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The current information on the cloning and sequencing of four alkaline phosphatase genes (PLAP, GCAP, IAP, TNAP) has been reviewed. It has provided insights into their evolutionary history and the mechanisms of catalysis and of uncompetitive inhibition. The oncodevelopmental biology of the germ cell and its excessive GCAP eutopic expression in neoplasia are noted, and there is reason to suggest that the enzyme may serve to guide migratory cells and to transport specific molecules such as fat and immunoglobulins across membranes. The hyperexpression of all four genes has been observed in various human tumors and in their cell lines, particularly cancers of the testis and ovary. The membrane APs have been investigated as targets for immunolocalization and immunotherapy.

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This is the history of discoveries of several enzyme tumor markers in the awardees laboratory. The first, beta-glucuronidase, was originally related to the physiological actions of estrogens and androgens. Perfection of histochemical techniques based on new substrates demonstrated the dual localization of beta-glucuronidase in endoplasmic reticulum and lysosomes. Tumor tissues, in general, are enriched with beta-glucuronidase. Next, acid phosphatase of the prostate gland possesses the distinctive property of undergoing inhibition by L-tartrate. This organ-specific inhibitor was incorporated into the Fishman-Lerner method for measuring serum acid phosphatase of prostatic origin. This significantly increased the specificity of the measurement of serum acid phosphatase for prostatic cancer. Finally, the discovery of the Regan Isoenzyme, placental alkaline phosphatase (PLAP) in a patient with disseminated lung cancer provided a tumor marker useful in the management of gonadal tumors, in particular. Closely related to PLAP is germ cell alkaline phosphatase which is eutopically expressed in seminoma. Finally, radioimmunolocalization and radioimmunotherapy of PLAP in these tumors have been achieved by others.

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The past few years have witnessed the reports of significant new events in alkaline phosphatase (AP) isozymes. The cloning of the relevant genes and their nucleotide sequencing have all been accomplished. As a group, the genes for the intestinal, germ cell and placental isozymes have considerable sequence similarity; it is noteworthy that they occupy vicinal positions on chromosome 2, while the tissue unspecific AP gene is located on chromosome 1. The latter makes evolutionary lineage and instances of coordinate expression understandable. Another new development is the demonstration of a phosphatidyl inositol glycan tail on the C-terminus of these chromosome-2 AP genes. This is the major membrane insertion mechanism for AP, which is a cell surface membrane enzyme. This information may be helpful in understanding the phenomenon of the depletion of intestinal mucosal AP during fat absorption. Finally, a discussion has been focussed on recent studies on seminoma and AP, including immunodetection and immunoradiotherapy.

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In 1930 the determination of serum alkaline phosphatase in patients with bone or liver disease ushered in the era of clinical enzymology. The association of elevated (bone) alkaline phosphatase in serum of patients with osteogenic sarcoma was the first evidence that tumor cells themselves produced the enzyme. It became clear, however, in the 1960s that the serum alkaline phosphatase was not a single enzyme but consisted of a family of isozymes originating from liver, bone, intestine, and placenta. This was a consequence of the introduction of a combination of electrophoretic separations, heat inactivation, and organ-specific amino acid inhibitors. This combination of measurements made possible the demonstration of a serum alkaline phosphatase of lung cancer origin, as confirmed by the histochemical visualization in lung cancer of the Regan Isozyme (placental alkaline phosphatase-PLAP). At present, the measurement of PLAP has its greatest utility as a tumor marker in seminoma and ovarian cancer. A PLAP-like isozyme in normal testis and ovary is expressed in these and other neoplasias and appears to be related to rare alleles of placental alkaline phosphatase. Current studies have utilized a panel of monoclonal antibodies to detect useful epitopes that suggest that PLAP and PLAP-like isozymes are coded by different genes. The PLAP gene has now been cloned and sequenced by Millan and others. This fundamental new information is providing a base line that will make it possible to explain the overlapping specificities of intestinal and placental isozymes, the degree of uniqueness of the PLAP-like isozyme, the precise mechanism of uncompetitive inhibition by L-phenylalanine and the evolutionary history of the alkaline phosphatases.(ABSTRACT TRUNCATED AT 250 WORDS)

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Two sets of monoclonal antibodies (mAbs) specific for human placental alkaline phosphatase (PLAP) were compared. One set of four mAbs was generated against solubilized and purified PLAP; the other set of seven mAbs was generated against the malignant cell line Hela TCRC-1 in which PLAP is an ectopically synthesized membrane-bound enzyme. Double immunodiffusion and competitive enzyme-linked immunosorbent assays were used to examine the relative spatial arrangement of the antigenic determinants to which each of the eleven mAbs binds. Significant differences in immunoreactivity of the antibodies were demonstrated. The mAbs to the solubilized and purified enzyme bound in either of two regions of the molecule. By contrast, all of the mAbs to PLAP as presented on the tumor cell surface bound in only one of these two regions. One of the major antigenic determinants on the solubilized enzyme is apparently unavailable for recognition by immunoreactive cells during immunization with whole cells. Furthermore, when mAbs are generated to this region using purified PLAP as the immunogen, they do not recognize membrane-bound PLAP. The 'hidden' determinant can be exposed in vitro after partial solubilization using butanol to extract the enzyme from HeLa TCRC-1 cells and subsequent treatment with 0.5% Nonidet P-40 detergent. The results of this study have implications for the potential use of mAbs in studies of other cell surface antigens and in tumor immunolocalization and drug targeting.

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Three monoclonal antibodies with distinct antigenic specificities were examined by electron microscopy for their binding to three common genetic variants (SS, FS, and FF) of human placental alkaline phosphatase. In the reaction with the monoclonal antibody H5, all three variants of human placental alkaline phosphatase preferentially formed circular immune complexes composed of two antibodies and two enzyme molecules. In separate reactions with the F11 and B2 monoclonal antibodies, the SS variant formed circular complexes and the FS variant formed Y-shaped complexes composed of one antibody and two enzyme molecules, whereas the FF variant scarcely reacted. These results confirm immunochemical data showing that H5 binds to both S and F subunits with similar affinities, whereas F11 and B2 bind the S subunit with markedly higher affinity than they do the F subunit. Furthermore, the formation of circular complexes in the reaction of the mixture of the two antibodies, F11 and B2, with FS molecules suggests that these two antibodies bind to different sites on the S subunit. Therefore, the F and S subunits differ from one another at more than one site. This is the first indication that alleles of human placental alkaline phosphatase may result from more than just single point mutations in the gene encoding them.

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The cell-surface distribution of human placental alkaline phosphatase (PLAP) on cultured cancer cells, A431 and HeLa TCRC-1, and on normal syncytial cells of placental tissue was examined in immunoelectron transmission microscopy using the gold-labeling technique. Chemically fixed cells were reacted with affinity-purified rabbit polyclonal antibodies to PLAP, and the antibodies were visualized using gold particles tagged with goat antirabbit IgG. On all cells PLAP was observed in clusters distributed throughout the membrane surface, including microvilli, but it was not expressed in desmosomes or along other dense regions on the membrane. Previous histochemical and immunochemical techniques failed to demonstrate clusters. The results show that (1) the gold-labeling technique allows a more precise localization of PLAP on the cell surface than previously employed methods, and (2) the distribution of the enzyme is the same on cultured cancer cells and on normal placental syncytial cells. The clustered distribution of PLAP is thus a general phenomenon and is probably influenced by the physiological function of the enzyme, which has yet to be defined.

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The HeLa TCRC-1 human adenocarcinoma cell line expresses a form of alkaline phosphatase that is similar to the common S-variant of placental alkaline phosphatase (PLAP) on the basis of electrophoretic mobility, catalytic properties, and reactivity with polyclonal antibodies. More sensitive probes of changes in protein structure than polyclonal antibodies are monoclonal antibodies (MAbs) which recognize individual antigenic sites on molecules. Therefore, we produced MAbs to HeLa TCRC-1 cells and selected those which bound to the alkaline phosphatase expressed by the cancer cells. Seven MAbs were obtained and characterized by (a) fine specificity analysis using allelic variants of PLAP and other human alkaline phosphatase isozymes, (b) immunoglobulin isotype, and (c) relative binding affinities to PLAP from two sources, placental tissue and HeLa TCRC-1 cells. The seven MAbs bind the enzymes from both sources with equal affinity indicating a high degree of structural homology if not identity between the normal S-variant of PLAP and its cancer-associated counterpart. We note that most of the MAbs to cancer cell surface-bound PLAP express either Ig (immunoglobulin) G2a or IgG2b heavy-chain isotypes, a higher incidence of these classes of IgG than has been observed with the purified and soluble PLAP immunogen which yields MAbs predominantly of the IgG1 isotype. Finally, some of these antibodies, like the ones prepared from purified PLAP, recognize differences between allelic variants.

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A431 human epidermoid carcinoma cells monophenotypically express the placental alkaline phosphatase (PLAP)-like enzyme shown by its catalytic and antigenic characteristics, properties which are shared by the Nagao isozyme. More specifically, it is L-leucine sensitive just as is the rare placental D-variant of PLAP and the testicular heat-stable enzyme. Collectively, these are all referred to as PLAP-like enzymes. The enzyme was localized to the surface of the plasma membrane since it was released in an active form by bromelain treatment of cells. The number of molecules per A431 cell was estimated by radioimmunoassay at 7.5 X 10(5), a value significantly higher than that observed for HeLa TCRC-1 cells (5 X 10(4) which express the S-variant of PLAP, also referred to as the Regan isozyme. The quantity of the enzyme was increased significantly (10-fold) by treating the cells with modulating agents including sodium butyrate, prednisolone, and hyperosmolar sodium chloride. The identification of a cell line such as A431 with enhanced expression in the amount of the PLAP-like enzyme and which can be further enhanced by modulating agents will facilitate studies of the differences and the similarities between this protein and other variants of PLAP. The A431 cell line now takes its place with other cell lines which are phenotypically restricted in their expression of alkaline phosphatase. Finally, the A431 cell line is also shown here to be a suitable model system for in vivo tumor studies such as immunolocalization.

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Certain monoclonal antibodies (mAbs) to human placental alkaline phosphatase (PLAP) block bromelain cleavage of a 2-kDa segment from each of the two polypeptide chains of PLAP. These mAbs also prevent the release of PLAP from cultured cancer cell surfaces by bromelain. Such proteolysis-blocking mAbs serve as tools to specifically modify the molecular topography of cell surfaces by protease treatment.

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The two most common homologous phenotypes (SS and FF) of human placental alkaline phosphatase were purified and observed in the electron microscope by rotary shadowing and negative staining techniques. In the rotary shadowing technique, the molecules of the two phenotypes appeared to be approximately elliptical with slit-like structures in the center of the molecules, suggestive of the groove between two subunits. The dimensions of the rotary-shadowed molecules were calculated as 10.1 nm X 5.7 nm for SS and 10.1 nm X 5.6 nm for FF phenotypes. The negative staining technique delivered more fine detail of the molecules than rotary shadowing. The predominant shape of the molecules in this method appeared to be rectangular, with a longitudinal stain-filled groove and with each of the half molecules (presumably 65,000 Mr subunit) very often appearing bi-lobed. This accounts for the molecules which appear to have four pronounced electron-transparent regions. The dimensions of the negatively stained rectangular-shaped molecules were measured as 7.5 nm X 5.5 nm for SS and 7.6 nm X 5.4 nm for FF phenotypes. No significant difference in electron microscopic appearance between the SS and FF phenotypes were observed.

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Increased levels of PLAP in the serum of patients with certain types of cancer, particularly ovarian cancer and seminoma, indicate that PLAP may be a useful marker for detection of those tumors. Our goal was to further examine the usefulness of PLAP as a tumor marker by investigating the possibility that monoclonal antibodies binding PLAP may be useful for detection of the growth and metastasis of tumors which express the enzyme. Thus, we developed an experimental system where both a PLAP- positive and PLAP- negative tumor were grown in nude mice. The mice were then injected with radioactively-labeled F(ab)'2 fragments of a monoclonal antibody. The results are in accord with those of others and indicate that the monoclonal antibody localized in the PLAP-positive tumor 10 times or more often than it localized in the PLAP-negative tumor or in normal mouse tissues. Therefore, PLAP would appear to be a useful marker for the immunodetection of certain tumors in humans by external scintigraphy. PLAP may similarly be effective as a target for the delivery of toxic reagents to tumor cells in vivo using drugs conjugated to PLAP-specific monoclonal antibodies.

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The production of early and term placental alkaline phosphatase (ALP), human chorionic gonadotropin beta-subunit (beta-hCG) and pregnancy-specific beta 1-glycoprotein (SP1) was confirmed in the newly established uterine cervical cancer call line SKG-IIIa. Treatment of these cells in culture with sodium butyrate caused an increase of all of these oncodevelopmental proteins. On the other hand, prednisolone treatment enhanced only term placental ALP, while it reduced early placental ALP, beta-hCG and SP1. These data suggested that such oncotrophoblastic proteins as early placental ALP, beta-hCG and SP1 were concordantly modulated by butyrate and prednisolone. These findings may support the possibility of the reexpression of sets of development phase-specific genes in cancer cells.

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Enzyme induction of HeLa cell placental alkaline phosphatase with various agents such as prednisolone, sodium butyrate, hyperosmolality (NaCl), or combination of these inducers resulted in the appearance of enzyme activity in the rough endoplasmic reticulum, nuclear envelope, Golgi apparatus, and plasma membrane. In the Golgi apparatus, intense reaction product deposits tended to be concentrated on its trans side, with small vesicles and granules also being positively stained. Inhibition of protein synthesis with cycloheximide was followed by the disappearance of enzyme activity from these cytoplasmic organelles but not from the plasma membrane. Treatment with monensin, a secretory protein transport inhibitor, uniformly increased activity in the rough endoplasmic reticulum while causing marked dilatation of the intensely positive Golgi cisternae. These results suggest that intracellular alkaline phosphatase is newly synthesized in the endoplasmic reticulum and then passes en route through the Golgi apparatus to the plasma membrane. Accordingly, the present system could represent the biosynthesis, transport, and incorporation of the model cell surface enzyme protein to add to the vesicular stomatitus virus glyco-1 (VSV-G) protein and acetylcholine receptor model systems for studying the dynamics of cell surface protein genesis, transport, and membrane integration.

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The production of Regan isoenzyme (heat-stable, L-phenylalanine-sensitive term-placental alkaline phosphatase), human chorionic gonadotropin beta-subunit, and pregnancy-specific beta 1-glycoprotein by newly characterized human uterine cervical cancer cell lines, SKG-IIIa and SKG-IIIb, is reported. These cell lines were derived from a moderately differentiated epidermoid cancer partially mixed with epidermoid clear-cell components. At the end of the first 4 months in culture 2 sublines with different morphologies were identified. In nude mice, SKG-IIIa produce clear-cell epidermoid cancer with much glycogen, while SKG-IIIb grew as a moderately differentiated epidermoid cancer rich in tonofilaments. The presence of Regan isoenzyme was established by biochemistry, enzyme cytochemistry, immunocytochemistry, and immunoelectrophoresis. However, the copresence of small amounts of early placental alkaline phosphatase was also demonstrated. The alkaline phosphatase specific activities of SKG-IIIa cells and SKG-IIIb cells were 3.7 and 1.4 nmol per mg protein per min, respectively. The existence was proven by radioimmunoassay of human chorionic gonadotropin beta-subunit (SKG-IIIa, 5.0 mlU/mg protein; SKG-IIIb, 4.4 mlU/mg protein), pregnancy-specific beta 1-glycoprotein (SKG-IIIa, 0.7 ng/mg protein) in the culture media as a tumor cell product. The described cell lines may serve as a more representative model system for studies of regulation of oncodevelopmental genes in gynecological tumors in general and in epidermoid cervical cancer in particular.

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The techniques for measurement of biosynthetic rates and intracellular transit times of an integral membrane protein isoenzyme have now been validated. Thus, induction of placental alkaline phosphatase in cultured HeLa cells by prednisolone and by butyrate is shown to result in its increased biosynthesis as measured by uptake of [35S]methionine into immunoprecipitated cell-surface placental alkaline phosphatase. The cell-surface placental alkaline phosphatase is liberated from the cells by proteolytic cleavage by bromelain, which results in a decrease of the placental alkaline phosphatase subunit mass from 64,000 to 62,000 daltons. The time of transit of new placental alkaline phosphatase molecules from their ribosomal site of synthesis to their terminal cell-surface, bromelain-sensitive site is approximately 55 min. This system may be useful in studies of regulation of intracellular protein processing and transport to the cell surface of proteins destined to become integral membrane proteins.

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