Prognostic value of Dicer expression in human breast cancers and association with the mesenchymal phenotype.

BACKGROUND: Dicer, a ribonuclease, is the key enzyme required for the biogenesis of microRNAs and small interfering RNAs and is essential for both mammalian development and cell differentiation. Recent evidence indicates that Dicer may also be involved in tumourigenesis. However, no studies have examined the clinical significance of Dicer at both the RNA and the protein levels in breast cancer. METHODS: In this study, the biological and prognostic value of Dicer expression was assessed in breast cancer cell lines, breast cancer progression cellular models, and in two well-characterised sets of breast carcinoma samples obtained from patients with long-term follow-up using tissue microarrays and quantitative reverse transcription-PCR. RESULTS: We have found that Dicer protein expression is significantly associated with hormone receptor status and cancer subtype in breast tumours (ER P=0.008; PR P=0.019; cancer subtype P=0.023, luminal A P=0.0174). Dicer mRNA expression appeared to have an independent prognostic impact in metastatic disease (hazard ratio=3.36, P=0.0032). In the breast cancer cell lines, lower Dicer expression was found in cells harbouring a mesenchymal phenotype and in metastatic bone derivatives of a breast cancer cell line. These findings suggest that the downregulation of Dicer expression may be related to the metastatic spread of tumours. CONCLUSION: Assessment of Dicer expression may facilitate prediction of distant metastases for patients suffering from breast cancer.

p53 induction prevents accumulation of aberrant transcripts in cancer cells.

Loss of fidelity of the splicing process occurs during tumor progression and can have a deleterious effect on genes like tumor suppressor genes. It was reported recently that the presence of aberrant transcripts of the TSG101 gene in breast cancer cells was associated with the mutation of the p53 tumor suppressor gene. On the basis of this observation, we have analyzed TSG101 transcript patterns in p53-active and p53-inactive cells. Using several isogenic cellular models, we demonstrate that the induction of p53 in cancer cells leads to a significant decrease of aberrant transcripts levels. This indicates a novel implication of p53 in the regulation of the splicing process.

Absence of p53-dependent induction of the metastatic suppressor KAI1 gene after DNA damage.

The p53 tumor suppressor protein functions to monitor the integrity of the genome. If a damage is detected, p53 binds tightly to specific sequence elements in the DNA and induces the transactivation of genes involved in various growth regulatory processes such as cell cycle progression, DNA repair and apoptosis. A p53-binding site was recently identified in the promoter region of the metastatic suppressor KAI1 gene, suggesting that this gene was a direct transcriptional target of p53. To test the relevance of this hypothesis, we studied the endogenous KAI1 expression in a series of human cell lines with varying p53 status in response to genotoxic treatment as well as in different cellular models exhibiting an inducible p53 activity. Overall, our data indicate that KAI1 expression is not significantly modulated by p53. This observation provides a direct evidence that the presence of a p53-binding site in regulatory domains is not a sufficient criteria to define a p53-transcriptional target gene.

BTG gene expression in the p53-dependent and -independent cellular response to DNA damage.

Exposure of mammalian cells to genotoxic agents evokes a complex cellular response. An ordered series of molecular events is necessary to sense DNA damage, transduce the signal, and ultimately delay the cell cycle or trigger apoptosis. Recently, we have shown that BTG2/TIS21 gene expression was induced in response to DNA damage through a p53-dependent pathway. This gene belongs to a newly identified family of structurally related genes whose other known human members are BTG1, BTG3, and Tob. To define the respective involvement of these four related genes in the cellular response to DNA damage, we studied their expression in human cell lines after a variety of genotoxic treatments. Our results demonstrated that were BTG1, BTG2/TIS21, and Tob genes the DNA damage--inducible genes. However, BTG2/TIS21 appeared to be the only p53-transcriptional target gene. We speculate that BTG proteins may play a coordinate role in a general transduction pathway that is induced in response to DNA damage. It has been previously described that recombinant BTG1 and BTG2/TIS21 can physically interact with PRMT1, an arginine methyl transferase, suggesting that BTG1 and BTG2/TIS21 induction may lead to posttranslational modifications of cellular proteins. In support of this hypothesis, we showed that the endogenous induction of BTG1 and BTG2 after genotoxic treatment was correlated with a modulation of protein methylation.

Relevance of mandates, notifications and threads in the management of continuity of care.

Availability of electronic healthcare records (EHCR) and geographical networks allows nowadays to realise a set of functionalities to support continuity of care. Actual exchange of complete clinical information, common centralised records, common decisions within an agreed protocols are not mandatory. Sub-optimal alternatives, involving easier management, are possible. In fact, the crucial challenge for continuity of care is the mutual awareness of the multiple perspectives by the actors contributing to patient's care. The various actors should timely know changes in the status of: mandates, i.e. who is involved in the care provision and thus is responsible for a "local" record. knowledge about the patient, i.e. active and inactive problems, impressions, relevant findings. provision of healthcare activities, i.e. plans, orders and performed activities. The decisions on the actual implementation depend on the healthcare context, as implemented within the information system. For example, the mechanism for notifications involves decisions on the quality and quantity of information that must be exchanged, as well as on the modalities for the exchange, regulated by individual user's profiles. Each notification could be sent as a message to a central repository, and then each authorized user could select within the repository the pertaining messages. Or a notification could be sent directly to the list of professionals involved in the care of a patient, that asked for it in their profile. Mandates may be used to regulate the access of the users to the patient's information. This approach was embedded in a European standard under development in CEN/TC251 (CONTSYS--"System of concepts to support continuity of care").

A hypermedia tool to support the interaction between radiologists and clinicians.

In this work we discuss problems related to the radiological reporting process and attempt to identify the ones which emerge during clinician-radiologist communication. We propose a hyper-reporting system, which allows the clinician to share the knowledge and critical factors that prompt the radiologist's diagnostic hypothesis during the discussion of results. This is possible by creating hypermedia links among different elements: reporting text, images, comments and the graphic models library. These elements make radiological knowledge explicit and clarify to the clinician the logical-diagnostic path traveled by the radiologist. A questionnaire, a survey of clinicians' and radiologists' needs allowed us to define some aspects of a hypermedia demo interface. Finally, we describe an example of a working session with the use of a few explanatory cards.

A hypermedia radiological reporting system.

Report is the main phase of a diagnostic process by images. The product of the process is the diagnostic report. We are proposing an hypermedia structure of diagnostic report in radiology, in order to facilitate exchange between radiologist and clinician (specialist in internal medicine or surgeon) on a clinical case, without anymore charge on the side of the radiologist but with an 'off-line' consultation. An hypermedia radiological report software will produce further advantages in many aspects: radiologist and clinician could access patient's data directly from DB on patients; radiologist could check DB on exemplary cases real-time; clinician could read preliminary and final reports available in network and make requests online. The proposed hyper-report system is modular. Starting from the 'report text' writing, edited by the radiologist on the basis of most significative images, it is possible to insert comments in text, drawing and 'external' images form.

Resistance of MCF7 human breast carcinoma cells to TNF-induced cell death is associated with loss of p53 function.

We have investigated the relationship between the development of tumor resistance towards the cytotoxic action of tumor necrosis factor-alpha (TNF) and p53 function, using the TNF-sensitive MCF7 human breast adenocarcinoma cell line and two TNF-resistant sublines, MCF7/R-A1 and MCF7/Adr. Use of single-strand conformation polymorphism (SSCP) analysis and DNA sequencing shows that MCF7 has a wild-type p53 gene, whereas both TNF-resistant sublines exhibit mutant p53. This includes a point mutation R280K in MCF7/R-A1 cells, and a point mutation at the splicing acceptor site on the upstream border of exon 5 resulting in a 21 pb deletion in MCF7/Adr cells. These mutations result in loss of p53 capacity to transactivate FASAY (functional assay in yeast). In contrast to what is observed for parental MCF7 cells, treatment of resistant sublines with TNF or gamma-irradiation fails neither to induce the expression of the p53-regulated gene products p21waf1/CIP1 and MDM2, nor to arrest the cells in the G1 phase of the cell cycle. Disruption of p53 wild-type function in MCF7 cells by transfection with human papillomavirus type-16 E6 gene, leads to abrogation of the cytotoxic, but not the cytostatic activity of TNF. Altogether, our results strongly suggest that wild-type p53 is involved in cytotoxic action of TNF, and point out that loss of p53 function contributes to resistance of tumor cell to TNF-induced killing.

Identification of BTG2, an antiproliferative p53-dependent component of the DNA damage cellular response pathway.

Cell cycle regulation is critical for maintenance of genome integrity. A prominent factor that guarantees genomic stability of cells is p53 (ref. 1). The P53 gene encodes a transcription factor that has a role as a tumour suppressor. Identification of p53-target genes should provide greater insight into the molecular mechanisms that mediate the tumour suppressor activities of p53. The rodent Pc3/Tis21 gene was initially described as an immediate early gene induced by tumour promoters and growth factors in PC12 and Swiss 3T3 cells. It is expressed in a variety of cell and tissue types and encodes a remarkably labile protein. Pc3/Tis21 has a strong sequence similarity to the human antiproliferative BTG1 gene cloned from a chromosomal translocation of a B-cell chronic lymphocytic leukaemia. This similarity led us to speculate that BTG1 and the putative human homologue of Pc3/Tis21 (named BTG2) were members of a new family of genes involved in growth control and/or differentiation. This hypothesis was recently strengthened by the identification of a new antiproliferative protein, named TOB, which shares sequence similarity with BTG1 and PC3/TIS21 (ref. 7). Here, we cloned and localized the human BTG2 gene. We show that BTG2 expression is induced through a p53-dependent mechanism and that BTG2 function may be relevant to cell cycle control and cellular response to DNA damage.

A hypermedia system for parasite identification.

In this paper a hypermedia system for parasite identification is described. The knowledge base is relative to the class of the Trematoda parasites and reports agent, vector, disease, related category of the International Classification of Diseases and geographic area. A graphic user-friendly human-machine interface has been realized for this system.

Context trees: representing co-operative healthcare activities in IREP.

The IREP Project is based on the concept of 'rehabilitation programme' (LLP--Life Long Programme). It consists in a global vision of the patient's therapeutic-rehabilitative path, that is realised by the description, selection, adaptation and evaluation of generalised protocols. This paper discusses the issues arising in the definition of multidisciplinary rehabilitation programmes involving the co-operation of independent teams. A methodology to formalise and collect descriptions of programmes is presented. A programme is considered as a set of interconnected activities, represented as a graph of Activity Modules, organised in Context Trees; the resulting structured representation is the framework to implement timely and effective communication services in a global information system on rehabilitation. Based on this experience, a set of possible requirements for software tools to formalise and manage programmes have been suggested; it was the basis for the realisation of a first prototype under field test in a rehabilitation environment by other partners of the IREP Project.

Genomic stability and wild-type p53 function of lymphoblastoid cells with germ-line p53 mutation.

Increased cancer risk associated with germ-line p53 mutation was linked to a deficit in the ability to maintain genomic stability. Accordingly, normal fibroblasts from cancer-prone individuals accumulate genomic aberrations with concomitant loss of wild-type p53 allele during in vitro culture. We tested whether such changes also occur in EBV-immortalized lymphoblastoid cells. Both normal and p53 germ-line mutant lymphoblastoid cells maintained functional p53 and genomic stability during long term in vitro culture. These unexpected differences between fibroblastic and lymphoblastic cells suggest that phenotypic expression of p53 deficiency is cell type specific. This could contribute to selective tissular localization of tumours observed in patients with Li-Fraumeni syndrome despite the presence of a mutant p53 allele in all cells.

ras, p53 and HPV status in benign and malignant prostate tumors.

To study the role of ras, p53 genes and HPV virus (16 and 18) in the development of prostate cancer, we analyzed tissue sections from 27 patients affected with carcinomas (stages A to D) and from 24 patients with adenomas. Mutations of H, K and N-ras and p53 (exons 2-9) were studied by SSCP and DNA sequencing. Accumulation of p53 protein was studied by immunohistochemistry on tissue sections. Tumors were also analyzed for the presence of HPV16 and -18 sequences by PCR and DNA hybridization with sequence-specific oligonucleotides. No mutation was found in the three ras genes studied, either in carcinomas or adenomas. By SSCP analysis we identified p53 mutations in only 2 of 19 carcinomas studied, both in exon 7. Immunohistochemical results strongly correlate with the SSCP results: p53 protein was positive in tumors with p53 mutation but not in others; 32% of studied adenomas had detectable HPV16 DNA, while 53% of carcinomas were HPV16+. Among these I presented a p53 mutation. No HPV18 E6 sequence could be detected. Our data show that in prostate tumors from France, mutations of p53 and ras are rare events but that these tumors display detectable HPV16 DNA at a high frequency. The low incidence of p53 mutation, associated to a significant proportion of tumors showing HPV16 DNA, could suggest that in prostate cancer HPV16 infection could participate in p53 inactivation by E6.

Modeling the management of protocols as the kernel of a healthcare information system.

A conceptual model of a health care structure is described. The management of protocols viewed as structured, flexible, and coherent descriptions of activities aimed to solve specific problems in the system is the kernel of the model. A global approach to the protocols is proposed. Each protocol is modeled from two points of view: the nature of the actions (clinical, administrative, programming, and control) and the life cycle of protocols (theoretical, customized, and performed). In our approach the patient folder not only records information about the patient, but also contains and controls the execution of the protocols planned to be performed on the patient. As a consequence, all aspects related to protocol execution are chronologically reported in the folder in a federated manner. In this way the patient folder becomes a complex, multifaceted object, able to capture all information needed to evaluate the patient evolution in a given period.

A model for the structured description of healthcare activities and related data.

Rehabilitation involves long-term, interdisciplinary processes. A model was developed, for the structured description of typical healthcare activities. Telematic services based on this model can support accurate data acquisition and communication among healthcare teams. Presentation of data within their context and according to the specific user's view is envisaged, based on deviations from the typical behaviours. Benefits are also expected in better understanding of the care processes themselves, easier comparison of different approaches, and diffusion of consensus-based knowledge.

Two germ-line mutations affecting the same nucleotide at codon 257 of p53 gene, a rare site for mutations.

Codon 257 of the p53 gene is an extremely rare target for somatic mutations (accounting for only two of 1600 published mutations). We report here two constitutional mutations both affecting the second nucleotide of codon 257. A thymine to adenine transversion resulting in an amino acid change from leucine to glutamine was found in one proband who developed multiple independent malignant tumors (osteosarcoma, phyllodes tumor, soft-tissue sarcoma). Her mother died of early-onset breast cancer. In the other case, a deletion resulting in a frameshift in the C-terminal coding region of p53 was found in a woman who was diagnosed with breast cancer at age 34. This woman belongs to a family with features of Li-Fraumeni syndrome. In both cases, the p53 mutations identified in the proband was found in other members of the family. Codon 257, even if rarely mutated in somatic cells, may thus be an important target for germ-line mutations.