Biomedical subjects

A Boninsegna

Publications and source records attributed to A Boninsegna.

At least 19 recordsLinked to original sources

PubMed2006

Analysis of dystroglycan regulation and functions in mouse mammary epithelial cells and implications for mammary tumorigenesis.

Abnormalities in the interactions of cells with the extracellular matrix (ECM) play an important role in the development and progression of many types of cancer and are a hallmark of malignant transformation. The dystroglycan (DG) complex is a transmembrane glycoprotein that forms a continuous link from the ECM to the actin cytoskeleton, providing structural integrity and perhaps transducing signal, in a manner similar to integrins. Deregulated expression of DG has been reported in a variety of human malignancies and related to tumor differentiation and aggressiveness. In breast cancer, reduced DG expression has been associated with patient survival and with loss of differentiation of tumor cells. Limited data are available on DG physiology in epithelial cells. In this study, we used the HC11 spontaneously immortalized murine mammary epithelial cells to study DG function(s) and regulation in normal cells. We found that expression of DG protein and mRNA is cell-cycle and cell-density regulated in these cells. Moreover, expression of both DG subunits increased upon lactogenic differentiation of the HC11 cells. The turnover of cell-surface-expressed DG was evaluated in the same cells and half-life of DG subunits was evaluated to be about 12 h. DG-specific small inhibitory RNAs were used to analyze the effects of a reduced expression of DG in these cells. Cells in which DG expression was suppressed were growth inhibited, accumulated in the S-phase of the cell cycle, failed to undergo lactogenic differentiation, and displayed an increase in the percentage of apoptotic cells. Moreover, changes were observed in the expression and/or activity of several molecules involved in cell growth control. These results demonstrate that DG expression is tightly regulated in normal mammary epithelial cells and support the hypothesis that DG is involved in several functions other than structural integrity in these cells. This finding provides new insight into the roles played by DG in epithelial cell physiology and will contribute to our understanding of its involvement in the process of epithelial cell transformation.

Animals↗

PubMed2005

Lycopene induces apoptosis in immortalized fibroblasts exposed to tobacco smoke condensate through arresting cell cycle and down-regulating cyclin D1, pAKT and pBad.

There is a lot of interest in the health benefits of dietary carotenoids and on the relationship of these compounds with smoke. In particular, it is unknown if the enhanced cancer risk observed in smokers following beta-carotene supplementation can be also found using other carotenoids. Here, we studied the effects of the tomato carotenoid lycopene on molecular pathways involved in cell cycle progression, apoptosis and survival in immortalized RAT-1 fibroblasts exposed to cigarette smoke condensate (TAR). Lycopene (0.5-2.0 microM) inhibited cell growth in a dose-and time-dependent manner, by arresting cell cycle progression and by promoting apoptosis in cells exposed to TAR. The arrest of cell cycle was independent of p53 and of 8-OH-dG DNA damage and related to a decreased expression of cyclin D1. Moreover, the carotenoid up-regulated apoptosis and down-regulated the phosphorylation of AKT and Bad in cells exposed to TAR. Such an effect was associated to an inhibition of TAR-induced expression of Cox-2 and hsp90, which is known to maintain AKT activity. This study suggests that lycopene, differently from beta-carotene, can exert protective effects against cigarette smoke condensate.

8-Hydroxy-2'-Deoxyguanosine↗

PubMed2001

Redox regulation of cell proliferation by pyrrolidine dithiocarbamate in murine thymoma cells transplanted in vivo.

Pyrrolidine dithiocarbamate (PDTC) is a synthetic compound largely used in cell biological studies and known to exert either antioxidant or pro-oxidant effects. Recently, its antitumoral activity has been proposed on the basis of its antioxidant and proapoptotic effects. In the present study, we evaluated the effect of increasing i.p. doses of PDTC on the growth of a strain of highly malignant thymoma cells inoculated in the peritoneum of inbred Balb/c mice. PDTC treatment increased the number of thymoma cells in a dose-dependent manner, enhancing the percentage of proliferating tumor cells. PDTC exerted regulatory effects on cell cycle distribution, decreasing the expression of cell cycle inhibitors. Alterations in the production of intracellular reactive oxygen species, levels of oxidized glutathione, and intracellular levels of the redox-active metals iron and copper were also observed. The above results represent the first evidence that PDTC may induce in vivo cell proliferation in a murine thymoma cell model. In addition, we suggest that the ability of PDTC to bind and transport metals inside the cell and its pro-oxidant property may be factors underlying its effects on thymoma cell proliferation and cell cycle distribution.

Animals↗

PubMed2001

Resveratrol, a natural phenolic compound, inhibits cell proliferation and prevents oxidative DNA damage.

Resveratrol (3,4',5-trihydroxystilbene) is a naturally occurring phenolic compound which is present at high levels in wine and has been recently proposed as a potential cancer chemopreventive and chemoterapeutic agent. In this study, we evaluated the antiproliferative activity of resveratrol on a panel of cell lines of various histogenetic origin, including normal rat fibroblasts and mouse mammary epithelial cells compared to human breast, colon and prostate cancer cells. The concentration of resveratrol inhibiting cell growth by 50% (IC(50)) ranged from about 20 to 100 microM. At such concentration, we were unable to detect a significant increase in the apoptotic index in most of the cell lines analyzed. We also studied the effects of resveratrol on cell cycle distribution. The most striking effect was a reduction in the percentage of cells in the G2/M phase which was most frequently associated with an increase of cells in the S phase of the cell cycle. We also found that resveratrol is able to prevent the increase in reactive oxygen species (ROS) following exposure to oxidative agents (i.e. tobacco-smoke condensate (TAR) and H(2)O(2)). Resveratrol also reduced nuclear DNA fragmentation, as assessed by single cell gel electrophoresis (comet test). Taken together our results suggest that resveratrol can act as an antimutagenic/anticarcinogenic agent by preventing oxidative DNA damage which plays a pivotal role in the carcinogenic activity of many genotoxic agents.

Animals↗

PubMed2001

Isolation of normal epithelial cells adapted to grow at nonphysiological concentration of magnesium.

Extracellular magnesium (Mg) depletion inhibits the growth of the HC11 normal mammary epithelial cells. In this study we found that an acute increase in extracellular Mg generally exerts a positive effect on the growth of these cells. We also isolated and characterized two derivatives adapted to grow and proliferate at nonphysiological concentration of Mg. The growth properties of the HC-LMg cells at 25 microM Mg were comparable to those of the parental HC11 cells in the regular medium (0.5 mM Mg) despite an increased expression of the CDK inhibitor p27(Kip1). They also showed a reduced dependence from serum to grow. The HC-HMg cells have been adapted to grow and proliferate at an increased (45 mM) Mg concentration. Cell total Mg content was 19.6, 9.7, and 20.1 nmol/mg protein in the HC11, HC-LMg, and HC-HMg cells, respectively. Thus, we have isolated derivatives of normal epithelial cells which are able to maintain Mg content in a physiological range in the face of different extracellular concentration gradients and will be a valuable tool for further studies on the regulation of Mg homeostasis in eukaryotic cells.

Animals↗

PubMed2001

DNA oxidative damage during differentiation of HL-60 human promyelocytic leukemia cells.

DNA oxidative damage was measured in human promyelocytic leukemia HL-60 cells, in the same cells committed to granulocytic differentiation with dimethyl sulfoxide (DMSO) or all-trans-retinoic acid (RA) and in mature human peripheral granulocytes (HPG). DNA damage was evaluated as single strand breaks and 8-OHdG adducts, measured by single cell electrophoresis or by monoclonal antibodies, respectively. The basal levels of either marker of DNA damage were higher in undifferentiated HL-60 cells than in HPG and DMSO- or RA-differentiated cells. Treatment with H(2)O(2) increased 8-OHdG formation in all cells, but the levels of DNA damage remained higher in undifferentiated cells as compared to the differentiated ones. Three lines of evidence suggested that the higher levels of DNA damage observed in undifferentiated cells were at least in part attributable to a reduced detoxification of reactive oxygen species (ROS). First, undifferentiated cells were shown to accumulate higher levels of dichlorodihydrofluorescein-detectable ROS than HPG and DMSO- or RA-differentiated cells. Second, undifferentiated HL-60 cells were characterized by reduced levels of GSH and lower GSH/GSSG ratios as compared to the differentiated cells. Third, pretreatment of undifferentiated HL-60 cells with antioxidants such as alpha-tocopherol or beta-carotene suppressed the elevation of ROS and the formation of 8-OHdG induced by H(2)O(2). Further evidence for the importance of the oxidant/antioxidant balance was obtained by modulating the iron-catalyzed decomposition of H(2)O(2) to hydroxyl radicals in undifferentiated HL-60 cells. In fact, pretreatment with FeSO(4) increased the formation of 8-OHdG induced by H(2)O(2), whereas pretreatment with the iron chelator deferoxamine produced the opposite effect. These results illustrate correlations between the oxidant/antioxidant balance and DNA damage and suggest that the capability of a cell population to withstand oxidative stress and DNA damage may depend on its degree of differentiation.

Antibodies, Monoclonal↗

PubMed2000

8-hydroxy-2'-deoxyguanosine in cervical cells: correlation with grade of dysplasia and human papillomavirus infection.

In this study, the 8-hydroxy-2'-deoxyguanosine (8-OHdG) level was assessed in human cervical cells by an immunoperoxidase method and was related to the presence of human papillomavirus (HPV) infection and precancerous lesions. After optimizing the immunohistochemical method of detecting oxidative DNA damage in whole cells, we have used this technique to estimate the oxidative damage in cervical cells collected during a routine PAP test. The analysis of variance (ANOVA) of the data from human samples showed significant differences in the 8-OHdG content among normal, low-grade and high-grade squamous intraepithelial lesion (SIL, HGSIL and LGSIL, respectively; P < 0.001). In the comparison of the three groups, statistically significant differences were detected between normal SIL and HGSIL (P < 0.001) and between LGSIL and HGSIL (P = 0.003), whereas no statistically significant difference was found between normal SIL and LGSIL (P = 0.1). Grouping observations by HPV status, no significant difference was detected in 8-OHdG levels between HPV(+) and HPV(-) subjects (P = 0.8). The polytomous and proportional odds models, extensions of the logistic regression analysis, showed that the effect of 8-OHdG levels in rising the risk of dysplasia was roughly constant through SIL grades. In conclusion, the immunoperoxidase method, applied to single human cervical cells, provides clear evidence that significant differences exist in 8-OHdG content between normal and dysplastic cells and that oxidative DNA damage might play an important role in cervical carcinogenesis.

8-Hydroxy-2'-Deoxyguanosine↗

PubMed1999

Polycyclic aromatic hydrocarbon-DNA adducts in cervical smears of smokers and nonsmokers.

OBJECTIVES: The aim of this study was to detect polycyclic aromatic hydrocarbon-DNA (PAH-DNA) adducts in single cervical cells collected during a routine Papanicolaou smear and to relate this carcinogen exposure dose marker to smoking habit. METHODS: An immunohistochemical assay, using a polyclonal antiserum raised against benzo[a]pyrene diol epoxide-DNA adducts, was performed to evaluate PAH-DNA adducts in cervical cells collected from 16 volunteers who smoked at least 20 cigarettes/day and 16 nonsmokers. RESULTS: The mean adduct level, determined as relative staining intensity by an optical density image analyzer, was significantly higher in smokers compared to nonsmokers (AOD x 1000 +/- SD = 98 +/- 32 and 73 +/- 25, respectively) (P = 0.04). CONCLUSIONS: These results demonstrate that this immunohistochemical assay, much simpler than other methodologies used to evaluate PAH-DNA adducts in cervical tissue, is sufficiently sensitive for quantitative adduct evaluation in single epithelial cervical cells, as already verified for other exfoliated material. This work thus confirms that tobacco smoke is a risk factor for genotoxic damage generation in cervical cells and indicates a procedure likely adaptable to a large population screening.

Adult↗

PubMed1999

Evaluation of polycyclic aromatic hydrocarbon-DNA adducts in exfoliated oral cells by an immunohistochemical assay.

Polycyclic aromatic hydrocarbon-DNA adducts were evaluated in oral cells from 98 healthy volunteers by an immunohistochemical method using a specific antiserum against benzo(a)pyrene-DNA adducts revealed by the immunoperoxidase reaction. Mean adduct content, determined as relative staining intensity by absorbance image analyzer, was significantly higher in the cells from tobacco smokers compared with nonsmokers (330 +/- 98, n = 33 versus 286 +/- 83, n = 64, respectively) with a P = 0.013 obtained by two-sample t test with equal variances. We found that in the smoker group, the PAH-DNA adduct content increases with the number of cigarettes. Thus, the relative staining intensity was 305 +/- 105 in the group smoking 1-10 cigarettes/day (n = 16), 347 +/- 77 in the 11-20 group (n = 14), and 386 +/- 112 in the group smoking more than 20 cigarettes/day (n = 3; P = 0.03 by nonparametric test for trend). No significant association was detected between PAH-DNA adducts in oral cells and variables such as residential area, oral infections, alcohol or vitamin intake, grilled food consumption, and professional activity. This work confirms and extends previous data suggesting that this immunohistochemical method might be used as a valuable dosimeter of genotoxic damage in a carcinogen-exposed population, although further studies are needed to verify the applicability of the test in high-risk populations other than smokers.

Adolescent↗

PubMed1998

4-Aminobiphenyl-DNA adducts in laryngeal tissue and smoking habits: an immunohistochemical study.

4-Aminobiphenyl (4-ABP)-DNA adducts and p53 overexpression were evaluated in laryngeal biopsies from 38 patients by immunohistochemical methods. Samples were categorized as tumors (n = 9), polyps (n = 28) or normal tissue (n = 1). 4-ABP-DNA adducts were evaluated with a quantitative immunoperoxidase method using monoclonal antibody 3C8 in both the lesion and adjacent tissue. Relative staining intensity data showed a log-normal distribution and values found in adjacent tissue from smokers were significantly higher (median: 173.5, geometric mean: 159.9) than those measured in adjacent tissue from non-smokers (median: 75.5, geometric mean: 7.40). Statistical significance was assessed both by non-parametric testing on raw data (P = 0.0007 on rank sum test) and by parametric testing on log-transformed data (P = 0.0002 on an unpaired t-test). Furthermore, relative staining intensity in the lesional tissue showed the same significant difference between smokers and non-smokers in patients affected by polyps, whereas no significant difference was detected in patients with laryngeal tumors. Overexpression of p53, also measured with an immunoperoxidase method, was observed in 44% of the malignant tumors and in 3.5% of the polyps. This work demonstrates that 4-ABP-DNA adducts can be evaluated in laryngeal tissue and are related to smoking exposure.

Adult↗

PubMed1997

Immunohistochemical analysis of p53 protein in transplant recipients with Kaposi's sarcoma.

PURPOSE: Kaposi's sarcoma (KS) is a proliferative process of suspected viral aetiology associated with immune deficiency. In transplanted patients, lesions regress on discontinuation of immunosuppressive therapy. The purpose of this work was to analyse the expression of the p53 oncosuppressor gene product, a proliferation regulator overexpressed in both malignant and non-malignant conditions, with the aim of better qualifying KS proliferation characteristics. METHODS: We analysed p53 expression in a group of transplanted, cyclosporin A-treated, KS patients by immunohistochemistry, utilizing the DO-7 (with and without the antigen retrieval pretreatment), and the PAb 240 monoclonal anti-p53 antibodies, the latter of which is able to detect a mutated epitope, and evaluating staining intensity and localization, whether cytoplasmic or nuclear. RESULTS: Seventy five percent of KS lesions from transplanted patients presented both nuclear and cytoplasmic positive p53 immunostaining with DO-7 antibody, thus demonstrating a presumably functional inactivation; one case also presented immunoreactivity with the PAb 240 antibody. CONCLUSIONS: On the basis of the results obtained and in the presence of lesion regression upon immunosuppression withdrawal, it may be concluded that KS in transplanted patients can be considered a non-malignant proliferative process, and that the cytoplasmic expression of p53 may stand for a functional inactivation pattern.

Female↗

PubMed1996

Prognostic significance of cytoplasmic p53 overexpression in colorectal cancer. An immunohistochemical analysis.

p53 overexpression was studied by immunohistochemistry in 96 consecutive colorectal cancer patients, subdividing positive specimens according to two staining patterns: cytoplasmic or nuclear. Forty-seven per cent of the cases were p53 positive, a significant correlation being found with Dukes' stage (P = 0.0036). A prevalence of nuclear staining was observed in Dukes' B and cytoplasmic in Dukes' D stages. After 36 months, 23% of the patients had a recurrence, and 45% were p53 positive, all Dukes' C-D stage with cytoplasmic staining. The Kaplan-Meier curve showed a significant correlation between p53 cytoplasmic staining and disease-free survival period (P = 0.002). With respect to disease-free survival, the Cox proportional hazard regression test, comparing p53 positivity with Dukes' stage, showed the latter to be the most significant variable. In our series of patients, advanced Dukes' stage tumours were localised in the right colon, where a higher percentage of p53 positivity (67% versus 40% of the left side), as well as a higher frequency of cytoplasmic staining was observed. In conclusion, from the data obtained, a strong correlation between p53 cytoplasmic staining and patient prognosis is clearly indicated.

Adult↗

PubMed1991

[Lipolysis and ketosis during swimming in thermal water].

Plasma free fatty acids, blood glucose, beta-hydroxybutyrate and acetoacetate variations were studied in rats during swimming. Rats were forced to swim for 30 min in thermal water (source of Abano Terme) at 35 degrees and in normal water at 25 degrees. During swimming in thermal water plasma free fatty acids were increased, the glycemia remained unaffected, the beta-hydroxybutyrate and acetoacetate decreased. The swim in normal water induced a sharp increase of plasma free fatty acids, a decrease of blood glucose, an increase of blood beta-hydroxybutyrate and a marked decrease of acetoacetate. From these data, some indications of clinical interest are presented and discussed.

3-Hydroxybutyric Acid↗

PubMed1991

[Beneficial effect of swimming in thermal waters on muscle glycogen depletion].

The effect of swimming in the termal water on muscle glycogen stores was studied. After 30 min the muscle glycogen results in a diminution, but it is not depleted. On the contrary, 30 min of swimming in normal water results in a depletion of muscle glycogene stores. The glycemic homeostasis is well maintained in thermal water, and hypoglicemia occurs only after swimming in normal water.

Animals↗

PubMed1990

Effect of swimming in thermal water on skeletal muscle, liver and heart glycogen.

Skeletal muscle, liver and heart glycogen variations, induced by swimming in thermal water (at 35 degrees C) as a model of physical exercise for clinical use, were studied. Muscle and liver glycogen moderately decreases after a 30-min period of swimming and comes near to depletion after 60 min. Heart glycogen decreases only slightly after 60 min. Blood glucose and plasma insulin decrease only after 60 min of swimming. A 30-min swim in thermal water, cooled to 25 degrees C, depletes muscle and liver glycogen and slightly decreases heart glycogen. Under these conditions, plasma insulin decreases and hypoglycemia occurs. The results seem to indicate some advantages of swimming in hot thermal water in order to prevent glycogen store depletion as the physiological prerequisite for a physical exercise of clinical interest to obtain therapeutical benefits, avoiding premature fatigue and exhaustion.

Animals↗

PubMed1985

The effect of portocaval shunt on hepatic glycogen stores during fasting.

The effect of a prolonged fast was studied in surgically portocaval shunted (PCS) rats. This shunt excludes the liver from the direct effect of pancreatic and enteric hormones, thus facilitating the study of the biochemical and metabolic effects of these hormones. In portocaval shunted rats, liver glycogen was lower than that of control rats, and remained unaffected during fasting. No remarkable difference was observed in blood glucose, plasma and liver free fatty acids and blood ketone bodies. Among blood nitrogen compounds, total protein, alanine and urea did not show any significant variation, while, in PCS rats, the initial low levels of creatinine resulted in an increase after fast. Skeletal muscle protein decreased only slightly in control rats, while their loss was remarkable in PCS rats. The possibility of a differential activation of gluconeogenesis and glycogenolysis in control and PCS rats is discussed.

Animals↗

PubMed1983

A simplified and computerizable regulatory enzyme model.

The concept of enzymes as simple biological catalysts has become too limiting and the kinetic behaviour has evidenced that the interaction of an enzyme with its substrate is more complex and can be influenced by various regulatory effectors. The resulting activity should be exactly what is required for the control of the overall flux through the metabolic pathway. A simplified enzyme model which can explain the sigmoidicity and the heterotropic effect involves the introduction of a limited number of kinetic parameters to obtain the best fit from v and s experimental values. This results in a more useful way to evaluate the possible regulatory effects of various substances such as metabolites, hormones and drugs.

Computers↗

PubMed1983

Kinetic alterations of the divalent cation-dependent ATPase activities of human erythrocyte membranes induced by blocking the membrane amino groups.

Treatment of erythrocyte membrane fragments with the amino group reagent sodium trinitrobenzenesulphonate (TNBS) leads to significant alteration of the kinetic properties of the membrane-bound (Ca2+ + Mg2+) and Mg2+-dependent ATPases, which appear to increase their affinity towards divalent cations and to decrease their maximal rates. Although it has not been possible to ascertain whether the amino groups involved in the TNBS effect belong to the membrane phospholipids or to the ATPase proteins, it appears that such groups play an essential role in the hydrolytic activity of the divalent cation-dependent ATPases, either by affecting the enzymes' microenvironment or by being directly involved in the enzymes'catalytic mechanism.

Adenosine Triphosphatases↗