Silvia Viaggi

Mutation frequencies of GNAQ, GNA11, BAP1, SF3B1, EIF1AX and TERT in uveal melanoma: detection of an activating mutation in the TERT gene promoter in a single case of uveal melanoma

Background:Uveal melanoma is the most frequent primary tumour of the eye. It is molecularly clearly distinct from cutaneous melanoma and shows a different pattern of driver mutations. The influence of sunlight ultraviolet (UV) exposure on the aetiology of uveal melanoma is a matter of debate. The recent identification of driver mutations in the promoter of the telomerase reverse transcriptase (TERT) gene with UV-induced cytidine-to-thymidine transitions in cutaneous melanoma prompted us to investigate whether these mutations also occur in uveal melanoma.Methods:We analysed 50 cases of uveal melanoma obtained from enucleation surgery for mutations in the genes GNAQ, GNA11, BAP1, SF3B1, EIFAX1 and TERT, measured gene expression using microarrays and analysed gene copy numbers by SNP arrays.Results:We detected a TERT mutation in only one case of a 57-year-old white male patient with clinical and histopathological features typical for uveal melanoma. The tumour showed mutations in GNA11 and EIF1AX that are typical for uveal melanoma and absent from cutaneous melanoma. No mutations were detected in GNAQ, BAP1 and SF3B1 that are frequently mutated in uveal melanoma. Both copies of chromosome 3 were retained. Several tumours among which the one carrying the TERT promoter mutation showed elevated TERT expression. Consistent with previous reports, GNAQ is inversely associated with chromosome 3 monosomy and metastasis. BAP1 mutations are significantly associated with chromosome 3 monosomy but not with relapse.Conclusion:These data indicate that TERT mutations are rare in uveal melanoma. No conclusion can be drawn on their potential influence on tumour progression.

Chapter 9 Measurement of Micronuclei by Flow Cytometry

Publisher Summary This chapter describes a technique for obtaining a suspension of micronuclei and nuclei for flow cytometric measurement of the frequency of micronuclei per main nuclei in cell cultures or human lymphocytes exposed to ionizing radiation or chemicals. The two-step method includes a treatment of the cells with salt solution I, which contains a detergent, followed by an additional treatment with solution II containing citric acid and sucrose. Both solutions contain ethidium bromide (EB) as a DNA-specific fluorescent dye; in some cases Hoechst 33258 (HO) is used additionally. By this two-step treatment the cellular membrane and the cytoplasm are destroyed and nuclei and micronuclei are released in suspension. No mechanical treatment or centrifugation steps are necessary to obtain a solution of main nuclei and micronuclei, because mechanical treatment would induce cellular or nuclear debris that could overlap the small micronuclei during flow cytometric measurement. The nuclear membrane is maintained and mitotic cells that could release chromosomes in the suspension are usually not destroyed. With this technique it is additionally possible to measure the DNA content of micronuclei and main nuclei for a simultaneous analysis of the cell-cycle distribution (main nuclei) and the DNA distribution of micronuclei. Analysis of the DNA distribution of micronuclei can give additional information on the mechanisms of action of the inducing agent.

The yeast p53 functional assay: a new tool for molecular epidemiology. Hopes and facts.

The assumption of molecular epidemiology that carcinogens leave fingerprints has suggested that analysis of the frequency, type, and site of mutations in genes frequently altered in carcinogenesis may provide clues to the identification of the factors contributing to carcinogenesis. In this mini-review, we revise the development, and validation of the yeast-based p53 functional assay as a new tool for molecular epidemiology. We show that this assay has some very interesting virtues but also has some drawbacks. The yeast functional assay can be used to determine highly specific mutation fingerprints in the human p53 cDNA sequence. Discrimination is possible when comparing mutation spectra induced by sufficiently different mutagens. However, we also reported that the same carcinogen may induce distinguishable mutation spectra due to known influencing factors.

Kinetics of radiation- and cytochrome c-induced modifications in liposomes analysed by FT–Raman spectroscopy

Fourier transform Raman spectroscopy on artificial lipid membranes was used to study radiation-induced peroxidation processes as a function of time after radiation exposure. The time dependent intensity changes of the Raman lines of various C=C bondings were compared to results obtained by measuring conjugated dienes and by the thiobarbituric acid test for malondialdehydes. The results show that mainly the cis C=C bonds of the lipid chains are involved and, therefore, indicate that gamma-radiation induces conformational changes in the lipid chain while the mobility of the lipid chains is reduced. New Raman bands can be assigned to aldehyde products induced at the end of the peroxidation process. The immediate decrease of the =CH vibration lines was directly correlated with the formation of conjugated C=C double bonds suggesting that these vibration lines are in contrast to the C=C lines solely Raman active, when isolated C=C bonds are present. Cytochrome c (ox.) incorporated into the bilayer of the artificial membranes induced autooxidation processes not influenced by gamma-radiation. It was observed that cytochrome c (ox.)-induced changes of the relative intensity of the C=C bonds differ from those induced by gamma-radiation. These results of cytochrome c together with the inhibitory effects of the antioxidant alpha-tocopherol suggest that the radical species involved in the cytochrome c induced process might be different from the free radicals involved in the gamma-radiation-induced process.

Exposure of human lymphocytes and lymphoblastoid cells to simulated microgravity strongly affects energy metabolism and DNA repair

Exposure of freshly drawn lymphocytes and lymphoblastoid cells (LB and COR3) to simulated microgravity decreased the intracellular ATP concentration to 50%–40% of the value found in normal growth conditions. The decrease was reversible although recovery to normal values occurred only slowly both in lymphocytes and in lymphoblastoid cells. Poly(ADP‐ribose) polymerase (PARP ) activity was increased indicating that cells exposed to conditions of reduced gravitation experience stress. Exposure to microgravity forces cells into a condition of metabolic quiescence in which they appear to be particularly sensitive to subsequent exposures to a genotoxic agent. Thus, treatment of cells with the strong redox agent potassium bromate under microgravity conditions, indicated an impairment in repair of DNA 8‐hydroxy‐2′‐deoxyguanosine (8‐OHdG), an oxidized derivative of deoxyguanosine. We conclude that gravitational modulation of the kind routinely obtained under laboratory conditions and during spaceflights is a stressful process to which cells appear to be extremely sensitive. These effects may reflect the physiological alterations observed in astronauts and in animals following spaceflights or exposure to conditions of simulated microgravity.

New insights into redox response modulation in Fanconi's anemia cells by hydrogen peroxide and glutathione depletors.

Fanconi’s anemia (FA) patients face severe pathological consequences. Bone marrow failure, the major cause of death in FA, accounting for as much as 80–90% of FA mortality, appears to be significantly linked to excessive apoptosis of hematopoietic cells induced by oxidative stress. However, 20–25% of FA patients develop malignancies of myeloid origin. A survival strategy for bone marrow and hematopoietic cells under selective pressure evidently exists. This study reports that lymphoblastoid cell lines derived from two FA patients displayed significant resistance to oxidative stress induced by treatments with H2O2 and various glutathione (GSH) inhibitors that induce production of reactive oxygen species, GSH depletion and mitochondrial membrane depolarization. Among the various GSH inhibitors employed, FA cells appear particularly resistant to menadione (5 μm) and ethacrynic acid (ETA, 50 μm), two drugs that specifically target mitochondria. Even after pre‐treatment with buthionine sulfoximine, a GSH synthesis inhibitor that induces enhanced induction of reactive oxygen species, FA cells maintain significant resistance to these drugs. These data suggest that the resistance to oxidative stress and the altered mitochondrial and metabolic functionality found in the FA mutant cells used in this study may indicate the survival strategy that is adopted in FA cells undergoing transformation. The study of redox and mitochondria regulation in FA may be of assistance in diagnosis of the disease and in the care of patients.

Identification of a novel set of genes reflecting different in vivo invasive patterns of human GBM cells

BackgroundMost patients affected by Glioblastoma multiforme (GBM, grade IV glioma) experience a recurrence of the disease because of the spreading of tumor cells beyond surgical boundaries. Unveiling mechanisms causing this process is a logic goal to impair the killing capacity of GBM cells by molecular targeting.We noticed that our long-term GBM cultures, established from different patients, may display two categories/types of growth behavior in an orthotopic xenograft model: expansion of the tumor mass and formation of tumor branches/nodules (nodular like, NL-type) or highly diffuse single tumor cell infiltration (HD-type).MethodsWe determined by DNA microarrays the gene expression profiles of three NL-type and three HD-type long-term GBM cultures. Subsequently, individual genes with different expression levels between the two groups were identified using Significance Analysis of Microarrays (SAM). Real time RT-PCR, immunofluorescence and immunoblot analyses, were performed for a selected subgroup of regulated gene products to confirm the results obtained by the expression analysis.ResultsHere, we report the identification of a set of 34 differentially expressed genes in the two types of GBM cultures. Twenty-three of these genes encode for proteins localized to the plasma membrane and 9 of these for proteins are involved in the process of cell adhesion.ConclusionsThis study suggests the participation in the diffuse infiltrative/invasive process of GBM cells within the CNS of a novel set of genes coding for membrane-associated proteins, which should be thus susceptible to an inhibition strategy by specific targeting.Massimiliano Monticone and Antonio Daga contributed equally to this work

The induction of numerical chromosome aberrations in human lymphocyte cultures and V79 Chinese hamster cells by diethyl sulfate

Human lymphocyte cultures were treated with increasing concentrations of diethyl sulfate (DES) at different times after stimulation with phytohemagglutinin (0, 24 and 45 h) and were scored for numerical chromosomal aberrations at different culture times (52, 72 and 96 h). A total of 3500 metaphases were analyzed. A statistically significant (p less than 0.01) increase in hypodiploid and hyperdiploid metaphases was found throughout the tested dose range (0.1-3 mM DES); the increase in polyploid metaphases was statistically significant at 1 mM (p less than 0.05) and 3 mM (p less than 0.01) DES. In human lymphocytes treated in Go, DES also induced chromatid breaks as well as micronuclei. In V79 Chinese hamster cells, DES induced micronuclei and polyploidy.

Uncommon cytogenetic findings in a case of splenic marginal zone lymphoma with aggressive clinical course

The majority of splenic marginal zone lymphoma (SMZL) patients experience an indolent clinical course; however, some cases transform to a high-grade lymphoma. Cytogenetic analyses have shown that chromosome 7 is the most frequently altered chromosome and, in some cases, 7q deletion has been found as a single aberration, suggesting its association with the development of SMZL. We studied one patient showing clinical features of SMZL with an aggressive course. Immunophenotypic, conventional and molecular cytogenetic techniques were applied to support the diagnosis. The immunophenotype of peripheral blood mononuclear cells showed the presence of 90% B-lymphocytes. Cytogenetic analysis indicated the presence of a stem-line lacking normal chromosomes 7, but showing a der(7) and a ring, and a side-line with additional aberrations: t(2;22), add(8). Fluorescence in situ hybridization analysis revealed a loss of the 7q32 region. Nonclonal rearrangements involving chromosome 7 were also detected. Chromosome 7 rearrangements were studied to investigate their evolution during the development of the pathology. We have shown that in this patient both chromosomes 7 underwent different rearrangements leading to a loss of the 7q32 region and that the ring chromosome originated from chromosome 7 and was associated with a t(7;7) (p22;q31). We conclude that not only the 7q deletion but also the proneness of chromosome 7 to rearrange might have played a role in the progression of this SMZL.

Malignant pleural mesothelioma: time for translational research

7from our group have showed that MPM expresses a cholinergic system involved in the regulation of both cell growth and the apoptotic machinery. These data now need to be reconciled to construct preclinical models and to approach clinical research from a translational perspective.