Biomedical subjects

Raffaele A Calogero

Publications and source records attributed to Raffaele A Calogero.

9 recordsLinked to original sources

PubMed2006

Comparative gene expression profiling reveals partially overlapping but distinct genomic actions of different antiestrogens in human breast cancer cells.

Antiestrogens used for breast cancer (BC) treatment differ among each other for the ability to affect estrogen receptor (ER) activity and thereby inhibit hormone-responsive cell functions and viability. We used high-density cDNA microarrays for a comprehensive definition of the gene pathways affected by 17beta-estradiol (E2), ICI 182,780 (ICI), 4OH-tamoxifen (Tamoxifen), and raloxifene (RAL) in ER-positive ZR-75.1 cells, a suitable model to investigate estrogen and antiestrogen actions in hormone-responsive BC. The expression of 601 genes was significantly affected by E2 in these cells; in silico analysis reveals that 86 among them include one or more potential ER binding site within or near the promoter and that the binding site signatures for E2F-1, NF-Y, and NRF-1 transcription factors are significantly enriched in the promoters of genes induced by estrogen treatment, while those for CAC-binding protein and LF-A1 in those repressed by the hormone, pointing to novel transcriptional effectors of secondary responses to estrogen in BC cells. Interestingly, expression of 176 E2-regulated mRNAs was unaffected by any of the antiestrogens tested, despite the fact that under the same conditions the transcriptional and cell cycle stimulatory activities of ER were inhibited. On the other hand, of 373 antiestrogen-responsive genes identified here, 52 were unresponsive to estrogen and 25% responded specifically to only one of the compounds tested, revealing non-overlapping and clearly distinguishable effects of the different antiestrogens in BC cells. As some of these differences reflect specificities of the mechanism of action of the antiestrogens tested, we propose to exploit this gene set for characterization of novel hormonal antagonists and selective estrogen receptor modulators (SERMs) and as a tool for testing new associations of antiestrogens, more effective against BC.

Breast Neoplasms↗

PubMed2006

Selection of suitable reference genes for accurate normalization of gene expression profile studies in non-small cell lung cancer.

BACKGROUND: In real-time RT quantitative PCR (qPCR) the accuracy of normalized data is highly dependent on the reliability of the reference genes (RGs). Failure to use an appropriate control gene for normalization of qPCR data may result in biased gene expression profiles, as well as low precision, so that only gross changes in expression level are declared statistically significant or patterns of expression are erroneously characterized. Therefore, it is essential to determine whether potential RGs are appropriate for specific experimental purposes. Aim of this study was to identify and validate RGs for use in the differentiation of normal and tumor lung expression profiles. METHODS: A meta-analysis of lung cancer transcription profiles generated with the GeneChip technology was used to identify five putative RGs. Their consistency and that of seven commonly used RGs was tested by using Taqman probes on 18 paired normal-tumor lung snap-frozen specimens obtained from non-small-cell lung cancer (NSCLC) patients during primary curative resection. RESULTS: The 12 RGs displayed showed a wide range of Ct values: except for rRNA18S (mean 9.8), the mean values of all the commercial RGs and ESD ranged from 19 to 26, whereas those of the microarray-selected RGs (BTF-3, YAP1, HIST1H2BC, RPL30) exceeded 26. RG expression stability within sample populations and under the experimental conditions (tumour versus normal lung specimens) was evaluated by: (1) descriptive statistic; (2) equivalence test; (3) GeNorm applet. All these approaches indicated that the most stable RGs were POLR2A, rRNA18S, YAP1 and ESD. CONCLUSION: These data suggest that POLR2A, rRNA18S, YAP1 and ESD are the most suitable RGs for gene expression profile studies in NSCLC. Furthermore, they highlight the limitations of commercial RGs and indicate that meta-data analysis of genome-wide transcription profiling studies may identify new RGs.

Carcinoma, Non-Small-Cell Lung↗

PubMed2006

The Hay Wells syndrome-derived TAp63alphaQ540L mutant has impaired transcriptional and cell growth regulatory activity.

p63 mutations have been associated with several human hereditary disorders characterized by ectodermal dysplasia such as EEC (ectrodactyly, ectodermal dysplasia, clefting) syndrome, ADULT (acro, dermato, ungual, lacrimal, tooth) syndrome and AEC (ankyloblepharon, ectodermal dysplasia, clefting) syndrome (also called Hay-Wells syndrome). The location and functional effects of the mutations that underlie these syndromes reveal a striking genotype-phenotype correlation. Unlike EEC and ADULT that result from missense mutations in the DNA-binding domain of p63, AEC is solely caused by missense mutations in the SAM domain of p63. In this paper we report a study on the TAp63alpha isoform, the first to be expressed during development of the embryonic epithelia, and on its naturally occurring Q540L mutant derived from an AEC patient. To assess the effects of the Q540L mutation, we generated stable cell lines expressing TAp63alpha wt, DeltaNp63alpha or the TAp63alpha-Q540L mutant protein and used them to systematically compare the cell growth regulatory activity of the mutant and wt p63 proteins and to generate, by microarray analysis, a comprehensive profile of differential gene expression. We found that the Q540L substitution impairs the transcriptional activity of TAp63alpha and causes misregulation of genes involved in the control of cell growth and epidermal differentiation.

Cell Line, Tumor↗

PubMed2005

Oxysterol-induced up-regulation of MCP-1 expression and synthesis in macrophage cells.

To investigate the proinflammatory potential of cholesterol and cholesterol oxidation products (oxysterols), which are present in oxidized low-density lipoproteins, foam cells, and fibrotic plaque, we used an in vitro model mimicking the challenge of macrophage cells by the cholesterol accumulating within the central core of atheroma. A biologically representative oxysterol mixture was shown to be potentially able to sustain a chronic inflammatory process within the vascular wall by up-regulating the expression of defined proinflammatory genes. In particular, expression and synthesis of the major chemokine for monocytes/macrophages, namely monocyte chemotactic protein-1 (MCP-1), were consistently increased when cells of the macrophage lineage (U937 cell line) were incubated with this mixture. On the contrary, an identical concentration of unoxidized cholesterol in no case modified expression or synthesis of the chemokine. Up-regulated expression and synthesis of MCP-1 by the oxysterol mixture was clearly dependent on a net increment of phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) and nuclear factor kappaB (NF-kappaB) nuclear binding. The results indicate that cholesterol may contribute to the progression of atherosclerotic lesions by strongly up-regulating crucial proinflammatory factors like MCP-1, but only after having been oxidized to oxysterols.

Active Transport, Cell Nucleus↗

PubMed2005

Immune prevention of mammary carcinogenesis in HER-2/neu transgenic mice: a microarray scenario.

Neoplastic transformation is a multistep process in which gene products of specific regulatory pathways are involved at each stage. Identification of these overexpressed or mutated gene products provides an unprecedented opportunity to address the immune system against defined antigens and eliminate transformed cells. Mice transgenic for these oncogenes (e.g. HER-2/neu, a prototype of deregulated oncogenic protein kinase membrane receptors) are ideal experimental models for assessing the potential of active immunization. The demonstration that vaccines can cure HER-2/neu transplantable tumors, prevent their onset and delay the progression of preneoplastic lesions in mice at risk suggests that efficient immunological inhibition of HER-2/neu carcinogenesis can be achieved by specific vaccination. To further explore this issue, halting of tumor progression in the mammary glands of BALB-neuT mice with two immunization protocols in two laboratories has been studied independently by DNA microarray analysis. Combination of the two sets of results revealed a clear correlation between them when the tumor mass was titrated by transcription profiling. It was also clear that both protocols induced a strong, polyclonal antibody response and halted tumor growth at a condition very similar to that at which the vaccination began. Differences in the expression profiles were mainly related to the expression levels of a few chemokines and T-cell-specific genes that may be in some way correlated with the efficacy of the vaccination. Last, combination of the expression data with the protection results indicated that chronic vaccination is needed to maintain an active IFN-gamma-mediated response in the mammary gland.

Animals↗

PubMed2004

Towards a long-lasting immune prevention of HER2 mammary carcinomas: directions from transgenic mice.

Several theoretical and practical issues differentiate immune prevention from tumor immune therapy. The latter seeks to induce a rapid reaction against a life-threatening tumor, whereas prevention is dictated by the need to maintain constant surveillance of a situation in which an event is foreseen, but may not occur. The time frame of successful prevention is therefore long and often lifelong. Time itself is thus a key factor in the elaboration of vaccines to prevent tumor growth and its great length in preventive management poses new immunological problems that cannot be studied in short-term vaccination-challenge experiments. Many recent data indicate that HER2 receptor displays several features of an ideal tumor associated antigen and that an immune response can significantly alter HER2 tumor progression. We are thus using vaccination in the immune prevention and cure of carcinomas in HER2 transgenic mice in the search for a rationale for the application of preventive and curative vaccination for patients with HER2/ErbB-2 neoplastic lesions or at risk of recurrence after successful surgery. The design of effective immunopreventive approaches that can be translated to human situations is an important issue. A molecularly defined, effective and validated anti HER2 vaccine and the definition of immune mechanisms leading to the inhibition of HER2-driven neoplastic proliferation may provide a new way of treating these patients.

Animals↗

PubMed2004

Purification of recombinant proteins with high isoelectric points.

The use of recombinant antigens is essential for the construction of robust and sensitive diagnostic assays. A critical step in the preparation of recombinant antigens is protein purification. Purification problems may be very different for related structural proteins expressed in the same host or for the same protein expressed in different hosts, because the biochemical characteristics of a recombinant protein, expressed in a heterologous system, are unique. In this chapter we make a brief introduction to protein purification procedures and we present a quick purification process suitable for the isolation of recombinant protein having high isoelectric points encoding non-conformational epitopes.

Antibodies, Viral↗

PubMed2004

Microarray data analysis and mining.

DNA microarray is an innovative technology for obtaining information on gene function. Because it is a high-throughput method, computational tools are essential in data analysis and mining to extract the knowledge from experimental results. Filtering procedures and statistical approaches are frequently combined to identify differentially expressed genes. However, obtaining a list of differentially expressed genes is only the starting point because an important step is the integration of differential expression profiles in a biological context, which is a hot topic in data mining. In this chapter an integrated approach of filtering and statistical validation to select trustable differentially expressed genes is described together with a brief introduction on data mining focusing on the classification of co-regulated genes on the basis of their biological function.

Cluster Analysis↗

PubMed2001

The human MDM2 oncoprotein increases the transcriptional activity and the protein level of the p53 homolog p63.

Genetic alteration of the p53 tumor suppressor gene, which monitors DNA damage and operates cell cycle checkpoints, is a major factor in the development of human malignancies. The p53 protein belongs to a family that also includes two structurally related proteins, p63 and p73. Although all three proteins share similar transcriptional functions and antiproliferative effects, each of them appears to play a distinct role in development and tumor suppression. One of the principal regulators of p53 activity is the MDM2 protein. The interaction of MDM2 with p53 inhibits p53 transcriptional activity and targets p53 for ubiquitin-dependent degradation. The ability of MDM2 to inhibit p53 functions is antagonized by the ARF oncosuppressor protein. We show here that like p53, the p63alpha and p63gamma isoforms are able to associate with human MDM2 (HDM2). Overexpression of HDM2 increased the steady-state level of intracellular p63 and enhanced its transcriptional activity. Both effects appeared to be counteracted by ARF coexpression. These data indicate that p63 can be activated by HDM2 under conditions in which p53 is inhibited. Therefore, HDM2 expression could support p63-specific transcriptional functions on a common set of genes, keeping interference by p53 at a minimum.

Animals↗