p16INK4a Prevents Centrosome Dysfunction and Genomic Instability in Primary Cells
Figure 2
Centrosome Duplication Is Uncoupled from DNA Replication in vHMECs
(A) Schematic of the centrosome duplication cycle (interior, green) in relation to the DNA replication cycle (exterior, blue). Each daughter cell inherits one copy of the DNA and one centrosome (G1 phase of the cell cycle). Centrosomes (small, green circles) begin duplication at the same point as when DNA replication is initiated (S phase of the cell cycle). Following duplication and maturation of the centrosome, the two centrosomes separate and migrate to opposite poles during early M (mitosis).
(B) Analysis of mononucleated cells with more than two centrosomes in HMECs (black: RM9 [5 PD], RM16 [less than 4 PD]), early-passage vHMECs (red: RM9 [21 PD], RM16 [7 PD]), and low FSC/SSC sorted vHMECs (blue: RM16 [17 and 30 PD]) untreated (−HU) or exposed to HU (+HU).
(C–E) Examples of normal centrosome numbers in HMECs (C) and vHMECs (D) and more than two centrosomes in vHMECs (E).
(F) Early-passage vHMECs (RM9 [14 PD] and RM16 [17 PD]) that were exposed to HU were stained with an antibody recognizing γ-tubulin and with PI (DNA counterstain), and the DNA content of each nucleus was measured by quantitative immunofluorescence microscopy. Cells were classified as having 2N to 4N (diploid) or more than 4N (polyploidy) DNA content. The centrosome number of each cell was correlated to the DNA content of that cell. Analysis included 100 to 200 cells (excluding binucleated cells). *Statistical significance (p < 0.005) based on comparison of −HU and +HU experiments.