Gene electrotransfer of IL-2 and IL-12 plasmids effectively eradicated murine B16.F10 melanoma.

Gene therapy has become an important approach for treating cancer, and electroporation represents a technology for introducing therapeutic genes into a cell. An example of cancer gene therapy relying on gene electrotransfer is the use of immunomodulatory cytokines, such as interleukin 2 (IL-2) and 12 (IL-12), which directly stimulate immune cells at the tumour site. The aim of our study was to determine the effects of gene electrotransfer with two plasmids encoding IL-2 and IL-12 in vitro and in vivo. Two different pulse protocols, known as EP1 (600 V/cm, 5 ms, 1 Hz, 8 pulses) and EP2 (1300 V/cm, 100 µs, 1 Hz, 8 pulses), were assessed in vitro for application in subsequent in vivo experiments. In the in vivo experiment, gene electrotransfer of pIL-2 and pIL-12 using the EP1 protocol was performed in B16.F10 murine melanoma. Combined treatment of tumours using pIL2 and pIL12 induced significant tumour growth delay and 71% complete tumour regression. Furthermore, in tumours coexpressing IL-2 and IL-12, increased accumulation of dendritic cells and M1 macrophages was obtained along with the activation of proinflammatory signals, resulting in CD4 + and CD8 + T-lymphocyte recruitment and immune memory development in the mice. In conclusion, we demonstrated high antitumour efficacy of combined IL-2 and IL-12 gene electrotransfer protocols in low-immunogenicity murine B16.F10 melanoma.

Transcriptomic analysis of nickel exposure in Sphingobium sp. ba1 cells using RNA-seq.

Nickel acts as cofactor for a number of enzymes of many bacteria species. Its homeostasis is ensured by proteins working as ion efflux or accumulation systems. These mechanisms are also generally adopted to counteract life-threatening high extra-cellular Ni2+ concentrations. Little is known regarding nickel tolerance in the genus Sphingobium. We studied the response of the novel Sphingobium sp. ba1 strain, able to adapt to high Ni2+ concentrations. Differential gene expression in cells cultured in 10 mM Ni2+, investigated by RNA-seq analysis, identified 118 differentially expressed genes. Among the 90 up-regulated genes, a cluster including genes coding for nickel and other metal ion efflux systems (similar to either cnrCBA, nccCBA or cznABC) and for a NreB-like permease was found. Comparative analyses among thirty genomes of Sphingobium species show that this cluster is conserved only in two cases, while in the other genomes it is partially present or even absent. The differential expression of genes encoding proteins which could also work as Ni2+-accumulators (HupE/UreJ-like protein, NreA and components of TonB-associated transport and copper-homeostasis systems) was also detected. The identification of Sphingobium sp. ba1 strain adaptive mechanisms to nickel ions, can foster its possible use for biodegradation of poly-aromatic compounds in metal-rich environments.

RNA editing signature during myeloid leukemia cell differentiation.

Adenosine deaminases acting on RNA (ADARs) are key proteins for hematopoietic stem cell self-renewal and for survival of differentiating progenitor cells. However, their specific role in myeloid cell maturation has been poorly investigated. Here we show that ADAR1 is present at basal level in the primary myeloid leukemia cells obtained from patients at diagnosis as well as in myeloid U-937 and THP1 cell lines and its expression correlates with the editing levels. Upon phorbol-myristate acetate or Vitamin D3/granulocyte macrophage colony-stimulating factor (GM-CSF)-driven differentiation, both ADAR1 and ADAR2 enzymes are upregulated, with a concomitant global increase of A-to-I RNA editing. ADAR1 silencing caused an editing decrease at specific ADAR1 target genes, without, however, interfering with cell differentiation or with ADAR2 activity. Remarkably, ADAR2 is absent in the undifferentiated cell stage, due to its elimination through the ubiquitin-proteasome pathway, being strongly upregulated at the end of the differentiation process. Of note, peripheral blood monocytes display editing events at the selected targets similar to those found in differentiated cell lines. Taken together, the data indicate that ADAR enzymes play important and distinct roles in myeloid cells.

BCR/ABL1 fusion transcripts generated from alternative splicing: implications for future targeted therapies in Ph+ leukaemias.

Philadelphia (Ph+) positive leukaemias are an example of haematological malignant diseases where different chromosomal rearrangements involving both BCR and ABL1 genes generate a variety of chimeric proteins (BCR/ABL1 p210, p190 and p230) which are considered pathological "biomarkers". In addition to these three, there is a variety of fusion transcripts whose origin may depend either on diverse genetic rearrangement or on alternative/atypical splicing of the main mRNAs or on the occurrence of single-point mutations. Although the therapy of Ph+ leukaemias based on Imatinib represents a triumph of medicine, not all patients benefit from such drug and may show resistance and intolerance. Furthermore, interruption of Imatinib administration is often followed by clinical relapse, suggesting a failure in the eradication of residual leukaemic stem cells. Therefore, while the targeted therapy is searching for new and implemented pharmacological inhibitors covering all the possible mutations in the kinase domain, there is urge to identify alternative molecular targets to develop other specific and effective therapeutic approaches. In this review we discuss the importance of recent advances based on the discovery of novel BCR/ABL1 variants and their potential role as new targets/biomarkers of Ph+ leukaemias in the light of the current therapeutic trends. The limits of the pharmacological inhibitors used for treating the disease can be overcome by considering other targets than the kinase enzyme. Our evaluations highlight the potential of alternative perspectives in the therapy of Ph+ leukaemias.

ASPicDB: a database resource for alternative splicing analysis.

MOTIVATION: Alternative splicing has recently emerged as a key mechanism responsible for the expansion of transcriptome and proteome complexity in human and other organisms. Although several online resources devoted to alternative splicing analysis are available they may suffer from limitations related both to the computational methodologies adopted and to the extent of the annotations they provide that prevent the full exploitation of the available data. Furthermore, current resources provide limited query and download facilities. RESULTS: ASPicDB is a database designed to provide access to reliable annotations of the alternative splicing pattern of human genes and to the functional annotation of predicted splicing isoforms. Splice-site detection and full-length transcript modeling have been carried out by a genome-wide application of the ASPic algorithm, based on the multiple alignments of gene-related transcripts (typically a Unigene cluster) to the genomic sequence, a strategy that greatly improves prediction accuracy compared to methods based on independent and progressive alignments. Enhanced query and download facilities for annotations and sequences allow users to select and extract specific sets of data related to genes, transcripts and introns fulfilling a combination of user-defined criteria. Several tabular and graphical views of the results are presented, providing a comprehensive assessment of the functional implication of alternative splicing in the gene set under investigation. ASPicDB, which is regularly updated on a monthly basis, also includes information on tissue-specific splicing patterns of normal and cancer cells, based on available EST sequences and their library source annotation. AVAILABILITY: www.caspur.it/ASPicDB

Statistical assessment of functional categories of genes deregulated in pathological conditions by using microarray data.

MOTIVATION: A major challenge in current biomedical research is the identification of cellular processes deregulated in a given pathology through the analysis of gene expression profiles. To this end, predefined lists of genes, coding specific functions, are compared with a list of genes ordered according to their values of differential expression measured by suitable univariate statistics. RESULTS: We propose a statistically well-founded method for measuring the relevance of predefined lists of genes and for assessing their statistical significance starting from their raw expression levels as recorded on the microarray. We use prediction accuracy as a measure of relevance of the list. The rationale is that a functional category, coded through a list of genes, is perturbed in a given pathology if it is possible to correctly predict the occurrence of the disease in new subjects on the basis of the expression levels of the genes belonging to the list only. The accuracy is estimated with multiple random validation strategy and its statistical significance is assessed against a couple of null hypothesis, by using two independent permutation tests. The utility of the proposed methodology is illustrated by analyzing the relevance of Gene Ontology terms belonging to biological process category in colon and prostate cancer, by using three different microarray data sets and by comparing it with current approaches. AVAILABILITY: Source code for the algorithms is available from author upon request. SUPPLEMENTARY INFORMATION: Colon cancer data set and a complete description of experimental results are available at: ftp://bioftp:76bioftpxxx@marx.ba.issia.cnr.it/supp-info.htm.

On the statistical assessment of classifiers using DNA microarray data.

BACKGROUND: In this paper we present a method for the statistical assessment of cancer predictors which make use of gene expression profiles. The methodology is applied to a new data set of microarray gene expression data collected in Casa Sollievo della Sofferenza Hospital, Foggia--Italy. The data set is made up of normal (22) and tumor (25) specimens extracted from 25 patients affected by colon cancer. We propose to give answers to some questions which are relevant for the automatic diagnosis of cancer such as: Is the size of the available data set sufficient to build accurate classifiers? What is the statistical significance of the associated error rates? In what ways can accuracy be considered dependant on the adopted classification scheme? How many genes are correlated with the pathology and how many are sufficient for an accurate colon cancer classification? The method we propose answers these questions whilst avoiding the potential pitfalls hidden in the analysis and interpretation of microarray data. RESULTS: We estimate the generalization error, evaluated through the Leave-K-Out Cross Validation error, for three different classification schemes by varying the number of training examples and the number of the genes used. The statistical significance of the error rate is measured by using a permutation test. We provide a statistical analysis in terms of the frequencies of the genes involved in the classification. Using the whole set of genes, we found that the Weighted Voting Algorithm (WVA) classifier learns the distinction between normal and tumor specimens with 25 training examples, providing e = 21% (p = 0.045) as an error rate. This remains constant even when the number of examples increases. Moreover, Regularized Least Squares (RLS) and Support Vector Machines (SVM) classifiers can learn with only 15 training examples, with an error rate of e = 19% (p = 0.035) and e = 18% (p = 0.037) respectively. Moreover, the error rate decreases as the training set size increases, reaching its best performances with 35 training examples. In this case, RLS and SVM have error rates of e = 14% (p = 0.027) and e = 11% (p = 0.019). Concerning the number of genes, we found about 6000 genes (p < 0.05) correlated with the pathology, resulting from the signal-to-noise statistic. Moreover the performances of RLS and SVM classifiers do not change when 74% of genes is used. They progressively reduce up to e = 16% (p < 0.05) when only 2 genes are employed. The biological relevance of a set of genes determined by our statistical analysis and the major roles they play in colorectal tumorigenesis is discussed. CONCLUSIONS: The method proposed provides statistically significant answers to precise questions relevant for the diagnosis and prognosis of cancer. We found that, with as few as 15 examples, it is possible to train statistically significant classifiers for colon cancer diagnosis. As for the definition of the number of genes sufficient for a reliable classification of colon cancer, our results suggest that it depends on the accuracy required.

Discovery of 342 putative new genes from the analysis of 5'-end-sequenced full-length-enriched cDNA human transcripts.

In this work we describe the process that, starting with the production of human full-length-enriched cDNA libraries using the CAP-Trapper method, led us to the discovery of 342 putative new human genes. Twenty-three thousand full-length-enriched clones, obtained from various cell lines and tissues in different developmental stages, were 5'-end sequenced, allowing the identification of a pool of 5300 unique cDNAs. By comparing these sequences to various human and vertebrate nucleotide databases we found that about 40% of our clones extended previously annotated 5' ends, 662 clones were likely to represent splice variants of known genes, and finally 342 clones remained unknown, with no or poor functional annotation. cDNA-microarray gene expression analysis showed that 260 of 342 unknown clones are expressed in at least one cell line and/or tissue. Further analysis of their sequences and the corresponding genomic locations allowed us to conclude that most of them represent potential novel genes, with only a small fraction having protein-coding potential.

Long-branch attraction phenomenon and the impact of among-site rate variation on rodent phylogeny.

The phylogenetic relationships among major lineages of rodents is one of the issues most debated by both paleontologists and molecular biologists. In the present study, we have analyzed all complete mammalian mitochondrial genomes available in the databases, including five rodent species (rat, mouse, dormouse, squirrel and guinea-pig). Phylogenetic analyses were performed on H-strand amino acid sequences by means of maximum-likelihood and on H-strand protein-coding and ribosomal genes by means of distance methods. Also, log-likelihood ratio tests were applied to different tree topologies under the assumption of rodent monophyly, paraphyly or polyphyly. The analyses significantly rejected rodent monophyly and showed the existence of two differentiated clades, one containing non-murids (dormouse, squirrel and guinea-pig) and the other containing murids (rat and mouse). Long-branch attraction between murids and the outgroups could not be responsible for the existence of two different rodent clades, as no significant differences in evolutionary rate have been observed, except in the case of the squirrel, which shows a lower rate. The impact of among-site rate variation models on the phylogeny of rodents has been evaluated using the gamma distribution model. Results have shown that relationships among rodents remained unchanged, and the general topology of the tree was not affected, even though some branches were not properly resolved, most likely due to a lack of fit between estimated and real rate heterogeneity parameters.

Guinea pig p53 mRNA: identification of new elements in coding and untranslated regions and their functional and evolutionary implications.

We report the sequence of the guinea pig p53 cDNA. The comparative analysis of the coding and noncoding regions of p53 cDNAs of all available complete vertebrate sequences has allowed us to single out new conserved signals possibly involved in p53 functional activity. We have focused our attention on the most variable region of the protein, the proline (P)-rich domain, suggested to play a fundamental role in antiproliferative pathways. In this domain we have identified the PXXXXP repeated motif and singled out a common consensus sequence that can be considered a signature for mammalian p53: PXXXXPX{0,4}PX{0,9}PA(T,P,I,)(S,P)WPL. We have demonstrated the significance of the PXXXXP motif in SH3-binding protein and suggested its structure to be a loop. Also, the 5' and 3' untranslated regions (UTRs) of the guinea pig were sequenced, and this study represents the first detailed structural analysis of the UTRs of the p53 mRNAs available in literature. The 5' UTR of guinea pig (233 nt) can be folded into a stable secondary structure resembling that predicted in mouse. The 3' UTR of guinea pig is 771 nt long and shows higher similarity with human than with rodent sequences, having a region of about 350 nt that is deleted in rat and mouse. In the 3' UTR we have identified the presence of a mammalian-wide interspersed repeat sequence and of a cytoplasmic polyadenylation element, which could be involved in translational activation by promoting polyadenylation of mRNA, providing information about a possible mechanism of regulation of p53 expression mediated by the 3' UTR of the mRNA. The observations presented here could open new avenues to targeted mutations and experimental approaches useful in investigating new regulation mechanisms of p53 translation, activity, and stability.

The members of the RH gene family (RH50 and RH30) followed different evolutionary pathways.

The evolution of the RH gene family is characterized by two major duplication events, the first one originating the RH50 and RH30 genes and the second one giving rise to RHCE and RHD, the two paralogous RH30 genes which encode the Rh blood group antigens in human. The new sequence data obtained here for mouse RH50 and RH30 and for macaque RH50 allowed us to compare the evolutionary rates of the two genes and to show that RH50 evolved about 2.6 times more slowly than RH30 at nonsynonymous positions. This result implies that Rh50 proteins were evolutionarily more conserved compared to Rh30 polypeptides, thus being indicative of the functional significance of the former protein in species as distantly related as sponge and human. The duplication event leading to RH50 and RH30 genes was estimated to have occurred between 250 and 346 million years ago. Moreover, we could also estimate that the duplication event producing the RHCE and RHD genes occurred some 8.5 +/- 3.4 million years ago, in the common ancestor of human, chimpanzee, and gorilla. Interestingly, this event seems to coincide with the appearance in these species of a G-to-T mutation in the RH50 gene which created a stop codon in the corresponding transcript. This led to an Rh50 C-terminal cytoplasmic domain shorter than that found in orangutan and early primates.

The 67-kDa laminin receptor originated from a ribosomal protein that acquired a dual function during evolution.

The 67-kDa laminin receptor (67LR) is a nonintegrin cell surface receptor that mediates high-affinity interactions between cells and laminin. Overexpression of this protein in tumor cells has been related to tumor invasion and metastasis. Thus far, only a full-length gene encoding a 37-kDa precursor protein (37LRP) has been isolated. The finding that the cDNA for the 37LRP is virtually identical to a cDNA encoding the ribosomal protein p40 has suggested that 37LRP is actually a component of the translational machinery, with no laminin-binding activity. On the other hand, a peptide of 20 amino acids deduced from the sequence of 37LR/p40 was shown to exhibit high laminin-binding activity. The evolutionary relationship between 23 sequences of 37LRP/p40 proteins was analyzed. This phylogenetic analysis indicated that all of the protein sequences derive from orthologous genes and that the 37LRP is indeed a ribosomal protein that acquired the novel function of laminin receptor during evolution. The evolutionary analysis of the sequence identified as the laminin-binding site in the human protein suggested that the acquisition of the laminin-binding capability is linked to the palindromic sequence LMWWML, which appeared during evolution concomitantly with laminin.

Lineage-specific evolution of echinoderm mitochondrial ATP synthase subunit 8.

Peculiar evolutionary properties of the subunit 8 of mitochondrial ATP synthase (ATPase8) are revealed by comparative analyses carried out between both closely and distantly related species of echinoderms. The analysis of nucleotide substitution in the three echinoids demonstrated a relaxation of amino acid functional constraints. The deduced protein sequences display a well conserved domain at the N-terminus, while the central part is very variable. At the C-terminus, the broad distribution of positively charged amino acids, which is typical of other organisms, is not conserved in the two different echinoderm classes of the sea urchins and of the sea stars. Instead, a motif of three amino acids, so far not described elsewhere, is conserved in sea urchins and is found to be very similar to the motif present in the sea stars. Our results indicate that the N-terminal region seems to follow the same evolutionary pattern in different organisms, while the maintenance of the C-terminal part in a phylum-specific manner may reflect the co-evolution of mitochondrial and nuclear genes.

Glutamine synthetase gene evolution in bacteria.

The evolution of the prokaryotic glutamine synthase (GS) genes, namely the GSI and GSII isoforms, has been investigated using the second codon positions, which have previously proven to behave as a good molecular clock. Our data confirm the early divergence between prokaryotic and eukaryotic GSII before the splitting between plants and animals. The phylogenetic tree of the GSI isoforms shows Archaebacteria to be more closely related to Eubacteria than to Eukaryotes. This finding is confirmed by the phylogenetic analysis carried out on both large and small subunits of rRNA. However, differently from the rRNA analyses, Crenarchaeota and Euryarchaeota Archaebacteria, as well as high- and low-GC gram-positive bacteria, appear to be polyphyletic. We provide evidence that the observed polyphyly of Archaebacteria might be only apparent, resulting from a gene duplication event preceding the split between Archaebacteria and Eubacteria and followed by the retention of only one isoform in the extant lineages. Both gram-negative bacteria and high-GC gram-positive bacteria, which appear closely related, have GS activity regulated by an adenylylation/deadenylylation mechanism. A lateral gene transfer from Archaebacteria to low-GC eubacteria is invoked to explain the observed polyphyly of gram-positive bacteria.

The peculiar evolution of apolipoprotein(a) in human and rhesus macaque.

Apo(a) is a low density lipoprotein homologous to plasminogen and has been shown to be involved in coronary atherosclerosis. In the present paper we will try to analyze the interesting evolutionary pattern of Apo(a). The plasminogen gene contains 5 cysteine-rich sequences, called kringles, followed by a protease domain. Apo(a), probably arisen by duplication of an ancestral plasminogen gene, contains many tandemly repeated copies of a sequence domain similar to the fourth kringle of plasminogen, 37 in human and at least 10 in the partially sequenced gene of rhesus, and the protease domain. We have found that the upstream kringles of apo(a) undergo Molecular Drive-like processes that produce high intraspecies similarity, whereas the downstream kringles evolve in a molecular clock-like manner and show an high interspecies sequence similarity. The latter regions are obviously suitable for dating the duplication event by which Apo(a) arose from plasminogen, but only if they evolve at the same rate in the two genes. Thus, we propose a "Molecular Clock Test" for assessing whether the comparison of two paralogous genes (or gene regions) can give reliable information on the dating of their origin by duplication. Applying this test to the kringle-4 domain of apo(a) and plasminogen gene, we demonstrate that the separation between the two genes by duplication dates back at about 90 Mya immediately before the radiation of mammals.