Cell division at the end of mitosis and G1 is controlled by Cdh1/Hct1, an activator of the E3-ubiquitin ligase anaphase-promoting complex (APC) that promotes the ubiquitylation and degradation of mitotic cyclins and other substrates. Cdh1-APC is active in postmitotic neurons, where it regulates axonal growth and patterning in the developing brain. However, it remains unknown whether Cdh1-APC is involved in preventing cell-cycle progression in terminally differentiated neurons. To address this issue, we used the small hairpin RNA strategy to deplete Cdh1 in postmitotic neurons. We observed that Cdh1 silencing rapidly triggered apoptotic neuronal death. To investigate the underlying mechanism, we focused on cyclin B1, a major Cdh1-APC substrate. Our results demonstrate that Cdh1 is required to prevent the accumulation of cyclin B1 in terminally differentiated neurons. Moreover, by keeping cyclin B1 low, Cdh1 prevented these neurons from entering an aberrant S phase that led to apoptotic cell death. These results provide an explanation for the mechanism of cyclin B1 reactivation that occurs in the brain of patients suffering from neurodegenerative diseases, such as Alzheimer's disease.

Anillin, an actin-binding protein localized at the cleavage furrow, is required for cytokinesis. Through an in vitro expression screen, we identified anillin as a substrate of the anaphase-promoting complex/cyclosome (APC/C), a ubiquitin ligase that controls mitotic progression. We found that the levels of anillin fluctuate in the cell cycle, peaking in mitosis and dropping drastically during mitotic exit. Ubiquitination of anillin required a destruction-box and was mediated by Cdh1, an activator of APC/C. Overexpression of Cdh1 reduced the levels of anillin, whereas inactivation of APC/C(Cdh1) increased the half-life of anillin. Functionally, anillin was required for the completion of cytokinesis. In anillin knockdown cells, the cleavage furrow ingressed but failed to complete the ingression. At late cytokinesis, the cytosol and DNA in knockdown cells underwent rapid myosin-based oscillatory movement across the furrow. During this movement, RhoA and active myosin were absent from the cleavage furrow, and myosin was redistributed to cortical patches, which powers the random oscillatory movement. We concluded that anillin functions to maintain the localization of active myosin, thereby ensuring the spatial control of concerted contraction during cytokinesis.

The anaphase-promoting complex/cyclosome (APC/C) regulates sister chromatid segregation and the exit from mitosis. Selection of most APC/C substrates is controlled by coactivator subunits (either Cdc20 or Cdh1) that interact with substrate destruction motifs--predominantly the destruction (D) box and KEN box degrons. How coactivators recognize D box degrons and how this is inhibited by APC/C regulatory proteins is not defined at the atomic level. Here, from the crystal structure of S. cerevisiae Cdh1 in complex with its specific inhibitor Acm1, which incorporates D and KEN box pseudosubstrate motifs, we describe the molecular basis for D box recognition. Additional interactions between Acm1 and Cdh1 identify a third protein-binding site on Cdh1 that is likely to confer coactivator-specific protein functions including substrate association. We provide a structural rationalization for D box and KEN box recognition by coactivators and demonstrate that many noncanonical APC/C degrons bind APC/C coactivators at the D box coreceptor.

The p27(Kip1) ubiquitin ligase receptor Skp2 is often overexpressed in human tumours and displays oncogenic properties. The activity of SCF(Skp2) is regulated by the APC(Cdh1), which targets Skp2 for degradation. Here we show that Skp2 phosphorylation on Ser64/Ser72 positively regulates its function in vivo. Phosphorylation of Ser64, and to a lesser extent Ser72, stabilizes Skp2 by interfering with its association with Cdh1, without affecting intrinsic ligase activity. Cyclin-dependent kinase (CDK)2-mediated phosphorylation of Skp2 on Ser64 allows its expression in mid-G1 phase, even in the presence of active APC(Cdh1). Reciprocally, dephosphorylation of Skp2 by the mitotic phosphatase Cdc14B at the M ->> G1 transition promotes its degradation by APC(Cdh1). Importantly, lowering the levels of Cdc14B accelerates cell cycle progression from mitosis to S phase in an Skp2-dependent manner, demonstrating epistatic relationship of Cdc14B and Skp2 in the regulation of G1 length. Thus, our results reveal that reversible phosphorylation plays a key role in the timing of Skp2 expression in the cell cycle.

SCF-type (SCF: Skp1-Cullin-F-box protein complex) E3 ligases regulate ubiquitin-dependent degradation of many cell cycle regulators, mainly at the G1/S transition. Here, we show that SCF(Grr1) functions during cytokinesis by degrading the PCH protein Hof1. While Hof1 is required early in mitosis to assemble a functional actomyosin ring, it is specifically degraded late in mitosis and remains unstable during the entire G1 phase of the cell cycle. Degradation of Hof1 depends on its PEST motif and a functional 26S proteasome. Interestingly, degradation of Hof1 is independent of APC(Cdh1), but instead requires the SCF(Grr1) E3 ligase. Grr1 is recruited to the mother-bud neck region after activation of the mitotic-exit network, and interacts with Hof1 in a PEST motif-dependent manner. Our results also show that downregulation of Hof1 at the end of mitosis is necessary to allow efficient contraction of the actomyosin ring and cell separation during cytokinesis. SCF(Grr1)-mediated degradation of Hof1 may thus represent a novel mechanism to couple exit from mitosis with initiation of cytokinesis.

An unselected series of 310 colorectal carcinomas, stratified according to microsatellite instability (MSI) and DNA ploidy, was examined for mutations and/or promoter hypermethylation of five components of the WNT signaling cascade [APC, CTNNB1 (encoding beta-catenin), AXIN2, TCF4, and WISP3] and three genes indirectly affecting this pathway [CDH1 (encoding E-cadherin), PTEN, and TP53]. APC and TP53 mutations were each present more often in microsatellite-stable (MSS) tumors than in those with MSI (P < .001 for both). We confirmed that the aneuploid MSS tumors frequently contained TP53 mutations (P < .001), whereas tumors with APC mutations and/or promoter hypermethylation revealed no associations to ploidy. Mutations in APC upstream of codons 1020 to 1169, encoding the beta-catenin binding site, were found in 15/144 mutated tumors and these patients seemed to have poor clinical outcome (P = .096). Frameshift mutations in AXIN2, PTEN, TCF4, and WISP3 were found in 20%, 17%, 46%, and 28% of the MSI tumors, respectively. More than half of the tumors with heterozygote mutations in AXIN2 were concurrently mutated in APC. The present study showed that more than 90% of all samples had alteration in one or more of the genes investigated, adding further evidence to the vital importance of activated WNT signaling in colorectal carcinogenesis.

OBJECTIVE

In an effort to additionally determine the global patterns of CpG island hypermethylation in sporadic breast cancer, we searched for aberrant promoter methylation at 10 gene loci in 54 primary breast cancer and 10 breast benign lesions.

METHODS

Genomic DNA sodium bisulfate converted from benign and malignant tissues was used as template in methyl-specific PCR for BRCA1, p16, ESR1, GSTP1, TRbeta1, RARbeta2, HIC1, APC, CCND2, and CDH1 genes.

RESULTS

The majority of the breast cancer (85%) showed aberrant methylation in at least 1 of the loci tested with half of them displaying 3 or more methylated genes. The highest frequency of aberrant promoter methylation was found for HIC1 (48%) followed by ESR1 (46%), and CDH1 (39%). Similar methylation frequencies were detected for breast benign lesions with the exception of the CDH1 gene (P = 0.02). The analysis of methylation distribution indicates a statistically significant association between methylation of the ESR1 promoter, and methylation at CDH1, TRbeta1, GSTP1, and CCND2 loci (P < 0.03). Methylated status of the BRCA1 promoter was inversely correlated with methylation at the RARbeta2 locus (P < 0.03).

CONCLUSIONS

Our results suggest a nonrandom distribution for promoter hypermethylation in sporadic breast cancer, with tumor subsets characterized by aberrant methylation of specific cancer-related genes. These breast cancer subgroups may represent separate biological entities with potential differences in sensitivity to therapy, occurrence of metastasis, and overall prognosis.

Genetic alterations occur during the adenoma-carcinoma sequence of colon cancer formation and drive the initiation and progression of colon cancer formation. The aberrant methylation of genes is an alternate, epigenetic mechanism for silencing tumor suppressor genes in colon cancer. The aim of this study was to determine on a global and gene-specific level the role of CpG island methylation in the initiation and progression of colon cancer. Consequently, we assessed the frequency of gene methylation in tumors representative of the commonly recognized histological steps of the adenoma-carcinoma progression sequence through the analysis of eight genes previously identified to be methylated in colon cancer, MGMT, HLTF, MLH1, p14(ARF), CDKN2A, TIMP3, THBS1, and CDH1. We observed that the proportion of tumors carrying methylated alleles increased from adenomas to adenocarcinomas but that the proportion of tumors with methylated alleles was not different between adenocarcinomas and metastases (69% versus 90%, P = 0.01 and 90% versus 81%, P>> 0.05). The most substantial difference occurred between early and advanced adenomas (47% versus 84%, P = 0.018). Furthermore, we observed that the frequency of gene methylation at the different steps of the progression sequence varied between genes. Thus, the aberrant methylation of genes appears to increase most significantly during the progression of early adenomas to advanced adenomas, and the frequency of specific gene methylation at the different steps of the adenoma-carcinoma progression sequence varies in a gene-specific fashion.

How hypermethylation and hypomethylation of different parts of the genome in cancer are related to each other and to DNA methyltransferase (DNMT) gene expression is ill defined. We used ovarian epithelial tumors of different malignant potential to look for associations between 5'-gene region or promoter hypermethylation, satellite, or global DNA hypomethylation, and RNA levels for ten DNMT isoforms. In the quantitative MethyLight assay, six of the 55 examined gene loci (LTB4R, MTHFR, CDH1CDH1, and IGSF4) were significantly hypermethylated relative to the degree of malignancy (after adjustment for multiple comparisons; P < 0.001). Importantly, hypermethylation of these genes was associated with degree of malignancy independently of the association of satellite or global DNA hypomethylation with degree of malignancy. Cancer-related increases in methylation of only two studied genes, LTB4R and MTHFR, which were appreciably methylated even in control tissues, were associated with DNMT1 RNA levels. Cancer-linked satellite DNA hypomethylation was independent of RNA levels for all DNMT3B isoforms, despite the ICF syndrome-linked DNMT3B deficiency causing juxtacentromeric satellite DNA hypomethylation. Our results suggest that there is not a simple association of gene hypermethylation in cancer with altered DNMT RNA levels, and that this hypermethylation is neither the result nor the cause of satellite and global DNA hypomethylation.

E-Cadherin alterations have been reported frequently in sporadic diffuse type gastric and lobular breast carcinomas. Germline mutations of this gene have been identified recently in several gastric cancer families. We analyzed seven patients with a family history of the disease who had diffuse type gastric cancer diagnosed before the age of 45 for germline mutations in CDH1, the gene encoding the E-cadherin protein. We identified a frameshift mutation in exon 3 in one patient with a strong family history of gastric cancer. The same germline mutation was found in the patient's mother, who had metachronous development of lobular breast and diffuse type gastric carcinomas. Immunohistochemistry for E-cadherin protein expression revealed an abnormal staining pattern in both of these tumors, suggesting complete inactivation of the cell adhesion molecule. Thus, our finding suggests that besides diffuse type gastric cancer, lobular breast carcinomas may be associated with germline CDH1 mutations.

OBJECTIVE

To identify germ line CDH1 mutations in hereditary diffuse gastric cancer (HDGC) families and develop guidelines for management of at risk individuals.

METHODS

We ascertained 31 HDGC previously unreported families, including 10 isolated early-onset diffuse gastric cancer (DGC) cases. Screening for CDH1 germ line mutations was done by denaturing high-performance liquid chromatography and automated DNA sequencing.

RESULTS

We identified eight inactivating and one missense CDH1 germ line mutation. The missense mutation conferred in vitro loss of protein function. Two families had the previously described 1003C>T nonsense mutation. Haplotype analysis revealed this to be a recurrent and not a founder mutation. Thirty-six percent (5 of 14) of the families with a documented DGC diagnosed before the age of 50 and other cases of gastric cancer carried CDH1 germ line mutations. Two of 10 isolated cases of DGC in individuals ages <35 years harbored CDH1 germ line mutations. One mutation positive family was ascertained through a family history of lobular breast cancer (LBC) and another through an individual with both DGC and LBC. Occult DGC was identified in five of six prophylactic gastrectomies done on asymptomatic, endoscopically negative 1003C>T mutation carriers.

CONCLUSIONS

In addition to families with a strong history of early-onset DGC, CDH1 mutation screening should be offered to isolated cases of DGC in individuals ages <35 years and for families with multiple cases of LBC, with any history of DGC or unspecified GI malignancies. Prophylactic gastrectomy is potentially a lifesaving procedure and clinical breast screening is recommended for asymptomatic mutation carriers.

Cell cycle progression is driven by waves of cyclin expression coupled with regulated protein degradation. An essential step for initiating mitosis is the inactivation of proteolysis mediated by the anaphase-promoting complex/cyclosome (APC/C) bound to its regulator Cdh1p/Hct1p. Yeast APC(Cdh1) was proposed previously to be inactivated at Start by G1 cyclin/cyclin-dependent kinase (CDK). Here, we demonstrate that in a normal cell cycle APC(Cdh1) is inactivated in a graded manner and is not extinguished until S phase. Complete inactivation of APC(Cdh1) requires S phase cyclins. Further, persistent APC(Cdh1) activity throughout G1 helps to ensure the proper timing of Cdc20p expression. This suggests that S phase cyclins have an important role in allowing the accumulation of mitotic cyclins and further suggests a regulatory loop among S phase cyclins, APC(Cdh1), and APC(Cdc20).

The Anaphase-Promoting Complex/Cyclosome (APC/C) ubiquitin ligase mediates degradation of cell cycle proteins during mitosis and G1. Cdc20/Fzy and Cdh1/Fzr are substrate-specific APC/C activators. The level of mammalian Cdh1 is high in mitosis, but it is inactive and does not bind the APC/C. We show that when Cdh1 is active in G1 and G0, its levels are considerably lower and almost all of it is APC/C associated. We demonstrate that Cdh1 is subject to APC/C-specific degradation in G1 and G0, and that this degradation depends upon two RXXL-type destruction boxes. We further demonstrate that addition of Cdh1 to Xenopus interphase extracts, which have an inactive APC/C, activates it to degrade Cdh1. These observations indicate that Cdh1 mediates its own degradation by activating the APC/C to degrade itself. Elevated levels of Cdh1 are deleterious for cell cycle progression in various organisms. This auto-regulation of Cdh1 could thus play a role in ensuring that the level of Cdh1 is reduced during G1 and G0, allowing it to be switched off at the correct time.

Ulcerative colitis (UC) and Crohn's disease (CD) are related polygenic inflammatory bowel diseases (IBDs), with distinct and overlapping susceptibility loci. Recently, hypothesis-free genome-wide association (GWA) studies have revolutionized the field of complex disease genetics. Substantial advances have been achieved in defining the genetic architecture of IBD. To date, over 60 published IBD susceptibility loci have been discovered and replicated, of which approximately a third are associated with both UC and CD, although 21 are specific to UC and 23 to CD. In CD, the breakthrough identification of NOD2 as a susceptibility gene was followed by a rapid phase of gene discovery from GWA studies between 2006 and 2008. Progress in UC was slower; however, by initially testing hits for CD in UC, and later scanning larger UC cohorts, significant new loci for UC have been discovered, with exciting novel insights into disease pathogenesis. Notably, genes implicated in mucosal barrier function (ECM1, CDH1, HNF4α, and laminin B1) confer risk of UC; furthermore, E-cadherin is the first genetic correlation between colorectal cancer and UC. Impaired IL10 signaling has reemerged as a key pathway in intestinal inflammation, and is perhaps the most amenable to therapeutic intervention in UC. Collaborative international efforts with large meta-analyses of GWA studies and replication will yield many new UC genes. Furthermore, a large effort is required to characterize the loci found. Fine-mapping, deep resequencing, and functional studies will be critical to translating these gene discoveries into pathogenic insights, and ultimately into clinical insights and novel therapeutics.

Exit from mitosis requires the degradation of regulatory proteins including the mitotic cyclins and securin through ubiquitination by the anaphase promoting complex (APC) bound to Cdc20 or Cdh1. Cdc20-APC is regulated through inhibition by the spindle assembly checkpoint protein MAD2. Knowledge of Cdh1-APC regulation is limited to the phosphorylation-dependent dissociation of Cdh1 from APC. We report a novel means of regulating Cdh1 by the MAD2-related gene, MAD2L2. MAD2L2 specifically binds and inhibits Cdh1-APC, paralleling the effect of MAD2 on Cdc20. We suggest that MAD2L2 and MAD2 inhibit the release of substrates from APC and propose a mechanism of inhibition.

The purpose was to validate the use of RUNX3 as a potential biomarker for detection of cancer in serum samples and to determine its sensitivity alone and in combination with p16, RASSF1A and CDH1 using methylation-specific polymerase chain reaction (MSP). We examined the promoter methylation status of RUNX3, p16, RASSF1A and CDH1 by MSP using the serum of 70 metastatic breast, non-small cell lung, gastric, pancreatic, colorectal or hepatocellular carcinomas. The DNA from 10 healthy serum controls was used to determine the specificity of methylation. According to our results, promoter hyper-methylation of RUNX3 was detected in the serum of 44 patients comprising breast 9/19 (47%), non-small cell lung 11/20 (55%), gastric 4/4 (100%), pancreatic 2/2 (100%), colorectal 11/17 (65%) and liver 7/8 (88%) carcinomas. Comparative figures for the other genes were as follows: p16 - 39/70 (7/19, 10/20, 2/4, 0/2, 12/17, 8/8); RASSF1A - 24/70 (8/19, 6/20, 1/4, 1/2, 4/17, 4/8); CDH1 - 10/70 (0/19, 4/20, 1/4, 1/2, 3/17, 1/8). Using a panel of four genes, hypermethylation of one or more genes was found in 62/70 samples (15/19, 19/20, 4/4, 2/2, 14/17, 8/8). A panel of three genes omitting RUNX3 detected hyper-methylation in only 50/70 samples. No methylation was detected in the 10 healthy serum controls. Thus, RUNX3 can be detected in the serum of a high proportion of advanced cancers. This suggests that serum hypermethylation of RUNX3 is at least as, or possibly more sensitive a marker, than other tumor suppressor genes currently under investigation. Inclusion of RUNX3 in gene panels can potentially increase the sensitivity of such panels for serum diagnosis of malignancies and warrants further study.

Cdh1p is a substrate-specific subunit of the anaphase-promoting complex (APC/C), which functions as an E3 ubiquitin ligase to degrade the mitotic cyclin Clb2p and other substrates during the G(1) phase of the cell cycle. Cdh1p is phosphorylated and thereby inactivated at the G(1)/S transition predominantly by Cdc28p-Clb5p. Here we show that Cdh1p is nuclear during the G(1) phase of the cell cycle, but redistributes to the cytoplasm between S phase and the end of mitosis. Nuclear export of Cdh1p is regulated by phosphorylation and requires active Cdc28p kinase. Cdh1p binds to the importin Pse1p and the exportin Msn5p, which is necessary and sufficient to promote efficient export of Cdh1p in vivo. Although msn5delta cells are viable, they are sensitive to Cdh1p overexpression. Likewise, a mutant form of Cdh1p, which is constitutively nuclear, prevents accumulation of Clb2p and leads to cell cycle arrest when overexpressed in wild-type cells. Taken together, these results suggest that phosphorylation-dependent nuclear export of Cdh1p by Msn5p contributes to efficient inactivation of APC/C(Cdh1).

Aberrant DNA methylation is recognized as being a common feature of human neoplasia.CpG island hypermethylation and global genomic hypomethylation occur simultaneously in the cancer cell. However, very little is known about the interindividual inherited susceptibility to these epigenetic processes. To address this matter, we have genotyped in 233 cancer patients (with colorectal, breast, or lung tumors), four germ-line variants in three key genes involved in the metabolism of the methyl group, methylene-tetrahydrofolate reductase, methionine synthase, and cystathionine beta-synthase, and analyzed their association with DNA methylation parameters. The epigenetic features analyzed were the 5-methylcytosine content in the genome of the tumors and their normal counterparts, and the presence of CpG island hypermethylation of tumor suppressor genes (p16(INK4a), p14(ARF), hMLH1, MGMT, APC, LKB1, DAPK, GSTP1, BRCA1, RAR beta 2, CDH1, and RASSF1). Two positive associations were found. First, carriers of genotypes containing the methylene-tetrahydrofolate reductase 677T allele show constitutive low levels of 5-methylcytosine in their genomes (P = 0.002), and tumors in these patients do not achieve severe degrees of global hypomethylation (P = 0.047). Second, tumors occurring in homozygous carriers of the methionine synthase 2756G allele show a lower number of hypermethylated CpG islands of tumor suppressor genes (P = 0.029). The existence of these associations may provide another example of the interplay between genetic and epigenetic factors in the cancer cell.

Endoscopic mucosal resection (EMR), endoscopic submucosal dissection (ESD), surgical gastrectomy, and chemotherapy are therapeutic options of gastric cancer; how-ever, prognosis of advanced gastric cancer patients is still poor. Gastric cancer cells with fibroblastoid morphological changes show increased motility and invasiveness due to decreased cell-cell adhesion, which are reminiscent of epithelial-mesenchymal transition (EMT) during embryonic development. Here, EMT signaling networks in gastric cancer were reviewed. E-cadherin at adherens junction is a key molecular target of EMT. CDH1 gene at human chromosome 16q22.1 encodes E-cadherin. Familial diffuse type gastric cancer occurs due to germ-line mutations of the CDH1 gene. Down-regulation of E-cadherin function due to mutation, deletion, CpG hyper-methylation, and SNAIL (SNAI1)- or SIP1-mediated transcriptional repression of the CDH1 gene leads to EMT in gastric cancer. Amplification of ERBB2, MET, FGFR2, PIK3CA, AKT1 genes, up-regulation of WNT2, WNT2B, WNT8B, and down-regulation of SFRP1 lead to EMT in gastric cancer through GSK3beta inhibition and following SNAIL-mediated CDH1 repression. Claudin (CLDN) and PAR3/PAR6/aPKC complex at tight junction are other key molecular targets of EMT. CLDN23 gene is down-regulated in intestinal type gastric cancer. Down-regulation of PAR3/PAR6/aPKC complex also leads to EMT. Single nucleotide polymorphisms (SNPs) and copy number polymorphisms (CNPs) of genes encoding EMT signaling molecules will be identified as novel risk factors of gastric cancer. In addition, antibodies, RNAi compounds, and small molecular inhibitors for EMT signaling molecules will be developed as novel therapeutic agents for gastric cancer. Personalized medicine based on the combination of genetic screening and novel therapeutic agents could dramatically improve the prognosis of gastric cancer patients in the future.

E-cadherin is involved in the formation of cell-junctions and the maintenance of epithelial integrity. Direct evidence of E-cadherin mutations triggering tumorigenesis has come from the finding of inactivating germline mutations of the gene (CDH1) in hereditary diffuse gastric cancer (HDGC). We screened a series of 66 young gastric cancer probands for germline CDH1 mutations, and two novel missense alterations together with an intronic variant were identified. We then analysed the functional significance of the two exonic missense variants found here as well as a third germline missense variant that we previously identified in a HGDC family. cDNAs encoding either the wild-type protein or mutant forms of E-cadherin were stably transfected into CHO (Chinese hamster ovary) E-cadherin-negative cells. Transfected cell-lines were characterized in terms of aggregation, motility and invasion. We show that a proportion of apparently sporadic early-onset diffuse gastric carcinomas are associated with germline alterations of the E-cadherin gene. We also demonstrate that a proportion of missense variants are associated with significant functional consequences, suggesting that our cell model can be used as an adjunct in deciding on the potential pathogenic role of identified E-cadherin germline alterations.

Bilaterality of breast cancer is an indicator of constitutional cancer susceptibility; however, the molecular causes underlying this predisposition in the majority of cases is not known. We hypothesize that epigenetic misregulation of cancer-related genes could partially account for this predisposition. We have performed methylation microarray analysis of peripheral blood DNA from 14 women with bilateral breast cancer compared with 14 unaffected matched controls throughout 17 candidate breast cancer susceptibility genes including BRCA1, BRCA2, CHEK2, ATM, ESR1, SFN, CDKN2A, TP53, GSTP1, <em>CDH1</em>, <em>CDH1</em>3, HIC1, PGR, SFRP1, MLH1, RARB and HSD17B4. We show that the majority of methylation variability is associated with intragenic repetitive elements. Detailed validation of the tiled region around ATM was performed by bisulphite modification and pyrosequencing of the same samples and in a second set of peripheral blood DNA from 190 bilateral breast cancer patients compared with 190 controls. We show significant hypermethylation of one intragenic repetitive element in breast cancer cases compared with controls (P = 0.0017), with the highest quartile of methylation associated with a 3-fold increased risk of breast cancer (OR 3.20, 95% CI 1.78-5.86, P = 0.000083). Increased methylation of this locus is associated with lower steady-state ATM mRNA level and correlates with age of cancer patients but not controls, suggesting a combined age-phenotype-related association. This research demonstrates the potential for gene-body epigenetic misregulation of ATM and other cancer-related genes in peripheral blood DNA that may be useful as a novel marker to estimate breast cancer risk. ACCESSION NUMBERS: The microarray data and associated .BED and .WIG files can be accessed through Gene Expression Omnibus accession number: GSE14603.

The cyclosome/anaphase promoting complex (APC) is a multisubunit ubiquitin ligase that targets mitotic regulators for degradation in exit from mitosis. It is activated at the end of mitosis by phosphorylation and association with the WD-40 protein Cdc20/Fizzy and is then kept active in the G1 phase by association with Cdh1/Hct1. The mitotic checkpoint system that keeps cells with defective spindles from leaving mitosis interacts with Cdc20 and prevents its stimulatory action on the cyclosome. The activity of Cdh1 is negatively regulated by phosphorylation, while the abundance of Cdc20 is cell cycle regulated, with a peak in M-phase. Cdc20 is also phosphorylated in G2/M and in mitotically arrested cells, but the role of phosphorylation remained unknown. Here we show that phosphorylation of Cdc20 by Cdk1/cyclin B abrogates its ability to activate cyclosome/APC from mitotic HeLa cells. A nonphosphorylatable derivative of Cdc20 stimulates cyclin-ubiquitin ligation in extracts from nocodazole-arrested cells to a much greater extent than does wild-type Cdc20. It is suggested that inhibitory phosphorylation of Cdc20/Fizzy may have a role in keeping the cyclosome inactive in early mitosis and under conditions of mitotic checkpoint arrest.

Progression through mitosis is controlled by protein degradation that is mediated by the anaphase-promoting complex/cyclosome (APC/C) and its associated specificity factors. In budding yeast, APC/C(Cdc20) promotes the degradation of the Pds1p anaphase inhibitor at the metaphase-to-anaphase transition, whereas APC/C(Cdh1) promotes the degradation of the mitotic cyclins at the exit from mitosis. Here we show that Pds1p has a novel activity as an inhibitor of mitotic cyclin destruction, apparently by preventing the activation of APC/C(Cdh1). This activity of Pds1p is independent of its activity as an anaphase inhibitor. We propose that the dual role of Pds1p as an inhibitor of anaphase and of cyclin degradation allows the cell to couple the exit from mitosis to the prior completion of anaphase. Finally, these observations provide a novel regulatory paradigm in which the sequential degradation of two substrates is determined by the substrates themselves, such that an early substrate inhibits the degradation of a later one.