Proteomic patterns of cultured breast cancer cells and epithelial mammary cells.

Breast cancer is one of the leading causes of death from cancer among women in western countries. The different types of breast cancer are grouped into invasive and noninvasive forms. Among the invasive types, ductal infiltrating carcinoma (DIC) is the most common and aggressive form. Using an in vitro model consisting of a DIC-derived cell line (8701-BC) and a nontumoral mammary epithelial cell line (HB2), we used the proteomics approach to search for homology and differences in protein expression patterns between tumoral and nontumoral phenotypes. Within an analysis window comprising 1,750 discernible spots we have currently catalogued 140 protein spots of potential interest. Fifty-eight of them were identified by gel matching with reference maps, immunodetection, or N-terminal microsequencing and classified into four functional groups. Twelve proteins were found differentially expressed in two cell lines: four were uniquely present in the neoplastic cell proteome and eight in epithelial cells. In addition, 53 proteins displayed different relative expression levels between the two cell lines, that is, 44 were more elevated in cancer cells and 9 in HB2 cells. Among proteins with greater relative abundance in cancer cells we identified glycolytic enzymes (or their isoforms), which may indicate that the known metabolic dysregulation in cancer can reflect oncogenic-related defects of glycolytic gene expression

A contribution to breast cancer cell proteomics: detection of new sequences

Ductal infiltrating carcinoma (DIC) of the breast is the most common and potentially aggressive form of cancer. Knowledge of proteomic profiles, attained both in vivo and in vitro, is fundamental to acquire as much information as possible on the proteins expressed in these pathologic conditions. We used the breast cancer cell line 8701-BC, established from a primary DIC, with the aim of contributing to the databases on mammary cancer cells, which in turn will be very useful for the identification of differentially expressed proteins in normal and neoplastic cells. Within an analysis window comprising about 1750 discernible spots, we have at present catalogued 84 protein spots. The proteins for which an identity was assigned were identified essentially using gel comparison, N-terminal (Nt) microseqencing and immune detection. Among the protein spots Nt-microsequenced, sixteen corresponded to known proteins, four resulted as modified, relative to matching sequences deposited on databases, and seven were unknown. These modified or novel sequences are thus of potential interest to the knowledge of breast cancer proteomics and its applications

A survey of grapevine fanleaf nepovirus isolates for the presence of satellite RNA

Grapevine fanleaf virus (GFLV) isolates from different geographical origins were surveyed for natural occurrence of satellite RNA. The results of molecular hybridization assays indicated that 5 isolates out of 34 tested, support the multiplication of a satellite RNA, both in Chenopodium quinoa and grapevine. The satellite molecules appear to have a high degree of sequence homology with, and the same size of, the satellite RNA supported by GFLV-F13 strain, isolated and characterized in France

Viroids of grapevines in Italy

Rebviroide in ItalienEs wird über das Vorkommen niedermolekularer RNAs bei 48 Vitis-vinifera-Stämmen und 15 amerikanischen Vitis-Arten und Kreuzungen aus Italien, Osteuropa, Mittelmeer- und Nahost-Ländern berichtet. In den Sämlingen zweier Sorten wurden keine derartigen RNAs gefunden. Aufgrund ihres elektrophoretischen Verhaltens wurden diese RNAs vorläufig identifiziert als Grapevine yellow speckle-Viroid (GYSVd), Grapeivine-Viroid 2 (GVd2) und Hop stunt-Viroid (HSVd). Das letztere Viroid löste bei künstlich infizierten Pflanzen von Tomate cv. Rutgers und Gurke cv. Suyo Befallssymptome aus. HSVd, GYSVd und GVd2 wurden in 97, 92 bzw. 11 % der untersuchten Proben wiedergefunden. In der Regel lagen Mischinfektionen vor, wobei die Kombination von HSVd mit GYSVd überwog. Diese beiden Viroide kamen regelmäßig in Reben mit den Symptomen von Yellow speckle oder Vein banding vor. Es wurde keine eindeutige Beziehung zwischen dem Vorkommen eines der Viroide und Rebkrankheiten mit unklarer Ätiologie, wie Vein necrosis oder Fleck, gefunden

Further studies on the use of molecular probes to grapevine closterovirus A

Two cloned cDNA probes to genomic RNA of grapevine closterovirus A (GVA) were utilized successfully for the detection of viral sequences in infected herbaceous hosts (Nicotiana benthamiana) and grapevines. One of the probes (pGA112) was complementary to the central part of the viral genome and gave light false positive signals with healthy grapevine extracts, whereas the other (pGA240), which is presumably colinear with the 3' terminus, was virus-specific and hybridized only with infected sample extracts. The two probes recognized smaller than genome RNAs in electrophoresed N. benthamiana extracts and hybridized differentially with the bands, thus suggesting that these represent subgenomic RNAs. Probe pGA240 may be used for GVA detection, but the preparation of samples for hybridization needs further improvement for routine testing.Weitere Untersuchungen über die Verwendung von Molekülsonden beim Grapevine-Closterovirus AZwei klonierte cDNA-Sonden für genomische RNA des Grapevine-Closterovirus A (GVA} wurden mit Erfolg zum Nachweis von Virussequenzen in infizierten krautigen Wirtspflanzen (Nicotiana benthamiana) und Reben benützt. Die eine Sonde (pGA112} war komplementär zum zentralen Teil des Virusgenoms und lieferte schwache falsch-positive Nachweisreaktionen mit Extrakten aus gesunden Reben; die andere Sonde (pGA240}, die vermutlich kolinear mit dem 3'-Terminus ist, war dagegen virusspezifisch und hybridisierte nur mit Extrakten aus infizierten Proben. In elektrophoretisch aufgetrennten N. benthamiana-Extrakten "erkannten" die beiden Sonden RNAs von weniger als Genomgröße; sie hybridisierten differenziert mit den Banden, so daß diese subgenomische RNAs darstellen könnten. Die Sonde pGA240 kann für den GVA-Nachweis verwendet werden; für Routinetests muß die Vorbereitung der Proben für die Hybridisierung noch verbessert werden

Detection of grapevine closterovirus A in infected grapevine tissue by reverse transcription-polymerase chain reaction

Reverse transcription-polymerase chain reaction (RT-PCR) was successfully applied to detection of GVA RNA in nucleic acid extracts of infected grapevines. In particular, an artificially synthesized DNA primer set designed to amplify a GVA cDNA fragment of 430 base pairs, specifically detected GVA RNA sequences in extracts from infected grapevine tissues such as leaves from in vitro-grown explants, leaves from greenhouse-grown rooted cuttings, and bark scrapings of mature canes from field-grown vines. The detection limit of GVA RNA by RT-PCR was estimated to be 200 fold higher than that obtained by molecular hybridization or ELISA

Decorin transfection induces proteomic and phenotypic modulation in breast cancer cells 8701-BC

Decorin is a prototype member of the small leucine-rich proteoglycan family widely distributed in the extracellular matrices of many connective tissues, where it has been shown to play multiple important roles in the matrix assembly process, as well as in some cellular activities. A major interest for decorin function concerns its role in tumorigenesis, as growth-inhibitor of different neoplastic cells, and potential antimetastatic agent. The aim of our research was to investigate wide-ranged effects of transgenic decorin on breast cancer cells. To this purpose we utilized the well-characterized 8701-BC cell line, isolated from a ductal infiltrating carcinoma of the breast, and two derived decorin-transfected clones, respectively, synthesizing full decorin proteoglycan or its protein core. The responses to the ectopic decorin production were examined by studying morphological changes, cell proliferation rates, and proteome modulation. The results revealed new important antioncogenic potentialities, likely exerted by decorin through a variety of distinct biochemical pathways. Major effects included the downregulation of several potential breast cancer biomarkers, the reduction of membrane ruffling, and the increase of cell-cell adhesiveness. These results disclose original aspects related to the reversion of malignant traits of a prototype of breast cancer cells induced by decorin. They also raise additional interest for the postulated clinical application of decori

Serological detection of Grapevine rupestris stem pitting-associated virus (GRSPa V) by a polyclonal antiserum to recombinant virus coat protein

The coat protein gene of Grapevine rupestris stem pitting-associated virus (GRSPaV) was amplified with primers based on the completely sequenced Californian GRSPaV isolate, The protein expressed in Escherichia coli was used to raise an antiserum in rabbit. This antiserum was successfully used to detect virus coat protein in infected grapevine extracts either spotted on polyvinyl difluoride membranes (dot immunobinding) or blotted on membranes after gel separation (Western blot). The antiserum titre was 1:5,000 in Western blot. GRSPaV was detected in leaf petioles and cortical scrapings from dormant canes during the whole vegetative season. Several accessions of Vitis rupestris, currently used as presumptive virus-free indicators of Rupestris stem pitting, were found to be infected by this virus. While the application of the antiserum in ELISA was ineffective, the availability of similarly simple and effective serological tools, such as dot immunobinding, may allow a wide survey for GRSPaV

Proteomic profiling of 13 paired ductal infiltrating breast carcinomas and non-tumoral adjacent counterparts.

According to recent statistics, breast cancer remains one of the leading causes of death among women in Western countries. Breast cancer is a complex and heterogeneous disease, presently classified into several subtypes according to their cellular origin. Among breast cancer histotypes, infiltrating ductal carcinoma represents the most common and potentially aggressive form. Despite the current progress achieved in early cancer detection and treatment, including the new generation of molecular therapies, there is still need for identification of multiparametric biomarkers capable of discriminating between cancer subtypes and predicting cancer progression for personalized therapies. One established step in this direction is the proteomic strategy, expected to provide enough information on breast cancer profiling. To this aim, in the present study we analyzed 13 breast cancer tissues and their matched non-tumoral tissues by 2-DE. Collectively, we identified 51 protein spots, corresponding to 34 differentially expressed proteins, which may represent promising candidate biomarkers for molecular-based diagnosis of breast cancer and for pattern discovery. The relevance of these proteins as factors contributing to breast carcinogenesis is discussed

Integrated multi-omics investigations of metalloproteinases in colon cancer: Focus on MMP2 and MMP9

Colorectal cancer (CRC) develops by genetic and epigenetic alterations. However, the molecular mechanisms underlying metastatic dissemination remain unclear and could benefit from multi-omics investigations of specific protein families. Matrix metalloproteinases (MMPs) are proteolytic enzymes involved in ECM remodeling and the processing of bioactive molecules. Increased MMP expression promotes the hallmarks of tumor progression, including angiogenesis, invasion, and metastasis, and is correlated with a shortened survival. Nevertheless, the collective role and the possible coordination of MMP members in CRC are poorly investigated. Here, we performed a multi-omics analysis of MMP expression in CRC using data mining and experimental investigations. Several databases were used to deeply mine different expressions between tumor and normal tissues, the genetic and epigenetic alterations, the prognostic value as well as the interrelationships with tumor immune-infiltrating cells (TIICs). A special focus was placed on to MMP2 and MMP9: their expression was correlated with immune markers and the interaction network of co-expressed genes disclosed their implication in epithelial to mesenchymal transition (EMT) and immune response. Finally, the activity levels of MMP2 and MMP9 in a cohort of colon cancer samples, including tissues and the corresponding sera, was also investigated by zymography. Our findings suggested that MMPs could have a high potency, as they are targeted in colon cancer, and might serve as novel biomarkers, especially for their involvement in the immune response. However, further studies are needed to explore the detailed biological functions and molecular mechanisms of MMPs in CRC, also in consideration of their expression and different regulation in several tissues