Alkaline Phosphatase Type Research Articles

Alkaline Phosphatase: A Marker of Alveolar Type II Cell Differentiation

In an effort to identify type II cells by a method independent of staining phospholipid inclusions, we evaluated a histochemical technique for alkaline phosphatase activity in normal rat lung, in freshly isolated type II cells, and in primary culture of type II cells. In the adult rat alveolus, alkaline phosphatase staining selectively identified type II cells, although nonciliated bronchiolar (Clara) cells and loose perivascular connective tissue also stained for alkaline phosphatase activity. In cell suspensions of type II cells and other dissociated lung cells, alkaline phosphatase staining correlated closely with the modified Papanicolaou technique and was particularly useful in distinguishing type II cells from alveolar macrophages. To determine if alkaline phosphatase was related to the differentiated phenotype of type II cells, we studied conditions known to affect other type II cell functions. When type II cells were cultured on plastic substrata, the intensity of alkaline phosphatase staining decreased with increasing time in culture. To quantitate the apparent decrease in alkaline phosphatase activity, we used a biochemical assay to study the expression of alkaline phosphatase by type II cells. The specific activity of alkaline phosphatase in type II cells declined with increasing time in tissue culture on plastic substrata. Alkaline phosphatase activity was maintained, however, by culturing cells on Englebreth-Holm-Swarm (EHS) tumor matrix. Cells that had reduced levels of alkaline phosphatase activity following 48 h of culture on plastic substrata could be "rescued" by removing them from the plastic substratum and reculturing them for 48 h on EHS matrix. Alkaline phosphatase activity was also increased by culturing type II cells in the presence of cAMP or sodium butyrate.(ABSTRACT TRUNCATED AT 250 WORDS)

Isoenzymes of alkaline phosphatase in germinoma cells.

Utilizing 18 germinomas, the characteristics of isoenzymes of alkaline phosphatase (ALP) in germinoma cells were examined by light and electron microscopic enzymohistochemical and immunohistochemical techniques and biochemical methods including inhibition tests, electrophoresis and electrosyneresis. ALP in germinoma cells was generally located on the cell surface. In a few germinoma cells, however, ALP was seen not only on the cell membrane but also in the endoplasmic reticulum. Enzymohistochemically as well as biochemically, ALP in germinoma cells was little sensitive to L-phenylalanine, moderately sensitive to L-homoarginine and somewhat resistant to heat in its inhibition tests. Immunohistochemical examinations and electrosyneresis showed the presence of the placental type of ALP in germinoma cells and biochemical analyses revealed that the heat-stable component of ALP in germinoma cells was consistent with the D-variant of the placental type of ALP. Therefore, germinoma cells possessed mixed isoenzymes of ALP, consisting mainly of the liver/bone type and a small amount of D-variant of the placental type on the cell surface. The prominent expression of ALP in germinoma cells may be due to the enhanced expression of gonadal genes active in the germinoma genome.

Placental type alkaline phosphatase in ovarian and other tumours

SummaryA highly specific solid phase enzyme immunoassay based on a mouse monoclonal antibody (H317), specific for placental type alkaline phosphatase, has been used to investigate the occurrence of this enzyme in the serum of patients with malignant and benign pathology. Serum levels >0.1 u/1 were found in 57 per cent of ovarian cancer patients, with a mean value of 0.62 u/1; in 14 per cent of benign ovarian tumour patients, with a mean value of 0.15 u/1; and in 6 per cent of patients with malignant tumours other than ovarian. No patients with benign chronic diseases had detectable serum placental type alkaline phosphatase. In a group of patients who had been treated with chemotherapy for ovarian carcinoma, only 4 per cent had detectable placental alkaline phosphatase.

Isolation and purification of placental type alkaline phosphatase from a seminoma.

An alkaline phosphatase (EC 3.1.3.1) of the placental type was isolated from a seminoma type of human testicular cancer tissue and was purified to homogeneity by sulfate-mediated chromatography on a column of Cibacron Blue Sepharose 4B. The purified enzyme had a specific activity of 40.6 kU per gram of protein and was obtained in a yield of 37%. The purification procedure used was simple and economical, and may be used to purify alkaline phosphatase isoenzymes from other cancer tissues. This is the first report of the purification of the enzyme in seminoma. Inhibition studies suggest that this enzyme is a Nagao variant rather than the Regan type reported in several cancer tissues.

INGUINAL SWEAT GLAND CARCINOMA WITH CHORIOCARCINOMATOUS DIFFERENTIATION

An inguinal sweat gland carcinoma is described including the unusual occurrence of alpha hCG. The alpha hCG was demonstrated in the tumor presenting choriocarcinomatous differentiation and undifferentiated polygonal cells in ordinary tumor nests. beta hCG and SP-1 were weakly and sporadically demonstrated in a small area of the tumor presenting choriocarcinomatous differentiation. On the other hand, hPL and histochemical reaction of placental type alkaline phosphatase were not observed in the choriocarcinomatous and undifferentiated tumor cells. CEA was observed in both ordinary sweat gland carcinoma nests and choriocarcinomatous differentiated regions. In the ordinary tumor nests, enzyme histochemical reactions of phosphorylase and SDH were positive. And S 100 protein was sporadically demonstrated. In spite of chemotherapy and radiotherapy, the patient died after a short duration of the disease.

Characterization of alkaline phosphatase from primordial germ cells and ontogenesis of this enzyme in the mouse

The physical characteristics of nonspecific alkaline phosphatase (ALP) from both mouse primordial germ cells (PGCs) and gonads were compared with corresponding samples from other organs at different developmental stages. Combining a cytochemical approach with polyacrylamide gel electrophoresis and the use of specific inhibitors, as well as neuraminidase treatment, heat sensitivity tests, and molecular-mass criteria, it was found that only one ALP isoenzyme was present in all organs up to day 14 of gestation. Distinct ALP isoenzymes first appeared in the small intestine on day 15 and, thereafter, in all other tissues except the gonads. In these organs, the embryonal ALP isoenzyme seemed to be retained until adulthood. Although the placenta had a different ALP isoenzyme than the embryo at all stages, this isoenzyme was found to be similar to that in the maternal decidual tissues. Therefore, we conclude that the mouse embryo only expresses one type of ALP that can be considered “embryonal”, regardless of the organ in which it first appears, and that this ALP is conserved in the gonads.

Clinical significance of heat labile Regan isoenzyme variant in Hodgkin's disease.

The activity of a heat labile variant of Regan type alkaline phosphatase (HLRI) was studied in the serum of 38 histologically proved patients with Hodgkin's disease. The activity was also studied in 40 normal healthy adults matched for age and sex as control. HLRI was significantly elevated in patients with Hodgkin's disease in comparison with normal controls (1.12 +/- 0.12 units/ml vs 0.09 +/- 0.01 units/ml; P less than 0.001). The mean values of the isoenzyme tended to increase progressively with increasing stages of disease, reflecting a significant linear trend (P less than 0.001). The serial measurement of this variant may serve as a useful parameter in assessing disease activity and response to chemotherapy.

A diploid epithelial cell line from normal adult rat liver with phenotypic properties of ‘oval’ cells

A diploid epithelial cell line (termed WB-F344) was isolated from the liver of an adult male Fischer-344 rat and the phenotypic characteristics of the cells were studied. These cells measure approximately two-fifths the volume of freshly isolated hepatocytes. They are histochemically negative for glucose-6-phosphatase and weakly positive for γ-glutamyl transpeptidase. They produce extensive intercellular reticulin fibers which stain immunocytochemically for fibronectin, and they synthesize both α-fetoprotein and albumin, but they do not accumulate glycogen particles. Ultrastructurally, they are polygonal cells with numerous intercellular desmosomes and nexus junctions, and they are partially surrounded by basement membrane-like material. Cytoplasmic organelles include few, but sometimes dilated profiles of rough endoplasmic reticulum, lysosomes, abundant free ribosomes, sparse smooth endoplasmic reticulum and Golgi membranes, microbodies, and small, pleomorphic mitochondria. They express A and C isozymes of aldolase, K isozyme of pyruvate kinase, LDH 2 to LDH 5 isozymes of lactate dehydrogenase, and ‘fetal liver’-type alkaline phosphatase isozyme. When compared with the phenotypes of isolated and purified normal hepatocytes, biliary epithelial (ductular) cells and ‘oval’ cells isolated from livers treated with chemical carcinogens, the phenotypic properties of the liver epithelial cell line in culture most resemble those of the ‘oval’ cells.

Serum placental type alkaline phosphatase in cigarette smokers.

By means of enzyme immunoassays based on two monoclonal antibodies with specificities for distinct forms of placental type alkaline phosphatase (Pl-ALP), together with L-leucine inhibition studies, it has been possible to distinguish the Nagao type carcinoplacental enzyme from other placental type alkaline phosphatases. This approach has shown that it is the Nagao type (placental like) enzyme which is detectable in small amounts in the plasma of healthy individuals, particularly cigarette smokers.

Placental type alkaline phosphatase in possibly premalignant alterations of human gastric mucosa.

The occurrence of placental type alkaline phosphatase (Regan enzyme) was studied in various pathological conditions of the human stomach using polyacrylamide gel electrophoresis and various inhibition techniques. Placental type alkaline phosphatase could be demonstrated in 16 out of 22 gastric carcinomas, in 3 out of 11 moderate and 6 out of 6 severe dysplasias, we failed however to demonstrate it in any other pathological conditions of the stomach, i.e. in intestinal metaplasia. There was no correlation between the occurrence of Regan enzyme and histological type of gastric carcinomas. The appearance of placental type alkaline phosphatase in gastric dysplasias may be suggestive of malignant transformation and its determination in repeated biopsy samples may be a help in the follow-up of patients with abnormal gastroscopic findings.

Characterization of a new human cell line derived from a xenografted embryonal carcinoma.

A human cell line has been established from a transplantable xenografted human testicular tumor, which, both in the original tumor and in the xenograft, exhibited the histological characteristics of an undifferentiated malignant teratoma (embryonal cell carcinoma). The cells in culture were undifferentiated by biochemical, morphological, and ultrastructural criteria, growing as small islands of cells that tended to form aggregates at high density. The cells showed some variation in chromosome number with 30 to 40% of the cells having a normal human karyotype. The cells expressed high levels of alkaline phosphatase, which by heat inactivation and inhibition studies was 40 to 50% placental type alkaline phosphatase. None of the cultures produced human chorionic gonadotrophin, alphafetoprotein, carcinoembryonic antigen, or fibronectin, although at high cell densities plasminogen activator could be detected at low levels. Cell surface studies showed that the cells shared antigens with the murine embryonal carcinoma cell line F9, expressed beta 2-microglobulin at very low and variable levels, and bound the lectin peanut agglutinin. These studies suggest that this cell line has some of the characteristics described for murine embryonal carcinoma cell lines.

Clinical Evaluation of Intestinal Type Alkaline Phosphatase in Diabetic Coma

Clinical Evaluation of Intestinal Type Alkaline Phosphatase in Diabetic Coma

Alkaline phosphatase isozymes as possible markers of differentiation in human testicular teratocarcinoma cell lines

The alkaline phosphatase (ALP) activity in eight independent cell lines derived from human testicular germ cell tumors was characterized. Seven out of eight of the lines had high ALP levels, and most of the activity in each case was of the liver/bone/kidney ALP type, as judged by thermostability, inhibition, and electrophoretic studies. Low levels of a heat stable, placental-like ALP were also present in these seven lines; only a small subpopulation of the cells of each line reacted strongly with an anti-placental ALP monoclonal antibody. The heat-stable, placental-like isozyme characteristic of these lines differed from the normal placental ALP in its inhibition profile. Thus it is possible that a subpopulation of the cells in these lines expresses a new embryonic ALP form.

Immunology and biochemistry of the Regan isoenzyme.

The Regan isoenzyme is a placental-type alkaline phosphatase that is expressed in a number of human tumors, particularly in gonadal and urologic cancers. Attention is given to the unique gene that codes for placental alkaline phosphatase and the similarities and differences in the tumor and placental gene products. The separation and identification of individual organ-specific isoenzymes is accomplished by a variety of biochemical, immunologic, and electrophoretic techniques and the correlation of the Regan isoenzyme, non-Regan isoenzyme, and Nagao isoenzyme, and the Kasahara isoenzyme is made with their developmental counterparts. The L-leucine-sensitivity phenotypes of placental and tumor alkaline phosphatases and the non-Regan early placental type alkaline phosphatases appear to be developmental phase-specific. Oncotrophoblast gene expression has been investigated with monophenotypic cell culture lines as a consequence of modulation by prednisolone and hyperosmolarity. Finally, general discussion of oncodevelopmental proteins as tumor markers precedes a current opinion of Regan isoenzyme as a tumor marker. Evidence now points to seminoma as a consistent producer of Regan isoenzyme although much more work will be required to establish its clinical utility.

Clinical Significance of Intestinal Type Alkaline Phosphatase in Diabetes Mellitus

Clinical Significance of Intestinal Type Alkaline Phosphatase in Diabetes Mellitus

Human endodermal sinus tumour in nude mice and its markers for diagnosis and management.

Two human endodermal sinus tumours (yolk sac tumours) were transplanted successfully into nude mice. The transplanted tumours maintained not only morphological characters, such as Schiller-Duval bodies, but also the ability to synthesise alpha-fetoprotein, lactic dehydrogenase 1, liver and bone type alkaline phosphatase, and some human serum proteins. Since these tumours produced lactic dehydrogenase 1 but not the other four isozymes of lactic dehydrogenase, this isozyme, like alpha-fetoprotein, seems to be a good marker for the diagnosis and management of cases of endodermal sinus tumour. One of the two tumours produced another fetal antigen or carcinoembryonic antigen in addition to alpha-fetoprotein. These two endodermal sinus tumours, with their various markers in nude mice, will be useful in studies on diagnostic markers.

Fetal Isoenzyme Expression in Human Tumor Xenografts1

It has been shown previously that a human cancer cell line (HeLa TCRC‐1) which produces only the Regan isoenzymes of alkaline phosphatase in culture, elaborates a different isoenzyme form when grown in an animal host. This alteration of isoenzyme expression results in the production of an isoenzyme form referred to as oncoamniotic (FL), since it shares many of the characteristics of the enzyme form produced by the FL amnion cell line.Here we shall report on the alteration of isoenzyme expression in a non‐Regan producing cell line heterotransplanted into an immunosuppressed animal host. HeLa TCRC‐2 is a cell line cloned for the exclusive production of an isoenzyme form which is most similar to adult bone type alkaline phosphatase. This cell line growing in an animal host produces an additional isoenzyme form which appears to be similar to the amniotic (FL) type. The original non‐Regan alkaline phosphatase of this cell line persists, and is co‐expressed during in vivo growth.When HeLa TCRC‐2 is removed from the animal and placed back into culture, the exclusive production of the adult bone type alkaline phosphatase is restored.The present report indicates the dramatic alterations in gene expression observed when well characterized cultured cells are heterotransplanted into immunosuppressed animals.

Plasma 25-hydroxyvitamin D and alkaline phosphatase isoenzymes in hyperthyroidism.

Plasma 25-hydroxyvitamin D (25-OHD) values in 22 hyperthyroid patients did not differ significantly from those of controls matched for age, sex, and the time of year that plasma samples were taken. In the hyperthyroid group, plasma alkaline phosphatase was significantly higher than in controls, and an increase in bone type alkaline phosphatase occurred in 50%. In the 18 female hyperthyroid patients the plasma gamma-glutamyl transferase was significantly increased compared with controls. Mean values for plasma albumin, alanine transferase, and calcium showed no significant difference between hyperthyroid and control patients.

Impact of heat stress and strain on health

To investigate the influence of the same mechanical loading on osteogenesis and osteoclastogenesis in vitro.Primary osteoblasts, bone marrow-derived mesenchymal stem cells (BMSCs, cultured in osteoinductive medium) and RAW264.7 cells cultured in osteoclast inductive medium were all subjected to a 1000 μstrain (μs) at 1 Hz cyclic mechanical stretch for 30 min (twice a day).After mechanical stimulation, the alkaline phosphatase (ALP) activity, osteocalcin protein level of the osteoblasts and BMSCs were all enhanced, and the mRNA levels of ALP and collagen type I increased. Additionally, extracellular-deposited calcium of both osteoblasts and BMSCs increased. At the same time, the activity of secreted tartrate-resistant acid phosphatase, the number of tartrate-resistant acid phosphatase-positive multinucleated cells, matrix metalloproteinase-9 protein levels of RAW264.7 cells and the extracellular calcium solvency all decreased.The results demonstrated that 1000 μs cyclic mechanical loading enhanced osteoblasts activity, promoted osteoblastic differentiation of BMSCs and restrained osteoclastogenesis of RAW264.7 cells in vitro.

Kinetics of Substrate Hydrolysis by Molecular Variants of Escherichia coli Alkaline Phosphatase

Abstract Two isozymes of Escherichia coli alkaline phosphatase have been compared in the stopped flow spectrophotometer with respect to the kinetics of hydrolysis of two phosphate esters. Hydrolysis of 2,4-dinitrophenyl phosphate at pH 5.5 is biphasic; release of chromophoric alcohol occurs in a rapid burst followed by a slower steady state. The two isozymes show similar burst rates and amplitudes but their steady state rates differ by a factor of 2. Hydrolysis of p-nitrophenyl phosphate at pH 8.0 shows no significant burst and the homogeneous steady state rate is essentially the same for both isozymes. These observations and the ionic strength dependence of the rates of these reaction steps support the hypothesis, proposed previously in the literature, that the molecular process which determines the steady state rate at pH 8.0 is responsible for the presteady state burst at pH 5.5. The steady state process at pH 5.5 must be a subsequent step in the catalytic mechanism. A molecular hybrid containing one subunit of wild type alkaline phosphatase and one subunit from an inactive mutationally altered alkaline phosphatase shows one-half the normal burst amplitude at pH 5.5. All transient and steady state rates observed for the hybrid equal those seen for wild type enzyme, once corrected for the halved number of active sites inferred from the burst amplitude. This finding casts doubt on catalytic mechanisms involving alternately functioning subunits that several workers have proposed for E. coli alkaline phosphatase.