Growth factor receptor/steroid receptor cross talk in trastuzumab-treated breast cancer.

Treatment with tyrosine kinase inhibitors (TKIs) including trastuzumab has revolutionized the management of HER2-positive breast cancer. Recent evaluation of clinical trial data suggests that a subset of HER2/ER double-positive cancers may not receive significant benefit from the TKI therapy. Here we investigate the cross talk between HER2 and ER in breast cancer and monitor the effect of trastuzumab on the tyrosine kinase effector transcription factor Myc. In HER2-positive breast cancer patients treated with neoadjuvant trastuzumab, steroid receptor-negative status (ER and PR negative) of pre-treatment biopsies predicted pathological complete response (pCR) (n=31 patients, P=0.0486), whereas elevated Myc protein inversely associated with pCR (P=0.0446). Liquid chromatography mass spectrometry identified the corepressor SMRT as a novel Myc-interacting protein. Trastuzumab treatment enhanced Myc-SMRT interactions in HER2-overexpressing breast cancer cells (LCC1) and inhibited expression of the Myc target gene survivin. In HER2-low, ER-positive steroid-dominant cells (MCF7), trastuzumab therapy repressed Myc-SMRT interactions and upregulated survivin expression. Trastuzumab treatment induced ER-CBP interactions, enhanced ER transcriptional activity and upregulated expression of the ER target gene pS2. The absence of pS2 expression in pre-treatment biopsies predicted pCR to neoadjuvant trastuzumab in breast cancer patients (n=25, P=0.0089) and pS2 expression associated with residual cancer burden (P=0.0196). Furthermore, metastatic tissues from patients who had failed trastuzumab therapy were pS2 positive. In HER2-overexpressing cells, trastuzumab treatment can repress Myc transcriptional activity and clinical response is favorable. However, with co-expression of the steroid pathway, this inhibition is lost and response to treatment is often poor.

Platelets increase survival of adenocarcinoma cells challenged with anticancer drugs: mechanisms and implications for chemoresistance.

BACKGROUND AND PURPOSE: Cancer cells grow without the restraints of feedback control mechanisms, leading to increased cancer cell survival. The treatment of cancer is often complicated by the lack of response to chemotherapy leading to chemoresistance and persistent survival of tumour cells. In this work we studied the role of platelets in chemotherapy-induced cancer cell death and survival. EXPERIMENTAL APPROACH: Human adenocarcinoma cells, colonic (Caco-2) and ovarian (59 M) cells, were incubated with 5-fluorouracil (1-300 µg·mL(-1) ) or paclitaxel (1-200 µg·mL(-1) ) in the presence or absence of platelets (1.5 × 10(8) mL(-1) ) for 1, 24 or 72 h. Following incubation, cancer cells were harvested and cell survival/death was assayed using flow cytometry, Western blotting, real-time PCR, TaqMan® Gene Expression Assays and proteomics. KEY RESULTS: Human platelets increased the survival of colonic and ovarian adenocarcinoma cells treated with two standard anticancer drugs, 5-fluorouracil and paclitaxel. In the presence of platelets, cancer cells up-regulated anti-apoptotic and down-regulated pro-apoptotic genes, increased the number of cells in the synthesis of DNA and decreased the number in the quiescent phase, increased expression of cyclins, DNA repair proteins and MAPKs. The analysis of platelet-Caco-2 secretome demonstrated the release of the chemokine RANTES, thrombospondin-1, TGF-ß and clusterin. Finally, human recombinant RANTES and thrombospondin-1 improved survival of Caco-2 cells challenged with paclitaxel. CONCLUSIONS AND IMPLICATIONS: These data demonstrate that platelets increase adenocarcinoma cells survival, proliferation and chemoresistance to standard anticancer drugs. Modulating cancer cell-platelet interactions may offer a new strategy to improve the efficacy of chemotherapy.

Neoplasms escape selective COX-2 inhibition in an animal model of breast cancer.

BACKGROUND: Cyclo-oxygenase-2 (COX-2) is up-regulated in malignant tumours rendering it an attractive target for cancer therapeutics. However, whether long-term antagonism maintains its initial efficacy on established tumours is unclear. METHODS: 4T1 cells were injected into the mammary fat pad of BALB/c mice (n = 8). Once tumour deposits were established, animals were randomized into two equal groups to receive either a selective COX-2 inhibitor (SC-236) or a drug vehicle. Further animals similarly treated (n = 7) were studied in diuresis cages allowing urine capture and analysis by mass spectrometry to determine Prostaglandin F-1 levels (PGF-1). In addition, both wild-type receiving SC-236 and COX-2 knockout mice receiving either SC 236 or vehicle were subjected to the same studies to determine whether tumour-derived or host-derived (stromal) COX-2 was the critical element. Finally, BALB/c mice with 4T1 tumours (n = 7) were treated with a combination of COX-2 and lipoxygenase (LOX) inhibition to attenuate this escape phenomenon. RESULTS: While selective COX-2 inhibition initially retarded tumour growth, a rapid increase in tumour growth rate occurred later (day 9). This escape phenomenon correlated with an increase in urinary PGF-1 levels. An identical trend was also observed whether COX-2 knockout mice received SC-236 or not, suggesting that this effect is due to increased tumour-derived COX-2 production rather than recovery of host COX-2 functional capacity. Finally, dual inhibition of COX and LOX pathways attenuated this escape process. CONCLUSION: The anti-neoplastic effects of selective COX-2 inhibition may not be sustained as tumours demonstrate an escape capacity. However, this phenomenon maybe attenuated by a combination of COX/LOX inhibitors.

The glycosylation of the variant surface glycoproteins and procyclic acidic repetitive proteins of Trypanosoma brucei.

Trypanosoma brucei, in common with the other African trypanosomes, exhibits unusual cell-surface molecular architecture. The bloodstream form of the parasite is coated with a continuous layer of approximately five million variant surface glycoprotein (VSG) dimers that provide the parasite with a macromolecular diffusion barrier to guard against lysis by the alternative complement pathway. The procyclic form of the parasite has a more diffuse cell-surface coat made up of approximately 2.5 million copies of procyclic acidic repetitive protein (PARP). Within the VSG and PARP coats exist lower-abundance surface glycoproteins such as receptors and nutrient transporters. Both the VSG molecules and the PARP molecules are attached to the membrane via glycosylphosphatidylinositol (GPI) membrane anchors and the VSGs and one form of PARP are N-glycosylated. In this article, the structures of the N-glycans and the GPI anchors of T. brucei VSGs and PARPs are reviewed and simple models of the surfaces of bloodstream and procyclic trypomastigotes are presented.

Natural processing sites for human cathepsin E and cathepsin D in tetanus toxin: implications for T cell epitope generation.

Cathepsin E is an aspartic proteinase that has been implicated frequently in Ag processing for presentation on class II MHC molecules, but no information exists on its cleavage specificity within Ags in relation to known T cell epitopes. We have analyzed the processing by cathepsin E of a large C-terminal domain of tetanus toxin (residues 872-1315), and we have compared the processing products with those liberated by cathepsin D, a related aspartic proteinase also thought to be involved in class II MHC-restricted Ag processing. Processing products were analyzed by N-terminal Edman degradation and mass spectrometry following reverse-phase HPLC separation of peptides. A total of 28 cleavage sites was identified, 11 of which were recognized by both cathepsins E and D. Most, although not all, sites were between pairs of hydrophobic residues and were located within the 200-amino-acid C terminal region known to contain several human T cell epitopes. Previously described T cell epitopes, for example, between residues 1273 and 1284, were flanked by cathepsin E and D cleavage sites. These data are consistent with an important role for cathepsins E and/or D in Ag processing in the human immune system.

Structural characterisation of two forms of procyclic acidic repetitive protein expressed by procyclic forms of Trypanosoma brucei.

A procyclic acidic repetitive protein (PARP) fraction was purified from long-term cultures of Trypanosoma brucei procyclic forms by a solvent-extraction and reverse phase chromatography procedure. The PARP fraction yielded small quantities of a single N-linked oligosaccharide with the structure Man alpha1-6(Man alpha1-3)Man alpha1-6(Man alpha1-3)Manbeta1-4GlcNAcbeta1-4GlcNAc (Man5GlcNAc2). Fractionation of PARP on Con A-Sepharose revealed that the majority (80 to 90%) of the PARP fraction did not bind to Con A and was composed of the parpA alpha gene product that contains repeats of -Glu-Pro-Pro-Thr- (GPEET-PARP) and that lacks an N-glycosylation site. This form of PARP has not been previously identified at the protein-level. The minor Con-A-binding fraction was shown to be rich in the previously described form of PARP, encoded by the parpAbeta and/or parpB alpha genes, that contains a -Glu-Pro- repeat domain (EP-PARP) and an N-glycosylation site. Analysis of longer and shorter-term cultures suggested that procyclic cells initially express predominantly EP-PARP that is gradually replaced by GPEET-PARP. Both forms of PARP were shown to contain indistinguishable glycosylphosphatidylinositol (GPI) membrane anchors, where the conserved GPI core structure is substituted by heterogeneous sialylated branched polylactosamine-like structures that are predicted to form a dense surface glycocalyx above which the polyanionic -Glu-Pro-Pro-Thr- and -Glu-Pro- repeat domains are displayed. The phosphatidylinositol (PI) component of the GPI anchor was shown to be a mixture of 2-O-acyl-myo-inositol-1-HPO4-(sn-1-stearoyl-2-lyso-glycerol) and 2-O-acyl-myo-inositol-1-HPO4-(sn-1-octadecyl-2-lyso-glycerol), where the acyl chain substituting the inositol ring showed considerable heterogeneity. Mass spectrometric and light scattering experiments both suggested an average mass of approximately 15 kDa for GPEET-PARP, with individual glycoforms ranging from about 12 kDa to 20 kDa, that is consistent with its amino acid and carbohydrate composition. A measured translational diffusion coefficient of 3.9 x 10(7) cm2 s(-1) indicates that this molecule has a highly elongated shape. The possible functions of these unusual glycoproteins are discussed.

Structure of the N-linked oligosaccharide of the main diagnostic antigen of the pathogenic fungus Paracoccidioides brasiliensis.

The major diagnostic antigen of Paracoccidioides brasiliensis is the exocellularly secreted 43,000 Da glycoprotein (gp43) which contains a single N-linked oligosaccharide chain. This oligosaccharide, although poorly immunogenic in man, is responsible for the cross-reactivity of the gp43 with sera from patients with histoplasmosis, and may have a role in fungal virulence. It contains a neutral high-mannose core (Man7GlcNAc2) to which a (1-->6)-linked alpha-D-Manp chain of variable length, substituted at the 2-O positions by single alpha-D-Manp residues, is attached. A terminal unit of beta-D-galactofuranose is (1-->6)-linked to one of the (1-->2)-linked mannosyl residues, either in the C or in the A arm of the oligosaccharide. The heterogeneity of the oligosaccharide is determined by the different sizes of the A arm and the sites of insertion of the beta-galactofuranosyl unit. The complete structure was determined by methylation analysis, 1H-NMR, mass spectrometry, acetolysis and mannosidase degradation. Electrospray mass spectrometry showed that the oligosaccharide comprises several subtypes ranging from Hex18GlcNAc2 to Hex10GlcNAc2 which accounts for the diffuse migration of the gp43 in polyacrylamide gels. The average size of the most frequent subtype is Hex13.6GlcNAc2. Dilute acid treatment to remove beta-D-Galf reduced the molecular masses of the majority of the subtypes by a single sugar unit.

Molecular species analysis and quantification of the glycosylphosphatidylinositol intermediate glycolipid C from Trypanosoma brucei.

The precursor of the glycosylphosphatidylinositol membrane anchor of the variant surface glycoprotein of Trypanosoma brucei is known as glycolipid A and it has the structure: EtN-PO4-6Man alpha 1-2Man alpha 1-6Man alpha 1-4GlcN alpha 1-6myo-inositol-PO4-(sn-1,2-dimyristoylglycerol). This precursor exists in equilibrium with its inositol-acylated form known as glycolipid C that contains a fatty acid attached to the inositol ring. In this study, we describe the purification to homogeneity of glycolipid C, its precise quantification and the analysis of the molecular species of glycolipid C by electrospray ionisation mass spectrometry. The results show that glycolipid C is present at 160000 copies per cell, that glycolipid C is acylated on the 2-position of the myo-inositol ring and that glycolipid C is heterogeneous with respect to the acyl chain attached to the inositol ring. The implications of these results with respect to the nature of the trypanosome inositol acyltransferase are discussed.

Structural studies on a lipoarabinogalactan of Crithidia fasciculata.

The monosaccharide D-arabinopyranose has only been found in glycoconjugates of the trypanasomatid parasites Leishmania major, Endotrypanum schaudinni and Crithidia fasciculata. The donor molecule for the relevant arabinosyltransferases is known to be GDP-alpha-D-Arap in L. major and C. fasciculata, and the latter organism is being used to study the biosynthesis of GDP-alpha-D-Arap. In this study, we describe the structure of the terminal product of arabinose metabolism in C. fasciculata, namely lipoarabinogalactan. This molecule was purified by hydrophobic-interaction chromatography and studied by a variety of techniques, including gas chromatography-mass spectrometry, electrospray mass spectrometry and chemical and enzymic digestions. These data show that lipoarabinogalactan contains a previously described D-arabino-D-galactan polysaccharide component covalently attached to a glycosylphosphatidylinositol type of membrane anchor that is similar to, but not identical with, that found in the lipophosphoglycans of the Leishmania.

Primary structure of CD52.

The CD52 antigen was extracted from human spleens with organic solvents and purified by immunoaffinity and reverse-phase chromatography. The latter step resolved two CD52 species, called CD52-I and CD52-II. Both species were found to contain similar N-linked oligosaccharides and glycosylphosphatidylinositol (GPI) anchor glycans. The N-linked oligosaccharides were characterized by methylation linkage analysis and, following exhaustive neuraminidase and endo-beta-galactosidase digestion, by the reagent array analysis method. The results showed that the single CD52 N-glycosylation site is occupied by large sialylated, polylactosamine-containing, core-fucosylated tetraantennary oligosaccharides. The locations of the phosphoryl substituents on the GPI anchor glycan were determined using a new and sensitive method based upon partial acid hydrolysis of the GPI glycan. The difference between CD52-I and CD52-II was in the phosphatidylinositol (PI) moieties of the GPI anchors. The phosphatidylinositol-specific phospholipase C-sensitive CD52-I contained exclusively distearoyl-PI, while the PI-phospholipase C-resistant CD52-II contained predominantly a palmitoylated stearoyl-arachidonoyl-PI, as judged by electrospray ionization mass spectrometry. Tandem mass spectrometric studies indicated that the palmitoyl residue of the CD52-II anchor is attached to the 2-position of the myo-inositol ring. Both the CD52-I and CD52-II PI structures are unusual for GPI anchors and the possible significance of this is discussed. The alkali-lability of the CD52 epitope recognized by the Campath-1H monoclonal antibody was studied. The data suggest that the alkali-labile hydroxyester-linked fatty acids of the GPI anchor are necessary for antibody binding.

Separation of cardiac glycosides by micellar electrokinetic capillary electrophoresis.

The separation of mixtures of primary and secondary cardiac glycosides by micellar electrokinetic capillary electrophoresis modified by cyclodextrins, urea and sodium cholate proved to be suitable for the determination of these hydrophobic compounds. It was possible to distinguish the two anomeric cardenolides glucodigifucoside and glucodigiglucomethyloside with all three buffer systems. Electropherograms of crude plant cell extracts from Digitalis lanata were obtained with this method.

Valproic acid-induced carbamazepine-10,11-epoxide toxicity in children and adolescents.

The study of 14 children and adolescents shows that the addition of carbamazepine (CBZ) to a basic valproic acid (VPA) therapy can result in unexpectedly high concentrations of carbamazepine-10,11-epoxide (CE) in the serum (up to 13 micrograms/ml). These concentrations were associated with marked side effects, especially vomiting and tiredness. The concentrations of CBZ were within the therapeutic range. Very high CE concentrations can largely be avoided at the commencement of the CBZ treatment if the CBZ dose is slowly increased. But high CE concentrations (4-8 micrograms/ml) associated with side effects can also be reached in later stages during the build up of CBZ treatment and under steady state conditions. The determination of the CE concentration is important when VPA and CBZ are administered together, especially when side effects occur.