Loss of Rb proteins causes genomic instability in the absence of mitogenic signaling

Abstract

Loss of G1/S control is a hallmark of cancer, and is often caused by inactivation of the retinoblastoma pathway. However, mouse embryonic fibroblasts lacking the retinoblastoma genes RB1, p107, and p130 (TKO MEFs) are still subject to cell cycle control: Upon mitogen deprivation, they enter and complete S phase, but then firmly arrest in G2. We now show that G2-arrested TKO MEFs have accumulated DNA damage. Upon mitogen readdition, cells resume proliferation, although only part of the damage is repaired. As a result, mitotic cells show chromatid breaks and chromatid cohesion defects. These aberrations lead to aneuploidy in the descendent cell population. Thus, our results demonstrate that unfavorable growth conditions can cause genomic instability in cells lacking G1/S control. This mechanism may allow premalignant tumor cells to acquire additional genetic alterations that promote tumorigenesis.

Figure 1.

Inhibition of the DDR results in accelerated cell cycle re-entry of G2-arrested TKO-Bcl2 MEFs after serum readdition. (A,B) Cell cycle distribution and percentage of mitotic cells as measured by MPM2 positivity in TKO-Bcl2 MEFs cultured in the presence or absence (w/o) of 10% FCS (for 7 d) (A), and in serum-starved TKO-Bcl2 MEF cultures restimulated with 10% FCS for 15, 18, 21, and 24 h in the presence (bottom part) or absence (top part) of caffeine (B). (C) Percentage of mitotic cells in TKO-Bcl2 MEFs at different time points after serum readdition in either the presence (black bars) or absence (white bars) of caffeine. Graph represents average values of two independent experiments; error bars show standard deviations. (D) p27^Kip1 and p21^Cip1 protein levels in TKO-Bcl2 MEFs at different time points after serum readdition in either the presence (lanes 7–11) or absence (lanes 2–6) of caffeine. Anti-γ-tubulin was used as loading control.

Figure 2.

Serum-deprived TKO-Bcl2 MEFs contain DSBs. (A) Immunofluorescent images of TKO-Bcl2 and wild-type MEFs cultured in the presence or absence of 10% FCS (for 7 d) to detect γ-H2AX and Rad51 foci. DNA was labeled with TOPRO-3. In the merge picture, DNA is blue, γ-H2AX is green, Rad51 is red, and colocalization of γ-H2AX and Rad51 is seen as yellow foci. (B) Quantification of γ-H2AX/Rad51 focus formation in TKO-Bcl2 MEFs cultured without 10% FCS for 1, 4, and 7 d. Cells were considered positive when they contained five or more superimposed γ-H2AX and Rad51 foci. At least 100 cells were counted for each condition. (C) Representative comets of nuclei of TKO-Bcl2 and wild-type Bcl2 MEFs stained with propidium iodide cultured in the presence or absence of 10% FCS (for 7 d). Cells exposed to 0.5 or 20 Gy of γ-irradiation served as controls. (D) Tail moments obtained from TKO-Bcl2 and wild-type Bcl2 MEFs cultured in the presence (untreated or γ-irradiated with 0.5 or 20 Gy) or absence of 10% FCS (for 7 d). Box plots represent interquartile ranges, horizontal bars denote the median, plus (+) indicates the mean value, and points indicate outliers. For each condition, 50 cells were analyzed using the CASP software. (E) Cell cycle distribution as determined by propidium iodide staining of TKO-Bcl2 and wild-type MEFs cultured in the presence or absence of 10% FCS (for 7 d).

Figure 3.

γ-H2AX/Rad51 foci partially dissolve when serum-restimulated TKO-Bcl2 MEFs re-enter the cell cycle. (A) Cell cycle distribution and percentage of mitotic cells in TKO-Bcl2 MEF cultures at different time points after FCS readdition. The percentages depicted in this graph represent five independent experiments. (B) Quantification of γ-H2AX/Rad51 foci in TKO-Bcl2 MEFs after FCS readdition. Cells were considered positive when they contained five or more foci positive for both γ-H2AX and Rad51. At least 100 cells were counted for each condition.

Figure 4.

Live-cell imaging of TKO-Bcl2 MEFs expressing 53BP1-GFP. (A) 53BP1-GFP foci in mitogen-deprived TKO-Bcl2 MEFs restimulated with serum for the indicated times. TKO-Bcl2 MEFs that entered mitosis are marked with ^ and +. (B) 53BP1-GFP-positive TKO-Bcl2 MEFs (marked with asterisk [*]) going through mitosis cultured in the presence of 10% FCS continuously. (C) Quantification of the number of serum-restimulated TKO-Bcl2 MEFs containing 53BP1-GFP foci before and after M phase.

Figure 5.

Serum deprivation of TKO-Bcl2 MEFs results in chromosomal aberrations. (A,B) Quantification of chromosomal breakage events in proliferating TKO-Bcl2 MEFs and wild-type MEFs (A) and serum-restimulated (21 h) TKO-Bcl2 MEFs after 7 d of mitogen deprivation and serum-restimulated wild-type MEFs after 3 d of mitogen deprivation (B). (C,D) Quantification of loss of centromeric cohesion in proliferating TKO-Bcl2 MEFs and wild-type MEFs (C) and serum-restimulated (21 h) TKO-Bcl2 MEFs after 7 d of mitogen deprivation and serum-restimulated wild-type MEFs after 3 d of mitogen deprivation (D). (A–D) Per condition, 50 metaphases were evaluated. (E,F) Chromosome spread of serum-restimulated wild-type MEFs after 3 d of mitogen deprivation (E) and serum-restimulated (21 h) TKO-Bcl2 MEFs after 7 d of mitogen deprivation (F). Arrows indicate chromatid breaks, and arrowheads indicate railroad chromosomes.

Figure 6.

Single-cell clones derived from serum-restimulated TKO-Bcl2 MEFs contain CNAs. (A,B) aCGH profiles of clones derived from TKO-Bcl2 cell cultured in 10% FCS continuously (A) or serum-restimulated TKO-Bcl2 cell (plated 48 h after serum readdition) (B). Log₂ hybridization ratios are plotted for 2803 BAC clones, represented on the CGH microarray, at their genomic position. Red dots represent amplifications >0, and green dots represent deletions <0 (Rosetta error model; P < 0.01). (C,D) M-FISH analysis of cells used in A and B, respectively.