Extended evaluation of the efficacy of a proactive forced oscillation technique-based auto-CPAP algorithm.

PURPOSE: Previous studies revealed substantially varying therapy efficacy of automatic continuous positive airway pressure (APAP) devices in the treatment of obstructive sleep apnea (OSA). We evaluated the efficacy of a new APAP device using the forced oscillation technique (FOT) to evaluate upper airway obstruction during apneas and flow contour analyses during hypopneas. METHODS: Forty-six initially diagnosed OSA patients were included and the pressure range was set from 5 to 20 hPa. Therapy efficacy was assessed based on the reduction of apnea-hypopnea index (AHI), improvement of objective sleep quality parameters, and the appropriateness of the device's pressure regulation. RESULTS: AHI and arousal index significantly decreased during APAP therapy (median [interquartile range]: AHI 36 [23-55] vs. 2 [1-6]/h, arousal index 30 [22-45] vs. 15 [10-19]/h, both p < 0.001). The amount of slow wave sleep (SWS) and rapid-eye-movement (REM) sleep significantly increased (SWS 20 [14-29] vs. 29 [19-34]%, REM 16 [11-21] vs. 24 [14-30]%, both p < 0.01). Most residual respiratory events during therapy were of central etiology and attributable to five patients, who presented with treatment-emergent central sleep apnea. The device's pressure regulation abolished most obstructive respiratory events (n = 6.7 residual obstructive events per patient). Of central respiratory events, 534/646 (83%) did not lead to pressure increases. CONCLUSION: This pilot study provides a proof of concept that the APAP device combining FOT and evaluation of flow contour allows for the suppression of obstructive events without relevant false reactions.

The role of CSA in the response to oxidative DNA damage in human cells.

Cockayne syndrome (CS) is a rare genetic disease characterized by severe growth, mental retardation and pronounced cachexia. CS is most frequently due to mutations in either of two genes, CSB and CSA. Evidence for a role of CSB protein in the repair of oxidative DNA damage has been provided recently. Here, we show that CSA is also involved in the response to oxidative stress. CS-A human primary fibroblasts and keratinocytes showed hypersensitivity to potassium bromate, a specific inducer of oxidative damage. This was associated with inefficient repair of oxidatively induced DNA lesions, namely 8-hydroxyguanine (8-OH-Gua) and (5'S)-8,5'-cyclo 2'-deoxyadenosine. Expression of the wild-type CSA in the CS-A cell line CS3BE significantly decreased the steady-state level of 8-OH-Gua and increased its repair rate following oxidant treatment. CS-A cell extracts showed normal 8-OH-Gua cleavage activity in an in vitro assay, whereas CS-B cell extracts were confirmed to be defective. Our data provide the first in vivo evidence that CSA protein contributes to prevent accumulation of various oxidized DNA bases and underline specific functions of CSB not shared with CSA. These findings support the hypothesis that defective repair of oxidative DNA damage is involved in the clinical features of CS patients.

Characterization of the ultraviolet B and X-ray response of primary cultured epidermal cells from patients with disseminated superficial actinic porokeratosis.

BACKGROUND: Disseminated superficial actinic porokeratosis (DSAP) is the most common porokeratosis and is characterized by multiple keratotic lesions which tend to occur at sun-exposed sites. A mild hypersensitivity to X-rays has been reported for DSAP-derived fibroblasts and frequent over-expression of p53 has been found in lesional epidermis. OBJECTIVES: In order to clarify whether genome maintenance mechanisms might be compromised in this disease the following approaches were undertaken: (i) primary cultured keratinocytes and fibroblasts from DSAP patients were characterized for ultraviolet (UV) B and X-ray response; (ii) 15 lesions were studied for p53 mutations, and (iii) the differentiation status of DSAP-derived keratinocytes was evaluated. METHODS: Primary cultures of keratinocytes and fibroblasts were established from lesional and nonlesional skin biopsies of two subjects with DSAP. p53 mutations were analysed by DNA sequencing of the conserved region of the TP53 gene. Differentiation was evaluated both in stratified epithelial sheets from confluent keratinocyte cultures and in organotypic skin cultures. RESULTS: The cytotoxic and apoptotic response to UVB or X-irradiation was similar in DSAP-derived keratinocytes and fibroblasts when compared with normal cells. Two of 15 lesions examined presented p53 mutations located at nondipyrimidine sites. A strikingly decreased expression of filaggrin was observed both in reconstructed epidermis and in reconstructed skin. CONCLUSIONS: The UVB and X-ray response of DSAP-derived keratinocytes and fibroblasts indicates that the actinic character of this skin pathology is not due to radiation hypersensitivity. In agreement with this finding, mutations in the p53 gene, which are often associated with UV-related skin carcinogenesis, were rarely detected in DSAP lesions and were not UV-specific. Reconstructed epidermis and reconstructed skin models successfully reproduced the main features of this genodermatosis, showing that DSAP-derived keratinocytes bear an inherent defect in the terminal differentiation programme.

UV mutation signature in tumor suppressor genes involved in skin carcinogenesis in xeroderma pigmentosum patients.

Molecular analysis of p53 and patched (PTCH), two candidate tumor suppressor genes for non-melanocytic skin cancer, was performed in skin tumors from six patients affected by the cancer-prone disease xeroderma pigmentosum (XP). UV-specific p53 mutations were detected at a frequency of 38-50% in all the tumor types analysed, including melanomas. Additional analysis of PTCH mutations in the subset of eight basal call carcinomas (BCC) revealed a very high mutation frequency of this gene (90%) which exceeded that detected in the p53 gene in the same tumors (38%). PTCH mutations were predominantly UV-specific C>T transitions. This mutation pattern is different from that reported in BCC from normal donors where PTCH mutation frequency is 27% and mutations are frequently deletions and insertions. These findings suggest that PTCH mutations represent an earlier event in BCC development than p53 alterations and that the inability of XP patients to repair UV-induced PTCH mutations might significantly contribute to the early and frequent appearance of BCC observed in these patients.

Factors that influence the DNA repair capacity of normal and skin cancer-affected individuals.

DNA repair capacity (DRC) was studied in 49 patients affected by basal cell carcinoma (BCC) and 68 cancer-free controls belonging to a larger case-control population enrolled for studying BCC risk factors. DRC was measured in the subjects' peripheral blood lymphocytes by using a host-cell reactivation assay that measures cellular activation of a reporter gene irradiated with UV light. A statistically significant age-related decline in DRC was observed in the controls from 20 to 70 years of age but not in the BCC cases. When the DRC values of the BCC patients and controls were compared by age, young BCC cases (age, < or =40 year) repaired less than the controls, although the difference was not statistically significant. Conversely, older BCC patients (age, >40 years) presented an enhanced repair capacity (P < 0.001) as compared with their controls. The search for possible factors associated with the high repair rate of elderly BCC cases revealed that both target cell physiology and life-style habits may affect host DNA repair. Smoking was the variable that explained most of the increase in DRC among older patients. The understanding of how these factors affect host DRC will be relevant for a correct use of this biomarker.

p53 mutations and chromosome instability in basal cell carcinomas developed at an early or late age.

Tumor DNA from 45 primary basal cell carcinoma (BCC) biopsies was screened for p53 gene mutations, chromosome 9 allele loss, and microsatellite instability. p53 mutation frequency increased significantly as a function of the age at BCC onset ranging from 6% (1/16) in early BCC (before age 40 years) to 35% (10/29) in late BCC. All p53 mutations found implicated sunlight as the mutagen. Chromosome 9 instability (allele loss or microsatellite instability) was detected at high frequency (38%) independently of age at tumor onset. Allelic loss was confined to chromosome 9q, whereas microsatellite instability was observed prevalently on chromosome 9p often in association with a replication error (RER+) phenotype. Most of our late BCC patients reported occupational sun exposure, while early BCC patients recalled childhood (0-20 years) recreational sun exposure. These data suggest that chronic exposure to sunlight is responsible for accumulation of p53 mutations and thus for late BCC appearance, whereas acute UV exposure in childhood and adolescence leads to early skin cancer development in genetically susceptible individuals via a p53-independent pathway.

Misincorporation rate and type on the leading and lagging strands of UV-damaged DNA.

We have examined the fidelity of replication of the leading and lagging strands of UV-irradiated DNA by using an EBV-derived shuttle vector system which contains as marker gene for mutation analysis the bacterial gpt gene in both orientations relative to the EBV oriP. Human cells stably transformed with this vector were UV irradiated and gpt mutation rate and type were analysed. An increased mutagenicity associated with UV irradiation was observed, but the average error frequency was unaffected by the direction of replication of the target gene. Some variability by position and sequence context of leading and lagging strand errors was detected, suggesting that the different architecture of the replication complex for the two strands might, to some extent, affect mutation spectra. The comparable fidelity of translesion replication on the leading and lagging strands is in agreement with the current model for eukaryotic replication that postulates the simultaneous synthesis of both strands by a DNA polymerase with a proof-reading exonuclease.

Genetic alterations in skin cancer.

Cancer is a multi-stage process in which the accumulation of genetic changes allows clonal expansion of abnormal cells that will eventually form a tumor. Skin cancer is the most common malignancy affecting human beings. Mutations of the tumor suppressor gene p53 are often found in non-melanoma skin cancer and pre-invasive lesions, like actinic keratosis. The type of mutations detected in the p53 gene strongly indicate UV light as the initiating and promoting agent in skin cancer development. Chromosome instability is also an early event in skin tumor formation. However, despite the huge amount of information available in the literature on molecular markers of skin cancers, much remains to be uncovered about the progression of genetic events that separate normal sun-exposed epidermis from skin cancer. In this paper the following issue will be addressed: how far are we from being able to define a human model for multistage skin carcinogenesis in humans?

Strand bias of ultraviolet light-induced mutations in a transcriptionally active gene in human cells.

Ultraviolet (UV)-induced repair and mutational spectra were analyzed in an inducible marker gene, the metallothionein-l/guamine-xanthine phosphoribosyl transferase (gpt) fusion gene, carried by an Epstein-Barr virus-derived shuttle vector episomically maintained in human cells. The repair rate of UV photodimers from the shuttle-vector molecules was typical of transcriptionally active sequences, 70% of the dimers being removed within 8 h after irradiation. The spectrum obtained under basal gene transcription was compared with that obtained under induced transcription. In both cases, base substitutions at dipyrimidine sequences predominated. Multiple mutations and deletions probably due to recombinational events induced by UV damage were also observed. Most of the UV-mutated dipyrimidine sites were located in the transcribed strand and were independent of the transcriptional activity of the target gene. In contrast, the distribution of mutations throughout the coding region of the gpt gene was affected by transcription, with a preferential clustering of mutations occurring in the 3' half of the gene after transcription induction. The strand bias observed in the UV spectra most likely reflects selection for nonfunctional gpt protein.

Molecular analysis of mutations induced by chemical carcinogens in mammalian cells. II. The use of recombinant DNA vectors.

One approach to molecular and mechanistic studies of mutagenesis in mammalian cells is to introduce a mutational target gene into the cells as part of a shuttle vector which is capable of replication in both mammalian cells and bacteria. Following mutagenesis in the mammalian cell host, the shuttle vector sequences are recovered from the mammalian cells and introduced into bacteria, where large amounts of the mutant gene can be produced for sequence analysis. The variety of shuttle vector systems which have been developed for this purpose will be described. Shuttle vectors have been widely used for the molecular analysis of mutations induced by physical and chemical agents and to investigate the factors which modulate mutation fixation. The data regarding chemically induced mutational spectra will be reviewed with particular emphasis on the studies aimed to dissect the complex process which lead from DNA lesion to mutation.

Mutagenic processing of ethylation damage in mammalian cells: the use of methoxyamine to study apurinic/apyrimidinic site-induced mutagenesis.

The aldehyde reagent methoxyamine is able to interact with apurinic/apyrimidinic sites formed in vivo within cells and displays both an anti-cytotoxic and an antimutagenic activity on N-ethyl-N'-nitro-N-nitrosoguanidine-induced DNA damage in Chinese hamster ovary cells. To clarify the underlying mechanism we have examined the mutational spectra induced by N-ethyl-N'-nitro-N-nitrosoguanidine alone and in the presence of methoxyamine in the hypoxanthine-guanine phosphoribosyltransferase gene of Chinese hamster ovary cells. In both cases all mutations were base pair substitutions, and their distribution among various classes did not differ significantly. Almost 60% were transitions, predominantly GC to AT, and the remaining 40% were transversions, mainly at AT base pairs. The analysis of the proportion of the different types of mutations showed that in the presence of methoxyamine, GC to AT transitions decreased by a factor of 1.8, and AT to CG transversions were reduced by a factor of 13. These data indicate that in mammalian cells the fixation of ethylation damage into mutations occurs by both (a) direct mutagenesis likely driven by O6-ethylguanine adducts and to a minor extent by O4-ethylthymine and (b) apurinic/apyrimidinic site-mediated mutagenesis. These apurinic/apyrimidinic sites are formed during the processing of ethylation at critical sites and are likely to involve O6-ethylguanine and O2-ethylthymine adducts.

Quantitative relationship between ethylated DNA bases and gene mutation at two loci in CHO cells.

In a previous study we showed that the formation of O6-ethylguanine (O6-EtGua) in the DNA of CHO cells in culture correlated with mutations induced by ethylnitrosourea (ENU) and diethylsulfate (DES) at the hypoxanthine-guanine-phosphoribosyltransferase (hprt) locus but not at the Na, K-ATPase locus. This study was extended to another ethylating agent, ethyl methanesulfonate (EMS). DNA adduct formation and induction of mutation at the two gene loci were determined simultaneously in CHO cells after EMS exposure. The extent of ethylation at the N7 and O6 positions of guanine and at the N3 site of adenine were measured and the possible correlations with 6-thioguanine resistance (6-TGr) and ouabain resistance (ouar) mutations were investigated. A good correlation between the levels of ethylation at O6 guanine and mutation frequency at hprt gene by all three ethylating agents was observed. In the case of the ouar locus, the frequency of O6-EtGua adducts correlated with mutation induction by EMS and ENU but not by DES. Although both EMS and DES have similar reaction mechanisms, these results highlight differences in their mutational specificity. The comparison of this type of analysis with mutational spectra revealed that correlation studies may be inadequate to analyse multicomponent phenomena like mutation formation.

N-methyl-N-nitrosourea-induced mutations in human cells. Effects of the transcriptional activity of the target gene.

In this study we addressed the question as to whether the mutagenesis by methylating agents is affected by the transcriptional activity of the damaged gene. An Epstein-Barr virus (EBV)-derived shuttle vector system was developed where the genetic target for mutation analysis, the bacterial gpt gene, is under the control of an eukaryotic inducible promoter in plasmid pF1-EBV and lacks the eukaryotic promoter in plasmid pF2-EBV. Two human cell lines that episomically maintain these shuttle vectors were established. In clone 6NT cells, which contain pF1-EBV plasmid, the gpt gene is actively transcribed and the transcription rate is regulated by zinc ions. In clone 3 cells, which harbor pF2-EBV plasmid, the gpt gene is not transcribed. Following treatment of both cell lines with the potent alkylating carcinogen N-methyl-N-nitrosourea (MNU), G.C to A.T transitions were the major mutagenic event, consistent with the miscoding potential of O6-methylguanine. The mutations were predominantly generated in the non-transcribed DNA strand of the active gpt gene. The same strand-bias was observed when the gpt gene was transcriptionally inactive, indicating that MNU-induced strand-specific formation of mutations is not due to transcription. Our data identify as major determinants of this phenomenon the sequence-specificity of MNU mutagenesis and the conformational properties of the target protein. Differences in mutation distribution were observed between the transcriptionally active and inactive gpt gene. This finding suggests that the organization of active genes in chromatin might modulate DNA alkylation and/or DNA repair.

Methoxyamine modification of abasic sites protects CHO cells from the cytotoxic and mutagenic effects of oxygen alkylation.

The biological effects of the interaction of methoxyamine (MX) with apurinic/apyrimidinic (AP) sites produced in CHO cells by treatment with alkylating agents were examined. A decrease in cytotoxicity was observed after a 10 min treatment with the SN1 alkylating agents ethylnitrosourea (ENU), N-ethyl-N'-nitro-N-nitrosoguanidine (ENNG) and N-methyl-nitrosourea when MX was present in the culture medium. Furthermore MX reduced the number of mutations to 6-thioguanine resistance induced by ENU and ENNG and the number of sister chromatid exchanges induced by ENU. In contrast, no protective effect of MX on survival was observed after a 10 min treatment with the SN2 alkylating agents diethylsulfate (DES), ethyl methane sulfonate and methyl methane sulfonate. A 3 h exposure to MX abolished the protective effect of MX on ENU-induced cytotoxicity and increased the cytotoxicity of DES. In vitro studies with synthetic oligonucleotides containing a single AP site opposite a normal guanine or O6-methylguanine showed that MX inhibits the cleavage of AP sites by the CHO AP endonuclease(s). A model is proposed in which different DNA lesions are involved in AP site formation after treatment with SN2 or SN2 alkylating agents. The involvement of specific alkylation products in cytotoxicity and mutagenesis is also discussed.

Inhibition of replicative DNA synthesis and induction of DNA repair in human fibroblasts by the intercalating drugs proflavine and 9-aminoacridine.

The induction of unscheduled DNA synthesis (UDS) and the alteration of semiconservative DNA replication by the structurally related intercalating agents proflavine and 9-aminoacridine were studied in MRC-5 human fibroblasts in culture. Autoradiographic determinations of both parameters were carried out simultaneously in the same culture specimens. Proflavine affected DNA synthesis, but did not elicit any UDS. 9-Aminoacridine inhibited DNA synthesis only at the highest concentration and caused UDS to a low but significant extent. These results suggest that the ability to induce UDS is not a general property of the intercalating agents and that the alterations of the DNA structure, typical of the "pure" intercalative process, are not handled by pathways involving unscheduled synthesis.

[ST-T segment alternans in ventricular tachycardia associated with inversion of the U wave in Prinzmetal angina during exercise test. Description of a clinical case]. / Alternanza del sopraslivellamento di ST-T durante tachicardia ventricolare associata ad inversione dell'onda U nell'angina di prinzmetal durante test da sforzo. Descrizione di un caso clinico.

A case of ST segment alternans during effort angina is described. This is the seventh such case in the literature. Here the alternans appeared during a ventricular tachycardia. As with the other cases in literature, the alternans took place during an ischemic attack which affected the anterior wall.

The origin of DNA single strand breaks induced by ethylating agents in mammalian cells.

Chinese hamster ovary (CHO) cells were treated with two ethylating agents, N-ethyl-N-nitrosourea (ENU) and diethylsulfate (DES), and the kinetics of DNA single strand break (ssb) induction and rejoining were determined in parallel with DNA adduct formation and removal. In the case of DES, DNA ssb as determined by alkaline elution (AE) were repaired very slowly with more than 50% of the lesions still present on DNA 3 h after treatment. In contrast, 45% of ENU-induced ssb were repaired within 10 min. From the relative concentration of the different ethylated products and their repair rates as measured by high performance liquid chromatography (HPLC) analysis of the ethylated DNA, a theoretical function was constructed that describes the number of ssb expected at each time point after exposure to the mutagen. DES-induced ssb are explained by excision repair processes active on the ethylated purines, mainly 3-ethyladenine (3-EtAde) and 7-ethylguanine (7-EtGua). On the same basis, the rapidly repaired ENU-induced ssb remain unexplained. These results are also discussed in relation to the sensitivity of the two techniques, AE and HPLC, for detecting DNA damage.

Molecular approaches to the study of chemical mutagenesis.

The use of shuttle vectors has been applied in recent years to develop a better understanding of the molecular mechanisms of mutagenesis in mammalian cells. These recombinant DNA molecules replicate together with the host eukaryotic cells and can be retrieved in bacteria for rapid detection and analysis of mutation. Two approaches based on the use of shuttle vectors for studying the mutagenic effects of DNA lesions induced by alkylating agents are presented.