Spontaneous and pronase‐induced HER2 truncation increases the trastuzumab binding capacity of breast cancer tissues and cell lines

A subgroup of HER2‐overexpressing breast tumours co‐expresses p95 $^{{\rm{HER2}}}$ , a truncated HER2 receptor that retains a functional HER2 kinase domain but lacks the extracellular domain, thus impairing trastuzumab binding. We evaluated p95 $^{{\rm{HER2}}}$ expression in 99 frozen breast carcinoma samples by western blot analysis. The HER2‐positive cell line BT474 treated with pervanadate or pronase was used as a positive control for p95 $^{{\rm{HER2}}}$ expression. Immunohistochemistry was performed on parallel formalin‐fixed, paraffin‐embedded sections of the same case series using antibodies directed against either the intra‐ or extra‐cellular binding domain of HER2. In particular, biotinylated trastuzumab (BiotHER) was used to evaluate the binding capacity of the humanized antibody. To avoid a subjective evaluation of the score values and the percentage of immunostained cells, the slides were scanned and automatically analysed. The number of cases with HER2 overexpression (score 3+) and HER2 gene amplification was higher in the p185 $^{{\rm{HER2}}}$ ‐positive/p95 $^{{\rm{HER2}}}$ ‐positive samples than in the p185 $^{{\rm{HER2}}}$ ‐positive/p95 $^{{\rm{HER2}}}$ ‐negative group. Automated analysis confirmed a significantly higher percentage of 3+ scored cells in p95 $^{{\rm{HER2}}}$ ‐positive cases. Conversely, the percentage of 2+ scored cells was higher in p95 $^{{\rm{HER2}}}$ ‐negative cases. The status of the HER2 extracellular domain was then studied using flow cytometry on BT474 cells after pronase enzymatic digestion using trastuzumab and pertuzumab, while the presence of HER2‐HER3 dimers was studied using a proximity‐ligation assay. In vitro experiments showed that short‐term pronase digestion of BT474 cells produced two HER2 fragments (of 95 and 150 kDa, detectable in tissue specimens as well), increased the binding affinity of trastuzumab, reduced the rate of HER2–HER3 dimers, and did not interfere with pertuzumab‐binding capacity. In conclusion, the presence of p95 $^{{\rm{HER2}}}$ as detected by western blot analysis does not compromise the immunohistochemical detection of HER2. Our data suggest that a reduction of the receptor steric hindrance as induced by enzymatic shedding may facilitate the binding capacity of trastuzumab. Copyright © 2012 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

An Access Route to Polyferrocenes via Modular Conjugation

The application of copper‐catalyzed azide/alkyne cycloaddition to monomers containing a ferrocene unit leads to polyferrocenes with ferrocene in the backbone. Catalyst performance and kinetic studies of a model system comprising 1,1'‐bis(azidoethyl)ferrocene and propargyl ether revealed the usage of CuI and DBU as the catalyst in DMF at 50 °C as efficient reaction conditions for the synthesis. The resulting polymers display molecular weight‐averages up to $\overline {M} _{{\rm n}} $ = 10 000 and $\overline {M} _{{\rm w}} $ = 33 000. The polyaddition of monomers both containing ferrocene units led to polyferrocenes with $\overline {M} _{{\rm n}} $ between 4 000 and 11 000 ($\overline {M} _{{\rm w}} $ up to 78 000). The present study thus provides a pathway to polyferrocenes based on modular ligation chemistry.

     

Enhancing the Activity and Thermal Stability of Iron Precatalysts Using 2‐(1‐{2,6‐bis[bis(4‐fluorophenyl)methyl]‐4‐methylphenylimino}ethyl)‐6‐[1‐(arylimino)ethyl]pyridines

A series of 2‐(1‐{2,6‐bis[bis(4‐fluorophenyl)methyl]‐4‐methylphenylimino}ethyl)‐6‐ [1‐(arylimino)ethyl]pyridine ligands is synthesized and fully characterized. The corresponding iron complexes are prepared and characterized by single‐crystal X‐ray diffraction for representative iron complexes, among other methods, revealing a pseudo square‐pyramidal geometry at the iron center. Upon activation with MMAO, all iron precatalysts exhibit high activity in ethylene polymerization, producing linear polyethylene. The observed activity is the highest reported for iron‐based precatalysts of this type. The polymerization parameters are shown to strongly affect the catalytic behavior, and both the activity and the polymer properties (i.e. $ \overline{M}_{\rm w}$ or $ \overline{M}_{\rm w}$ /$ \overline{M}_{\rm n}$ ) can be controlled.     

Characterization of the timing and prevalence of receptor tyrosine kinase expression changes in oesophageal carcinogenesis

Despite being common in epithelial malignancies, the timing of receptor tyrosine kinase (RTK) up‐regulation is poorly understood and therefore hampers the identification of the receptor to target for effective treatment. We aimed to determine if RTK expression changes were early events in carcinogenesis. Oesophageal adenocarcinoma and its pre‐invasive lesion, Barrett's oesophagus, were used for immunohistochemical analysis of the RTK panel, EGFR, ErbB2, ErbB3, Met, and FGFR2, by utilizing a cohort of patients with invasive disease ($n = 367$ ) and two cohorts with pre‐invasive disease, one cross‐sectional ($n = 110$ ) and one longitudinal in time ($n = 91$ ). The results demonstrated that 51% of oesophageal adenocarcinomas overexpressed at least one of the RTK panel, with 21% of these overexpressing multiple receptors. Up‐regulation of RTK expression was an early event corresponding with low‐grade dysplasia development (25% in areas without dysplasia versus 63% in low‐grade dysplasia, $p < 0.001$ ). There was a trend for an increase in the prevalence of concomitant overexpression of multiple receptors as intestinal metaplasia progressed to low‐grade dysplasia, 7% versus 10%; and from low‐grade dysplasia to high‐grade dysplasia, 10% versus 19% ($p = 0.06$ and 0.24, respectively). The timing of receptor up‐regulation varied; FGFR, ErbB2, and Met overexpression occurred as dysplasia first developed, whilst EGFR overexpression was predominately seen in invasive disease and ErbB3 overexpression was uniformly rare. We provide evidence for a frequent and early role for multiple different RTKs in oesophageal carcinogenesis. Given the early timing of receptor deregulation, inhibiting RTKs in pre‐invasive disease may also represent a novel and effective chemopreventive strategy. Copyright © 2012 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Molar Mass and Microstructure Analysis of PI‐b‐PMMA Copolymers by SEC‐NMR

The online coupling of size‐exclusion chromatography and NMR is used to characterize block copolymers consisting of polyisoprene (PI) and poly(methyl methacrylate) (PMMA) regarding their distributions of molar mass (MMD) and chemical composition (CCD). Using the CCD, $\overline{M}_{\rm n}$ and $\overline{M}_{\rm w}$ are calculated on the basis of PI and PMMA homopolymer calibrations. The microstructure distribution of PMMA and the distribution of isomeric units of PI in dependence of molar mass is also demonstrated. Furthermore, a simulation analysis is presented for a bimodal eluting sample. It allows for full separation, quantification and molar mass determination of the coeluting co‐ and homopolymer fractions. The quantification of the fractions is verified by liquid chromatography at critical conditions.     

Thermally Curable Polyoxanorbornene by Ring Opening Metathesis Polymerization

A novel norbornene monomer bearing a thermally curable benzoxazine group is synthesized and polymerized by ROMP using the Grubbs first‐generation ruthenium catalyst. The $\overline {M} _{{\rm w}} $ of the side‐chain benzoxazine functionalized polyoxanorbornene can be up to 87 000 Da with unimodal molecular‐weight distribution and narrow polydispersity between 1.18 and 1.65. Thermally activated ring‐opening polymerization of benzoxazine moieties leads to the formation of highly cross‐linked structures. The effects of molecular weight on the thermal ROP of polybenzoxazine precursors are examined.

     

Bismuth‐Triflate‐Catalyzed Polymerizations of ε‐Caprolactone

εCL was polymerized using the triflates of lanthanum, samarium, magnesium, aluminum, scandium, and bismuth as catalysts. Bismuth triflate proved to be extraordinarily reactive, and catalyzed polymerizations of εCL even at 20 °C. Adding DTBMP reduced the polymerization rate only slightly. Furthermore, no evidence of a cationic mechanism was found by end‐group analyses. Polymerization at 20 °C either in bulk or in solution only yielded polyesters of low or medium molecular weights ($\overline {M} _{{\rm n}} $ up to 30 000 Da). Yet addition of alcohols allowed for a proper control of molecular weight and end‐groups. Additionally, low catalyst concentrations and low temperature resulted in narrow molecular weight distributions and polylactones almost free of cyclic compounds.

     

Parallel Synthesis of Polymer Libraries Using Atom Transfer Radical Polymerization (ATRP)

Parallel atom transfer radical polymerization (ATRP) of styrene and t‐butyl acrylate (t‐BA) was investigated. A series of ATRP polymerizations were carried out in parallel with varying targeted molecular weight ( ) and showed excellent reproducibility of , polydispersity, and conversion. In addition, polymerizations were done to determine the effect of inhibitor on styrene polymerization, yielding similar results. In synthesizing a library of polymers having varying using ATRP, polymers having lower reach their target values earlier and the others continue to react under heating, resulting in peak broadening for the low polymers. Reinitiation experiments indicated that termination reactions were taking place resulting in “dead” polymer chains which were unable to reinitiate polymerization.

     

Polymer Loops vs. Brushes on Surfaces: Adsorption,Kinetics, and Viscoelastic Behavior of α,ω‐Thiol Telechelics on Gold

Telechelic thiopolymers were explored towards formation of doubly‐bound (polymer loops) vs. singly‐bound (polymer brushes) chains on gold substrates. The conformation, adsorption kinetics, and viscoelastic properties of the α,ω‐dithiol and monothiol PS telechelics were investigated by means of XPS and QCM‐D techniques. The oxidation behavior of the free thiol proved to be important for distinguishing doubly‐bound vs. singly‐bound chains. The results show a critical dependence of $\overline {M} _{{\rm n}} $ on the ability to obtain polymer loops. Comparison with unperturbed dimensions obtained by scaling theory show that the loop is less stretched and occupies more lateral space than the brush. The results are important in demonstrating the different and perhaps superior properties of polymer loops vs. singly‐bound polymer brushes.

     

Enzymatic Synthesis and Chemical Recycling of Polythiocaprolactone

High‐molecular‐weight polythiocaprolactone (PTCL) was prepared in a green process via lipase‐catalyzed ROP of a cyclic 6‐mercaptohexanoic acid (6MH) oligomer. PTCL was readily depolymerized by lipase to cyclic 6MH in dilute toluene solution, which was then readily repolymerized by the same lipase to produce PTCL with the same $\overline {M} _{{\rm w}} $ as the initial PTCL in a chemical recycling process. The T_m of PTCL was higher than that of the corresponding PCL. A P(TCL‐co‐CL) copolymer with 60 mol‐% TCL (6MH) units showed a higher T_m as the PCL homopolymer. Similar apparent K_m values were obtained for the cyclic 6MH oligomers and caprolactone oligomers, however, the V_max of cyclic 6MH oligomers was significantly lower than that of the corresponding caprolactone oligomers.

     

Amphiphilic PEG‐b‐PMCL‐b‐PDMAEMA Triblock Copolymers: From Synthesis to Physico‐Chemistry of Self‐Assembled Structures

A synthetic route toward a new family of amphiphilic mPEG‐b‐PMCL‐b‐PDMAEMA triblock copolymers is reported. Chemical structures and compositions are confirmed by ¹H NMR and SEC. Polydispersity indices are typically <1.4, indicating good control of the reactions. The physicochemical parameters associated with mPEG‐b‐PMCL‐b‐PDMAEMA self‐assembled structures are investigated. Nanoparticles are prepared via a co‐solvent method, and parameters such as nanoparticle $\overline {M} _{{\rm w}} $ , N_agg, A₂, and R_h are calculated based on static and dynamic light scattering data. Critical aggregation concentrations for the polymers are determined by measuring surface tensions of polymer solutions. TEM is employed to visualize the morphology of the assemblies.

     

Spontaneous Deposition of FITC‐Labeled Dextran into Covalently Assembled (PGMA/PAH)4 Microcapsules

Spontaneous deposition of fluorescein isothiocyanate labeled dextran (FITC‐dextran) in covalently assembled poly(glycidyl methylacrylate) (PGMA)/poly(allylamine hydrochloride) (PAH) microcapsules was studied and its mechanism was proposed. Incubation of the microcapsules in the FITC‐dextran solution resulted in strong fluorescence emission from the capsule interiors. The deposition was found to be molecular weight ( ) dependent and the number of capsules filled with FITC‐dextran was increased along with the of FITC‐dextran. Furthermore, the correlation between the deposited substances and the charged species in the capsules was explored quantitatively through UV–Vis and fluorescence spectroscopy.

     

Controlled RAFT Polymerization of N‐Vinylphthalimide and its Hydrazinolysis to Poly(vinyl amine)

Polymerization of NVPI was carried out by a RAFT process using five xanthate‐type, a dithiocarbamate‐type, and a dithioester‐type CTA. The xanthate‐type [O‐ethyl‐S‐(1‐ethoxy carbonyl) ethyl dithiocarbonate and O‐ethyl‐S‐(1‐ethoxycarbonyl‐1‐methyl)ethyl dithiocarbonate] and the dithiocarbamate‐type CTA (benzyl‐1‐pyrrolecarbodithioate) were the most efficient to obtain poly(NVPI) with controlled molecular weights ( = 4 100–13 000) and low polydispersities ( = 1.29–1.38). The effects of parameters such as solvent, temperature, and CTA‐to‐initiator molar ratio, were examined in order to determine the conditions leading to optimal control of the polymerization.

     

Influence of Molecular Weight on Physical and Mechanical Properties of Linear Symmetric S‐(S/B)‐S Triblock Copolymers

Symmetric linear S‐(S/B)‐S triblock copolymers with 80 wt.‐% of PS having a block composition of 20‐60‐20 and a S/B ratio of 70:30 in the middle block are studied with varying molecular weights ( ). Increase in the leads to a change in the morphology from disordered to ordered microphase separated structure, as characterised by TEM and SAXS. Two distinct glass transitions for PS‐ and PB‐rich phases are observed from DMA measurements, which are due to phase separation at high . Rheological studies (master curves) reveal an extended rubbery plateau and a delay in the terminal response with an increase in , whereas a terminal flow behaviour is observed for materials having a disordered nature at low . A brittle‐to‐tough transition is observed from tensile tests with the increase in .

     

p53 mutation and loss have different effects on tumourigenesis in a novel mouse model of pleomorphic rhabdomyosarcoma

Pleomorphic rhabdomyosarcoma is the most common variant of this tumour in adults and has a very poor outcome. Two genes which are known to play a role in rhabdomyosarcoma development are KRas and p53. In the majority of human tumours, p53 abnormalities are point mutations that result in the expression of a mutant form of the protein. It is now hypothesized that these mutant forms of p53 may be playing an oncogenic role, over and above simple loss of the wild‐type function. In this study, we use Cre‐LoxP technology to develop a novel mouse model of rhabdomyosarcoma, crossing mice expressing a common KRas mutation (G12V) with mice that either lose p53 expression or express a mutant form of p53. We use this model to explore the different effects of p53 loss and mutation in the setting of an activating KRas mutation. We found that either complete loss of p53 (p53 ) or the expression of one mutant p53 allele with concomitant loss of the second allele (p53 ) resulted in the rapid development of rhabdomyosarcoma in 15/16 and 19/19 mice, respectively. In contrast, there was a marked difference between mice which lose a single copy of p53 (p53 ) and mice expressing a single copy of mutant p53 (p53 ). Fourteen out of 16 p53 mice developed rhabdomyosarcoma, compared with two out of 31 p53 mice. As a consequence of this, p53 mice had a median lifespan nearly double that of the p53 mice. To underline the enhanced effect of p53 mutation in tumour progression, metastases were seen only in those mice which expressed the mutant form. These data demonstrate that mutant p53 can co‐operate with activated, mutant KRas to influence tumourigenesis and metastatic potential, over and above simple loss of normal protein function. Copyright © 2010 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Long‐Chain Branches in Syndiotactic Polypropene Induced by Vinyl Chloride

Syndiotactic polypropene was synthesized using a metallocene catalyst system and traces of VC. The use of VC was found to decrease the molar mass of the samples distinctly and linearly, while retaining the molar mass distribution, the crystallinity, and the syndiotacticity. The VC, however, initiates the formation of long‐chain branches, which were detected by GPC‐MALLS and rheology. Besides their effect on the viscosity and modulus functions, a thermorheologic complexity can be observed. Also the flow activation energy E_a was found to be increased. The modified samples did not obey the relation for linear sPP, indicating the presence of LCBs.

     

Direct Monitoring of the Cross‐Over from Diffusion‐Controlled to Decomposition‐Controlled Initiation in Free Radical Polymerization

High‐resolution free radical polymerization kinetics was obtained using automatic continuous online monitoring of polymerization reactions (ACOMP). A sharp cross‐over from diffusion‐controlled initiation at low [monomer] to initiator decomposition control at higher [monomer] was found, and agrees with the quasi‐steady state approximation (QSSA). The cross‐over was also measurable within individual experiments. The kinetic implications for polymer weight average molecular weight and intrinsic viscosity (η)_w were analyzed, and the QSSA‐predicted trends for the ratios of final to initial , and (η)_w, confirmed. Analytical expressions for conversion are contrasted, and it was found that first‐order fits, while not fully justified theoretically, nonetheless are robust, which simplifies calculations needed for controlling molecular weight distributions in “semi‐batch” reactions, where reagents are fed to the reactor through programmable flow profiles. At the low monomer concentrations used, there was no evidence that propagation or termination rate coefficients changed during the reactions.

     

Acetyl Halide Initiator Screening for the Cationic Ring‐Opening Polymerization of 2‐Ethyl‐2‐Oxazoline

Kinetic investigations on the cationic ring‐opening polymerization of 2‐ethyl‐2‐oxazoline were conducted using acetyl chloride, acetyl bromide, and acetyl iodide as initiators. Various polymerization temperatures ranging from 80 to 220 °C were applied under microwave irradiation. The resulting polymerization mixtures were characterized with GC and GPC for the determination of monomer conversion and molecular weight distribution, respectively. Well defined polymers with narrow molecular weight distributions ( = 6 000 Dalton, PDI ≈ 1.10) were obtained with all three initiators.

     

The Structure and Aggregation of Hydrogen‐Bonded Interpolymer Complexes of Poly(acrylic acid) With Poly(N‐vinylpyrrolidone) in Dilute Aqueous Solution

Hydrogen‐bonded complexation between high‐molecular‐weight poly(N‐vinylpyrrolidone) (PVP, $\overline{M}_{\rm w}$ = 650 kDa) and poly(acrylic acid) (PAA) with weight‐average molar weights between 0.9 and 50 kDa, is studied in dilute, salt‐free aqueous solution. The onset of complexation, aggregation, and the structure of the formed particles are strongly influenced by PAA molecular weight, solution composition, and the method of sample preparation. With increasing poly(acrylic acid) chain length, IPCs show higher stability against aggregation. IPCs based on high‐molecular‐weight PAA show core‐corona architecture ensuring good sterical stabilization, whereas for those containing the shortest polyacid a loose soft‐sphere structure without distinct borders of different chain densities is found.     

AB2 Functional Polyesters via Ring Opening Polymerization: Synthesis and Characterization

Aliphatic AB₂ functional polyesters were conveniently prepared by the ring opening polymerization of ε‐caprolactone and L ‐lactide in the presence of the AB₂ functional initiator 2,2‐bis(hydroxymethyl)propionic acid (bis‐MPA) and Sn(Oct)₂ as the catalyst. In L ‐lactide polymerization, both bis‐MPA hydroxyl groups initiated the polymerization reaction, but for ε‐caprolactone polymerization this depended on the monomer to initiator to catalyst ratio. Initiation at two hydroxyl groups occurred at high monomer to initiator ([M]/[I]) ratio and at high Sn(Oct)₂ to monomer ratio. The melting temperatures of the AB₂‐functional PLLA and PCL polymers were comparable to linear polymers with a equal to the per arm in the polymer.