p16(INK4a) Prevents Centrosome Dysfunction and Genomic Instability in Primary Cells

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

Loss of chromosomal integrity in human mammary epithelial cells subsequent to escape from senescence

The genomic changes that foster cancer can be either genetic or epigenetic in nature. Early studies focused on genetic changes and how mutational events contribute to changes in gene expression. These point mutations, deletions and amplifications are known to activate oncogenes and inactivate tumor suppressor genes. More recently, multiple epigenetic changes that can have a profound effect on carcinogenesis have been identified. These epigenetic events, such as the methylation of promoter sequences in genes, are under active investigation. In this review we will describe a methylation event that occurs during the propagation of human mammary epithelial cells (HMEC) in culture and detail the accompanying genetic alterations that have been observed

Loss of p16^INK4a Results in Centrosome Dysfunction and the Subsequent Generation of Aneuploid HMF and HeLa Cells

<div><p>(A) Western blot analysis of p16^INK4a expression in HMF and HeLa cells infected with vector-only (vector) or shRNA directed against p16^INK4a (p16^INK4a shRNA).</p> <p>(B) Examples of more than two centrosomes in HMF (p16^INK4a shRNA) and HeLa (p16^INK4a shRNA) cells. Centrosome number was determined by immunocytochemistry with an antibody recognizing the centrosome-associated γ-tubulin protein.</p> <p>(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 p16^INK4a 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 (<i>p</i> < 0.005) based on comparison of −HU and +HU experiments.</p></div

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

<div><p>(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 (<i>p</i> < 0.005) based on comparison of −HU and +HU experiments.</p> <p>(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).</p> <p>(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.</p></div

vHMECs Accumulate Mitotic and Centrosome Abnormalities

<div><p>(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 (<i>p</i> < 0.005) based on comparison of HMECs to vHMECs.</p> <p>(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).</p> <p>(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.</p></div