Intracellular alkaline phosphatase activity in cultured human cancer cells

Summary The effect of saponin treatment in demonstrating intracellular portion of alkaline phosphatase activity in human cancer cell lines was evaluated. Previous reports using standard lead-salt techniques visualized enzyme almost exclusively on the plasma membrane and sometimes in the lysosomes. However, by treating cells with saponin before or during the cytochemical incubation, intracellular alkaline phosphatase became demonstrable at the endoplasmic reticulum, Golgi apparatus, Golgi-derived vesicles and mitochondria as well as lysosomes and plasma membrane. These intracellular catalytic activities were significantly inhibited by the specific amino acid inhibitors characteristic for each cell line, and this suggested that intracellular alkaline phosphatase is the same isoenzyme as that present in the plasma membrane. The results of our current and previous studies therefore indicate that saponin reveals latent intracellular alkaline phosphatase activity by changing the membrane's physical state; thereby increasing the availability of both catalytic and antigenic sites of the enzyme to substrate and to antibody respectively.This work was supported by National Institutes of Health Grant No. CA 21967     

Effect of water-soluble matrix fraction extracted from the nacre of Pinctada maxima on the alkaline phosphatase activity of cultured fibroblasts

A new approach to the isolation of the water-soluble factors from nacre without any demineralization is described and examined their effect on fibroblast cells in culture. The soluble matrix in pure water from the nacre of Pinctada maxima was analysed by size-exclusion HPLC. Four fractions (SE1-SE4) of the water-soluble matrix (WSM) were further analysed by anion-exchange HPLC. The amino acid composition of the WSM showed that it is mainly composed of glycine and alanine. SE1 and SE4 had different amino acid compositions from the whole WSM. The WSM and SE4 tested on a culture of human foetus lung tissue fibroblasts increased the alkaline phosphatase (ALP) activity. SE1 caused a decrease in ALP activity. Our results support the hypothesis that WSM promotes the differentiation of cells in vitro.     

Dependence of alkaline phosphatase activity on bioecological parameters of Black Sea fish

The data on alkaline phosphatase activity in the liver of eight dominant species of Black Sea fishes are presented. The enzyme activity varies within a wide range; significant sexual differences have not been registered. The activity of the enzyme is similar in specimens of different age groups, but it decreases in old fishes. The enzyme activity increases in prespawning and spawning, which evidences for its participation in the process of sexual maturation of fish. Based on our and published data on the dependence of alkaline phosphatase activity on the degree of the environmental pollution, the enzyme can be recommended as a biomarker for bioindication, biotesting, and ichthyological monitoring.     

Reduction of alkaline phosphatase activity in aged human osteogenic periodontal ligament fibroblasts exhibiting short telomeres

The osteogenic cell type of human periodontal ligament fibroblasts (PDLF) undergoes senescence at finite population doubling numbers unrelated to donor ages. This study investigated telomere lengths of osteogenic PDLF from differently aged donors and alterations of the osteoblast-like properties in the aged PDLF with short telomeres. Telomere lengths of osteogenic PDLF were biased towards long or short among all 15- to 51-year-old individuals, and did not show a normal distribution by Pearsons test or a correlation to donor age by simple regression analysis. In osteogenic PDLF, senescence-associated -galactosidase was expressed in 78.5% of cells in the clones with short telomeres (mean 3.02 kbp), and in 9.4% of cells in the clones with long telomeres (mean 13.06 kbp). These results suggest that human periodontium comprises aged osteogenic PDLF without correlation to age. Osteogenic PDLF with long telomeres strongly expressed alkaline phosphatase (ALPase) activity whereas cells with short telomeres expressed ALPase activity to a weaker extent. Total activity of ALPase in the clones of osteogenic PDLF with long telomeres was significantly higher than that in the clones with short telomeres. The produced amounts of both osteopontin and osteocalcin in the clones of osteogenic PDLF with long telomeres were slightly but statistically significantly smaller than those in the clones with short telomeres. These findings suggest that aged osteogenic PDLF reduce the expression of ALPase activity but that there is not a critical alteration in bone-associated protein production. Aged osteogenic PDLF may impair the ability to induce ALPase-dependent calcification.This work was supported by a grant-in-aid for Scientific Research (B) (2) from the Ministry of Education, Science, Sports, and Culture of Japan (No. 12470379).     

Properties of alkaline phosphatase in crude homogenates of human diploid fibroblasts

The kinetic properties of the "constitutive" and the "induced" alkaline phosphatase in diploid fibroblasts are compared with those of the enzymes in crude tissue homogenates. Both the constitutive as the induced enzyme have properties comparable with those of the liver-bone-kidney group. The induced alkaline phosphatase clearly differs from the "constitutive" alkaline phosphatase concerning the effect of high concentrations of L-phenylalanine and the effect of Mg2+ ions. The induced alkaline phosphatase seems to be identical with the enzyme in liver, but the constitutive alkaline phosphatase could not be identified.     

Lysosomal glycosidase activity in cultured human fibroblasts

A study was made of the activity of 3 lysosomal glycosidases -beta-D-galactosidase (K. P. 3.2.1.23), alpha-L-fucosidase (K. P. 3.2.1.51), N-acetyl-beta-D-hexosoaminidase (K. P. 3.2.1.52) depending on the time after subcultivation and duration of the passage of human skin embryonal and postembryonal fibroblasts. It was established that changes in the specific activity of the enzymes should be calculated with reference to the cell rather than to protein whose amount might vary considerably. It was also found that for measuring the specific activity of enzymes, of great importance are the procedures of cell removal from the base layer (by mechanical scraping off or by trypsin solution) and the regimen of the homogenization of cell preparations.     

Pseudohypophosphatasia: aberrant localization and substrate specificity of alkaline phosphatase in cultured skin fibroblasts.

We explored the biochemical basis for the disorder pseudohypophosphatasia (PsHYPT) in one patient by examining the substrate specificity and localization of alkaline phosphatase (ALP) in cultured dermal fibroblasts. Despite substantial ALP activity, in cell homogenates, toward the artificial substrate 4-methyl-umbelliferyl phosphate (4-MUP), there was a marked deficiency in ALP activity toward the natural substrates pyridoxal 5'-phosphate (PLP) and phosphoethanolamine (PEA), indicating altered substrate specificity. Furthermore, although 4-MUP phosphatase (4-MUP-P'tase) activity was predominantly localized as an ecto-enzyme, the small amount of PLP phosphatase (PLP-P'tase) activity was intracellular. This differential localization was apparent in intact cells, since (1) brief acidification of the medium at 4 degrees C inactivated a majority of the 4-MUP-P'tase activity but only 15% of the PLP-P'tase activity (in contrast to greater than 85% inactivation of both in homogenates), (2) greater than 70% of the 4-MUP-P'tase activity but only 30% of the PLP-P'tase activity was released by phosphatidylinositol-specific phospholipase C (PI-PLC) digestion, and (3) degradation of extracellular PLP was less than 35% of that of disrupted cells. Both 4-MUP- and PLP-P'tase activities were predominantly in a lipid-anchored form that could be converted to a soluble, lipid-free form by PI-PLC digestion. Our findings suggest that the clinical and biochemical presentation of this PSHPT patient results from the production of two aberrant ALP species. One form of ALP has appropriate ectoorientation but is preferentially deficient in activity toward natural substrates; the other ALP species has appropriate substrate specificity but is sequestered from substrates by its intracellular location.     

Induction of alkaline phosphatase in cultured human fibroblasts: Comparison of normal cells and those from patients with cystic fibrosis

The membrane glycoprotein enzyme, alkaline phosphatase was induced in cultured human fibroblasts by dibutyryl cyclic AMP, sodium butyrate, the serum glycoprotein fetuin, the Tamm-Horsfall urinary glycoprotein, and by a number of inhibitors of DNA synthesis. The uninduced basal enzyme activity increased at later stages of growth when the cells became confluent. Induction by dibutyryl cyclic AMP or fetuin was most effective when the agents were added after the cells had reached stationary phase and was maximal after at least two days of exposure. The levels of induction resulting from the addition of pairs of the agents, dibutyryl cyclic AMP, n-butyrate and fetuin were additive indicating that these have different modes of action. The inhibitors of DNA synthesis, cytosine arabinoside, hydroxyurea, and methothrexate were less effective inducers. Bromodeoxyuridine which also has non-DNA mediated effects induced to the same extent as dibutyryl cyclic AMP.Similar experiments with sex- and age-matched cell strains derived from patients with cystic fibrosis failed to detect differences in the levels of induction from those observed in normal cells. In addition, the combined inductive effects of Tamm-Horsfall glycoprotein, isoproterenol and theophylline, were similar with normal and cystic fibrosis cells.     

Phospholipase C activity in cultured human skin fibroblasts

In this paper we report the detection of phospholipase C activity in cultured human skin fibroblasts by a rapid, sensitive method. Sonicates of fibroblasts were incubated with L-3-phosphatidyl-[U-14C]-inositol and the incubation mixture extracted with chloroform/methanol. The solvent components were then separated into 2 phases by the addition of 2 M KCl. Phospholipase C activity, determined from the amount of [14C] in the aqueous phase, agreed well with the enzyme activity assessed by other methods. The optimum pH for the enzyme was 7.0 and the enzyme was found to be dependent on Ca2+ and deoxycholate for optimal activity. The demonstration of phospholipase C activity by this method in cultured skin fibroblasts provides a useful means with which to study, in human tissues, the physiological control of this enzyme and its derangements in disease states in a controlled fashion.     

Differences in phosphatidate hydrolytic activity of human alkaline phosphatase isozymes

Hydrolytic activities of human alkaline phosphatase isozymes were investigated using phosphatidases with various fatty acyl chains (egg phosphatidate and dioleoyl, distearoyl, dipalmitoyl, dimyristoyl and dilauroyl phosphatidates). In the presence of sodium deoxycholate, purified human placental and intestinal alkaline phosphatases hydrolyzed all the phosphatidates examined. The hydrolytic activity was maximal in the presence of 10 g/l sodium deoxycholate. Of the phosphatidates, dilauroyl phosphatidate was the best substrate. Using the same unit of the enzyme, the phosphatidate hydrolytic activity of placental alkaline phosphatase was 2- to 3-times higher than that of the intestinal enzyme. In contrast, liver alkaline phosphatase did not hydrolyze phosphatidates with long fatty acyl chains (C16-18) even in the presence of sodium deoxycholate. The liver enzyme hydrolyzed dimyristoyl and dilauroyl phosphatidates very slowly. These results show that the phosphatidates with long fatty acyl chains were useful to differentiate placental and intestinal alkaline phosphatases from the liver enzyme, and suggest that the former enzymes play a different physiological role from the liver enzyme.     

Induction of alkaline phosphatase in cultured human fibroblasts. Comparison of normal cells and those from patients with cystic fibrosis.

The membrane glycoprotein enzyme, alkaline phosphatase was induced in cultured human fibroblasts by dibutyryl cyclic AMP, sodium butyrate, the serum glycoprotein fetuin, the Tamm-Horsfall urinary glycoprotein, and by a number of inhibitors of DNA synthesis. The uninduced basal enzyme activity increased at later stages of growth when the cells became confluent. Induction by dibutyryl cyclic AMP or fetuin was most effective when the agents were added after the cells had reached stationary phase and was maximal after at least two days of exposure. The levels of induction resulting from the addition of pairs of the agents, dibutyryl cyclic AMP, n-butyrate and fetuin were additive indicating that these have different modes of action. The inhibitors of DNA synthesis, cytosine arabinoside, hydroxyurea, and methothrexate were less effective inducers. Bromodeoxyuridine which also has non-DNA mediated effects induced to the same extent as dibutyryl cyclic AMP. Similar experiments with sex- and age-matched cell strains derived from patients with cystic fibrosis failed to detect differences in the levels of induction from those observed in normal cells. In addition, the combined inductive effects of Tamm-Horsfall glycoprotein, isoproterenol and theophylline, were similar with normal and cystic fibrosis cells.     

Adipose conversion of mouse bone marrow fibroblasts in vitro: their alkaline phosphatase activity

By an in vitro colony assay and cytochemical staining, we investigated the capacity of mouse bone marrow fibroblasts to differentiate into adipocytes and to express alkaline phosphatase (ALP) activity. Glucocorticoids enhanced colony formation of the fibroblasts, and stimulated their adipose conversion (55-65% of the colonies became adipocyte-positive), but they did not affect ALP activity. The fibroblasts became heterogeneous in size and morphology after growing in vitro then differentiated into adipocytes. All the cell types had ALP activity, and more than 95% of the colonies contained ALP-positive cells. ALP staining was strongest in cells in the early stage of adipose conversion, gradually decreasing with maturation. Our results indicate that the majority of the mouse bone marrow fibroblasts that formed colonies under our culture conditions are preadipocytes. We conclude that these fibroblasts originate from adventitial reticular cells present in bone marrow stroma because reticular cells have been reported to possess high ALP activity and have been suggested to differentiate into adipocytes.     

Variability of alkaline phosphatase activity in diploid human cells in vitro

Experiments were made with 19 strains obtained from different tissues (skin, lungs, muscles) of 8-10-week-old medical abortions and skin biopsies of healthy donors to study the manifestations of alkaline phosphatase (AP) activity in human diploid cells in vitro. Based on the data obtained it is concluded that AP activity is marked by demonstrable intra- and interstrain variability. The spectrum of "AP activity" trait variability is broader for transformed cells than for human diploid cells.     

Phosphoprotein-phosphatase activity associated with human placental alkaline phosphatase.

Human placental alkaline phosphatase, a marker protein for some nontrophoblastic neoplasms, was found to have phosphoprotein phosphatase activity. This was demonstrated by the dephosphorylation of ³²P-labeled histones, protamine, glycogen synthetase, casein, and phosvitin at various pH values. Unlike the general phosphoprotein phosphatase, the placental alkaline phosphatase does not have phosphorylase $\text{a}$ phosphatase activity.     

Synthesis of first trimester placental alkaline phosphatase in cultured human term placental cells

Alkaline phosphatase activity in human placental cells transformed by a tsA mutant of simian virus 40 (SV40) can be greatly induced by growing these cells at 40 degrees C, the temperature at which the tsA transformants regain their nontransformed phenotype. The induction of alkaline phosphatase in these cells requires the synthesis of both RNA and protein. The induced alkaline phosphatase from a SV40 tsA30 mutant-transformed term placental cell line (TPA30-1) was purified, characterized, and compared with alkaline phosphatase from term placenta and first trimester placenta. The form of alkaline phosphatase found in TPA30-1 cells differs from the phosphatase of term placenta in physiochemical and immunological properties. The TPA30-1 phosphatase is, however, indistinguishable from the alkaline phosphatase of human first trimester placenta by several criteria, including electrophoretic mobility, apparent molecular weight (Mr = 165,000), size of monomeric subunit (Mr = 77,000), heat lability, and sensitivity to inhibition by amino acids and EDTA. In addition, alkaline phosphatase from both TPA30-1 cells and first trimester placenta can be inactivated by antiserum to liver alkaline phosphatase but not by antiserum to term placental alkaline phosphatase. The induction of first trimester phosphatase in cells derived from term placenta provides a system for the study of alkaline phosphatase gene regulation in human placenta.