Peter J. Wirth

Identification of gel-separated tumor marker proteins by mass spectrometry

Two‐dimensional gel electrophoresis with subsequent analysis by mass spectrometry was applied to study differences in protein expression between benign and malignant solid tumors from human beast, lung and ovary cells. Cells from freshly resected clinical material were lysed and the extracts were subjected to isoelectric focusing with immobilized pH gradients followed by second‐dimensional separation on 10—13% sodium dodecyl sulfate (SDS)/polyacrylamide gels. Polypeptides were identified using matrix‐assisted laser desorption/ionization and electrospray ionization mass spectrometry after in‐gel protein digestion. Some of the upregulated polypeptides in malignant cells are of potential importance as markers of tumor proliferation. Twenty such proteins were identified, ten constituting novel identifications and ten sequence verifications of previously gel‐matched proteins. The proteins identified span a wide range of functions, but several cases of protein truncation were found. Truncated forms of cytokeratins 6D and 8, and of cathepsin D were identified. Truncated froms of these overexpressed proteins support the presence of proteolytic processing steps in tumor material. The protein processing and the difference between protein and mRNA abundancies in tumors of different malignancy and origin suggest that studies at the protein level are important for an understanding of tumor phenotypes.

Specific polypeptide differences in normal versus malignant human breast tissues by two-dimensional electrophoresis

SummaryPostmitochondrial and cytosolic polypeptides were extracted from human breast tumors and non-malignant breast tissue and analyzed using high resolution two-dimensional polyacrylamide gel electrophoresis (2D-PAGE). Approximately 800–1000 postmitochondrial and 600–800 silver stained cytosolic polypeptides were detected over the pH range of 4.8 to 7.5 and molecular weight range of 18–120 kDa. The 2D-PAGE patterns of polypeptides from normal and malignant tissue were very similar, although both qualitative and quantitative polypeptide differences were noted. Six cytosolic polypeptides (pI/molecular weight × 10−3) 5.20/80 kDa, 5.75/43, 6.25/40, 5.43/35, 5.45/34.5, 5.50/34 and 6.15/24 were expressed only in malignant tissues. One constitutive polypeptide, 7.25/52, was not detected in any of the malignant tissue samples. Quantitatively, marked differences in spot density were noted in polypeptides localized mainly in the molecular weight ranges of 22–40 kDa and pI ranges of 5.65–7.00. A general increase in polypeptide expression was noted in malignant tissues as compared to normal. Twenty-two polypeptides were significantly and consistently increased in tumor samples while only one polypeptide was decreased. One polypeptide, p24 (6.15/24) was expressed in greatest concentrations in tumors which also expressed the greatest estrogen receptor content. Expression of p24 was markedly reduced in normal tissue and malignant tissues expressing low levels of estrogen and progesterone receptors.

Influence of conjugation reactions on the mutagenicity of aromatic amines

2-Acetylaminofluorene (AAF) and 2-aminofluorene (AF), as well as their N-hydroxylated metabolites, N-OH-AAF and N-OH-AF, were studied for mutagenic effects in Salmonella typhimurium with rat- and mouse-liver S9 and microsomal subfractions in the presence of cofactors for glucuronidation and glutathione (GSH) transfer. Addition of UDPGA did not affect the mutagenicity of AAF, AF or N-OH-AAF under any experimental condition. Addition of GSH, on the other hand, markedly inhibited AAF, AF and N-OH-AAF. This seemed to be due to the direct effect of GSH, and not through an enzyme-catalyzed conjugation. Further, GSH inhibited the direct mutagenicity of N-OH-AF.

Guanethidine N-oxide formation as a measure of cellular flavin-containing monooxygenase activity

The usefulness of guanethidine N-oxide formation as a measure of cellular flavin-containing monooxygenase activity was assessed using the purified hog liver enzyme, rat liver microsomes and hepatocytes. The apparent Km and Vmax for this reaction in hepatocytes were 0.30 +/- 0.20 mM and 0.81 +/- 0.36 nmole per 10(6) cells min-1 respectively. The Km for the purified enzyme was 0.31 mM and the Vmax was 0.56 nmole per microgram enzyme min-1. 2-Diethylaminoethyl-2,2-diphenyl valerate (SKF-525A) at a concentration of 0.5 mM had no effect on guanethidine N-oxide formation by either rat liver microsomes or the purified enzyme. In contrast 2,4-dichloro-6-phenylphenoxyethylamine (DPEA) at the same concentration caused greater than a 100% increase in the microsomal production of guanethidine N-oxide. The tertiary amines imipramine, chloropromazine and methylpyrilene inhibited N-oxide formation by both hepatocytes and the purified enzyme. These data indicate that guanethidine N-oxide formation can be used as a measure of cellular flavin-containing monooxygenase activity.

Induction by phenobarbital in McA-RH7777 rat hepatoma cells of a polycyclic hydrocarbon inducible cytochrome P450

The metabolism of 2-acetylaminofluorene (AAF) to its six oxidative metabolites has been used to study cytochrome P450 monooxygenase activity in two rat hepatoma cell lines, McA-RH7777 and Reuber H4-II-E. McA-RH7777 cells exhibited considerably higher basal activities than H4-II-E cells for all metabolic pathways studied. Phenobarbital induced AAF metabolite formation in McA-RH7777 cells to a similar extent as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), but was only a weak inducer of these activities in H4-II-E cells. Northern blot analysis utilizing specific phenobarbital or 3-methylcholanthrene inducible cytochrome P450 cDNA probes indicated that there was at least a 10-fold increase in a 3-methylcholanthrene inducible cytochrome P450 transcript in phenobarbital treated McA-RH7777 cells. These data suggest that in this transformed cell line phenobarbital behaves as a polycyclic hydrocarbon-like inducer.

The role of biliary excretion in the hepatotoxicity of furosemide in the mouse

Abstract [ 35 S]Furosemide, administered iv or ip, was shown by autoradiography to accumulate in the intestinal tract, indicating that excretion through the biliary system occurs. Direct measurement of furosemide in bile showed this process to be both dose dependent and saturable, T m = 350 μg/min/kg. Pretreatment with sulfobromophthalein (BSP) increased the hepatic necrosis of furosemide by blocking its biliary excretion. BSP, when equilibrated with serum, did not alter furosemide binding to serum proteins. Furosemide is tightly bound to serum proteins, showing two classes of binding sites, K D = 7.5 × 10 4 and 1.1 × 10 3 . Increases in the total metabolism of furosemide and in the extent of covalent binding of furosemide metabolites to liver proteins in vivo paralleled the increases in hepatic necrosis observed at high doses. It is concluded that biliary excretion of furosemide in the mouse is an important elimination pathway and that, when the biliary excretion is saturated by high doses of furosemide, or partially blocked by BSP, hepatic necrosis occurs.

Structural studies and two-dimensional gel electrophoresis of γ-glutamyl transpeptidase

Abstract The heterodimeric enzyme γ-glutamyl transpeptidase (EC 2.3.2.2) was isolated from adult rat kidney and purified to homogeneity for structural studies using papain solubilization and multiple chromatographies. Two-dimensional gel electrophoresis was found to resolve the active papain-purified enzyme into at least 18 components. Seven components with apparent molecular masses of 23,000–26,000 and isoelectric point range of 5.4–7.0 constitute the light subunit, and 11 components with apparent molecular mass of 51,000–53,000 and isoelectric point range of 5.8–7.1 constitute the heavy subunit. Immunoblot analysis of two-dimensional gels showed that all of these components are immunoreactive with a mixture of the two antibodies generated separately against the light and heavy subunits. Preparative subunit separation was achieved using reversephase HPLC under acidic but nonreducing conditions. N-Terminal amino acid sequencing of the separated subunits of the papain-purified enzyme yielded sequence information for the first 32 residues of the heavy chain with the N-terminal starting sequence Gly LysProAspHisValTyrSerArgAla, and for the first 36 residues of the light subunit with the N-terminal starting sequence ThrAlaHisLeuSerValValSerGluAsp.