Heparansulfate proteoglycans (HSPG) play an important role in cell–cell and cell–matrix interactions and signaling, and one of the key enzymes in heparansulfate biosynthesis is d-glucuronyl C5-epimerase (GLCE). A tumor suppressor function has been demonstrated for GLCE in breast and lung carcinogenesis; however, no data are available as to the expression and regulation of the gene in prostate cancer. In this study, decreased GLCE expression was observed in 10% of benign prostate hyperplasia (BPH) tissues and 53% of prostate tumors, and increased GLCE mRNA levels were detected in 49% of BPH tissues and 21% of tumors. Statistical analysis showed a positive correlation between increased GLCE expression and Gleason score, TNM staging, and prostate-specific antigen (PSA) level in the prostate tumors (Pearson correlation coefficients GLCE/Gleason = 0.56, P < 0.05; GLCE/TNM = 0.62, P < 0.05; and GLCE/PSA = 0.88, P < 0.01), suggesting GLCE as a candidate molecular marker for advanced prostate cancer. Immunohistochemical analysis revealed an intratumoral heterogeneity of GLCE protein levels both in BPH and prostate cancer cells, resulting in a mixed population of GLCE-expressing and nonexpressing epithelial cells in vivo. A model experiment on normal (PNT2) and prostate cancer (LNCaP, PC3, DU145) cell lines in vitro showed a 1.5- to 2.5-fold difference in GLCE expression levels between the cancer cell lines and an overall decrease in GLCE expression in cancer cells. Methyl-specific polymerase chain reaction (PCR), bisulfite sequencing, and deoxy-azacytidin (aza-dC) treatment identified differential GLCE promoter methylation (LNCaP 70–72%, PC3 32–35%, DU145, and PNT2 no methylation), which seems to contribute to heterogeneous GLCE expression in prostate tumors. The obtained results reveal the complex deregulation of GLCE expression in prostatic diseases compared with normal prostate tissue and suggest that GLCE may be used as a potential model to study the functional role of intratumor cell heterogeneity in prostate cancer progression.
The molecular mechanisms of intratumour heterogeneity of cancer cells, contributing to tissue malignisation, remain unclear. This study reveals the complex deregulation of d-glucuronyl C5-epimerase (GLCE) expression in benign prostatic hyperplasia and prostate tumours, and the high intratumour heterogeneity of prostate cancer cells in terms of GLCE expression and promoter methylation. The results suggest that GLCE may be used as a potential target gene to study the functional role of cancer cell heterogeneity in disease progression and treatment.
d-Glucuronyl C5-epimerase; expression; heparansulfate proteoglycan; heterogeneity; methylation; prostate cancer
D-glucuronyl C5-epimerase (GLCE) is a potential tumor-suppressor gene involved in heparan sulfate biosynthesis. GLCE expression is significantly decreased in breast tumors; however, the underlying molecular mechanisms remain unclear. This study examined the possible epigenetic mechanisms for GLCE inactivation in breast cancer. Very little methylation of the GLCE promoter region was detected in breast tumors in vivo and in breast cancer cells (MCF7 and T47D) in vitro and GLCE expression in breast cancer cells was not altered by 5-deoxyazacytidine (5-aza-dC) treatment, suggesting that promoter methylation is not involved in regulating GLCE expression. Chromatin activation by Trichostatin A (TSA) or 5-aza-dC/TSA treatment increased GLCE expression by two to 3-fold due to an increased interaction between the GLCE promoter and the TCF4/β-catenin transactivation complex, or H3K9ac and H3K4Me3 histone modifications. However, ectopic expression of TCF4/β-catenin was not sufficient to activate GLCE expression in MCF7 cells, suggesting that chromatin structure plays a key role in GLCE regulation. Although TSA treatment significantly repressed canonical WNT signaling in MCF7 cells, it did not influence endogenous TCF4/β-catenin mRNA levels and activated TCF4/β-catenin-driven transcription from the GLCE promoter, indicating GLCE as a novel target for TCF4/β-catenin complex in breast cancer cells. A correlation was observed between GLCE, TCF4 and β-catenin expression in breast cancer cells and primary tumors, suggesting an important role for TCF4/β-catenin in regulating GLCE expression both in vitro and in vivo. Taken together, the results indicate that GLCE expression in breast cancer is regulated by a combination of chromatin structure and TCF4/β-catenin complex activity.
D-glucuronyl C5-epimerase; GLCE; heparan sulphate proteoglycan; biosynthesis; tumor-suppressor gene; hypermethylation; chromatin structure; WNT signaling; TCF4/β-catenin target; breast cancer
In HIV-1-infected patients a long lasting CD4+ cell decline influences the host-EBV balance and thereby increases the risk for EBV related malignancies. In spite of a world-wide access to combination antiretroviral therapy (cART) there are still a considerable number of HIV-1-infected patients who will develop severe immunodeficiency. These undiagnosed HIV-1 infected patients, so called late testers, demonstrate an increased lymphoma risk, compared to patients diagnosed early. Consecutive individual screening for EBV DNA-load in late testers might be a useful predictor of emerging EBV-malignancy.
Patient biopsies and ascites were analyzed morphologically, by immuncyto-histochemistry and in-situ hybridization. Viral DNA and RNA load were quantified by PCR. Cell lines from primary tumor and from ascites, were established in vitro and further analyzed.
We here report on a case of EBV-positive lymphoma in an AIDS patient, first presenting with pleural effusion and ascites and was thus initially considered a primary effusion lymphoma (PEL) but was later diagnosed as a plasmablastic lymphoma (PBL). The patient had responded to cART with undetectable HIV-RNA and increased CD4 cell count one year prior to lymphoma presentation. At the time of lymphoma diagnosis the HIV-RNA values were <50 RNA-copies per mL blood (undetectable) and the CD4-positive cell count 170 ×106/L. The lymphoma was CD45-negative and weakly CD22- and CD30-positive. The patient had a history of Kaposi sarcoma and HHV-8 seropositivity. The lymphoma biopsies, and three cell lines derived on different occasions from the tumor cell effusion, were all EBV-positive but HHV-8 negative.
A noticeable EBV-DNA load decline was observed during the remission of the lymphoma following CHOP-therapy. The EBV-DNA load increased dramatically at the time of recurrence.
EBV DNA load might be useful in monitoring the effect of lymphoma treatment as well as in estimating the risk of EBV-associated lymphoma in HIV-1 infected patients with pronounced immunosuppression.
EBV; HIV-1; HHV-8; DNA load; Plasmablastic lymphoma
To investigate—by molecular, classical and functional methods—the microbiota in biopsies and faeces from patients with active Crohn's disease (CD) and controls.
The microbiota in biopsies was investigated utilizing a novel molecular method and classical cultivation technology. Faecal samples were investigated by classical technology and four functional methods, reflecting alterations in short chain fatty acids pattern, conversion of cholesterol and bilirubin and inactivation of trypsin.
By molecular methods we found more than 92% similarity in the microbiota on the biopsies from the two groups. However, 4.6% of microbes found in controls were lacking in CD patients. Furthermore, NotI representation libraries demonstrate two different clusters representing CD patients and controls, respectively. Utilizing conventional technology, Bacteroides (alt. Parabacteroides) was less frequently detected in the biopsies from CD patients than from controls. A similar reduction in the number of Bacteroides was found in faecal samples. Bacteroides is the only group of bacteria known to be able to inactivate pancreatic trypsin. Faecal tryptic activity was high in CD patients, and inversely correlated to the levels of Bacteroides.
CD patients have compositional and functional alterations in their intestinal microbiota, in line with the global description hypothesis rather than the candidate microorganism theory. The most striking functional difference was high amount of faecal tryptic activity in CD patients, inversely correlated to the levels of Bacteroides in faeces.
Non-Hodgkin lymphoma (NHL) is a frequent cancer and incidence rates have increased markedly during the second half of the 20th century; however, the few established risk factors cannot explain this rise and still little is known about the aetiology of NHL. Spatial analyses have been applied in an attempt to identify environmental risk factors, but most studies do not take human mobility into account. The aim of this study was to identify clustering of NHL in space and time in Denmark, using 33 years of residential addresses. We utilised the nation-wide Danish registers and unique personal identification number that all Danish citizens have to conduct a register-based case-control study of 3210 NHL cases and two independent control groups of 3210 each. Cases were identified in the Danish Cancer Registry and controls were matched by age and sex and randomly selected from the Civil Registration System. Residential addresses of cases and controls from 1971 to 2003 were collected from the Civil Registration System and geocoded. Data on pervious hospital diagnoses and operations were obtained from the National Patient Register. We applied the methods of the newly developed Q-statistics to identify space-time clustering of NHL. All analyses were conducted with each of the two control groups, and we adjusted for previous history of autoimmune disease, HIV/AIDS or organ transplantation. Some areas with statistically significant clustering were identified; however, results were not consistent across the two control groups; thus we interpret the results as chance findings. We found no evidence for clustering of NHL in space and time using 33 years of residential histories, suggesting that if the rise in incidence of NHL is a result of risk factors that vary across space and time, the spatio-temporal variation of such factors in Denmark is too small to be detected with the applied method.
Zinc finger, DHHC-type containing 2 (ZDHHC2), originally named as reduced expression associated with metastasis protein (REAM), has been proposed as a putative tumor/metastasis suppressor gene and is often aberrantly decreased in human cancers. However ZDHHC2 expression pattern and its clinical significance have not yet been investigated in gastric adenocarcinoma.
Quantitative Real-Time PCR (qRT-PCR) and immunostaining were performed to detect ZDHHC2 expression in gastric adenocarcinoma, and then the correlation between ZDHHC2 expression and clinicpathologic parameters, and patient survival was analyzed. Compared to the adjacent normal tissues, ZDHHC2 expression was significantly reduced in gastric tumor tissues as shown by qRT-PCR and immunostaining. Low expression of ZDHHC2 was observed in 44.7% (211/472) of gastric adenocarcinoma patients, and was associated significantly with lymph node metastasis (p<0.001) and histological grade (p<0.001). Multivariate Cox regression analysis indicated that ZDHHC2 expression had a significant, independent predictive value for survival of gastric cancer patients (HR = 0.627, p = 0.001).
Our data suggest that reduced ZDHHC2 expression is associated with lymph node metastasis and independently predicts an unfavorable prognosis in gastric adenocarcinoma patients.
Increasing evidence demonstrated that inactivation of tumor suppressor genes (TSGs) by aberrant promoter methylation is an early event during carcinogenesis. Aiming at developing early diagnostic or prognostic tools for various tumors, we took an EBV-associated tumor, nasopharyngeal carcinoma (NPC), as a model and developed a powerful assay based on “multiplex methylation specific-PCR (MMSP)”. The MMSP assay was designed to detect tumor-specific methylation status of several NPC-related genes and was capable of acquiring multiplex information simultaneously through a single PCR reaction with the tiny tumor DNA derived from the direct body fluid close to the primary tumor. In this study, we collected paired nasopharyngeal (NP) swabs and NPC biopsies from 49 NPC patients and twenty noncancerous controls. A panel of markers including two EBV, and two cellular TSG markers were applied in this NPC-specific-MMSP assay. We optimized the working condition of MMSP so that it provides information equal to that from the corresponding separate PCRs. The results showed that MMSP patterns of NPC swab were largely consistent with those of corresponding biopsies and significantly distinguished themselves from those of 20 noncancerous volunteers. Among the 69 samples (49 NPCs and 20 normal controls), the sensitivity of detecting NPC from NP swabs is 98%. The specificity is as high as 100%. In conclusion, being characterized by its noninvasiveness, high reproducibility and informativeness, MMSP assay is a reliable and potential diagnostic tool for NPC. It paves the way for the development of population screening and early diagnosis approaches for various tumor types.
microRNAs (miRNAs) are key posttranscriptional regulators of gene expression. In the present study, regulation of tumor-suppressor gene D-glucuronyl C5-epimerase (GLCE) by miRNA-218 was investigated. Significant downregulation of miRNA-218 expression was shown in primary breast tumors. Exogenous miRNA-218/anti-miRNA-218 did not affect GLCE mRNA but regulated GLCE protein level in MCF7 breast carcinoma cells in vitro. Comparative analysis showed a positive correlation between miRNA-218 and GLCE mRNA, and negative correlation between miRNA-218 and GLCE protein levels in breast tissues and primary tumors in vivo, supporting a direct involvement of miRNA-218 in posttranscriptional regulation of GLCE in human breast tissue. A common scheme for the regulation of GLCE expression in normal and tumor breast tissues is suggested.
miRNA; D-glucuronyl C5-epimerase; GLCE; tumor-suppressor gene; expression; heparan sulphate proteoglycan; biosynthesis; breast cancer
The B lymphotrophic γ-herpesvirus EBV is associated with a variety of lymphoid- and epithelial-derived malignancies, including B cell lymphomas in immunocompromised and immunosuppressed individuals. The primary oncogene of EBV, latent membrane protein 1 (LMP1), activates the PI3K/Akt pathway to induce the autocrine growth factor, IL-10, in EBV-infected B cells, but the mechanisms underlying PI3K activation remain incompletely understood. Using small molecule inhibition and siRNA strategies in human B cell lines expressing a chimeric, signaling-inducible LMP1 protein, nerve growth factor receptor (NGFR)-LMP1, we show that NGFR-LMP1 utilizes Syk to activate PI3K/Akt signaling and induce IL-10 production. NGFR-LMP1 signaling induces phosphorylation of BLNK, a marker of Syk activation. Whereas Src kinases are often required for Syk activation, we show here that PI3K/Akt activation and autocrine IL-10 production by NGFR-LMP1 involves the Src family kinase Fyn. Finally, we demonstrate that NGFR-LMP1 induces phosphorylation of c-Cbl in a Syk- and Fyn-dependent fashion. Our results indicate that the EBV protein LMP1, which lacks the canonical ITAM required for Syk activation, can nevertheless activate Syk, and the Src kinase Fyn, resulting in downstream c-Cbl and PI3K/Akt activation. Fyn, Syk, and PI3K/Akt antagonists thus may present potential new therapeutic strategies that target the oncogene LMP1 for treatment of EBV+ B cell lymphomas.
Nasopharyngeal carcinoma (NPC) is a common cancer in Southeast Asia, particularly in southern regions of China. EBV infection is closely associated with NPC and has long been postulated to play an etiological role in the development of NPC. However, the role of EBV in malignant transformation of nasopharyngeal epithelial cells remains enigmatic. The current hypothesis of NPC development is that premalignant nasopharyngeal epithelial cells harboring genetic alterations support EBV infection and expression of EBV genes induces further genomic instability to facilitate the development of NPC. The latent membrane protein 1 (LMP1) is a well-documented EBV-encoded oncogene. The involvement of LMP1 in human epithelial malignancies has been implicated, but the mechanisms of oncogenic actions of LMP1, particularly in nasopharyngeal cells, are unclear. Here we observed that LMP1 expression in nasopharyngeal epithelial cells impaired G2 checkpoint, leading to formation of unrepaired chromatid breaks in metaphases after γ-ray irradiation. We further found that defective Chk1 activation was involved in the induction of G2 checkpoint defect in LMP1-expressing nasopharyngeal epithelial cells. Impairment of G2 checkpoint could result in loss of the acentrically broken chromatids and propagation of broken centric chromatids in daughter cells exiting mitosis, which facilitates chromosome instability. Our findings suggest that LMP1 expression facilitates genomic instability in cells under genotoxic stress. Elucidation of the mechanisms involved in LMP1-induced genomic instability in nasopharyngeal epithelial cells will shed lights on the understanding of role of EBV infection in NPC development.
Latent membrane protein 2A (LMP2A), expressed in most Epstein-Barr virus (EBV)-associated malignancies, has been demonstrated to be responsible for the maintenance of latent infection and epithelial cell transformation. Besides, it could also act as the target for a CTL-based therapy for EBV-associated malignancies. In the present study, sequence variations of LMP2A in EBV-associated gastric carcinoma (EBVaGC) and healthy EBV carriers from Guangzhou, southern China, where nasopharyngeal carcinoma (NPC) is endemic, were investigated. Widespread sequence variations in the LMP2A gene were found, with no sequence identical to the B95.8 prototype. No consistent mutation was detected in all isolates. The immunoreceptor tyrosine-based activation motif (ITAM) and PY motifs in the amino terminus of LMP2A were strictly conserved, suggesting their important roles in virus infection; while 8 of the 17 identified CTL epitopes in the transmembrane region of LMP2A were affected by at least one point mutation, which may implicate that the effect of LMP2A polymorphisms should be considered when LMP2A-targeted immunotherapy is conducted. The polymorphisms of LMP2A in EBVaGC in gastric remnant carcinoma (GRC) were for the first time investigated in the world. The LMP2A sequence variations in EBVaGC in GRC were somewhat different from those in EBVaGC in conventional gastric carcinoma. The sequence variations of LMP2A in EBVaGC were similar to those in throat washing of healthy EBV carriers, indicating that these variations are due to geographic-associated polymorphisms rather than EBVaGC-associated mutations. This, to our best knowledge, is the first detailed investigation of LMP2A polymorphisms in EBVaGC in Guangzhou, southern China, where NPC is endemic.
We propose an innovative, integrated, cost-effective health system to combat major non-communicable diseases (NCDs), including cardiovascular, chronic respiratory, metabolic, rheumatologic and neurologic disorders and cancers, which together are the predominant health problem of the 21st century. This proposed holistic strategy involves comprehensive patient-centered integrated care and multi-scale, multi-modal and multi-level systems approaches to tackle NCDs as a common group of diseases. Rather than studying each disease individually, it will take into account their intertwined gene-environment, socio-economic interactions and co-morbidities that lead to individual-specific complex phenotypes. It will implement a road map for predictive, preventive, personalized and participatory (P4) medicine based on a robust and extensive knowledge management infrastructure that contains individual patient information. It will be supported by strategic partnerships involving all stakeholders, including general practitioners associated with patient-centered care. This systems medicine strategy, which will take a holistic approach to disease, is designed to allow the results to be used globally, taking into account the needs and specificities of local economies and health systems.
The role of microRNA-155 (miR-155) has been associated with oncogenesis of several human tumors. However the expression pattern of miR-155 has not been investigated in nasopharyngeal carcinoma (NPC). The present study was to assess miR-155 expression pattern and its possible function in NPC, to identify its targets and evaluate their clinical applications in NPC. MiR-155 was found to be upregulated in two Epstein-Barr virus (EBV) negative NPC derived cell lines CNE1 and TW03, as well as in NPC clinical samples by quantitative Real-time PCR and in situ hybridization detection. EBV encoded LMP1 and LMP2A could further enhance the expression of miR-155 in NPC CNE1 and TW03 cells. JMJD1A and BACH1 were identified as putative targets of miR-155 in a bioinformatics screen. Overexpression of miR-155 downregulated a luciferase transcript fused to the 3′UTR of JMJD1A and BACH1. MiR-155 mimic could downregulate the expression of JMJD1A and BACH1, while miR-155 inhibitor could upregulate JMJD1A expression in NPC cell lines. Moreover, downregulation of JMJD1A was significantly correlated with N stage in TNM classification (p = 0.023), a lower five-year survival rate (p = 0.021), and a lower five-year disease-free survival rate (p = 0.049) of NPC patients. Taken together, up-regulation of miR-155 in NPC is partly driven by LMP1 and LMP2A, and results in downregulation of JMJD1A, which is associated with N stage and poor prognosis of NPC patients. The potential of miR-155 and JMJD1A as therapeutic targets in NPC should be further investigated.
A goal of systems biology is to analyze large-scale molecular networks including gene expressions and protein-protein interactions, revealing the relationships between network structures and their biological functions. Dividing a protein-protein interaction (PPI) network into naturally grouped parts is an essential way to investigate the relationship between topology of networks and their functions. However, clear modular decomposition is often hard due to the heterogeneous or scale-free properties of PPI networks.
To address this problem, we propose a diffusion model-based spectral clustering algorithm, which analytically solves the cluster structure of PPI networks as a problem of random walks in the diffusion process in them. To cope with the heterogeneity of the networks, the power factor is introduced to adjust the diffusion matrix by weighting the transition (adjacency) matrix according to a node degree matrix. This algorithm is named adjustable diffusion matrix-based spectral clustering (ADMSC). To demonstrate the feasibility of ADMSC, we apply it to decomposition of a yeast PPI network, identifying biologically significant clusters with approximately equal size. Compared with other established algorithms, ADMSC facilitates clear and fast decomposition of PPI networks.
ADMSC is proposed by introducing the power factor that adjusts the diffusion matrix to the heterogeneity of the PPI networks. ADMSC effectively partitions PPI networks into biologically significant clusters with almost equal sizes, while being very fast, robust and appealing simple.
Cell lineage commitment and differentiation are governed by a complex gene regulatory network. Disruption of these processes by inappropriate regulatory signals and by mutational rewiring of the network can lead to tumorigenesis. Cancer cells often exhibit immature or embryonic traits and dysregulated developmental genes can act as oncogenes. However, the prevailing paradigm of somatic evolution and multi-step tumorigenesis, while useful in many instances, offers no logically coherent reason for why oncogenesis recapitulates ontogenesis. The formal concept of “cancer attractors”, derived from an integrative, complex systems approach to gene regulatory network may provide a natural explanation. Here we present the theory of attractors in gene network dynamics and review the concept of cell types as attractors. We argue that cancer cells are trapped in abnormal attractors and discuss this concept in the light of recent ideas in cancer biology, including cancer genomics and cancer stem cells, as well as the implications for differentiation therapy.
Protection of the large intestine with its enormous amount of commensal bacteria is a challenge that became easier to understand when we recently could describe that colon has an inner attached mucus layer devoid of bacteria (Johansson et al. (2008) Proc. Natl. Acad. Sci. USA 105, 15064–15069). The bacteria are thus kept at a distance from the epithelial cells and lack of this layer, as in Muc2-null mice, allow bacteria to contact the epithelium. This causes colitis and later on colon cancer, similar to the human disease Ulcerative Colitis, a disease that still lacks a pathogenetic explanation. Dextran Sulfate (DSS) in the drinking water is the most widely used animal model for experimental colitis. In this model, the inflammation is observed after 3–5 days, but early events explaining why DSS causes this has not been described.
When mucus formed on top of colon explant cultures were exposed to 3% DSS, the thickness of the inner mucus layer decreased and became permeable to 2 µm fluorescent beads after 15 min. Both DSS and Dextran readily penetrated the mucus, but Dextran had no effect on thickness or permeability. When DSS was given in the drinking water to mice and the colon was stained for bacteria and the Muc2 mucin, bacteria were shown to penetrate the inner mucus layer and reach the epithelial cells already within 12 hours, long before any infiltration of inflammatory cells.
DSS thus causes quick alterations in the inner colon mucus layer that makes it permeable to bacteria. The bacteria that reach the epithelial cells probably trigger an inflammatory reaction. These observations suggest that altered properties or lack of the inner colon mucus layer may be an initial event in the development of colitis.
We evaluated the effect of combination anti-retroviral treatment (cART) on the host control of EBV infection in moderately immunosuppressed HIV-1 patients. Twenty HIV-1 infected individuals were followed for five years with repeated measurements of EBV DNA load in peripheral blood lymphocytes in relation to HIV-RNA titers and CD4+ cell counts. Individuals with optimal response, i.e. durable non-detectable HIV-RNA, showed a decline of EBV load to the level of healthy controls. Individuals with non-optimal HIV-1 control did not restore their EBV control. Long-lasting suppression of HIV-replication after early initiation of cART is a prerequisite for re-establishing the immune control of EBV.
Epstein-Barr virus; HIV-1; antiretroviral treatment; host virus relationship
Hepatitis C Virus (HCV) infection is a leading indication for liver transplantation. HCV infection reoccurs almost universally post transplant, decreasing both graft longevity and patient survival. The immunosuppressant, cyclosporine A (CsA) has potent anti-HCV activity towards both HCV replicons and the genotype 2a cell culture infectious virus. Previously, we isolated mutations in the 1bN replicon with less sensitivity to CsA that mapped to both NS5A and NS5B regions of the virus. Mutations in NS5A alone conferred decreased CsA susceptibility regardless of NS5B mutations.
We examined the mechanisms by which NS5A mutations contribute to CsA resistance and if they are strain dependent. Using in vitro mutagenesis, the amino acid position 321 mutation of NS5A was restored to the wild-type tyrosine residue conferring partial CsA susceptibility on the mutant replicon. The 321 mutation also alters CsA susceptibility of the JFH cell culture virus. Additionally, we demonstrated a novel CsA-sensitive interaction between NS5A and both cyclophilin A and B. Both the mutant NS5A and wild type NS5A bind cyclophilin in vitro. The NS5A: cyclophilin interaction requires both the NS5A region identified by the resistance mutants and cyclophilin catalytic residues. In cell culture, NS5A from CsA resistant mutant has an enhanced interaction with cyclophilin B. Additionally; NS5B facilitates a stronger binding of mutant NS5A to endogenous cyclophilin B than wild-type in cell culture.
Collectively, this data suggests direct interactions between cyclophilins and NS5A are critical to understand for optimal use of cyclophilin inhibitors in anti-HCV therapy.
Lymphocyte function-associated antigen 1 (LFA-1), a member of β2-integrin family, exerts multiple roles in host T cell immunity and has been identified as a useful drug-development target for inflammatory and autoimmune diseases. Applying the findings that primary resting T cells absorb nanometric membrane vesicles derived from antigen presenting cells (APC) via dual receptor/ligand interactions of T cell receptor (TCR) with cognate peptide-major histocompatibility complex (MHC) complex (pMHC) and LFA-1 with its ligand, intercellular adhesion molecule-1 (ICAM-1), and that signaling cascades triggered by TCR/pMHC interaction take a part in the vesicle-absorption, we established a cell-based high throughput assay for systematic investigation, via isolation of small molecules modulating the level of vesicle-absorption, of molecular mechanisms underlying the T cell absorption of APC-derived vesicles, i.e., structural basis of TCR/pMHC and LFA-1/ICAM-1 interactions and TCR-mediated LFA-1 activation. As primary T cells along with physiological ligands expressed in biological membrane are used and also individual cells in assay samples are analyzed by flow cytometry, results obtained using the assay system hold superior physiological and therapeutic relevance as well as statistical precision.
Many different genetic alterations are observed in cancer cells. Individual cancer genes display point mutations such as base changes, insertions and deletions that initiate and promote cancer growth and spread. Somatic hypermutation is a powerful mechanism for generation of different mutations. It was shown previously that somatic hypermutability of proto-oncogenes can induce development of lymphomas.
We found an exceptionally high incidence of single-base mutations in the tumor suppressor genes RASSF1 and RBSP3 (CTDSPL) both located in 3p21.3 regions, LUCA and AP20 respectively. These regions contain clusters of tumor suppressor genes involved in multiple cancer types such as lung, kidney, breast, cervical, head and neck, nasopharyngeal, prostate and other carcinomas. Altogether in 144 sequenced RASSF1A clones (exons 1–2), 129 mutations were detected (mutation frequency, MF = 0.23 per 100 bp) and in 98 clones of exons 3–5 we found 146 mutations (MF = 0.29). In 85 sequenced RBSP3 clones, 89 mutations were found (MF = 0.10). The mutations were not cytidine-specific, as would be expected from alterations generated by AID/APOBEC family enzymes, and appeared de novo during cell proliferation. They diminished the ability of corresponding transgenes to suppress cell and tumor growth implying a loss of function. These high levels of somatic mutations were found both in cancer biopsies and cancer cell lines.
This is the first report of high frequencies of somatic mutations in RASSF1 and RBSP3 in different cancers suggesting it may underlay the mutator phenotype of cancer. Somatic hypermutations in tumor suppressor genes involved in major human malignancies offer a novel insight in cancer development, progression and spread.
The role of the MYC oncogene in the apoptotic pathways is not fully understood. MYC has been reported to protect cells from apoptosis activation but also to sensitize cells to apoptotic stimuli. We have previously demonstrated that the down-regulation of Myc protein activates apoptosis in melanoma cells and increases the susceptibility of cells to various antitumoral treatments. Beyond the well-known role in the G1→S transition, MYC is also involved in the G2-M cell cycle phases regulation.
In this study we have investigated how MYC could influence cell survival signalling during G2 and M phases. We used the microtubules damaging agent paclitaxel (PTX), to arrest the cells in the M phase, in a p53 mutated melanoma cell line with modulated Myc level and activity. An overexpression of Myc protein is able to increase endoreduplication favoring the survival of cells exposed to antimitotic poisoning. The PTX-induced endoreduplication is associated in Myc overexpressing cells with a reduced expression of MAD2, essential component of the molecular core of the spindle assembly checkpoint (SAC), indicating an impairment of this checkpoint. In addition, for the first time we have localized Myc protein at the spindle poles (centrosomes) during pro-metaphase in different cell lines.
The presence of Myc at the poles during the prometaphase could be necessary for the Myc-mediated attenuation of the SAC and the subsequent induction of endoreduplication. In addition, our data strongly suggest that the use of taxane in antitumor therapeutic strategies should be rationally based on the molecular profile of the individual tumor by specifically analyzing Myc expression levels.
CD44 splice variants are long-known as being associated with cell transformation. Recently, the standard form of CD44 (CD44s) was shown to be part of the signature of cancer stem cells (CSCs) in colon, breast, and in head and neck squamous cell carcinomas (HNSCC). This is somewhat in contradiction to previous reports on the expression of CD44s in HNSCC. The aim of the present study was to clarify the actual pattern of CD44 expression in head and neck epithelia.
Expression of CD44s and CD44v6 was analysed by immunohistochemistry with specific antibodies in primary head and neck tissues. Scoring of all specimens followed a two-parameters system, which implemented percentages of positive cells and staining intensities from − to +++ (score = %×intensity; resulting max. score 300). In addition, cell surface expression of CD44s and CD44v6 was assessed in lymphocytes and HNSCC.
In normal epithelia CD44s and CD44v6 were expressed in 60–95% and 50–80% of cells and yielded mean scores with a standard error of a mean (SEM) of 249.5±14.5 and 198±11.13, respectively. In oral leukoplakia and in moderately differentiated carcinomas CD44s and CD44v6 levels were slightly increased (278.9±7.16 and 242±11.7; 291.8±5.88 and 287.3±6.88). Carcinomas in situ displayed unchanged levels of both proteins whereas poorly differentiated carcinomas consistently expressed diminished CD44s and CD44v6 levels. Lymphocytes and HNSCC lines strongly expressed CD44s but not CD44v6.
CD44s and CD44v6 expression does not distinguish normal from benign or malignant epithelia of the head and neck. CD44s and CD44v6 were abundantly present in the great majority of cells in head and neck tissues, including carcinomas. Hence, the value of CD44s as a marker for the definition of a small subset of cells (i.e. less than 10%) representing head and neck cancer stem cells may need revision.
Revealing the spectrum of combinatorial regulation of transcription at individual promoters is essential for understanding the complex structure of biological networks. However, the computations represented by the integration of various molecular signals at complex promoters are difficult to decipher in the absence of simple cis regulatory codes. Here we synthetically shuffle the regulatory architecture — operator sequences binding activators and repressors — of a canonical bacterial promoter. The resulting library of complex promoters allows for rapid exploration of promoter encoded logic regulation. Among all possible logic functions, NOR and ANDN promoter encoded logics predominate. A simple transcriptional cis regulatory code determines both logics, establishing a straightforward map between promoter structure and logic phenotype. The regulatory code is determined solely by the type of transcriptional regulation combinations: two repressors generate a NOR: NOT (a OR b) whereas a repressor and an activator generate an ANDN: a AND NOT b. Three-input versions of both logics, having an additional repressor as an input, are also present in the library. The resulting complex promoters cover a wide dynamic range of transcriptional strengths. Synthetic promoter shuffling represents a fast and efficient method for exploring the spectrum of complex regulatory functions that can be encoded by complex promoters. From an engineering point of view, synthetic promoter shuffling enables the experimental testing of the functional properties of complex promoters that cannot necessarily be inferred ab initio from the known properties of the individual genetic components. Synthetic promoter shuffling may provide a useful experimental tool for studying naturally occurring promoter shuffling.
The Epstein-Barr virus is widespread in all human populations and is strongly associated with human disease, ranging from infectious mononucleosis to cancer. In infected cells the virus can adopt several different latency programs, affecting the cells' behaviour. Experimental results indicate that a specific genetic switch between viral latency programs, reprograms human B-cells between proliferative and resting states. Each of these two latency programs makes use of a different viral promoter, Cp and Qp, respectively. The hypothesis tested in this study is that this genetic switch is controlled by both human and viral transcription factors; Oct-2 and EBNA-1. We build a physico-chemical model to investigate quantitatively the dynamical properties of the promoter regulation and experimentally examine protein level variations between the two latency programs.
Our experimental results display significant differences in EBNA-1 and Oct-2 levels between resting and proliferating programs. With the model we identify two stable latency programs, corresponding to a resting and proliferating cell. The two programs differ in robustness and transcriptional activity. The proliferating state is markedly more stable, with a very high transcriptional activity from its viral promoter. We predict the promoter activities to be mutually exclusive in the two different programs, and our relative promoter activities correlate well with experimental data. Transitions between programs can be induced, by affecting the protein levels of our transcription factors. Simulated time scales are in line with experimental results.
We show that fundamental properties of the Epstein-Barr virus involvement in latent infection, with implications for tumor biology, can be modelled and understood mathematically. We conclude that EBNA-1 and Oct-2 regulation of Cp and Qp is sufficient to establish mutually exclusive expression patterns. Moreover, the modelled genetic control predict both mono- and bistable behavior and a considerable difference in transition dynamics, based on program stability and promoter activities. Both these phenomena we hope can be further investigated experimentally, to increase the understanding of this important switch. Our results also stress the importance of the little known regulation of human transcription factor Oct-2.
Nasopharyngeal carcinoma (NPC) is one of the most common cancers in southern China. In addition to environmental factors such as Epstein-Barr virus infection and diet, genetic susceptibility has been reported to play a key role in the development of this disease. The x-ray repair cross-complementing group 1 (XRCC1) gene is important in DNA base excision repair. We hypothesized that two common single nucleotide polymorphisms of XRCC1 (codons 194 Arg→Trp and 399 Arg→Gln) are related to the risk of NPC and interact with tobacco smoking.
We sought to determine whether these genetic variants of the XRCC1 gene were associated with the risk of NPC among the Cantonese population in a hospital-based case control study using polymerase chain reaction-restriction fragment length polymorphism analysis. We conducted this study in 462 NPC patients and 511 healthy controls.
After adjustment for sex and age, we found a reduced risk of developing NPC in individuals with the Trp194Trp genotype (OR = 0.48; 95% CI, 0.27–0.86) and the Arg194Trp genotype (OR = 0.79; 95% CI, 0.60–1.05) compared with those with the Arg194Arg genotype. Compared with those with the Arg399Arg genotype, the risk for NPC was not significantly different in individuals with the Arg399Gln genotype (OR = 0.82; 95% CI, 0.62–1.08) and the Gln399Gln genotype (OR = 1.20; 95% CI, 0.69–2.06). Further analyses stratified by gender and smoking status revealed a significantly reduced risk of NPC among males (OR = 0.32; 95% CI, 0.14–0.70) and smokers (OR = 0.34; 95% CI, 0.14–0.82) carrying the XRCC1 194Trp/Trp genotype compared with those carrying the Arg/Arg genotype. No association was observed between Arg399Gln variant genotypes and the risk of NPC combined with smoking and gender.
Our findings suggest that the XRCC1 Trp194Trp variant genotype is associated with a reduced risk of developing NPC in Cantonese population, particularly in males and smokers. Larger studies are needed to confirm our findings and unravel the underlying mechanisms.