Nimai K. Ghosh

A study of adenosine 3':5'-cyclic monophosphate, sodium butyrate and cortisol as inducers of HeLa alkaline phosphatase.

Abstract The role of adenosine 3′:5′-cyclic monophosphate in the cortisol-mediated induction of HeLa 65 alkaline phosphatase was investigated. Although growth of these cells with 0.5–1.0 m m N 6 , O 2 ′-dibutyryl adenosine 3′:5′-cyclic monophosphate induces a 5- to 8-fold increase in cellular phosphatase activity after 72 hr, neither cAMP nor theophylline induce at concentrations up to 1 m m . Sodium butyrate induces the enzyme as well as dibutyryl cAMP. Moreover, induction kinetics show sodium butyrate to be a more efficient inducer than dibutyryl cAMP, inducing activity as quickly as cortisol. This suggests that the butyric acid cleaved from dibutyryl cAMP by HeLa cells is the mediator of induction when the cyclic nucleotide derivative is used.

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. Km 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 (k3) 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 (Ki) 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.

HORMONALLY INDUCED MODIFICATION OF HELA ALKALINE PHOSPHATASE WITH INCREASED CATALYTIC ACTIVITY

ABSTRACT. Alkaline phosphatase (AP) activity of HeLa cells is increased 5 to 20 fold during growth in medium with Cortisol. The kinetics of induction show a 12 to 20 hr lag period after adding hormone, followed by a rapid linear increase of enzyme activity which reaches a plateau after 60 to 90 hr. RNA and protein synthesis are required for “induction”. However, physical and immunological methods show that the amount of AP protein in induced cells is not significantly increased, suggesting that an enhanced catalytic efficiency is responsible for the increase in enzyme activity.

Characterization of monosaccharides in human placental alkaline phosphatase by gas—liquid chromatography

Abstract Gas—liquid chromatography of hydrolysates of highly purified human placental alkaline phosphatase (orthophosphoric-monoester phosphohydrolase, EC 3.1.3.1) demonstrated the presence of monosaccharide residues, mannose, galactose, glucose and fucose. This enzyme, therefore, is a sialoglycoprotein.

Comparison of aldolase isozymes in placenta, HeLa cells, and human fibroblast cultures.

The aldolase specific activity of the human carcinoma cell line, HeLa, against fructose 1,6-diphosphate as substrate is 4- to 5-fold greater than the specific activity of diploid human fibroblast cultures derived from skin and lung. HeLa aldolase is isozyme is predominantly the A type and its substrate preferences resemble human placenta. These findings provide further support for the oncofetal enzyme consitution of HeLa cells.

Effects of prednisolone and butyrate on agglutinability of HeLa cells by concanavalin A

Agglutinability by concanavalin A was measured with HeLa65 cells grown with prednisolone or sodium butyrate, 2 compounds that increase the activity of the carcinoplacental form of alkaline phosphatase, an enzyme localized in membranes. Prednisolone enhanced concanavalin A agglutination approximately 3-fold while sodium butyrate had no effect.

Drug-induced biochemical markers of cancer in cervical carcinoma cells

The elevation in the serum level of CEA in cancer patients undergoing treatment with 5-FU and other antitumor drugs has been reported. In the present study, the ectopic synthesis of multiple carcinoplacental markers has been observed to be induced (10- to 264-fold) simultaneously in the same cervical carcinoma cells (HeLa65, HeLa71 and HeLa2.2) by hydroxyurea and sodium butyrate. Among the drug-induced biochemical markers observed in HeLa cells are four sialopeptides. Regan Isoenzyme (Placental Isoenzyme of Alkaline Phosphatase), HCT-Beta, FSH-Beta, HCG-Alpha and also a steroid hormone, Progesterone. The peptide and steroid hormones were quantitated by specific radioimmunoassays (RIA), in cultured cells, media, and homogenates of tumor tissues. The induction of biochemical markers was observed also with lung carcinoma cells. That multiple polypeptides, or steroids regulated by them, are simultaneously inducible in the same cancer cells, suggest the proximity on the DNA strand of several oncofetal and oncoplacental genes derepressed by antineoplastic drugs. This fundamental study has had important clinical ramifications. The results may be used to recognize the retention by cancer patients of occult malignancy after radiotherapy or surgery. The unsuspected metastasis may be reflected by a transient rise in the serum level of these markers during chemotherapy with anticancer drugs, which specifically inhibit DNA replication without interfering with the transcription of messenger-RNA and subsequent translation of proteins. The drug-induced protein-hormones, observed in this study, are the products of activated trophoblastic/pituitary genes in the nondividing DNA of neoplastic cells.

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.