Alkaline Phosphatase In HeLa Cells Research Articles

Long Wavelength TCF-Based Fluorescent Probe for the Detection of Alkaline Phosphatase in Live Cells.

A long wavelength TCF-based fluorescent probe (TCF-ALP) was developed for the detection of alkaline phosphatase (ALP). ALP-mediated hydrolysis of the phosphate group of TCF-ALP resulted in a significant fluorescence “turn on” (58-fold), which was accompanied by a colorimetric response from yellow to purple. TCF-ALP was cell-permeable, which allowed it to be used to image ALP in HeLa cells. Upon addition of bone morphogenic protein 2, TCF-ALP proved capable of imaging endogenously stimulated ALP in myogenic murine C2C12 cells. Overall, TCF-ALP offers promise as an effective fluorescent/colorimetric probe for evaluating phosphatase activity in clinical assays or live cell systems.

Iodinated and fluorinated steroid 2′-aryl-[3,2-c] pyrazoles as potential glucocorticoid receptor imaging agents

We have synthesized several halogenated steroids as potential glucocorticoid receptor mediated imaging agents. These compounds are analogs of aryl-pyrazolo steroids, similar to the potent glucocorticoid, cortivazol. Compounds containing the halogens, iodine, bromine, and fluorine, as well as the E- and Z-iodovinyl side chain at the para position of 2′-phenyl-11β,17,21-trihydroxy-16α-methyl-20-oxo-pregn-4-eno[3,2-c] pyrazole were prepared. They were tested as ligands for the glucocorticoid receptor by competition for the binding of [ 3H]dexamethasone and for glucocorticoid potency by the induction of alkaline phosphatase in HeLa cells. None of the iodinated steroids were good ligands for the glucocorticoid receptor or potent glucocorticoids. The bromo analog was only slightly better than the iodinated steroids as a ligand, and it had a potency in the HeLa cell assay about half that of dexamethasone. The fluoro analog showed good binding to the glucocorticoid receptor and was a very potent glucocorticoid, approximately seven times that of dexamethasone. Consequently, it appears that the fluoro steroid, 2′-(4-fluorophenyl)-11β,17,21-trihydroxy-16α-methyl-20-oxo-pregn-4-eno[3,2-c] pyrazole, when labeled with 18F, would make an excellent glucocorticoid receptor-mediated imaging agent for positron emission tomography.

Effect of pyrimidine deoxynucleosides and sodium butyrate on expression of the glycoprotein hormone α-subunit and placental alkaline phosphatase in HeLa cells

Production of the glycoprotein hormone common α-subunit and placental alkaline phosphatase activity can be modulated in HeLa cells by a variety of deoxynucleosides. Dose response curves for thymidine (Thd), fluorodeoxyuridine (FdUrd), bromodeoxyuridine (BrdUrd) and iododeoxyuridine (IdUrd) demonstrate that, in general, alkaline phosphatase was increased by lower concentrations of inducer than was α-subunit. The deoxynucleosides were not as effective as sodium butyrate as inducers of either protein. Whereas Thd and the halogenated dUrd derivatives enhanced protein expression, deoxycytidine (dCyd) had negative effects. Induction by deoxynucleosides of both alkaline phosphatase and α-subunit was inhibited by dCyd, but induction of alkaline phosphatase by butyrate was more sensitive to dCyd inhibition than was the buryrate-mediated induction of α-subunit. These results suggest that the two proteins are not regulated in a coordinate manner. Reversal of alkaline phosphatase induction by dCyd was not observed in cells preincubated with sodium butyrate for 6–24 h before the addition of dCyd, indicating that the deoxynucleoside interferes with an early event in the butyrate-mediated response. Combinations of butyrate with Thd, BrdUrd or IdUrd were synergistic with respect to the induction of HeLa-α. It is concluded that incorporation of the deoxynucleosides into DNA may not be required for the synergistic response since 2′,5′-dideoxythymidine was an effective as Thd. Cytoplasmic dot hybridizations demonstrate that a primary effect of the various effectors is to increase the steady-state levels of α-subunit mRNA. There was a good correlation between α-subunit accumulation and corresponding levels of α-mRNA, suggesting that regulation occurs at a pretranslational site. Although the mechanism(s) is not understood, these data provide evidence that nucleosides or their derivatives can significantly affect gene expression.

Measurement of biosynthetic rates and intracellular transit times for a cell-surface membrane glycoprotein, alkaline phosphatase in HeLa cells

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.

Induction of alkaline phosphatase activity in HeLa cells by sodium butyrate

The induction of HeLa cell alkaline phosphatase activity by sodium butyrate could be inhibited by the coadministration of caffeine or theophylline. The inhibitions were dose dependent, and at any given concentration the potency was theophylline greater than caffeine. Although the induction by sodium butyrate was more sensitive to the inhibition by the xanthines than was that produced by 5-iodo-2'-deoxyuridine, the magnitudes of the increases in cyclic AMP concentrations after treatment with the xanthines were similar in the inhibition of both types of induction. The induction of alkaline phosphatase activity by sodium butyrate also produced a shift in the thermostability pattern of the enzyme, with a proportionately greater increase in the heat-labile, rather than heat-stable, form of the activity.

The Ontogony of the Human Placental Glucocorticoid Receptor and Inducibility of Heat-Stable Alkaline Phosphatase*

The human placenta was found to contain a cytosol receptor for glucocorticoids. The concentration of this receptor in term placenta was 27-fold higher than that found in cytosol from first trimester placenta. The levels of cytosol glucocorticoid receptor in three trophoblastic cell lines (JAr, BeWo, and JEG) were also determined and all were found to be low. The ability of prednisolone, a potent glucocorticoid, to stimulate heat-stable alkaline phosphatase activity found in these cells was tested. Although control experiments demonstrated that the conditions were adequate to stimulate HeLa cell alkaline phosphatase, none of the trophoblastic lines responded to prednisolone administration. This result may be explained by the observation that the JAr cells lacked any detectable glucocorticoid receptor and the receptor levels in cytosol prepared from JEG and BeWo cells were 12% and 2%, respectively, of those measured in HeLa cytosol. Our studies also suggest that the increase in serum levels of heat-stable alkaline phosphatase observed during pregnancy may reflect increasing placental sensitivity to glucocorticoids as a result of increased receptor levels.

Modulation of HeLa Cell Alkaline Phosphatase Isoenzyme Expression by Prednisolone

Various HeLa cell lines have been examined in order to determine the effect of hormone treatment upon alkaline phosphatase isoenzyme expression. Both biochemical and immunoelectrophoretic analyses show that three cell lines demonstrating activity of the placental‐type (Regan) isoenzyme in absence of prednisolone are induced to much higher levels after growth in hormone‐containing medium for 72 hours. Prednisolone‐treatment resulted in increased enzyme activity but no change in the isoenzyme profile as determined by microzone and polyacrylamide disc gel electrophoresis. On the other hand, the majority of the activity in HeLa line D98AH2 in absence of hormone was found to be heat‐sensitive and inhibited by phenylalanine. Immunoelectrophoretic analysis indicated that amnion/intestinal isoenzyme was expressed. No appreciable change in total enzyme activity occurred in presence of hormone despite the induction of the Regan isoenzyme and the diminution of amnion/intestinal type. Thus, treatment of this cell line with hormone resulted in differential expression of the amnion/intestinal and placental isoenzyme components. These results support a regulatory function of prednisolone with respect to expression of alkaline phosphatase isoenzymes in tumor cell lines and identify a HeLa cell line, D98AH2, which elaborates the amnion/intestinal isoenzyme.

An immunological and biochemical analysis of alkaline phosphatase in HeLa cells exposed to 5-iodo-2′-deoxyuridine

As ascertained by antibody titration, 5-iodo-2'-deoxyuridine (IUdR) appears not to increase alkaline phosphatase activity by increasing the concentration of the enzyme in HeLa cells but rather more probably by increasing the catalytic activity of the enzyme molecule. However, continued induction of activity ceases when protein synthesis is inhibited. The specific activity attained by the combined use of IUdR and hydrocortisone was equal to or less than the sum of specific activities obtained when either agent was used alone. The moles of phosphate bound per mole of alkaline phosphatase was similar for the enzyme from control cells or cells treated with IUdR or hydrocortisone. Even though each of several compounds tested inhibited alkaline phosphatase activity by 50 per cent at a different concentration, there was no difference between the concentrations needed for each compound to cause 50 per cent inhibition of the enzyme from control or IUdR-treated cells.

Rapid cell surface-related stimulation of alkaline phosphatase in HeLa cells by dimethyl DL-2,3-distearoylotypropyl-2'-hydroxyethylammonium acetate (Rosenthal's inhibitor).

Cultures of HeLa cells, strains S3G (HeLa65) and S3K (HeLa71) were grown in plastic dishes until firmly attached and were then treated with sonic dispersions of Rosenthal's phospholipase inhibitor. A rapid increase in alkaline phosphatase activity occurred following this treatment in the S3G strain (low inducible alkaline phosphatase) but not in the S3K strain (high constitutive alkaline phosphatase). The stimulatory effect was dependent on the presence of bovine serum in the medium. No stimulation of alkaline phosphatase was observed in a variety of soluble preparations of this enzyme.

Substrate specificities of base-level and induced forms of oncofetal alkaline phosphatase in HeLa cells.

HeLa65 cells contain an oncofetal form of alkaline phosphatase that exhibits an increase in catalytic activity when cultures are grown in medium containing adrenal glucocorticoid hormones such as hydrocortisone or its analogue prednisolone. The increase in alkaline phosphatase specific activity in cells grown with hormone is apparently the result of a reduced phosphate content of the enzyme which alters the transition state of the enzyme-substrate complex with an increase in catlytic efficiency. Fourteen different substrates possessing aliphatic, aromatic and heterocyclic configurations were studied with respect to the initial velocities of hydrolysis catalyzed by the base-level and induced forms of the enzyme. The magnitude of increase in catalytic activity was not substantially influenced by the structure of the nonphosphoryl moiety of substrates.

Plasma membrane localization of alkaline phosphatase in HeLa cells.

The localization of alkaline phosphatase in HeLa cells was examined by electron microscopic histochemistry and subcellular fractionation techniques. Two monophenotypic sublines of HeLa cells which respectively produced Regan and non-Regan isoenzymes of alkaline phosphatase were used for this study. The electron microscopic histochemical results showed that in both sublines the major location of alkaline phosphatase is in the plasma membrane. The enzyme reaction was occasionally observed in some of the dense body lysosomes. This result was supported by data obtained from a subcellular fractionation study which showed that the microsomal fraction rich in plasma membrane fragments had the highest activity of alkaline phosphatase. The distribution of this enzyme among the subcellular fractions closely paralleled that of the 5'-nucleotidase, a plasma membrane marker enzyme. Characterization of the alkaline phosphatase present in each subcellular fraction showed identical enzyme properties, which suggests that a single isoenzyme exists among fractions obtained from each cell line. The results, therefore, confirm the reports suggesting that plasma membrane is the major site of alkaline phosphatase localization in HeLa cells. The absence of any enzyme reaction in the perimitochondrial space in these cultured tumor cells also indicates that the mitochondrial localization of the Regan isoenzyme reported in ovarian cancer may not be a common phenomenon in Regan-producing cancer cells.

Alkaline phosphatase in HeLa cells: Stimulation by phospholipase A 2 and lysophosphatidylcholine

Treatment of homogenates and plasma membrane preparations from HeLa cells with phospholipase A 2 (EC 3.1.1.4) caused a 50% increase in activity of membrane-associated alkaline phosphatase. Lysophosphatidylcholine, dispersed in 0.15 M KCl, affected alkaline phosphatase in a similar fashion by releasing the enzyme from particulate fractions into the incubation medium and by elevating its specific activity. Higher concentrations of lysophosphatidylcholine solubilized additional protein from particulate fractions but did not further increase the specific activity of the released alkaline phosphatase. Particulate fractions from HeLa cells were exposed to the effects of liposomes prepared from lysophosphatidylcholine and cholesterol. The ratio of particulate protein/lysophosphatidylcholine (by weight) required for optimal activation of alkaline phosphatase was one. Kinetic studies indicated that phospholipase A 2 and lysophosphatidylcholine enhanced the apparent V of the enzyme but did not significantly alter its apparent K m . The increased release of alkaline phosphatase from the particulate matrix by lysophosphatidylcholine was confirmed by disc electrophoresis. The release of the enzyme by either phospholipase A 2 or by lysophosphatidylcholine appeared to be followed by the formation of micelles that contained lysophosphatidylcholine. The new complexes had relatively less cholesterol and more lysophosphatidylcholine than the native membranes. The possibility that lysophosphatidylcholine formed a lipoprotein complex with the solubilized alkaline phosphatase was indicated by a break point in the Arrhenius plot which was evident only in the lysophosphatidylcholine-solubilized enzyme but could not be demonstrated in alkaline phosphatase that had been released with 0.15 M KCl alone.

A study of protein profiles in two strains of HeLa cells including membrane preparations. Lack of prednisolone effects.

Steroid-mediated induction of alkaline phosphatase in HeLa cells could not be ascribed to detectable qualitative or quantitative changes in cell proteins separable by disc gel electrophoresis. Similarly, neither qualitative nor quantitative differences in membrane proteins were observed after HeLa cell strains with either inducible or suppressible alkaline phosphatases were exposed to prednisolone. However, differences in protein profiles of the two cell strains were present.

Studies on hormonal induction of alkaline phosphatase in HeLa cell cultures. Kinetic, thermodynamic and electrophoretic properties of induced and base-level enzymes

As previously described the “induction” of a 5- to 20-fold increase in alkaline phosphatase (orthophosphoric monoester phosphohydrolase, EC 3.1.3.1) activity by hydrocortisone in HeLa 65 cell cultures requires both RNA and protein synthesis; however, immunological methods clearly show that the amount of enzyme protein is not increased. The present study compares the catalytic properties of the base-level and induced alkaline phosphatases. K m values for these enzymes are similar, however, the maximal velocity ( V) of induced alkaline phosphatase (27 nmoles/min) is considerably higher than that of base level (1.3 nmoles/min). The first-order rate constant ( k 3) for decomposition of enzyme substrate ( ES) complex to products, of induced alkaline phosphatase is 7- to 17-fold higher than that of base-level alkaline phosphatase. Enzyme kinetics are similar with both phenylphosphate and p-nitrophenyl-phosphate as substrates. Hormone-induced alkaline phosphatase in HeLa 65 cells possesses higher catalytic activity presumably because it can bind with phosphomonoesters to produce a more rapid decomposition of the ES complex. HeLa cell alkaline phosphatase is similar to the placental enzyme with respect to inhibition by L-tryptophan and L-phenylalanine. Inhibition of both base-level and induced HeLa alkaline phosphatase by these L-amino acids is stereospecific, uncompetitive and homosteric. The inhibitor constants ( K i ) for these L-amino acids increases with increasing temperature. Values for change of enthalpy ( ΔH), free energy ( ΔF), and entropy ( ΔS) for binding of L-tryptophan to base-level and induced HeLa alkaline phosphatase are similar and both enzymes resemble the placental alkaline phosphatase. Alkaline phosphatase prepared from HeLa 65 cells grown in the presence and absence of hydrocortisone exhibits three isozymes as detected by electrophoretic mobility on starch gel. The isozymes are molecular weight variants that can be separated by Sephadex G-200 gel filtration. A HeLa 71 cell with high constitutive levels of alkaline phosphatase possesses five isozymes on electrophoresis. Multiple molecular forms of base-level and induced alkaline phosphatase are similar and closely resemble those of human placental enzyme.

Production of fetal-like alkaline phosphatase by HeLa cells.

Alkaline phosphatase produced by HeLa cells differs in its chemical and physical properties from the enzyme found in adult organs and tissues (Cox and Griffin, 1967). In the present study HeLa cell alkaline phosphatase was compared to a fetal form of the enzyme found in human placenta. Both enzymes have approximately the same molecular weight as judged by sucrose density gradients, and the chemical and physical properties of these alkaline phosphatases are similar. The electrophoretic pattern of the HeLa cell enzyme resembles the placental alkaline phosphatase of the heterozygous FS phenotype except that it is slower moving. Double immunodiffusion using an antibody against HeLa cell alkaline phosphatase and placental and HeLa cell enzymes as antigens shows a single line of partial identity between the two enzymes, with a small spur suggesting additional antigenic sites on the HeLa cell enzyme. The data suggest that malignant cells in culture, HeLa, are producing a fetal-like alkaline phosphatase probably by “derepression of the genome.” However, the electrophoretic and immunological characteristics of the enzyme are altered sufficiently so that it can be distinguished from the normally produced fetal enzyme.

Serum-dependent depression of alkaline phosphatase in HeLa cells.

AbstractReduction in alkaline phosphatase activity was observed when HeLa S3 cells were grown in Puck's medium containing high concentrations of human serum. This effect was not seen with the enzyme of Chang liver 8A cells. The induction of increased alkaline phosphatase in HeLa S3 by prednisolone or by osmolality changes was not prevented by serum. The concentration of serum in the culture medium had no influence on acid phosphatase activity.

Relationships between steroid binding and elevation of alkaline phosphatase in HeLa cells

1. 1. Elevation of alkaline phosphatase (orthophosphoric monoester phosphohydrolase, EC 3.1.3.1) by prednisolone in growing cultures of HeLa-S3 cells was partially inhibited by several C 21 steroids. The requirement necessary to bring about this effect included the 11-deoxy structure of the steroid. The reversal was observed also with those 11-hydroxysteroids which were inactive as alkaline phosphatase inducers because they lacked a hydroxyl in position 17 ( e.g. corticosterone). In contrast, the 11-ketosteroids were inactive as inhibitors of alkaline phosphatase induction. The reduced derivatives of cortisol ( e.g. tetrahydrocortisol) were similarly inactive. 2. 2. Ability to reverse elevation of alkaline phosphatase seemed to be related to the interference with binding of cortisol at the cell surface. This was evidenced by the lack of specific binding competition in cells detached from glass by treatment trypsin or by mechanical removal.

A chemical method for the isolation of HeLa cell nuclei and the nuclear localization of HeLa cell alkaline phosphatase.

A chemical method for the isolation of HeLa cell nuclei and the nuclear localization of HeLa cell alkaline phosphatase.

Effects of Cations on Activity and Thermostability of Alkaline Phosphatase in HeLa Cells.

SummaryIn deoxycholate lysates from HeLa cells Zn+ + reversed inhibition of alkaline phosphatase by glycine. Low concentrations of this cation were found to reactivate dialyzed preparations of the same enzyme. Mg+ + retarded thermal inactivation of alkaline phosphatase. Delay in heat inactivation was observed in alkaline phosphatase preparations from both prednisolone treated and control cultures of HeLa cells.

Effect of corticosteroids on the formation of alkaline phosphatase in HeLa cells

Studies of the enzymatic make-up in established lines of mammalian cells have revealed remarkable similarity among cells of diverse origins (1). Alkaline phosphatase was found to constitute an exception since it varied in activity not only from one strain to another but also within the same strain of cells, depending upon the clone being studied (2,3). Although little is known about the factors controlling the levels of this enzyme in cell cultures, a recent study of Cox and MacLeod (3) has shown that prednisolone increased the level of alkaline phosphatase in HeLa cells. The work reported in this communication primarily involved a study of the relationship between the steroid structure and its ability to stimulate alkaline phosphatase.