G. Michael Hass

Expression of the human cachexia-associated protein (HCAP) in prostate cancer and in a prostate cancer animal model of cachexia

Prostate cancer (CaP) patients with disseminated disease often suffer from severe cachexia, which contributes to mortality in advanced cancer. Human cachexia‐associated protein (HCAP) was recently identified from a breast cancer library based on the available 20‐amino acid sequence of proteolysis‐inducing factor (PIF), which is a highly active cachectic factor isolated from mouse colon adenocarcinoma MAC16. Herein, we investigated the expression of HCAP in CaP and its potential involvement in CaP‐associated cachexia. HCAP mRNA was detected in CaP cell lines, in primary CaP tissues and in its osseous metastases. In situ hybridization showed HCAP mRNA to be localized only in the epithelial cells in CaP tissues, in the metastatic foci in bone, liver and lymph node, but not in the stromal cells or in normal prostate tissues. HCAP protein was detected in 9 of 14 CaP metastases but not in normal prostate tissues from cadaveric donors or patients with organ‐confined tumors. Our Western blot analysis revealed that HCAP was present in 9 of 19 urine specimens from cachectic CaP patients but not in 19 urine samples of noncachectic patients. HCAP mRNA and protein were also detected in LuCaP 35 and PC‐3M xenografts from our cachectic animal models. Our results demonstrated that human CaP cells express HCAP and the expression of HCAP is associated with the progression of CaP and the development of CaP cachexia.

Interference in immunoassays by human anti-mouse antibodies

Mogensen and Moller [l] described interference in the Abbott CA 125 enzyme immunoassay (EIA) by putative human antimouse antibodies (HAMA).and demonstrated that this interference could be reduced by diluting the specimen with mouse serum. HAMA are predominantly IgGs that arise in response to immunizing doses of mouse monoclonal antibodies (Mabs) [2, 31. The high titres often found in HAMA sera should be distinguished from the low levels of anti-mouse activity resulting from heterophile antibodies, mostly IgM [4-81. Since HAMA are polyclonal and of complex specificity, many different types of interference can be observed in immunoassays with HAMAcontaining sera. The type of interference is dictated by the assay configuration (i.e. competitive vs. sandwich, one-step vs. twostep), the antibodies used (specificity, isotype and use as capture or probe reagent), the analyte and the subclass or isotype of the Mab used to immunize the patient. On injection of a patient with a Mab, HAMA are elicited that typically exhibit both anti-isotype and anti-idiotype specificities. The anti-isotype component may well bind to Mabs in an immunoassay and interfere, especially when the injected Mab and the Mabs used in the assay are of the same isotype. Elevated carcinoembryonic antigen (CEA) values were obtained in a patient who had been injected with mouse Mab B72.3 upon assay with the Abbott list 5863 product, which is a double monoclonal sandwich EIA in a two-step format (Table 1). That these increased values resulted from positive interference by HAMA was shown by reassay after removal of HAMA by either heat treatment or chromatography on immobilized protein A (patient 1, Table 2). Similar positive interferences in immunoassays have been reported for other commercially available immunoassays [2, 3, 9, lo]. These false positives, and presumably those described by Mogensen and Moller [ 11, result from HAMA bridging the probe and capture antibodies and were only partly corrected by diluting the specimen with mouse serum. Sandwich assay formats that we have evaluated and found to be most resistant to HAMA effects are double polyclonal assays or monoclonaYpolyclonal assays, in which the Mab is used as probe. Our CEA EIA One-Step (list 4439), which uses guineapig anti-CEA as capture antibody and a Mab as probe, yielded an appropriate value on the high titre HAMA specimen (Table 1). Efficient recovery of CEA (103%) added to this specimen demonstrated that false negatives were not produced. HAMA may also produce false negatives. For example, in immunoassays with mouse Mabs as capture antibodies and probes that are not mouse Mabs, HAMA binds to the solid phase Mab and sterically blocks capture of the antigen from the specimen, but does not recognize the probe. The addition of

The amino acid sequence of the activation peptide of bovine pro-carboxypeptidase A

The amino acid sequence of the activation peptide of bovine pro-carboxypeptidase A subunit I has been determined by automated Edman degradation of the cyanogen bromide fractions derived from the precursor protein. The activation peptide contains 94 amino acid residues in a unique sequence which precedes directly the amino-terminal alanine residue of carboxypeptidase A alpha. A notable feature of the activation peptide is the presence of acidic amino acid residues immediately preceding the site of activation. The amino acid sequence of the activation peptide of bovine pro-carboxypeptidase A shows extensive similarity to those of the corresponding porcine and rat enzymes.

Identification of the target of monoclonal antibody A6H as dipeptidyl peptidase IV/CD26 by LC MS\MS.

The monoclonal antibody (MAb) A6H, originally developed to fetal renal tissues, was found to be highly reactive to renal cell carcinoma and was subsequently demonstrated to co-stimulate a subpopulation of T cells. The A6H antigen had not been identified heretofore. Antigen from detergent extracts of renal cell carcinoma cells (7860) was immunoabsorbed with A6H-agarose, and the resin-bound proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The antigen had a molecular weight of approximately 120 kDa as determined by Western blots. The 120-kDa protein band was excised and subjected to in-gel tryptic digestion, and the resulting peptides were separated and analyzed by liquid chromatography tandem mass spectrometry (LC MS\MS). The tandem mass spectra of the eluting peptides were used in combination with the SEQUEST computer program to search a human National Cancer Institute (NCI) protein database for the identity of the protein. The target antigen was shown to be dipeptidyl peptidase IV (DPP IV), which is also known as the cluster differentiation antigen CD26. Flow analysis of the expression of the A6H antigen and of CD26 on 7860 cells and on peripheral blood lymphocytes supported the identification of the A6H antigen as DPP IV. Recognition that the A6H antigen is DPP IV/CD26 afforded the opportunity to compare previous studies on A6H with those on other anti-CD26 antibodies in terms of expression in cancer cell lines and various tissues and as co-stimulators of T-cell activation.

Modification of the acetyl and nitro derivatives of carboxypeptidase A by N-bromoacetyl-N-methyl-L-phenylalanine.

Glutamic acid 270 and tyrosine 248 have been implicated in the catalytic mechanism of carboxypeptidase A. The y-carboxylate of glutamic acid 270 is believed to function as nucleophile in the hydrolysis of peptide and ester substrates [l] . Modification of glutamic acid 270 either by Woodward reagent K [2,3] or by the affinity label, N-bromoacetyl-Nmethyl-Lphenylalanine (BrAcN(Me)Phe) [4,5] produces loss of enzymatic activity. The reaction of carboxypeptidase A with BrAcN(Me)Phe obeys saturation kinetics allowing separate estimation of the contributions of binding and reactivity (i.e. nucleophilicity and orientation) to the observed rate of inactivation. Modification of tyrosine 248 by several reagents, including N-acetylimidazole [7] produces enzyme derivatives with greatly reduced peptidase activity. Lipscomb has proposed that the phenolic hydroxyl of tyrosine 248 serves as proton donor in the hydrolysis of peptide substrates based both on the effect of chemical modification of tyrosine 248 and upon the location of this residue relative to the susceptible peptide bond as determined by X-ray crystallographic analysis [ 11. Alternatively, tyrosine 248 could function by orienting the substrate for nucleophilic attack by glutamic acid 270 rather than by direct participation in catalysis. The affinity labeling of the nitro and acetyl deriva-

Kinetics of the inactivation of trypsinogen by methanesulfonyl fluoride in the pH-stat.

Abstract Equations are derived describing the pseudo first-order inactivation of trypsinogen by excess methanesulfonyl fluoride in the pH-stat. From this analysis, the second-order rate constant for the reaction of trypsinogen and methanesulfonyl fluoride has been determined. The method described is widely applicable for the kinetic analysis of pseudo first-order reactions of enzymes and zymogens with hydrolyzable inhibitors in the pH-stat.