Journal of Nucleic Acids Investigation

P53 and microRNAs in chronic lymphocytic leukemia

Tue, 12 Jul 2011 00:00:00 +0000

There has been considerable progress in characterising chronic lymphocytic leukemia with respect to the underlying biology and the corresponding clinical presentation. Most patients with chronic lymphocytic leukemia live for years without the need for therapy. This does not hold true for patients that exhibit inactivation of the tumour suppressor p53 or, possibly, its pathway members. Deletions of chromosome 17p and mutations of TP53 are the main characteristic of aggressive and refractory disease. In this review we will outline the current understanding of mechanisms known to contribute to dysfunctional p53 and its connection to microRNAs.

HER2 splice variants and their relevance in breast cancer

Wed, 06 Jul 2011 00:00:00 +0000

The HER2 gene amplification occurs in 20-30% of breast cancer and is correlated with a poorer prognosis compared to HER2-negative disease due to increased proliferation and metastatic potential. Two major types of receptor inhibitors have been developed for therapy and one for each categories is currently used in clinic: i) the humanized monoclonal antibody trastuzumab, directed against the HER2 extracellular domain; and ii) the EGFR/HER2 dual tyrosine kinase inhibitor lapatinib. However, patients may develop resistance to drugs and show disease progression. Several resistant mechanisms have been explored and are still under investigation. Here, we focus our attention on the role played by the alternative splicing forms of HER2 in mediating HER2 oncogenic activity and in conditioning the response to HER2 therapies. Three HER2 splice variants have been described so far; the p100 and the herstatin gave raised to two secreted proteins of 100 kd and 68 kd, respectively that act as cell growth inhibitors. Herstatin has been described for its ability to interrupt the constitutive HER2 activation, but also for its capacity to hamper HER2 dimerization with the others HER receptors. Interestingly, herstatin, present as mRNA and protein in non cancerous tissue in areas adjacent to breast carcinoma, is absent as protein in 75% of mammary tumors, which indicates that cancer cells are protected by some intrinsic mechanism against the putative growth-inhibitory effects of this naturally occurring molecule. The third splice form of HER2 gene is the Δ16HER2, encoding for a receptor lacking exon16, whose absence determines a constitutive active dimers with transforming activity in vitro and in vivo. The Δ16HER2 binds to trastuzumab to a less extend, due to conformational changes of the extracellular domain. The Δ16HER2 accounts for almost 9% of the total HER2 transcripts in human breast cancers and, additionally, Δ16HER2 levels are supposed to increase proportionally at the increasing of the HER2 wild-type copy numbers in human primary breast cancers. The availability of a specific assay to determine and quantify the expression levels of this splicing form and the availability of Δ16HER2 transgenic mice models made this variant as the most promising for the development of biodrugs.

Finally, HER2 carboxy-terminal fragments (CTFs), generated by alternative initiation of translation, were observed in breast cancer patients. In particular, 611-CTF was described to activate multiple signaling pathways since it is expressed as a constitutively active homodimer. Expression of 611-CTF led to development of aggressive and invasive mammary tumors and it was suggested to be a potent oncogene capable of promoting mammary tumor progression and metastasis.

High mobility group A-interacting proteins in cancer: focus on chromobox protein homolog 7, homeodomain interacting protein kinase 2 and PATZ

Monica Fedele, Giovanna Maria Pierantoni, Pierlorenzo Pallante, Alfredo Fusco — Fri, 16 Mar 2012 00:00:00 +0000

The High Mobility Group A (HMGA) proteins, a family of DNA architectural factors, by interacting with different proteins play crucial roles in neoplastic transformation of a wide range of tissues. Therefore, the search for HMGA-interacting partners was carried out by several laboratories in order to investigate the mechanisms underlying HMGA-dependent tumorigenesis. Three of the several HMGA-binding proteins are discussed in this review. These are the Chromobox family protein (chromobox protein homolog 7, CBX7), the homeodomain interacting protein kinase 2 (HIPK2) and the POZ/domain and Kruppel zinc finger family member, PATZ. All of them play a critical role in tumorigenesis, and may also be independent markers of cancer. Their activities are linked to cell cycle, apoptosis and senescence. In this review, we discuss the properties of each protein, including their effect on HMGA1 functions, and propose a model accounting for how their activities might be coordinated.

Predictive molecular markers for EGFR-TKI in non-small cell lung cancer patients: new insights and critical aspects

Paola Ulivi, Daniele Calistri, Wainer Zoli, Dino Amadori — Thu, 15 Jul 2010 00:00:00 +0000

In recent years, a number of novel agents have been investigated that target specific molecular pathways in non-small cell lung cancer (NSCLC). A great deal of effort has been focused on identifying specific markers that predict treatment response, given that a tailored approach would maximize both the therapeutic index and the cost-effectiveness. The epidermal growth factor receptor (EGFR) pathway has emerged as a key regulator of cancer cell proliferation and invasion, and several specific EGFR inhibitors have been examined. Gefitinib and erlotinib are selective EGFR tyrosine kinase inhibitors (EGFR-TKIs), demonstrating good results in selected cases both in terms of objective response rate and of overall survival. At present, EGFR gene mutations are the best positive predictive factors for TKI therapy, and a number of other potential biomarkers are being investigated as additional positive or negative predictors of response. The correct selection of patients that could benefit from these innovative therapies, based on an accurate molecular characterization, is mandatory to provide the best clinical management. Currently, the main factor limiting the characterization of metastatic NSCLC patients is the small quantity of tumor cells available for molecular analysis. In this paper we provide an overview of the most important molecular predictive markers for EGFR-TKIs therapy in NSCLC patients, and focus attention on biological samples suitable for analysis and alternative sampling approaches such as plasma- or serum-derived DNA.

Survival of the fittest or best adapted: HLA-dependent tumor development

Thu, 27 May 2010 00:00:00 +0000

The major histocompatibility complex (MHC) comprises a set of genes that are critical to immunity and surveillance against neoplastic transformation. There is increasing evidence that the MHC antigens not only regulate antitumor immune responses in experimental animal models but directly correlate with survival and prognosis of patients with diverse types of cancers. MHC antigens may in the future function as potential biomarkers for prognosis and allow selection of cancer patients for intensive therapy.

Cancer immunoediting and dioxin-activating aryl hydrocarbon receptor: a missing link in the shift toward tumor immunoescape?

Ruggero Ridolfi, Massimo Guidoboni, Laura Ridolfi — Wed, 19 May 2010 00:00:00 +0000

The aryl hydrocarbon receptor (AhR), a member of the PAS protein family, is found in organisms as diverse as Drosophila melanogaster, nematodes, and mammals. While several reviews have reported that AhR, once activated by agonist ligands, causes long-term effects such as modification of cell growth through cell cycle control, there is also recent evidence of its decisive role in immunosuppression. The most widely studied AhR agonist is 2,3,7,8-tetrachlorodibenzo-p-dioxin, which binds AhR with the highest known affinity, leading to profound suppression of both humoral and cellular immune responses, with praecox thymus involution, consequent thymocyte loss, and induction of T-cell apoptosis. Dioxin-AhR binding causes a decline in the number of dendritic cells and enhances apoptosis following their inappropriate activation. Dioxin-mediated activation of AhR also has a direct influence on the expansion of regulatory T-cells CD4+CD25+ FoxP3+ (T-regs) and an adverse affect on CD8+ T-cell responses. Dioxin released from industrial and waste incinerators over the last few decades has caused widespread contamination of food, leading to its accumulation in fatty tissue in animals and humans. The elimination half-life of dioxin in humans (7-10 years) may favor the potentially continuous and long-lasting activation of AhR, leading to perpetual immune suppression and facilitating the onset, growth, and diffusion of tumors, especially in young people. In the cancer immunoediting hypothesis, which subdivides the relationship between tumor and immune system into three phases: elimination, equilibrium, and escape, it is thought that dioxin accumulation may cause an inevitable shift toward tumor escape.

Induced pluripotent stem cells: the long-expected revolution in medical science and practice?

Antonio Sorrentino — Mon, 01 Mar 2010 00:00:00 +0000

Within the matter of a few years, development of the somatic reprogramming technology to generate induced pluripotent stem (iPS) cells has contributed enormously to the stem cell research field. We learned that differentiated adult cells possess an unrestricted plasticity that allows them to be driven back to their embryonic or pluripotent state, but owing to the juvenile nature of this novel science chapter, there are many unanswered questions and dilemmas. It is indisputable, however, that iPS cells potentially could represent the jack-of-all-trades remedy in areas of medicine ranging from toxicology screening to regenerative medicine. In this review I will summarize the current strategies employed to reprogram somatic cells and the major promises and hurdles for the future of iPS cells.

A new entropy model for RNA: part I. A critique of the standard Jacobson-Stockmayer model applied to multiple cross links

Wayne Dawson, Kenji Yamamoto, Gota Kawai — Tue, 24 Jul 2012 00:00:00 +0000

The Jacobson-Stockmayer (JS) model is used in a number of standard programs for calculating the conformational entropy of RNA (and proteins). However, it is shown in this study that, in certain limiting cases, the current form of this model can lead to highly unphysical conclusions. The origin of this behavior can be traced to misunderstandings that occurred during the development of the model as applied to folded, single-stranded RNA. Here we show that an alternative model known as the cross linking entropy (CLE) model can overcome these issues. The principal object that causes entropy loss on a global scale in the CLE model is the stem, the primary measure of structural order in such coarse-grained calculations. The principal objects in the JS-model are various types of loops, and, with the exception of the hairpin loop, they are topologically local in character. To extract experimentally measurable variables, a simplified version of the CLE model is developed that resembles many features of the contact order model used in RNA and protein folding. These modifications are then applied to single molecule force-extension experiments (molecular tweezers) to extract quantitative information. It is further shown that a crude derivative of the CLE model itself can be derived directly from the JS-model when the misunderstandings are examined and corrected.

A new entropy model for RNA: part II. Persistence-related entropic contributions to RNA secondary structure free energy calculations

Wayne Dawson, Kenji Yamamoto, Kentaro Shimizu, Gota Kawai — Fri, 15 Mar 2013 00:00:00 +0000

In previous work, we have shown that the entropy of a folded RNA molecule can be divided into local and global contributions using the cross-linking entropy (CLE) model, where, in the case of RNA, the cross- links are the base-pair stacking interactions. The local contribution to the CLE is revealed in the Kuhn length (a measure of the stiffness of the RNA). The Kuhn length acts as a scaling parameter. When the size of the system is rescaled, the relationship between local and global free energy must be renormalized to reflect this rescaling. In this renormalization process, the Kuhn length increases, the local entropy also increases due to freezing out of the local conformational degrees of freedom. At the same time, as the number of degrees of freedom decrease, there is a significant reduction in the global entropy. Here we present a method, based on the concepts of renormalization theory, to quantitatively estimate the size of the contribution from the local entropy as a function of the Kuhn length. The local entropy correction is used to predict the current empirically derived constant in the Jacobson-Stockmayer equation. The variation in the Kuhn length is shown to be largely influenced by the length of the double-stranded RNA stems formed in the secondary structure of folded RNA. This result is used to test the resulting entropy under a variable Kuhn length in stem-loop structures. Comparisons between a variable Kuhn length and a static Kuhn length on a short stem-loop of RNA are also examined. The model is quite general and is also directly applicable to protein structure and folding problems.

A new entropy model for RNA: part III. Is the folding free energy landscape of RNA funnel shaped?

Wayne Dawson, Toshikuni Takai, Nobuharu Ito, Kentaro Shimizu, Gota Kawai — Mon, 03 Nov 2014 00:00:00 +0000

The concept of a free energy (FE) landscape, in which the conformations of a polymer progressively take on the structure of the native state while spiraling down a FE surface that resembles the shape of a funnel, has long been viewed as the reason why a complex protein structure forms so rapidly compared to the number of conformations available to it. On the other hand, this landscape picture is less clear with RNA due to the multiplicity of conformations and the uncertainties in the current thermodynamics. It is therefore sometimes proposed that within the ensemble of suboptimal states of the RNA molecule, the vast majority of those states all closely resemble the native state and therefore simply overwhelm the few states that represent the global minimum FE. However, calculations of the free energy of observed structures often suggest that the most frequently observed cluster of structures are far from the minimum FE, particularly in the case of long sequences. If so, then such a FE surface is unlikely to be funnel shaped. We have been developing a version of vsfold that can evaluate the suboptimal structures of the FE surface (through a modified version called vs_subopt). Here we show that the ensemble of suboptimal structures for a number of known RNA structures can actually be both close to the minimum FE and also be the dominant observed structure when a proper Kuhn length is selected. Two state aptamers known as riboswitches can show neighboring FE states in the suboptimal structures that match the observed structures and their relative difference in FE is well within the range of the binding free energy of the metabolite. For the riboswitches and other short RNA sequences (less than 250 nt), the flow of the suboptimal structures (including pseudoknots) tended to resemble a rock rolling down a hill along the reaction coordinate axis. An important insight yielded by the cross-linking entropy (CLE) model is that the global entropy limits the size of domains. Hence, based on the CLE model, Levinthal’s paradox is overcome by the funnel shape in the FE, by a reduction in the number of degrees of freedom due to Kuhn length, and by limits on the size of the domains that can form. These concepts are also applicable to calculating transition rates between different suboptimal structures.

A new entropy model for RNA: part IV, The Minimum Free Energy (mFE) and the thermodynamically most-probable folding pathway (TMPFP)

Wayne Dawson, Gota Kawai — Mon, 13 Jul 2015 00:00:00 +0000

Here we discuss four important questions (1) how can we be sure that the thermodynamically most-probable folding-pathway yields the minimum free energy for secondary structure using the dynamic programming algorithm (DPA) approach, (2) what are its limitations, (3) how can we extend the DPA to find the minimum free energy with pseudoknots, and finally (4) what limitations can we expect to find in a DPA approach for pseudoknots. It is our supposition that some structures cannot be fit uniquely by the DPA, but may exist in real biology situations when disordered regions in the biomolecule are necessary. These regions would be identifiable by using suboptimal structure analysis. This grants us some qualitative tools to identify truly random RNA sequences, because such are likely to have greater degeneracy in their thermodynamically most-probable folding-pathway.

A new entropy model for RNA: part V, Incorporating the Flory-Huggins model in structure prediction and folding

Wayne Dawson, Gota Kawai — Mon, 13 Jul 2015 00:00:00 +0000

The effect of solvent-biopolymer interactions is hardly negligible. Whereas the ideal (non-interacting) polymer consisting of N monomers in an ideal solvent is expected to have the terminal ends of its chain with a root-mean-squared (rms) end-to-end separation distance (rms) proportional to the square root of N, real interactions of a rms polymer both with itself and with the solvent often tend to strongly perturb rms. In rms poor solvent, the biopolymer can collapse into a small globule much smaller than the ideal rms due to excluding solvent. In good solvent, the biopolymer can swell to a size much larger than the ideal r due to favoring solvent. These effects require rms corrections to an ideal polymer equation. We have been developing the cross linking entropy (CLE) model in this series. The model attempts find the maximum entropy of a folded polymer by taking into account the correlation caused by bonding and other interactions of the structure. In RNA, this mostly occurs in the stems. Here we adapt CLE model to handle polymer swelling and collapse for RNA molecules both in good and in poor solvent. This work is intended to introduce this type of study and to allow its systematic application in problems of RNA folding and structure prediction. The current study suggests that there may be some tendency for RNA to behave as a polymer in poor solvent and that this collapse may happen in sequences longer than 50 nt.

Computational prediction of candidate miRNAs and their targets from the completed Linum ussitatissimum genome and EST database

Tiffanie Y. Moss, Christopher A. Cullis — Tue, 05 Jun 2012 00:00:00 +0000

Flax is an important agronomic crop grown for its fiber (linen) and oil (linseed oil). In spite of many thousands of years of breeding some fiber varieties have been shown to rapidly respond to environmental stress with heritable changes to its genome. Many miRNAs appear to be induced by abiotic or biotic conditions experienced through the plant life cycle. Computational miRNA analysis of the flax genome provides a foundation for subsequent research on miRNA function in Linum usitatissimum and may also provide novel insight into any regulatory role the RNAi pathway may play in generating adaptive structural variation in response to environmental stress. Here a bioinformatics approach is used to screen for miRNAs previously identified in other plant species, as well as to predict putative miRNAs unique to a particular species which may not have been identified as they are less abundant or dependent upon a specific set of environmental conditions. Twelve miRNA genes were identified in flax on the basis of unique pre-miRNA positions with structural homology to plant pre-miRNAs and complete sequence homology to published plant miRNAs. These miRNAs were found to belong to 7 miRNA families, with an additional 2 matches corresponding to as yet unnamed poplar miRNAs and a parologous miRNA with partial sequence homology to mtr-miR4414b. An additional 649 novel and distinct flax miRNA genes were identified to form from canonical hairpin structures and to have putative targets among the ~30,000 flax Unigenes.

The effect of in vitro exposure to antisense oligonucleotides on macrophage morphology and function

Fri, 18 Nov 2011 00:00:00 +0000

Antisense oligonucleotides (AON) delivered via inhalation are in drug development for respiratory diseases. In rodents and monkeys, repeated exposure to high doses of inhaled phosphorothioate (PS) AON can lead to microscopic changes in the lungs, including accumulation of alveolar macrophages in the lower airway that have a foamy appearance. The functional consequences that result from this morphological change are unclear as there is controversy whether the vacuoles/inclusion bodies reflect normal clearance of the inhaled AON or are early indicators of lung toxicity. The morphological and functional responses of macrophage to PS AON were characterized in vitro using the comparator drug amiodarone, as a known inducer of foamy macrophages. Morphological changes of increased vacuolization with the presence of lamellated structures were observed in macrophages in response to both amiodarone and AON treatment. Functional responses to the drugs clearly differed with amiodarone treatment leading to apoptosis of cells and cell death, release of proinflammatory mediators IL-1RA, MIP-1α and TNFα, decrease in IP-10, a cytokine shown to be involved in protection against pulmonary fibrosis and altered phagocytosis capacity of the cells. In contrast, AON in concentrations up to 30 μM, had no effect on cell viability or apoptosis, had minimal effects on pro-inflammatory cytokines, increased IP-10 levels and did not alter the phagocytic capacity of the cells. Exposure of macrophages to AON in vitro, led to morphological changes of increased vacuolization, but did not lead to functional consequences which were observed with another vacuolization-inducing drug, suggesting that the in vivo phenotypic changes observed following inhalation of AON may be consistent with a clearance mechanism and not an activation or impairment of macrophages.

Unusual 5'-regulatory structure and regulation of the murine Mlc1 gene: lack of promoter-specific functional elements

Sat, 22 Oct 2011 00:00:00 +0000

The MLC1 gene is involved in an autosomal recessive neurological disorder, megalencephalic leucoencephalopathy with subcortical cysts (MLC), which is characterized by macrocephaly during the first year of life and swollen white matter (leucoencephaly). Variants of MLC1 have also been associated with psychiatric disorders such as schizophrenia, major depression and bipolar disorder. Currently, little is known about the encoded protein (MLC1). Judging from its similarity to other known proteins, it may serve as a trans-membrane transporter. However, the function of the encoded protein and its gene regulation has not been investigated successfully so far. We investigated the 5’ region of the murine Mlc1 with respect to regulatory elements for gene expression. A promoter search and an in silico analysis were conducted. Luciferase reporter gene constructs with potential promoter regions were created to study promoter activity in vitro. We found two alternative first exons for the murine Mlc1 but were not able to detect any promoter activity for the investigated reporter gene constructs in different cell lines, thus pointing to the presence of essential cis-acting elements far outside of the region. In silico analysis indicated an uncommon promoter structure for Mlc1, with CCAAT-boxes representing the only noticeable elements.

Development of network inference among LexA/RecA-dependent manner genes in the SOS response

Sachiyo Aburatani — Thu, 25 Nov 2010 00:00:00 +0000

The expression of many genes varies under different conditions. The relationships among genes can be identified from DNA microarray data. To better understand the SOS response, I developed a network model of whole LexA/RecA-dependent manner genes in two genotypes of Escherichia coli from expression data after ultraviolet (UV) irradiation. First, all LexA/RecA-dependent manner genes were estimated by a combination of the Mann-Whitney U test and hierarchical clustering. Second, the relationships between genes were inferred from a graphical Gaussian model combined with hierarchical clustering. Here, I incorporate a step-by-step procedure of the method developed in my previous study to identify interactions within larger clusters. The analysis suggested the presence of a sequential relationship between LexA/RecA-dependent manner gene groups in which the association between neighboring groups was positive while that between non-neighboring groups was negative. I suggest the inferred network provides information on the functional role of poorly defined genes whose expression patterns change according to LexA/RecA. Although relationships in the network require biological validation, the model presented here provides insights into potential LexA/RecA regulation following UV irradiation.

Silencing of hpv16 e6 and e7 oncogenic activities by small interference RNA induces autophagy and apoptosis in human cervical cancer cells

Tue, 30 Aug 2011 00:00:00 +0000

Cervical cancer is the second most common form of death by cancer in women worldwide and has special attention for the development of new treatment strategies. Human Papilloma Virus (HPV) persistent infection is the main etiological agent of this neoplasia, and the main cellular transformation mechanism is by disruption of p53 and pRb function by interaction with HPV E6 and E7 oncoproteins. This generates alterations in cellular differentiation and cellular death inhibition. Thus, HPV E6 and E7 oncogenes represent suitable targets for the development of gene therapy strategies against cervical cancer. An attractive technology platform is developing for post-transcriptional selective silencing of gene expression, using small interference RNA. Therefore, in the present study, we used SiHa cells (HPV16+) transiently transfected with specific siRNA expression plasmids for HPV16 E6 and E7 oncogenes. In this model we detected repression of E6 and E7 oncogene and oncoprotein expression, an increase in p53 and hypophosphorylated pRb isoform protein expression, and autophagy and apoptosis morphology features. These findings suggest that selective silencing of HPV16 E6 and E7 oncogenes by siRNAs, has significant biological effects on the survival of human cancer cells and is a potential gene therapy strategy against cervical cancer.

GyrB vs. 16s rRna sequencing for the identification of Campylobacter jejuni, Campylobacter coli, and Campylobacter lari

Nereus William Gunther IV, Jonnee Almond, Xianghe Yan, David S Needleman — Thu, 05 May 2011 00:00:00 +0000

Species of the genus Campylobacter are responsible for the largest number of bacterial food-borne illness cases occurring yearly in the developed world. The majority of disease is caused by three of the thermotolerant Campylobacter species: Campylobacter jejuni, Campylobacter coli, and Campylobacter lari. An inability to differentiate these three species using the commonly employed 16S rRNA sequencing procedure has led to the development of alternative methods to identify these bacteria. Some of these methods include the utilization of the gyrB gene. A reliable method was developed for the differentiation of C. jejuni, C. coli, and C. lari employing the gyrB gene. It involves amplification and sequencing of a species-variable region of the gene with a single pair of DNA primers. The method works well for the separation and organization of the three Campylobacter strains as well as satisfying the suggested guidelines for sequence based identification for most strains investigated.

FHIT suppresses inflammatory carcinogenic activity by inducing apoptosis in esophageal epithelial cells

Thu, 20 May 2010 00:00:00 +0000

We focused on the mechanism by which FHIT suppresses neoplastic transformation in normal but damaged esophageal epithelial cells exposed to inflammatory stimuli in vivo and to chemo-radiotherapy in clinical samples. For in vitro analysis, Adenoviral-FHIT (Ad-FHIT) in TE4 and TE2 were used for microarray analysis. For in vivo analysis, wild-type (WT) FHIT and FHIT-deficient (KO) C57BL/6 mice were exposed to N-nitrosomethylbenzylamine (NMBA) and to a cyclooxygenase-2 inhibitor (COXI). Considering DNA damage on clinical samples, expressions of FHIT, BAX and PCNA were evaluated by comparing between 3 cases of esophageal cancer cases of the chemo-radiotherapy responder and 7 cases of the non-responder. In in vitro analysis, we listed the down-regulated genes in Ad-FHIT that significantly control Lac-Z infected cells, such as prostaglandin E receptor 4, cyclooxygenase-1 and cyclooxygenase-2. In in vivo analysis, FHIT-KO mice were much more susceptible to tumorigenesis than were FHIT-WT mice. A significant difference in PGE2 activation was observed between FHIT-WT mice (5.2 ng/mL) and FHIT-KO mice (28.4 ng/mL) after exposure to NMBA in the absence of COXI as determined by ELISA assay (P less than 0.01). BAX expression was significantly higher in FHIT-WT (1.0±0.43) than in FHIT-KO (0.17±0.17) (P less than 0.05). The IHC score for FHIT and BAX expression was significantly higher in responders than the others (P less than 0.05). FHIT possesses tumor suppressor activity by induction of apoptosis in damaged cells after exposure to inflammatory carcinogens and DNA damaging chemo-radiotherapy.

A novel detection method for heteroduplex DNA using carbodiimide-induced interrupted primer extension

Tania Tabone, Richard Cotton, Ninan Mathew, J. Des Parkin, Judy Savige — Thu, 03 Jun 2010 00:00:00 +0000

Direct sequencing may be problematic in demonstrating mutations where inherited disease results from multiple different heterozygous variants in large genes. We describe here a novel mutation screening method based on the ability of carbodiimide to bind mismatched DNA and interrupt primer extension thereby identifying both a heterozygous variant and its location. This assay detected all four classes of DNA mismatch in 550 bp engineered plasmid fragments and in two dominantly inherited renal diseases. In patients with thin basement membrane nephropathy, the method demonstrated multiple variants within a single amplicon including some close to the primer binding site. This method also detected a complex mutation in medullary cystic kidney disease type 2 (c.278-289 del/insCCGGCTCCT) as multiple termination events and, furthermore, correctly identified five affected and 28 unaffected family members. Carbodiimide-induced interrupted primer extension identifies heterozygous variants in large or multiexonic genes, where the variants differ in each family, their locations are unknown, and even if there are multiple known non-pathogenic variants within the same amplicon. This assay incorporates a “universal” protocol that detects all types of mutations without the need for further optimization, and potentially detects mutations where the proportion of heteroduplex is less than 50%.

Robust hybridization-based genotyping probes for HPV 6, 11, 16 and 18 obtained via in vitro selection

Mon, 12 Apr 2010 00:00:00 +0000

This paper describes the technical and analytical performance of a novel set of hybridization probes for the four GARDASIL^® vaccine-relevant HPV types (6, 11, 16 and 18). These probes are obtained through in vitro selection from a pool of random oligonucleotides, rather than the traditional “rational design” approach typically used as the initial step in assay development. The type-specific segment of the HPV genome was amplified using a GP5⁺/6⁺ PCR protocol and 39 synthetic oligonucleotide templates derived from each of the HPV types, as PCR targets. The robust performance of the 4 selected hybridization probes was demonstrated by monitoring the preservation of the specificity and sensitivity of the typing assay over all 39 HPV types, using a different spectrum of HPV (genome equivalent: 103-109) and human DNA concentrations (10-100 ng) as well as temperature and buffer composition variations. To the Authors’ knowledge, this is a unique hybridization-based multiplex typing assay. It performs at ambient temperatures, does not require the strict temperature control of hybridization conditions, and is functional with a number of different non-denaturing buffers, thereby offering downstream compatibility with a variety of detection methods. Studies aimed at demonstrating clinical performance are needed to validate the applicability of this strategy.

The mitochondrial housekeeping gene 16S is inappropriate as an internal standard in comparative studies of rare mitochondrial transcripts using S1-nuclease protection assays

Sandra Ebert, Line Breumlund, Roland Nau, Uwe Michel — Wed, 14 Apr 2010 00:00:00 +0000

The analysis of rare mitochondrial transcripts derived from the L-strand of the mitochondrial genome requires a sensitive method such as the S1-nuclease protection assay. We examined whether the ribosomal mitochondrial transcript 16S is suitable as an internal standard in a multiplex S1-nuclease protection assay for the measurement of different mitochondrial transcripts. For reliable quantification of rare mitochondrial transcripts with the RNase protection assay, a minimum of 2 μg of total RNA is necessary. Standard curves of 16S RNA produced with total RNA from human kidney, liver, brain, and a human neuroblastoma cell line (SH-SY5Y) revealed dose-response relationships that were saturated already at less than 0.5 μg of total RNA. Therefore, 16S is inappropriate as an internal standard for analyzing mitochondrial transcripts with RNase protection assays when more than 0.5 μg of total RNA have to be analyzed.

MicroRNAs, SNPs and cancer

Angela V. Vitale, Huiping Tan, Peng Jin — Mon, 11 Apr 2011 00:00:00 +0000

MiRNAs are probable regulators of cell events such as differentiation, propagation and apoptosis. These cellular phenomena are also associated with benign and malignant tumor cells, therefore, it is presumed that miRNAs act as natural oncogenes or tumor suppressor genes. Whether a particular miRNA serves as either could almost be moot when the additional problems of SNPs enter the fray. A miRNA involved with SNPs (miR-SNPs) on any regulatory level, whether naturally cancerinducing or not, could easily undergo an oncogenic transformation. This work reviews targets of miRNAs and the miRNAs themselves frequently containing SNPs reflecting different risks and markers of cancer with emphasis on familial groups and populations of shared heredity.

MicroRNAs as biomarkers for the diagnosis and prognosis of human cancer

Daniela Asslaber, Josefina Piñón Hofbauer, Richard Greil, Alexander Egle — Mon, 13 Dec 2010 00:00:00 +0000

miRNAs are small-noncoding RNA molecules that regulate gene expression on a posttranscriptional level. A number of oncogenes and tumor suppressors were found to be targets of miRNAs and global miRNA expression signatures were able to distinguish between cancerous and non-cancerous tissues. Therefore it was not surprising that some miRNAs could be linked to the pathogenesis of cancer. In this review we provide an overview of the use of microRNAs as diagnostic and prognostic tools in cancer and focus on the use of miRNA expression as biomarker for disease activity.

Expression of microRNA during bovine adipogenesis

Scott L. Pratt, T. Ashley Burns, Erin Curry, Susan K. Duckett — Thu, 11 Nov 2010 00:00:00 +0000

Studies have recently indicated that the adipogenic process and the expression of genes involved in lipid metabolism may be regulated in part at the post-transcriptional level by a class of small RNA called microRNA (miRNA). The objectives of this study were to i) determine if miRNAs are differentially expressed, and ii) evaluate expression of known miRNA targets in bovine adipocytes. Bovine adipose samples were collected from castrated males fattened on a high concentrate diet (C) or pasture (PA) and were frozen in liquid nitrogen and stored at -80°C, or used to generate primary stromal-vascular cells (SV). SV cells were cultured to confluence (Control) or differentiated at confluence and harvested 2 (D2), 6 (D6), or 12 (D12) days post-confluence. A 3x3 microarray analysis was performed comparing Control and differentiated samples. miR-21, -221, and -222 (P less than 0.05) were differentially expressed. qRT-PCR was conducted using the in vitro samples, and all three miRNAs were down regulated on D2 (P less than 0.05). miR-221 and -222 were decreased on D6 compared to Control (P less than 0.05), but only miR-222 expression was decreased at D12 (P less than 0.05) compared to Control. miR-21 increased in expression compared to Control on D12 (P less than 0.05). In vivo, only miR-21 expression was affected and it was reduced in PA compared to C fat samples (P less than 0.05). Two targets of miR-21 are Programmed Cell Death Protein 4 (PDCD4) and Phosphatase and Tensin Homolog (PTEN), and neither messenger RNA was differentially expressed in vitro (P greater than 0.05), but both messenger RNAs were elevated for PA compared to C (P less than 0.05). These data show that miRNAs are differentially expressed in adipose cells and tissue, and that miR-21 may be involved in adipocyte function by regulating the translation of PDCD4 and PTEN.

Non-coding RNAs and cancer: microRNAs and beyond

Rachel Marie Raia, George Adrian Calin — Fri, 11 Mar 2011 00:00:00 +0000

Non-coding RNAs were previously thought to have little importance because they are not directly translated into a protein like their coding counterparts. However, it was recently found that non-coding RNAs do in fact have a much bigger role than previously thought. They are involved in cancer predisposition, development and progression. MicroRNAs, very short non-coding RNAs, are abnormally expressed in cancer and some harbor mutations that affect expression levels. MicroRNA alterations have been observed in all forms of cancer that have been researched to the current date. MicroRNAs are also located in cancer- associated genomic regions, which have been previously shown to affect gene expression leading to the activation or inhibition of cancer growth. Single-nucleotide polymorphisms within microRNAs can predispose someone to cancer. MicroRNAs have been shown to target both tumor suppressors, inhibiting cancer development, as well as oncogenes, stimulating cancer development. Some microRNAs can switch between these two functions and behave as a tumor suppressor at one time and an oncogene at another time. MicroRNAs can be used for diagnostic purposes as well as prognostic evaluations. Outside of microRNAs, ultraconserved genes, another group of non-coding RNAs, also express differently in cancer patients. Large intervening non-coding RNAs, specifically one termed HOTAIR, have been quantified in very high levels in cancer cells and have been implicated in metastasis. Further research into noncoding RNAs may allow for the development of therapies that will target non-coding RNAs creating better treatment options for cancer patients, improving their prognosis. This review discusses the most current discoveries about non-coding RNAs, revealing their associations with cancer.

Molecular markers for prediction of risk of radiation-related injury to normal tissue

Marco Ghilotti, Marco Alessandro Pierotti, Manuela Gariboldi — Mon, 11 Oct 2010 00:00:00 +0000

Radiotherapy is one of the most effective methods for the treatment of cancer, but occurrence of adverse reactions developing in the co-irradiated normal tissue can be a threat for patients. Identification of individuals at risk of severe reaction is very difficult and considerable efforts have been made to correlate normal tissue toxicity with cellular responses to ionizing radiation. Genetic markers enabling to identify hyper-sensitive patients prior to treatment would considerably improve its outcome. Gene association studies should help to identify such markers. Expression levels of specific transcripts could be putative markers; in fact, different studies found associations between gene expression profiles in normal cells and the reaction of normal tissues to radiation therapy. The finding that ionizing radiation induces the deregulation of a high number of genes suggests that also microRNAs that affect the expression of a large number of target genes may be involved. This review briefly introduces the mechanisms of radiation-induced normal tissue toxicity and summarizes clinical research focused on the evaluation of molecular biomarkers for predicting risk of injury to normal tissue, mainly describing gene transcripts alterations.

Online resources for microRNA analysis

Panagiotis Alexiou, Manolis Maragkakis, Artemis G Hatzigeorgiou — Fri, 25 Feb 2011 00:00:00 +0000

The use of online tools for bioinformatics analyses is becoming increasingly widespread. Resources specific to the field of microRNAs are available, varying in scope and usability. Online tools are the most useful for casual as well as power users since they need no installation, are hardware independent and are used mostly through graphic user interfaces and links to external sources. Here, we present an overview of useful online resources that have to do with microRNA genomics, gene finding, target prediction and functional analysis.

MicroRNAs in mouse models of lymphoid malignancies

Tue, 25 May 2010 00:00:00 +0000

The discovery of microRNAs (miRNAs) has revealed a new layer of gene expression regulation that affects many normal and pathologic biological systems. Among the malignancies affected by the dysregulation of miRNAs there are cancers of lymphoid origin, in which miRNAs are thought to have tumor suppressive or tumor promoting activities, depending on the nature of their specific targets. In the last 4-5 years, the experimental field that provided the deepest insights into the in vivo biology of miRNAs is that of mouse modeling in which transgenic and knockout animals mimic, respectively, over-expression or down-regulation of specific miRNAs involved in human leukemia/lymphoma. This review discusses recent advances in our understanding of lymphoid malignancies based on the natural and engineered mouse models of three different miRNAs, miR-15a/16-1 cluster, miR-155, and miR-17-92 cluster.

Therapeutic implications of microRNAs in human cancer

Michael Rossbach — Mon, 07 Feb 2011 00:00:00 +0000

MicroRNAs (miRNAs) are a class of highly evolutionarily conserved non-coding RNAs (ncRNAs) that modulate gene expression. Several studies have shown that the expression of miRNAs is deregulated in human malignancies. For ncRNAs and miRNAs, such gene-profiling studies in tumorigenic tissues have identified significant signatures that are of both diagnostic and prognostic value. Addressing the functions of ncRNAs not only give insights into the molecular mechanisms that underlie complex genetic processes, but may also elucidate novel mechanisms that contribute to early stages of tumor development, progression and metastasis. MiRNA-based novel approaches target the ncRNAome, including, for instance, miRNA expression levels and improved designs of miRNA-mimics or more precise target-predictions, prevent off-target effects of novel drugs and make miRNAs become a highly efficient class of therapeutics. For miRNA-based therapeutic studies two direct strategies are currently under investigation, viz. (i) the overexpression of given miRNAs to inhibit the expression of protein-coding genes or (ii) the inhibition of target miRNAs with antisense constructs like antagomiRs. Indirect strategies include the use of novel drugs that modulate miRNA expression levels by directly targeting their processing or transcription. Further, miRNA-based biomarkers have a significant impact on the development of both therapeutic and diagnostic agents, a concept known as theranostics and are highly relevant for drug development and personalized medicine.

Role of microRNAs in solid tumors

Rie Hamano, Hideshi Ishii, Hiroshi Miyata, Yuichiro Doki, Masaki Mori — Tue, 19 Apr 2011 00:00:00 +0000

Accumulating experimental evidence indicates that microRNAs play important roles in various biological processes, such as cell differentiation, proliferation, metabolism and apoptosis. In addition, several reports concluded that altered expression of specific microRNA genes contributes to the initiation and progression of cancer. Here, we summarize the current knowledge about aberrant expression of various microRNAs in human solid cancers (e.g., lung, breast, and gastric cancers), their target proteins, and the relationship between their expression and response to chemotherapies. We also review the potential for using microRNAs as biomarkers for the diagnosis and cancer therapy. The development of treatment strategies against human solid cancers based on the profile and/or certain features of microRNAs is promising.

Body fluid micro(mi)RNAs as biomarkers for human cancer

Edward R. Sauter, Neel Patel — Mon, 17 Jan 2011 00:00:00 +0000

Accepted tools for early cancer detection run the gamut from Pap staining to detect cervical cancer detection to colonoscopy and biopsy for colorectal cancer detection to imaging (mammogram and, in high risk women, magnetic resonance imaging) and biopsy for breast cancer detection. These modalities use standard cytopathologic assessment to determine if disease is present. There are few biologic (DNA, RNA, protein or carbohydrate) markers (biomarkers) that are in general use for the detection of any cancer, or to identify individuals at increased cancer risk. Biomarkers have been identified that provide information to physicians on disease prognosis. Panels of biomarkers are being developed to predict response to treatment in individuals with known cancer, and some are currently in use. Nonethless, there is a great need to identify accurate biomarkers for the early detection of a new cancer, to identify individuals at increased cancer risk, and among those with cancer, to determine their likelihood of responding to a given treatment, risk of disease relapse and death. Micro (mi)RNAs hold promise as biomarkers for determining at risk individuals, for early cancer detection, and among those with cancer, to assess how likely a person is to respond a given treatment, their risk of disease recurrence and death.

Role of microRNAs in the molecular diagnosis of cancer

Simona Giglio, Andrea Vecchione — Mon, 10 May 2010 00:00:00 +0000

MicroRNAs (miRNAs) are evolutionarily conserved, endogenous, small non-coding RNA molecules of about 22 nucleotides in length that function as posttranscriptional gene regulators. They are involved in numerous cellular processes including development, cell differentiation, cell cycle regulation and apoptosis. There is increasing evidence to show that miRNAs are mutated or differentially expressed in many types of cancer and specific functions of the miRNAs are now becoming apparent. Here we discuss the current literature on potential usefulness of miRNAs as diagnostic markers, emphasizing the involvement of specific miRNAs in particular tumor types, highlighting their potential role in distinguishing benign from malignant tissues and/or the different subtypes of the same tumor and/or in diagnosis and classification of tumor of unknown origin.

A modified Phenol-chloroform extraction method for isolating circulating cell free DNA of tumor patients

Clemens Hufnagl, Markus Stöcher, Martin Moik, Roland Geisberger, Richard Greil — Mon, 11 Mar 2013 00:00:00 +0000

Searching for new cancer biomarkers, circulating cell-free DNA (cfDNA) has become an appealing target of interest as an elevated level of cfDNA has been detected in the circulation of cancer patients in comparison with healthy controls. Since cfDNA can be isolated from the circulation and other body fluids of patients without harming their physical condition, cfDNA is becoming a promising candidate as a novel non-invasive biomarker for cancer. The challenge in the diagnostic analysis of cfDNA is its very low presence in human plasma/serum and its partially strong fragmentation. Here we evaluated a modified phenol/chloroform extraction method for the isolation of cfDNA and compared it with published standard methods for cfDNA isolation.

Practical strategies for the evaluation of high-affinity protein/nucleic acid interactions

Sarah E. Altschuler, Karen A. Lewis, Deborah S. Wuttke — Fri, 20 Sep 2013 00:00:00 +0000

The quantitative evaluation of binding interactions between proteins and nucleic acids is highly sensitive to a variety of experimental conditions. Optimization of these conditions is critical for obtaining high quality, reproducible data, particularly in the context of very high affinity interactions. Here, we discuss the practical considerations involved in optimizing the apparent binding constant of an interaction as measured by two common quantitative assays, electrophoretic mobility shift assay and double-filter binding when measuring extremely tight protein/nucleic acid interactions with sub-nanomolar binding affinities. We include specific examples from two telomere end-binding protein systems, Schizosaccharomyces pombe Pot1 and Saccharomyces cerevisiae Cdc13, to demonstrate potential experimental pitfalls and some useful strategies for optimization.