Janet Weinstock

Antitumor efficacy of cytotoxic drugs and the monoclonal antibody 806 is enhanced by the EGF receptor inhibitor AG1478

Blockade of epidermal growth factor receptor (EGFR) signaling with specific inhibitors of the EGFR tyrosine kinase retards cellular proliferation and arrests the growth of tumor xenografts. AG1478, an inhibitor of the EGFR tyrosine kinase, is used in laboratory studies; however, its therapeutic potential has not been elucidated. Therefore, we evaluated an aqueous form of AG1478 for its antitumor activity in mice bearing human xenografts expressing the WT EGFR or a naturally occurring ligand-independent truncation of the EGFR [delta2–7 (de2–7) EGFR or EGFRvIII]. Parenteral administration of soluble AG1478 blocked phosphorylation of the EGFR at the tumor site and inhibited the growth of A431 xenografts that overexpress the WT EGFR and glioma xenografts expressing the de2–7 EGFR. Strikingly, even subtherapeutic doses of AG1478 significantly enhanced the efficacy of cytotoxic drugs, with the combination of AG1478 and temozolomide displaying synergistic antitumor activity against human glioma xenografts. AG1478 was also examined in combination with mAb 806, an anti-EGFR antibody that was raised against the de2–7 EGFR but unexpectedly also binds a subset of the EGFR expressed in cells exhibiting amplification of the EGFR gene. The combination of AG1478 and mAb 806 displayed additive, and in some cases synergistic, antitumor activity against tumor xenografts overexpressing the EGFR. Here, we demonstrate that different classes of inhibitors to the EGFR can have synergistic antitumor activity in vivo. These results establish the antitumor efficacy of the EGFR inhibitor AG1478 and provide a rationale for its clinical evaluation in combination with both chemotherapy and other EGFR therapeutics.

Influence of Interleukin-6 (IL-6) Dimerization on Formation of the High Affinity Hexameric IL-6·Receptor Complex

The high affinity interleukin-6 (IL-6) signaling complex consists of IL-6 and two membrane-associated receptor components: a low affinity but specific IL-6 receptor and the affinity converter/signal transducing protein gp130. Monomeric (IL-6M) and dimeric (IL-6D) forms of Escherichia coli-derived human IL-6 and the extracellular (“soluble”) portions of the IL-6 receptor (sIL-6R) and gp130 have been purified in order to investigate the effect of IL-6 dimerization on binding to the receptor complex. Although IL-6D has a higher binding affinity for immobilized sIL-6R, as determined by biosensor analysis employing surface plasmon resonance detection, IL-6M is more potent than IL-6D in a STAT3 phosphorylation assay. The difference in potency is significantly less pronounced when measured in the murine 7TD1 hybridoma growth factor assay and the human hepatoma HepG2 bioassay due to time-dependent dissociation at 37°C of IL-6 dimers into active monomers. The increased binding affinity of IL-6D appears to be due to its ability to cross-link two sIL-6R molecules on the biosensor surface. Studies of the IL-6 ternary complex formation demonstrated that the reduced biological potency of IL-6D resulted from a decreased ability of the IL-6D·(sIL-6R)2 complex to couple with the soluble portion of gp130. These data imply that IL-6-induced dimerization of sIL-6R is not the driving force in promoting formation of the hexameric (IL-6·IL-6R·gp130)2 complex. A model is presented whereby the trimeric complex of IL-6R, gp130, and IL-6M forms before the functional hexamer. Due to its increased affinity for the IL-6R but its decreased ability to couple with gp130, we suggest that a stable IL-6 dimer may be an efficient IL-6 antagonist.

High-performance liquid chromatographic analysis of the tyrphostin AG1478, a specific inhibitor of the epidermal growth factor receptor tyrosine kinase, in mouse plasma

The tyrphostin 4-(3-chloroanilino)-6,7-dimethoxyquinazoline (AG1478) is undergoing evaluation as a potential new anticancer agent. We have developed a specific and sensitive reversed-phase HPLC assay for AG1478 in mouse plasma. The method involves a rapid and simple extraction process followed by separation on a Symmetry C8 stationary phase with a gradient of acetonitrile in ammonium acetate buffer. A linear response was achieved over the concentration range of 0.2-100 microM using multilevel calibration with internal standard method of calculation. Inter- and intra-assay accuracy and precision were better than +/-10%. The limit of quantitation was 0.2 microM. We have used this method to study the preclinical pharmacokinetics of this new agent in mice.

ISOLATION AND PARTIAL AMINO ACID SEQUENCE OF A 78 KDA PORCINE GASTRIN-BINDING PROTEIN

1. A 78 kDa protein (p78) has been partially purified from washed membranes isolated from the corpus of porcine gastric mucosa. The purification was monitored by covalent cross-linking of iodinated [Nle15]-gastrin. 2. A single N-terminal sequence extending for 33 amino acids was obtained from the p78 preparation. Partial sequences totalling 192 amino acids were also obtained from 14 tryptic and 3 Staphylococcal V8 peptides. 3. 10 peptides plus the N-terminal sequence were derived from a previously unsequenced protein which was distantly related to the product of the E. coli fadB gene (Baldwin G. S. (1993) Comp. Biochem. Physiol. 104B, 55-61). The remaining 7 peptides were derived from the beta-subunit of the gastric H+/K(+)-ATPase. 4. The gastrin-binding activity remained in association with p78, and could be separated from the beta-subunit of the gastric H+/K(+)-ATPase, during chromatography on tomato lectin-Sepharose. 5. We conclude that p78 binds gastrin, and is a novel member of the enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase family of enzymes.

Binding of gastrin to gastric transferrin

Binding of 125I‐gastrin2,17 to porcine gastric transferrin has been demonstrated by covalent cross‐linking with disuccinimidyl suberate. The concentration of gastrin17 required to reduce cross‐linking by 50% was approx. 100 μM. The occurrence of both gastrin and gastric transferrin in porcine gastric mucosa and lumen suggests a novel synergistic role for the observed interaction in the uptake of dietary iron.

Isolation of transferrin from porcine gastric mucosa: Comparison with porcine serum transferrin

1. An iron-binding glycoprotein has been purified to homogeneity from porcine gastric mucosa. 2. The molecular weight (80,000), amino acid composition, carbohydrate content, N-terminal amino acid sequence, tryptic map, stoichiometry of iron binding (2 mol/mol), visible absorption spectrum of the ferric complex and chromatographic behaviour of the gastric protein are all strikingly similar to the corresponding properties of porcine serum transferrin. 3. The quantity of the gastric protein (1.3 mg/g wet weight) present in the gastric mucosa suggests that it is not serum transferrin (plasma concentration 1.8 mg/ml) contaminating the tissue. 4. A role for transferrin in the uptake of dietary iron by the gastrointestinal tract is proposed.

Colorectal Cancer Cell Line Proteomes are Representative of Primary Tumors and Predict Drug Sensitivity

BACKGROUND AND AIMS Proteomics holds promise for individualizing cancer treatment. We analyzed to what extent the proteomic landscape of human colorectal cancer (CRC) is maintained in established CRC cell lines and the utility of proteomics for predicting therapeutic responses. METHODS Proteomic and transcriptomic analyses were performed on 44 CRC cell lines, compared against primary CRCs (n=95) and normal tissues (n=60), and integrated with genomic and drug sensitivity data. RESULTS Cell lines mirrored the proteomic aberrations of primary tumors, in particular for intrinsic programs. Tumor relationships of protein expression with DNA copy number aberrations and signatures of post-transcriptional regulation were recapitulated in cell lines. The 5 proteomic subtypes previously identified in tumors were represented among cell lines. Nonetheless, systematic differences between cell line and tumor proteomes were apparent, attributable to stroma, extrinsic signaling, and growth conditions. Contribution of tumor stroma obscured signatures of DNA mismatch repair identified in cell lines with a hypermutation phenotype. Global proteomic data showed improved utility for predicting both known drug-target relationships and overall drug sensitivity as compared with genomic or transcriptomic measurements. Inhibition of targetable proteins associated with drug responses further identified corresponding synergistic or antagonistic drug combinations. Our data provide evidence for CRC proteomic subtype-specific drug responses. CONCLUSIONS Proteomes of established CRC cell line are representative of primary tumors. Proteomic data tend to exhibit improved prediction of drug sensitivity as compared with genomic and transcriptomic profiles. Our integrative proteogenomic analysis highlights the potential of proteome profiling to inform personalized cancer medicine.

Purification and partial amino acid sequence of annexin V from porcine gastric mucosal membranes

1. Annexin V has been purified from Triton X-100 extracts of porcine gastric mucosal membranes by a combination of chromatography on concanavalin A-Sepharose and DEAE-Sepharose, and preparative gel electrophoresis. 2. No N-terminal amino acid sequence was detected. 3. The sequences of 11 tryptic peptides were determined, amounting to a total of 121 amino acids, or 38% of the molecule. 4. When the peptides were compared with the cDNA-derived sequence of human annexin V, only three substitutions were observed. 5. Human and porcine annexin V are 97% homologous within the sequenced regions.