Fusion Gene Pax3-fkhr Transfected Rhabdomyosarcoma Rd Cell Differentiation And Mrf Expression

The Expression of a number of human paired box-containing (PAX) genes has been correlated with various types of tumors. Muscle development is accurately controlled by a complicated regulating net during embryonal development Myogenic regulatory factors (MRF) are skeletal muscle-specific transcription factors, which are essential for muscle deteiminafion and differentiation. Pax3 is a important evolutionarily conserved transcription factor, which is to be upstream of controlling net in myogenic process. it is essential for the muscle precuror cells of the lateral dermomyotome to migrate to the limb buds and to contain the primary differentiation. Chromosomal t (2; 13 ) (q35; q14 ) franslocalion fuses the DNA binding domain of PAX3 with the trans-activation domain of FKHR which is a member of the forkhead domain gene family. The alveolar rhabdomyosarcoma ( ARMS ) is characterized by the specific PAX3-FKI{R chimera. In present study, influences of PAX3-FKHR on differentiation and expression of myogenic regulatory factor in human rhabdomyosarcoma were investigated after PAX3-FKHR MyoD and MRF4 cDNA were tmnsfected into human rhabdomyosarcoma cells RD. Methods: Cloning the PAX3-FKIIR cDNA into the eukaiyocyte expression vector pIRES2-EGFP; Extracting and purifying pEMSV-MyoD pEMSV-MRF4 and PEPF (pIRES2- EGFP-PAX3-FKHR) ; The pEMSV-MyoD pEMSV-MRF4 and PEPF was frnsfected by lipofectin methods into cultured rhabdomyosarcoma cells RI); MyoD mRNA and MRF4 mRNA were measured by in situ hybridization, PAX3-FKHR mRNA was measured by reverse transctiption-PCR, MHC (myosin heavy chain) and a -actin were assayed by cell immimocytochemisUy methods and semi-quantitative assay to image methods; The variation of cells growth and morphologic were obsezved by light micmscope. Result 1. Expression of MyoD and MRF4 in RD cells transfected by MyoD and MRF4 markedly elevated, cells were more regularly arranged, most cells looked like long fusiform, volume increaseed, density of cytoplasmic particle was increased, growth of cells was repressed, expression of MHC and a -actin compared with the control were significantly increased (PO.05). 3. Cells transfected with PAX3-FKHR and expressing it after expressing MyoD orMRF4 ware disorder and poly-layer, grew quick]); shapes of most cells were multi-angle shape or small round shape, density of cytoplasmic particle was decreased, expression of MyoD or MRF4 ware not inhibited, MHC and a -actin expressed hardly. No level was sigriifical higher in it than in the control (PO.O5). Conclusion: 1. Differentiation of rhabdomyosareoma cells RD could be promoted by transfected with MvoD or MRF4. 2. PAX3-FKHR were able to inhibit myogenic differentiation of MyoD/MRF4-expressing RD cells. 3. PAX3-FKI-IR didn't inhibit...