Altered JS-2 expression in colorectal cancers and its clinical pathological relevance

JS-2 is a novel gene located at 5p15.2 and originally detected in primary oesophageal cancer. There is no study on the role of JS-2 in colorectal cancer. The aim of this study is to determine the gene copy number and expression of JS-2 in a large cohort of patients with colorectal tumours and correlate these to the clinicopathological features of the cancer patients. We evaluated the DNA copy number and mRNA expression of JS-2 in 176 colorectal tissues (116 adenocarcinomas, 30 adenomas and 30 non-neoplastic tissues) using real-time polymerase chain reaction. JS-2 expression was also evaluated in two colorectal cancer cell lines and a benign colorectal cell line. JS-2 amplification was noted in 35% of the colorectal adenocarcinomas. Significant differences in relative expression levels for JS-2 mRNA between different colorectal tissues were noted (p = 0.05). Distal colorectal adenocarcinoma had significantly higher copy number than proximal adenocarcinoma (p = 0.005). The relative expression level of JS-2 was different between colonic and rectal adenocarcinoma (p = 0.007). Mucinous adenocarcinoma showed higher JS-2 expression than non-mucinous adenocarcinoma (p = 0.02). Early T-stage cancers appear to have higher JS-2 copy number and lower expression of JS-2 mRNA than later stage cancers (p = 0.001 and 0.03 respectively). Colorectal cancer cell lines showed lower expression of JS-2 than the benign colorectal cell line. JS-2 copy number change and expression were shown for the first time to be altered in the carcinogenesis of colorectal cancer. In addition, genetic alteration of JS-2 was found to be related to location, pathological subtypes and staging of colorectal cancer.

Signet-ring cell carcinoma of colorectum : current perspectives and molecular biology

PURPOSE Colorectal signet-ring cell carcinoma (SRCC) is rare, and very little detailed information on the molecular biology of the disease is available. METHODS The literature on the clinical, pathological and, in particular, the molecular biology of this rare entity was critically reviewed. The reviewed articles take into account a total of 1,817 cases of SRCC, but only 143 cases have molecular data available. The characteristics of two patients with colorectal SRCC were also discussed. RESULTS Colorectal SRCC mostly occurs in younger patients, is larger and has different site predilection compared with conventional colorectal adenocarcinoma. It can occur as one of the synchronous cancers in the colorectum. The cancer is usually diagnosed at advanced stages because of the late manifestation of symptoms, and aggressive treatment strategy is required. Limited reports in the literature have shown that the variant of colorectal cancer demonstrated a different pattern of genetic alterations of common growth kinase-related oncogenes (K-ras, BRAF), tumour suppressor genes (p53, p16), gene methylation and cell adhesion-related genes related to the Wingless signalling pathway (E-cadherin and beta-catenin) from conventional colorectal adenocarcinoma. Colorectal SRCC also showed high expression of mucin-related genes and genes related to the gastrointestinal system. There was also a higher prevalence of microsatellite instability-high tumours and low Cox-2 expression in colorectal SRCC as opposed to conventional adenocarcinoma. CONCLUSIONS Colorectal SRCC has unique molecular pathological features. The unique molecular profiles in SRCC may provide molecular-based improvements to patient management in colorectal SRCC.

Control of cell cycle progression by phosphorylation of cyclin-dependent kinase (CDK) substrates

The eukaryotic cell cycle is a fundamental evolutionarily conserved process that regulates cell division from simple unicellular organisms, such as yeast, through to higher multicellular organisms, such as humans. The cell cycle comprises several phases, including the S-phase (DNA synthesis phase) and M-phase (mitotic phase). During S-phase, the genetic material is replicated, and is then segregated into two identical daughter cells following mitotic M-phase and cytokinesis. The S- and M-phases are separated by two gap phases (G1 and G2) that govern the readiness of cells to enter S- or M-phase. Genetic and biochemical studies demonstrate that cell division in eukaryotes is mediated by CDKs (cyclin-dependent kinases). Active CDKs comprise a protein kinase subunit whose catalytic activity is dependent on association with a regulatory cyclin subunit. Cell-cycle-stage-dependent accumulation and proteolytic degradation of different cyclin subunits regulates their association with CDKs to control different stages of cell division. CDKs promote cell cycle progression by phosphorylating critical downstream substrates to alter their activity. Here, we will review some of the well-characterized CDK substrates to provide mechanistic insights into how these kinases control different stages of cell division.

Yhh1p/Cft1p directly links poly(A) site recognition and RNA polymerase II transcription termination

RNA polymerase II (pol II) transcription termination requires co‐transcriptional recognition of a functional polyadenylation signal, but the molecular mechanisms that transduce this signal to pol II remain unclear. We show that Yhh1p/Cft1p, the yeast homologue of the mammalian AAUAAA interacting protein CPSF 160, is an RNA‐binding protein and provide evidence that it participates in poly(A) site recognition. Interestingly, RNA binding is mediated by a central domain composed of predicted β‐propeller‐forming repeats, which occurs in proteins of diverse cellular functions. We also found that Yhh1p/Cft1p bound specifically to the phosphorylated C‐terminal domain (CTD) of pol II in vitro and in a two‐hybrid test in vivo. Furthermore, transcriptional run‐on analysis demonstrated that yhh1 mutants were defective in transcription termination, suggesting that Yhh1p/Cft1p functions in the coupling of transcription and 3′‐end formation. We propose that direct interactions of Yhh1p/Cft1p with both the RNA transcript and the CTD are required to communicate poly(A) site recognition to elongating pol II to initiate transcription termination.

Independent functions of yeast Pcf11p in pre-mRNA 3' end processing and in transcription termination

Pcf11p, an essential subunit of the yeast cleavage factor IA, is required for pre‐mRNA 3′ end processing, binds to the C‐terminal domain (CTD) of the largest subunit of RNA polymerase II (RNAP II) and is involved in transcription termination. We show that the conserved CTD interaction domain (CID) of Pcf11p is essential for cell viability. Interestingly, the CTD binding and 3′ end processing activities of Pcf11p can be functionally uncoupled from each other and provided by distinct Pcf11p fragments in trans. Impaired CTD binding did not affect the 3′ end processing activity of Pcf11p and a deficiency of Pcf11p in 3′ end processing did not prevent CTD binding. Transcriptional run‐on analysis with the CYC1 gene revealed that loss of cleavage activity did not correlate with a defect in transcription termination, whereas loss of CTD binding did. We conclude that Pcf11p is a bifunctional protein and that transcript cleavage is not an obligatory step prior to RNAP II termination.

Head and neck squamous-cell cancer and its association with polymorphic enzymes of xenobiotic metabolism and repair

Tobacco smoking, alcohol drinking, and occupational exposures to polycyclic aromatic hydrocarbons are the major proven risk factors for human head and neck squamous-cell cancer (HNSCC). Major research focus on gene-environment interactions concerning HNSCC has been on genes encoding enzymes of metabolism for tobacco smoke constituents and repair enzymes. To investigate the role of genetically determined individual predispositions in enzymes of xenobiotic metabolism and in repair enzymes under the exogenous risk factor tobacco smoke in the carcinogenesis of HNSCC, we conducted a case-control study on 312 cases and 300 noncancer controls. We focused on the impact of 22 sequence variations in CYP1A1, CYP1B1, CYP2E1, ERCC2/XPD, GSTM1, GSTP1, GSTT1, NAT2, NQO1, and XRCC1. To assess relevant main and interactive effects of polymorphic genes on the susceptibility to HNSCC we used statistical models such as logic regression and a Bayesian version of logic regression. In subgroup analysis of nonsmokers, main effects in ERCC2 (Lys751Gln) C/C genotype and combined ERCC2 (Arg156Arg) C/A and A/A genotypes were predominant. When stratifying for smokers, the data revealed main effects on combined CYP1B1 (Leu432Val) C/G and G/G genotypes, followed by CYP1B1 (Leu432Val) G/G genotype and CYP2E1 (-70G>T) G/T genotype. When fitting logistic regression models including relevant main effects and interactions in smokers, we found relevant associations of CYP1B1 (Leu432Val) C/G genotype and CYP2E1 (-70G>T) G/T genotype (OR, 10.84; 95% CI, 1.64-71.53) as well as CYP1B1 (Leu432Val) G/G genotype and GSTM1 null/null genotype (OR, 11.79; 95% CI, 2.18-63.77) with HNSCC. The findings underline the relevance of genotypes of polymorphic CYP1B1 combined with exposures to tobacco smoke.

A high cell count cascade full bridge converter for wide bandwidth ultrasonic transducer excitation

A nine level modular multilevel cascade converter (MMCC) based on four full bridge cells is shown driving a piezoelectric ultrasonic transducer at 71 and 39 kHz, in simulation and experimentally. The modular cells are small stackable PCBs, each with two fully integrated surface mount 22 V, 40 A MOSFET half-bridge converters, and include all control signal and power isolation. In this work, the bridges operate at 12 V and 384 kHz, to deliver a 96 Vpp 9 level waveform with an effective switching frequency of 3 MHz. A 9 pH air cored inductor forms a low pass filter in conjunction with the 3000 pF capacitance of the transducer load. Eight equally phase-displaced naturally sampled pulse width modulation (PWM) drive signals, along with the modulating sinusoid, are generated using phase accumulation techniques in a dedicated FPGA. Experimental time domain and FFT plots of the multilevel and transducer output waveforms are presented and discussed.

Offspring of mothers fed a high fat diet display hepatic cell cycle inhibition and associated changes in gene expression and DNA methylation

The association between an adverse early life environment and increased susceptibility to later-life metabolic disorders such as obesity, type 2 diabetes and cardiovascular disease is described by the developmental origins of health and disease hypothesis. Employing a rat model of maternal high fat (MHF) nutrition, we recently reported that offspring born to MHF mothers are small at birth and develop a postnatal phenotype that closely resembles that of the human metabolic syndrome. Livers of offspring born to MHF mothers also display a fatty phenotype reflecting hepatic steatosis and characteristics of non-alcoholic fatty liver disease. In the present study we hypothesised that a MHF diet leads to altered regulation of liver development in offspring; a derangement that may be detectable during early postnatal life. Livers were collected at postnatal days 2 (P2) and 27 (P27) from male offspring of control and MHF mothers (n = 8 per group). Cell cycle dynamics, measured by flow cytometry, revealed significant G0/G1 arrest in the livers of P2 offspring born to MHF mothers, associated with an increased expression of the hepatic cell cycle inhibitor Cdkn1a. In P2 livers, Cdkn1a was hypomethylated at specific CpG dinucleotides and first exon in offspring of MHF mothers and was shown to correlate with a demonstrable increase in mRNA expression levels. These modifications at P2 preceded observable reductions in liver weight and liver:brain weight ratio at P27, but there were no persistent changes in cell cycle dynamics or DNA methylation in MHF offspring at this time. Since Cdkn1a up-regulation has been associated with hepatocyte growth in pathologic states, our data may be suggestive of early hepatic dysfunction in neonates born to high fat fed mothers. It is likely that these offspring are predisposed to long-term hepatic dysfunction.

PTRF/cavin-1 neutralizes non-caveolar caveolin-1 microdomains in prostate cancer

Caveolin-1 has a complex role in prostate cancer and has been suggested to be a potential biomarker and therapeutic target. As mature caveolin-1 resides in caveolae, invaginated lipid raft domains at the plasma membrane, caveolae have been suggested as a tumor-promoting signaling platform in prostate cancer. However, caveola formation requires both caveolin-1 and cavin-1 (also known as PTRF; polymerase I and transcript release factor). Here, we examined the expression of cavin-1 in prostate epithelia and stroma using tissue microarray including normal, non-malignant and malignant prostate tissues. We found that caveolin-1 was induced without the presence of cavin-1 in advanced prostate carcinoma, an expression pattern mirrored in the PC-3 cell line. In contrast, normal prostate epithelia expressed neither caveolin-1 nor cavin-1, while prostate stroma highly expressed both caveolin-1 and cavin-1. Utilizing PC-3 cells as a suitable model for caveolin-1-positive advanced prostate cancer, we found that cavin-1 expression in PC-3 cells inhibits anchorage-independent growth, and reduces in vivo tumor growth and metastasis in an orthotopic prostate cancer xenograft mouse model. The expression of α-smooth muscle actin in stroma along with interleukin-6 (IL-6) in cancer cells was also decreased in tumors of mice bearing PC-3-cavin-1 tumor cells. To determine whether cavin-1 acts by neutralizing caveolin-1, we expressed cavin-1 in caveolin-1-negative prostate cancer LNCaP and 22Rv1 cells. Caveolin-1 but not cavin-1 expression increased anchorage-independent growth in LNCaP and 22Rv1 cells. Cavin-1 co-expression reversed caveolin-1 effects in caveolin-1-positive LNCaP cells. Taken together, these results suggest that caveolin-1 in advanced prostate cancer is present outside of caveolae, because of the lack of cavin-1 expression. Cavin-1 expression attenuates the effects of non-caveolar caveolin-1 microdomains partly via reduced IL-6 microenvironmental function. With circulating caveolin-1 as a potential biomarker for advanced prostate cancer, identification of the molecular pathways affected by cavin-1 could provide novel therapeutic targets.

Human CD141(+) (BDCA-3)(+) dendritic cells (DCs) represent a unique myeloid DC subset that cross-presents necrotic cell antigens

The characterization of human dendritic cell (DC) subsets is essential for the design of new vaccines. We report the first detailed functional analysis of the human CD141(+) DC subset. CD141(+) DCs are found in human lymph nodes, bone marrow, tonsil, and blood, and the latter proved to be the best source of highly purified cells for functional analysis. They are characterized by high expression of toll-like receptor 3, production of IL-12p70 and IFN-beta, and superior capacity to induce T helper 1 cell responses, when compared with the more commonly studied CD1c(+) DC subset. Polyinosine-polycytidylic acid (poly I:C)-activated CD141(+) DCs have a superior capacity to cross-present soluble protein antigen (Ag) to CD8(+) cytotoxic T lymphocytes than poly I:C-activated CD1c(+) DCs. Importantly, CD141(+) DCs, but not CD1c(+) DCs, were endowed with the capacity to cross-present viral Ag after their uptake of necrotic virus-infected cells. These findings establish the CD141(+) DC subset as an important functionally distinct human DC subtype with characteristics similar to those of the mouse CD8 alpha(+) DC subset. The data demonstrate a role for CD141(+) DCs in the induction of cytotoxic T lymphocyte responses and suggest that they may be the most relevant targets for vaccination against cancers, viruses, and other pathogens.

Comparison of ovine mesenchymal cell and osteoblast osteogeic capacity in 2D and 3D

This thesis explored the different bone-forming potential of specific bone cells with differing embryological origin, on conventional culture platforms compared to 3D biocompatible scaffolds in vitro. Bone mesenchymal stem cells, mandibular osteoblasts and long bone osteoblasts from adult and juvenile sheep were compared in the study, as the embryological origin of the osteoblasts from the craniofacial and appendicular skeleton differs. The study demonstrated differing characteristics of the various cell types when cultured on the two different platforms compared and this may have an impact on future research into cell seeded tissue scaffolds to aid in vivo tissue regeneration.

Oncogenic BRAF induces melanoma cell invasion by downregulating the cGMP-specific phosphodiesterase PDE5A

We show that in melanoma cells oncogenic BRAF, acting through MEK and the transcription factor BRN2, downregulates the cGMP-specific phosphodiesterase PDE5A. Although PDE5A downregulation causes a small decrease in proliferation, its major impact is to stimulate a dramatic increase in melanoma cell invasion. This is because PDE5A downregulation leads to an increase in cGMP, which induces an increase in cytosolic Ca2+, stimulating increased contractility and inducing invasion. PDE5A downregulation also this leads to an increase in short-term and long-term colonization of the lungs by melanoma cells. We do not observe this pathway in NRAS mutant melanoma or BRAF mutant colorectal cells. Thus, we show that in melanoma cells oncogenic BRAF induces invasion through downregulation of PDE5A.