MicroRNAs targeting mutant K-ras by electrotransfer inhibit human colorectal adenocarcinoma cell growth in vitro and in vivo

Mutations of K-ras have been found in 30-60% of colorectal carcinomas and are believed to be associated with tumor initiation, tumor progression and metastasis formation. Therefore, silencing of mutant K-ras expression has become an attractive therapeutic strategy for colorectal cancer treatment. The aim of our study was to investigate the effect of microRNA (miRNA) molecules directed against K-ras (miRNA-K-ras) on K-ras expression level and the growth of colorectal carcinoma cell line LoVo in vitro and in vivo. In addition, we evaluated electroporation as a gene delivery method for transfection of LoVo cells and tumors with plasmid DNA encoding miRNA-K-ras (pmiRNA-K-ras). Results of our study indicated that miRNAs targeting K-ras efficiently reduced K-ras expression and cell survival after in vitro electrotransfection of LoVo cells with pmiRNA-K-ras. In vivo, electroporation has proven to be a simple and efficient delivery method for local administration of pmiRNA-K-ras molecules into LoVo tumors. This therapy shows pronounced antitumor effectiveness and has no side effects. The obtained results demonstrate that electrogene therapy with miRNA-K-ras molecules can be potential therapeutic strategy for treatment of colorectal cancers harboring K-ras mutations. © 2010 Nature Publishing Group All rights reserved.

Novel use of faecal sterols to assess human faecal contamination in Antarctica : a likelihood assessment matrix for environmental monitoring

Wastewater containing human sewage is often discharged with little or no treatment into the Antarctic marine environment. Faecal sterols (primarily coprostanol) in sediments have been used for assessment of human sewage contamination in this environment, but in situ production and indigenous faunal inputs can confound such determinations. Using gas chromatography with mass spectral detection profiles of both C27 and C29 sterols, potential sources of faecal sterols were examined in nearshore marine sediments, encompassing sites proximal and distal to the wastewater outfall at Davis Station. Faeces from indigenous seals and penguins were also examined. Faeces from several indigenous species contained significant quantities of coprostanol but not 24-ethylcoprostanol, which is present in human faeces. In situ coprostanol and 24-ethylcoprostanol production was identified by co-production of their respective epi isomers at sites remote from the wastewat er source and in high total organic matter sediments. A C 29 sterols-based polyphasic likelihood assessment matrix for human sewage contamination is presented, which distinguishes human from local fauna faecal inputs and in situ production in the Antarctic environment. Sewage contamination was detected up to 1.5 km from Davis Station.

Molecular profiling of human mammary gland links breast cancer risk to a p27(+) cell population with progenitor characteristics

Early full-term pregnancy is one of the most effective natural protections against breast cancer. To investigate this effect, we have characterized the global gene expression and epigenetic profiles of multiple cell types from normal breast tissue of nulliparous and parous women and carriers of BRCA1 or BRCA2 mutations. We found significant differences in CD44+ progenitor cells, where the levels of many stem cell-related genes and pathways, including the cell-cycle regulator p27, are lower in parous women without BRCA1/BRCA2 mutations. We also noted a significant reduction in the frequency of CD44+p27+ cells in parous women and showed, using explant cultures, that parity-related signaling pathways play a role in regulating the number of p27+ cells and their proliferation. Our results suggest that pathways controlling p27+ mammary epithelial cells and the numbers of these cells relate to breast cancer risk and can be explored for cancer risk assessment and prevention.

Human single-stranded DNA binding protein 1 (hSSB1/NABP2) is required for the stability and repair of stalled replication forks

Aberrant DNA replication is a primary cause of mutations that are associated with pathological disorders including cancer. During DNA metabolism, the primary causes of replication fork stalling include secondary DNA structures, highly transcribed regions and damaged DNA. The restart of stalled replication forks is critical for the timely progression of the cell cycle and ultimately for the maintenance of genomic stability. Our previous work has implicated the single-stranded DNA binding protein, hSSB1/NABP2, in the repair of DNA double-strand breaks via homologous recombination. Here, we demonstrate that hSSB1 relocates to hydroxyurea (HU)-damaged replication forks where it is required for ATR and Chk1 activation and recruitment of Mre11 and Rad51. Consequently, hSSB1-depleted cells fail to repair and restart stalled replication forks. hSSB1 deficiency causes accumulation of DNA strand breaks and results in chromosome aberrations observed in mitosis, ultimately resulting in hSSB1 being required for survival to HU and camptothecin. Overall, our findings demonstrate the importance of hSSB1 in maintaining and repairing DNA replication forks and for overall genomic stability.

Minimum cost of transport in human running is not ubiquitous

Purpose This study explores recent claims that humans exhibit a minimum cost of transport (CoTmin) for running which occurs at an intermediate speed, and assesses individual physiological, gait and training characteristics. Methods Twelve healthy participants with varying levels of fitness and running experience ran on a treadmill at six self-selected speeds in a discontinuous protocol over three sessions. Running speed (km[middle dot]hr-1), V[spacing dot above]O2 (mL[middle dot]kg-1[middle dot]km-1), CoT (kcal[middle dot]km-1), heart rate (beats[middle dot]min-1) and cadence (steps[middle dot]min-1) were continuously measured. V[spacing dot above]O2 max was measured on a fourth testing session. The occurrence of a CoTmin was investigated and its presence or absence examined with respect to fitness, gait and training characteristics. Results Five participants showed a clear CoTmin at an intermediate speed and a statistically significant (p < 0.05) quadratic CoT-speed function, while the other participants did not show such evidence. Participants were then categorized and compared with respect to the strength of evidence for a CoTmin (ClearCoTmin and NoCoTmin). The ClearCoTmin group displayed significantly higher correlation between speed and cadence; more endurance training and exercise sessions per week; than the NoCoTmin group; and a marginally non-significant but higher aerobic capacity. Some runners still showed a CoTmin at an intermediate speed even after subtraction of resting energy expenditure. Conclusion The findings confirm the existence of an optimal speed for human running, in some but not all participants. Those exhibiting a COTmin undertook a higher volume of running, ran with a cadence that was more consistently modulated with speed, and tended to be aerobically fitter. The ability to minimise the energetic cost of transport appears not to be ubiquitous feature of human running but may emerge in some individuals with extensive running experience.

miR-126 in human cancers : clinical roles and current perspectives

miR-126 has been implicated in the processes of inflammation and angiogenesis. Through these processes, miR-126 is implicated in cancer biology, but its role there has not been well reviewed. The aim of this review is to examine the molecular mechanisms and clinicopathological significance of miR-126 in human cancers. miR-126 was shown to have roles in cancers of the gastrointestinal tract, genital tracts, breast, thyroid, lung and some other cancers. Its expression was suppressed in most of the cancers studied. The molecular mechanisms that are known to cause aberrant expression of miR-126 include alterations in gene sequence, epigenetic modification and alteration of dicer abundance. miR-126 can inhibit progression of some cancers via negative control of proliferation, migration, invasion, and cell survival. In some instances, however, miR-126 supports cancer progression via promotion of blood vessel formation. Downregulation of miR-126 induces cancer cell proliferation, migration, and invasion via targeting specific oncogenes. Also, reduced levels of miR-126 are a significant predictor of poor survival of patients in many cancers. In addition, miR-126 can alter a multitude of cellular mechanisms in cancer pathogenesis via suppressing gene translation of numerous validated targets such as PI3K, KRAS, EGFL7, CRK, ADAM9, HOXA9, IRS-1, SOX-2, SLC7A5 and VEGF. To conclude, miR-126 is commonly down-regulated in cancer, most likely due to its ability to inhibit cancer cell growth, adhesion, migration, and invasion through suppressing a range of important gene targets. Understanding these mechanisms by which miR-126 is involved with cancer pathogenesis will be useful in the development of therapeutic targets for the management of patients with cancer.

Clinicopathological relevance of BRAF mutations in human cancer

BRAF represents one of the most frequently mutated protein kinase genes in human tumours. The mutation is commonly tested in pathology practice. BRAF mutation is seen in melanoma, papillary thyroid carcinoma (including papillary thyroid carcinoma arising from ovarian teratoma), ovarian serous tumours, colorectal carcinoma, gliomas, hepatobiliary carcinomas and hairy cell leukaemia. In these cancers, various genetic aberrations of the BRAF proto-oncogene, such as different point mutations and chromosomal rearrangements, have been reported. The most common mutation, BRAF V600E, can be detected by DNA sequencing and immunohistochemistry on formalin fixed, paraffin embedded tumour tissue. Detection of BRAF V600E mutation has the potential for clinical use as a diagnostic and prognostic marker. In addition, a great deal of research effort has been spent in strategies inhibiting its activity. Indeed, recent clinical trials involving BRAF selective inhibitors exhibited promising response rates in metastatic melanoma patients. Clinical trials are underway for other cancers. However, cutaneous side effects of treatment have been reported and therapeutic response to cancer is short-lived due to the emergence of several resistance mechanisms. In this review, we give an update on the clinical pathological relevance of BRAF mutation in cancer. It is hoped that the review will enhance the direction of future research and assist in more effective use of the knowledge of BRAF mutation in clinical practice.

Clinical impacts of mammalian target of rapamycin expression in human colorectal cancers

This study investigated the clinicopathologic roles of mammalian target of rapamycin (mTOR) expression and its relationship to carcinogenesis and tumor progression in a colorectal adenoma-adenocarcinoma model. Two colon cancer cell lines with different pathologic stages (SW480 and SW48) and 1 normal colonic epithelial cell line (FHC) were used, in addition to 119 colorectal adenocarcinomas and 32 adenomas. mTOR expression profiles at messenger RNA (mRNA) and protein levels were investigated in the cells and tissues using real-time quantification polymerase chain reaction and immunohistochemistry. The findings were correlated with the clinicopathologic features of the tumors. The colon cell line from stage III cancer (SW48) showed higher expression of mTOR mRNA than that from stage II cancer (SW480). At the tissue level, mTOR showed higher mRNA and protein expression in colorectal carcinoma than in adenoma. The mRNA and protein expression was correlated with each other in approximately one-third of the carcinomas and adenomas. High levels of mTOR mRNA expression were noted more in carcinoma or adenoma arising from the distal portion of the large intestine (P = .025 and .019, respectively). Within the colorectal cancer population, a high level of expression of mTOR mRNA was related to the presence of lymph node metastases (P = .031), advanced pathologic stage (P = .05), and presence of persistent disease or tumor recurrence (P = .035). To conclude, the study has indicated that mTOR is likely to be involved in the development and progression of colorectal cancer and is linked to cancer initiation, invasiveness, and progression.

Telomerase activity : detection and clinical implications in human cancer

Telomerase is an extremely important enzyme required for the immortalisation of tumour cells. Because the gene is activated in the vast majority of tumour tissues and remains unused in most somatic cells, it represents a marker with huge diagnostic, prognostic and treatment implications in cancer. This article summarises the basic structure and functions of telomerase and considers its clinical implications in colorectal and other cancers.

Genome-wide analysis in a murine Dnmt1 knockdown model identifies epigenetically silenced genes in primary human pituitary tumors

DNA methylation at promoter CpG islands (CGI) is an epigenetic modification associated with inappropriate gene silencing in multiple tumor types. In the absence of a human pituitary tumor cell line, small interfering RNA-mediated knockdown of the maintenance methyltransferase DNA methyltransferase (cytosine 5)-1 (Dnmt1) was used in the murine pituitary adenoma cell line AtT-20. Sustained knockdown induced reexpression of the fully methylated and normally imprinted gene neuronatin (Nnat) in a time-dependent manner. Combined bisulfite restriction analysis (COBRA) revealed that reexpression of Nnat was associated with partial CGI demethylation, which was also observed at the H19 differentially methylated region. Subsequent genome-wide microarray analysis identified 91 genes that were significantly differentially expressed in Dnmt1 knockdown cells (10% false discovery rate). The analysis showed that genes associated with the induction of apoptosis, signal transduction, and developmental processes were significantly overrepresented in this list (P < 0.05). Following validation by reverse transcription-PCR and detection of inappropriate CGI methylation by COBRA, four genes (ICAM1, NNAT, RUNX1, and S100A10) were analyzed in primary human pituitary tumors, each displaying significantly reduced mRNA levels relative to normal pituitary (P < 0.05). For two of these genes, NNAT and S100A10, decreased expression was associated with increased promoter CGI methylation. Induced expression of Nnat in stable transfected AtT-20 cells inhibited cell proliferation. To our knowledge, this is the first report of array-based "epigenetic unmasking" in combination with Dnmt1 knockdown and reveals the potential of this strategy toward identifying genes silenced by epigenetic mechanisms across species boundaries.

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.

Assessment of Geogenic Contaminants in Water Co-Produced with Coal Seam Gas Extraction in Queensland, Australia : Implications for Human Health Risk

Organic compounds in Australian coal seam gas produced water (CSG water) are poorly understood despite their environmental contamination potential. In this study, the presence of some organic substances is identified from government-held CSG water-quality data from the Bowen and Surat Basins, Queensland. These records revealed the presence of polycyclic aromatic hydrocarbons (PAHs) in 27% of samples of CSG water from the Walloon Coal Measures at concentrations <1 µg/L, and it is likely these compounds leached from in situ coals. PAHs identified from wells include naphthalene, phenanthrene, chrysene and dibenz[a,h]anthracene. In addition, the likelihood of coal-derived organic compounds leaching to groundwater is assessed by undertaking toxicity leaching experiments using coal rank and water chemistry as variables. These tests suggest higher molecular weight PAHs (including benzo[a]pyrene) leach from higher rank coals, whereas lower molecular weight PAHs leach at greater concentrations from lower rank coal. Some of the identified organic compounds have carcinogenic or health risk potential, but they are unlikely to be acutely toxic at the observed concentrations which are almost negligible (largely due to the hydrophobicity of such compounds). Hence, this study will be useful to practitioners assessing CSG water related environmental and health risk.

Intramacrophage survival of uropathogenic Escherichia coli : differences between diverse clinical isolates and between mouse and human macrophages

Uropathogenic E. coli (UPEC) are the primary cause of urinary tract infections. Recent studies have demonstrated that UPEC can invade and replicate within epithelial cells, suggesting that this bacterial pathogen may occupy an intracellular niche within the host. Given that many intracellular pathogens target macrophages, we assessed the interactions between UPEC and macrophages. Colonization of the mouse bladder by UPEC strain CFT073 resulted in increased expression of myeloid-restricted genes, consistent with the recruitment of inflammatory macrophages to the site of infection. In in vitro assays, CFT073 was able to survive within primary mouse bone marrow-derived macrophages (BMM) up to 24 h post-infection. Three additional well-characterized clinical UPEC isolates associated with distinct UTI symptomatologies displayed variable long-term survival within BMM. UPEC strains UTI89 and VR50, originally isolated from patients with cystitis and asymptomatic bacteriuria respectively, showed elevated bacterial loads in BMM at 24 h post-infection as compared to CFT073 and the asymptomatic bacteriuria strain 83972. These differences did not correlate with differential effects on macrophage survival or initial uptake of bacteria. E. coli UTI89 localized to a Lamp1+ vesicular compartment within BMM. In contrast to survival within mouse BMM, intracellular bacterial loads of VR50 were low in both human monocyte-derived macrophages (HMDM) and in human T24 bladder epithelial cells. Collectively, these data suggest that some UPEC isolates may subvert macrophage anti-microbial pathways, and that host species differences may impact on intracellular UPEC survival.