Eric H. Holmes

Current evaluation of the tissue localization and diagnostic utility of prostate specific membrane antigen

Current statistics indicate that prostate carcinoma is the most common form of cancer diagnosed in American men, resulting in the second highest cancer death rate. Early diagnosis and accurate staging are imperative given that there is little effective treatment for metastatic disease, especially after androgen deprivation therapy fails. Identification of new biochemical markers for disease progression will provide important tools for diagnosis and monitoring. One such potential marker is prostate specific membrane antigen (PSMA).

Influence of Lewis α1-3/4-L-Fucosyltransferase (FUT3) Gene Mutations on Enzyme Activity, Erythrocyte Phenotyping, and Circulating Tumor Marker Sialyl-Lewis a Levels

Fucosylated glycoproteins carrying α1-4 fucose residues are of importance for cell adhesion and as tumor markers. The Lewis gene, FUT3, encodes the only known α1-4-fucosyltransferase (FucT), and individuals who are deficient in this enzyme type as Lewis-negative on erythrocytes. We examined the mutational spectrum of the Lewis gene in Denmark and found 6 different mutations. Five, T59G, T202C, C314T, G508A, and T1067A, were frequent, and one, C445A, was only detected in one out of 40 individuals. Allele-specific polymerase chain reaction as well as cloning of FUT3 alleles showed that the 202 and 314 mutations were co-located on the same allele. COS7 cells transfected with an allele having the 202/314 mutations lacked enzyme activity. Polymerase chain reaction-cleavage assays were established for the genotyping of healthy individuals as well as 20 genuine Lewis-negative cancer patients and 10 non-genuine. The latter have Lewis-negative erythrocytes but saliva α1-4FucT activity. The genuine Lewis-negative individuals had mutations on both FUT3alleles. In 66 healthy individuals, a gene dosage effect was detected as FUT3 heterozygous individuals had a lower α1-4FucT activity in saliva than did homozygous wild-type individuals. The lower enzyme level in heterozygous individuals resulted in a significantly (p < 0.04) lower level of circulating sialyl-Lewis a structure in serum. This has the clinical impact that cut-off levels in tumor marker assays should be defined on the basis of genotyping. In the group of non-genuine Lewis-negative cancer patients, whose erythrocytes convert from Lewis-positive to Lewis-negative during the disease, FUT3 heterozygosity was significantly (p < 0.05) more common.

Structure-Function Analysis of Human 13Fucosyltransferases A GDP-FUCOSE-PROTECTED, N-ETHYLMALEIMIDE-SENSITIVE SITE IN FucT-III AND FucT-V CORRESPONDS TO Ser IN FucT-IV

Human α1→3fucosyltransferases constitute a family of closely related membrane-bound enzymes distinguished by differences in acceptor specificities and inherent protein biochemical properties. One such biochemical property is sensitivity to enzyme inactivation by sulfhydral-group modifying reagents such as N-ethylmaleimide. The basis for this property has been studied using a fusion protein of FucT-III and FucT-V composed of Protein A coupled to the catalytic domain of the enzyme. The results indicate that modification of FucT-V by 5,5′-dithiobis(2-nitrobenzoic acid) resulted in efficient enzyme inactivation that could be reversed by excess thiol reagent suggesting that the free sulfhydral group on the enzyme was required for activity. Recombinant forms of both FucT-III and FucT-V were irreversibly inactivated by N-ethylmaleimide and could be effectively protected from inactivation by GDP-fucose and GDP but not by UDP-galactose, fucose, or N-acetyllactosamine. Analysis of the distribution of Cys residues in aligned sequences of cloned human α1→3fucosyltransferases indicated one site, Cys143 of FucT-III and Cys156 of FucT-V, corresponded to the highly conservative replacement of Ser178 in FucT-IV, an enzyme insensitive to N-ethylmaleimide. A site-directed mutagenesis experiment was performed to replace Ser178 of FucT-IV with a Cys residue. The mutant FucT-IV enzyme was active; however, the Km for GDP-fucose was increased about 3-fold compared to the native enzyme to 28 ± 3 μM. This enzyme was N-ethylmaleimide sensitive and could be partially protected by GDP-fucose but not N-acetyllactosamine. These results support the importance of Ser178 of FucT-IV in donor substrate binding and strongly suggest analogous Cys residues are the GDP-fucose protectable, N-ethylmaleimide-sensitive sites present in FucT-III and -V.

Analysis of glycosylation of prostate-specific membrane antigen derived from LNCaP cells, prostatic carcinoma tumors, and serum from prostate cancer patients.

Prostate‐specific membrane antigen (PSMA) has been detected in human prostatic cancer tissues, serum, and seminal fluid based on Western blot data with the monoclonal antibody 7E11.C5. The reactive protein is very similar in size to that from human prostatic carcinoma LNCaP cells and corresponds to a protein with a molecular size of about 110,000 daltons. Given that PSMA is known to be a 750 amino acid protein of about 84,000 daltons, a substantial portion, perhaps 20–25% of the native molecular weight, is composed of carbohydrates.

Comparison of serum PSMA, PSA levels with results of cytogen-356 ProstaScint® scanning in prostatic cancer patients

Stored serum from clinical trial cases undergoing ProstaScint® (CYT‐356) scanning were available for Prostate Specific Membrane Antigen (PSMA) assay. Prostate Specific Antigen (PSA) levels had already been determined. This provided an opportunity to see what correlations existed between the serum markers and the ProstaScint® scan. A group of patients had the studies preprostatectomy, whereas another group had the studies postprostatectomy.

Measurement of serum prostate-specific membrane antigen, a new prognostic marker for prostate cancer

OBJECTIVES To describe current results with Western blot assay for prostate specific membrane antigen (PSMA) using 7E11.C5 antibody and the development of an additional antibody measurement for PSMA by a new sandwich immunoassay. METHODS A population of patients from a screening group, from a difficult diagnostic group, from a pre- and postoperative radical prostatectomy group, and from a group with metastatic disease followed for a serial period, provided the serum values for a prospective assessment of PSMA by Western blot assay. A new monoclonal antibody was sought, reacting to the C-terminal region of PSMA in order to develop a sandwich radioimmunoassay. RESULTS PSMA values in screened patients correlate with the more advanced stage of the cancers determined. In postprostatectomy patients, the PSMA value corresponds more with preoperative values and with the values of those with a poor clinical course. In difficult diagnostic cases, the PSMA value is increased, specifically in hormone-refractory cases and particularly in those cases judged by other criteria, such as the National Prostatic Cancer Project, to be in clinical progression compared with those judged to be in clinical remission. The level of PSMA value appears to be independent of homogeneous tumor volume and to be more related to that of prior hormone treatment, or to where prostate cancer cells can be documented to be outside the prostate. A new monoclonal antibody, 3F5.4G6, reacts with the extracellular domain of PSMA near the C-terminal region. This is in contrast to the previously measured antibody 7E11.C5, which reacts with an N-terminal epitope. 3F5.4G6 recognizes the same PSMA protein as does 7E11.C5. The epitopes are essentially at opposite ends of the molecule. The 3F5.4G6 antibody reacts with the LNCaP line but not with DU145, or PC3. These two antibodies to PSMA are well suited for use in a new sandwich immunoassay. CONCLUSIONS PSMA provides a prostatic cancer serum test by using Western blot, which suggests a clinical prognostic value not seen with other markers. New antibodies, such as 3F5.4G6, reacting with the extracellular domain of PSMA combined with 7E11.C5, appear to offer an opportunity for a new sandwich immunoassay.

Monoclonal antibody 7E11.C5 staining of viable LNCaP cells

Prostate‐specific membrane antigen (PSMA) is a transmembrane glycoprotein defined by the monoclonal antibody 7E11.C5. The 7E11.C5 antibody forms the basis of an in vivo diagnostic imaging agent (ProstaScint, Cyt‐356) for identification of metastatic prostate cancer. The epitope on PSMA recognized by 7E11.C5 has been determined to be the first 6 amino acids from the N‐terminal, expressed on the cytoplasmic side of the plasma membrane. Thus, the basis for 7E11.C5 specificity in imaging studies remains unclear.

Characterization and membrane organization of β1 → 3- and β1 → 4-galactosyltransferases from human colonic adenocarcinoma cell lines Colo 205 and SW403: Basis for preferential synthesis of type 1 chain lacto-series carbohydrate structures☆

Evidence indicates that activation of a beta 1----3N-acetylglucosaminyltransferase is responsible for accumulation of large quantities of lacto-series tumor-associated antigens in human colonic adenocarcinomas. Expression of type 1 and 2 core chain derivatives characterize human colonic adenocarcinomas, whereas normal adult colonic epithelial cells express detectable quantities of only type 1 chain derivatives. The basis for preferential synthesis of type 1 chain lacto-series carbohydrate structures characteristic of normal colonic mucosa and human colonic adenocarcinoma Colo 205 cells has been studied. The beta 1----3- and beta 1----4galactosyltransferase enzymes associated with synthesis of type 1 and 2 core chain structures, respectively, have been separated from a Triton X-100 solubilized membrane fraction of Colo 205 cells by chromatography on an alpha-lactalbumin-Sepharose column and their properties studied. Optimal transfer of beta 1----3-linked galactose to acceptor Lc3 occurred in the presence of 0.1% Triton CF-54 with Triton X-100 providing 75% of maximal activity. The enzyme was active over a broad pH range from 6.5 to 7.5 and had a near absolute requirement for Mn2+. The Km values for donor UDPgalactose and acceptor Lc3 were determined to be 48 and 13 microM, respectively. In contrast, the beta 1----4galactosyltransferase required taurodeoxycholate for maximal activity and the Km for Lc3 was found to be 20-fold higher than that for the beta 1----3-specific enzyme under the same assay conditions. Studies with membrane-bound beta 1----3- and beta 1----4galactosyltransferases as found in Golgi-rich membrane fractions of SW403 and Colo 205 adenocarcinoma cells showed that preferential synthesis of type 1 chain structures occurs under conditions similar to those in vivo for biosynthesis of lacto-series core chains. The results suggest that both the higher affinity of the beta 1----3galactosyltransferase for acceptor Lc3 and the membrane organizational features result in preferential synthesis of type 1 chain structures.

Method for identifying prostate cells in semen using flow cytometry

Prostate‐specific antigen (PSA) cannot differentiate benign prostatic hyperplasia (BPH), from prostatitis, or prostate cancer in the range of 4.0–10 ng/ml. An accurate cytologic or histologic assessment is necessary to confirm the proper diagnosis. The nature of a biopsy tends to make it a selective test not frequently repeated. We are reporting a technique employing semen as a source for the differential diagnosis of prostate epithelial cells.

Biosynthesis of fucose containing lacto-series glycolipids in human colonic adenocarcinoma Colo 205 cells☆

Biosynthesis of fucose containing lacto-series glycolipids has been studied in human colonic adenocarcinoma Colo 205 cells. Transfer of fucose in both alpha 1----3 linkage to type 2 chain acceptors and alpha 1----4 linkage to type 1 chain acceptors was demonstrated with a Triton X-100 solubilized membrane fraction. The enzyme was found to be highly active over a broad pH range between 6.0 and 7.5. Kinetics of the transfer reactions were studied and indicated that the enzyme had an apparent Km for GDPfucose of 53 and 49 microM with acceptors nLc4 and Lc4, respectively. The apparent Km values for acceptors Lc4, nLc4, and IV3NeuAcnLc4 were determined to be 42, 18, and 26 microM, respectively. Transfer of fucose to the type 1 chain acceptor Lc4 alone and in the presence of increasing concentrations of the type 2 chain acceptor IV3NeuAcnLc4 or Gb3 suggested that both type 1 and 2 acceptors were alternate acceptors for a single enzyme. This was further established by the finding that IV3NeuAcnLc4 behaved as a competitive inhibitor of fucose transfer with respect to Lc4. Conditions were defined for preparative scale in vitro synthesis of fucosylated products of nLc6 catalyzed by the Colo 205 cell enzyme. Yields of the monofucosyl derivative of 2.5 mg (46%) and 1 mg (17%) of the difucosyl derivative were obtained from 5 mg of original nLc6. The structures of these biosynthetic products were carefully studied by 1H NMR, +FAB-MS, and methylation analysis. These studies revealed extremely high purity products composed of III3FucnLc6 and III3V3Fuc2nLc6. The significance of the nature of these products and enzymatic properties is discussed.