Serum protein signature may improve detection of ductal carcinoma in situ of the breast.

Ductal carcinoma in situ (DCIS) of the breast is part of a spectrum of preinvasive lesions that originate within normal breast tissue and progress to invasive breast cancer. The detection of DCIS is important for the reduction of mortality from breast cancer, but the diagnosis of preinvasive breast tumors is hampered by the lack of an adequate detection method. To identify the changes in protein expression during the initial stage of tumorigenesis and to identify the presence of new DCIS markers, we analysed serum from 60 patients with breast cancer and 60 normal controls using mass spectrometry. A 23-protein index was generated that correctly distinguishes the DCIS and control groups with sensitivities and specificities in excess of 80% in two independent cohorts. Two candidate peptides were purified and identified as platelet factor 4 (PF-4) and complement C3a(desArg) anaphylatoxin (C3a(desArg)) using liquid chromatography-tandem mass spectrometry (LC-MS/MS). In an independent serum set of 165 patients, PF-4 and C3a(desArg) were significantly upregulated in DCIS compared with non-cancerous controls, as validated using western blot and enzyme-linked immunosorbent assay. We conclude that our serum protein-based test, used in conjunction with image-based screening practices, could improve the sensitivity and specificity of breast cancer detection.

In vivo restoration of RhoB expression leads to ovarian tumor regression.

Ovarian cancers are very aggressive cancers most often diagnosed when metastasis has already occurred in the entire peritoneal cavity. Ovarian adenocarcinoma cells present an undetectable level of RhoB GTPase. Using preclinical ovarian cancer models, we aimed to evaluate the potential use of RhoB cDNA as a tumor suppressor gene in gene therapy. RhoB restoration in vitro, through recombinant adenovirus transduction, resulted in the apoptosis of endogenous RhoB protein low-expressing cell lines (OVCAR-3 and IGROV-1) through the activation of the intrinsic apoptotic caspase cascade. We showed that a single injection of 10(8) p.f.u. of adenoviral vector encoding a reporter gene into the peritoneal cavity of ovarian tumor bearing mice can induce the gene modification of a large quantity of cells throughout the cavity. We thereby tested the effect of AdRhoB injections to treat ovarian cancer-bearing mice. The ectopic expression of RhoB, following its introduction via viral transduction into nude mice in vivo, was highly effective in suppressing tumor growth of ovarian cancer xenografts. Therapeutic agents designed to correct defects of RhoB at the molecular level may thereby provide innovative treatment options for patients not responding to standard therapies.

Selective inhibition of HER2 inhibits AKT signal transduction and prolongs disease-free survival in a micrometastasis model of ovarian carcinoma.

Although first-line chemotherapy induces complete clinical remission in many cases of epithelial ovarian cancer, relapse usually occurs 18-28 months from diagnosis owing to micrometastases. The present study aimed to evaluate the effect of trastuzumab on disease-free and overall survival in a specially designed murine model of ovarian cancer (OVCAR-3), which mimicked the natural history of human micrometastatic disease. Trastuzumab can cure the mice if started soon after induction chemotherapy. It can modestly inhibit the proliferation through mitogen-activated protein kinase signal transduction and clearly inhibit AKT phosphorylation, which is involved in survival pathway. As OVCAR-3 cell lines show no HER2 amplification or overexpression, these results warrant further studies to assess the efficacy of trastuzumab in the early stage of relapse in cancer models other than those overexpressing HER2.

RhoB prenylation is driven by the three carboxyl-terminal amino acids of the protein: evidenced in vivo by an anti-farnesyl cysteine antibody.

Protein isoprenylation is a lipid posttranslational modification required for the function of many proteins that share a carboxyl-terminal CAAX motif. The X residue determines which isoprenoid will be added to the cysteine. When X is a methionine or serine, the farnesyl-transferase transfers a farnesyl, and when X is a leucine or isoleucine, the geranygeranyl-transferase I, a geranylgeranyl group. But despite its CKVL motif, RhoB was reported to be both geranylgeranylated and farnesylated. Thus, the determinants of RhoB prenylation appear more complex than initially thought. To determine the role of RhoB CAAX motif, we designed RhoB mutants with modified CAAX sequence expressed in baculovirus-infected insect cells. We demonstrated that RhoB was prenylated as a function of the three terminal amino acids, i.e., RhoB bearing the CAIM motif of lamin B or CLLL motif of Rap1A was farnesylated or geranylgeranylated, respectively. Next, we produced a specific polyclonal antibody against farnesyl cysteine methyl ester allowing prenylation analysis avoiding the metabolic labeling restrictions. We confirmed that the unique modification of the RhoB CAAX box was sufficient to direct the RhoB distinct prenylation in mammalian cells and, inversely, that a RhoA-CKVL chimera could be alternatively prenylated. Moreover, the immunoprecipitation of endogenous RhoB from cells with the anti-farnesyl cysteine antibody suggested that wild-type RhoB is farnesylated in vivo. Taken together, our results demonstrated that the three last carboxyl amino acids are the main determinants for RhoB prenylation and described an anti-farnesyl cysteine antibody as a useful tool for understanding the cellular control of protein farnesylation.

RhoA prenylation is required for promotion of cell growth and transformation and cytoskeleton organization but not for induction of serum response element transcription.

The importance of post-translational geranylgeranylation of the GTPase RhoA for its ability to induce cellular proliferation and malignant transformation is not well understood. In this manuscript we demonstrate that geranylgeranylation is required for the proper cellular localization of V14RhoA and for its ability to induce actin stress fiber and focal adhesion formation. Furthermore, V14RhoA geranylgeranylation was also required for suppressing p21(WAF) transcription, promoting cell cycle progression and cellular proliferation. The ability of V14RhoA to induce focus formation and enhance plating efficiency and oncogenic Ras anchorage-dependent growth was also dependent on its geranylgeranylation. The only biological activity of V14RhoA that was not dependent on its prenylation was its ability to induce serum response element transcriptional activity. Furthermore, we demonstrate that a farnesylated form of V14RhoA was also able to bind RhoGDI-1, was able to induce cytoskeleton organization, proliferation, and transformation, and was just as potent as geranylgeranylated V14RhoA at suppressing p21(WAF) transcriptional activity. These results demonstrate that RhoA geranylgeranylation is required for its biological activity and that the nature of the lipid modification is not critical.

The farnesyltransferase inhibitor FTI-277 suppresses the 24-kDa FGF2-induced radioresistance in HeLa cells expressing wild-type RAS.

In this paper, we describe the effect of the inhibitor of farnesyltransferase (FTI-277) on radioresistance induced by the 24-kDa isoform of FGF2 in human cells expressing wild-type RAS. Treatment with FTI-277 (20 microM) for 48 h prior to irradiation led to a significant decrease in survival of radioresistant cells expressing the 24-kDa isoform (HeLa 3A) but had no effect on the survival of control cells (HeLa PINA). The radiosensitizing effect of FTI-277 is accompanied by a stimulation of postmitotic cell death in HeLa 3A cells and by a reduction in G(2)/M-phase arrest in both cell types. These results clearly demonstrate that at least one farnesylated protein is involved in the regulation of the radioresistance induced by the 24-kDa isoform of FGF2. Furthermore, the radiation-induced G(2)/M-phase arrest is also under the control of farnesylated protein. This work also demonstrates that FTase inhibitors may be effective radiosensitizers of certain human tumors with wild-type RAS.

Epidermal growth factor stimulates 3-hydroxy-3-methylglutaryl-coenzyme A reductase expression via the ErbB-2 pathway in human breast adenocarcinoma cells.

HMG-CoA reductase is the key enzyme for the biosynthesis of isoprenoid compounds essential for cell growth and differentiation. Its tyrosine kinase-dependent modulation has recently been suggested and described in the ErbB-2 overexpressing cell line SKBR-3 [Asslan et al. (1998) Biochem. J. 330, 241-246]. Epidermal growth factor (EGF) increased the HMG-CoA reductase activity, protein, and mRNA levels only in ErbB-2-expressing cells (SKBR-3 and MCF-7) but not in MDA-MB-468 cells that do not express ErbB-2 even though their EGF receptor was efficiently phosphorylated. Tyrphostin AG 879, a specific inhibitor of ErbB-2 tyrosine kinase activity, decreased HMG-CoA reductase activity only in cells that expressed ErbB-2. A functional EGF receptor appeared to be necessary since its inhibition by the specific tyrphostin AG 1478 abolished the EGF effects. Phosphatidylinositol 3-kinase (PI 3-kinase) might be a crucial enzyme in the signaling pathway since the specific inhibitor, LY 294002, was shown to inhibit HMG-CoA reductase activity and to completely abolish the stimulation by EGF in SKBR-3 cells.

SupraMolecular BioVectors (SMBV) improve antisense inhibition of erbB-2 expression.

New therapeutic strategies are now being developed against adenocarcinoma associated with erbB-2 amplification, particularly by inhibiting p185erbB-2 expression. Antisense oligodeoxynucleotides seem promising for this purpose as long as they are efficiently protected against degradation and targeted into the cells. We present antisense oligonucleotide carriers, the supramolecular biovectors (SMBVs), for which we have already demonstrated the ability to improve both cellular uptake and protection of oligodeoxynucleotide. The present work demonstrates that SMBVs elicit a specific and non-toxic action of antisense compounds in a cell model, irrespective of their sensitivity to nucleases. This is a major point, considering the specificity problems associated with the use of nuclease-resistant phosphorothioate oligodeoxynucleotide. SMBVs improve antisense efficiency of oligodeoxynucleotide designed against p185erbB-2, with a complete growth arrest of SK-Br-3, human adenocarcinoma mammary cells that overexpress p185erbB-2 and no effect on MCF-7 cells that normally express p185erbB-2. The comparison of SMBVs with DOTAP reveals the statistically higher efficiency of SMBVs, which allows the antisense inhibition of p185erbB-2 expression in 65-75% of SK-Br-3 cells (P < 0.05). The efficiency and controlled synthesis of SMBVs underline their potentialities as oligodeoxynucleotide carriers for in vivo experiments.

Central cardiovascular effects of tacrine in the conscious dog: a role for catecholamines and vasopressin release.

Centrally acting cholinergic agents are currently reported to increase blood pressure in various species through the stimulation of muscarinic cholinoceptors. Moreover, several cardiovascular adverse effects have been reported from clinical studies. The aim of this study was to investigate the effects of tacrine, an acetylcholinesterase inhibitor which has been reported to have therapeutic potential in Alzheimer's disease, on blood pressure and two vasopressor systems (sympathetic and vasopressinergic) in Beagle dogs. Intravenous (i.v.) tacrine (2 mg kg(-1)) induced, in conscious and anesthetized dogs, an increase in systolic and diastolic blood pressure, accompanied by bradycardia. This increase was dose-dependent with a peak effect at 1.5 min following administration. Tacrine also induced an increase in noradrenaline, adrenaline and vasopressin plasma levels. Pretreatment with the muscarinic receptor antagonist, atropine (2 mg kg(-1), i.v.), abolished the pressor response to i.v. injection of tacrine while pretreatment with the peripheral muscarinic receptor antagonist, methylscopolamine (0.2 mg kg(-1), i.v.), did not alter the increase in blood pressure. Similarly, noradrenaline and adrenaline changes in plasma levels were not modified by methylscopolamine but were abolished by atropine pretreatment. A similar tendency although not significant was observed for vasopressin plasma levels. The present results demonstrate that in dogs, tacrine (2 mg kg(-1), i.v.) stimulates central muscarinic cholinoceptors to increase blood pressure through activation of the two components of the sympathetic nervous system (i.e., neuroneuronal noradrenergic and the neurohormonal adrenergic pathways) as well as through increasing noradrenaline, adrenaline and vasopressin plasma levels.

Salmeterol-induced desensitization, internalization and phosphorylation of the human beta2-adrenoceptor.

1. Partial agonists of the beta2-adrenoceptor which activate adenylyl cyclase are widely used as bronchodilators for the relief of bronchoconstriction accompanying many disease conditions, including bronchial asthma. The bronchodilator salmeterol has both a prolonged duration of action in bronchial tissue and the ability to reassert this activity following the temporary blockade of human beta2-adrenoceptors with antagonist. 2. We have compared the activation and desensitization of human beta2-adrenoceptor stimulation of adenylyl cyclase induced by salmeterol, adrenaline and salbutamol in a human lung epithelial line, BEAS-2B, expressing beta2-adrenoceptor levels of 40-70 fmol mg(-1), and in human embryonic kidney (HEK) 293 cell lines expressing 2-10 pmol mg(-1). The efficacy observed for the stimulation of adenylyl cyclase by salmeterol was only approximately 10% of that observed for adrenaline in BEAS-2B cells expressing low levels of beta2-adrenoceptor, but similar to adrenaline in HEK 293 cells expressing very high levels of receptors. Salmeterol pretreatment of these cells induced a rapid and stable activation of adenylyl cyclase activity which resisted extensive washing and beta2-adrenoceptor antagonist blockade, consistent with binding to a receptor exosite and/or to partitioning into membrane lipid. 3. The desensitization and internalization of beta2-adrenoceptors induced by the partial agonists salmeterol and salbutamol were considerably reduced relative to the action of adrenaline. Consistent with these observations, the initial rate of phosphorylation of the receptor induced by salmeterol and salbutamol was much reduced in comparison to adrenaline. 4. Our data suggest that the reduction in the rapid phase of desensitization of beta2-adrenoceptors after treatment with salmeterol or salbutamol is caused by a decrease in the rate of beta2-adrenoceptor kinase (betaARK) phosphorylation and internalization. In contrast, the rate of cyclic AMP-dependent protein kinase (PKA)-mediated phosphorylation by these partial agonists appears to be similar to adrenaline.

Improved oligonucleotide uptake and stability by a new drug carrier, the SupraMolecular Bio Vector (SMBV).

Antisense oligodeoxynucleotides are potential therapeutic agents, but their development is still limited by both a poor cellular uptake and a high degradation rate in biological media. The strategy that we propose to face these problems is to use small synthetic carriers, around 30 nm diameter, the SupraMolecular Bio Vectors (SMBV). We used positively charged SMBV and settled the ionic incorporation of negatively charged oligonucleotides into these carriers. A minimal leakage of 10% of total incorporated oligonucleotides was then measured during two months. Both protection and uptake of oligonucleotides were then analyzed. On the one hand, we showed that the incorporation of oligonucleotides into the selected SMBV allows to significantly increase, 8 times, their half-life, in cell growth medium. On the other hand, the internalization of the SMBV, into cells, by an endosomal pathway has been characterized. The essential point is that the SMBV uptake elicits the simultaneous oligonucleotide uptake. The oligonucleotide amount that goes through cells within 5 h can be up to 30 times higher than for free oligonucleotides and the fraction of oligonucleotides that is present in the cytosol is increased up to 10 fold after incorporation into the SMBV. This study demonstrates the ability of SMBV to improve oligonucleotide cellular behaviour.

Stable activation and desensitization of beta 2-adrenergic receptor stimulation of adenylyl cyclase by salmeterol: evidence for quasi-irreversible binding to an exosite.

The relaxation of tracheal smooth muscle by the beta 2-adrenergic receptor (beta AR) agonist salmeterol displays several unusual properties: (i) slow onset of action (t1/2 = 5-15 min), (ii) prolonged activation (t1/2 = 8-14 hr), and (iii) the ability to recover from beta AR blockade. These properties led to the hypothesis that salmeterol binds with very high affinity to an exosite in addition to the beta AR activating site. Despite extensive characterization of salmeterol-induced bronchodilation, little is known about the molecular actions of salmeterol. We report the unique properties of salmeterol binding to the beta AR, activation of adenylyl cyclase, and desensitization of the hamster beta AR expressed in L cells. First, we found that salmeterol activation of adenylyl cyclase, although rapid and potent (low EC50 relative to epinephrine), was nevertheless remarkably inefficient relative to the full agonist epinephrine. Reduced coupling efficiency of salmeterol was demonstrated using formulations recently introduced by our group. Second, we found that pretreatment of L cells with salmeterol led to a stable activation of adenylyl cyclase that survives extensive wash procedures and sucrose step gradient purification of plasma membrane fractions. This activation of basal adenylyl cyclase did not require salmeterol binding to the classic active site during pretreatment, as it occurred in the presence of an excess of a beta AR antagonist. Third, we found that the rapid phase of salmeterol-induced desensitization was much reduced relative to epinephrine, consistent with its poor coupling efficiency and with its prolonged activation of adenylyl cyclase. These unique properties of salmeterol support the proposal that it binds reversibly to the activating or active site and as well to an extremely high affinity exosite from which it has access to the active site.

[Rapid micromethod for CH50 determination using an algorithm]. / Détermination rapide par logiciel de la CH50 en microméthode.

The titration of haemolytic complement in biological liquid cause inconvenient in calculating concentration, it was long and fastidious. We report a fast technic based on measure of hemolysis in microplate++ method, which exploitation of results, it does with an appropriate algorithm.

[Immunoenzymatic determination of free immunoglobulin light chains in the cerebrospinal fluid: its value in the biological diagnosis of multiple sclerosis]. / Dosage immunoenzymatique des chaînes légères libres d'immunoglobulines dans le liquide céphalorachidien: intérêt dans le diagnostic biologique de la sclérose en plaques.

Based on previous data on free immunoglobulin light chains in the cerebrospinal fluid (CSF) of patients with multiple sclerosis (MS), we evaluated free kappa and lambda light chains in the CSF from 3 patients groups: (a) 42 with MS, (b) 16 with other neurological inflammatory diseases (ONID) and (c) 42 with non inflammatory neurological diseases (NIND) used as control. The kappa and lambda light chains contents were evaluated using a specific and sensitive (0.5 mcg/ml) enzyme linked immunosorbent assay (ELISA). Whole CSF immunoglobulins G (CSF-IgG) concentrations were determined by an immuno-nephelometric method and CSF oligoclonal banding was assessed by agarose gel electrophoresis. Elevated free kappa light chains levels were found in 36 of the 42 (85.7%) MS-CSF, and only in 2 of the 16 (12.5%) ONID-CSF. Moreover, all MS-CSF with oligoclonal banding (21/42) exhibited detectable free kappa light chains. In contrast, elevated free lambda light chains or whole CSF-IgG concentrations were found not so specific. These results suggest that free kappa light chains measurement in MS-CSF may have an important diagnostic usefulness. This assay offers a reliable alternative to other available procedures and may constitute complementary technique in routine investigation of MS-CSF.

[Rapid and sensitive micromethod for protein determination by the Coomassie-blue technique]. / Micro-methode rapide et sensible de dosage des proteines par la technique au bleu de Coomassie.

The Coomassie blue method of proteins quantitation initially reported by Bradford and modified by Macart et al. appears to be simple, fast, sensitive and of low cost. In this study, we report an improved micromethod derived from the technic described by Macart. Applied to the protein quantitation of 100 cerebrospinal fluid (CSF) specimens and 50 serum specimens our method showed a very good correlation with the macromethod of Macart (r = 0.985 for CSF proteins, r = 0.944 for serum proteins) and also with the Lowry technic (r = 0.986 for CSF proteins, r = 0.898 for serum proteins). Our technic may be used with various biological fluids (urines, tears, saliva...) and seems to be of particular interest for low protein level fluids and for little volume samples (it needs only 20 mcl).

[Detection of oligoclonal immunoglobulins in the cerebrospinal fluid using agar gel immunoelectrophoresis with silver salt staining]. / Détection des immunoglobulines oligoclonales dans le l. c. r. par IEF sur gel d'agarose et coloration par sel d'argent.

Detection of oligoclonal immunoglobulins in multiple sclerosis cerebrospinal fluid (MS-CSF) seems to be an important test in the biological diagnosis of the disease. In our laboratory, the classical agarose gel electrophoretic technic allowed the detection of CSF oligoclonal bands in only 40% of the MS patients. This relatively low percentage in comparison with those obtained by other investigators led us to develop a much more resolving electrophoretic technic: an agarose gel isoelectric focusing (IEF) with silver staining. By this method, we detected oligoclonal immunoglobulins in 43 (43%) of 100 MS-CSF exhibiting only polyclonal patterns on classical electrophoresis. Oligoclonal banding appeared particularly in patients with inflammatory type profile (36/60) according to Schuller's classification, but also in those with normal (5/26) or inflammatory transudative (2/8) type profiles. MS-CSF with oligoclonal immunoglobulins on IEF had a higher IgG/Albumin ratio (p = 0.01) than those with polyclonal immunoglobulins, while the mean IgG levels were not significantly different (p = 0.07). These results support the diagnostic usefulness of the IgG/Albumin ratio. Agarose IEF appears to be a useful technic in the detection of oligoclonal immunoglobulins and may be applied at least to CSF of patients with clinical signs of multiple sclerosis.

Depression of reticulo-ruminal motor functions through the stimulation of alpha 2-adrenoceptors.

Inhibition of the cyclical contractions of the reticulum and the rumen by detomidine (10-40 micrograms/kg, i.v.), xylazine (20-80 micrograms/kg, i.v.) and clonidine (2.5-10 micrograms/kg, i.v.) were compared in sheep and cattle housed individually in box stalls. Two alpha 2-adrenergic receptor blocking agents, tolazoline and yohimbine, were administered intravenously for prevention (0.1-0.4 mg/kg) or reversal (0.4-1.2 mg/kg) of these effects. Continuous recording of the reticuloruminal contractions and measurement of the volume of ruminal gas eliminated through the upper respiratory tract indicated that the three alpha 2-agonists inhibited the primary ruminal contractions associated with the reticular contractions. The occurrence of secondary ruminal contractions was also blocked in sheep, but only suppressed in cattle. The inhibition of reticulo-ruminal contractions was prevented or reversed competitively by the two alpha 2-blocking agents, suggesting an alpha 2-adrenoceptor mediation of the inhibition of cyclical motor activity of the reticulo-rumen. In contrast with tolazoline, yohimbine was unable to alleviate the accumulation of gas resulting from inhibition of the secondary ruminal contractions.

Alleviation of excessive gas accumulation in the ruminant stomach by ritanserin.

The relationships between forestomach motility and eructation rate were studied in sheep and cattle. Three ewes and 2 heifers were implanted with strain gauges on the reticulo-rumen and fitted with a cannula in the dorsal sac of the rumen. Studies were performed in sheep after induction of hypocalcemia by Na2EDTA infusion and cattle were studied after ruminal distension. Experiments were performed by measuring the rate and volume of eructated ruminal gases, using a technique by which the trachea is transected. The frequency of reticulo-ruminal contractions decreased 40% within 30 minutes of Na2EDTA infusion to the sheep. The volume of eructated gas (for 30-minute periods) decreased from 10.7 L to 5.5 L at the end of the 60-minute infusion period. Pretreatment with ritanserin (0.1 mg/kg, subcutaneously) not only prevented bloating during the ruminal stasis induced by hypocalcemia, but also significantly increased the eructated volume of gas. In cattle, ritanserin given at the same dose level (0.1 mg/kg, subcutaneously) significantly increased the volume of eructated gas after ruminal distension. This study supports the hypothesis that the caudal esophageal sphincter has a role in the rate of ruminal gas eructation and indicates that its relaxation may be due to a 5-hydroxytryptamine antagonist.