. In cell clones expressing c-myb antisense RNA, levels of the c-myb protein were down-regulated and the proliferation rate was slower than that of cells transfected with sense constructs or the untransfected parental cell line. Treatment of neuroblastoma and neuroepithelioma cell lines with a c-myb antisense oligodeoxynucleotide strongly inhibited cell growth. These data indicate a definite involvement of c-myb in the proliferation of neuroectodermal tumor cells extending the role of this protooncogene beyond the hematopoietic system. The availability of cell clones that transcribe c-myb antisense RNA provides a useful tool to study the involvement of other genes in the proliferation and differentiation of neuroblastoma cells.

Animals↗
PubMed1992

Fc gamma receptors are expressed on human neuroblastoma cell lines: lack of correlation with N-myc oncogene activity.

FcRs (Fc Receptors) have been detected on the cell surface of two human neuroblastoma cell lines; IMR 32 and SK-N-SH, by immunocytochemistry and flow cytometric analysis, using a previously characterized polyclonal antiserum raised against the Fc gamma R isolated from a human CLL line (Gorini, Medgyesi, Garavini, Dorrington and Down, 1987; Rozsnay, Sarmay, Szabo, Medgyesi, Gorini and Gergely, 1990). FcR is expressed on all the cells of both lines at least at the same level as on the HL60 promyelocyte cell line used as positive control. Two electrophoretic components displaying apparent molecular masses of 70 and 43 kDa respectively have been identified by SDS-PAGE followed by Western blotting analysis of crude cell membranes. In addition, "in situ" hybridization experiments seem to exclude a correlation between FcR expression and N-myc oncogene activity. The presence of FcR in neuroblastoma could be related to a possible functional role even on these cells which do not belong to the immune system; moreover, they could also be exploited for a diagnostic characterization of this tumor.

Blotting, Western↗
PubMed1991

Transcription of N-myc and proliferation-related genes is linked in human neuroblastoma.

A definite association between the transcription of N-myc oncogene and proliferation-related genes, histone H3, c-myc and p53, was found in a set of 12 primary untreated neuroblastomas and a metastasis of one of these at relapse. Molecular analysis allowed us to discriminate between actually proliferating and non-proliferating tumors, and suggested a link between N-myc and proliferation. Flow cytometric analysis of DNA distribution was less reliable for assessing tumor proliferative activity. Our data also seem to indicate a down-regulation of c-myc by N-myc in human neuroblastoma.

Blotting, Northern↗
PubMed1991

Decrease of proliferation rate and induction of differentiation by a MYCN antisense DNA oligomer in a human neuroblastoma cell line.

The effects of an antisense oligodeoxynucleotide to codons 2-7 of the oncogene MYCN on the human neuroblastoma cell line LAN-5 were studied. Treated cells showed a decreased MYCN protein expression and synthesis by immunoperoxidase staining and immunoprecipitation. At the same time, the replication rate was inhibited, and the phenotype was modified toward a more differentiated type. Our data suggest the involvement of oncogene MYCN in both proliferative and differentiative processes.

Base Sequence↗