Obiettivo The differential diagnosis of neuroblastoma in pathological specimens can be extremely difficult yet crucial for the choice of the therapeutic strategy. The aims of this project are :- To develop immunohistological and molecular biology based tests for the diagnosis of neuroblastoma;- To quantitate the state of differentiation of neuroblastoma tumours by observer-independent methods;- To investigate the use of non hydrolyzable antisense oligonucleotides as potential inducers of neuronal differentiation of this neoplasia in animal models.Expected Outcome- Improved diagnostic tools for the pathological diagnosis of neuroblastoma tumours;- Most sensitive detection of neuroblastoma specific mRNAs prior bo bone marrow transplantations;- Potential application of antisense oligonucleotides as therapeutical agents to release the block of differentiation in this type of tumour. Results so far- Characterisation of LAN-1 cells during neuronal differentiation (WIS) During the last year, the group in the Weizmann Institute has worked out conditions for the growth of neuroblastoma cells and procedures for the induction of differentiation in these cells based on humoral and stationary inducing signals. Our results confirm that treatment with inducing agents yields highest differential capacity when laminin and/or fibronectin were used as coating material. Therefore, experiments to follow the expression of MAP proteins were performed under standard conditions using 50 mM dbcAMP as inducer and laminin / fibronectin coated culture dishes. LAN-1 cells express in addition to MAP2 variant low amounts of tau MAP protein. We have followed their abundance after differentiation both on RNA and proteins' levels by Northern blots and RNase protection assays. The level of tau mRNA increases by 5-fold following induction, while the protein level increases only 2-fold. Experiments to determine stabilisation effects on mRNA levels and efficiency of translation are planned to resolve this discrepancy.- Primary structure of neuroblastoma specific MAP2 (MPI) During the last year, the group in the Max-Planck-Institute (MPI) has identified the junctional region in the neuroblastoma specific MAP2 species and thus reached the most relevant milestone for the first year of this project. The PCR experiments provided the following results : - from calculations that were based on the prior knowledge, PCR with primer pairs 9B/9F should result only in a single band of 634 bp size, 10B/9F of 1022 bp and MTB-B/9F of 1364 bp. But in all 3 PCRs appeared and additional band, each about 340 bp smaller;- a Southern blot with these PCR gels gave two strong signals after hybridising with 32P-labelled MAP2 cDNA;- subcloning in pBluescript and sequencing of the lower band finally revealed a new splice variant of MAP2 in human neuroblastoma cells, in which nucleotides 4169 to 4509 were missing.Thus, we have identified a dimorphism in the lengths of the amplification products only with oligonucleotides hybridising 3' to position 4509 and 5' of position 4169. The larger amplification product was of the expected size for human MAP2 (Albala et al., 1994), the lower band was about 340 bp smaller. Subcloning and sequencing of both amplification products showed that both fragments were derived from human MAP2 transcripts and that indeed 340 bp are missing in the smaller amplification product. These results suggest that NB-MAP2 arises as an alternative splice form of the normal high molecular weight MAP2 isoform in neuroblastoma cells.- Production and identification monoclonal antibody directed against the NB-MAP2 specific epitope Computational translation of the human neuroblastoma specific MAP2 cDNA sequence revealed that the splice junction of human neuroblastoma specific MAP2 mRNA encodes a unique amino acid sequence motif (W V D T Q A A G G E) which is not found in normal human MAP2. We then planned to generate antibodies specific for this amino acid motif. We have generated recombinant fusion proteins harbouring the NB-MAP2 specific amino acid motif in the immunological background of mouse dehydrofolate reductase. These fusion proteins were utilised as antigens for the immunisation of mice. From the spleen cells of these mice, we generated about 20 different monoclonal antibodies specific for NB-MAP2 that have to be characterised and improved in the next year.- Outlook Elucidation of the primary structure of NB-MAP2 is a prerequisite for the following studies that are envisaged within the next years : - generation of specific monoclonal antibodies against this isoform which fulfil all the criteria required for a diagnostic tool for pathological diagnosis;- construction of oligonucleotide primers for the polymerase chain reaction to allow the most sensitive detection of transcripts encoding the neuroblastoma-specific MAP2 isoform;- to investigate in animal models whether inhibition of the expression of the neuroblastoma-specific MAP2 isoform by antisense oligonucleotides can induce differentiation of the tumour and thereby improve the patient's prognosis.The diagnostic tests are both based on the discovery that human neuroblastoma tumours express a unique microtubule-associated protein 2 (MAP2) component of 250 kDa that is not found in normal tissues or other types of tumours of neuroectodermal origin. The activities are therefore focused on :- Elucidation of the primary structure of the neuroblastoma-specific MAP2 isoform;- Generation of specific monoclonal antibodies against this isoform which fulfil all the requirements of a diagnostic tool for the pathological diagnosis;- Construction of oligonucleotide primers for the use in polymerase chain reactions to allow the most sensitive detection of transcripts encoding the neuroblastoma-specific MAP2 isoform;- Investigate in animal models whether inhibition of the expression of the neuroblastoma-specific MAP2 isoform by antisense oligonucleotides can induce differentiation of the tumour and thereby improve the patient's prognosis. Programma(i) IC-ISC C - Activity (Euratom, EEC) on cooperation in science and technology, 1984- Argomento(i) Data not available Invito a presentare proposte Data not available Meccanismo di finanziamento CSC - Cost-sharing contracts Coordinatore MAX-PLANCK-GESELLSCHAFT ZUR FOERDERUNG DER WISSENSCHAFTEN E.V. Contributo UE Nessun dato Indirizzo Deutschordenstrasse 46 60528 FRANKFURT AM-MAIN Germania Mostra sulla mappa Costo totale Nessun dato
