Establishment And Function Analysis Of PARP-1 Mutant Medulloblastoma Cell Lines

Objective:To establish and characterize mouse medulloblastoma(MB) cell lines with PARP-1 mutation,in order to explore the molecular machanism of MB,and to investigate the impact of PARP-1 on DNA repair proteins including Brca1,Nbs1,Ku70,Ku80 and RAD51 in the MB cells.Method:Primary culture of MB cells from PARP-1/p53 double mutant mouse MBs.Enhanced green fluorescent protein plasmid and a fluorescent PARP-1-GFP fusion protein plasmid were transfected into the MB cells using cationic liposome Lipofectamine 2000TM protocal.Cells with strong fluorescence was screened by limiting dilution for several times and then by cloning culture,stable MB cell lines expressing EGFP and PARP-1-EGFP fusion proteins was established.Immunofluorescence assay for neuronal cell-specific markers was analyzed,and cell growth,chromosome number,and the role of PARP-1 on the expression levels of protein or RNA of Brca1,Nbs1,Ku80 and Rad51 were investigated by RT-PCR and Western blot after cell cycle block.Results:The MB cell lines showed positive immunoactivity for the neuronal-specific markers such as Vimentin,Dcx andβⅢ-Tubulin,with aneuploid chromosome,and were negative for PARP-1 protein.Exogenous PARP-1 expression was visualized by immunofluorescence and Western blot analysis after pEGFP-C1-hPARP-1 transfection.In mouse MB cells,high level expression of Rad51 was showed in S and G2/M phase due to deletion of PARP-1 while the protein level of Nbs1,Ku70 and Ku80 keeps unchanged.The RNA level of Brca1 was increased in DNA synthetic phase in MB cell and PARP-1 can inhibit the transcription level of Brca1.Summary:We have established the mouse MB cell lines with defective DNA damage response and MB cell lines with stable expression of EGFP and PARP-1-EGFP fusion proteins.Activation of Rad51 and Brca1 was involved in the PARP-1 deficient MB formation.