ABSTRACT
When explanted into culture, normal human cells exhibit a finite number of cell divisions before entering a proliferative arrest termed replicative senescence. To identify genes essential for entry into replicative senescence, we performed an RNA interference (RNAi)-based loss-of-function screen and found that suppression of the Never in Mitosis Gene A (NIMA)-related protein kinase gene NEK4 disrupted timely entry into senescence. NEK4 suppression extended the number of population doublings required to reach replicative senescence in several human fibroblast strains and resulted in decreased transcription of the cyclin-dependent kinase inhibitor p21. NEK4-suppressed cells displayed impaired cell cycle arrest in response to double-stranded DNA damage, and mass spectrometric analysis of Nek4 immune complexes identified a complex containing DNA-dependent protein kinase catalytic subunit [DNA-PK(cs)], Ku70, and Ku80. NEK4 suppression causes defects in the recruitment of DNA-PK(cs) to DNA upon induction of double-stranded DNA damage, resulting in reduced p53 activation and H2AX phosphorylation. Together, these observations implicate Nek4 as a novel regulator of replicative senescence and the response to double-stranded DNA damage.
Subject(s)
Cellular Senescence , DNA Damage , Fibroblasts/cytology , Protein Serine-Threonine Kinases/metabolism , Cell Cycle Checkpoints , Cell Line , Cell Proliferation , Cells, Cultured , Cyclin-Dependent Kinase Inhibitor p21/genetics , DNA/metabolism , DNA-Activated Protein Kinase/metabolism , Down-Regulation , Fibroblasts/metabolism , Humans , NIMA-Related Kinases , Protein Interaction Maps , Protein Serine-Threonine Kinases/genetics , Telomerase/metabolism , Telomere/metabolism , Transcription, GeneticABSTRACT
POT1 is a 3' telomeric single-stranded overhang binding protein that has been implicated in chromosome end protection, the regulation of telomerase function, and defining the 5' chromosome terminus. In human cancer cells that exhibit constitutive hTERT activity, hPOT1 exerts control over telomere length. Primary human fibroblasts express low levels of catalytically active hTERT in an S-phase-restricted manner that fails to counteract telomere attrition with cell division. Here, we show that diploid human fibroblasts in which hPOT1 expression has been suppressed harbor telomeres that are longer than control cells. This difference in telomere length delays the onset of replicative senescence and is dependent on S-phase-restricted hTERT expression. These findings are consistent with the view that hPOT1 promotes a nonextendable telomere state resistant to extension by S-phase-restricted telomerase. Manipulating this function of hPOT1 may thus hasten the cytotoxic effects of telomerase inhibition.
Subject(s)
Diploidy , Fibroblasts/enzymology , Suppression, Genetic , Telomerase/metabolism , Telomere-Binding Proteins/metabolism , Telomere/metabolism , Cell Proliferation , Cells, Cultured , Cellular Senescence , Fibroblasts/cytology , Gene Expression Regulation , Humans , Male , RNA, Messenger/genetics , RNA, Messenger/metabolism , S Phase , Shelterin Complex , Telomerase/antagonists & inhibitors , Telomere-Binding Proteins/genetics , Time FactorsABSTRACT
The transition of ductal carcinoma in situ (DCIS) to invasive carcinoma is a poorly understood key event in breast tumor progression. Here, we analyzed the role of myoepithelial cells and fibroblasts in the progression of in situ carcinomas using a model of human DCIS and primary breast tumors. Progression to invasion was promoted by fibroblasts and inhibited by normal myoepithelial cells. Molecular profiles of isolated luminal epithelial and myoepithelial cells identified an intricate interaction network involving TGFbeta, Hedgehog, cell adhesion, and p63 required for myoepithelial cell differentiation, the elimination of which resulted in loss of myoepithelial cells and progression to invasion.
