Digital PCR-based plasma cell-free DNA mutation analysis for early-stage pancreatic tumor diagnosis and surveillance.

BACKGROUND: Cell-free DNA (cfDNA) shed from tumors into the circulation offers a tool for cancer detection. Here, we evaluated the feasibility of cfDNA measurement and utility of digital PCR (dPCR)-based assays, which reduce subsampling error, for diagnosing pancreatic ductal adenocarcinoma (PDA) and surveillance of intraductal papillary mucinous neoplasm (IPMN). METHODS: We collected plasma from seven institutions for cfDNA measurements. Hot-spot mutations in KRAS and GNAS in the cfDNA from patients with PDA (n = 96), undergoing surveillance for IPMN (n = 112), and normal controls (n = 76) were evaluated using pre-amplification dPCR. RESULTS: Upon Qubit measurement and copy number assessment of hemoglobin-subunit (HBB) and mitochondrially encoded NADH:ubiquinone oxidoreductase core subunit 1 (MT-ND1) in plasma cfDNA, HBB offered the best resolution between patients with PDA relative to healthy subjects [area under the curve (AUC) 0.862], whereas MT-ND1 revealed significant differences between IPMN and controls (AUC 0.851). DPCR utilizing pre-amplification cfDNA afforded accurate tumor-derived mutant KRAS detection in plasma in resectable PDA (AUC 0.861-0.876) and improved post-resection recurrence prediction [hazard ratio (HR) 3.179, 95% confidence interval (CI) 1.025-9.859] over that for the marker CA19-9 (HR 1.464; 95% CI 0.674-3.181). Capturing KRAS and GNAS could also provide genetic evidence in patients with IPMN-associated PDA and undergoing pancreatic surveillance. CONCLUSIONS: Plasma cfDNA quantification by distinct measurements is useful to predict tumor burden. Through appropriate methods, dPCR-mediated mutation detection in patients with localized PDA and IPMN likely to progress to invasive carcinoma is feasible and complements conventional biomarkers.

Long term outcomes from lymphatic venous anastomosis after total hysterectomy to prevent postoperative lymphedema in lower limb.

BACKGROUND: Lymphedema in lower limb is one of major postoperative complications followed by a total hysterectomy with lymph node dissection. The objective of this report is to examine a long-term result of lymphaticovenous anastomosis procedure as a preventive surgery. METHODS: Sixteen patients with endometrial cancer underwent an abdominal hysterectomy with a bilateral salpingo-oophorectomy. Just after pelvic lymph node dissection, either end-to-end or sleeve anastomosis utilizing venules and suprainguinal lymph vessels was performed. During the observation period from 4 to 13 years, the symptom of lymphedema in lower extremities has been assessed. RESULTS: Among 16 patients, 1 presented postoperative lymphedema grade 3 (CTCAE (Common Terminology Criteria for Adverse Events) Ver. 4.0, 10025233) in lower limb, and a second surgery at 7 years after the first one was required. Other 6 patients showed non-severe symptoms of lymphedema, diagnosed as grade 1. The rest 9 patients did not show any symptoms of postoperative lymphedema in a long term (up to 13 years). CONCLUSION: From the long term outcomes of our 16 cases, we propose that a direct lymphaticovenous microsurgery immediately after a hysterectomy with lymphadenectomy of external inguinal lymph node is one of the appropriate therapeutic choices to prevent severe lymphedema in lower limb.

Transthyretin cardiac amyloidosis: an update on diagnosis and treatment.

Transthyretin cardiac amyloidosis (ATTR-CA) demonstrates progressive, potentially fatal, and infiltrative cardiomyopathy caused by extracellular deposition of transthyretin-derived insoluble amyloid fibrils in the myocardium. Two distinct types of transthyretin (wild type or variant) become unstable, and misfolding forms aggregate, resulting in amyloid fibrils. ATTR-CA, which has previously been underrecognized and considered to be rare, has been increasingly recognized as a cause of heart failure with preserved ejection fraction among elderly persons. With the advanced technology, the diagnostic tools have been improving for cardiac amyloidosis. Recently, the efficacy of several disease-modifying agents focusing on the amyloidogenic process has been demonstrated. ATTR-CA has been changing from incurable to treatable. Nevertheless, there are still no prognostic improvements due to diagnostic delay or misdiagnosis because of phenotypic heterogeneity and co-morbidities. Thus, it is crucial for clinicians to be aware of this clinical entity for early diagnosis and proper treatment. In this mini-review, we focus on recent advances in diagnosis and treatment of ATTR-CA.

Programmed expression of pro-apoptotic BMCC1 during apoptosis, triggered by DNA damage in neuroblastoma cells.

BACKGROUND: The multi-functional BMCC1 (BCH motif-containing molecule at the carboxyl terminal region 1)/PRUNE2 plays a clear role in suppression of tumor activity. In the patients with neuroblastoma (NB), reduced expression of BMCC1 in primary tumor tissues was associated with poor prognosis. By contrast, enforced expression of BMCC1 as well as elevated expression of BMCC1 in response to DNA-damage promotes apoptosis by abrogating Akt-mediated survival pathways. METHODS: We addressed molecular mechanisms underlying changes in regulation of BMCC1 expression during the process of apoptosis, which was promoted by a DNA-damaging drug Cisplatin (CDDP), in NB-derived cells. RESULTS: Elevated expression of BMCC1 was identified as an early response to DNA damage, which is accompanied by phosphorylation of ataxia telangiectasia mutated kinase (ATM) and accumulation of E2F1. Indeed, inhibition of ATM using an ATM inhibitor resulted in a decrease in expression of BMCC1 at mRNA levels. In addition, an E2F-binding sight was required for activation of BMCC1 promoter in response to DNA damage. On the other hand, knockdown of E2F1 yielded abrogated induction of BMCC1 in the cells after treatment with CDDP, suggesting that BMCC1 accumulation was caused by ATM-E2F1-dependent transcription. Finally, we demonstrated that full-length BMCC1 was proteolytically cleaved by apoptosis-activated caspase-9 during advanced stages of apoptosis in SK-N-AS cells. CONCLUSIONS: In this study, we demonstrated the programmed expression of full-length BMCC1 in human NB cells undergoing DNA damage-induced apoptosis. The elucidation of the molecular mechanisms controlling the regulation of BMCC1 during apoptosis initiated by DNA damage provides useful information for understanding drug resistance of tumor cells and spontaneous regression of NB.

Tumor suppressor KIF1Bß regulates mitochondrial apoptosis in collaboration with YME1L1.

KIF1Bß, a member of the kinesin superfamily of motor proteins, is a haploinsufficient tumor suppressor mapped to chromosome 1p36.2, which is frequently deleted in neural crest-derived tumors, including neuroblastoma and pheochromocytoma. While KIF1Bß acts downstream of the nerve growth factor (NGF) pathway to induce apoptosis, further molecular functions of this gene product have largely been unexplored. In this study, we report that KIF1Bß destabilizes the morphological structure of mitochondria, which is critical for cell survival and apoptosis. We identified YME1L1, a mitochondrial metalloprotease responsible for the cleavage of the mitochondrial GTPase OPA1, as a physical interacting partner of KIF1Bß. KIF1Bß interacted with YME1L1 through its death-inducing region, as initiated the protease activity of YME1L1 to cleave the long forms of OPA1, resulting in mitochondrial fragmentation. Overexpression of YME1L1 promoted apoptosis, while knockdown of YME1L1 promoted cell growth. High YME1L1 expression was significantly associated with a better prognosis in neuroblastoma. Furthermore, in NGF-deprived PC12 cells, KIF1Bß and YME1L1 were upregulated, accompanied by mitochondrial fragmentation and apoptotic cell death. Small interfering RNA-mediated knockdown of either protein alone, however, remarkably inhibited the NGF depletion-induced apoptosis. Our findings indicate that tumor suppressor KIF1Bß plays an important role in intrinsic mitochondria-mediated apoptosis through the regulation of structural and functional dynamics of mitochondria in collaboration with YME1L1. Dysfunction of the KIF1Bß/YME1L1/OPA1 mechanism may be involved in malignant biological features of neural crest-derived tumors as well as the initiation and progression of neurodegenerative diseases.

Hereditary cardiac amyloidosis associated with Pro24Ser transthyretin mutation: a case report.

BACKGROUND: Transthyretin amyloidosis is a systemic disorder caused by extracellular deposition of insoluble amyloid fibrils in peripheral and autonomic nerves, heart, kidney, gastrointestinal tract, and other organs. Hereditary transthyretin amyloidosis is an autosomal dominant disease. More than 120 mutations have been reported in the transthyretin gene with considerable phenotypic heterogeneity and geographic diversity. Among them, a sporadic case of hereditary transthyretin amyloidosis with cardiac-predominant phenotype is very rare, progressive, and potentially fatal if left undiagnosed. However, a clinical diagnosis of cardiac amyloidosis still remains challenging due to non-specific symptoms, and less sensitivity and specificity of medical examinations. CASE PRESENTATION: A 60-year-old Japanese man with a history of embolic stroke and hypertrophic cardiomyopathy visited our department for heart failure. The present case exhibited only cardiomyopathy without any clinical signs of systemic amyloidosis manifested as carpal tunnel syndrome, polyneuropathy, or autonomic dysfunction. An echocardiogram revealed severe asymmetric left ventricular hypertrophy, biatrial dilatation, pericardial effusion, and preserved left ventricular ejection fraction of 50% with severe diastolic dysfunction. Technetium pyrophosphate scintigraphy indicated marked diffuse myocardial uptake of technetium pyrophosphate, strongly suggesting transthyretin cardiac amyloidosis, which was firmly confirmed by a left ventricular endomyocardial biopsy. Genetic analysis demonstrated a transthyretin C70T (Pro24Ser) heterozygous mutation. Tafamidis, a transthyretin stabilizer, was started. His cardiac symptoms remained unchanged for 12 months. CONCLUSIONS: Here we report the case of a patient with hereditary cardiac amyloidosis associated with a Pro24Ser mutation in transthyretin, which is the first case reported in Japan. Technetium pyrophosphate scintigraphy was extremely useful for definitive diagnosis. Thus, we propose that the nuclear imaging technique should be taken into account even for an exploratory diagnosis of transthyretin cardiac amyloidosis.

A case of biopsy-proven eosinophilic myocarditis related to tetanus toxoid immunization.

Vaccine-associated myocarditis is an extremely rare, yet potentially lethal disease, which requires early diagnosis and prompt treatment. However, its pathogenesis remains elusive. We report the first case of biopsy-proven eosinophilic myocarditis related to tetanus toxoid immunization, with unique histopathologic findings, characterized by perivascular eosinophilic infiltrates with myocyte necrosis and abundant interstitial lymphocytic infiltrates with myocyte necrosis, separately. A systemic high-dose corticosteroid treatment had a dramatic beneficial effect on hemodynamic instability and resulted in complete recovery. This case highlights the value of endomyocardial biopsy in establishing a definite diagnosis and understanding the pathogenesis of vaccine-associated myocarditis.

An improved digital polymerase chain reaction protocol to capture low-copy KRAS mutations in plasma cell-free DNA by resolving 'subsampling' issues.

Genetic alterations responsible for the initiation of cancer may serve as immediate biomarkers for early diagnosis. Plasma levels of cell-free DNA (cfDNA) in patients with cancer are higher than those in healthy individuals; however, the major technical challenge for the widespread implementation of cfDNA genotyping as a diagnostic tool is the insufficient sensitivity and specificity of detecting early-stage tumors that shed low amounts of cfDNA. To establish a protocol for ultrasensitive droplet digital polymerase chain reaction (ddPCR) for quantification of low-frequency alleles within a limited cfDNA pool, two-step multiplex ddPCR targeting eight clinically relevant mutant KRAS variants was examined. Plasma samples from patients with colorectal (n = 10) and pancreatic cancer (n = 9) were evaluated, and cfDNA from healthy volunteers (n = 50) was utilized to calculate reference intervals. Limited cfDNA yields in patients with resectable colorectal and pancreatic cancers did not meet the requirement for efficient capture and quantification of rate mutant alleles by ddPCR. Eight preamplification cycles followed by a second-run ddPCR were sufficient to obtain approximately 5000-10 000 amplified copies per ng of cfDNA, resolving the subsampling issue. Furthermore, the signal-to-noise ratio for rare mutant alleles against the extensive background presented by the wild-type allele was significantly enhanced. The cutoff limit of reference intervals for mutant KRAS was determined to be ~ 0.09% based on samples from healthy individuals. The modification introduced in the ddPCR protocol facilitated the quantification of low-copy alleles carrying driver mutations, such as oncogenic KRAS, in localized and early-stage cancers using small blood volumes, thus offering a minimally invasive modality for timely diagnosis.

Retroperitoneal perforation arising from duodenal diverticulum treated by endoscopic drainage: a case report.

Retroperitoneal perforation of duodenal diverticula around the papilla of Vater is relatively rare. In this report, we describe retroperitoneal abscess, which was successfully treated by endoscopic drainage. Thus, endoscopic approach for retroperitoneal perforation caused by diverticulum is one of the treatment options in addition to surgery.

Transcriptional regulation of BMCC1 mediated by E2F1 in neuroblastoma cells.

BCH motif-containing molecule at the carboxyl terminal region 1 (BMCC1)/PRUNE2 is highly expressed in patients with favorable neuroblastoma (NB), encoding a multifunctional scaffold protein that modulates several signaling networks including RhoA and AKT pathways. Accumulating evidence suggests that BMCC1 acts as a tumor-suppressor. In this study, we addressed molecular mechanism underlying transcriptional regulation of BMCC1 in NBs. We found that transcription factor E2F1 was recruited to E2F-binding site in the promoter region of BMCC1 gene. Indeed, overexpression of E2F1 resulted in an increase in the expression level of BMCC1 in NB cell lines. On the other hand, knockdown of E2F1 in NB cells yielded down-regulation of BMCC1. Also, we showed that BMCC1 and E2F1 were simultaneously induced at G1 to S phase transition. Therefore, we conclude that E2F1 directly facilitated BMCC1 transcription. Taking together, these results suggest that BMCC1 induced by E2F1 acts as a tumor suppressor through its pro-apoptotic function, resulted in favorable prognosis of NB.

Tracking the Clonal Evolution of Adenosquamous Carcinoma, a Rare Variant of Intraductal Papillary Mucinous Neoplasm of the Pancreas.

Adenosquamous carcinoma (ASC) is an uncommon variant of pancreatic neoplasm. We sought to trace the mode of tumor progression using specimens of ASC associated with intraductal papillary mucinous neoplasm (IPMN) of the pancreas. A resected specimen of the primary pancreatic ASC, developed in a 72-year-old man, was subjected to mutation profiling using amplicon-targeted sequencing and digital polymerase chain reaction. DNA was isolated from each histological compartment including noninvasive IPMN, squamous cell carcinoma (SCC), and adenocarcinoma (AC). Histologically, an IPMN with a large mural nodule was identified. The invasive tumor predominantly consisted of SCC, and a smaller AC was found around the lesion. Squamous metaplasias were sporadically distributed within benign IPMNs. Mutation alleles KRAS and GNAS were identified in all specimens of IPMN including the areas of squamous metaplasia. In addition, these mutations were found in SCC and AC. Clear transition from flat/low-papillary IPMN to SCC indicated a potent invasion front, and the SCC compartment was genetically unique, because the area has a higher frequency of mutation KRAS. The invasive tumors with distinct histological appearances shared the form of noninvasive IPMN as a common precursor, rather than de novo cancer, suggesting the significance of a genetic profiling scheme of tumors associated with IPMN.

Pathogenesis, Surgical Treatment, and Cure for SUNCT Syndrome.

BACKGROUND: Short-lasting unilateral neuralgiform headache attacks with conjunctival injection and tearing (SUNCT) are characterized by attacks of moderate to severe stabbing pain, strictly unilateral, with periorbital or temporal distribution, associated with cranial autonomic symptoms, such as lacrimation and redness of the ipsilateral eye. METHODS: To obtain mechanistic insights into the pathogenesis of SUNCT syndrome, more than 800 cases treated in our institution during the last 7 years were retrospectively reviewed. Two patients showed typical autonomic symptoms of SUNCT. RESULTS: Magnetic resonance imaging suggested potential compression of the trigeminal nerve by the intracranial artery in these cases and complete remission was achieved by microvascular decompression. CONCLUSIONS: Microvascular decompression provides an appropriate therapeutic choice if vascular compression of the trigeminal nerve is identified. From our 2 cases, we propose that, in some cases of SUNCT diagnosed previously, characteristic symptoms were induced by compression of the side surface of the first branch of the trigeminal nerve at the root exit zone by the intracranial artery.

Identification of novel candidate compounds targeting TrkB to induce apoptosis in neuroblastoma.

Neuroblastoma (NB) is one of the most frequent solid tumors in children and its prognosis is still poor. The neurotrophin receptor TrkB and its ligand brain-derived neurotrophic factor (BDNF) are expressed at high levels in high-risk NBs and are involved in defining the poor prognosis of the patients. However, the TrkB targeting therapy has never been realized in the clinic. We performed an in silico screening procedure utilizing an AutoDock/grid computing technology in order to identify novel small chemical compounds targeting the BDNF-binding domain of TrkB. For the first screening, a library of three million synthetic compounds was screened in silico and was ranked according to the Docking energy. The top-ranked 37 compounds were further functionally screened for cytotoxicity by using NB cell lines. We have finally identified seven compounds that kill NB cells with the IC50 values of 0.07-4.6 µmol/L. The terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay showed that these molecules induce apoptosis accompanied by p53 activation in NB cell lines. The candidate compounds and BDNF demonstrated an antagonistic effect on cell growth, invasion, and colony formation, possibly suggesting competition at the BDNF-binding site of TrkB. The candidate compounds had tumor-suppressive activity in xenograft and in vivo toxicity tests (oral and intravenous administrations) using mice, and did not show any abnormal signs. Using in silico Docking screening we have found new candidate TrkB inhibitors against high-risk NBs, which could lead to new anti-cancer drugs.

Novel adaptor protein Shf interacts with ALK receptor and negatively regulates its downstream signals in neuroblastoma.

Our neuroblastoma cDNA project previously identified Src homology 2 domain containing F (Shf) as one of the genes expressed at high levels in favorable neuroblastoma. Shf is an adaptor protein containing four putative tyrosine phosphorylation sites and an SH2 domain. In this study, we found that Shf interacted with anaplastic lymphoma kinase (ALK), an oncogenic receptor tyrosine kinase in neuroblastoma. Real-time PCR analysis showed that Shf mRNA is highly expressed in non-metastatic neuroblastomas compared to metastatic tumor samples (P < 0.030, n = 106). Interestingly, patients showing high ALK and low Shf mRNA expressions showed poor prognosis, whereas low ALK and high Shf expressions were related to better prognosis (P < 0.023, n = 38). Overexpression of ALK and siRNA-mediated knockdown of Shf yielded similar results, such as an increase in cellular growth and phosphorylation of ALK, in addition to Erk1/2 and signal transducer and activator of transcription 3 (STAT3) that are downstream signals of the ALK-initiated phospho-transduction pathway. Knockdown of Shf also increased the cellular mobility and invasive capability of neuroblastoma cells. These results suggest that Shf interacts with ALK and negatively regulates the ALK-initiated signal transduction pathway in neuroblastoma. We thus propose that Shf inhibits phospho-transduction signals mediated by ALK, which is one of the major key players on neuroblastoma development, resulting in better prognosis of the tumor.

LMO3 interacts with p53 and inhibits its transcriptional activity.

High expression of LMO3 contributes to the development and aggressiveness of neuroblastoma. LMO3 belongs to the LIM-only protein family, in which de-regulation of its members is implicated in human carcinogenesis. However, the molecular mechanism of LMO3 activity in oncogenesis remained poorly characterized. We found that LMO3 is a direct interacting partner of p53 both in vitro and in vivo. The DNA-binding domain of p53 is required for this interaction. Furthermore, expression of LMO3 repressed p53-dependent mRNA expression of its target genes by suppressing promoter activation. Interestingly, chromatin immunoprecipitation assay showed that LMO3 facilitated p53 binding to its response elements. This suggests that LMO3 acts as a co-repressor of p53, suppressing p53-dependent transcriptional regulation without inhibition of its DNA-binding activity.

G9a selectively represses a class of late-replicating genes at the nuclear periphery.

We have investigated the role of the histone methyltransferase G9a in the establishment of silent nuclear compartments. Following conditional knockout of the G9a methyltransferase in mouse ESCs, 167 genes were significantly up-regulated, and no genes were strongly down-regulated. A partially overlapping set of 119 genes were up-regulated after differentiation of G9a-depleted cells to neural precursors. Promoters of these G9a-repressed genes were AT rich and H3K9me2 enriched but H3K4me3 depleted and were not highly DNA methylated. Representative genes were found to be close to the nuclear periphery, which was significantly enriched for G9a-dependent H3K9me2. Strikingly, although 73% of total genes were early replicating, more than 71% of G9a-repressed genes were late replicating, and a strong correlation was found between H3K9me2 and late replication. However, G9a loss did not significantly affect subnuclear position or replication timing of any non-pericentric regions of the genome, nor did it affect programmed changes in replication timing that accompany differentiation. We conclude that G9a is a gatekeeper for a specific set of genes localized within the late replicating nuclear periphery.

ReplicationDomain: a visualization tool and comparative database for genome-wide replication timing data.

BACKGROUND: Eukaryotic DNA replication is regulated at the level of large chromosomal domains (0.5-5 megabases in mammals) within which replicons are activated relatively synchronously. These domains replicate in a specific temporal order during S-phase and our genome-wide analyses of replication timing have demonstrated that this temporal order of domain replication is a stable property of specific cell types. RESULTS: We have developed ReplicationDomain http://www.replicationdomain.org as a web-based database for analysis of genome-wide replication timing maps (replication profiles) from various cell lines and species. This database also provides comparative information of transcriptional expression and is configured to display any genome-wide property (for instance, ChIP-Chip or ChIP-Seq data) via an interactive web interface. Our published microarray data sets are publicly available. Users may graphically display these data sets for a selected genomic region and download the data displayed as text files, or alternatively, download complete genome-wide data sets. Furthermore, we have implemented a user registration system that allows registered users to upload their own data sets. Upon uploading, registered users may choose to: (1) view their data sets privately without sharing; (2) share with other registered users; or (3) make their published or "in press" data sets publicly available, which can fulfill journal and funding agencies' requirements for data sharing. CONCLUSION: ReplicationDomain is a novel and powerful tool to facilitate the comparative visualization of replication timing in various cell types as well as other genome-wide chromatin features and is considerably faster and more convenient than existing browsers when viewing multi-megabase segments of chromosomes. Furthermore, the data upload function with the option of private viewing or sharing of data sets between registered users should be a valuable resource for the scientific community.

Global reorganization of replication domains during embryonic stem cell differentiation.

DNA replication in mammals is regulated via the coordinate firing of clusters of replicons that duplicate megabase-sized chromosome segments at specific times during S-phase. Cytogenetic studies show that these "replicon clusters" coalesce as subchromosomal units that persist through multiple cell generations, but the molecular boundaries of such units have remained elusive. Moreover, the extent to which changes in replication timing occur during differentiation and their relationship to transcription changes has not been rigorously investigated. We have constructed high-resolution replication-timing profiles in mouse embryonic stem cells (mESCs) before and after differentiation to neural precursor cells. We demonstrate that chromosomes can be segmented into multimegabase domains of coordinate replication, which we call "replication domains," separated by transition regions whose replication kinetics are consistent with large originless segments. The molecular boundaries of replication domains are remarkably well conserved between distantly related ESC lines and induced pluripotent stem cells. Unexpectedly, ESC differentiation was accompanied by the consolidation of smaller differentially replicating domains into larger coordinately replicated units whose replication time was more aligned to isochore GC content and the density of LINE-1 transposable elements, but not gene density. Replication-timing changes were coordinated with transcription changes for weak promoters more than strong promoters, and were accompanied by rearrangements in subnuclear position. We conclude that replication profiles are cell-type specific, and changes in these profiles reveal chromosome segments that undergo large changes in organization during differentiation. Moreover, smaller replication domains and a higher density of timing transition regions that interrupt isochore replication timing define a novel characteristic of the pluripotent state.

Replication labeling with halogenated thymidine analogs.

In this unit, several basic protocols to identify sites of DNA replication utilizing incorporation of halogenated thymidine analogs into DNA, followed by immunofluorescent imaging are described. Antibodies specific for halogenated thymidine analogs such as bromodeoxyuridine (BrdU), chlorodeoxyuridine (CldU), and iododeoxyuridine (IdU) can provide a rapid, nonhazardous, and sensitive method for detecting DNA replication in single cells, in a manner analogous to the traditional use of tritiated thymidine. In combination with different techniques to prepare the DNA template, a variety of DNA replication-related events can be examined by conventional fluorescence-microscopic approaches. Because origin firing and the progression of replication forks are regulated in the context of subnuclear compartments through protein-protein interactions, chromatin modifications, and subnuclear localization of replication clusters, visualizing replication foci significantly facilitates understanding of nuclear dynamics during S-phase.

Functional contribution of Pds5 to cohesin-mediated cohesion in human cells and Xenopus egg extracts.

Sister chromatid cohesion is essential for proper segregation of the genome in mitosis and meiosis. Central to this process is cohesin, a multi-protein complex conserved from yeast to human. Previous genetic studies in fungi have identified Pds5/BimD/Spo76 as an additional factor implicated in cohesion. Here we describe the biochemical and functional characterization of two Pds5-like proteins, Pds5A and Pds5B, from vertebrate cells. In HeLa cells, Pds5 proteins physically interact with cohesin and associate with chromatin in a cohesin-dependent manner. Depletion of the cohesin subunit Scc1 by RNA interference leads to the assembly of chromosomes with severe cohesion defects. A similar yet milder set of defects is observed in cells with reduced levels of Pds5A or Pds5B. In Xenopus egg extracts, mitotic chromosomes assembled in the absence of Pds5A and Pds5B display no discernible defects in arm cohesion, but centromeric cohesion is apparently loosened. Unexpectedly, these chromosomes retain an unusually high level of cohesin. Thus, Pds5 proteins seem to affect the stable maintenance of cohesin-mediated cohesion and its efficient dissolution during mitosis. We propose that Pds5 proteins play both positive and negative roles in sister chromatid cohesion, possibly by directly modulating the dynamic interaction of cohesin with chromatin. This idea would explain why cells lacking Pds5 function display rather complex and diverse phenotypes in different organisms.