Studio del gene bin1 nel rabdomiosarcoma

Rhabdomyosarcoma (RMS) is the most common pediatric soft-tissue sarcoma. Although the precise cell type from which the tumor originates is still a matter of debate, the evidence points towards the myogenic lineage. RMS are divided in two main subgroups on the basis of histology: alveolar (ARMS) and embryonal (ERMS). ARMS, which is associated with a relatively high frequency of metastatic disease and needs an intensive therapeutic regimen, is the rhabdomyosarcoma subtype that carries the poorest prognosis. The most relevant feature of ARMS subtype is the presence of one of two possible chromosomal translocations, t(2;13)(q35;q14) and t(1;13)(p36;q14), which result in the expression of the chimeric PAX3/FKHR and PAX7/FKHR transcription factors, respectively. In contrast to ARMS, no specific genetic lesions have been identified in ERMS. Several studies tried to identify novel candidate genes in carcinogenesis using array-based analysis and serial analysis of gene expression. Our previous meta-analysis on four microarray and two SAGE datasets of gene expression of RMS identified common regulatory pathways that could be responsible of tumor growth. In the list of common differential expressed genes, bin1 was under-expressed in all of the studies. The bin1 (amphiphysin II) gene maps to the long arm of human chromosome 2 (2q14) and is characterized by 19 exons, six of which are alternately spliced. At least 10 different splice variants have been identified that differ widely in subcellular localization, tissue distribution, and ascribed functions. bin1 has properties of a soppressor or negative modifier in cancer: its expression is often attenuated or abolished in breast and prostate cancers, colon cancer, astrocytomas, neuroblastomas and malignant melanoma, where its ectopic expression can inhibit proliferation and/or promote apoptosis. These bin1 functions may depend on its interaction with the N-terminal region of Myc. Many studies suggest that bin1 may restrain cellular proliferation and survival in a contextual manner that is dependent on some features of neoplastic pathophysiology. While a simple and readily classifiable function has yet to emerge for BIN1 tumor soppressor proteins, current information argues that they interact with vesicular membranes and act as scaffolds to integrate celluler signalling and trafficking. My research focused on the study of bin1 gene in RMS tumor. bin1 expression level was evaluated in 8 biopsies of ARMS, which were analyzed in our previous study of expression profiling in RMS, by Sybr-Green based Real Time PCR, confirming its under-expression. Then we tested total transcripts levels in 7 RMS cell lines, 4 ARMS and 3 ERMS, and found that bin1 was significantly under-expressed respect to fetal skeletal muscle. Because different isoforms derived from bin1 gene have been described, to examine patterns of bin1 splicing, RT-PCR, cloning and sequencing techniques were performed using RNAs isolated by RC2, RH30 and RD cell lines. They represent ARMS positive for t(1;13), ARMS positive for t(2;13) and ERMS, respectively. We identified several isoforms and subsequentaly we analized protein expression levels. Previous work had indicated that bin1 mRNA and protein levels in murine skeletal muscle were higher than in most other tissues. Thus we examined BIN1 expression in 7 RMS cell lines and in C1C12 cells, a non transformed myoblast cell line derived from murine skeletal muscle, by western blot analysis. We revealed an intermediate level of BIN1 expression in RMS cell lines with respect to proliferating or differentiated C2C12. To identify the protein variants expressed in RMS cells, 293T were transiently transfected with vectors for 3 BIN1 splice isoforms and protein lysates were analysed, by immunoblotting, with 2 different BIN1 antibodies, 99D and 2F11. The second recognizes all the BIN1 isoforms while the first recognizes alternately spliced isoforms, characterized by exon 13. Only 2 variants were detected, the ubiquitous isoform BIN1-10 and the isoform associated to aberrant splicing BIN1+12A. Because specific BIN1 proteins are associated to different localization in the cell we used sub-fraction analysis and immunofluorescence technique in 7 and 2 RMS cell lines respectively and showed a BIN1 expression in the nuclear soluble fraction. Because RH30 cell line has high levels of NMYC and BIN1 interacts with c-MYC in a common portion of both proteins, we studied gene expression profiling of NMYC positive RH30 cells after infection with MSCVBIN-10 retrovirus. We identified a group 582 differentially expressed genes, 75% of them are down-regulated. The Database for Annotation, Visualization and Integrated Discovery (DAVID) visualized enriched functional-related gene groups correlated with inflammation, immunity response and cell migration. Thus BIN1-10 overexpression may have an anti-tumor role in RMS reducing inflammation and tumor invasivity and modulating immunity response by indo gene level. Other studies are warranted to confirm and integrate these data.