An abnormal chromosome number, aneuploidy, is a common characteristic of tumor cells. Boveri proposed nearly 100 years ago that aneuploidy causes tumorigenesis, but this has remained untested due to the difficulty of selectively generating aneuploidy. Cells and mice with reduced levels of the mitosis-specific, centromere-linked motor protein CENP-E are now shown to develop aneuploidy and chromosomal instability in vitro and in vivo. An increased rate of aneuploidy does drive an elevated level of spontaneous lymphomas and lung tumors in aged animals. Remarkably, however, in examples of chemically or genetically induced tumor formation, an increased rate of aneuploidy is a more effective inhibitor than initiator of tumorigenesis. These findings reveal a role of aneuploidy and chromosomal instability in preventing tumorigenesis.

Cetuximab is a monoclonal antibody that targets the human epidermal growth factor receptor (EGFR). Although approved for use in EGFR-overexpressing advanced colorectal cancer, recent studies have shown a lack of association between EGFR overexpression and cetuximab response, requiring the identification of novel biomarkers predictive of response to this agent. To do so, 22 colon cancer cell lines were screened for cetuximab response in vitro and sensitive and resistant lines were identified. In sensitive cell lines, cetuximab induced a G 0 -G 1 arrest without inducing apoptosis. Notably, cetuximabsensitive but not cetuximab-resistant cell lines were preferentially responsive to EGF-stimulated growth. Whereas neither EGFR protein/mRNA expression nor gene copy number correlated with cetuximab response, examination of the mutation status of signaling components downstream of EGFR showed that cell lines with activating PIK3CA mutations or loss of PTEN expression (PTEN null) were more resistant to cetuximab than PIK3CA wild type (WT)/PTENexpressing cell lines (14 F 5.0% versus 38.5 F 6.4% growth inhibition, mean F SE; P = 0.008). Consistently, PIK3CA mutant isogenic HCT116 cells showed increased resistance to cetuximab compared with PIK3CA WT controls. Furthermore, cell lines that were PIK3CA mutant/PTEN null and Ras/BRAF mutant were highly resistant to cetuximab compared with those without dual mutations/PTEN loss (10.8 F 4.3% versus 38.8 F 5.9% growth inhibition, respectively; P = 0.002), indicating that constitutive and simultaneous activation of the Ras and PIK3CA pathways confers maximal resistance to this agent. A priori screening of colon tumors for PTEN expression status and PIK3CA and Ras/BRAF mutation status could help stratify patients likely to benefit from this therapy.

Nucleolar segregation is observed under some physiological conditions of transcriptional arrest. This process can be mimicked by transcriptional arrest after actinomycin D treatment leading to the segregation of nucleolar components and the formation of unique structures termed nucleolar caps surrounding a central body. These nucleolar caps have been proposed to arise from the segregation of nucleolar components. We show that contrary to prevailing notion, a group of nucleoplasmic proteins, mostly RNA binding proteins, relocalized from the nucleoplasm to a specific nucleolar cap during transcriptional inhibition. For instance, an exclusively nucleoplasmic protein, the splicing factor PSF, localized to nucleolar caps under these conditions. This structure also contained pre-rRNA transcripts, but other caps contained either nucleolar proteins, PML, or Cajal body proteins and in addition nucleolar or Cajal body RNAs. In contrast to the capping of the nucleoplasmic components, nucleolar granular component proteins dispersed into the nucleoplasm, although at least two (p14/ARF and MRP RNA) were retained in the central body. The nucleolar caps are dynamic structures as determined using photobleaching and require energy for their formation. These findings demonstrate that the process of nucleolar segregation and capping involves energy-dependent repositioning of nuclear proteins and RNAs and emphasize the dynamic characteristics of nuclear domain formation in response to cellular stress. INTRODUCTIONThe nucleus is a dynamic organelle consisting of interacting chromosomal and protein compartments. One of the major pathways of nuclear translocation is the movement of preribosomal particles from the nucleolus into the cytoplasm for the assembly of functional ribosomes. The main nucleolar functions involve RNA polymerase (pol) I transcription, posttranscriptional maturation of pre-rRNA transcripts and their subsequent assembly with ribosomal proteins into preribosomal particles. Other functions have been attributed to the nucleolus (for reviews, see Carmo-Fonseca et al., 2000;Olson, 2004b) and include the processing of RNA pol III transcripts, RNA editing, sequestration of cell cycle components in yeast, and Mdm2 protein in mammalian cells. The localization of telomere proteins and telomerase RNA in nucleoli suggests a role for the nucleolus in aging.Nucleolar components are found in all cells and tissues although the size, shape, and number of nucleoli may change depending on the species, cell type, and functional state. Transmission electron microscopy (TEM) has revealed three major structures within nucleoli: fibrillar centers (FC), dense fibrillar components (DFC), and the granular component (GC; for reviews, see Busch and Smetana, 1970;Goessens, 1984;Shaw and Jordan, 1995;Scheer and Hock, 1999). rDNA transcription units are found in the FC and consist of tandem repeats of these genes. rRNAs are harbored within the DFC and are processed there. It is therefore thought that rRNA transcription occurs at the interface betw...

The distribution of cytosine methylation in 6.2 Mb of the mouse genome was tested using cohybridization of genomic representations from a methylation-sensitive restriction enzyme and its methylation-insensitive isoschizomer. This assay, termed HELP (HpaII tiny fragment Enrichment by Ligation-mediated PCR), allows both intragenomic profiling and intergenomic comparisons of cytosine methylation. The intragenomic profile shows most of the genome to be contiguous methylated sequence with occasional clusters of hypomethylated loci, usually but not exclusively at promoters and CpG islands. Intergenomic comparison found marked differences in cytosine methylation between spermatogenic and brain cells, identifying 223 new candidate tissue-specific differentially methylated regions (T-DMRs). Bisulfite pyrosequencing confirmed the four candidates tested to be T-DMRs, while quantitative RT-PCR for two genes with T-DMRs located at their promoters showed the HELP data to be correlated with gene activity at these loci. The HELP assay is robust, quantitative, and accurate and is providing new insights into the distribution and dynamic nature of cytosine methylation in the genome.

Our results suggest that pretherapeutic gene expression profiling may assist in response prediction of rectal adenocarcinomas to preoperative chemoradiotherapy. The implementation of gene expression profiles for treatment stratification and clinical management of cancer patients requires validation in large, independent studies, which are now warranted.

SUMMARY Acquired chromosomal instability and copy number alterations are hallmarks of cancer. Enzymes capable of promoting site-specific copy number changes have yet to be identified. Here, we demonstrate that H3K9/36me3 lysine demethylase KDM4A/JMJD2A overexpression leads to localized copy gain of 1q12, 1q21, and Xq13.1 without global chromosome instability. KDM4A amplified tumors have increased copy gains for these same regions. 1q12h copy gain occurs within a single cell cycle, requires S phase and is not stable but regenerated each cell division. Sites with increased copy number are re-replicated and have increased KDM4A, MCM and DNA polymerase occupancy. Suv39h1/KMT1A or HP1γ overexpression suppresses the copy gain, while H3K9/K36 methylation interference promotes gain. Our results demonstrate that overexpression of a chromatin modifier results in site-specific copy gains. This begins to establish how copy number changes could originate during tumorigenesis and demonstrates that transient overexpression of specific chromatin modulators could promote these events.

Even though hematopoietic stem cell (HSC) dysfunction is presumed in myelodysplastic syndrome (MDS), the exact nature of quantitative and qualitative alterations is unknown. We conducted a study of phenotypic and molecular alterations in highly fractionated stem and progenitor populations in a variety of MDS subtypes. We observed an expansion of the phenotypically primitive long-term HSCs (lineage ؊ /CD34 ؉ /CD38 ؊ /CD90 ؉ ) in MDS, which was most pronounced in higher-risk cases. These MDS HSCs demonstrated dysplastic clonogenic activity. Examination of progenitors revealed that lower-risk MDS is

Patients with multiple endocrine neoplasia type 1 (MEN1) develop multiple endocrine tumors, primarily affecting the parathyroid, pituitary, and endocrine pancreas, due to the inactivation of the MEN1 gene. A conditional mouse model was developed to evaluate the loss of the mouse homolog, Men1, in the pancreatic beta cell. Men1 in these mice contains exons 3 to 8 flanked by loxP sites, such that, when the mice are crossed to transgenic mice expressing cre from the rat insulin promoter (RIP-cre), exons 3 to 8 are deleted in beta cells. By 60 weeks of age, >80% of mice homozygous for the floxed Men1 gene and expressing RIP-cre develop multiple pancreatic islet adenomas. The formation of adenomas results in elevated serum insulin levels and decreased blood glucose levels. The delay in tumor appearance, even with early loss of both copies of Men1, implies that additional somatic events are required for adenoma formation in beta cells. Comparative genomic hybridization of beta cell tumor DNA from these mice reveals duplication of chromosome 11, potentially revealing regions of interest with respect to tumorigenesis.Multiple endocrine neoplasia type 1 (MEN1; Online Mendelian Inheritance in Man no. 131100) is a familial tumor syndrome characterized primarily by multiple endocrine tumors of the parathyroids, anterior pituitary, and pancreas. The gene responsible for MEN1, located on chromosome 11q13 (21), was identified in 1997 by positional cloning (5), and germ line mutations in MEN1 have been found in the majority of MEN1 kindreds (1,4,27). Somatic MEN1 mutations in sporadic parathyroid adenomas, pituitary tumors, insulinomas, gastrinomas and lung carcinoids have also been reported (7,9,16,33,34). The protein product of MEN1, termed menin, is ubiquitously expressed, is targeted to the nucleus (13), and has been reported to interact with a variety of proteins, including JunD (2), Smad3 (19), Pem (22), Nm23 (25), NF-B (15), and RPA2 (32).The mouse Men1 gene has also been characterized (12, 31), and the protein product has been found to be 97% identical to human menin. As with many tumor suppressor gene knockouts, homozygous Men1 knockout mice die in utero between embryonic days 11.5 and 12.5. These mice exhibit delayed development and craniofacial abnormalities, perhaps associated with neural tube closure defects (6). This lethality has made it impossible to study the tumorigenic effects following germ line loss of both Men1 alleles. Heterozygote Men1 knockout mice develop an endocrine tumor spectrum similar to the human MEN1 phenotype, including pancreatic, pituitary, and parathyroid lesions (6).To overcome the early lethality of homozygotes and to study the function of Men1 in both the developing mouse pancreas and during tumorigenesis, we engineered specific deletion of exons 3 to 8 of the Men1 gene in the pancreatic beta cells using the cre-loxP system. Mice in which exons 3 to 8 of Men1 were flanked by loxP sites (floxed) were generated. Exons 3 to 8 were then deleted by breeding the mice with one of three independ...