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CDKN2A locus on chromosome 9p21 encodes two tumour suppressor proteins pl6INK4A, which is a regulator of the retinoblastoma (RB) protein, and p14ARF, which is involved in the ARF–Mdm2–p53 pathway. The aim of this study was to determine if CDKN2A gene products are implicated in differentiated thyroid carcinogenesis and progression. We used real-time quantitative RT–PCR and immunohistochemistry to assess both transcripts and proteins levels in 60 tumours specimens. Overexpression of p14ARF and pl6INK4A was observed in follicular adenomas, follicular carcinomas and papillary carcinomas, while downregulation was found in oncocytic adenomas compared to nontumoral paired thyroid tissues. These deregulations were statistically significant for pl6INK4a (P=0.006) in follicular adenomas and close to statistical significance for p14ARF in follicular adenomas (P=0.06) and in papillary carcinomas (P=0.05). In all histological types, except papillary carcinomas, we observed a statistically significant relationship between p14ARF and E2F1 (r=0.64 to 1, P

Resume MGMT (O6 methyl-guanine methyl-transferase) est un gene qui participe a la reparation de l’ADN et qui, par son mecanisme d’action, va pouvoir supprimer les groupements alkyls formes par les chimiotherapies alkylantes, et ainsi induire des chimio-resistances. Les etudes recentes semblent montrer que l’expression de ce gene est liee a la methylation de son promoteur qui pourrait donc donner des indications sur une chimio-sensibilite eventuelle. L’etude de MGMT pourrait avoir un interet therapeutique et pronostique. On retrouve, cependant, peu de series d’oligodendrogliomes dont les resultats restent contradictoires. Ceci est probablement du a l’heterogeneite tumorale, mais aussi au caractere certainement multifactoriel de la chimio-sensibilite. Il apparait donc difficile, pour l’instant, de definir le choix d’un traitement complementaire en fonction du seul statut de MGMT.

During the last years, spectacular progress in the field of molecular biology raised the hope that it might replace or at least improve significantly the World Health Organization classification of tumors of the central nervous system. Although up to now, this has not been the case, a few biomarkers have found their place as prognostic factors for certain tumor types on the one hand, and as predictive factors of response to treatments on the other hand. The present chapter will deal with the following tumor types: astrocytic tumors (including Diffuse astrocytoma, Anaplastic astrocytoma, Glioblastoma, and Pilocytic astrocytoma), Oligodendroglial tumours (including Oligodendroglioma and Anaplastic oligodendroglioma), and mixed gliomas (including Oligoastrocytoma and Anaplastic oligodendroglioma). The current biomarkers are not decision-making parameters on a case by case basis, but they have progressively emerged as cornerstone indicators as far as stratification in clinical studies is concerned. As a consequence, during therapeutic trials, it can be considered that groups of patients whose biomarkers anticipate a poor response to standard treatment might be offered the opportunity of innovative therapies. In this context, the most recent developments of molecular biology culminating in integrated molecular analysis of tumors and the possibilities offered by neurosphere cultures established from glioblastoma multiforme that recapitulate the tumor allow to anticipate a growing importance of a series of new biomarker in the years to come.

International audience; This study assessed the prognostic value of several markers involved in gliomagenesis, and compared it with that of other clinical and imaging markers already used. Four-hundred and sixteen adult patients with newly diagnosed glioma were included over a 3-year period and tumour suppressor genes, oncogenes, MGMT and hTERT expressions, losses of heterozygosity, as well as relevant clinical and imaging information were recorded. This prospective study was based on all adult gliomas. Analyses were performed on patient groups selected according to World Health Organization histoprognostic criteria and on the entire cohort. The endpoint was overall survival, estimated by the Kaplan-Meier method. Univariate analysis was followed by multivariate analysis according to a Cox model. p14(ARF), p16(INK4A) and PTEN expressions, and 10p 10q23, 10q26 and 13q LOH for the entire cohort, hTERT expression for high-grade tumours, EGFR for glioblastomas, 10q26 LOH for grade III tumours and anaplastic oligodendrogliomas were found to be correlated with overall survival on univariate analysis and age and grade on multivariate analysis only. This study confirms the prognostic value of several markers. However, the scattering of the values explained by tumour heterogeneity prevents their use in individual decision-making.

Glial tumours are a devastating, poorly understood condition carrying a gloomy prognosis for which clinicians sorely lack reliable predictive parameters facilitating a sound treatment strategy. Tp73, a p53 family member, expresses two main classes of isoforms--transactivatory activity (TA)p73 and DeltaTAp73--exhibiting tumour suppressor gene and oncogene properties, respectively. The authors examined their expression status in high- and low-grade adult gliomas. Isoform-specific real-time reverse transcription-polymerase chain reaction was used for the analysis of Tp73 isoform transcript expression in a series of 51 adult patients harbouring glial tumours, in order to compare tumour grades with each other, and with non-tumoural samples obtained from epileptic patients as well. Our data demonstrate increase of TAp73 and DeltaTAp73 transcript levels at onset and early stage of the disease. We also show that DeltaEx2-3 isoform expression in low-grade tumours anticipates clinical and imaging progression to higher grades, and correlates to the patients' survival. Expression levels of P1 promoter generated Tp73 isoforms--and particularly DeltaEx2-3--indeed allow for prediction of the clinical progression of low-grade gliomas in adults. Our data are the first such molecular biology report regarding low-grade tumours and as such should be of help for sound decision-making.

Aim This study was aimed to determine p73 status in thyroid tumours. Methods Differential expression of the TAp73, ΔTAp73 transcripts was measured in a panel of 60 thyroid malignancies by quantitative RT-PCR. Results By comparison to normal thyroid tissue surrounding the tumours, we observed significant downregulation of TP73 transcripts in adenomas and in differentiated carcinomas. Correlations were found in normal tissue specimens between the expression of TAp73 and ΔNp73 transcripts and that of p53, p14 ARF p16 INK4a , but these correlations were lost in carcinomas (PTC or FTC). Conclusions We have found significant variations of TAp73, ΔNp73, p53, p14 ARF p16 INK4a , expressions and correlations between the expressions of those different genes in thyroid cancer.

The INK4 family of proteins p15INK4b, p14ARF and p16INK4a function as cell cycle inhibitors where they are involved in the inhibition of G1 phase progression. Methylation of the p15INK4b promoter never seems to occur in solid tumors but is a major gene silencing mechanism in hematological malignancies. p14ARF and p16INK4a promoter methylation often occurs in solid tumors but also in leukemias and lymphomas. In chronic myelogenous leukemia (CML), only a few reports have been published regarding INK4 methylation and the results of the literature are discordant. Thus clearly, more works on large series have to be performed independently.

In an effort to extend the potential relationship between the methylation status of MGMT promoter and response to CENU therapy, we examined the methylation status of MGMT promoter in 44 patients with glioblastomas. Tumor specimens were obtained during surgery before adjuvant treatment, frozen and stored at -80 degrees C until for DNA extraction process. DNA methylation patterns in the CpG island of the MGMT gene were determined in every tumor by methylation specific PCR (MSP). These results were then related to overall survival and response to alkylating agents using statistical analysis. Methylation of the MGMT promoter was detected in 68% of tumors, and 96.7% of methylated tumors exhibited also an unmethylated status. There was no relationship between the methylation status of the MGMT promoter and overall survival and response to alkylating agents. Our observations do not lead us to consider promoter methylation of MGMT gene as a prognostic factor of responsiveness to alkylating agents in glioblastomas.

Background. Altered topoisomerase II α (Topo II α) expression and telomerase activity (TA) reflect tumour cell growth and malignant transformation. Methods. We examined TA by using a TRAP assay and expression of Topo II α by immunohistochemical analysis in a series of 27 cases of papillary thyroid carcinoma (PTC). Results. Topo II α labelling index (LI) ranged from 0.1 to 4.2% and was significantly associated with patient age ( r =−0.42, p =0.003), with higher levels of Topo II α in patients under 40 years. There was no relationship between Topo II α LI, AGES score or other clinical outcome. TA was detected in 14 PTC, with relative levels ranging from 1.2 to 102 units. A significant positive correlation between the multiplicity of tumoral foci and the TA levels ( p −2 ) was noted. Conclusion. We concluded that Topo II α cannot be used as a marker of tumour aggressiveness. Furthermore, enhanced Topo II α expression in PTCs from patients less than 40 years old suggests that this age group might benefit from Topo II inhibitor chemotherapy.

Antibodies present in sera of patients with auto immune diseases (systemic Lupus erythematosus, mixed connective tissue disease) were used to react with nuclear ribonucleoproteins (HnRNPs) from normal cells and cells infected with retroviruses. Only antibodies directed against Sm and RNP antigens precipitated particles with definite spectra of small nuclear RNAs (SnRNA) and proteins. No difference could be found between infected and uninfected cells, suggesting that virus replication is dependent on normal cellular fonctions.

A Friend mink cell focus-inducing (Fr-MCF) virus isolated from a Friend tumor cell line was able to induce acute erythroleukemia associated with polycythemia when injected as a Friend murine ecotropic leukemia virus (F-MuLV) pseudotype into adult Swiss and ICFW mice. One virus isolate recovered from leukemic cells and designated as FV-F3 presented the following properties: (i) persistence of the same leukemogenic power when propagated in vivo and in vitro; (ii) in vivo spleen focus-forming (SFFV) capacity; (iii) presence of erythropoietin (EPO)-independent CFU-E in leukemic animals; (iv) expression of a 32 RNA specifically recognized by a SFFV probe, in FV-F3 infected cells; and (v) expression in FV-F3-infected cell of polypeptides in the range of gp52 SFFV. Peptide analysis of these products revealed close similarities with the parental MCF virus. These data suggest that a SFFV genome arose by genetic recombinational events involving MCF virus.

Oncornaviral particles on the way of their synthesis have been isolated from cytoplasm of a Friend virus-producing murine cell line. By use of electron microscopy and retrotranscriptase assay, it was found that the majority of the viral structures is pelleted by sedimentation at 90.000 g. The Poly (A) RNA of this pellet contains principally 70S and 35S RNA species which, as judged by size determination and hybridization with Friend-virus c-DNA are the species embedded into the virions. We verified that conditions which were used in the course of isolation, effectively dissociate polysomes and prevent them from co-sedimenting with viruses.

Summary Unesterified cellulose selectively binds natural or artificial poly-adenylate-containing Polynucleotides. This property was used for separating Poly-(A)-rich RNA from other species existing in mammalian cells. RNA was characterized for Poly-(A) presence and size. From these studies, it was concluded that chromatography on cellulose column is one of the simplest tools for isolating m-RNA of eucaryotic cells on a large scale.

RNA was prepared from the murine leukemia virus pseudotype of mouse sarcoma virus. On polyacrylamide gels, 70 S single-stranded RNA was detected as well as smaller RNA species of 28 S, 18 S, and 4 S. In addition, two components of 8 S were discovered. These 8 S RNA molecules may correspond to the 7 S RNA of Rous sarcoma virus and to the 9 S of avian myeloblastosis virus.

Two tumor Friend cell lines producing anemia-inducing virus (TF-A line) or polycythemia-inducing virus (TF-P line) were compared for their viral-encoded glycoproteins. The envelope glycoproteins of the two viral populations differ by their electrophoretic mobilities. The gpr env precursors also differ by their relative mobilities. The TF-P cells contain the typical gp50–52 molecular species, which is coded for by Spleen Focus Forming sequences (SFFV) present in the genome of the polycythemia-inducing virus. The TF-A cells do not contain the gp50–52, but express in small amounts a species with a higher apparent molecular weight. This species which has been named FV-A gp55 could be equivalent to the gp50–52 coded for by the SFFV sequences. Very similar results were obtained with leukemic cells prepared from enlarged spleens of mice infected with the anemic or polycythemic Friend viruses.

Ribonucleoproteins were isolated from the cytoplasm of Friend-Eveline cells which produce the Friend virus complex, after a short labelling with [ 3 H] uridine. These particles moved with a sedimentation coefficient of 53S in sucrose gradient and had a buoyant density of 1.46 g/cm 3 in CsCl gradient. Analysis of their RNA content showed that they possessed a 35S major species having the size of the viral genome subunit. Moreover, a positive hybridization was observed when RNA of the 53S particles was annealed with viral complementary DNA. No such particles were found in cultures of uninfected murine cells suggesting that 53S RNPs have a viral origin.

A 55 Kd nuclear antigen, designated PSL has been found using a human autoimmune serum. This antigen accumulates in the nucleus, during the S phase, transiently associates with chromatin and possesses a DNA-binding ability. It appears to be highly conserved among eukariotic species including mammals, birds and insects.

An S phase-related nuclear 55K antigen, also designated PSL, has been characterized in various mammalian cells, using a human serum from a patient with autoimmune disorders (Barque et al., EMBO j 2 (1983) 743) [6]. In this report, we show by immunoelectron microscopy that the p55 protein associates in situ with the chromatin of rat hepatocytes. This association is a transient one, as indirect immunofluorescence studies show that PSL does not bind to individualized metaphase chromosomes. Furthermore, immunoprecipitation tests indicate that the majority of PSL is in the non-chromosomal cell fraction. These results suggest that this nuclear antigen is directly involved in the DNA replication process.

Friend murine erythroleukemia is a model of virus-induced acute leukemia. RNA and protein viral expression were simultaneously studied in a Friend erythroleukemia murine cell line (HFL/b cells, Freedman et al. (1975) J. expl. Med.142, 212–223), which had lost its capacity of producing infectious virus and was found to contain neither structural p30 nor gp70 envelope-glycoprotein by radioimmunoassay. Presence of the spleen focus-forming virus (SFFV) in these cells was demonstrated by its biological rescue in vivo and in vitro with Moloney-helper leukemia virus (M-MuLV). Molecular hybridization using a DNA complementary to Friend virus complex revealed the presence in cytoplasmic Poly(A) RNA of a major species with the size of the 32S SFFV genome. No. 38S helper Friend Murine Leukemia Virus (F-MuLV) RNA was detected. Viral-encoded polypeptide expression was studied by immunoprecipitation of cell extracts. A first class of polypeptides constituted by two species of 50,000 and 52,000 daltons was precipitated by a goat anti Rauscher gp70 serum and corresponded to the glycoprotein encoded by the SFFV genome. A second class of polypeptides with apparent mobilities of 48,000 and 50,000 daltons was precipitated by anti p15 and anti p12 sera and to a lesser extent by an anti p30 serum. These products were glycosylated in part. Moreover two p37 and p39 products, which disappeared more rapidly than p48gag + p50gag in pulse-chase labeling experiments were also precipitated by anti-gag sera. No other gag-product such as pr65gag was detected, confirming a negative expression of the F-MuLV genome and supporting the idea that all the gag-related products expressed in these cells were coded by SFFV virus.