Mitotic chromosome condensation mediated by the retinoblastoma protein is tumor-suppressive

Abstract

Condensation and segregation of mitotic chromosomes is a critical process for cellular propagation, and, in mammals, mitotic errors can contribute to the pathogenesis of cancer. In this report, we demonstrate that the retinoblastoma protein (pRB), a well-known regulator of progression through the G1 phase of the cell cycle, plays a critical role in mitotic chromosome condensation that is independent of G1-to-S-phase regulation. Using gene targeted mutant mice, we studied this aspect of pRB function in isolation, and demonstrate that it is an essential part of pRB-mediated tumor suppression. Cancer-prone Trp53(-/-) mice succumb to more aggressive forms of cancer when pRB's ability to condense chromosomes is compromised. Furthermore, we demonstrate that defective mitotic chromosome structure caused by mutant pRB accelerates loss of heterozygosity, leading to earlier tumor formation in Trp53(+/-) mice. These data reveal a new mechanism of tumor suppression, facilitated by pRB, in which genome stability is maintained by proper condensation of mitotic chromosomes.

Figure 1.

Rb1^ΔL/ΔL cells display aberrant chromosome condensation and segregation. (A) Metaphase chromosome spreads were prepared from MEFs or ESCs of the indicated genotypes. Chromosomes were stained with DAPI (blue) and a probe for major satellite repeats (green) to mark centromeres. Red arrows indicate contact between centromeres from different chromosomes, and yellow arrows indicate centromere contact involving three or more chromosomes. Inlays highlight expanded views of select chromosomes. Bars, 25 μm. (B) The frequency of centromere interactions per mitosis is plotted for each genotype (Rb1^ΔL/ΔL and Rb1^+/+) and cell type (MEF and ESC). (Right graph) In addition, the number of centromeres involved in each interaction was determined for ESC metaphase spreads, and is displayed as the frequency of multiple chromosome interactions. (C) Video microscopy was performed on MEFs expressing an H2B-GFP reporter by capturing phase-contrast and GFP images every 3 min over a 15-h time course. The images shown begin with the onset of chromatin condensation in prophase as the left-most panel. The last image of the metaphase plate before the onset of anaphase is indicated along with the elapsed time since the onset of prophase (in minutes). The right-most image shows resolved daughter (or binucleated) cells. The numbers in the left-most image correspond to references in the Supplemental Material and Supplemental Movies. Bars, 50 μm.

Figure 2.

Defective loading of Condensin II complexes on RB1^ΔL/ΔL chromosomes. (A) Chromatin fractions were prepared from MEFs that were either proliferating asynchronously or enriched for M-phase cells. Protein content in these fractions was analyzed by SDS-PAGE, followed by Coomassie staining for histone proteins, or Western blotting for the indicated components of the Cohesin and Condensin complexes. (B) Wild-type MEFs were transduced with retroviruses expressing the indicated shRNAs and H2B-GFP. Cell extracts were analyzed by Western blotting for CAP-D3 and Actin. (UT) Untransduced cells. (C) Video microscopy was performed on cells expressing either a control luciferase shRNA, or shRNAs directed against CAP-D3 (sh63 and sh64). Phase-contrast and GFP images were taken every 3 min for 15 h. Representative pictures include the onset of prophase in the left-most panel, and the last view of the metaphase plate before anaphase along with the time elapsed from prophase. The last frame on the right shows the cells after either cytokinesis (shLuc), or failure to resolve as two daughter cells (resulting in binucleation [sh63]) or as persistent anaphase bridges (sh64). The numbers in the left-most image correspond to references in the Supplemental Material and Supplemental Movies. Bars, 50 μm. (D) Extracts were prepared from MEFs of the indicated genotypes. Chromatin fractions from these cells were then subjected to immunoprecipitation with anti-pRB antibodies. The relative amount of CAP-D3, precipitated with wild-type and mutant pRB, was detected by Western blotting. Input levels of relevant proteins from chromatin fractions are shown, and the CAP-D3 blot is overexposed to demonstrate that Condensin II complexes are present in the Rb1 mutant input.

Figure 3.

More aggressive tumors in Rb1^ΔL/ΔL; Trp53^−/− mice. (A) Kaplan-Meier survival proportions are shown for Rb1^ΔL/ΔL; Trp53^−/− (n = 45) and Trp53^−/− (n = 35) mice. (B–I) Representative images of cancers found in Trp53^−/− control and Rb1^ΔL/ΔL; Trp53^−/− compound mutants. (B) This necropsy reveals lymphoma with multiple affected lymph nodes, as indicated by arrows. (C) H&E staining of a tissue section from one of the affected lymph nodes from B. (D) Mouse with thymic lymphoma and a sarcoma near its left forelimb; both are indicated by arrows. (E) Histological analysis of the sarcoma in D reveals extensive infiltration of this tumor by cells from the neighboring lymphoma. (F) Necropsy demonstrates an enlarged liver in this Rb1^ΔL/ΔL; Trp53^−/− mutant mouse. (G) Histology of the liver in F reveals extensive infiltration by lymphocytes, indicative of metastasis. (H) Necropsy of a Trp53^−/− control mouse shows an enlarged thymus that is typical of these mice. (I) This micrograph shows H&E staining of a thymic lymphoma from a Trp53^−/− mouse. Bars: B,D,F,H, 1 cm; C,E,G,I, 100 μm.

Figure 4.

Increased genomic instability in Rb1^ΔL/ΔL; Trp53^−/− thymic lymphoma. (A) Kaplan-Meier survival proportions are shown for Rb1^ΔL/ΔL; Trp53^−/− (n = 18) and Trp53^−/− (n = 32) mice that succumbed to thymic lymphoma. (B) Schematic diagram of the T-cell receptor β (TCR_β) locus that was PCR-amplified to assess clonality of thymic lymphomas. Primer pairs 1 and 4 and 2 and 3 were used in a nested strategy to amplify rearranged forms of this gene found in tumor samples, as described in the Materials and Methods. (C) Agarose gel electrophoresis of T-cell receptor β (TCR_β) PCR, including a water-only negative control, and three normal thymus samples as positive controls. Four-digit numbers correspond to ear tag numbers for individual mice, and are present to allow correlations with pathology and array data in Supplemental Tables 1 and 2. The asterisks indicate samples that were used for aCGH analysis. (D) Control, or tumor DNA versus control, was used for hybridization to whole-genome arrays to determine regions of gain or loss in thymic lymphoma samples. Representative graphs depicting log₂ ratio values plotted against chromosome number are shown. Data points from individual chromosomes are shown in different colors. (E) Whole-chromosome gains and losses were inferred by differences in an entire chromosome and were compared with controls. The number of whole-chromosome changes for each tumor is plotted against their genotypes. The control male versus control male hybridization is shown in blue, the male versus female hybridizations are shown in yellow, and Trp53^−/− and Rb1^ΔL/ΔL; Trp53^−/− samples are denoted by red and green, respectively. The analysis of all chromosomes, or autosomes alone, are shown. The mean number of changes was compared between genotypes using a t-test.

Figure 5.

Accelerated loss of heterozygosity in Rb1^ΔL/ΔL; Trp53^+/− mice. (A) Kaplan-Meier survival proportions are shown for Rb1^ΔL/ΔL; Trp53^+/− (n = 24) and Trp53^+/− (n = 25) mice that succumbed to detectable cancers. (B) Southern blot analysis of tumors from Rb1^ΔL/ΔL; Trp53^+/− and Trp53^+/− mice was performed to assess the relative abundance of wild-type and null Trp53 alleles. Four-digit numbers correspond to ear tags for individual mice to allow correlation with pathology data in Supplemental Table 1. The ratio of mutant to wild-type allele abundance was determined by phosphorimaging, and is displayed below each lane. (C) Model of the Rb1^ΔL mutation's role in cancer susceptibility of these mice. Prior reports establish that Trp53^+/− mice succumb to cancer after a long latency, and that it is accompanied by loss of heterozygosity at the Trp53 locus. The age at which cancer initiates in Rb1^ΔL/ΔL; Trp53^+/− mice and the loss of the wild-type Trp53 locus in these tumors suggests that chromosome instability, caused by the Rb1^ΔL mutation, induces loss of heterozygosity more rapidly, causing an earlier onset of cancer.