Expression of miR-487b and miR-410 encoded by 14q32.31 locus is a prognostic marker in neuroblastoma.

BACKGROUND: Combination of age at diagnosis, stage and MYCN amplification stratifies neuroblastoma into low-risk and high-risk. We aimed to establish whether a microRNA (miRNA) signature could be associated with prognosis in both groups. METHODS: Microarray expression profiling of human miRNAs and quantitative reverse-transcriptase PCR of selected miRNAs were performed on a preliminary cohort of 13 patients. Results were validated on an independent cohort of 214 patients. The relationship between miRNA expression and the overall or disease-free survival was analysed on the total cohort of 227 patients using the log-rank test and the multivariable Cox proportional hazard model. RESULTS: A total of 15 of 17 miRNAs that discriminated high-risk from low-risk neuroblastoma belonged to the imprinted human 14q32.31 miRNA cluster and two, miR-487b and miR-410, were significantly downregulated in the high-risk group. Multivariable analyses showed miR-487b expression as associated with overall survival and disease-free survival in the whole cohort, independently of clinical covariates. Moreover, miR-487b and miR-410 expression was significantly associated with disease-free survival of the non-MYCN-amplified favourable neuroblastoma: localised (stage 1, 2 and 3) and stage 4 of infant <18 months. CONCLUSION: Expression of miR-487b and miR-410 shows predictive value beyond the classical high-/low-risk stratification and is a biomarker of relapse in favourable neuroblastoma.

Genomic and allelic expression status of the p73 gene in human neuroblastoma.

BACKGROUND AND PROCEDURE: The p53 gene homologue, p73, is located on the 1p36-3 locus, which is frequently deleted in human neuroblastoma (NB). A survey of 61 NB showed that among 33% of informative cases, p73 loss of heterozygosity (LOH) occurred in 7 of 20 (35%). RESULTS: LOH pattern of vicinal markers suggested that the p73 gene could not be considered as the candidate NB suppressor gene. Moreover, comparative measurements of allelic expression in tumors and corresponding patient lymphocytes indicate that pure biallelism is much more frequent in lymphocytes than in tumors (71% vs 30%, P= 0.05), which suggests that disequilibrated allelic expression is associated with NB disease. CONCLUSION: Therefore, in the p73 LOH NBs, the p73 gene could be altered in the maintained allele not by mutations [Ishimiya et al.: Med Pediatr Oncol, this issue], but rather by an abnormal transcription.

[A functional gene map is required to adapt therapy of metastatic neuroblastoma]. / Vers une carte génétique fonctionnelle des neuroblastomes métastiques pour une thérapeutique adaptée.

Neuroblastoma is a very common solid tumor which arises in childhood and shows an extreme heterogeneity at the clinical, histological and genetic levels. Besides age and stage, N-myc amplification and 1p deletion are prognostic factors of the disease: in Europe, these genetic markers are used to conduct therapy. In France, N-myc amplification is a factor of bad prognosis which leads, in all forms of the disease including localised forms and metastatic forms of children aged of less than 1 year, to a myeloablative treatment with autologous hematopoietic stem cells transplantation. By contrast, N-myc amplification has no impact on the survival of children aged of more than 1 year with a poor prognosis (30% overall survival, 5 years) but this genetic abnormality is taken into account to treat primary tumor of these patients. In an attempt to find out prognostic factors of these aggressive forms of the disease, various pathways (apoptosis, differentiation angiogenesis, detoxication, immune response) have been recently surveyed, but studies have been carried out on a limited number of genes. Moreover, experimental models of human metastatic neuroblastoma have been obtained in which variations of genes transcript levels involved in these pathways, are observed. The current break-through of cDNA microarrays allows to develop a dynamic transcriptomic scanning of these models as well as of tumors and bone marrows from patients upon conventional chemotherapy. This technology will enable: i) to define molecular entities of the metastatic disease; ii) to apply adapted treatment; iii) to develop new therapeutic strategies.

A Ku80 fragment with dominant negative activity imparts a radiosensitive phenotype to CHO-K1 cells.

DNA non-homologous end joining, the major mechanism for the repair of DNA double-strands breaks (DSB) in mammalian cells requires the DNA-dependent protein kinase (DNA-PK), a complex composed of a large catalytic subunit of 460 kDa (DNA-PKcs) and the heterodimer Ku70-Ku80 that binds to double-stranded DNA ends. Mutations in any of the three subunits of DNA-PK lead to extreme radiosensitivity and DSB repair deficiency. Here we show that the 283 C-terminal amino acids of Ku80 introduced into the Chinese hamster ovary cell line CHO-K1 have a dominant negative effect. Expression of Ku(449-732) in CHO cells was verified by northern blot analysis and resulted in decreased Ku-dependent DNA end-binding activity, a diminished capacity to repair DSBs as determined by pulsed field gel electrophoresis and decreased radioresistance determined by clonogenic survival. The stable modifications observed at the molecular and cellular level suggest that this fragment of Ku80 confers a dominant negative effect providing an important mechanism to sensitise radioresistant cells.

Transfer of Ku86 RNA antisense decreases the radioresistance of human fibroblasts.

Ku86 has been shown to be involved in DNA double-strand break (DSB) repair and radiosensitivity in rodents, but its role in human cells is still under investigation. The purpose of this study was to evaluate the radiosensitivity and DSB repair after transfection of a Ku86-antisense in a human fibroblast cell line. Simian virus 40-transformed MRC5V1 human fibroblasts were transfected with a vector (pcDNA3) containing a Ku86-antisense cDNA. The main endpoints were Ku86 protein level, Ku DNA end-binding and DNA protein kinase activity, clonogenic survival, and DSB repair kinetics. After transfection of the Ku86-antisense, decreased Ku86 protein expression, Ku DNA end-binding activity, and DNA protein kinase activity were observed in the uncloned cellular population. The fibroblasts transfected with the Ku86-antisense showed also a radiosensitive phenotype, with a surviving fraction at 2 Gy of 0.29 compared with 0.75 for the control and 20% of unrepaired DSB observed at 24 hours after irradiation compared with 0% for the control. Several clones were also isolated with a decreased level of Ku86 protein, a surviving fraction at 2 Gy between 0.05 and 0.40, and 10-20% of unrepaired DSB at 24 hours. This study is the first to show the implication of Ku86 in DSB repair and in the radiosensitivity of human cells. This investigation strongly suggests that Ku86 could constitute an appealing target for combining gene therapy and radiation therapy.

c-myc, p53 and bcl-2, apoptosis-related genes in infiltrating breast carcinomas: evidence of a link between bcl-2 protein over-expression and a lower risk of metastasis and death in operable patients.

Apoptosis is an important physiological process controlled by multiple genes, including c-myc, p53 and bcl-2, and its inhibition may lead to the development of human cancers. In this study, we analyzed expression of the c-myc gene using Northern blot and of the p53 and bcl-2 proteins by immuno-histochemistry in 175 breast tumor specimens obtained from patients with operable breast cancer. We evaluated the relation between expression of these 3 genes and (i) the main usual prognostic factors (tumor size, histo-prognostic grade, hormone receptors and number of positive nodes); (ii) the risk of death and relapse, taking into account these 4 factors, after a mean period of follow-up of 9.5 years (SD 2 years). Over-expression of c-myc, p53 and bcl-2 was observed in 35%, 23% and 63% of tumors, respectively. Over-expression of c-myc was strongly linked to the number of positive nodes (p = 0.0005). p53 protein expression was associated with both high-grade (p = 0.0001) and hormone receptor-negative (p = 0.0001) tumors. In contrast, bcl-2 protein over-expression was associated with the main favorable prognostic factors and, more particularly, with hormone receptor-positive tumors (p = 0.0001). Multivariate analysis, using the Cox model, showed that only 2 factors were independently linked to the risk of death: number of positive nodes, which increased the risk (p = 0.0001), and bcl-2 protein over-expression, which decreased the risk (p = 0.008). When bcl-2 over-expression was studied in relation to nodal status, hormone receptor status and chemo- and hormone therapy, no significant difference was observed between different subgroups of patients. bcl-2 expression was also associated with a significantly lower risk of distant metastasis (p = 0.04). In conclusion, bcl-2 expression characterizes a particular phenotype of breast cancer with a favorable prognosis, and it may therefore be used as a marker of long-term survival. Int. J. Cancer (Pred. Oncol.) 84:562-567, 1999.

[High incidence of p53 mutations in primary and metastatic head and neck tumors. Frequent protein overexpression in normal epithelium]. / Incidence élevée des mutations p53 dans les tumeurs primitives et métastases des voies aérodigestives supérieures et fréquente surexpression en protéine dans les épithéliums normaux.

Mutation of the p53 tumor suppressor gene is the most commonly observed gene alteration in human cancers. In order to identify new prognostic factors and tumor aggressiveness in squamous cell head and neck carcinomas, we analyzed 50 node metastases and 28 primary tumors including 13 matched specimens for p53 alterations. Mutations were found in 54 (69%) tumors, 76% of which were missense, 9% were nonsense and 15% were microdeletions or microinsertions. Twenty-five mutations were transitions mostly G-->A (40%) and 20 were transversions mostly G-->T (25%) thus confirming the role of tobacco carcinogens in the induction of these mutations. For eight patients mutations were observed in matched primary tumors and metastases, indicating clonal dissemination of tumor cells in most of these carcinomas. Furthermore the incidence of mutations was not different in primary tumors and node metastases indicating that this gene alteration was not related to the metastatic dissemination. No correlation was found between mutation and clinical parameters, the 8-year survival rates were not different (log rank test: P = 0.49) in patients with and without mutation. There was a good correlation between p53 mutation and protein overexpression (Fisher's exact test: P < 10(-4). Interestingly, immunostaining was also observed in basal cells from normal mucosa and in early lesions adjacent to the primary tumor in 11/15 specimens irrespective of the presence of mutation in the corresponding tumors. p53 protein overexpression may therefore constitute a biomarker for early stages of carcinogenesis of the head and neck epithelium.

High incidence of loss of heterozygosity and abnormal imprinting of H19 and IGF2 genes in invasive cervical carcinomas. Uncoupling of H19 and IGF2 expression and biallelic hypomethylation of H19.

The few imprinted genes characterized so far include the insulin-like growth factor-2 gene (IGF2) coding for a foetal growth factor and the H19 gene whose normal function is unknown but which is likely to act as an RNA with an antitumour effect. IGF2 is expressed by the paternal allele and H19 by the maternal allele. This reciprocal expression is quite interesting because both H19 and IGF2 genes are located close to each other on chromosome 11p15.5 in a region subject to loss of heterozygosity (LOH). Moreover, loss of imprinting (LOI) or biallelic expression has been proposed as an epigenetic mechanism for tumorigenesis in a variety of human cancers including Wilms' tumour. In this study we report the LOH, LOI and methylation status of H19 and IGF2 genes in 29 invasive cervical carcinomas of different clinical stages. Fourteen (48%) and 13 (45%) tumours were heterozygous for H19 and IGF2 respectively. LOH for H19 and IGF2 genes were found in 2 of 14 (14%) and 3 of 13 (23%) informative tumours, respectively. LOI of H19 and IGF2 was detected in 2 of 12 (17%) and 5 of 10 (50%) tumours with no LOH, respectively. More interestingly, monoallelic expression of the otherwise silent H19 allele (allele switch) was observed in 2 of 12 (17%) tumours and biallelic expression of IGF2 was detected in one specimen of normal cervix adjacent to the tumour. The expressing H19 allele, and to a lower degree also the silent allele, were hypomethylated in tumours suggesting that demethylation of both H19 alleles may be associated with an early step of imprinting alteration. In cervical cancer H19 and IGF2 expressions could be independently regulated. In conclusion, our data suggest that H19 and IGF2 genes, via deletions and/or abnormal imprinting, could play a crucial role in a large proportion (58%) of cervical cancers where they may be associated with disease progression.

High incidence of p53 alterations (mutation, deletion, overexpression) in head and neck primary tumors and metastases; absence of correlation with clinical outcome. Frequent protein overexpression in normal epithelium and in early non-invasive lesions.

We have analysed 78 head and neck carcinomas (50 node metastases and 28 primary tumors including 13 matched specimens) in 65 patients for p53 alterations. Mutations were found in 54 (69%) tumors. Of the 53 mutations within exons, 40 (76%) were missense, five (9%) nonsense and eight (15%) microdeletions or microinsertions. Twenty-five (47%) mutations were transitions mostly G-->A (40%) and 20 (38%) were transversions, mostly G-->T (25%), thus confirming the role of tobacco carcinogens in the induction of these mutations. The incidence of mutations was not different in primary tumors (68%) and node metastases (70%) indicating that this gene alteration was not related to the metastatic dissemination. For eight patients, mutations were observed in matched primary tumors and metastases, indicating clonal dissemination of tumor cells in most of these carcinomas. There was a good correlation between mutations and protein overexpression (Fisher's exact test P < 10(-4). Immunostaining was also observed in basal cells from normal epithelium and in early lesions adjacent to the primary tumor in 11/15 (73%) specimens irrespective of the presence of mutation in the corresponding tumors. These data confirm that p53 overexpression is an early event in the multistep process of epithelial cell carcinogenesis. Loss of heterozygosity for the TP53 locus was detected in 54% of tumors but no association was found with mutation (Fisher's exact test P = 0.14). No mdm-2 amplification was detected in any tumors. No correlation was found between mutation and clinical parameters, the 5-year survival rates were not different (log rank test P = 0.39) in patients with and without mutation. In conclusion, we have shown that p53 gene mutations and deletions and protein overexpression are frequent in the most aggressive head and neck carcinomas but are not associated with disease progression. The presence of protein in normal mucosa and in non-invasive lesions may constitute a biomarker for early stages of carcinogenesis.

Association of c-erbB2-gene amplification with poor prognosis in non-inflammatory breast carcinomas but not in carcinomas of the inflammatory type.

It is now accepted that c-erbB2-gene amplification is correlated with poor clinical outcome for patients, mainly when axillary nodes are invaded. We have confirmed this result by multivariate analysis in 178 patients with non-inflammatory breast cancer followed up for a mean period of 6.8 years (SD, 1.6 years). In addition, we have shown that c-erbB2 amplification, found in 30 (17%) specimens, was associated with a high risk of multiple metastases developing simultaneously. In contrast, for the 67 patients with inflammatory breast carcinoma, the most aggressive type of breast carcinoma, the c-erbB2 amplification detected in 24 (36%) specimens was not found to be associated with a higher risk of death, suggesting that the c-erbB2 gene plays a different role in the progression of these 2 types of breast cancer. Furthermore, our data stress the importance of the methodological approach used to determine gene amplification. Although Southern blot hybridization is a tumour- and time-consuming method not easy to adopt in routine clinical practice, this method remains a reference quantitative method.

Chromatin reconstitution on small DNA rings. IV. DNA supercoiling and nucleosome sequence preference.

Nucleosome formation on inverted repeats or on some alternations of purines and pyrimidines can be inhibited in vitro by DNA supercoiling through their supercoiling-induced structural transitions to cruciforms or Z-form DNA, respectively. We report here, as a result of study of single nucleosome reconstitutions on a DNA minicircle, that a physiological level of DNA supercoiling can also enhance nucleosome sequence preference. The 357 base-pair minicircle was composed of a promoter of phage SP6 RNA polymerase joined to a 256 base-pair fragment containing a sea urchin 5 S RNA gene. Nucleosome formation on the promoter was found to be enhanced on a topoisomer with in vivo superhelix density when compared to topoisomers of lower or higher superhelical densities, to the nicked circle, or to the linear DNA. In contrast, nucleosomes at other positions appeared to be insensitive to supercoiling. This observation relied on a novel procedure for the investigation of nucleosome positioning. The reconstituted circular chromatin was first linearized using a restriction endonuclease, and the linear chromatin so obtained was electrophoresed as nucleoprotein in a polyacrylamide gel. The gel showed well-fractionated bands whose mobilities were a V-like function of nucleosome positions, with the nucleosome near the middle migrating less. This behavior is similar to that previously observed for complexes of sequence-specific DNA-bending proteins with circularly permuted DNA fragments, and presumably reflects the change in the direction of the DNA axis between the entrance and the exit of the particle. Possible mechanisms for such supercoiling-induced modulation of nucleosome formation are discussed in the light of the supercoiling-dependent susceptibility to cleavage of the naked minicircle with S1 and Bal31 nucleases; and a comparison between DNase I cleavage patterns of the modulated nucleosome and of another, non-modulated, overlapping nucleosome.

Protein-induced unwinding of DNA: measurement by gel electrophoresis of complexes with DNA minicircles. Application to restriction endonuclease EcoRI, catabolite gene activator protein and lac repressor.

An electrophoretic procedure for the measurement of the helix unwinding induced by a sequence-specific protein is described. The method, which was applied here to EcoR I, CAP and lac repressor, involved the migration of the complexes with positively and negatively supercoiled DNA minicircles carrying a single protein binding site. Mobility shifts of complexes relative to naked DNAs appeared to be a result of i) the unwinding; of ii) an increase in the molecular frictional coefficient, which led to a retardation; of iii) bending, in the particular case of CAP, which induced an acceleration; and of iv) looping, in the case of lac repressor, which also resulted in an acceleration. Under conditions where the migration of the naked topoisomers was V-like (topoisomer mobility showed the same linear increase with both negative and positive supercoilings; Zivanovic et al. (1986) J. Mol. Biol., 192, 645-660), the protein unwinding contribution to mobility was assumed to be identical to that experimentally observed in the case of a thermal unwinding: all negatively supercoiled topoisomers were retarded and all positively supercoiled topoisomers were accelerated to the same extent. In contrast, the mobility contribution of the frictional term, as well as those of bending and looping, appeared to vary strongly with the magnitude of the supercoiling, but only weakly with its polarity. As a consequence, these latter contributions may approximately cancel when one is measuring the difference between the shifts observed for two comigrating, negatively and positively supercoiled, topoisomers, allowing the unwinding to be calculated. While estimates obtained for EcoR I, 23 +/- 3 degrees, and CAP, about 29 degrees, were in good agreement with previous measurements using topoisomerase I, the value found for lac repressor, 13 to 16 degrees, was significantly smaller.

ATP-independent DNA topoisomerase II as potential drug target in trypanosomes.

Two categories of trypanosomal type II topoisomerases have been isolated from trypanosomes: one is unique since it is able to realize DNA topoisomerization reactions in the absence of ATP, in contrast to the other enzyme and mammalian topoisomerase II. The biochemical properties of ATP-independent topoisomerase II from Trypanosoma cruzi are described in this report. The enzyme can decatenate trypanosome kinetoplast DNA networks, catenate supercoiled DNA molecules, unknot P4 phage DNA, and cleave double-stranded DNA. The enzyme is inhibited by various classes of drugs and is more sensitive than mammalian topoisomerase II. Therefore, trypanosome ATP-independent topoisomerase II provides a potential target for chemotherapy.

ATP-independent type II topoisomerase from trypanosomes.

We have characterized in Trypanosoma cruzi a DNA topoisomerase capable of decatenating complex trypanosomal kinetoplast DNA networks in the absence of ATP. The enzymatic activity requires Mg2+ and K+. Using a defined DNA topoisomer we showed that the linking number changes by steps of 2, which characterizes the enzyme as a type II topoisomerase. The enzyme can catenate supercoiled DNA molecules, unknot DNA, and cleave double-stranded DNA. The enzyme has no ATPase activity. The native enzyme has an Mr of about 200,000. Crude extracts and partially purified fractions contain an aggregating factor that can substitute spermidine in catenating reactions. Because of the presence of this factor, the kinetoplast DNA can only be decatenated by purified fractions. The enzyme is inhibited by certain drugs and provides a potential target for chemotherapy. Such an enzyme was also characterized in Trypanosoma equiperdum.

[A DNA topoisomerase from trypanosomes able to catenate, decatenate and unknot DNA without ATP]. / Une ADN-topoisomérase de trypanosome capable de caténer, décaténer et dénouer l'ADN sans ATP.

A novel DNA-topoisomerase from Trypanosoma cruzi was partially purified. The enzyme, without ATP addition, catalyzes decatenation of kinetoplast DNA, catenation of circular supercoiled DNAs and unknotting of P4 phage DNA. The presence of Mg++ is required as well as a suitable concentration of KCl. In stoichiometric conditions the trypanosome enzyme induces double-strand DNA cleavage. The reaction is highly stimulated by some chemicals. Such characteristics allow to include this enzyme into the type II class of DNA-topoisomerases.

[Amplification of the expression of the c-myc oncogene in bronchial epidermoid carcinoma in man]. / Augmentation de l'expression de l'oncogène c-myc dans des carcinomes épidermoïdes des bronches chez l'homme.

Squamous cell lung carcinomas from 10 untreated patients were examined for the state of the oncogene c-myc. Blot hybridization experiments have demonstrated the amplification of the oncogene of about six fold in only one tumor. The oncogene amplification was not detected in normal tissues of patients. The analysis of RNA by Northern blot revealed the presence in the seven tumors examined of a 2.4 kb c-myc RNA band. The level of c-myc expression evaluated by dot blot analysis was 5 to 14 fold greater in tumors than that of histologically normal lung of the same patients.

Inhibition of the reactions catalysed by a type I topoisomerase and catenating enzyme of Trypanosoma cruzi by DNA-intercalating drugs. Preferential inhibition of the catenating reaction.

A catenating enzyme and a type I topoisomerase were purified from Trypanosoma cruzi. We investigated the inhibitory effect of DNA-intercalating drugs on topoisomerisations catalysed by these enzymes. Inhibition of catenation was detected by electrophoretic analysis in neutral agarose gels. However, the inhibition of relaxation was not readily detectable in these gels since supercoiled DNA, which was relaxed in the presence of an intercalating drug, returned to a supercoiled state when the drug was removed. Thus electrophoretic analyses were made in gels containing chloroquine so that unreacted DNA could be distinguished from DNA relaxed by the enzyme. The results show that the catenation was more sensitive to DNA-intercalating drugs than the relaxation.

A specific inhibitor of type I DNA-topoisomerase of Trypanosoma cruzi: dimethyl-hydroxy-ellipticinium.

2-6 dimethyl-9-hydroxyellipticinium inhibited the relaxation of supercoiled DNA by the type I topoisomerase of T. cruzi. Since DNA relaxed in the presence of an intercalating drug prior to electrophoresis became supercoiled when the ligand was removed, we analysed the topoisomerisation in gels containing another ligand, chloroquine. The inhibition which is reported here, concerning a type I topoisomerase, is of an exceptional efficiency.