Serum alkaline phosphatase in hypophosphatasia.

It is recognized that serum alkaline phosphatase may reflect enzyme contributions from bone, liver, and intestine. We have investigated serum alkaline phosphatases in two siblings with hypophosphatasia. After administration of long-chain triglycerides, the major alkaline phosphatase component of their sera was shown to be of intestinal origin on the basis of inhibition by l-phenylalanine. Starch block electrophoresis suggested that there were other regions of l-phenylalanine-sensitive alkaline phosphatase in addition to the major slow-moving intestinal band. Medium-chain triglycerides which are absorbed by the portal route did not cause a similar augmentation of intestinal alkaline phosphatase activity. These studies indicate that serum levels of intestinal alkaline phosphatase are increased normally after long-chain fat feeding in hypophosphatasia and may be the major component of total serum alkaline phosphatase activity.

Activation of developmental genes in neoplastic transformation.

The view is advanced that it is important to fix the stage of development corresponding to the pattern of expression of oncodevelopmental proteins in neoplastic transformation and neoplasia. From a map of development focused on events beginning with the zygote and ending with the establishment of fetus and placenta, it is possible to explain why certain developmental gene products are restricted to fetus or placenta or distributed in both. It also suggests which products can be expected to be expressed concordantly in neoplastic transformation and in neoplasia.

Regulatory controls of oncotrophoblast proteins and developmental alkaline phosphatases in cancer cells.

The two oncotrophoblast proteins, Regan isoenzyme (placental-type alkaline phosphatase) and human chorionic gonadotrophin, are readily studied oncodevelopmantal gene products in human cancer patients and in three experimental model systems. The latter consists of (a) HeLa sublines TCRC-1 and TCRC-2, which produce Regan and non-Regan isoenzymes, (b) HEp-2 and FL amnion cell lines as models for the reciprocal expression of developmental genes, and (c) modulation in vivo of developmental gene expression in HeLa cells. In the case of the third model, for example, HeLa TCRC-1 cells grow in immunosuppressed rats to form a tumor nodule, which expresses a new oncoamnion (FL) isoenzyme, while the Regan isoenzyme disappears. Return of the tumor cells to cell culture medium results in a disappearance of the oncoamnion (FL) species and the reappearance of Regan isoenzyme. This interesting model is expected to bridge the interpretation of experiments done in cell culture with observations made on tumors of cancer patients. Most helpful in interpretation of all these studies has been a chronology of early development. It appears that the counterparts of a number of tumor proteins appear as early as gametogenesis and as late as 10 weeks of gestation.

Presence of the rare D-variant heat-stable, placental-type alkaline phosphatase in normal human testis.

In 11 adult testes studied, about 0.3 to 4.6% of the total alkaline phosphatase activity was heat stable and L-phenylalanine sensitive but L-homoarginine insensitive. The testicular heat-stable enzyme was more susceptible to inhibition by L-leucine and ethylenediaminetetraacetate than were the normal placental and intestinal enzymes. By antibody-directed enzyme inhibition test, the testicular heat-stable enzyme cross-reacted completely with normal placental enzyme but clearly distinguished itself from a heat-stable component of normal intestinal enzyme. Thus, placental alkaline phosphatase D-variant is synthesized in testis, indicating that the gene for elaborating this placental protein is probably already active in the testicular cells. The high incidence of this protein in cancers of testis and ovary is probably due to its increased production by gonadal genes present in the genome of these particular tumors.

Characterization of the placental alkaline phosphatase-like (Nagao) isozyme on the surface of A431 human epidermoid carcinoma cells.

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.

Evidence for homology of normal and neoplastic human placental alkaline phosphatases as determined by monoclonal antibodies to the cancer-associated enzyme.

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.

Developmental phase-specific alkaline phosphatase isoenzymes of human placenta and their occurrence in human cancer.

Alkaline phosphatase electrophoretic patterns characteristic of three phases in early human trophoblast development are described in this preliminary communication. Phase 1 (6 to 10 weeks) consists entirely of two heat-sensitive, L-homoarginine-inhibited bands, the slower one of which possesses antigenic determinants of live-bone-type alkaline phosphatase, whereas the fast band lacks any of the known alkaline phosphatase antigenic determinants. Phase 2 pattern (11 to 13 weeks) is that of a mixture of Phase 1 and Phase 3 isozyme components, the latter exhibiting two isozyme bands with the characteristics of term placental alkaline phosphatases correspond in order to non-Regan isoenzyme, a mixture of Regan and non-Regan isoenzymes and Regan isoenzyme in a variety of human cancer tissues. The biochemical profile characteristic of trophoblast developmental Phase 1 alkaline phosphatase is expressed as 78.5% heat-sensitive inhibition (5 min at 65 degrees), 66.3% L-homoarginine inhibition, and 17.3% L-phenylalanine inhibition where n = 12. It is hypothesized that the alkaline phosphatase of human tumor tissues reflects the expression of placental genes corresponding to one or more phases of trophoblastic development.

A simple radioimmunoassay of human placental alkaline phosphatase (Regan isoenzyme) using specific antibody polymers.

Rabbit antiserum against highly purified high-molecular-weight B-variant of human placental alkaline phosphatase (M.W. 200,000) was rendered monospecific by absorption with polymerized pooled male serum proteins; the absorbed antiserum was then polymerized with ethyl chloroformate and used in radioimmunoassay as a stable solid-phase immunoabsorbent. Homogeneous preparation of the enzyme, with a specific activity of 477 mumoles phenol per mg per min, was also obtained by absorbing the chromatographically purified enzyme with polymerized rabbit antiserum directed to whole human serum proteins; the pure enzyme was then labeled with 125-I as the tracer retaining at least 80% of its antigenicity. Only a minute quantity of the polymerized antibody particles is required for each assay in admixture with the labeled and unlabeled enzyme. By adding a small amount of starch-gel particles before low-speed centrifugation, complete phase separation was achieved. The radioimmunoassay could detect 0.4 to 0.8 ng enzyme protein per tube, which is comparable to the sensitivity achieved by enzymic assays. However, radioimmunoassay is advantageous over the enzymic assay in being direct, specific (no interference by the nonplacental-type alkaline posphatase), and capable of detecting both catalytically active and inactive forms of the enzyme. Native variants of placental-type alkaline phosphatase including Regan isoenzyme and Nagao isoenzyme (D-phenotype of normal placental alkaline phosphatase), could thus be directly determined by this procedure in the clinical specimens.

Characterization and use of an allotype-specific monoclonal antibody to placental alkaline phosphatase in the study of cancer-related phosphatase polymorphism.

The hybridoma technique was used to produce an allotype-specific monoclonal antibody (F11) that reacts with the products of the S, I, and D alleles of PLAP but not of the F allele. Serum and ascites samples from patients with different cancers containing high levels of PLAP were tested for reactivity with F11. These tumor-derived PLAPs were of the Nagao type as shown by their sensitivity to inhibition by L-leucine. This type of inhibition is exhibited also by the rare D allelic variant of PLAP but not by the common forms. Thus, it has been proposed that the Nagao enzyme represents reexpression of the D allele of PLAP. F11 reactive and nonreactive samples as well as samples with intermediate reactivity were found among the cancer sera and ascites. Our results show tha tumor-derived Nagao enzyme does not represent the reexpression of the D allele but instead, in spite of its distinct inhibition pattern, expresses the same genetic polymorphism that is found in the placenta.

Newly established uterine cervical cancer cell line (SKG-III) with Regan isoenzyme, human chorionic gonadotropin beta-subunit, and pregnancy-specific beta 1-glycoprotein phenotypes.

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.

Placental alkaline phosphatase as a tumor marker for seminoma.

A sensitive and specific enzyme-linked immunoabsorbent assay was used in a retrospective study of serum levels of placental alkaline phosphatase (PLAP) in testicular cancer. Sixteen of 28 men with active seminoma had elevated PLAP levels, and 71% had elevated levels of either PLAP, human chorionic gonadotropin, or both. Only four of 22 men with active nonseminomatous cancer had elevated PLAP levels, and the levels were normal in all control patients, including 33 men apparently cured of testicular cancer. In six of ten serial studies, PLAP levels provided information not otherwise available that would have been useful clinically, and the levels never were elevated inappropriately. Our data suggest that PLAP is a clinically useful serum tumor marker for seminoma.

Molecular dimensions of the SS and FF phenotypes of human placental alkaline phosphatase. Rotary shadowing and negative staining electron microscope measurements.

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.

Monoclonal antibodies block the bromelain-mediated release of human placental alkaline phosphatase from cultured cancer cells.

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.

Markers for ovarian cancer: regan isoenzyme and other glycoproteins.

Since embryonic genes are not generally active in normal adult subjects and because certain of these genes are activated in cancer leading to ectopic synthesis, it is the difference between the ectopic level and the normal adult concentrations of embryonic gene products which we seek in developing "markers" for ovarian cancer. The carcinoplacental alkaline phosphatases corresponding to the term gestational phenotypes correlate positively with ovarian cancer as does hCG. Other fetal and placental glycoproteins whose presence is noted in ovarian cancer include CEA, alpha-FP, and Björklund's antigen. Antigens of mucinous cystadenocarcinoma have not yet been examined for their possible fetal or placental origins. The degree of concordance of expression of Regan isoenzyme and hCG is variable. Profiles of glycoproteins would appear to offer an opportunity to inquire more deeply into the nature of ovarian cancer and from this inquiry, one can expect to develop a system of markers which can be of clinical use.

Complement fixation for study of placental-type alkaline phosphatase.

Preparations of human placental alkaline phosphatase differing in specific enzyme activities were compared by microcomplement fixation assays using monospecific antisera. While both specific enzyme activity and complement fixation units increased 15,000-fold upon purification, the ratio between these units remained constant. Separation of an alkaline phosphatase preparation into 'A' and 'B' forms by ampholine isoelectric focusing indicated that these forms also possessed the same ratio of immunoreactive enzyme protein to enzyme activity. The correspondence of complement fixation units with specific enzyme activity indicates that complement fixation with monospecific antisera can be used to analyze structural differences among alkaline phosphatase isoenzymes.

Alkaline phosphatase biosynthesis in the endoplasmic reticulum and its transport through the Golgi apparatus to the plasma membrane: cytochemical evidence.

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.

Demonstration of species difference of placental alkaline phosphatase isozymes in acetone-fixed paraffin-embedded tissues.

The best preservation and discrimination of alkaline phosphatase isozymes by means of amino acid inhibition and heat treatment was obtained in sections of acetone-fixed paraffin-embedded tissues as compared to tissues fixed either in 95% ethanol or standard formalin. The results are illustrated in a study differentiating the isozymes of human and mouse placenta.

Clustered distribution of human placental alkaline phosphatase on the surface of both placental and cancer cells. Electron microscopic observations using gold-labeled antibodies.

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.

Immunocytochemical demonstration of intracytoplasmic alkaline phosphatase in HeLa TCRC-1 cells.

The ultrastructural localization of alkaline phosphatase has been examined in cells of a HeLa subline (TCRC-1) that are monophenotypic for Regan isoenzyme expression. Enzyme activity was demonstrated at the cell surface plasma membrane and in certain lysosomes as revealed by the lead citrate method. The regular direct immunoperoxidase procedure utilizing antibodies in IgG or Fab' form showed the same distribution patterns of alkaline phosphatase. However, when the cell surface antigen was blocked in advance with specific unlabeled antibodies and direct immunocytochemistry performed in the presence of saponin, intracellular alkaline phosphatase antigen was observed in the perinuclear space, endoplasmic reticulum, and Golgi apparatus. The results appeared to be concordant with the current concept that membrane glycoproteins are formed in the endoplasmic reticulum, modified in the Golgi apparatus and then transported to the cell surface. Intracellular alkaline phosphatase was observed predominantly in some cell populations especially mitotic cells, suggesting that the enzyme protein was synthesized in and around the mitotic phase. Accordingly, this technique of differential membrane immunocytochemistry appears to provide an opportunity to follow ectopic gene expression as a function of cell cycle and enzyme induction.

Demonstration of lysosomal and extralysosomal sites for acid phosphatase in mouse kidney tubule cells with p-nitrophenylphosphate lead-salt technique.

Dual localization of acid phosphatase in lysosomal and extralysosomal sites of the tubule epithelial cells of normal mouse kidney was observed at the light and electron microscope level using a modified Gomori lead-salt method with p-nitrophenylphosphate (pNPP) as substrate. Based on previous biochemical and cytochemical findings, we developed optimal conditions for the enzyme activity in extralysosomal sites. The conditions used for the light microscopic level consisted of 1.5 mM PNPP, 2.0 MM Pb(NO3)2 and 0.05 M acetate buffer (pH 5.8). Those for the electron microscopic study required 3.0 mM PNPP, 3.6 MM Pb(NO3)2 and 0.1 M acetate buffer (pH 5.8). This modified lead-salt technique was highly specific and provided a suitable method for the demonstration of nonlysosomal as well as lysosomal sites of acid phosphatase activity in the tubule epithelial cells of normal mouse kidney. As expected, the enzyme activity appeared in the lysosomes, but the prominent reaction in the brush border, the rough endoplasmic reticulum and basal infolding plasma membranes was not anticipated. We were able to demonstrate in situ organelle precursors of microsomal acid phosphatase such as endoplasmic reticulum, plasma membrane and basal infolding membranes showing the same substrate preference, which had been observed previously in biochemical studies in our laboratory. Since the possible participation of alkaline phosphatases, K+-pNPPase or Na+-K+-adenosine triphosphatase was ruled out by use of appropriate inhibitors, the enzyme-reactive sites can be interpreted as reflecting nonspecific acid phosphatase.