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 À/À 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 +/À 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.[Keywords: Cohesion; condensation; chromosome instability; cell cycle; chromatin] Supplemental material is available at http://www.genesdev.org.

Genome instability is a characteristic of malignant cells; however, evidence for its contribution to tumorigenesis has been enigmatic. In this study, we demonstrate that the retinoblastoma protein, E2F1, and Condensin II localize to discrete genomic locations including major satellite repeats at pericentromeres. In the absence of this complex, aberrant replication ensues followed by defective chromosome segregation in mitosis. Surprisingly, loss of even one copy of the retinoblastoma gene reduced recruitment of Condensin II to pericentromeres and caused this phenotype. Using cancer genome data and gene-targeted mice, we demonstrate that mutation of one copy of RB1 is associated with chromosome copy-number variation in cancer. Our study connects DNA replication and chromosome structure defects with aneuploidy through a dosage-sensitive complex at pericentromeric repeats. SIGNIFICANCE:Genome instability is inherent to most cancers and is the basis for selective killing of cancer cells by genotoxic therapeutics. In this report, we demonstrate that instability can be caused by loss of a single allele of the retinoblastoma gene that prevents proper replication and condensation of pericentromeric chromosomal regions, leading to elevated levels of aneuploidy in cancer. Cancer Discov; 4(7); 840-53.

Transforming growth factor ␤ (TGF-␤) is a crucial mediator of breast development, and loss of TGF-␤-induced growth arrest is a hallmark of breast cancer. TGF-␤ has been shown to inhibit cyclin-dependent kinase (CDK) activity, which leads to the accumulation of hypophosphorylated pRB. However, unlike other components of TGF-␤ cytostatic signaling, pRB is thought to be dispensable for mammary development. Using gene-targeted mice carrying subtle missense changes in pRB (Rb1 ⌬L and Rb1 NF ), we have discovered that pRB plays a critical role in mammary gland development. In particular, Rb1 mutant female mice have hyperplastic mammary epithelium and defects in nursing due to insensitivity to TGF-␤ growth inhibition. In contrast with previous studies that highlighted the inhibition of cyclin/CDK activity by TGF-␤ signaling, our experiments revealed that active transcriptional repression of E2F target genes by pRB downstream of CDKs is also a key component of TGF-␤ cytostatic signaling. Taken together, our work demonstrates a unique functional connection between pRB and TGF-␤ in growth control and mammary gland development.

The concept that aneuploidy is a characteristic of malignant cells has long been known; however, the idea that aneuploidy is an active contributor to tumorigenesis, as opposed to being an associated phenotype, is more recent in its evolution. At the same time, we are seeing the emergence of novel roles for tumor suppressor genes and oncogenes in genome stability. These include the adenomatous polyposis coli gene (APC), p53, the retinoblastoma susceptibility gene (RB1), and Ras. Originally, many of these genes were thought to be tumor suppressive or oncogenic solely because of their role in proliferative control. Because of the frequency with which they are disrupted in cancer, chromosome instability caused by their dysfunction may be more central to tumorigenesis than previously thought. Therefore, this review will highlight how the proper function of cell cycle regulatory genes contributes to the maintenance of genome stability, and how their mutation in cancer obligatorily connects proliferation and chromosome instability.

Organization of chromatin structure is indispensible to the maintenance of genome integrity. The retinoblastoma tumor suppressor protein (pRB) mediates both transcriptional repression and chromatin organization, but the independent contributions of these functions have been difficult to study. Here, we utilize a synthetic Rb1 mutant allele (F832A) that maintains pRB association at cell cycle gene promoters, but disrupts a cyclin-dependent kinase (CDK)-resistant interaction with E2F1 to reduce occupancy of pRB on intergenic chromatin. Reduced pRB chromatin association increases spontaneous γH2AX deposition and aneuploidy. Our data indicates that the CDK-resistant pRB-E2F1 scaffold recruits Condensin II to major satellite repeats to stabilize chromatin structure in interphase and mitosis through mechanisms that are distinct from silencing of repetitive sequence expression.

A fundamental need in the analysis of the cell cycle is the ability to isolate relatively homogeneous populations of cells in different phases. This is complicated by the variable proliferative properties and responses to synchronizing methods of different cancer-derived cell lines. Paradoxically, cell lines with genetic defects in cell cycle control are sometimes chosen because they are amenable to chemical synchronization. Embryonic fibroblasts from mice present the opportunity to study the effects of defined genetic modifications on a normal cell cycle. However, synchronization of these cells has often been challenging. In this chapter we outline three basic protocols for isolating mouse fibroblasts at the G1-to-S-phase transition, in S phase, and during mitosis.

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<p>PDF file 3766K, Analysis of gammaH2AX distribution, chromosomal instability phenotypes in heterozygous cells, and verification of genotypes in human cells</p>