Biomedical subjects

E H Holmes

Publications and source records attributed to E H Holmes.

At least 19 recordsLinked to original sources

PubMed2000

Human alpha 1,3/4 fucosyltransferases. Characterization of highly conserved cysteine residues and N-linked glycosylation sites.

Human alpha1,3 fucosyltransferases (FucTs) contain four highly conserved cysteine (Cys) residues, in addition to a free Cys residue that lies near the binding site for GDP-fucose (Holmes, E. H., Xu, Z. , Sherwood, A. L., and Macher, B. A. (1995) J. Biol. Chem. 270, 8145-8151). The participation of the highly conserved Cys residues in disulfide bonds and their functional significance were characterized by mass spectrometry (MS) analyses and site-directed mutagenesis, respectively. Among the human FucTs is a subset of enzymes (FucT III, V, and VI) having highly homologous sequences, especially in the catalytic domain, and Cys residues in FucT III and V were characterized. The amino acid sequence of FucT III was characterized. Peptides containing the four conserved Cys residues were detected after reduction and alkylation, and found to be involved in disulfide bonds. The disulfide bond pattern was characterized by multiple stage MS analysis and the use of Glu-C protease and MS/MS analysis. Disulfide bonds in FucT III occur between Cys residues (Cys(81) to Cys(338) and Cys(91) to Cys(341)) at the N and C termini of the catalytic domain, bringing these ends close together in space. Mutagenesis of highly conserved Cys residues to Ser in FucT V resulted in proteins lacking enzymatic activity. Three of the four mutants have molecular weights similar to wild type enzyme and maintained an ability to bind GDP, whereas the other (Cys(104)) produced a series of lower molecular weight bands when characterized by Western blot analysis, and did not bind GDP. FucTs have highly conserved, potential N-linked sites, and our mass spectrometry analyses demonstrated that both N-linked sites are modified with oligosaccharides.

Amino Acid Sequence↗

PubMed2000

A GDP-fucose-protected, pyridoxal-5'-Phosphate/NaBH(4)-sensitive lys residue common to human alpha1-->3Fucosyltransferases corresponds to Lys(300) in FucT-IV.

Human alpha1-->3/4fucosyltransferases (FucTs) contain a common essential pyridoxal-5'-phosphate(PLP)/NaBH(4) reactive, GDP-fucose-protectable Lys. For identification, site-directed mutants at lysines of FucT-IV and -VII were prepared and tested. Non conserved lysine mutants K119Y and K394Q were similar to wild-type FucT-IV. However, mutants of conserved lysines K228R and K300R were distinct. The specific activity of K228R was 2- to 3-fold lower but retained K(m) values for donor and acceptor substrates as wild-type FucT-IV. The specific activity of K300R was reduced over 400-fold with an apparent K(m) for GDP-fucose over 200 microM. FucT-VII mutants K169R and K240R (equivalent to K228R and K300R for FucT-IV, respectively) were inactive. No change in PLP/NaBH(4) sensitivity occurred with K119Y, K228R, and K394Q compared to wild-type FucT-IV. These and previous results (A. L. Sherwood, A. T. Nguyen, J. M. Whitaker, B. A. Macher, M. R. Stroud, and E. H. Holmes, J. Biol. Chem. 273, 25256-25260, 1998) demonstrate that of three conserved lysines in FucT-IV, two (Lys(228) and Lys(283)) are not involved in substrate binding but perhaps in catalysis. The third site, Lys(300), is involved in GDP-fucose binding and PLP/NaBH(4) inactivation.

Amino Acid Sequence↗

PubMed2000

Isolation and characterization of monoclonal antibodies specific for protein conformational epitopes present in prostate-specific membrane antigen (PSMA).

Prostate-specific membrane antigen (PSMA) is a 750-amino acid glycoprotein highly expressed in malignant prostate tissues. PSMA reacts with the murine monoclonal antibody 7E11.C5, whose binding epitope has been mapped to the N-terminal of the protein distributed on the cytoplasmic side of the plasma membrane. We have developed murine monoclonal antibodies specific for extracellular epitopes of PSMA. Three of these antibodies--1G9, 3C6, and 4D4--display distinct binding properties consistent with their recognition of conformational epitopes within native PSMA. Results indicate this panel of antibodies binds to native full-length PSMA, but not to fusion proteins containing portions of the linear sequence of the protein. Antibody binding is greatly reduced upon heat denaturation of native PSMA, and these antibodies do not detect PSMA by Western blot. Immunoprecipitation experiments demonstrate the ability of each to bind to full-length PSMA as well as PSM', a form of the protein missing the first 57 amino acids. These results indicate each antibody is specific for an epitope within the extracellular domain, a region spanning residues 44-750. Flow cytometric experiments indicate strong specific binding to live LNCaP cells. Antibody inhibition studies demonstrate that these antibodies recognize at least two distinct epitopes. Taken together, the results demonstrate that these antibodies are specific for native protein conformational epitopes within the extracellular domain. Their properties, in particular strong binding to live cancer cells, make them ideal candidates that are clearly superior to linear sequence epitope specific antibodies for in vivo applications.

Animals↗

PubMed2000

Oxidative DNA damage precedes DNA fragmentation after experimental stroke in rat brain.

Experimental stroke using a focal cerebral ischemia and reperfusion (FCIR) model was induced in male Long-Evans rats by a bilateral occlusion of both common carotid arteries and the right middle cerebral artery for 30-90 min, followed by various periods of reperfusion. Oxidative DNA lesions in the ipsilateral cortex were demonstrated using Escherichia coli formamidopyrimidine DNA N-glycosylase (Fpg protein)-sensitive sites (FPGSS), as labeled in situ using digoxigenin-dUTP and detected using antibodies against digoxigenin. Because Fpg protein removes 8-hydroxy-2'-deoxyguanine (oh8dG) and other lesions in DNA, FPGSS measure oxidative DNA damage. The number of FPGSS-positive cells in the cortex from the sham-operated control group was 3 +/- 3 (mean +/- SD per mm(2)). In animals that received 90 min occlusion and 15 min of reperfusion (FCIR 90/15), FPGSS-positive cells were significantly increased by 200-fold. Oxidative DNA damage was confirmed by using monoclonal antibodies against 8-hydroxy-guanosine (oh8G) and oh8dG. A pretreatment of RNase A (100 microg/ml) to the tissue reduced, but did not abolish, the oh8dG signal. The number of animals with positive FPGSS or oh8dG was significantly (P<0.01) higher in the FCIR group than in the sham-operated control group. We detected few FPGSS of oh8dG-positive cells in the animals treated with FCIR of 90/60. No terminal UTP nicked-end labeling (TUNEL)-positive cells, as a detection of cell death, were detected at this early reperfusion time. Our data suggest that early oxidative DNA lesions elicited by experimental stroke could be repaired. Therefore, the oxidative DNA lesions observed in the nuclear and mitochondrial DNA of the brain are different from the DNA fragmentation detected using TUNEL.

Animals↗

PubMed2000

Toward optimal PT use.

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Centers for Medicare and Medicaid Services, U.S.↗

PubMed1999

Oxidative damage to the c-fos gene and reduction of its transcription after focal cerebral ischemia.

We investigated oxidative damage to the c-fos gene and to its transcription in the brain of Long-Evans rats using a transient focal cerebral ischemia and reperfusion (FCIR) model. We observed a significant (p < 0.001) increase in the immunoreactivity to 8-hydroxy-2'-guanine (oh8G) and its deoxy form (oh8dG) in the ischemic cortex at 0-30 min of reperfusion in all 27 animals treated with 15-90 min of ischemia. Treatment with a neuronal nitric oxide synthase (nNOS) inhibitor, 3-bromo-7-nitroindazole (60 mg/kg, i.p.), abolished the majority but not all of the oh8G/oh8dG immunoreactivity. Treatment with RNase A reduced the oh8G immunoreactivity, suggesting that RNA may be targeted. This observation was further supported by decreased levels of mRNA transcripts of the c-fos and actin genes in the ischemic core within 30 min of reperfusion using in situ hybridization. The reduction in mRNA transcription occurred at a time when nuclear gene damage, detected as sensitive sites to Escherichia coli Fpg protein in the transcribed strand of the c-fos gene, was increased 13-fold (p < 0.01). Our results suggest that inhibiting nNOS partially attenuates FCIR-induced oxidative damage and that nNOS or other mechanisms induce nuclear gene damage that interferes with gene transcription in the brain.

8-Hydroxy-2'-Deoxyguanosine↗

PubMed1999

Analysis of the expression and enzymatic properties of alpha1-->3fucosyltransferase from human lung carcinoma NCI-H69 and PC9 cells.

An analysis of alpha1-->3fucosyltransferase expression and enzyme properties has been conducted in human lung carcinoma NCI-H69 and PC9 cells. The results indicate that multiple forms of alpha1-->3 fucosyltransferase are found in these cells. RT-PCR experiments using total RNA from NCI-H69 and PC9 cells amplified transcripts for three of these enzymes, FucT-IV, -VI, and -VII. Fucose transfer into glycolipid acceptors mediated by truncated chimeric and full length recombinant FucT-IV and -VI enzymes was examined. Both enzymes were found to be type 2 chain specific, but only FucT-VI efficiently transferred fucose to both neutral and sialylated acceptors. A truncated recombinant form of FucT-VI was capable of fucose transfer to the internal Glc residue of a variety of glycolipid acceptors. This property was not observed with the recombinant full length enzyme, suggesting the N-terminal portion of the protein, composed of the intracellular domain, transmembrane domain, and a part of the stem region, is involved in interactions with glycolipid acceptors. Using taurodeoxycholate as the detergent, the distribution of initial fucose transfer into nLc6catalyzed by recombinant full length enzyme indicated 34% of the mono-fucosyl product was fucosylated at the III-GlcNAc and 66% at the V-GlcNAc for FucT-IV, and almost all of the FucT-VI mono-fucosyl product was III-GlcNAc fucosylated. Similar experiments with VI2NeuAcnLc6as the acceptor resulted in predominantly III-GlcNAc monofucosylation, although detectable V-GlcNAc monofucosylation was obtained with FucT-VI. When the cationic detergent G-3634-A was used, substantially greater initial transfer into the V-GlcNAc of both neutral and sialylated acceptors with FucT-VI was observed. Using nonsialylated acceptors, total alpha1-->3 fucosyltransferase activity in NCI-H69 cells was analyzed and found to be diminished 25-30% by exposure to 30 mM NEM, which can be attributed to FucT-VI inactivation. The remaining 70-75% of NEM-resistant activity is attributed to FucT-IV, an NEM-resistant enzyme form capable of fucosylating nonsialylated acceptors. These results suggest that multiple forms of alpha1-->3fucosyltransferase are expressed in NCI-H69 and PC9 cells, which may account for the observed properties of enzyme derived from these cell lines.

Animals↗

PubMed1998

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

BACKGROUND: 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). METHODS: A review was conducted to identify reports concerning evaluation of diagnostic applications of PSMA. RESULTS: PSMA is a membrane-bound glycoprotein that is highly restricted to the prostate. Immunohistochemical findings indicate that PSMA is a marker of epithelial cells of the prostate. This expression is increased in association with prostate carcinoma, particularly in hormone-refractory disease. Given its membrane-bound character, PSMA has been exploited as a marker for tumor detection by immunoscintiscanning with the 111In-labeled anti-PSMA monoclonal antibody 7E11.C5. Increased concentrations of 7E11.C5-reactive antigen are present in the serum of prostate carcinoma patients compared with healthy individuals; also, hematogenous circulating prostate carcinoma cells are detectable with reverse transcriptase-polymerase chain reaction analysis with primers specific for PSMA. New monoclonal antibodies specific for extracellular portions of the PSMA molecule currently are being utilized in applied studies. CONCLUSIONS: PSMA is a widely used marker for prostate epithelial cells. Its up-regulation in association with cancer, particularly in advanced cancer, is ideal for application as a prognostic marker. A variety of promising clinical applications utilizing PSMA have been or are being developed. In the future, these promise to have an important impact on cancer diagnosis and patient treatment.

Antigens, Surface↗

PubMed1998

Cloning of a novel member of the UDP-galactose:beta-N-acetylglucosamine beta1,4-galactosyltransferase family, beta4Gal-T4, involved in glycosphingolipid biosynthesis.

A novel putative member of the human UDP-galactose:beta-N-acetylglucosamine beta1,4-galactosyltransferase family, designated beta4Gal-T4, was identified by BLAST analysis of expressed sequence tags. The sequence of beta4Gal-T4 encoded a type II membrane protein with significant sequence similarity to other beta1,4-galactosyltransferases. Expression of the full coding sequence and a secreted form of beta4Gal-T4 in insect cells showed that the gene product had beta1,4-galactosyltransferase activity. Analysis of the substrate specificity of the secreted form revealed that the enzyme catalyzed glycosylation of glycolipids with terminal beta-GlcNAc; however, in contrast to beta4Gal-T1, -T2, and -T3, this enzyme did not transfer galactose to asialo-agalacto-fetuin, asialo-agalacto-transferrin, or ovalbumin. The catalytic activity of beta4Gal-T4 with monosaccharide acceptor substrates, N-acetylglucosamine as well as glucose, was markedly activated in the presence of alpha-lactalbumin. The genomic organization of the coding region of beta4Gal-T4 was contained in six exons. All intron/exon boundaries were similarly positioned in beta4Gal-T1, -T2, and -T3. beta4Gal-T4 represents a new member of the beta4-galactosyltransferase family. Its kinetic parameters suggest unique functions in the synthesis of neolactoseries glycosphingolipids.

Amino Acid Sequence↗

PubMed1998

Human alpha1,3/4-fucosyltransferases. I. Identification of amino acids involved in acceptor substrate binding by site-directed mutagenesis.

In a previous study (Xu, Z., Vo, L., and Macher, B. A. (1996) J. Biol. Chem. 271, 8818-8823), a domain swapping approach demonstrated that a region of amino acids found in human alpha1, 3/4-fucosyltransferase III (FucT III) conferred a significant increase in alpha1,4-FucT acceptor substrate specificity into alpha1, 3-fucosyltransferase V (FucT V), which, under the same assay conditions, has extremely low alpha1,4-FucT acceptor substrate specificity. In the current study, site-directed mutagenesis was utilized to identify which of the eight amino acids, associated with alpha1,4-FucT acceptor substrate specificity, is/are responsible for conferring this new property. The results demonstrate that increased alpha1,4-FucT activity with both disaccharide and glycolipid acceptors can be conferred on FucT V by modifying as few as two (Asn86 to His and Thr87 to Ile) of the eight amino acids originally swapped from FucT III into the FucT V sequence. Neither single amino acid mutant had increased alpha1,4-FucT activity relative to that of FucT V. Kinetic analyses of FucT V mutants demonstrated a reduced Km for Galbeta1,3GlcNAc (type 1) acceptor substrates compared with native FucT V. However, this was about 20-fold higher than that found for native FucT III, suggesting that other amino acids in FucT III must contribute to its overall binding site for type 1 substrates. These results demonstrate that amino acid residues near the amino terminus of the catalytic domain of FucT III contribute to its acceptor substrate specificity.

Amino Acid Sequence↗

PubMed1998

Human alpha1,3/4-fucosyltransferases. II. A single amino acid at the COOH terminus of FucT III and V alters their kinetic properties.

An analysis of the acceptor substrate specificity of domain swap mutants of human alpha1,3/4-fucosyltransferases (FucTs) III and V has been carried out. The results demonstrate that changing Asp336 of FucT III to Ala (as in FucT V) produced a protein (III/V1) with a reduced activity with a variety of acceptors. An analysis of the kinetic properties of FucT III and the III/V1 mutant demonstrated that III/V1 had a 40-fold reduction in its affinity for the H-type 1 acceptor substrate (Fucalpha1,2Galbeta1,3GlcNAc) and 4-fold reduction in its affinity for GDP-fucose when compared with FucT III. Further, the overall catalytic efficiency of III/V1 was approximately 100-fold lower than that of FucT III with an H-type 1 acceptor substrate. The complementary domain swap resulting from the change of Ala349 of FucT V to Asp (V/III1) produced a FucT that had higher enzyme activity with a range of acceptor substrates and had a higher affinity for an H-type 2 acceptor substrate (Fucalpha1, 2Galbeta1,4GlcNAc) with an 8-fold higher overall catalytic efficiency than that of FucT V. No significant change occurred in the Km for GDP-fucose for this protein when compared with FucT V. Kinetic parameters of two other FucT domain swaps (III8/V and V8/III), resulting in proteins that differed from FucT III and V at the NH2 terminus of their catalytic domain, were not significantly different from those of the parental enzymes when H-type 1 and H-type 2 acceptor substrates were utilized. Thus, substitution of an acidic amino acid for a nonpolar amino acid (i.e. Asp versus Ala) at the COOH terminus of FucTs produces an enzyme with enhanced enzyme activities. These results, together with the results presented in the accompanying papers (Nguyen, A. T., Holmes, E. H., Whitaker, J. M., Ho, S., Shetterly, S., and Macher, B. A. (1998) J. Biol. Chem. 273, 25244-25249; Sherwood, A. L., Nguyen, A. T., Whitaker, J. M., Macher, B. A., and Holmes, E. H. (1998) J. Biol. Chem. 273, 25256-25260), provide new insights into the structure/function relationships of human alpha1,3/4-FucT enzymes.

Amino Acid Sequence↗

PubMed1998

Human alpha1,3/4-fucosyltransferases. III. A Lys/Arg residue located within the alpha1,3-FucT motif is required for activity but not substrate binding.

Amino acid sequence alignment of human alpha1, 3/4-fucosyltransferases (FucTs) demonstrates that three highly conserved Lys residues are present in the catalytic domain of FucTs III, IV, V, and VI. Two of these sites are conserved in FucT VII, with the third located within the alpha1,3-FucT motif as a conservative change to Arg at position 223. Site-directed mutagenesis experiments were conducted to change Lys255 of FucT V (equivalent to Arg223 of FucT VII) to either Arg255 or Ala255. Enzyme assays demonstrate that the FucT V K255R mutant has a 34-fold lower specific activity than native FucT V and that the K255A mutant is inactive. Site-directed mutagenesis of FucT VII was also conducted to change Arg223 to Lys223 for analysis of the effect on enzyme kinetic parameters. No differences in acceptor specificities or Km values for either substrate were observed between native FucT VII and the R223K mutant; however, the purified R223K mutant enzyme had a 2-fold increased specific activity compared with purified native FucT VII. No change in GDP-fucose-protectable pyridoxal-P/NaBH4 inactivation was observed for native or mutant FucT V or VII, further supporting the absence of involvement of this residue in sugar nucleotide binding. The results indicate that a basic residue in this position is required for enzyme activity, with a Lys residue providing higher intrinsic activity. The lack of influence of this site on substrate binding parameters and its location within the alpha1,3-FucT motif suggest that at least some of the residues within this motif are involved in catalysis rather than substrate binding.

Amino Acid Sequence↗

PubMed1998

Method for identifying prostate cells in semen using flow cytometry.

BACKGROUND: 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. METHODS: Eleven vasectomized and nonvasectomized prostate cancer patients provided semen samples (stage T1 to T2). Two patients provided repeat samples. In addition, 15 vasectomized or nonvasectomized individuals without evidence of disease provided semen samples. Three million cells fixed with 50% ethanol were stained by an antibody (7E11.C5) to prostate-specific membrane antigen (PSMA), Hybritech Antibody (399) to PSA, and cytokeratin 8 and 18. In addition to the antibodies described, a DNA stain To-Pro 3 was used to identify 2n-4n DNA containing cells. A dual laser, Becton Dickinson FACSCaliber cytometer, was used to analyze the samples. RESULTS: All semen specimens contained diploid, cytokeratin 18-positive epithelial cells regardless of disease status. A clear difference between prostate cancer and normal prostate cell samples was observed using staining with 7E11.C5. The ratio of prostatic cells in the total epithelial cell population (PSMA:cytokeratin ratios) was calculated for each specimen. A retrospective study of sixteen semen samples from 11 prostate cancer patients had a mean PSMA:cytokeratin ratio of 0.57, whereas the samples from 15 patients without evidence of cancer had a mean PSMA:cytokeratin ratio of 0.11. This difference was significant. PSA staining was variable and inconsistent. CONCLUSIONS: This report demonstrates that human semen contains prostate cells that can be characterized and used in the clinical diagnosis of prostate cancer.

Adult↗

PubMed1998

Cloning and expression of the catalytic domain from rat hepatoma H35 cell GDP-fucose:GM1 alpha 1-->2fucosyltransferase, an enzyme which is activated during early stages of chemical carcinogenesis in rat liver.

A ganglioside GM1-specific alpha 1-->2fucosyltransferase is induced during the early stages of chemical carcinogenesis with N-2-acetylaminofluorene (AAF) in rat liver hepatocytes. The induction of this enzyme gives rise to the expression of a fucose-containing ganglioside with the same determinant structure as blood group B on a GM1 ganglioside core. Fucoganglioside synthesis is not found in normal rat liver but is elevated in premalignant liver and is often highly expressed in derived rat hepatoma cell lines. Based upon the consensus sequence from portions of previously cloned human, rabbit, and rat alpha 1-->2fucosyltransferase enzymes, primers were designed which were used in RT-PCR experiments with rat hepatoma H35 cell total RNA to generate cDNAs encoding the extracellular, catalytic domain of the H35 cell alpha 1-->2fucosyltransferase. Sequencing of these PCR fragments showed them to encode a novel enzyme with high homology to other cloned enzymes, particularly secretor alpha 1-->2fucosyltransferases. The derived sequence indicated that the 3' portion of the gene was virtually identical to the alpha 1-->2fucosyltransferase B (FTB) fragment reported earlier in rat PROb colon-adenocarcinoma cells (J-P. Piau et al. Biochem. J. 300, 623-626, 1994). A PCR product corresponding to the H35 cell alpha 1-->2fucosyltransferase was obtained from total RNA isolated from F344 rat liver after 0.03% N-2-acetylaminofluorene administration. No PCR product was obtained from total RNA isolated from normal F344 liver using PCR primers for the H35 cell alpha 1-->2fucosyltransferase. The H35 cell alpha 1-->2fucosyltransferase was expressed in the pPROTA vector and the derived fusion protein demonstrated the ability to transfer fucose to ganglioside GM1 but not to the neolacto-series acceptor nLcOse4Cer.

2-Acetylaminofluorene↗

PubMed1998

A family of human beta3-galactosyltransferases. Characterization of four members of a UDP-galactose:beta-N-acetyl-glucosamine/beta-nacetyl-galactosamine beta-1,3-galactosyltransferase family.

BLAST analysis of expressed sequence tags (ESTs) using the coding sequence of a human UDP-galactose:beta-N-acetyl-glucosamine beta-1, 3-galactosyltransferase, designated beta3Gal-T1, revealed no ESTs with identical sequences but a large number with similarity. Three different sets of overlapping ESTs with sequence similarities to beta3Gal-T1 were compiled, and complete coding regions of these genes were obtained. Expression of two of these genes in the Baculo virus system showed that one represented a UDP-galactose:beta-N-acetyl-glucosamine beta-1, 3-galactosyltransferase (beta3Gal-T2) with similar kinetic properties as beta3Gal-T1. Another gene represented a UDP-galactose:beta-N-acetyl-galactosamine beta-1, 3-galactosyltransferase (beta3Gal-T4) involved in GM1/GD1 ganglioside synthesis, and this gene was highly similar to a recently reported rat GD1 synthase (Miyazaki, H., Fukumoto, S., Okada, M., Hasegawa, T., and Furukawa, K. (1997) J. Biol. Chem. 272, 24794-24799). Northern analysis of mRNA from human organs with the four homologous cDNA revealed different expression patterns. beta3Gal-T1 mRNA was expressed in brain, beta3Gal-T2 was expressed in brain and heart, and beta3Gal-T3 and -T4 were more widely expressed. The coding regions for each of the four genes were contained in single exons. beta3Gal-T2, -T3, and -T4 were localized to 1q31, 3q25, and 6p21.3, respectively, by EST mapping. The results demonstrate the existence of a family of homologous beta3-galactosyltransferase genes.

Amino Acid Sequence↗

PubMed1998

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.

Antibodies, Monoclonal↗

PubMed1998

Isolation and characterization of monoclonal antibodies specific for the extracellular domain of prostate specific membrane antigen.

PURPOSE: Monoclonal antibodies specific for protein epitopes of prostate specific membrane antigen (PSMA) expressed on the external surface of prostatic epithelial cells were prepared to provide material for use in the diagnosis or treatment of prostatic cancer. MATERIALS AND METHODS: Mice were immunized with LNCaP cell membranes followed by purified PSMA before fusion. Hybridomas were screened by reactivity with purified PSMA. Resulting antibodies were characterized by enzyme-linked immunosorbent assay, Western blot and fluorescence-activated cell sorter analyses. RESULTS: Monoclonal antibody producing hybridomas designated 3E11, 3C2, 4E10-1.14, 3C9 and 1G3 were obtained which displayed specificities for differing regions of the extracellular domain of the PSMA protein. These antibodies reacted strongly with PSMA from multiple sources and specifically stained unfixed PSMA expressing cells by flow cytometric analysis. CONCLUSIONS: The antibodies obtained displayed strong reactivity and specificity for extracellular epitopes of PSMA. These antibodies will have value in future diagnostic and therapeutic applications focusing on PSMA as a target antigen.

Animals↗

PubMed1998

Prostate-specific membrane antigen: current and future utility.

Prostate cancer is the most common cancer and the second leading cause of cancer-related death among men. New prostatic markers are needed to increase diagnostic and prognostic effectiveness. One such new marker is prostate-specific membrane antigen (PSMA). PSMA is a highly prostate-restricted membrane glycoprotein that is expressed in normal prostatic epithelial cells and elevated in prostate cancers, especially in poorly differentiated, metastatic, and hormone refractory carcinomas. It has been measured in serum with immunocompetitive and Western blot assays, and its levels have been found to be correlated with the prediction of treatment failure and disease prognosis. Reverse transcriptase-polymerase chain reaction (RT-PCR) assays with primers specific for PSMA have been shown to be more effective than PSA-specific primers in detecting hematogenous circulating prostate cancer cells; however, no clear benefit in patient staging or utility as a predictor of clinical outcome or response to treatment has so far been obtained using RT-PCR methods. PSMA is currently utilized as an immunoscintigraphic target using the antibody conjugate CYT-356 (ProstaScint; Cytogen, Princeton, NJ) and has been shown to have clinical value, particularly in detecting occult prostate cancer. Another current application of PSMA is in immunotherapy of prostate cancer, in which promising results have been obtained in a phase I trial, and a phase II trial is underway. The research summarized in this article indicates that PSMA is an excellent target for diagnostic and therapeutic applications in prostate cancer.

Antigens, Neoplasm↗