Objective    To characterize the effects of TPP1 knockdown on Pot1a and Pot1b localization at telomeres and on the telomere end protection.     Methods    Knockdown of endogenous TPP1 in mouse embryonic fibroblasts (MEFs) with the retrovirus vector encoding shRNA targeting TPP1

IF/PNA-FISH was performed to determine the localization of Pot1a and Pot1b at telomeres

and TdT-FITC was applied t

o characterize the effects on the function of telomere end protection

cellular senescence was analyzed by SA-beta gal staining

and phosphorylated p53

ser18

and p21 were examined by Western blotting.      Results    Pot1a and Pot1b were unable to localize at telomeres in about 65% of MEFs with TPP1 knockdown

while that was found in less than 5% of MEFs without TPP1 knockdown (t=10.96

P0.01). TdT-FITC positive cells were detected in 50% of TPP1 knockdown MEFs

as compared with that of 4.7% in the control MEFs (t=14.37

P0.01). Furthermore

90% of TdT signals appeared to co-localize with telomeres in TPP1 knockdown cells

which was significantly higher than that of 3% in the control MEFs (t=17.59

P0.01). The SA-beta gal positive cells

a hallmark of cellular senescence

were found in 25.7% and 22.0% of TPP1 knockdown cells by shTPP1-1 or shTPP1-2

paralleled with marked induction of p53

ser18

phosphorylation and p21 expression in those cells. Whereas

cellular senescence occurred only in 1.7% of the control MEFs (t=18.23

P0.01; t=16.9

P0.01)

and increased p53

ser18

phosphorylation and p21 expression were not detected.     Conclusion    TPP1 is required for Pot1a and Pot1b localization at telomeres

and knockdown of TPP1 results in telomere end accessible and elicits p53-dependent cellular senescence.