PAX3 function in embryonic cell lines undergoing different differentiation pathways and their corresponding tumours


Year:

Session type:

Patricia Kumar

Manchester Metropolitan University, UK

Abstract

Background

PAX3 is a gene transiently expressed in the neural crest during embryonic development. It is abnormally re-expressed in several tumours derived from the neural crest and dorsal dermomyotome, including melanoma, neuroblastoma and rhabdomyosarcoma. In alveolar rhabdomyosarcoma it is translocated to another chromosome (t2q35:13q14), forming a hybrid PAX3-FKHR gene, which confers more aggressive properties than PAX3 overexpression alone.

We are studying PAX3 overexpression in the above tumours, with the objective of identifying the genes which it up-/down-regulates, in order to seek new molecular targets for tumour therapy. It might be expected that genes up-regulated by PAX3 in a non-malignant cell will be down-regulated after siRNA treatment of the corresponding tumour, if the genes are important to tumourigenesis. It might be expected also that PAX3 controls different genes in different cell differentiation pathways: melanocyte, neuronal or muscle. Genes having great significance for tumourigenesis might be expected to be regulated similarly in all three differentiation pathways. In this way we hope to find genes with fundamental significance for these tumours.

Method

We have isolated and cloned seven PAX3 splice variants. Three of these (PAX3c, e and g) have been transfected singly into murine melanocytes, embryonic stem cells and myoblasts, which represent non-malignant potential precursor cells for melanoma, neuroblastoma and rhabdomyosarcoma. We have used RNA interference (siRNA) to decrease PAX3 expression in the above tumours, followed by microarray analysis. Genes of interest have their status confirmed by semi-quantitative PCR and western blotting.

Results

Affymetrix microarrays have allowed us to compare cells before and after PAX3 transfection and identify genes up- or down-regulated by PAX3.

Their function will be investigated, by trial up-/downregulation, whichever is appropriate, in cell lines derived from melanoma, neuroblastoma and rhabdomyosarcoma, in order to discover their potential for tumour therapy.