(D) FACS analysis of G 1 and S phases in Sc and shLB1 cells. (C) FACS analysis of the complete cell cycle. Error bars represent standard deviations. Proliferation rates were determined as described in Materials and Methods. (B) The proliferation rates of shLB1 and Sc cells were compared for 5 days following silencing and selection. (A) LB1, LB2, LA, and LC protein levels were measured by immunoblotting at PD3 following silencing and selection. Reduction of LB1 levels slows proliferation and DNA replication in U-2-OS and HCT116 cell lines. Taken together, the results suggest that the maintenance of lamin B1 levels is required for DNA replication and repair through regulation of the expression of key factors involved in these essential nuclear functions.Ĭopyright © 2015, American Society for Microbiology. We further demonstrate that lamin B1 interacts directly with the promoters of some genes associated with DNA damage response and repair, including BRCA1 and RAD51. The expression of multiple factors involved in DNA replication and repair by both nonhomologous end joining and homologous repair is misregulated when lamin B1 levels are reduced. The double-strand DNA breaks resulting from replication fork collapse were inefficiently repaired, causing persistent DNA damage signaling and the assembly of extensive repair foci on chromatin. The S phase delay appears to be due to the stalling and collapse of replication forks. We demonstrate that reducing nuclear lamin B1 expression by short hairpin RNA-mediated silencing in cancer cell lines to approximately 50% of normal levels causes a delay in the cell cycle and accumulation of cells in early S phase. Nuclear lamins play important roles in the organization and structure of the nucleus however, the specific mechanisms linking lamin structure to nuclear functions are poorly defined.
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