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Biomedical subjects

A Rosolen

Publications and source records attributed to A Rosolen.

At least 19 recordsLinked to original sources
PubMed1998

Normal and rearranged PAX3 expression in human rhabdomyosarcoma.

PAX3, a member of the PAX-gene family, encodes a nuclear transcription factor that is transiently expressed in the neural tube and in muscle progenitor cells and regulates embryonal development in the mouse. Together with the FKHR gene it is involved in the t(2;13)(q35;q14), a specific translocation associated with alveolar rhabdomyosarcoma (ARMS). As a consequence of the rearrangement two chimeric transcripts originate: FKHR-PAX3 and PAX3-FKHR. We studied the expression of wild type PAX3 and the chimeric transcripts originating from the t(2;13) in a series of 23 rhabdomyosarcomas (RMS) of childhood, by reverse transcriptase polymerase chain reaction (RT-PCR). Wild type PAX3 was detected in 48% of the RMS, whereas another 39% were positive only after nested PCR. Normal adult-skeletal muscle showed a very weak expression of PAX3, but fetal muscle did not express PAX3. PAX3-FKHR was found in 11 of 15 alveolar RMS, 7 of which were positive also for the reciprocal transcript, whereas no RMS expressed FKHR-PAX3 alone. These results confirm that the PAX3-FKHR transcript is specifically associated with the alveolar RMS and that it is a more sensitive marker of the t(2;13) than the reciprocal product FKHR-PAX3. Furthermore, the finding of PAX3 expression with or without PAX3-FKHR transcript in the great majority of the cases raises the question of whether PAX3 expression could play a role in the pathogenesis of RMS.

Adult↗
PubMed1998

In vitro and in vivo antitumor effects of retrovirus-mediated herpes simplex thymidine kinase gene-transfer in human medulloblastoma.

Herpes simplex virus thymidine kinase gene (HSVtk) transfer together with treatment with the prodrug ganciclovir (GCV) represents the most commonly used suicide gene approach for the gene therapy of human central nervous system malignancies. Despite encouraging results reported in clinical trials conducted in adults, very little is known about the feasibility of this approach for the treatment of CNS tumors of childhood. We studied the effects of the HSVtk/GCV system on human medulloblastoma cells in vitro and in vivo. The transfer of tk gene to medulloblastoma cells was capable of mediating cell suicide in vitro and in vivo upon treatment with GCV, but the overall effect in vivo appeared to be suboptimal. The relatively low sensitivity of the medulloblastoma cells to viral infection and a limited bystander effect, coupled with a low expression of connexin-43 protein, might partially explain these results. Whether this is a peculiarity of the cell line studied or a general characteristic of medulloblastoma remains to be determined. These findings should be taken into account for the future planning of gene therapy trials for human medulloblastoma.

Cerebellar Neoplasms↗
PubMed1998

Neuroendocrine differentiation in Ewing's sarcomas and primitive neuroectodermal tumors revealed by reverse transcriptase-polymerase chain reaction of chromogranin mRNA.

Ewing's sarcomas (ESs), primitive neuroectodermal tumors (PNETs), and neuroblastomas (NBs) are closely related neoplasms supposedly derived from the neural crest and belonging to the family of the small blue round cell tumors of infancy and childhood. We investigated the expression of the neuroendocrine and neuroectodermal markers chromogranin A (CgA) and secretogranin II (SgII) in ESs, PNETs, and NBs, both in primitive tumors (five, nine, and four cases, respectively) and in established cell lines (three ES and two PNET cell lines). Different technical approaches, namely immunohistochemistry, Northern blot analysis, and reverse transcriptase-polymerase chain reaction (RT-PCR) were used in parallel. Chromogranin A and secretogranin II production was constantly detectable in NBs by all procedures. CgA mRNA was detectable in most ESs and PNETs only by RT-PCR, whereas SgII mRNA was detectable in some ESs and PNETs by Northern blot analysis and in all tumors by RT-PCR. CgA and SgII proteins were never detectable by immunohistochemistry in ESs and PNETs. We conclude that neuroendocrine differentiation is shared by all three tumor entities, being more overt in NBs and rudimentary in ESs and PNETs; traces of chromogranin mRNA are detectable only by a highly sensitive RT-PCR procedure.

Adolescent↗
PubMed1998

Effects of all-trans retinoic acid and interferon alpha in peripheral neuroectodermal tumor cell cultures and xenografts.

Peripheral neuroectodermal tumors (PNET) have an unsatisfactory outcome when treated with standard approaches. Among novel treatments, the use of biological response modifiers has rarely been reported in this group of malignancies. We have previously demonstrated that both all-trans retinoic acid (ATRA) and interferon á (IFNá) can inhibit proliferation of human PNET cells and that ATRA can up-regulate IFNá receptor expression in vitro. In this study we evaluated the anti-tumor effects of ATRA and IFNá in PNET cells in vitro and in a human PNET xenograft model, using CHP100 cells. A synergistic inhibitory effect of ATRA and IFNá was observed on CHP100 cells in vitro. On the contrary, a significant inhibition of tumor growth was observed in mice treated with ATRA alone, whereas neither IFNá nor the combination of ATRA and IFNá, reached a statistically significant anti-tumor effect. Histologic examination of tumors revealed the presence of necrosis upon treatment with IFNá, whereas almost no necrosis, but a more differentiated morphology, confirmed by electron microscopy analysis, was associated with the ATRA containing treatments. Taken together these data show an in vitro and in vivo anti-tumor activity of ATRA in human PNET cells, although no synergism of ATRA and IFNá was observed in our xenograft model.

Animals↗
PubMed1998

Detection of the MyoD1 transcript in rhabdomyosarcoma cell lines and tumor samples by reverse transcription polymerase chain reaction.

Rhabdomyosarcoma (RMS) is the most common soft-tissue sarcoma of childhood. Diagnosis of RMS can be difficult when it appears as a small round-cell tumor without evidence of differentiation. Recently, a set of regulatory proteins expressed during skeletal muscle development has been described. Among them, MyoD1 has been detected by Northern blot and immunohistochemical analyses in normal skeletal muscle and RMS. Given the relevance of this marker in the diagnosis of RMS, we developed an assay to evaluate the expression of MyoD1 mRNA in small tissue specimens by reverse transcription polymerase chain reaction. Specificity and sensitivity of the assay was determined in a series of 25 tumor cell lines and 39 pediatric tumor samples, including 35 RMSs. Subsequently, we studied the expression of MyoD1 in bone marrow and peripheral blood stem cell specimens. We detected the MyoD1 transcript in normal skeletal muscle and in almost all RMSs, whereas no expression was found in non-RMS samples or in normal hematopoietic tissues. This assay showed high sensitivity and specificity, and it could be a useful molecular tool for the diagnosis of RMS within small roundcell tumors of childhood and for the detection of minimal bone marrow and peripheral blood stem cell involvement in children with RMS, regardless of the histological subtype.

Bone Marrow↗
PubMed1997

Expression of type I interferon receptor in solid tumors of childhood.

Interferon alpha (IFN alpha) is used as an antineoplastic agent, both in hematopoietic malignancies and in solid tumors, because of its immunomodulatory action and direct antitumor activity. IFN alpha binds to specific cell-surface receptors that mediate its biologic activity. We studied the expression of IFN alpha receptors in pediatric solid tumors by use of the monoclonal antibody IFNaR3, which specifically recognizes the alpha subunit of the IFN Type I receptor. In three cell lines derived from those tumors, we determined the structure of the receptors by affinity cross-linking and immunoprecipitation techniques, and we determined their ability to mediate an antiproliferative effect. All of the tumor specimens studied by immunocytochemical analysis, including neuroblastomas, primitive neuroectodermal tumors, and rhabdomyosarcomas, stained positive for the IFN alpha receptor antibody, although in some cases immunoreactivity was weak. The three cell lines, derived from a neuroblastoma, a primitive neuroectodermal tumor, and a Ewing's sarcoma, respectively, showed the same pattern of IFN alpha receptor expression, both by affinity crosslinking and immunoprecipitation assays. Treatment with IFN alpha of those cell lines induces growth inhibition in vitro. These results suggest that IFN Type I receptor might be expressed in most solid tumors of childhood and that its structure is identical to the receptor expressed by the majority of hematologic malignancies.

Adolescent↗
PubMed1996

Analysis of cyclin-dependent kinase inhibitor genes (CDKN2A, CDKN2B, and CDKN2C) in childhood rhabdomyosarcoma.

p16INK4A, p15INK4B, and p18 proteins are highly specific inhibitors of cyclin-dependent serine/threonine kinase (CDK) activities required for GI-S transition in the eukaryotic cell division cycle. Mutations, mainly homozygous deletions, of the CDKN2A (p16INK4A/MTSI) gene have been recently found in tumor cell lines and in many primary tumors. We looked for homozygous deletions of CDKN2A, CDKN2B (p15INK4B), and CDKN2C (p18) in 12 primary rhabdomyosarcoma (RMS) specimens and in five cell lines established from this cancer type. By means of polymerase chain reaction (PCR) and PCR-single strand conformation polymorphism (PCR-SSCP), we analyzed the presence of biallelic gene deletion or point mutation causing gene function loss. All the examined tumor cell lines (100%) and three of 12 (25%) primary tumors showed homozygous deletion of CDKN2A. Furthermore, no aberrant bands in primary tumors were detected via SSCP, suggesting the absence of mutations in the coding region. In all cases the deleted area at 9p21 also involved the CDKN2B gene. Conversely, no homozygous deletion or point mutations were detected when CDKN2C was analyzed. Our results strongly indicate that the p16INK4A (and/or p15INK4B) protein plays a key role in the development and/or progression of childhood rhabdomyosarcoma and suggest that this CDK-inhibitor protein might control proliferation and/or differentiation of human muscle cells. Moreover, alteration of CDKN2C does not appear to be involved in the genesis of rhabdomyosarcoma.

Adolescent↗
PubMed1993

Effect of over-expression of bacterial ribonuclease H on the utility of antisense MYC oligodeoxynucleotides in the monocytic leukemia cell line U937.

RNase H has been clearly implicated in vitro in mediating some antisense effects. In vivo evidence is limited to experiments performed in Xenopus oocytes in which antisense oligonucleotides are microinjected. In other mammalian cell systems scant data have been obtained to support or deny a role for RNase H as an antisense mediator in vivo. These experiments were designed to test the hypothesis that RNase H mediates the MYC antisense-induced reduction in MYC protein observed in the human monocytic leukemia cell line U937. A bacterial RNase H-containing episomal replicon was constructed and stable transfectants were obtained which expressed E coli RNase H in their cytoplasm at a 10-fold higher level than endogenous RNase H. These cells failed to demonstrate heightened sensitivity to MYC antisense (phosphorothioate, end capped and phosphodiester) compared with untransfected or E coli RNase H antisense transfected cells. PCR analysis of each transfectant treated and untreated with MYC antisense failed to demonstrate the appearance of truncated MYC mRNA. These results do not support a role for RNase H in the mediation of MYC antisense-induced MYC protein reduction and growth inhibition in U937 cells.

Base Sequence↗
PubMed1992

Multichain structure of the IFN-alpha receptor on hematopoietic cells.

The structure of IFN-alpha receptor was studied by 1) developing antibodies against the receptor, and 2) screening a number of cell lines by affinity cross-linking to identify cells that express different IFN-alpha 2 receptor structures. We report that two different patterns of IFN-alpha 2 receptor are observed in human cells of hematopoietic origin. The predominant form of the IFN-alpha receptor is a multichain structure in which IFN-alpha 2 forms complexes of 110 and 130 kDa (alpha-subunit). A high Mr complex of 210 kDa results from the association of alpha-subunit and other receptor components. In contrast, another form of the receptor has been identified in the IFN-alpha-resistant U-937 cell line and in some cases of acute leukemia. This form of the IFN-alpha receptor is characterized by the presence of the alpha- subunit, and the absence of the 110- and 210-kDa bands. Also a novel 180-kDa complex and a more prominent 75-kDa band are observed. Functional studies performed in U-937 cells showed that this cell line is not only partially resistant to the antiproliferative and antiviral effects of IFN-alpha, but also fails to down-regulate the alpha-subunit of the IFN-alpha receptor upon IFN-alpha binding.

Cell Line↗
PubMed1992

Antisense inhibition of gene expression: a tool for studying the role of NMYC in the growth and differentiation of neuroectoderm-derived cells.

To understand the role of individual genes in regulating biological processes, one must be able to interfere specifically with either their expression or function. While monoclonal antibodies have proven very useful in studying cell surface proteins, the specific inhibition of intracellular proteins in viable cells is a much more difficult problem. The goal of antisense technology is to develop small oligonucleotides, plasmids, or retroviral vectors that can be introduced easily into viable cells in order to inhibit gene products specifically. In this report, we will describe our use of antisense DNA and RNA to study the role of the NMYC proto-oncogene in neuroectodermal cell growth and differentiation.

Animals↗
PubMed1992

Antisense inhibition of oncogene expression.

To understand the role of individual genes in regulating biological processes, one must be able to interfere specifically with either their expression or function. While monoclonal antibodies have proven very useful in studying cell surface proteins, the specific inhibition of intracellular proteins in viable cells is a much more difficult problem. The goal of antisense technology is to develop small oligonucleotides, plasmids, or retroviral vectors which can be readily introduced into living cells in order to inhibit specific gene expression. In this review, we briefly describe the principles of antisense usage, including problems of cellular uptake and intracellular distribution, mechanism of antisense action, and the properties of various oligonucleotide derivatives. In addition we present several examples of the biological effects of antisense administration used to study the role of specific genes in the regulation of cell growth and differentiation.

Animals↗
PubMed1992

A phase I study of interleukin-2 in children with cancer.

Recombinant interleukin-2 (IL-2) produces clinical responses in approximately 20% of adult patients with renal cell carcinoma and melanoma, with both high-dose bolus and continuous infusion regimens. Because of the lower toxicity of continuous infusion, we elected to investigate in a Phase I trial a 5-day continuous infusion repeated for three weeks in children with malignancies refractory to standard therapy. Nineteen children with solid tumors and eight children with hematologic malignancies were entered into the study. The maximum tolerated dose was 3 x 10(6) U/m2/day, with dose-limiting toxicities occurring in five of seven patients treated at the 5 x 10(6) U/m2/day dose level. Dose-limiting toxicities included hypotension, hyperbilirubinemia, thrombocytopenia, pulmonary/pleural effusion, and nephrotoxicity. Serum IL-2 levels were detectable at the higher dose levels and were comparable to those observed in adult patients. Hematologic changes at the higher dose levels included rebound lymphocytosis occurring within 48 h of discontinuation of IL-2, eosinophilia, and decreased platelet counts. No objective responses to therapy were seen. We have identified a dose and schedule of administration for IL-2 in pediatric patients that can be given without intensive care unit support. Pediatric Phase II trials examining the anti-tumor activity of IL-2 given by this schedule are in progress.

Adolescent↗
PubMed1991

In vivo modulation of N-myc expression by continuous perfusion with an antisense oligonucleotide.

In this study we investigated the in vivo efficacy of continuous subcutaneous perfusion of unmodified phosphodiester oligodeoxynucleotides. The in vitro sequelae of antisense inhibition of the target gene, N-myc, have been documented and include moderate growth inhibition without effects on myc expression, loss of secretogranin I expression, and morphologic alterations. We chose to use N-myc as a model target to determine if antisense effects observed in vitro can be reproduced in vivo. N-myc-expressing human neuroectodermal tumors were grown as subcutaneous xenografts in athymic mice. Antisense and sense oligodeoxynucleotides directed against N-myc were delivered to the vicinity of the tumor by a subcutaneously implanted microosmotic pump. Antisense treatment led to loss of N-myc protein from the tumor, as well as to the loss of the neuroendocrine differentiation marker protein secretogranin I. Myc protein expression remained unaffected. Mean tumor mass was reduced by 50% in antisense-treated animals, and antisense-treated tumors morphologically resembled antisense-transfected in vitro cell cultures. These results demonstrate that regional, in vivo perfusion of an unmodified oligonucleotide specifically downregulates gene expression in human tumor xenografts with concomitant effects on tumor phenotype and growth rate that correlate well with in vitro observations.

Base Sequence↗
PubMed1991

Episome-generated N-myc antisense RNA restricts the differentiation potential of primitive neuroectodermal cell lines.

Neuroectodermal tumors of childhood provide a unique opportunity to examine the role of genes potentially regulating neuronal growth and differentiation because many cell lines derived from these tumors are composed of at least two distinct morphologic cell types. These types display variant phenotypic characteristics and spontaneously interconvert, or transdifferentiate, in vitro. The factors that regulate transdifferentiation are unknown. Application of antisense approaches to the transdifferentiation process has allowed us to explore the precise role that N-myc may play in regulating developing systems. We now report construction of an episomally replicating expression vector designed to generate RNA antisense to part of the human N-myc gene. Such a vector is able to specifically inhibit N-myc expression in cell lines carrying both normal and amplified N-myc alleles. Inhibition of N-myc expression blocks transdifferentiation in these lines, with accumulation of cells of an intermediate phenotype. A concomitant decrease in growth rate but not loss of tumorigenicity was observed in the N-myc nonamplified cell line CHP-100. Vector-generated antisense RNA should allow identification of genes specifically regulated by the proto-oncogene N-myc.

Animals↗