[Intrinsic radiosensitivity: predictive assays that will change daily practice]. / Radiosensibilité individuelle : les tests vont changer nos pratiques.

The impact of curative radiotherapy depends mainly on the total dose delivered homogenously in the targeted volume. Nevertheless, the dose delivered to the surrounding healthy tissues may reduce the therapeutic ratio of many radiation treatments. In a same population treated in one center with the same technique, it appears that individual radiosensitivity clearly exists, namely in terms of late side effects that are in principle non-reversible. This review details the different radiobiological approaches that have been developed to better understand the mechanisms of radiation-induced late effects. We also present the possibilities of clinical use of predictive assays in the close future.

[Identification of predictive biomarkers to radiotherapy outcome through proteomics approaches]. / Identification de marqueurs prédictifs de la réponse à la radiothérapie par approche protéomique.

The success of radiotherapy mainly depends on the total administered dose. This dose must be homogenously delivered onto the tumor and must preserve the surrounding healthy tissue. However, several patients are hypersensitive to ionizing radiations and may develop important radiation-induced early and late side effects. The prediction of these side effects remains currently impossible, involving to limit the given dose with the risk to decrease the therapeutic benefit for patients. Therefore, one of the major challenges in radiobiology is to accurately predict tumour radioresistance and to determine normal tissue radiosensitivity to tailor treatment. Several studies have been carried out and different predictive assays have been described in this field. However, none of them showed significant results for clinical use. For several years, many technological advances in proteomic fields have been performed in order to identify new biomarkers. After a brief description of the main characteristics of tumor radioresistance and normal tissue radiosensitivity, we will develop in this review the different approaches proposed so far to identify predictive tools of radiotherapy outcome. We will then analyze in detail how proteomic studies can improve the understanding of mechanisms associated with radiosensitivity of healthy tissue and radioresistance of tumor cells and how they could highlight new predictive biomarkers in radiobiology.

Proteomic analysis of differently archived breast cancer tissues.

Currently, the most common practice of human breast tissue preservation is formalin fixation which ensures good quality for histopathological analyses but damages DNA, RNA, and proteins, impairing their usefulness for molecular analysis and biomarker investigations. We investigated the potential value of a non-toxic fixative for sparing proteins preserved in paraffin-embedded breast biopsies. Specimens were fixed in formalin-free fixative prior to paraffin embedding, and then processed for quality and quantity of protein conservation. Similar protein patterns were observed in formalin-free fixative and frozen tissues using mono- and bi-dimensional electrophoresis, as well as western blotting. Protein patterns assessed by mass spectrometric analysis were found to be identical for frozen and formalin-free-fixed tissues. Immunohistochemistry using various antibodies showed comparable results for both tissue storage methods. In conclusion, we believe that formalin-free fixative represents an easy-to-use alternative to formalin for archived tissue and for biomarker investigations, since it simultaneously protects both the histomorphology and the integrity of macromolecules.

Autoantibody signatures: progress and perspectives for early cancer detection.

Becoming invasive is a crucial step in cancer development, and the early spread of tumour cells is usually undetected by current imaging technologies. In patients with cancer and no signs of overt metastases, sensitive methods have been developed to identify circulating autoantibodies and their antigen counterparts in several cancers. These technologies are often based on proteomic approaches, and recent advances in protein and antibody microarrays have greatly facilitated the discovery of new antibody biomarkers in sera from cancer patients. Interestingly, in a clinical application setting, combinations of multiple autoantibody reactivities into panel assays have recently been proposed as relevant screening tests and validated in several independent trials. In addition, autoantibody signatures seem to be particularly relevant for early detection of cancer in high-risk cancer patients. In this review, we highlight the concept that immunogenic epitopes associated with the humoural response and key pathogenic pathways elicit serum autoantibodies that can be considered as relevant cancer biomarkers. We outline the proteomic strategies employed to identify and validate their use in clinical practice for cancer screening and diagnosis. We particularly emphasize the clinical utility of autoantibody signatures in several cancers. Finally, we discuss the challenges remaining for clinical validation.

Pemphigus vulgaris antigen mRNA quantification for the staging of sentinel lymph nodes in head and neck cancer.

BACKGROUND: Molecular diagnosis has been proposed to enhance the intra-operative diagnosis of sentinel lymph node (SLN) invasion in head and neck squamous cell carcinoma (HNSCC). Although cytokeratin (CK) mRNA quantification with real-time reverse transcriptase-PCR (QRT-PCR) has produced encouraging results, the more discriminating markers remain to be identified. METHODS: Pemphigus vulgaris antigen (PVA), squamous cell carcinoma antigen (SCCA), and CK17 mRNA were quantified using QRT-PCR, and the results were compared with an extensive histopathological examination of the entire SLNs on 78 SLNs harvested from 22 patients with HNSCC. RESULTS: SCCA and CK17 quantification showed significantly higher mRNA values for macrometastases (MAs) than for either negative or isolated tumour cell (ITC) SLNs (P<0.01). Pemphigus vulgaris antigen allowed the discrimination of all MAs and micrometastases from both negative and ITC SLNs (P<0.001). For the neck staging of patients, considering metastatic vs non-metastatic status, receiver-operating characteristic curve analysis found areas under the curve of 93.8, 97.9, and 100% for CK17, SCCA, and PVA, respectively. With PVA, a cutoff value of 562 copies per 100 ng of cDNA permitted the correct distinction between patients with positive as opposed to negative neck nodes in all cases. CONCLUSION: PVA seems to be a highly promising marker for accurate intra-operative SLN staging in HNSCC by QRT-PCR.

Serum protein signature may improve detection of ductal carcinoma in situ of the breast.

Ductal carcinoma in situ (DCIS) of the breast is part of a spectrum of preinvasive lesions that originate within normal breast tissue and progress to invasive breast cancer. The detection of DCIS is important for the reduction of mortality from breast cancer, but the diagnosis of preinvasive breast tumors is hampered by the lack of an adequate detection method. To identify the changes in protein expression during the initial stage of tumorigenesis and to identify the presence of new DCIS markers, we analysed serum from 60 patients with breast cancer and 60 normal controls using mass spectrometry. A 23-protein index was generated that correctly distinguishes the DCIS and control groups with sensitivities and specificities in excess of 80% in two independent cohorts. Two candidate peptides were purified and identified as platelet factor 4 (PF-4) and complement C3a(desArg) anaphylatoxin (C3a(desArg)) using liquid chromatography-tandem mass spectrometry (LC-MS/MS). In an independent serum set of 165 patients, PF-4 and C3a(desArg) were significantly upregulated in DCIS compared with non-cancerous controls, as validated using western blot and enzyme-linked immunosorbent assay. We conclude that our serum protein-based test, used in conjunction with image-based screening practices, could improve the sensitivity and specificity of breast cancer detection.

Humoral response to cancer as a tool for biomarker discovery.

There is an important need to find relevant biomarkers that show high sensitivity and specificity for early diagnosis and prognosis of cancer. An immune response to cancer is elicited in humans, as demonstrated in part by the identification of autoantibodies against a number of tumor-associated antigens in sera from patients with different types of cancer. Identification of tumor-associated antigens and their cognate autoantibodies is a promising strategy for the discovery of relevant biomarkers. During the past few years, proteomic approaches, including SEREX, SERPA and, more recently, protein microarrays, have been the dominant strategies used to identify tumor-associated antigens and their cognate autoantibodies. In this review, we aim to describe advantages, drawbacks, and recent improvements of these approaches for the study of humoral responses.

Proteomic analysis of RCL2 paraffin-embedded tissues.

Histopathological diagnosis in most of the world's hospitals is based upon formalin-fixed and paraffin-embedded (FFPE) tissues. Although this standard fixation and embedding procedure keeps the tissue in excellent form for morphological and immunohistological analysis, FFPE is inappropriate for nucleic acids and protein studies. We investigated the potential value of RCL2, a new non-toxic fixative, for sparing proteins preserved in paraffin-embedded tissues. Normal colonic mucosa tissue was fixed in RCL2 prior to paraffin embedding (RCL2P), and then processed for quality and quantity of protein conservation, as compared to frozen and FFPE tissues using complementary proteomic analysis approaches. Using 4 different protein extraction protocols, RCL2P tissue consistently showed the highest protein yield. Similar protein patterns were observed with RCL2P and frozen tissues using mono and bi-dimensional electrophoresis. Moreover, membrane, cytoplasmic and nuclear proteins, as well as phosphorylated proteins, were successfully detected using western-blot. Furthermore, protein patterns observed by mass spectrometry analysis after laser-captured microdissection were found to be identical for frozen and RCL2-fixed tissues. At last, immunohistochemistry using various antibodies showed comparable results between both tissue storage methods. We concluded that RCL2 has great potential for performing both morphological and molecular analyses on the same archival paraffin-embedded tissue sample, and can be a new method for investigating protein biomarkers.

Comprehensive proteomic analysis of the human milk proteome: contribution of protein fractionation.

In-depth analysis of the milk proteome by mass spectrometry is challenged by the presence of few high-abundance proteins that interfere with the detection of lower-abundance proteins. Here, we evaluated the proteomic analysis of milk samples following a strong anion exchange fractionation procedure using denaturating conditions ensuring the disruption of protein-protein interactions. Crude whey or skim milk and their different resulting fractions were analyzed by protein chip array mass spectrometry. Using protein chip array mass spectrometry, several high-abundance proteins were localized in distinct fractions increasing the total number of unique peptides and proteins detected. This total number increased by about 20-30% by combining different chromatographic surface arrays used for capture. Reproducible results were obtained in human skim milk and whey; however this approach was not successful with milk fat globule membrane and required refinement. Hence, milk profiling by anion exchange fractionation combined to protein chip array mass spectrometry represents a promising tool to detect unknown low-abundance milk proteins that may ultimately prove useful as biomarkers of diseases transmitted by breastfeeding.

[A novel fixed paraffin-embedded tissue processing for proteomic analysis]. / Etude d'un nouveau fixateur pour l'analyse du protéome tissulaire.

Human tissues are an important biological material for the discovery of biomarkers and identification of novel therapeutic targets. Formalin fixed and paraffin embedded (FFPE) tissue represents the most abundant supply of archival material for clinical and molecular analyses. Although FFPE preserves the cellular and architectural morphologic details in tissue sections, formalin facilitates the formation of protein-protein crosslinks rendering FFPE tissues refractory to many protein studies. The aim of this study was to assess the feasibility of proteomic investigations of a new non-toxic fixative using a comprehensive panel of proteomic methods. Tissues were processed for quality and quantity of protein conservation, as compared to frozen and FFPE tissues using complementary proteomic analysis approaches. Similar protein patterns were observed between our tissue fixative protocol and frozen tissues using mono and bidimensional electrophoresis and protein identification by mass spectrometry was not affected. Several proteins were successfully detected using western blot and immunohistochemistry showed comparable results between both tissue storage methods. We demonstrate that our new fixative protocol represents an easy-to-use alternative to FFPE compatible with both current diagnostic pathology practice and tissue proteomic investigations.

Proteomics-based identification of HSP60 as a tumor-associated antigen in early stage breast cancer and ductal carcinoma in situ.

The detection of autoantibodies in cancer patients has been shown to constitute an excellent tool for early diagnosis. Because breast cancer still lacks early diagnostic markers, we investigated novel tumor-associated antigens and related autoantibodies in sera from patients with early stage breast cancer compared to autoimmune disease, other cancers, and healthy volunteers, using a proteomics-based approach. Among the 26 protein antigens specifically recognized by early stage breast cancer sera, we focused on Heat Shock Protein 60 (HSP60). Using ELISA, we investigated the frequency of autoantibodies directed against this protein in the sera of 240 individuals, comprising patients with either ductal carcinoma in situ (DCIS) ( n = 49) or early stage breast cancer ( n = 58), other cancers ( n = 20), autoimmune disease ( n = 20), and healthy subjects ( n = 93). Autoantibodies directed against HSP60 were present in 16/49 (31%) early stage breast cancer and 18/58 (32.6%) DCIS patients, compared to 4/93 (4.3%) healthy subjects. In particular, autoantibodies were present in 11/23 patients (47.8%) with high-grade DCIS, compared to 5/26 (19.2%) with low-grade DCIS. HSP60 mRNA levels were significantly higher in primary breast cancer compared to healthy breast tissues. Using immunohistochemistry, we found that HSP60 expression gradually increases from normal through DCIS to invasive tissues. Our results indicate that HSP60 autoantibodies may be of interest in terms of clinical utility for the early diagnosis of breast cancer and more particularly in DCIS. Moreover, HSP60 overexpression during the first steps of breast carcinogenesis may be functionally correlated to tumor growth and/or progression.

Cell culture models of transmissible spongiform encephalopathies.

In this review, we describe the generation and use of cell culture models of transmissible spongiform encephalopathies, also known as prion diseases. These models include chronically prion-infected cell lines, as well as cultures expressing variable amounts of wild-type, mutated, or chimeric prion proteins. These cell lines have been widely used to investigate the biology of both the normal and the pathological isoform of the prion protein. They have also contributed to the comprehension of the pathogenic processes occurring in transmissible spongiform encephalopathies and in the development of new therapeutic approaches of these diseases.

Cleavage of the amino terminus of the prion protein by reactive oxygen species.

Relatively limited information is available on the processing and function of the normal cellular prion protein, PrP(C). Here it is reported for the first time that PrP(C) undergoes a site-specific cleavage of the octapeptide repeat region of the amino terminus on exposure to reactive oxygen species. This cleavage was both copper- and pH-dependent and was retarded by the presence of other divalent metal ions. The oxidative state of the cell also decreased detection of full-length PrP(C) and increased detection of amino-terminally fragmented PrP(C) within cells. Such a post-translational modification has vast implications for PrP(C), in its processing, because such cleavage could alter further proteolysis, and in the formation of the scrapie isoform of the prion protein, PrP(Sc), because abnormal cleavage of PrP(Sc) occurs into the octapeptide repeat region.

Prion infection impairs the cellular response to oxidative stress.

The molecular mechanism of neurodegeneration in transmissible spongiform encephalopathies remains uncertain. In this study, it was demonstrated that prion-infected hypothalamic neuronal GT1 cells displayed a higher sensitivity to induced oxidative stress over noninfected cells. In addition, the infected cells presented an increased lipid peroxidation and signs of apoptosis associated with a dramatic reduction in the activities of the glutathione-dependent and superoxide dismutase antioxidant systems. This study indicates for the first time that prion infection results in an alteration of the molecular mechanisms promoting cellular resistance to reactive oxygen species. This finding is vital for future therapeutic approaches in transmissible spongiform encephalopathies and the understanding of the function of the prion protein.

Amphotericin B inhibits the generation of the scrapie isoform of the prion protein in infected cultures.

Transmissible spongiform encephalopathies form a group of fatal neurodegenerative disorders that have the unique property of being infectious, sporadic, or genetic in origin. Although some doubts about the nature of the responsible agent of these diseases remain, it is clear that a protein called PrP(Sc) plays a central role. PrP(Sc) is a conformational variant of PrP(C), the normal host protein. Polyene antibiotics such as amphotericin B have been shown to delay the accumulation of PrP(Sc) and to increase the incubation time of the disease after experimental transmission in laboratory animals. Unlike for Congo red and sulfated polyanions, no effect of amphotericin B has been observed in infected cultures. We show here for the first time that amphotericin B can inhibit PrP(Sc) generation in scrapie-infected GT1-7 and N2a cells. Its activity seems to be related to a modification of the properties of detergent-resistant microdomains. These results provide new insights into the mechanism of action of amphotericin B and confirm the usefulness of infected cultures in the therapeutic research of transmissible spongiform encephalopathies.

A naturally occurring sequence variation that creates a YY1 element is associated with increased cystic fibrosis transmembrane conductance regulator gene expression.

We have identified previously a novel complex mutant allele in the cystic fibrosis transmembrane conductance regulator (CFTR) gene in a patient affected with cystic fibrosis (CF). This allele contained a mutation in CFTR exon 11 known to cause CF (S549R(T>G)), associated with the first alteration described so far in the minimal CFTR promoter region (-102T>A). Studies on genotype-phenotype correlations revealed striking differences between patients carrying mutation (S549R(T>G)) alone, who had a severe disease, and patients carrying the complex allele (-102(T>A)+S549R(T>G)), who exhibited milder forms of CF. We thus postulated that the sequence change (-102T>A) may attenuate the effects of the severe (S549R(T>G)) mutation through regulation of CFTR expression. Analysis of transiently transfected cell lines with wild-type and -102A variant human CFTR-directed luciferase reporter genes demonstrates that constructs containing the -102A variant (which creates a Yin Yang 1 (YY1) core element) increases CFTR expression significantly. Electrophoretic mobility shift assays indicate that the -102 site is located in a region of multiple DNA-protein interactions and that the -102A allele recruits specifically an additional nuclear protein related to YY1. The finding that the YY1-binding allele causes a significant increase in CFTR expression in vitro may allow a better understanding of the milder phenotype observed in patients who carry a severe CF mutation within the same gene.

Effect of Congo red on wild-type and mutated prion proteins in cultured cells.

Transmissible spongiform encephalopathies form a group of fatal neurodegenerative disorders that have the unique property of being infectious, sporadic, or genetic in origin. Although some doubts remain on the nature of the responsible agent of these diseases, it is clear that a protein called PrP(Sc) (which stands for the scrapie isoform of the prion protein) has a central role in their pathology. PrP(Sc) represents a conformational variant of a normal protein of the host: the cellular isoform of the prion protein, or PrP(C). Compounds such as glycosaminoglycans and Congo red (CR) have been shown to interfere with both in vitro and in vivo PrP(Sc) formation. It was hypothesized that CR acts by overstabilizing the conformation of PrP(Sc) molecules or by modifying trafficking of PrP(C). Using transfected cells expressing 3F4-tagged mouse PrPs, we show here that CR does not interfere with conversion of PrP molecules carrying pathogenic mutations. On the contrary, after incubation with the drug, some of their properties, such as insolubility and protease resistance, are enhanced and are even acquired by the wild-type molecule. This last observation suggests an alternative mechanism of action of CR and leads us to reconsider the relationship between the biochemical properties of PrP and conformational alteration of the protein.

Effect of amphotericin B on wild-type and mutated prion proteins in cultured cells: putative mechanism of action in transmissible spongiform encephalopathies.

Transmissible spongiform encephalopathies form a group of fatal neurodegenerative disorders that have the unique property of being infectious, sporadic, or genetic in origin. Although some doubts remain on the nature of the responsible agent of these diseases, it is clear that a protein called PrP(Sc) [the scrapie isoform of prion protein (PrP)] plays a central role. PrP(Sc) represents a conformational variant of PrP(C) (the cellular isoform of PrP), the normal host protein. Polyene antibiotics, such as amphotericin B, have been shown to delay the accumulation of PrP(Sc) and to increase the incubation time of the disease after experimental transmission in laboratory animals. Unlike agents such as Congo red, the inhibitory effect of amphotericin B on PrP(Sc) generation has not been observed in infected cultures. Using transfected cells expressing wild-type or mutated mouse PrPs, we show here that amphotericin B is able to interfere with the generation of abnormal PrP isoforms in culture. Its action seems related to a modification of PrP trafficking through the association of this glycosylphosphatidylinositol-anchored protein with detergent-resistant microdomains. These results represent a first step toward the comprehension of the mechanism of action of amphotericin B in transmissible spongiform encephalopathies.

Trafficking of the cellular isoform of the prion protein.

Transmissible spongiform encephalopathies form a group of fatal neurodegenerative disorders that have the unique property of being infectious, sporadic or genetic in origin. Although the nature of the responsible agent of these diseases is uncertain, it is clear that a protein called PrPSc has a central role in their pathology. PrPSc is a conformational variant of a normal protein called PrPC. Understanding the transition from PrPC to PrPSc is a major issue in the field. In this article, we will review what is known about the cell biology of PrPC, the understanding of which is crucial considering that trafficking of this molecule governs generation of PrPSc.

Comparison of Bombyx mori and Helicoverpa armigera cytoplasmic actin genes provides clues to the evolution of actin genes in insects.

The cytoplasmic actin genes BmA3 and BmA4 of Bombyx mori were found clustered in a single genomic clone in the same orientation. As a similar clustering of the two cytoplasmic actin genes Ha3a and Ha3b also occurs in another lepidopteran, Helicoverpa armigera, we analyzed the sequence of the pair of genes from each species. Due to the high conservation of cytoplasmic actins, the coding sequence of the four genes was easily aligned, allowing the detection of similarities in noncoding exon and intron sequences as well as in flanking sequences. All four genes exhibited a conserved intron inserted in codon 117, an original position not encountered in other species. It can thus be postulated that all of these genes derived from a common ancestral gene carrying this intron after a single event of insertion. The comparison of the four genes revealed that the genes of B. mori and H. armigera are related in two different ways: the coding sequence and the intron that interrupts it are more similar between paralogous genes within each species than between orthologous genes of the two species. In contrast, the other (noncoding) regions exhibited the greatest similarity between a gene of one species and a gene of the other species, defining two pairs of orthologous genes, BmA3 and HaA3a on one hand and BmA4 and HaA3b on the other. However, in each species, the very high similarities of the coding sequence and of the single intron that interrupts it strongly suggest that gene conversion events have homogenized this part of the sequence. As the divergence of the B. mori genes was higher than that of the H. armigera genes, we postulated that the gene conversion occurred earlier in the B. mori lineage. This leads us to hypothesize that gene conversion could also be responsible for the original transfer of the common intron to the second gene copy before the divergence of the B. mori and H. armigera lineages.