p16(INK4a) prevents centrosome dysfunction and genomic instability in primary cells - PubMed (original) (raw)

p16(INK4a) prevents centrosome dysfunction and genomic instability in primary cells

Kimberly M McDermott et al. PLoS Biol. 2006 Mar.

Abstract

Aneuploidy, frequently observed in premalignant lesions, disrupts gene dosage and contributes to neoplastic progression. Theodor Boveri hypothesized nearly 100 years ago that aneuploidy was due to an increase in centrosome number (multipolar mitoses) and the resultant abnormal segregation of chromosomes. We performed immunocytochemistry, quantitative immunofluorescence, karyotypic analysis, and time-lapse microscopy on primary human diploid epithelial cells and fibroblasts to better understand the mechanism involved in the production of supernumerary centrosomes (more than two microtubule nucleating bodies) to directly demonstrate that the presence of supernumerary centrosomes in genomically intact cells generates aneuploid daughter cells. We show that loss of p16(INK4a) generates supernumerary centrosomes through centriole pair splitting. Generation of supernumerary centrosomes in human diploid epithelial cells was shown to nucleate multipolar spindles and directly drive production of aneuploid daughter cells as a result of unequal segregation of the genomic material during mitosis. Finally, we demonstrate that p16(INK4a) cooperates with p21 through regulation of cyclin-dependent kinase activity to prevent centriole pair splitting. Cells with loss of p16(INK4a) activity have been found in vivo in histologically normal mammary tissue from a substantial fraction of healthy, disease-free women. Demonstration of centrosome dysfunction in cells due to loss of p16(INK4a) suggests that, under the appropriate conditions, these cells can become aneuploid. Gain or loss of genomic material (aneuploidy) may provide the necessary proproliferation and antiapoptotic mechanisms needed for the earliest stages of tumorigenesis.

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Figures

Figure 1

Figure 1. vHMECs Accumulate Mitotic and Centrosome Abnormalities

(A) The solid line graph represents the in vitro growth curves of both HMECs (black circles) and vHMECs (red squares) isolated from RM16. The bar graph represents analysis of mononucleated cells containing more than two centrosomes. Centrosome number was determined by immunocytochemistry with an antibody recognizing the centrosome-associated γ-tubulin protein (excluding multinucleated cells). Cells were analyzed at multiple points along the growth curves of two different individuals (RM9 and RM16). HMEC (black) (RM9 and RM16 [less than five PD]) and vHMECs (red) analyzed at early-passage (RM9 [14 PD] and RM16 [20 PD]), late-passage (RM9 [43 PD], RM9 [65 PD]), and the agonescence (RM9 [70 PD] and RM16 [50 PD]). *Statistical significance (p < 0.005) based on comparison of HMECs to vHMECs. (B) Example of a late-passage vHMEC with a tripolar mitotic metaphase. Examples of centrosomes of HMECs (C and E) and vHMECs (D and F) detected by immunocytochemistry with antibodies recognizing the centrosome-associated γ-tubulin (C and D) and centrin (E and F) proteins. Examples of HMECs representing normal centrosomes during the G1 phase of the cell cycle (C and E, first panel), during the S and G2 phases of the cell cycle (C and E, second panel), during the M phase with centrosomes migrating to opposite poles of the cell (C and E, third and fourth panels), and vHMECs containing cells with more than two centrosomes (D and F, arrowhead). (G) Agonescent vHMECs (RM16 [47 and 50 PD]) 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 (γ-tubulin signal) of each cell was linked to that individual nucleus. Analysis included 150 to 250 cells.

Figure 2

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.

Figure 3

Figure 3. Loss of p16INK4a Uncouples the Centrosome Duplication and DNA Replication Cycles in HMECs

(A) Western blot analysis of p16INK4a expression in HMECs parental, infected with vector-only (vector), or shRNA directed against p16INK4a (p16INK4a shRNA). (B, arrowhead) Examples of more than two centrosomes in HU-exposed HMECs (p16INK4a shRNA). (C) HMECs infected with vector-only (black: RM9 [6 and 7 PD]) or p16INK4a shRNA (gray: RM9 [7 and 8 PD]) were untreated (−HU) or exposed to HU (+HU). (D) Expression levels of p16INK4a (red) in HMECs, vHMECs transfected with vector-only or p16INK4a. (E) vHMECs infected with vector-only (red: RM15 [33 to 37 PD]) or p16INK4a (gray: RM15 [33 to 37 PD]) were untreated (−HU) or exposed to HU (+HU). Centrosome number was determined by immunocytochemistry with an antibody recognizing the centrosome-associated γ-tubulin protein. Analysis included at least 100 cells. *Statistical significance (p < 0.005) based on comparison of −HU and +HU experiments.

Figure 4

Figure 4. Generation of More Than Two Centrosomes in vHMECs following S Phase Arrest Is Due to Centriole Pair Splitting

(A and B) Analysis of the centriole number in of the supernumerary centrosomes of early-passage vHMECs (RM15 [19 PD]) that express EGFP-CETN2 (centriole marker, green) untreated (−HU) or exposed to HU (+HU). Centrosome number was determined by immunocytochemistry with an antibody recognizing the centrosome-associated γ-tubulin protein (centrosome marker, red). Analysis included at least 100 cells (excluding binucleated cells). *Statistical significance (p < 0.005) based on comparison of −HU and +HU experiments. (B) Examples of HU-exposed vHMECs with one centrosome (containing a pair of centrioles), two centrosomes (each containing one centriole), three centrosomes (one of the centrosomes contains a pair of centrioles and two of the centrosomes have only one centriole), and four centrosomes (each containing one centriole). (C) Examples of HU-exposed and released vHMECs that express EGFP-CETN2 (green) and have been stained with a γ-tubulin antibody that recognizes microtubule spindles (red) that have two centrosomes, each containing two centriole (top), and supernumerary centrosomes, each containing one centriole (bottom). Supernumerary centrosomes with one centriole (arrowhead) can nucleate microtubules to form a multipolar spindle apparatus.

Figure 5

Figure 5. Supernumerary Centrosomes Produce Multipolar Spindles and Correlate with Aneuploidy

(A) BrdU incorporation of HMECs (top) and vHMECs (bottom). Cell cycle analysis were performed using BrdU incorporation for cells that were untreated (−HU), treated for 48 h with HU (+HU), or treated for 48 h with HU, followed by release from HU treatment for 7 to 8 d (+HU → release). DNA replication resumes following release from 48 h of HU treatment. (B) Microtubule nucleation sites were determined by immunocytochemistry with an antibody recognizing γ-tubulin. (C) Analysis of HMECs (black: RM9 and RM26 [less than 4 PD]) and vHMECs (red: RM15 [25 PD], RM16 [13 PD]) for multipolar mitosis in untreated (−HU) or exposed to HU followed by release from HU treatment (+HU → release). (D) Analysis of HMECs (black: RM9 and RM15 [less than 5 PD]) and vHMECs (red: RM9 [21 PD], RM16 [17 PD]), HMECs infected with p16INK4a shRNA (gray) for genomic abnormalities in untreated (−HU) or exposed to HU followed by release from HU treatment (+HU → release). Types of chromosomal abnormalities represented include aneuploidy, structural abnormalities, and telomeric associations. Analysis included at least 100 metaphases. *Statistical significance (p < 0.005) based on comparison of −HU and +HU → release experiments.

Figure 6

Figure 6. Supernumerary Centrosomes Play a Causal Role in the Production of Aneuploid Cells

(A) Early-passage vHMECs (RM18, 13–33 PD) that express EGFP–γ-tubulin (green) and EGFP-H2B (green) were stained with antibody recognizing the centrosome associated γ-tubulin protein (red) and with DAPI to localize DNA (blue). The merged image demonstrates colocalization of EGFP–γ-tubulin with the centrosome and EGFP-H2B with the DNA. (B) Examples of the mitotic progression of cells that divide into two nuclei with two centrosomes (−HU and +HU → release; bipolar, two centrosomes), that divide into two nuclei with more than two centrosomes (+HU → release; bipolar, more than two centrosomes), and that divide into greater than two nuclei with more than two centrosomes (+HU → release; multipolar, with more than two centrosomes). Arrowhead points to the EGFP–γ-tubulin signal (centrosomes). The EGFP-H2B signal was selected (pastel-colored nuclei) and the total signal intensity was quantitated. Determining the fold difference (EGFP-H2B signal intensity of daughter cells 1/EGFP-H2B signal intensity of daughter cells 2) allowed us to determine whether cells had segregated their DNA equally (fold difference close to 1.00) or unequally (fold difference >1.00). (C) Bar graph of the individual (white, black, red, and red stripe) and mean (yellow) fold differences in EGFP-H2B signals intensity between daughter cells. Standard deviations represent analysis of up to ten time frames per mitotic event. The dashed line represents the average mean fold difference (1.08) of the normal mitosis (−HU and +HU → release; bipolar, two centrosomes). *Statistical significance (p < 0.05) based on comparison of the mean fold difference (yellow bars) of cells completing mitosis with two centrosomes (−HU and +HU → release; bipolar, two centrosomes) compared to the individual and mean fold differences of cells completing mitosis with more than two centrosomes (+HU → release; bipolar or multipolar, more than two centrosomes).

Figure 7

Figure 7. Loss of p16INK4a Results in Centrosome Dysfunction and the Subsequent Generation of Aneuploid HMF and HeLa Cells

(A) Western blot analysis of p16INK4a expression in HMF and HeLa cells infected with vector-only (vector) or shRNA directed against p16INK4a (p16INK4a shRNA). (B) Examples of more than two centrosomes in HMF (p16INK4a shRNA) and HeLa (p16INK4a shRNA) cells. Centrosome number was determined by immunocytochemistry with an antibody recognizing the centrosome-associated γ-tubulin protein. (C) Analysis of parental HMF (RM9 [1 PD], RM21 [3 PD]) and HeLa cells (black) and HMF and HeLa cells infected with vector-only (HMF: RM9 [7 PD], RM21 [11 PD]) (white) or HMF and HeLa cells infected with p16INK4a shRNA (HMF: RM9 [6 PD], RM21 [7 PD]) (gray) containing mononucleated cells with more than two centrosomes. Cells were untreated (−HU) or exposed to HU (+HU). Analysis included more than two HMF and HeLa cells. *Statistical significance (p < 0.005) based on comparison of −HU and +HU experiments.

Figure 8

Figure 8. Unregulated Kinase Activity Is Responsible for the Uncoupling of the Centrosome Duplication and DNA Replication Cycles

(A and B) vHMECs (RM9 [17 PD], RM16 [20 PD]) that were untreated (−HU) or treated for 6, 12, 24, 36, and 48 h with HU (+HU) were analyzed by (A) flow cytometry of PI and BrdU incorporation (% cells arrested in S phase) and (B) confocal microscopy for mononucleated cells with more than two centrosomes. The kinase inhibitors purvalanol A (Pur) and roscovitine (Rosc) were added following the addition of HU for 36 h and incubated in the presence of HU for 12 h (B). (C) Fibroblasts that express p21 (p21+/+) and that have p21 knocked out (p21−/−) were analyzed by confocal microscopy for mononucleated cells with more than two centrosomes. Centrosome number was determined by immunocytochemistry with an antibody recognizing the centrosome associated γ-tubulin protein. Centrosome number analysis included at least 200 cells per sample (excluding binucleated cells). *Statistical significance (p < 0.005) based on comparison of −HU and +HU experiments. (D) Model of two mechanisms by which p16INK4a can mediate Cdk2 activity. The p16INK4a protein inhibits the G1 kinase activity of the cyclinD1-Cdk4 complex by binding to Cdk4 and preventing its association with cyclinD1, thereby preventing transcriptional activation of the Cdk2 binding partners cyclin E/A (Mechanism 1). The p21 kinase inhibitor is required for assembly of and stabilization of the cyclinD1-Cdk4 complex. In the presence of p16INK4a, the cyclinD1-Cdk4 complex is disrupted, causing p21 to be released. When p21 accumulates to a critical threshold, it can bind to and inhibit the late G1 and S phase kinase activity of the cylinE/A-Cdk2 complex (Mechanism 2).

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