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>