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S Pepe

Publications and source records attributed to S Pepe.

At least 55 records · Page 3Linked to original sources
PubMed1997

'Cross talk' between opioid peptide and adrenergic receptor signaling in isolated rat heart.

BACKGROUND: Cardiac myocyte sarcolemma contains both catecholamine and opioid peptide receptors (OPRs). Opioid peptides are coreleased with catecholamines from nerve terminals in the heart. We investigated whether OPR stimulation influences the effects of beta-adrenergic receptor (beta-AR) stimulation in the isolated, isovolumic rat heart and whether the mechanism of such an interaction involves both beta-AR subtypes or an alteration in beta-AR-mediated increase in cAMP. METHODS AND RESULTS: Norepinephrine (NE, 10(-7) mol/L) increased peak left ventricular systolic pressure (LVSP) and cAMP more than twofold compared with controls. The delta-OPR agonist leucine-enkephalin (LE, 10(-8) mol/L) markedly inhibited the beta1-AR-induced positive inotropic effect and increase in cAMP but alone had no effect on basal LVSP or basal cAMP levels. The OPR antagonist naloxone 10(-8) mol/L added to LE+NE perfusate reversed the LE-induced decrease in cAMP and LVSP even though naloxone alone had no effect on LVSP and cAMP levels. LE could not counteract the twofold increase in LVSP produced by the nondegradable cAMP analog CPT-cAMP 2.3x10(-5) mol/L or a high concentration of forskolin (10(-7) mol/L) but did reverse the 173+/-11.8% and 135+/-13.6% increases in LVSP stimulated by 10(-8) and 0.5x10(-8) mol/L forskolin, respectively. LE inhibited cAMP production at all concentrations of forskolin (10(-7), 10(-8), and 0.5x10(-8) mol/L). Pertussis toxin (PTX) pretreatment abolished LE effects on beta1-AR stimulation. Zinterol 10(-5) and 10(-6) mol/L, a specific beta2-AR agonist that elicits a cAMP-independent inotropic effect in rat heart, caused 225+/-14% and 182+/-5% increases in LVSP that could not be reversed by addition of LE. CONCLUSIONS: Potent, inhibitory "cross talk" between delta-OPR and beta1-AR signaling pathways occurs via a PTX-sensitive G(i/o) protein involved in adenylyl cyclase inhibition in rat heart.

Adenylate Cyclase Toxin↗
PubMed1997

Opioid peptide receptor stimulation reverses beta-adrenergic effects in rat heart cells.

Opioid peptide receptor (OPR) agonists are co-released with the beta-adrenergic receptor (beta-AR) agonist norepinephrine (NE) from nerve terminals in the heart during sympathetic stimulation. Whereas recent studies indicate that OPR and beta-AR coexist on the surface of cardiac myocytes, whether significant "cross talk" occurs between OPR and beta-AR signaling cascades within heart cells is unknown. In the present study we demonstrate a marked effect of delta-OPR stimulation to modulate beta-adrenergic responses in single isolated rat ventricular myocytes. Nanomolar concentrations (10(-8) M) of the OPR agonist leucine enkephalin (LE), a naturally occurring delta-opioid peptide, inhibited NE-induced increases in sarcolemmal L-type Ca2+ current, cytosolic Ca2+ transient, and contraction. The antiadrenergic effect of LE was pertussis toxin sensitive and abolished by naloxone, an opioid receptor antagonist. In contrast, LE was unable to inhibit the positive inotropic effects induced by equipotent concentrations of 8-(4 chlorophenylthio)-adenosine 3',5'-cyclic monophosphate, a cell-permeant adenosine 3',5'-cyclic monophosphate analog, or by the non-receptor-induced increase in contraction by elevated bathing Ca2+ concentration. These results indicate that an interaction of the OPR and beta-AR systems occurs proximal to activation of the adenosine 3',5'-cyclic monophosphate-dependent protein kinase of the beta-AR intracellular signaling pathway. This modulation of beta-adrenergic effects by OPR activation at the myocyte level may have important implications in the regulation of cardiac Ca2+ metabolism and contractility, particularly during the myocardial response to stress.

Adrenergic beta-Agonists↗
PubMed1996

DNA flow cytometry in patients undergoing liver transplantation for hepatocellular carcinoma.

The purpose of the study was to analyse patterns of DNA content in hepatocellular carcinomas (HCC) submitted to orthotopic liver transplantation (OLT). Paraffin-embedded archival material from 15 patients (ten men, five women, mean age 51 +/- 1.78 years) transplanted in St-Roch Hospital between 1988 and 1991 was available for laboratory evaluation by flow cytometry. Five out of 15 were incidental HCC. The analysis was performed by a FACSscan flow cytometer coupled to a Hewlett-Packard computer. The cellular DNA content was defined as diploid or aneuploid in the presence of a single (DNA index of 1) or two distinct (DNA index different from 1) Gzero/G1 peaks, respectively. All incidental HCC (five patients) were diploid, the tumour size was 1.2 +/- 0.2 cm, the number of nodules was 1.4 +/- 0.24 and the mortality rate was 40%. No death in the incidental HCC group was related to neoplastic recurrence. In the remaining ten patients transplanted for HCC, we observed 50% diploid tumours, the tumour size was 5.2 +/- 1.55 cm and the number of nodules was 2.7 +/- 0.56. In this group six patients died of neoplastic recurrence (two were diploid and four aneuploid). The diameter of the neoplasm in diploid patients who died of neoplastic recurrence was over 5 cm and the number of nodules was over three. Moreover, in aneuploid patients who died of neoplastic recurrence, the diameter of the neoplasm was less than 5 cm in three cases and the number of nodules was less than three in two patients. This study indicates that incidental HCC may be a less aggressive malignancy and may have a better prognosis. In this group, no patient recurred after OLT and all tumours were diploid. Aneuploidy, tumour size (> 5 cm) and number of lesions (> 3) are prognostic indicators for neoplastic recurrence in patients transplanted for hepatocellular carcinoma.

Adult↗
PubMed1996

Dietary fish oil confers direct antiarrhythmic properties on the myocardium of rats.

This study tested the hypothesis that in vivo antiarrhythmic effects of dietary fish oil can be attributed directly to changes in myocardial properties. Sixty adult male rats were fed a fish oil diet (FO), an isoenergetic saturated fat diet (SAT) or a low fat reference diet (REF) for 16 wk. hearts isolated from these rats were perfused with washed porcine erythrocytes (0.4 hematocrit) in working heart mode. Dietary fish oil prevented reperfusion-induced ventricular fibrillation (VF) (% of rats with VF: REF 50%, SAT 80% P = 0.35, FO 0% P < 0.05 n = 10) and reduced arrhythmias in ischemia. In a separate set of hearts from rats fed the three diets, FO increased while SAT reduced the stimulation threshold for programmed electrical induction of VF during control perfusion compared with REF (mean +/- SD: REF 7.1 +/- 0.2 mA; SAT 5.8 +/- 0.2 mA, P < 0.001; FO 15.1 +/- 1.0 mA, P < 0.001, n = 10) and during subsequent ischemia (REF 5.9 +/- 0.2 mA; SAT 3.8 +/- 0.3 mA, P < 0.001; FO 8.9 +/- 0.2 mA, P < 0.001, n = 10). The isolated working heart model used physiological workload and oxygenation but excluded extracardiac influences. Dietary fish oil prevented the initiation and reduced the severity of arrhythmias in the isolated hearts in response to a variety of stimuli. These results establish that irrespective of any effects on blood pressure or platelet function in vivo, dietary fish oil directly affects myocardial properties which may contribute to observed clinical reductions in cardiac mortality associated with fish consumption.

Animals↗
PubMed1996

Differential sensitivity to non-major histocompatibility complex-restricted recombinant interleukin 2-activated lymphocyte killing of human mammary epithelial MCF-10A cells overexpressing oncogenes or protein kinase A subunits.

The sensitivity of human tumor cells to activated lymphocytes is considered to play an essential role in the antitumor activity of recombinant interleukin-2 (rIL-2)-based immunotherapy. We have investigated the effects of several genes involved in the regulation of cell growth and transformation on the sensitivity of human mammary epithelial MCF-10A cells to non-MHC-restricted, rIL-2-activated lymphocytes. Therefore, the lysability of MCF-10A cells overexpressing activated oncogenes (Ha-ras, erbB-2, and a mutated p53), growth factors [transforming growth factor alpha (TGFalpha)], or cAMP-dependent protein kinase A subunits (RIalpha, RIIbeta, and Calpha) was evaluated comparatively at different effector:target ratios by a 51Cr release assay. Parental MCF-10A, MCF-10A p53-mutated, and MCF-10A RIIbeta cells showed an intermediate sensitivity. Lysability was increased significantly in MCF-10A Ha-ras, MCF-10A TGFalpha, and MCF-10A RIalpha cells, reduced in MCF-10A Calpha cells, and completely abrogated in MCF-10A erbB-2 cells. These differences could not be explained by simple changes in the cell surface expression of MHC class I and intercellular adhesion molecule-1 proteins or by secretion of TGFbeta. Treatment with TAb 250, a mouse anti-p185(erbB-2) monoclonal antibody, or down-regulation of p185(erbB-2) expression resulted in circumvention of MCF-10A erbB-2 cell resistance. We conclude that molecular changes at the single-gene level resulting in alterations of intracellular signaling and/or cell transformation modulate sensitivity of human mammary epithelial cells to non-MHC-restricted, rIL-2-induced cytotoxicity, regardless of MHC class I and/or intercellular adhesion molecule-1 expression or TGFbeta secretion. Furthermore, anti-p185(erbB-2) monoclonal antibodies may be useful as adjuncts to rIL-2 treatment in patients with erbB-2-overexpressing tumors.

Animals↗
PubMed1995

NM23 gene expression correlates with cell growth rate and S-phase.

Two human NM23 genes have been identified: NM23.H1 and NM23.H2 coding for the A and B subunit of a nucleoside diphosphate kinase (NDPK), respectively. NM23.H1 gene has been proposed as a suppressor of metastatic ability in tumor cells, NM23.H2 is identical to the c-myc transcription factor, PuF. The NM23 coding sequence is strongly preserved through different species. Indirect evidence of various types has been accumulated and seems to support an implication of NM23 in cell proliferation. This report shows that the NM23 gene expression is strictly related to the growth state of the cells. Two different in vitro systems (human peripheral blood lymphocytes and human breast epithelial cell line MCF-10A) and one in vivo (human primary infiltrating ductal breast carcinomas) system have been investigated. The mRNA is present in PHA-stimulated peripheral blood lymphocytes, whereas it is nearly undetectable in their resting counterparts. The level of the NM23 gene expression parallels the fraction of cells incorporating thymidine (S-phase) in neoplastic mammary tissues. In synchronously cycling MCF-10A cells NM23.H1 mRNA reaches a maximum abundance in the S-phase and is absent or only present at very low levels during G0/G1 phase, whereas NM23.H2 is present in growth-arrested cells but is upregulated following serum growth stimulation.

Breast↗
PubMed1995

Epidermal growth factor receptor (EGFr) expression in non-small cell lung carcinomas correlates with metastatic involvement of hilar and mediastinal lymph nodes in the squamous subtype.

Epidermal growth factor receptor (EGFr) levels were evaluated in paraffin-embedded tumour specimens of non-small cell lung cancer (NSCLC) from 176 patients who underwent surgical resection. The EGFr expression was evaluated by immunocytochemical assay using a monoclonal antibody which recognises the external domain of the receptor. EGFr immunoreactivity was significantly higher in squamous than in non-squamous cell carcinomas (P = 0.0009). Hilar and/or mediastinal nodal involvement was found in 29 of 105 (27.4%) squamous cancers, and in this group of patients, the mean of EGFr positive cells was significantly higher than that of patients without nodal involvement (P = 0.01). No significant correlations were found between the expression of EGFr and other clinicopathological or biological parameters such as T-status, grading, proliferative activity. EGFR is suggested to represent a useful indicator of nodal metastasis in NSCLC.

Adenocarcinoma↗
PubMed1995

Bryostatin 1 enhances lymphokine activated killer sensitivity and modulates the beta 1 integrin profile of cultured human tumor cells.

Bryostatin 1 interferes with protein kinase C (PKC) signaling which is involved in the activation of human and murine cytotoxic T lymphocytes, and in the growth and differentiation of tumor cells. Bryostatin 1 has immunomodulating and antitumor properties as demonstrated by preclinical and clinical studies. Here we report that bryostatin 1 increases the susceptibility to lymphokine activated killers and modifies the pattern of beta 1 integrin expression of human tumor cells. On the basis of these results the use of bryostatin 1 in combination with immunostimulating cytokines such as interleukin-2 in the treatment of human cancer is suggested.

Adjuvants, Immunologic↗
PubMed1995

[Current clinical interest in monitoring digoxinemia].

After a short introduction about the current role of digitalis in the treatment of the supraventrical arrhythmias and about the factors that make often problematic the achievement of an optimal posology of the drug, the results relative to more recent 340 digoxinaemia determinations in patients of Policlinico in Palermo or in outpatients are presented. Just the 43.8% of the patients had a digoxinaemia value in the range considered therapeutic; just 45 patients (32.1%), out of the 140 in which the digoxinaemia had been monitored for, at least, 5 days, were in the therapeutic range at the first determination; the 47.8% of the patients were underdosed and the 38.8% of them showed higher values than the therapeutic range. Determination 5 or more days later showed digoxinaemia values in the therapeutic range in 112 patients (80%). According to the reported results, it may be presumed that the posology correction effectuated by the physician on these patients might have been driven by the digoxinaemia values, whose determination must be considered an unavoidable guide to the digitalis treatment.

Anti-Arrhythmia Agents↗
PubMed1995

Cyclic AMP-dependent protein kinase type I is involved in hypersensitivity of human breast cells to topoisomerase II inhibitors.

Topoisomerase II (Topo II) is an essential enzyme that catalyzes the breakage of double-strand DNA and is the target of several effective anticancer drugs, including the epipodophyllotoxins. The regulatory subunits of the cyclic AMP-dependent protein kinase are differentially expressed in normal and cancer cells. The RIalpha subunit is overexpressed in cells transformed by transforming growth factor-alpha (TGF-alpha) or Ha-ras oncogene. It has been shown that murine cells transformed by Ha-ras become hypersensitive to Topo II-targeting anticancer drugs. In this report we have tested whether any correlation exists between the expression of RIalpha protein and cellular sensitivity of Topo II-targeting drugs. Normal human breast MCF-10A cells and their derivatives overexpressing TGF-alpha, Ha-ras, or the different protein kinase subunits were treated with either Topo II inhibitors, such as etoposide, teniposide, or amsacrine, or with drugs which act independently of Topo II, such as bleomycin. Here we show that MCF-10A TGF-alpha and MCF-10A Ha-ras cells overexpress the RIalpha protein and become hypersensitive to epypodophyllotoxins and amsacrine but not to bleomycin. Direct introduction of the RIalpha gene into MCF-10A induces hypersensitivity to Topo II inhibitor drugs. In contrast, the overexpression of the other protein kinase subunits, RIIbeta or Calpha, does not modify the drug sensitivity of MCF-10A cells. No differences in the mRNA/protein content or in the activity of Topo II were found between hypersensitive cells and parental MCF-10A cells, suggesting that RIalpha may influence drug sensitivity via modulation of events downstream of the Topo II-DNA cleavable complex.

Amsacrine↗
PubMed1995

Phase I clinical study with 8-chloro-cAMP and evaluation of immunological effects in cancer patients

The site-selective cyclic AMP analogue 8-chloro-cAMP (8-Cl-cAMP) is able to inhibit the growth of a wide variety of cancer cell lines in vitro and in vivo. 8-Cl-cAMP has been extensively investigated as a new potential anticancer agent and, more recently, preclinical Phase I studies have been conducted in animal models to study its toxicity. We have conducted the first Phase I trial with 8-Cl-cAMP to define the maximum tolerated dose, toxicity, plasma drug levels, and immunological effects in patients with cancers refractory to standard treatments. We have administered 36 courses of 8-Cl-cAMP to 17 patients by continous i.v. infusion of the drug for 5 days/week for 2 weeks followed by a 1-week rest period. Six increasing dose levels, from 0.01 to 0.25 mg/kg/h, were explored. Drug plasma levels were determined and the expression of interleukin 2 receptor alpha amount of natural killer cells, and cytolytic activity against K562 cells were measured in peripheral blood lymphocytes. A grade 4 and a grade 3 increase in serum creatinine and a grade 2 increase in blood urea nitrogen observed in two patients were the dose-limiting toxicity. The maximum tolerated dose (0.2 mg/kg/h) determined a grade 1 increase in serum creatinine. An increase in calcium levels was observed in several patients. The 8-Cl-cAMP plasma concentrations obtained at the steady state were in the range previously shown to be effective for cancer cell growth inhibition in vitro. Interleukin 2 receptor alpha expression, natural killer cell number, and cytolytic activity from peripheral blood lymphocytes were markedly increased after 8-Cl-cAMP administration at all dose levels. In conclusion, at doses below the maximum tolerated dose, 8-Cl-cAMP was not toxic but reached plasma concentrations in the potential therapeutic range for growth inhibition. Moreover, 8-Cl-cAMP determined a marked biomodulatory effect and showed antitumor activity.

Journal Article↗
PubMed1994

Differential effects of protein kinase A sub-units on Chinese-hamster-ovary cell cycle and proliferation.

It has been shown that a marked increase in the levels of RI alpha sub-units and a decrease in RII beta sub-unit levels correlate with neoplastic transformation or with the mitogenic response of normal cells to hormones and growth factors. The selective down-regulation of RI alpha and the following induction of RII beta determine cell-growth arrest and differentiation of several cancer cells. To directly address the question whether the 2 protein-kinase-A(PKA) isoforms play different roles in the control of proliferation and cell-cycle distribution, we introduced and over-expressed the different PKA sub-units in Chinese-hamster-ovary (CHO) cells via retroviral-vector-mediated gene transfer. Whereas CHO cells treated with RI alpha anti-sense oligodeoxynucleotides were growth arrested and accumulated in the G0/G1 phases of the cell cycle, infection of CHO cells with a retroviral vector in order to over-express RI alpha determined growth advantages in monolayer conditions and substantially increased their cloning efficiency in soft agar. These events correlated with a sustained percentage of cells in S phase induced by RI alpha over-expression in the infected cells. In contrast, CHO cells infected with retroviral vectors over-expressing either a RII beta sub-unit or a C alpha catalytic sub-unit of PKA exhibited growth arrest within a few days of culture and accumulated in the G2-M phase of the cell cycle. The results of our study demonstrate that the different PKA sub-units play different and specific roles in the control of cell growth and cell-cycle distribution.

Animals↗
PubMed1994

Omega 3 polyunsaturated fatty acid modulates dihydropyridine effects on L-type Ca2+ channels, cytosolic Ca2+, and contraction in adult rat cardiac myocytes.

The effect of docosahexaenoic acid (DHA; C22:6) on dihydropyridine (DHP) interaction with L-type Ca2+ channel current (ICa), cytosolic Ca2+ (Cai), and cell contraction in isolated adult rat cardiac myocytes was studied. The DHP L-type Ca(2+)-channel blocker nitrendipine (10 nM) reduced peak ICa (measured by whole-cell voltage clamp from -45 to 0 mV) and reduced the amplitude of the Ca2+ transient (measured as the transient in indo-1 fluorescence, 410/490 nm) and the twitch amplitude (measured via photodiode array) during steady-state electrical stimulation (0.5 Hz). The DHP L-type Ca2+ channel agonist BAY K 8644 (10 nM) significantly increased ICa, the amplitude of the Cai transient, and contraction. When cells were exposed to DHA (5 microM) simultaneously with either BAY K 8644 or nitrendipine, the drug effects were abolished. Arachidonic acid (C20:4) at 5 microM did not block the inhibitory effects of nitrendipine nor did it prevent the potentiating effects of BAY K 8644. DHA modulation of DHP action could be reversed by cell perfusion with fatty acid-free bovine serum albumin at 1 mg/ml. Neither DHA nor arachidonic acid alone (5 microM) had any apparent effect on the parameters measured. DHA (5 microM) had no influence over beta-adrenergic receptor stimulation (isoproterenol, 0.01-1 microM)-induced increases in ICa, Cai, or contraction. The findings that DHA inhibits the effect of DHP agonists and antagonists on Ca(2+)-channel current but has no effect alone or on beta-adrenergic-induced increases in ICa suggests that DHA specifically binds to Ca2+ channels at or near DHP binding sites and interferes with ICa modulation.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed1994

Overexpression of the RI alpha subunit of protein kinase A confers hypersensitivity to topoisomerase II inhibitors and 8-chloro-cyclic adenosine 3'5'-monophosphate in Chinese hamster ovary cells.

We have shown that a mutant derivative of Chinese hamster ovary CHO-K1 cells, ADR-5, which shows hypersensitivity to topoisomerase II (topo II)-inhibitory drugs, is cross-sensitive to the site-selective cyclic AMP analogue 8-chloro-cyclic AMP. We tested the hypothesis that overexpression of the type I alpha regulatory subunit of protein kinase A may represent a common element conferring hypersensitivity to both topo II inhibitors and 8-chloro-cyclic AMP in ADR-5 cells. We have demonstrated that ADR-5 cells overexpress RI alpha protein, compared to parental CHO-K1 cells. Moreover, retroviral vector-mediated transfer of the RI alpha gene into CHO-K1 cells was able to confer a drug-hypersensitive phenotype similar to that exhibited by ADR-5 cells. Analysis of topo II protein levels and activity revealed no differences between parental and infected cells, suggesting that protein kinase A may be involved in the downstream processing of topo II-mediated events.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1994

Flow-cytometric detection of the RI alpha subunit of type I cAMP-dependent protein kinase in human cells.

cAMP-dependent protein kinase (PKA) is composed of two genetically distinct catalytic (C) and regulatory (R) subunits. There are two different classes of PKA, designated as type I and type II, which contain distinct R subunits (RI or RII, respectively) but share a common C subunit. Enhanced expression of type I PKA has been correlated with cell proliferation and neoplastic transformation. Detection of the different PKA subunits is usually performed by photoaffinity labeling with 8-N3-32P-cAMP or by radioimmunolabeling techniques. Both techniques are time consuming and require a high number of cells and the use of radioactive reagents. Using the MCF-10A normal human mammary cell line infected with a recombinant retroviral vector containing the human RI alpha gene (MCF-10A RI alpha), we have developed a flow-cytometric assay to detect the intracellular content of RI alpha protein in human cells. MCF-10A and MCF-10A RI alpha cells were fixed in 1.5% paraformaldehyde at 37 degrees C for 15 min and permeabilized by methanol and acetone (1:1) at -20 degrees C for 5 min before staining with a specific IgG2a MoAb followed by a FITC-conjugate rabbit-anti mouse IgG. This procedure was also successfully utilized to recognize RI alpha protein content in human peripheral blood lymphocytes. Flow-cytometric detection of the RI alpha subunit in human cells is feasible and allows the study of the role of type I PKA in cell growth and neoplastic transformation.

Breast↗