In the latent infection of Kaposi's sarcoma-associated herpesvirus (KSHV), its 160-kb circularized episomal DNA is replicated and maintained in the host nucleus. KSHV latency-associated nuclear antigen (LANA) is a key factor for maintaining viral latency. LANA binds to the terminal repeat (TR) DNA of the viral genome, leading to its localization to specific dot structures in the nucleus. In such an infected cell, the expression of the viral genes is restricted by a mechanism that is still unclear. Here, we found that LANA interacts with SUV39H1 histone methyltransferase, a key component of heterochromatin formation, as determined by use of a DNA pull-down assay with a biotinylated DNA fragment that contained a LANA-specific binding sequence and a maltose-binding protein pull-down assay. The diffuse localization of LANA on the chromosomes of uninfected cells changed to a punctate one with the introduction of a bacterial artificial chromosome containing most of the TR region, and SUV39H1 clearly colocalized with the LANA-associated dots. Thus, the LANA foci in KSHV-infected cells seemed to include SUV39H1 as well as heterochromatin protein 1. Furthermore, a chromatin immunoprecipitation assay revealed that the TR and the open reading frame (ORF) K1 and ORF50/ RTA genes, but not the ORF73/LANA gene, lay within the heterochromatin during KSHV latency. Taken together, these observations indicate that LANA recruits heterochromatin components to the viral genome, which may lead to the establishment of viral latency and govern the transcription program.Kaposi's sarcoma-associated herpesvirus (KSHV), also called human herpesvirus 8, was discovered in Kaposi's sarcoma (KS) lesions (8) and is strongly associated with multicentric Castleman's disease and primary effusion lymphoma (PEL), which are found predominantly in AIDS patients (6). KSHV belongs to the gammaherpesviruses, which have two distinct replication systems, lytic and latent. For lytic replication, the immediate-early gene product RTA (31), which functions as a strong transactivator that upregulates many viral genes, must be expressed. In PEL cells, KSHV is mainly in the latent state, during which RTA is not expressed (18). Thus, it is likely that KSHV has regulatory machinery that enables it to maintain the latent phase as a default state.The latency-associated nuclear antigen (LANA) is the 1,162-amino-acid (aa) product of the KSHV open reading frame 73 (ORF73) gene, which is expressed during latency (20, 37). LANA has an acidic amino acid repeat region in the middle and a DNA-binding/dimerization domain at its C terminus (42). During latency, LANA binds a DNA sequence in the terminal repeat (TR) region of the viral genome, which consists of repeated sequences made up of 801-bp units and contains an origin of replication (OriP) (2, 3, 13). Most cell lines derived from PELs carry a viral genome with a long TR sequence of more than 20 kb (17). Given LANA's functional similarity to EBNA1, which is encoded by another gammaherpesvirus, Epstein-Barr virus, it is lik...

During latency, Kaposi's sarcoma-associated herpesvirus (KSHV) is thought to replicate once and to be partitioned in synchrony with the cell cycle of the host. In this replication cycle, the KSHV terminal repeat (TR) sequence functions as a replication origin, assisted by the latency-associated nuclear antigen (LANA). Thus, TR seems to function as a cis element for the replication and partitioning of the KSHV genome. Viral replication and partitioning are also likely to require cellular factors that interact with TR in either a LANAdependent or -independent manner. Here, we sought to identify factors that associate with TR by using a TR DNA column and found that poly(ADP-ribose) polymerase 1 (PARP1) and known replication factors, including ORC2, CDC6, and Mcm7, bound to TR. PARP1 bound directly to a specific region within TR independent of LANA, and LANA was poly(ADP-ribosyl)ated by PARP1. Drugs such as hydroxyurea and niacinamide, which raise or lower PARP activity, respectively, affected the virus copy number in infected cells. Thus, the poly(ADPribosyl)ation status of LANA appears to affect the replication and/or maintenance of the viral genome. Drugs that specifically up-regulate PARP activity may lead to the disappearance of latent KSHV.Kaposi's sarcoma-associated herpesvirus (KSHV) or human herpesvirus 8 (HHV8) was discovered in Kaposi's sarcoma lesions (12) and is strongly associated with multicentric Castleman's disease and primary effusion lymphomas, which are predominantly found in AIDS patients (9). KSHV belongs to the gammaherpesviruses, and most of the virus is in the latent phase in these diseases (13).Epstein-Barr virus (EBV), another human gammaherpesvirus, also establishes latency and is associated with epithelial and many kinds of lymphoid tumors (28). Several genes with potential oncogenic activity, latency-associated nuclear antigen (LANA), K-cyclin (ORF72), vFLIP (ORF71), K15, and LANA2 (vIRF3) of KSHV (17,20,46,55) and EBNA1, LMP1, LMP2, and EBER of EBV (40), are expressed during latency, and the presence of the virus seems to be a minimum requirement for cancer formation (10) and to be tightly linked to the growth activity of the virus-positive B-cell lymphoma lines. Therefore, even in the context of serious disease, control of the latent infection itself could be an important strategy in treatment of these cancers.In latent replication, the KSHV genome is thought to replicate once per cell cycle, in synchrony with the host replication machinery, as reported for EBV (1,27,54). Recent studies of KSHV latent replication and maintenance have revealed that (i) the terminal repeat sequence functions as a replication origin (3, 4) and (ii) LANA binds to a specific sequence within the terminal repeat (TR) and is an essential viral gene product for latent viral replication (3,4,27). These two facts are important for understanding how the KSHV genome replicates in the latent phase. In fact, the two viral components TR and LANA appeared to be sufficient to execute viral replication in a transient t...

Amida was isolated by the yeast two-hybrid system as a novel protein which associated with Arc, a non-transcriptional immediate early gene specific to the brain. Amida was confirmed to be associated with Arc in vitro and in vivo. Amida shows no homology to known proteins. Amida is ubiquitously expressed, although it is abundant in the brain. A transfection study revealed that Amida was localized in the nucleus and after 72 h the transfected cells underwent apoptosis. Furthermore, we found two nuclear localization signals and a domain needed for interacting with Arc was encompassed by two nuclear localization signals. Co-transfection experiment with Amida and Arc suggested that Amida transported Arc into the nucleus and negatively regulated Amida-induced cell death. These results indicate that Arc together with Amida may modulate cell death in the brain.

The detection of colon cancer using endoscopy is widely used, but the interpretation of the diagnosis is based on the clinician's naked eye. This is subjective and can lead to false detection. Here we developed a rapid and accurate molecular fluorescence imaging technique using antibody-coated quantum dots (Ab-QDs) sprayed and washed simultaneously on colon tumor tissues inside live animals, subsequently excited and imaged by endoscopy. QDs were conjugated to matrix metalloproteinases (MMP) 9, MMP 14, or carcinoembryonic antigen (CEA) Abs with zwitterionic surface coating to reduce nonspecific bindings. The Ab-QD probes can diagnose tumors on sectioned mouse tissues, fresh mouse colons stained ex vivo and also in vivo as well as fresh human colon adenoma tissues in 30 min and can be imaged with a depth of 100 μm. The probes successfully detected not only cancers that are readily discernible by bare eyes but also hyperplasia and adenoma regions. Sum and cross signal operations provided postprocessed images that can show complementary information or regions of high priority. This multiplexed quantum dot, spray-and-wash, and endoscopy approach provides a significant advantage for detecting small or flat tumors that may be missed by conventional endoscopic examinations and bestows a strategy for the improvement of cancer diagnosis.

The replication and transcription activator (RTA) of Kaposi's sarcoma-associated herpesvirus (KSHV), or human herpesvirus 8, a homologue of Epstein-Barr virus BRLF1 or Rta, is a strong transactivator and inducer of lytic replication. RTA acting alone can induce lytic replication of KSHV in infected cell lines that originated from primary effusion lymphomas, leading to virus production. During the lytic replication process, RTA activates many kinds of genes, including polyadenylated nuclear RNA, K8, K9 (vIRF), ORF57, and so on. We focused here on the mechanism of how RTA upregulates the K9 (vIRF) promoter and identified two independent cis-acting elements in the K9 (vIRF) promoter that responded to RTA. These elements were finally confined to the sequence 5-TCTGGGACAGTC-3 in responsive element (RE) I-2B and the sequence 5-GTA CTTAAAATA-3 in RE IIC-2, both of which did not share sequence homology. Multiple factors bound specifically with these elements, and their binding was correlated with the RTA-responsive activity. Electrophoretic mobility shift assay with nuclear extract from infected cells and the N-terminal part of RTA expressed in Escherichia coli, however, did not show that RTA interacted directly with these elements, in contrast to the RTA responsive elements in the PAN/K12 promoter region, the ORF57/K8 promoter region. Thus, it was likely that RTA could transactivate several kinds of unique cis elements without directly binding to the responsive elements, probably through cooperation with other DNA-binding factors.

A WD increases the susceptibility to DSS-induced inflammation and accelerates the infiltration of macrophages. In turn, this resulted in the development and progression of colon cancer.

No abstract

Immunoprofiling is useful for predicting prognosis in various malignancies and provides targets for immunotherapy. Quantitative multispectral imaging system, which allows simultaneous detection of multiple immune markers, is a novel method for examining the tumor immune environment. We compared the expression levels of various surface markers in immune cells between colitis-associated cancer (CAC) and sporadic colorectal cancer (CRC) and evaluated the clinical usefulness of immunoprofiling in CRC. Tumor specimens from 24 CAC patients and 48 sporadic CRC patients, matched by age, sex, and tumor location to CAC, were included in the analysis. The expression levels of CD3, CD8, Foxp3, and programmed death-ligand 1 (PD-L1) in immune cells at the invasive margins of tumor tissues were evaluated by quantitative multispectral imaging. The CAC group had significantly less levels of cells expressing CD3, CD8, Foxp3, or PD-L1 (all, p < 0.01). In the CAC group, patients whose immune cells had high expression of CD3 + and CD8 + had better overall survival. The immune profiling patterns of CAC patients were significantly distinct from those of sporadic CRC patients, suggesting that CAC and sporadic CRC have distinct disease phenotypes. Immunoprofiling can be helpful for evaluation of clinical prognosis in CAC.