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.

Aims and methods. LUTS suggestive of BPH.

OBJECTIVE: Few epidemiological studies are available on Italian patients with lower urinary tract symptoms and their QoL. QUIBUS (QUality of life Investigated in BPH patients with Urinary Symptoms) is an observational longitudinal study aimed at evaluating symptoms and QoL in a large sample of Italian patients and investigating their correlation with demographic, social and clinical characteristics of BPH. PATIENTS AND METHODS: Patients with lower urinary tract symptoms and prostate enlargement suggestive of BPH (both old and new diagnosis) were enrolled between November 1998 and May 1999 in 31 Italian centers of urology. This longitudinal investigation consists of an enrollment visit, in which demographic, social and clinical aspects are recorded as baseline data, and a follow-up visit after 1 year of treatment freely assigned by the investigators. Symptoms and QoL are assessed by means of IPSS, ICS-BPH (at both visits) and SF-36 (only at the follow-up visit) questionnaires. RESULTS: 1,033 patients were enrolled. The follow-up visit is still under evaluation. In this series of papers the baseline results are presented and discussed in terms of (i) medical management, (ii) life-style, (iii) symptoms, bothersomeness and QoL, (iv) sexual function of a large and representative sample of Italian patients and (v) uroflowmetry.

Effects of simulated microgravity on metabolic activities related to DNA damage and repair in lymphoblastoid cells.

We adopted a simple experimental framework to follow the dependence of structural aberrations and the modifications in selected metabolic processes correlated with the exposure of cells to microgravity. Alterations to the cellular metabolism induced by exposure to microgravity are evidentiated in the modification of PARP activity (strongly dependent to the presence of DNA damages and to the altered gene expression), in the modification of the repair ability and in the cell's energy homeostasis (NAD and ATP). Cells are exposed continuously to microgravity in a Random Positioning Machine (RPM) in complete medium for 48 hours. At the end of this period a part of these cells are immediately analysed for the parameters reported above and the remaining were furtherly incubated in standard laboratory conditions to document eventual defects during the phases of the recovery process. A part of cells, just after exposure to microgravity, were also subjected to treatment with a strong damaging agent, KBrO3, and these cells were subsequently analyzed. This final treatment was meant to amplify the eventual deficiencies experienced by microgravity-exposed cells in the DNA repair process also in dependence with the alterated metabolic conditions resulting after the exposure to microgravity.

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.

Chromosomal aberrations evaluated by CGH, FISH and GTG-banding in a case of AIDS-related Burkitt's lymphoma.

BACKGROUND AND OBJECTIVE: We have previously reported on a complex chromosome rearrangement [der(17)] in a B-cell line, BRG A, established from an AIDS patient with Burkitt's lymphoma (BL). The aim of the present study was the definition of der(17) composition and the identification of complete or partial chromosome gains and losses in two cell clones (BRG A and BRG M) derived from this patient. DESIGN AND METHODS: We applied comparative genome hybridization (CGH) to detect the DNA misrepresentations in the genome of the two cell clones. Findings from CGH and banding analysis could then direct the choice of probes for chromosome painting experiments to elucidate der(17) composition. RESULTS: CGH analysis identified gains of chromosomes 1q, 7q, 12q, 13q, 15q, 17p, 20p,q and losses of chromosomes 3p and 5q in BRG A and gain of chromosome 1q and loss in chromosome 6q in BRG M. Some of the detected alterations had already been described in lymphomas, while others appeared to be new. The combination of these techniques allowed a precise definition of der(17), composed by translocated regions from chromosomes 12 and 15. INTERPRETATION AND CONCLUSIONS: We demonstrated CGH to be a powerful tool in the identification of recurrent chromosome aberrations in an AIDS-related BL and in ascertaining the origin of marker chromosomes. We were also able to identify a different pattern of aberrations and assess an independent sequence of events leading to the 1p gain in the two subclones.

p53 mutations experimentally induced by 8-methoxypsoralen plus UVA (PUVA) differ from those found in human skin cancers in PUVA-treated patients.

8-Methoxypsoralen (8-MOP) plus UVA irradiation (PUVA therapy) has been used for the treatment of psoriasis. PUVA therapy has been associated with an increased risk of developing skin squamous cell carcinoma (SCC). In order to determine the PUVA-induced p53 mutation spectrum, a yeast expression vector harbouring a human wild-type p53 cDNA was incubated with 8-MOP, and UVA irradiated in vitro. PUVA-damaged and undamaged DNA was transfected into a yeast strain containing the ADE2 gene regulated by a p53-responsive promoter. An 8-MOP concentration-dependent decrease in survival and increase in mutant frequency were observed. At a fixed 8-MOP concentration, survival decreased and mutant frequency increased as UVA irradiation increased. Eleven mutant clones contained 11 mutations: 10 were single base pair substitutions, the remaining one being a complex mutation. All eight T:A-targeted mutations were at 5'-TpA sites, hallmark mutations of PUVA mutagenesis. Through a rigorous statistical test, the PUVA-induced p53 mutation spectrum appears to differ significantly (P < 0.0002) from that observed in SCC in PUVA-treated patients. The present work demonstrates that a specific PUVA-induced mutational fingerprint could be obtained and recognized on human p53 cDNA. This result may suggest that PUVA therapy can be a risk factor for the development of SCC in psoriasis patients through a mechanism not involving the induction of p53 mutations.

N-(2-chloroethyl)-N-nitrosourea tethered to lexitropsin induces minor groove lesions at the p53 cDNA that are more cytotoxic than mutagenic.

Many different N-chloroethyl-N-nitrosourea (CENU) derivatives have been synthesized in an attempt to minimize carcinogenic activity while favoring antineoplastic activity. CENU derivatives linked to the dipeptide lexitropsin (lex) showed significant changes in groove- and sequence-selective DNA alkylation inducing thermolabile N3-alkyladenines (N3-Alkyl-As) at lex equilibrium binding sites. CENU-lex sequence specificity for DNA alkylation was determined using 32P-end-labeled restriction fragments of the p53 cDNA. The adducted sites were converted into single-strand breaks by sequential heating at neutral pH and exposure to piperidine. To establish the mutagenic and lethal properties of CENU-lex-specific lesions, a yeast expression vector harboring a human wild-type p53 cDNA was treated in vitro with CENU-lex and transfected into a yeast strain containing the ADE2 gene regulated by a p53-responsive promoter. p53 mutants were isolated from independent ade- transformants. The results revealed that: (a) CENU-lex preferentially induces N3-Alkyl-A at specific lex equilibrium binding sites, the formations of which are strongly inhibited by distamycin; (b) reactivity toward Gs is still present, albeit to a lesser extent when compared to N-(2-chloroethyl)-N-cyclohexyl-N-nitrosourea and to CENU; (c) 91% of the 49 CENU-lex p53 mutations (45 of 49) were bp substitutions, 29 of which were GC-->AT transitions, mainly at 5' purine G sites; (d) all AT-targeted mutations but one were AT-->TA transversions; (e) the distribution of the CENU-lex mutations along the p53 cDNA was not random, with position 273 (codon 91), where only GC-->AT transitions were observed, being a real (n = 3, P < 0.0002) CENU-lex mutation hot spot; and (f) a shift in DNA alkylation sites between lesion spectra induced by CENU-lex and N-(2-chloroethyl-N-cyclohexyl-N-nitrosourea was associated with an increased lethality and a decreased mutagenicity, whereas no dramatic change in mutational specificity was observed. Hence, it is tempting to conclude that, in this experimental system, N3-Alkyl-A is more lethal than mutagenic, whereas O6-alkylguanine is a common premutational lesion formed at non-lex binding sites. These results suggest that CENU derivatives with virtually absolute specificity for A residues would make targeting of lethal, nonmutagenic lesions at A+T-rich regions possible, and this may represent a new strategy for the development of new chemotherapeutic agents with a higher therapeutic index.

5-methylcytosine at HpaII sites in p53 is not hypermutable after UVC irradiation.

Using a yeast based p53 functional assay we previously demonstrated that the UVC-induced p53 mutation spectrum appears to be indistinguishable from the one observed in Non Melanoma Skin Cancer (NMSC). However, position 742 (codon 248, CpG site) represented the major hot spot in NMSC but was not found mutated in the yeast system. In order to determine whether UVC-induced mutagenic events may be facilitated at methylated cytosine (5mC), a yeast expression vector harbouring a human wild-type p53 cDNA (pLS76) was methylated in vitro by HpaII methylase. Methylation induced 98% protection to HpaII endonuclease. Unmethylated and methylated pLS76 vectors were then UVC irradiated (lambda(max): 254 nm) and transfected into a yeast strain containing the ADE2 gene regulated by a p53-responsive promoter. The results revealed that: (i) 5mC at HpaII sites did not cause any difference in the UVC-induced survival and/or mutagenicity; (ii) none of the 20 mutants derived from methylated pLS76 showed p53 mutations targeted at HpaII sites; (iii) the UVC-induced p53 mutation spectra derived from methylated and unmethylated pLS76 were indistinguishable not only when classes of mutations and hot spots were concerned, but also when compared through a rigorous statistical test to estimate their relatedness (P = 0.85); (iv) the presence of 5mC did not increase the formation of photo-lesions at codon 248, as determined by using a stop polymerase assay. Although based on a limited number of mutants, these results suggest that the mere presence of 5mC at position 742 does not cause a dramatic increase of its mutability after UVC irradiation. We propose that position 742 is a hot spot in NMSC either because of mutagenic events at 5mC caused by other UV components of solarlight and/or because not all the NMSC are directly correlated with UV mutagenesis but may have a "spontaneous" origin.

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.

Primary sclerosing cholangitis: clinical presentation, natural history and prognostic variables: an Italian multicentre study. The Italian PSC Study Group.

OBJECTIVE: Because large-scale reports of PSC in the Mediterranean area we are still lacking, in this study we evaluated by Kaplan-Meyer analysis the natural history of primary sclerosing cholangitis (PSC) in Italy and by means of other statistical methods we identified the variables most useful in predicting survival of such patients. DESIGN: Retrospective multicentre study of unselected patients with PSC. Several variables involving sex, age, associated diseases, clinical features, laboratory, cholangiographic and histological findings at presentation and clinical outcome at data recording were collected by means of a detailed questionnaire. SETTING: 16 Italian university and regional hospitals all over the country, thus giving a geographically representative population. PATIENTS: A total of 117 PSC patients (73 men and 44 women); median age 35 years. METHODS: Survival analysis was performed by the Kaplan-Meyer method; the prognostic influence on survival of collected data was evaluated by univariate chi(2) analysis with Wilcoxon and log-rank tests. The same prognostic variables were also evaluated by multivariate analysis (Cox model), using a stepwise regression procedure. All statistical analyses were performed using the SAS statistical software. RESULTS: At presentation 70% of patients were symptomatic; symptoms did not relate to liver histology. Both intra- and extrahepatic bile duct lesions were detected in 46% of patients at cholangiography. Inflammatory bowel disease was found in 54% of symptomatic patients, ulcerative colitis was 36% of total. Clinical outcome (91/117): 15 underwent liver transplantation or died from liver disease (cholangiocarcinoma). Survival at 10 years was 74%. Features of poor prognosis were cholesterol, aspartate aminotransferase (AST), haemoglobin and albumin. CONCLUSION: PSC in Italy mainly follows a benign course and among clinical features recorded at presentation, serum cholesterol, AST, haemoglobin and albumin may provide some objective criteria to assess disease severity.

Radiation-induced structural modifications in dsDNA analysed by FT-Raman spectroscopy.

Structural changes of double stranded DNA in aqueous solution induced by ionizing radiation were studied by Fourier-Transform-Raman spectroscopy. In addition to base damage, strand breaks, structural changes, i.e. unstacking of the bases, premelting effects and disordering of the B-form backbone could be observed. The amount of these different kinds of damage depended on the concentration of the DNA solution. Specifically, the following modifications were found depending on the gamma-ray dose and DNA concentration. (1) Intensity increase of the lines of dT (1240 cm-1) and dA (729, cm-1) indicating unstacking of these bases. (2) Intensity and frequency changes of the marker bands of all four bases indicating structural modifications. (3) Intensity decrease of the sugar marker lines showing change of the bonds in the deoxyribose and of bonds between the sugar moiety and the phosphodiester. (4) Intensity decrease of the lines of the phosphodiester groups (1094 and 790 cm-1) with simultaneous appearance of a difference peak at 1080 cm-1 and a new peak at 980 cm-1 suggesting the presence of strand breaks. (5) Intensity decrease of the B-form marker band (approximately 835 cm-1) and new lines at 876 cm-1), at approximately 660 cm-1 (C3'-endo/anti of dG) and at 1312 cm-1 (C3'endo/syn of dA) indicating decrease of the B-form conformation and the developing of partly new secondary forms of the DNA representing a helix-to-coil transition.

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.

Antikinetochore antibodies and flow karyotyping: new techniques to detect aneuploidy in mammalian cells induced by ionizing radiation and chemicals.

For a possible detection of aneuploidy induction by chemicals and ionizing radiation, fluorescein-bound antikinetochore antibodies (CREST-scleroderma antibodies) were used to discriminate between micronuclei deriving from acentric fragments or from chromosome loss induced in Chinese hamster cells. The cells were treated with aphidicolin, adriamycin, Hoechst 33258, colcemid, the alkylating agent diethyl sulfate, and ionizing radiation. The frequency of micronucleated cells, the fraction of kinetochore-positive and -negative micronuclei per cell, and the fraction of kinetochore-positive micronuclei was measured using immunofluorescence staining of kinetochores in micronuclei. Of the micronuclei and fragmented nuclei induced by colcemid, 99% contained kinetochores, whereas ionizing radiation induced only 4% of kinetochore-positive micronuclei. The other drugs induced variable, in some cases also cell-cycle-dependent, fractions of kinetochore positive micronuclei. With this technique a discrimination between clastogenic effects and effects that occur at the level of spindle formation of the agent studied seems to be possible. Flow karyotyping was used to study the induction of stable homogeneous and numerical aberrations in diploid Chinese hamster cell clones that had survived a dose of 15 Gy gamma-radiation. All analyzed clones showed deviations in their flow karyotypes: the mean number was 9.2 deviations per clone, compared to 1.1 deviations per clone in unirradiated control clones.