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Biomedical subjects

R Montesano

Publications and source records attributed to R Montesano.

At least 109 records · Page 6Linked to original sources
PubMed1993

Altered expression of the cyclin D1 and retinoblastoma genes in human esophageal cancer.

We have examined DNA from four human esophageal carcinoma cell lines and 50 primary esophageal carcinomas obtained from China, Italy, and France for amplification of the cyclin D1 gene. We also examined 36 of these 50 carcinomas for expression of the cyclin D1 and retinoblastoma (RB) proteins by immunohistochemistry. We found a 3- to 10-fold amplification of the cyclin D1 gene in 16 of the 50 (32%) tumors and in two of the four cell lines. Cyclin D1 protein was overexpressed in 12 of 13 tumors and the two cell lines that showed gene amplification when compared to normal controls. Studies on RB protein expression indicated that 6 of the 36 (17%) tumor samples examined and one cell line did not show detectable expression of this protein. The tumors and cell lines that had cyclin D1 gene amplification and overexpression exhibited normal levels of expression of RB protein. By contrast, the tumors and cell line that did not appear to express the RB protein did not show amplification of the cyclin D1 gene and expressed only low levels of the cyclin D1 protein (P = 0.03). These results suggest that the inhibitory effect of RB on cell cycle progression can be abrogated during tumor development either by loss of expression of the RB gene or by increased expression of the cyclin D1 gene.

Blotting, Southern↗
PubMed1993

Alkylation and oxidative-DNA damage repair activity in blood leukocytes of smokers and non-smokers.

The levels of 3 DNA repair enzymes involved in alkylation and oxidative DNA damage repair in human peripheral blood leukocytes were measured in 20 smokers and 17 non-smokers. No differences in O6-alkylguanine-DNA-alkyltransferase (AGT) activity were found between the 2 groups and the AGT distribution within the population appeared to be unimodal. In contrast, the mean activities of both the methylpurine (MeP)- and the 2-6-diamino-4-hydroxy-5N formamidopyrimidine (FaPy)-DNA glycosylases were higher in the smokers, although only the difference between the MeP-DNA glycosylase means was statistically significant. The standard deviations of these 2 enzymes were also higher in the smokers. The MeP-DNA glycosylase activity showed a bimodal distribution when all subjects were considered. This may in part be due to the smoking habit; 83% of the subjects with enzyme activities higher than 500 fmoles/mg protein were current smokers, whilst 85% of the non-smokers had lower enzyme activities. However, if the smokers were considered separately, a bimodal distribution of this enzyme activity could still be observed. No strong correlation was observed between enzyme activity and age, although the slopes of the regression lines of enzyme activity on age were all negative. The relationship between enzyme activities was studied by bivariate distribution and a strong correlation was only found between the MeP-DNA glycosylase and the FaPy-glycosylase, with the highest values of both enzyme activities being observed in the smokers and the lowest in the non-smokers. Our results suggest that the activity of certain DNA repair enzymes can be modulated by environmental exposure.

Adult↗
PubMed1993

Genistein, a dietary-derived inhibitor of in vitro angiogenesis.

Consumption of a plant-based diet can prevent the development and progression of chronic diseases that are associated with extensive neovascularization; however, little is known about the mechanisms. To determine whether prevention might be associated with dietary-derived angiogenesis inhibitors, we have fractionated urine of healthy human subjects consuming a plant-based diet and examined the fractions for their abilities to inhibit the proliferation of vascular endothelial cells. Using gas chromatography-mass spectrometry, we showed that one of the most potent fractions contained several isoflavonoids, which we subsequently synthesized. Of all synthetic compounds, the isoflavonoid genistein was the most potent and inhibited endothelial cell proliferation and in vitro angiogenesis at concentrations giving half-maximal inhibition of 5 and 150 microM, respectively. As we have previously demonstrated, genistein concentrations in urine of subjects consuming a plant-based diet are in the micromolar range, while those of subjects consuming a traditional Western diet are lower by a factor of > 30. The high excretion of genistein in urine of vegetarians and our present results suggest that genistein may contribute to the preventive effect of a plant-based diet on chronic diseases, including solid tumors, by inhibiting neovascularization. Thus, genistein may represent a member of a new class of dietary-derived anti-angiogenic compounds.

Adrenal Cortex↗
PubMed1993

p53 mutations and aflatoxin B1 exposure in hepatocellular carcinoma patients from Thailand.

The prevalence and type of mutations in the p53 tumour-suppressor gene have been determined in 15 hepatocellular carcinomas (HCC) originating from Thailand. Direct sequencing of exons 5-8 revealed 2 mutations, an AGG to AGT (Arg-->Ser) transversion at codon 249, and an ATC-->AAC (Ile-->Asn) transversion at codon 254. Samples from the Thai patients were analyzed for the presence of aflatoxin-liver DNA and aflatoxin-serum albumin adducts, and all but one were found negative. All the patients were genotyped for glutathione-S-transferase (GST) mu, an enzyme possibly involved in the detoxification of AFB1, and 12 out of 15 had the null genotype. In general, the level of aflatoxin-albumin adducts in sera and the prevalence of p53 mutation at codon 249 in HCC were lower than in other areas at high risk of HCC, including southern China and parts of Africa.

Adolescent↗
PubMed1993

Correlation of p53 mutations with epidermal growth factor receptor overexpression and absence of mdm2 amplification in human esophageal carcinomas.

Esophageal carcinomas from 24 patients, most of whom were smokers and consumed alcoholic beverages daily, were analyzed for mutations in exons 5-8 of the p53 tumor suppressor gene. Mutations were identified by polymerase chain reaction amplification and direct sequencing in 12 of 24 (50%) of the samples; almost half of the mutations were at A:T base pairs. Nuclear accumulation of p53 protein, determined by immunohistochemistry with the CM-1 polyclonal antibody, was observed in all cases in which a missense mutation in the p53 gene was detected. None of the 24 carcinomas had amplification of the mdm2 gene, an alternate pathway to p53 loss of function. Alterations involving three other cancer-related genes associated with human esophageal carcinogenesis, c-erbB-1/epidermal growth factor receptor (EGFR), c-myc, and retinoblastoma (Rb), were examined by Southern blot or immunohistochemical analysis in the same sample set to explore the possibility of a link between oncogene activation and loss of tumor suppressor function. While no associations were observed between amplification of the c-myc or EGFR genes and p53 abnormalities, a significant correlation (P < 0.01) was seen between the presence of p53 mutation and EGFR overexpression. Absence of Rb protein, measured immunohistochemically, was observed in four tumors, none of which had aberrations of the p53 gene.

Carcinoma, Squamous Cell↗
PubMed1993

Biphasic effect of transforming growth factor-beta 1 on in vitro angiogenesis.

Although the existence of an increasing number of angiogenesis-regulating cytokines is well documented, the response elicited by combinations of these cytokines is largely unknown. Using an in vitro model in which microvascular endothelial cells can be induced to form capillary-like tubes within three-dimensional collagen or fibrin gels, we have investigated the effect of transforming growth factor-beta 1 (TGF-beta 1) on basic fibroblast growth factor (bFGF)-induced and vascular endothelial growth factor (VEGF)-induced angiogenesis. Endothelial cell invasion and capillary lumen formation were inhibited by TGF-beta 1 at relatively high concentrations (5-10 ng/ml), while lower concentrations (100 pg/ml-1 ng/ml) of TGF-beta 1 potentiated the effect of bFGF- and VEGF-induced invasion. The optimal potentiating effect was observed at 200-500 pg/ml TGF-beta 1. At invasion-potentiating doses of TGF-beta 1, lumen size in fibrin gels was markedly reduced compared to that in cultures treated with bFGF alone. These results show that TGF-beta 1 exerts a biphasic effect on bFGF- and VEGF-induced angiogenesis in vitro. Our studies support the notion that the nature of the angiogenic response elicited by a specific cytokine is contextual, i.e., depends on the presence and concentration of other cytokines in the pericellular environment of the responding endothelial cell.

Adrenal Cortex↗
PubMed1993

Nonrandom distribution of O6-methylguanine in H-ras gene sequence from DNA modified with N-methyl-N-nitrosourea.

The distribution of O6-meG in the rat H-ras gene sequence was studied using PCR by transition of O6-meG to adenine during the reaction. In order to study the transition mutations the PCR product was cloned in a replicative form of phage M13mp18 and sequenced. The use of PCR for detection of O6-meG was validated by using oligonucleotides (61 bases) containing one O6-meG residue at a defined site. After treatment of rat liver DNA by N-methyl-N-nitrosourea in vitro, a striking nonrandom sequence distribution of O6-meG was observed. Sixty-eight per cent of O6-methylated Gs were found in the middle G of the sequences GGT and GGA in the H-ras gene whereas no methylation was found in the middle G of the sequences AGG, GGG, TGT, TGC, CGA and CGC. No O6-meG adduct was found in the 12th codon of H-ras (sequence GGA). The frequency of O6-meG formation as a function of two flanking nucleotides on each side of the target guanine was calculated as an approach to understanding more distant sequence effects. It was found that in the DNA sequence studied the formation of O6-meG was highest if the G was flanked by PyPu or PuPu on the 5' side (Py, pyrimidine and Pu, purine) whereas PuPu on the 3' side showed maximal inhibition of O6-meG formation.

Animals↗
PubMed1993

Upregulation of urokinase receptor expression on migrating endothelial cells.

One of the phenotypic hallmarks of migrating endothelial cells, both in vivo and in vitro, is expression of the urokinase-type plasminogen activator (u-PA), a key mediator of extracellular proteolysis. In the study reported here, we have used an in vitro model of endothelial cell migration to explore the mechanism of this phenomenon. We have found that wounding of an endothelial cell monolayer triggers a marked, rapid and sustained increase in expression of a specific high-affinity receptor for u-PA (u-PAr) on the surface of migrating cells. Migrating cells displayed an increase in the levels of u-PA and u-PAr mRNAs, and this increase was mediated by endogenous basic fibroblast growth factor (bFGF). We also show that the increase in u-PA activity on migrating cells can be accounted for by an increase in receptor-bound u-PA, and that the increase in activity is also dependent on endogenous bFGF. These results demonstrate that the expression of plasmin-mediated proteolytic activity by migrating endothelial cells is a consequence of increased production of both u-PA and its receptor, and that this in turn is mediated by endogenous bFGF. This suggests that u-PA, produced at increased levels by migrating cells, binds to u-PAr whose expression is upregulated on the same cells. These observations are in accord with the postulated role of u-PAr in mediating efficient and spatially restricted extracellular proteolysis, particularly in the context of cell migration.

Animals↗
PubMed1993

Quantification of 7-methyldeoxyguanosine using immunoaffinity purification and HPLC with electrochemical detection.

7-Methylguanine (7-meG) could be a useful marker of recent past exposure to environmental methylating agents for use in epidemiological studies. A method is described that is appropriate for such an application. 7-meG was released from DNA by thermal hydrolysis under conditions (pH 9, 70 degrees C, 8 h) that preferentially released the base from DNA rather than RNA and, following immunopurification using antibodies specific for this DNA adduct, quantification was achieved either by HPLC with electrochemical detection (ECD) or by ELISA. The detection limits of the two approaches were 0.5 and 2 pmol 7-meG/DNA sample respectively. 7-meG was analysed in DNA samples contaminated with known amounts of RNA to test the possible interference in the analysis by the minor modified nucleoside 7-methylguanine, which is present as a normal component of RNA. 7-meG levels measured in human pancreas and untreated rat liver DNA were between 2 and 7 pmol 7-meG/mumol guanosine and this level could not be explained by RNA contamination. The combination of immunoaffinity purification and HPLC with ECD provides a method that is sensitive and specific for 7-meG and suitable for integration into molecular epidemiological studies.

Animals↗
PubMed1993

Decreased dimethylnitrosamine-induced O6- and N7-methyldeoxyguanosine levels correlate with development and progression of lesions in rat hepatocarcinogenesis.

Formation and repair of O6-medG and N7-medG (O6- and N7-methyldeoxyguanosine) in glutathione S-transferase-P form (GST-P)-positive liver cell foci, nodules, primary hepatocellular carcinoma (HCC) and transplanted hepatocellular carcinoma (TRP) induced by N-ethyl-N-hydroxyethylnitrosamine (EHEN) were immunohistochemically assessed following a single exposure to dimethylnitrosamine (DMN). Male Fischer 344 rats received a 0.1% solution of EHEN as their drinking water for 4 weeks and were maintained on basal diet until week 40, when a single 50 mg/kg body weight dose of DMN was administered intraperitoneally. Nude rats (NIH rnu/rnu) bearing TRP were similarly treated. Sequential killing 6, 12, 24, 48 and 72 h thereafter revealed significantly decreased indices of cells binding antibodies to O6-medG and N7-medG adducts in GST-P-positive foci and nodules, and particularly HCC and TRP, as compared to background parenchyma values. Similarly, differences between foci/nodules and HCC/TRP were also significant, indicating that decrease in adduct formation is associated with further malignant conversion. The rate of DNA adduct repair in foci and nodules subsequent to the peak found at the 12 h time-point did not appear to be significantly different from that in the surrounding tissue at the dose of DMN studied. The results indicate decreased formation of DMN-associated DNA damage, in line with the known metabolic profile of carcinogen-induced focal liver lesions.

Animals↗
PubMed1993

Influence of aging on multi-organ carcinogenesis in rats induced by N-methyl-N-nitrosourea.

The effects of aging on the multi-organ carcinogenesis induced in rats by N-methyl-N-nitrosourea (MNU), a direct carcinogen which does not need metabolic activation to exert carcinogenicity, were examined in male F344 rats. In the first experiment, rats at 6, 52, and 98 weeks of age were treated with MNU (20 mg/kg body weight, i.p.) twice weekly for 6 weeks and then maintained without any further treatment for 24 weeks in the case of young and middle-aged rats and for 18 weeks in the case of the old rats. In young rats, malignant lymphomas, particularly thymic types, were observed at significantly high incidence. A striking result in the middle-aged rats was the significantly higher incidence of adenocarcinomas in the small intestine than in young or old animals. The induction of proliferative and neoplastic lesions of the large intestine also tended to be increased in middle-aged rats. In addition, epithelial hyperplasia of the tongue, but not the forestomach, occurred at the highest incidence in the middle-aged group. There were no differences in the induction of epithelial lesions in the urinary bladder among the groups. In a second experiment, investigation of DNA synthesis in the tongue, small and large intestines, urinary bladder and lymph nodes did reveal significant increases or tendency for increase in the MNU-treated groups, but without differences with age. In contrast, the thymus of young rats showed significantly increased incorporation of BrdU label after administration of MNU, whereas it was markedly reduced in middle-aged rats. In a third experiment, O6-methyldeoxyguanine (O6-medG) DNA adduct formation was immunohistochemically detected in various organs including the thymus, forestomach, and small intestine without any differences with age. Thus, the results demonstrated that while the target organs of MNU are modified by the age of the animals, levels of DNA synthesis and O6-medG DNA adduct formation in most cases can not explain the observed differences in carcinogenic susceptibility.

Adenocarcinoma↗
PubMed1993

Retinal degeneration induced by N-methyl-N-nitrosourea and detection of 7-methyldeoxyguanosine in the rat retina.

Retinal degeneration induced by a single parenteral dose (40 mg) of N-methyl-N-nitrosourea (MNU) was studied in rats and rabbits. Sequential observations showed that severe pathological changes, that is, complete or widespread destruction of the photoreceptor layer and the outer nuclear layer of the retina, occurred in both animals by 72 hr after the administration of MNU. At week 5, at the end stage of the retinal degeneration induced by MNU, thin remnants remained with the inner nuclear layer in direct contact with the pigment epithelium. An additional immunohistochemistry study using antibodies against 7-methyldeoxyguanosine (7-mdGua), revealed the presence of high levels of 7-mdGua DNA adducts in the nuclei of the outer nuclear layer cells. This finding suggests that the alkylation of the outer nuclear layer by MNU was the direct cause of the retinal degeneration seen in these animals.

Animals↗
PubMed1993

Paracrine induction of angiogenesis in vitro by Swiss 3T3 fibroblasts.

During angiogenesis, normally quiescent endothelial cells that line existing microvessels are induced to invade the surrounding extracellular matrix and to form capillary sprouts in response to paracrine factors released by neighboring cell types. In an attempt to mimic the physiological conditions under which angiogenesis is triggered in vivo, we have designed two co-culture systems that are suitable for the study of paracrine interactions between microvascular endothelial cells and cell types that might produce angiogenic factors. In the first model, cells to be co-cultured with endothelial cells were suspended within a collagen gel and overlaid with an additional collagen gel devoid of cells, onto which bovine microvascular endothelial cells were subsequently seeded and grown to confluence. In the second model, a small collagen gel disc containing a suspension of endothelial cells was embedded into a larger, cell-free collagen gel disc, which in turn was surrounded by an annular collagen gel containing other cell types. We show that Swiss 3T3 fibroblasts and their 3T3-L1 substrain induce the formation of capillary-like tubes by endothelial cells in these co-culture systems, whereas Balb 3T3 cells, as well as a number of other fibroblasts and epithelial cells, lack this ability. Thus, endothelial cells grown on a collagen gel containing Swiss 3T3 or 3T3-L1 cells invaded the underlying matrix to form a network of interconnecting tubules. In addition, Swiss 3T3 and 3T3-L1 cells stimulated extensive radial outgrowth of tubular sprouts from the periphery of endothelial-cell-containing collagen discs. Conditioned medium from Swiss 3T3 cells mimicked the effect of co-culture by inducing formation of capillary-like tubes, and also increased plasminogen activator activity in endothelial cells. Conditioned medium from Balb 3T3 cells, in contrast, lacked these activities. Preliminary evidence suggests that the factor(s) in Swiss 3T3 conditioned medium that induces tubule formation by endothelial cells may be different from a number of well-characterized angiogenic cytokines. The co-culture systems described here should prove to be useful for the identification of physiological regulators of angiogenesis produced by various cell types.

3T3 Cells↗
PubMed1993

Molecular dosimetry of aflatoxin exposure: contribution to understanding the multifactorial etiopathogenesis of primary hepatocellular carcinoma with particular reference to hepatitis B virus.

Aflatoxin exposure and hepatitis B virus infection have been implicated as major risk factors for primary hepatocellular carcinoma (PHC) in high-incidence regions of the world. Investigations using the assay of aflatoxin bound to peripheral blood albumin have shown that exposure can occur throughout the life span of the individual, including during the perinatal period, in high-incidence areas such as The Gambia, Senegal, Kenya, and The People's Republic of China. The possibility of measuring aflatoxin exposure at the individual level permits an investigation of the putative mechanisms of interaction of this carcinogen with HBV in the etiopathogenesis of PHC. Animal models, e.g., Pekin duck and HBV-transgenic mice, have also been used to study these questions, and the available data are reviewed.

Aflatoxin B1↗
PubMed1992

Potent synergism between vascular endothelial growth factor and basic fibroblast growth factor in the induction of angiogenesis in vitro.

Vascular endothelial growth factor (VEGF), also known as vascular permeability factor or vasculotropin, is a recently characterized endothelial-specific mitogen which is angiogenic in vivo. Here we demonstrate that VEGF is angiogenic in vitro: when added to microvascular endothelial cells grown on the surface of three-dimensional collagen gels, VEGF induces the cells to invade the underlying matrix and to form capillary-like tubules, with an optimal effect at approximately 2.2nM (100ng/ml). When compared to basic fibroblast growth factor (bFGF) at equimolar (0.5nM) concentrations, VEGF was about half as potent. The most striking effect was seen in combination with bFGF: when added simultaneously, VEGF and bFGF induced an in vitro angiogenic response which was far greater than additive, and which occurred with greater rapidity than the response to either cytokine alone. These results demonstrate that like bFGF, VEGF induces an angiogenic response via a direct effect on endothelial cells, and that by acting in concert, these two cytokines have a potent synergistic effect on the induction of angiogenesis in vitro. We suggest that the synergism between VEGF and bFGF plays an important role in the control of angiogenesis in vivo.

Adrenal Cortex↗
PubMed1992

Hepatocyte growth factor increases urokinase-type plasminogen activator (u-PA) and u-PA receptor expression in Madin-Darby canine kidney epithelial cells.

We have recently demonstrated that fibroblast-conditioned medium induces Madin-Darby canine kidney (MDCK) epithelial cells to form branching tubules when grown in three-dimensional collagen or fibrin gels (Montesano, R., Schaller, G., and Orci, L. (1991) Cell 66, 697-711), and that this morphogenetic effect is mediated by hepatocyte growth factor (HGF), also known as scatter factor (Montesano, R., Matsumoto, K., Nakamura, T., and Orci, L. (1991) Cell 67, 901-908). In fibrin gels, this effect is inhibited by addition of exogenous serine protease inhibitors, which suggests a role for plasminogen activators (PAs) in the matrix remodeling required for tubulogenesis. In the studies reported in this paper, we have investigated the effect of fibroblast-conditioned medium (CM) and HGF on the production of PAs by MDCK cells. We have found that urokinase-type PA (u-PA) activity and mRNA are increased 4.9-fold by CM from human MRC-5 fibroblasts, which has tubulogenic activity, but not by CM from human Detroit-550 fibroblasts, which lacks tubulogenic activity. The u-PA inductive property of MRC-5 CM was completely inhibited by preincubation with antibodies to recombinant human HGF (rhHGF). Exogenously added rhHGF also increased u-PA activity and mRNA 5.9-fold in MDCK cells, with an optimal effect at approximately 10 ng/ml. MRC-5 CM also increased u-PA receptor mRNA 34.9-fold in MDCK cells, an effect which was inhibited by 71% by preincubating the CM with antibodies to rhHGF, and which was mimicked by exogenously added rhHGF (31.3-fold increase). These results demonstrate that HGF, which induces tubulogenesis by MDCK cells in vitro, also increases u-PA and u-PA receptor expression in these cells. Taken together with our previous observations, this suggests that the resulting increase in extracellular proteolysis, appropriately localized to the cell surface, is required for epithelial morphogenesis.

Animals↗
PubMed1992

Plasminogen activator inhibitor-1 is induced in migrating endothelial cells.

It has been proposed that a finely tuned protease-anti-protease equilibrium must be maintained during processes of cell migration in order to limit extracellular proteolysis to the close proximity of the cell surface, and thereby to prevent excessive extracellular matrix degradation. We have previously shown that urokinase-type plasminogen activator (u-PA) activity is induced in microvascular endothelial cells migrating from the edges of a wounded monolayer in vitro (Pepper et al., J. Cell Biol., 105:2535-2541, 1987). By Northern analysis, we now demonstrate that plasminogen activator inhibitor 1 (PAI-1) mRNA is increased in multiple-wounded monolayers of bovine microvascular (BME) or aortic (BAE) endothelial cells, with a maximal 7- and 9-fold increase 4 h after wounding, respectively. By in situ hybridization, we demonstrate that the increase in PAI-1 mRNA is localized to cells at the edge of a wounded BME or BAE cell monolayer. The increase in PAI-1 mRNA observed in BME cells is independent of cell division and is inhibited by antibodies to basic fibroblast growth factor (bFGF), suggesting that PAI-1 induction in migrating cells is mediated by the autocrine activity of bFGF. Taken together with our previous observations on the induction of u-PA, these results support the hypothesis that the proteolytic balance in the pericellular environment of migrating cells is regulated through the concomitant production of proteases and protease inhibitors.

Animals↗