Alterations of ubiquitin ligases in human cancer and their association with the natural history of the tumor.

Protein ubiquitination is critical for many cellular processes, through its ability to regulate protein degradation and various signaling mechanisms. In the ubiquitin (Ub) system, substrate specificity is achieved through the E3 family of Ub ligases. Because alterations of the ubiquitination machinery have been reported in human cancers, the selective interference with Ub ligases might represent a powerful therapeutic tool. Here, we report the first wide survey of misregulation of Ub ligases in cancer. We analysed 82 Ub ligases in nine types of cancer by in situ hybridization on tissue microarrays. We found 27 instances in which an Ub ligase was altered in a given type of tumor, when compared with normal tissues: 21 cases of overexpression and 6 cases of underexpression. We further analysed selected Ub ligases in large cohorts of breast and non-small-cell lung carcinomas. In five, of six, of these extended analyses (HUWE1, CCNB1IP1, SIAH1 and SIAH2 in breast cancer and CCNB1IP1 in lung cancer), we found that the levels of Ub ligases correlated significantly with relevant prognostic factors, and with clinical outcome. Our findings show that the alteration of Ub ligases is a frequent event in cancer and identify candidate targets for molecular therapies.

Breast cancer metastases are molecularly distinct from their primary tumors.

Metastases have been widely thought to arise from rare, selected, mutation-bearing cells in the primary tumor. Recently, however, it has been proposed that breast tumors are imprinted ab initio with metastatic ability. Thus, there is a debate over whether 'phenotypic' disease progression is really associated with 'molecular' progression. We profiled 26 matched primary breast tumors and lymph node metastases and identified 270 probesets that could discriminate between the two categories. We then used an independent cohort of breast tumors (81 samples) and unmatched distant metastases (32 samples) to validate and refine this list down to a 126-probeset list. A representative subset of these genes was subjected to analysis by in situ hybridization, on a third independent cohort (57 primary breast tumors and matched lymph node metastases). This not only confirmed the expression profile data, but also allowed us to establish the cellular origin of the signals. One-third of the analysed representative genes (4 of 11) were expressed by the epithelial component. The four epithelial genes alone were able to discriminate primary breast tumors from their metastases. Finally, engineered alterations in the expression of two of the epithelial genes (SERPINB5 and LTF) modified cell motility in vitro, in accordance with a possible causal role in metastasis. Our results show that breast cancer metastases are molecularly distinct from their primary tumors.

Gene expression analysis of early and advanced gastric cancers.

Gastric carcinoma is one of the major causes of cancer mortality worldwide. Early detection results in excellent prognosis for patients with early cancer (EGC), whereas the prognosis of advanced cancer (AGC) patients remains poor. It is not clear whether EGC and AGC are molecularly distinct, and whether they represent progressive stages of the same tumor or different entities ab initio. Gene expression profiles of EGC and AGC were determined by Affymetrix technology and quantitative polymerase chain reaction. Representative regulated genes were further analysed by in situ hybridization (ISH) on tissue microarrays. Expression analysis allowed the identification of a signature that differentiates AGC from EGC. In addition, comparison with normal gastric mucosa indicated that the majority of alterations associated with EGC are retained in AGC, and that further expression changes mark the transition from EGC to AGC. Finally, ISH analysis showed that representative genes, differentially expressed in the invasive areas of EGC and AGC, are not differentially expressed in the non-invasive areas of the same tumors. Our data are more directly compatible with a progression model of gastric carcinogenesis, whereby EGC and AGC may represent different molecular stages of the same tumor. Finally, the identification of an AGC-specific signature might help devising novel therapeutic strategies for advanced gastric cancer.

[Erectile disfunction after non nerve-sparing radical pelvic surgery]. / Disfunzione erettiva da chirurgia pelvica demolitiva non nerve-sparing.

BACKGROUND: The patients who undergo radical pelvic surgery often found that sexual function is impaired. In this research hypothesis, we evaluated the efficacy of alternative therapy to conventional PGE 1 injections, such as the association of Sildenafil and L-Arginine. This association in based on the principle that L-Arginine, the precursor of nitric oxide, improves the effect of Sildenafil, which is effective in the presence of nitric oxide. METHODS: The experimental plan was to make a comparative study of 2 random groups of patients selected from those undergoing radical cystectomies and prostatectomies over the past three years. 116 patients were illegible (64 prostatectomies and 52 cystectomies). The fìrst random group was treated with Sildenafil alone and the second with Sildenafil and L-Arginine. The efficacy of treatment was evaluated by the Buckling test (pressure threshold of cavernous flexation at penile axial rigidity) once-after ambulatorial administration and then by telephonic interview (subjective evaluation) after home administration. RESULTS: The starter dose was 50 mg and was inefficient in both groups (Buckling test between 0 and 250). 100-mg doses gave significant results (Buckling test > 500) in both groups, especially the second. Cardiopathic patients, diabetics and patients with retinal disorders were excluded from the study. The mean age of patients was 65 years. CONCLUSIONS: The resumption of relatively satisfactory sexual activity was demonstrated using non-invasive pharmacological treatment.

[Erectile dysfunction after non-nerve sparing radical pelvic surgery. Therapeutical experience with sildenafil and L-arginine evaluated by Buckling test]. / Disfunzione erettiva da chirurgia pelvica demolitiva non nerve-sparing. Esperienza terapeutica con sildenafil e L-Aginina valutata con Buckling test.

BACKGROUND: Patients undergoing radical pelvic floor surgery are often find that sexual function is impaired. In this research hypothesis, we evaluated the efficacy of alternative therapy to conventional PGE 1 injections, such as the association of Sildenafil and L-Arginine. This association is based on the principle that L-Arginine, the precursor of nitric oxide, improves the effect of Sildenafil, which is effective in the presence of nitric oxide. METHODS: The experimental plan was to make a comparative study between 2 random groups of patients selected from those undergoing radical cystectomies and prostatectomies over the past three years. 116 patients were eligible (64 prostatectomies and 52 cystectomies). The first random group was treated with Sildenafil alone and the second with Sildenafil and L-Arginine. The efficacy of treatment was evaluated using the Buckling test (pressure threshold of cavernous flexation at penile axial rigidity) once after ambulatorial administration and then by telephone interview (subjective evaluation) after administration at domicile. RESULTS: The starter dose was 50 mg and was inefficient in both groups (Buckling test between 0 and 250). 100-mg doses gave significant results (Buckling test >500) in both groups, especially the second. Cardiopathic patients, diabetics and patients with retinal disorders or who were unmotivated were excluded from the study. The mean age of patients was 65. CONCLUSIONS: The resumption of relatively satisfactory sexual activity was demonstrated using non-invasive pharmacological treatment.

Evolution of Shc functions from nematode to human.

The Shc protein family is characterized by the (CH2)-PTB-CH1-SH2 modularity. Its complexity increased during evolution from one locus in Drosophila (dShc), to at least three loci in mammals (shc, rai and sli). The three mammalian loci encode, because of alternative initiation codon usage and splicing pattern, at least six Shc-like proteins. Genetic and biological evidence indicates that the mammalian Shc isoforms regulate functions as diverse as growth (p52/p46Shc), apoptosis (p66Shc) and life-span (p66Shc). Available structure-function data and analysis of sequence similarities of Shc-like genes and proteins suggest complex diversification of Shc functions during evolution. Notably, Ras activation, the best-characterized Shc activity, appears to be a recent evolutionary acquisition.

Tyrosine phosphorylation of Eps15 is required for ligand-regulated, but not constitutive, endocytosis.

Membrane receptors are internalized either constitutively or upon ligand engagement. Whereas there is evidence for differential regulation of the two processes, little is known about the molecular machinery involved. Previous studies have shown that an unidentified kinase substrate is required for endocytosis of the epidermal growth factor receptor (EGFR), the prototypical ligand-inducible receptor, but not of the transferrin receptor (TfR), the prototypical constitutively internalized receptor. Eps15, an endocytic protein that is tyrosine phosphorylated by EGFR, is a candidate for such a function. Here, we show that tyrosine phosphorylation of Eps15 is necessary for internalization of the EGFR, but not of the TfR. We mapped Tyr 850 as the major in vivo tyrosine phosphorylation site of Eps15. A phosphorylation-negative mutant of Eps15 acted as a dominant negative on the internalization of the EGFR, but not of the TfR. A phosphopeptide, corresponding to the phosphorylated sequence of Eps15, inhibited EGFR endocytosis, suggesting that phosphotyrosine in Eps15 serves as a docking site for a phosphotyrosine binding protein. Thus, tyrosine phosphorylation of Eps15 represents the first molecular determinant, other than those contained in the receptors themselves, which is involved in the differential regulation of constitutive vs. regulated endocytosis.

Eps15 is recruited to the plasma membrane upon epidermal growth factor receptor activation and localizes to components of the endocytic pathway during receptor internalization.

Eps15 is a substrate for the tyrosine kinase of the epidermal growth factor receptor (EGFR) and is characterized by the presence of a novel protein:protein interaction domain, the EH domain. Eps15 also stably binds the clathrin adaptor protein complex AP-2. Previous work demonstrated an essential role for eps15 in receptor-mediated endocytosis. In this study we show that, upon activation of the EGFR kinase, eps15 undergoes dramatic relocalization consisting of 1) initial relocalization to the plasma membrane and 2) subsequent colocalization with the EGFR in various intracellular compartments of the endocytic pathway, with the notable exclusion of coated vesicles. Relocalization of eps15 is independent of its binding to the EGFR or of binding of the receptor to AP-2. Furthermore, eps15 appears to undergo tyrosine phosphorylation both at the plasma membrane and in a nocodazole-sensitive compartment, suggesting sustained phosphorylation in endocytic compartments. Our results are consistent with a model in which eps15 undergoes cycles of association:dissociation with membranes and suggest multiple roles for this protein in the endocytic pathway.

Recognition specificity of individual EH domains of mammals and yeast.

The Eps homology (EH) domain is a recently described protein binding module that is found, in multiple or single copies, in several proteins in species as diverse as human and yeast. In this work, we have investigated the molecular details of recognition specificity mediated by this domain family by characterizing the peptide-binding preference of 11 different EH domains from mammal and yeast proteins. Ten of the eleven EH domains could bind at least some peptides containing an Asn-Pro-Phe (NPF) motif. By contrast, the first EH domain of End3p preferentially binds peptides containing an His-Thr/Ser-Phe (HT/SF) motif. Domains that have a low affinity for the majority of NPF peptides reveal some affinity for a third class of peptides that contains two consecutive amino acids with aromatic side chains (FW or WW). This is the case for the third EH domain of Eps15 and for the two N-terminal domains of YBL47c. The consensus sequences derived from the peptides selected from phage-displayed peptide libraries allows for grouping of EH domains into families that are characterized by different NPF-context preference. Finally, comparison of the primary sequence of EH domains with similar or divergent specificity identifies a residue at position +3 following a conserved tryptophan, whose chemical characteristics modulate binding preference.

Eps15R is a tyrosine kinase substrate with characteristics of a docking protein possibly involved in coated pits-mediated internalization.

eps15R was identified because of its relatedness to eps15, a gene encoding a tyrosine kinase substrate bearing a novel protein-protein interaction domain, called EH. In this paper, we report a biochemical characterization of the eps15R gene product(s). In NIH-3T3 cells, three proteins of 125, 108, and 76 kDa were specifically recognized by anti-eps15R sera. The 125-kDa species is a bona fide product of the eps15R gene, whereas p108 and p76 are most likely products of alternative splicing events. Eps15R protein(s) are tyrosine-phosphorylated following epidermal growth factor receptor activation in NIH-3T3 cells overexpressing the receptor, even at low levels of receptor occupancy, thus behaving as physiological substrates. A role for eps15R in clathrin-mediated endocytosis is suggested by its localization in plasma membrane-coated pits and in vivo association to the coated pits' adapter protein AP-2. Finally, we demonstrate that a sizable fraction of eps15R exists in the cell as a complex with eps15 and that its EH domains exhibit binding specificities that are partially distinct from those of eps15. We propose that eps15 and eps15R are multifunctional binding proteins that serve pleiotropic functions within the cell.

Binding specificity and in vivo targets of the EH domain, a novel protein-protein interaction module.

EH is a recently identified protein-protein interaction domain found in the signal transducers Eps15 and Eps15R and several other proteins of yeast nematode. We show that EH domains from Eps15 and Eps15R bind in vitro to peptides containing an asparagine-proline-phenylalanine (NPF) motif. Direct screening of expression libraries with EH domains yielded a number of putative EH interactors, all of which possessed NPF motifs that were shown to be responsible for the interaction. Among these interactors were the human homolog of NUMB, a developmentally reguated gene of Drosophila, and RAB, the cellular cofactor of the HIV REV protein. We demonstrated coimmunoprecipitation of Eps15 with NUMB and RAB. Finally, in vitro binding of NPF-containing peptides to cellular proteins and EST database screening established the existence of a family of EH-containing proteins in mammals. Based on the characteristics of EH-containing and EH-binding proteins, we propose that EH domains are involved in processes connected with the transport and sorting of molecules within the cell.

Neuropharmacological effects of low and high doses of repeated oral dexfenfluramine in rats: a comparison with fluoxetine.

The neuropharmacological effects of repeated oral doses of dexfenfluramine (DF; 1.25-10 mg/kg, twice daily for 21 days) were examined in rats and related to the drug brain levels. Results were compared with fluoxetine (FL) given at similar doses relative to its anorectic ED50. Both drugs dose-dependently slowed body weight gain and reduced brain serotonin (5-HT). However, at 1.25 mg/kg DF caused only a slight and transient decrease in cortical 5-HT. Comparable doses of FL (6.25-12.5 mg/kg) lowered 5-HT more than DF, besides slightly reducing striatal dopamine. At higher doses DF markedly reduced 5-HT in all regions, and to a lesser extent noradrenaline in hippocampus. There was a negative relationship between 5-HT and log total active drug levels and the indole was approximately halved at drug levels about 50 times lower with DF than FL. However, the ratio between drug levels causing marked 5-HT reductions and those considered anorectic was similar for DF and FL because brain levels at the anorectic ED50 were higher with FL than DF. Long-lasting reductions of 5-HT were also observed but recovery was only consistently slow beginning from 5 mg/ kg DF. Comparable doses of FL could not be used because its general toxicity leads to the death of rats after only 2-4 multiples of its anorectic ED50.

Eps15 is constitutively oligomerized due to homophilic interaction of its coiled-coil region.

Eps15 is a member of an emerging family of proteins containing a novel protein/protein interaction domain, the EH domain, of as yet unknown function. Recent findings of Eps15 association with clathrin adaptor complex AP-2 and its localization in clathrin-coated pits have implicated Eps15 in the regulation of vesicle trafficking. Here we show that Eps15 exists in several multimeric states in vivo. When purified recombinant Eps15 or lysates of NIH 3T3 cells were treated with cross-linking reagents, covalent dimers of Eps15 and larger covalent multimers were detected in high yield. Large Eps15 oligomers co-immunoprecipitated with AP-2 at an efficiency higher than that of Eps15 dimers. Furthermore, cross-linking of the membrane-bound fraction of Eps15 in mildly permeabilized cells was as efficient as that of the cytosolic fraction. Size-exclusion column chromatography of recombinantly produced Eps15 and of total cell lysates was performed to examine the equilibrium ratio of the monomers versus the aggregated forms of Eps15. These experiments showed that essentially all the Eps15 was aggregated, whereas monomers of Eps15 could be obtained only under strong denaturing conditions. To map the region of Eps15 responsible for dimerization, fusion proteins corresponding to the three structural domains of Eps15 were prepared. Cross-linking analysis revealed that the central portion of Eps15, which possesses a coiled-coil region (residues 321-520), serves as the interacting interface. The possibility that hetero-oligomeric complexes of Eps15 dimers and AP-2 function during the recruitment of proteins into coated pits is discussed.

Depletion and time-course of recovery of brain serotonin after repeated subcutaneous dexfenfluramine in the mouse. A comparison with the rat.

The indole-depleting effects of repeated subcutaneous doses of dexfenfluramine (D-F) (2.5, 5, 10, 20 and 40 mg/kg/day, for four days) in mice were examined with regard to the initial response and time-course of recovery and related to the pharmacokinetics of D-F and its active metabolite dexnorfenfluramine (D-NF). Steady-state plasma and brain concentrations of D-F rose dose-dependently with a metabolite-to-drug ratio averaging 0.4 in brain. This confirmed that in mice D-NF contributes less than in other species to the effects of D-F. Regional serotonin (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA) contents were decreased dose-dependently 4 hr after the last injection of D-F. However, two weeks after D-F (2.5-10 mg/kg/day) brain indoles had almost totally recovered, and the long-term effects of the 20 mg/kg/day dose were completely reversed by six weeks, when significant effects are still observable in rats. Although substantial recovery was evident even at 40 mg/kg/day, 5-HT but not 5-HIAA was still slightly reduced nine weeks later. Comparative studies in rats given 2.5-20 mg/kg/day D-F indicated much more severe initial indole depletions than in mice. Brain levels of D-F and D-NF were much higher in rats than in mice. The total active drug brain concentration (D-F + D-NF) was significantly correlated with 5-HT content in both species, with approx 20 nmol/g of total drug causing 50% reduction. These findings point to species differences in D-F kinetics as a main reason for differences in the neurochemical response, supporting the view that the recovery of indoles over time is related to the extent of initial depletion, which in turn depends on critical drug brain concentrations. In view of the qualitative and quantitative species differences in the pharmacodynamics and pharmacokinetics of D-F neither of these rodent species is a suitable model for predicting potential drug toxicity in humans.

Brain uptake and distribution of the potential memory enhancer CL 275,838 and its main metabolites in rats: relationship between brain concentrations and in vitro potencies on neurotransmitter mechanisms.

The kinetics, brain uptake and distribution of CL 275,838, a potential memory enhancer, and its main metabolites (II and IV) were evaluated in rats after intraperitoneal doses of 5, 10 and 20 mg/kg. Brain maximum concentrations (Cmax) of the three compounds after pharmacologically active doses were then related to the in vitro concentrations affecting some monoaminergic and amino acid receptor sites to examine the relative importance of these neurotransmitter systems in the pharmacological actions of CL 275,838. After 10 mg/kg CL 275,838, the unchanged compound rapidly entered the brain and distributed almost uniformly in various regions inside the blood-brain barrier. Its disappearance from brain and plasma was almost parallel with a comparable elimination half-life (t 1/2) of about 2 h. Metabolite II entered the brain and equilibrated with plasma more slowly than the parent compound, achieving mean Cmax (0.2 microM) within 3 h of dosing. Metabolite IV was rapidly detected in rat brain but hardly amounted to 10% (0.1 microM) of the parent compound Cmax (1 microM). There was a linear relationship between dose and plasma and brain concentrations of the three compounds up to 20 mg/kg CL 275,838. At micromolar concentrations the parent compound had affinity for serotonin (5-HT) uptake sites, 5-HT2 and dopamine (DA2) receptors. Only at much higher concentrations than those achieved in vivo after pharmacologically active doses did it increase the binding of 3H-glutamate to NMDA (N-methyl-D-aspartate) receptors. Metabolite II has a similar neurochemical profile.(ABSTRACT TRUNCATED AT 250 WORDS)

Multiple-dose pharmacokinetics and safety of a potential memory-enhancing compound, CL 275,838, in healthy male volunteers.

The pharmacokinetics and safety of CL 275,838, a new potential memory-enhancing compound, were examined after 14 daily doses (50 and 100 mg) in 16 healthy male volunteers, age 20 to 59 years, in a randomized, double-blind, placebo-controlled, parallel group study. Trough blood samples (predose) were collected on days 2, 4, 7, 10, and 14, and further samples were drawn after the final dose (day 14) to define the multiple-dose kinetics of the parent compound and its metabolites II and IV. Intercurrent clinical events, vital functions, EEG, ECG, and cognitive tests (attention, verbal memory, and spatial memory) were considered as outcome measures of safety. Performance in cognitive tests was also studied to collect preliminary information on possible therapeutic action. Predose plasma concentrations of the parent compound and its two metabolites increased approximately in proportion to the dose, and accumulation was complete within 7 days, regardless of the dose. At steady state, mean Cmax and AUC of the parent compound and its two metabolites were dose related. Mean wash-out t1/2 was 18 to 20 hours for the parent compound, 22-23 hours for metabolite II, and 28-33 hours for metabolite IV; these elimination t1/2 are comparable for the two doses, and are similar to those observed in single-dose studies. For the 50-mg-dose group, predicted and observed average plasma concentrations (Css) of CL 275,838 and its two metabolites did not differ significantly.(ABSTRACT TRUNCATED AT 250 WORDS)

[Infectious complications in oncologic-gynecologic surgery: clinical and economic evaluation]. / Morbosità infettiva in chirurgia oncologico-ginecologica: valutazione clinica ed economica.

In the Department of Obstetric and Gynecology, Faculty of Medicine-Varese, between March 1991 and June 1992, 74 consecutive patients undergoing elective oncologic surgery were evaluated in order to rationalize the use of antibiotics to decrease the costs of infectious complications. We divided the patients into two groups: a high infection risk group (in which every patient was submitted to antibiotic prophylaxis) and a low infection risk group (in which we didn't use any antibiotic prophylaxis). Our findings indicate that selection criteria for HIR patients are probably correct and in this group AP is necessary. In the LIR group, 45.5% of patients was not submitted to any antibiotic therapy. It's necessary to test the real efficacy of an AP in LIR patients in whom we had not a important incidence of infectious complications. In the LIR group AP should not exceed Lit. 23,251 per patient to be cost-effective.

Single-dose safety and pharmacokinetics of a potential cognition-enhancing compound, CL 275,838, in healthy volunteers.

The pharmacokinetics and safety of CL 275,838, a potential cognition-enhancing compound, were studied after single escalating oral doses first in young healthy male volunteers and then in old (60-74 years) and very old (over 75 years) volunteers of both sexes. In all age groups absorption of CL 275,838 was rapid as assessed by the mean time to reach maximum plasma concentrations (Cmax) which averaged 1-2 hr, regardless of the dose administered. In young male volunteers both Cmax and area under the curve (AUC) increased proportionally with dose from 10 to 100 mg. Mean elimination half-lives (t1/2) of the parent compound (18-21 hr) and of its circulating metabolites II (20-22 hr) and IV (27-30 hr) were well comparable for the doses tested (50 and 100 mg). Age did not appreciably affect plasma Cmax of CL 275,838 or its two metabolites. Mean AUC and elimination half-life did not appreciably differ between old and very old subjects given 50 mg CL 275,838, with the limitations dictated by the small number of elderly subjects examined. Compared with younger volunteers receiving comparable doses, however, the elderly had higher mean plasma AUC of the unchanged compound and its two metabolites, although the parameter varied widely between subjects. The mean elimination t1/2 (+/- SD) was longer in the elderly (38.8 +/- 19.6, 50.5 +/- 24.5 and 41.7 +/- 12.1 hr, respectively, for the parent compound and its metabolites II and IV) than in the young subjects. The cause(s) of these variations and the possible clinical implications remain to be established.(ABSTRACT TRUNCATED AT 250 WORDS)

Liquid chromatography of the potential memory-enhancing agent CL 275,838 and its main metabolites, using a post-column photochemical reactor and fluorimetric detection.

On irradiation with short-wavelength ultraviolet light, the potential memory-enhancing compound CL 275,838 (I) and its desbenzyl derivative CL 286,527 (metabolite II) are cleaved into the highly fluorescent derivative CL 228,346 (metabolite IV). This reaction was exploited for the sensitive and selective detection of these compounds in human and animal plasma, after reversed-phase high-performance liquid chromatography on a Supelco LC18 DB column (15 cm x 4.6 mm I.D.) at room temperature. The parent compound and its metabolites were isolated from plasma constituents using the Sep-Pak C18 Plus cartridge, with satisfactory recovery (76-90%) and selectivity. The detection limits were ca. 1.25, 5 and 0.3 ng/ml for I, II and IV, respectively, using 1 ml of plasma. The validation procedure, which includes analysis of multiple ascending calibration curves based on between-day values and replicate analysis of quality control samples analysed with each standard curve, indicated acceptable precision and accuracy of the method within the concentration ranges investigated, the overall coefficient of variation and relative error being less than 10%. The method was successfully applied to plasma samples from healthy volunteers and animals after single of multiple doses of compound I. Metabolites II and IV were detectable in plasma of all species, the former at higher concentrations than the parent compound and metabolite IV. Together with the fact that metabolite II retains much of the parent compound's biological activity in vivo and in vitro, this suggests that it may contribute to the pharmacological effects of compound I.