Naphthol AS-TR Phosphate Research Articles

A Novel Small-Molecule Inhibitor Targeting CREB-CBP Complex Possesses Anti-Cancer Effects along with Cell Cycle Regulation, Autophagy Suppression and Endoplasmic Reticulum Stress.

Lung adenocarcinoma, the most common subtype of lung cancer, is the leading cause of cancer death worldwide. Despite attempts for the treatment of lung cancer which have been accumulating, promising new therapies are still needed. Here, we found that cyclic-AMP response element-binding protein (CREB)-CREB binding protein (CBP) transcription factors complex inhibitor, Naphthol AS-TR phosphate (NASTRp), is a potential therapeutic agent for lung cancer. We show that NASTRp inhibited oncogenic cell properties through cell cycle arrest with concomitant suppression of tumor-promoting autophagy with down-regulations of Atg5-12 and Atg7, and accumulation of p62 in human lung cancer cell lines. In addition, NASTRp induced expression of endoplasmic reticulum stress markers such as DDIT3/CHOP, and led to apoptosis along with Bim induction. These findings suggest that transcription factor/co-activator complex, CREB-CBP, can be a potential therapeutic target and its inhibition could be a novel therapeutic strategy for lung cancer.

UV irradiation of a B-cell hybridoma increases expression of alkaline phosphatase: involvement in apoptosis

Expression of alkaline phosphatase (APase) by 7TD1 B-cell hybridoma was amplified by ultraviolet irradiation; cell growth was inhibited and cell death by apoptosis was increased. Irradiation induced high levels of APase activity in cycling as well as in apoptotic cells. In contrast, APase activity faded with time in nonirradiated cells and was no longer expressed in spontaneous apoptotic cells appearing after several days in culture. This was demonstrated by cell morphology, DNA fragmentation, and flow cytometry after simultaneous staining of DNA with Hoechst 33342 and APase with naphthol AS-TR phosphate--fast red RC fluorescent reagent. Levamisole, a specific inhibitor of APase activity, almost totally abrogated apoptosis induced by ultraviolet irradiation at doses that failed to affect 7TD1 cell survival. These data suggest that APase could play a role in the signalling cascade that mediates apoptosis in irradiated cells.

Histochemical localisation of acid phosphatase and non-specific esterase in the midguts of two species of tick,Boophilus microplus andRhipicephalus appendiculatus, as determined by light microscopy

Serial sections of glycol methacrylate-embedded and frozen midguts of Boophilus microplus and Rhipicephalus appendiculatus were studied histochemically by light microscopy. The use of the naphthol AS-TR phosphate technique combined with glycol methacrylate embedding enabled the precise localisation of lysosomal enzyme activity, despite the ubiquity of haematin granules in tick midgut epithelia. The presence of acid phosphatase and non-specific esterase activity in the same cells was observed in all of the various developmental stages and feeding phases of the ticks. The pattern of appearance of these cells paralleled the reported level of protease activity in the midgut lumen. The cells were found to be solitary, particularly during the slow digestive phases, and appeared to move into the lumen, where they eventually disintegrated. The cells therefore appear to function as holocrine secretory cells. This is the first report indicating the presence of such secretory cells in the midgut of unfed ticks. The disintegration of these cells in the lumen suggests that lumenal digestion may be more important than hitherto realised.

Characterization of cells isolated and cultured from human bone.

Cells isolated from samples of human iliac crest and human femoral heads by collagenase digestion have been successfully cultured in Fitton-Jackson modified BGJb culture medium supplemented with penicillin (100 units/ml), streptomycin (100 micrograms/ml), and fetal calf serum (10%). Although only a low proportion of the cells survived the initial plating (less than 1%), cells established in culture were readily passaged. Examination of cells obtained at intervals during the collagenase digestion showed that the percentage of cells that attached increased with time of digestion. Rapid sample preparation of rat bone did not substantially increase the number of cells attaching. Thus, it seems unlikely that the low survival was due to loss of viability during sample transportation and preparation. Of several media tested BGJb supplemented with 10% fetal calf serum supported the best growth. Population doubling time averaged 104 hr. Cultured human bone cells were assayed for alkaline phosphatase activity using the azo dye method with naphthol ASTR phosphate as the substrate. A portion of the cells (19%) demonstrated high activity in all cultures examined regardless of the passage number of the culture. Autoradiography of cells exposed to [3H]thymidine showed incorporation of the label into both alkaline phosphate-positive and -negative cells. The stimulation of cell proliferation by growth factors was studied by determining the incorporation of [3H]thymidine into DNA. The specific skeletal growth factor from human bone stimulated cell proliferation several-fold with a half-maximal effect at 5 micrograms/ml. Insulin, epidermal growth factor, and a crude preparation of somatomedin C also stimulated cell proliferation.

Immunocytochemical characterization of human blood cells.

A simple immunocytochemical method using calf intestinal alkaline phosphatase as the enzymatic indicator to demonstrate surface antigens on human blood cells has been developed. The blood cells were labeled with cell specific monoclonal antibodies followed by linkage with an antiimmunoglobulin alkaline phosphatase conjugate. Cytochemical demonstration of alkaline phosphatase activity on the blood cells reflects the presence of surface antigens on these cells. The effects on the cytochemical reaction of fixation, substrates, couplers, activators and inhibitors, and storage of cytologic materials have been examined systematically. The best staining conditions are to incubate labeled smears in a 0.04 M barbital buffer at pH 7.6 containing 30 mg% naphthol AS-TR phosphate, 40 mg% fast red ITR, and 1 mM levamisole. This method is both sensitive and specific and appears most practical for objective identification of the human blood cells.

Acid hydrolases in the suckling rat small intestine. II. On the importance of alkaline phosphatase inhibition in the histochemical localization of acid phosphatase activity

Phosphatase activities against beta-glycerophosphate, I-naphthyl phosphate and naphthol AS-TR phosphate were investigated, at acid and aldaline pH levels, using unfixed and fixed cryostat sections of suckling rat jejunum. The use of 10 mm EDTA and 10 mm NaF as inhibitors indicated that alkaline phosphate is predominantly located in the microvillous region of the adsorptive cells, while acid phosphatase is located in small particles distributed between the brush borders and the nuclei of these cells. Alkaline phosphatase activity was found to interfere with the localization of acid phosphatase unless EDTA was included in the incubation medium. A modified Gomori medium, containing 10 mm EDTA and additional lead nitrate, is described. Latency experiemtns using this medium, with unfixed sections, indicated the lysosomal nature of particulate acid phosphatase. The discussion stresses the importance of including an aldaline phosphatase inhibitor in incubation media designed to localize extralysosomal acid phosphatase activity.

Histochemical studies on alkaline phosphatases in mineralizing oral tissues in the mouse.

Activity of alkaline phosphatases in unfixed cold microtome setions from the lower first molar area of newborn mice was recorded by histochemical methods. A substrate specificity test included the following phosphate compounds: ATP, CTP, GTP, UTP, ADP, AMP, GP, PPi, MDP and naphthol AS-TR phosphate. Intense staining was obtained in osteoblasts, stratum intermedium of the enamel organ and odontoblasts with all the substrates, except PPi and MDP. Staining of skeletal muscle fibres was obtained only with triphosphates as substrates. Addition of-SH groups decreased the hydrolysis of triphosphate compounds in cells involved in mineralization while the hydrolysis of monophosphate was inhibited. In contrast triphosphatase activity in striated muscle was enhanced when-SH compounds were added. Demineralization with EDTA diminished the cytoplasmic staining but induced a nuclear staining in hard tissue forming cells when triphosphates were used as substrates. No cytoplasmic and only slight nuclear staining was seen with GP or AMP as substrates. The triphosphate hydrolyzing capacity of tongue muscle fibres was, however, increased after the decalcification treatment. Addition of Mg2+ ions to the incubation media distinctly lowered the hydrolysis of triphosphates in the investigated tissues whereas the hydrolysis of ADP, AMP, GP and naphthol AS-TR phosphate remained unchanged. In view of the findings the triphosphatase activities at alkaline pH of muscle fibres and of cells related to hard tissue formation are considered to be due to activity of separate enzymes. The orthophosphate liberating enzyme activities at alkaline pH in osteoblasts, stratum intermedium and odontoblasts may be expressions of the catalytic functions of one common enzyme. Furthermore, the results indicate that CaATP might be the substrate used by the alkaline ATPase in mineralizing areas.

The histochemical demonstration of hydrolytic enzymes in adjuvant-induced arthritis in rats.

Acid phosphatase, alkaline phosphatase, leucine naphthylamidase and β-glucuronidase were studied histochemically in unfixed, undemineralized affected paws from rats with adjuvant-induced arthritis. Sections were prepared in a newly designed bone cryostat and it was found that untreated tissue sections gave the most satisfactory results. Acid and alkaline phosphatase activities were studied using azo dye techniques and the substrate naphthol AS-TR phosphate gave the best results with regard to sensitivity and localization.

The histochemical differentiation of various phosphatases in a population of osteoclasts by a simultaneous coupling method using different diazonium salts, with observations on the presence of inhibitors in stable diazonium salts.

The numerous osteoclasts in a giant cell tumour of bone were found to possess at least two distinct phosphatases capable of hydrolysing naphthol AS-TR phosphate. An acid phosphatase, with optimum activity about pH 4.7, could be demonstrated by simultaneous coupling with Fast Bordeaux OL or Red Violet LB, but not with Fast Red TR. The last-named salt, on the other hand, could be used for demonstrating a phosphatase with an optimum pH of activity about 7.3, showing some activity as an ‘alkaline’ phosphatase at pH 8.3. This enzyme was markedly inhibited by zinc ions and could not be demonstrated by simultaneous coupling with diazonium salts stabilized with zinc chloride. The acid phosphatase was much less sensitive to zinc, but showed marked inhibition by aluminium, which had comparatively little effect on the other enzyme. Some discrepancies between the published formulae of stable diazonium salts and the substances found to be present in them are discussed.

Distribution of acid and alkaline phosphatase activity in undemineralized sections of the rat tibial diaphysis.

The distribution of acid and alkaline phosphatase activity in the rat tibia has been studied in transverse sections sawed from fresh undemineralized diaphyses. The endosteal resorbing surfaces had the highest acid phosphatase activity as demonstrated with 50 mM α-naphthol phosphate as the substrate. Strong activity was present not only in osteoclasts and adjacent osteocytes but extracellularly lining Howship's lacunae. In areas of bone formation, moderate acid phosphatase activity was present in osteoblasts and young osteocytes. In addition, a zone of activity was found at the mineralizing front where mineralization is initiated. This active zone ( a) was separated from the active periosteal cell layer by a zone of inactive osteoid and ( b) in formol-calcium-fixed demineralized sections extended into young bone. The identity of the mineralizing front was confirmed by tetracycline labeling. The activity at the mineralizing front had the properties of an enzyme. Alkaline phosphatase activity demonstrated with naphthol ASTR phosphate was largely associated with the osteoblasts and other mesenchymal cells at forming surfaces.

Histochemical detection of acid and alkaline phosphatases in periodontal tissues after experimental tooth movement.

Acid and alkaline phosphatases were demonstrated histochemically in periodontal structures and alveolar bone tissues after experimental tooth movement. Previous reporting has concerned periodontal structures during orthodontic tooth movement (C. SANDSTEDT, Nord Tandl Tidsk No. 4,5, 1905; A.E. ZAKI and G.V. HUYSEN, JDent Res 42:1373-1379, 1963). Sixty Wistar strain rats were used in this experiment. During ether anesthesia, a rubber dam (1.5 mm wide and 0.15 mm thick) was inserted between the first and second molars on the left side of the upper jaw. The right side was observed as a control (K. TAKIMOTO ET AL: | Dent Res 45:1473-1476, 1966). Rats were killed in groups of 20 after one, three, and five days. In rats killed after five days, the rubber dam was replaced after three days by two rubber strips to exert a constant amount of force. Blocks of fresh tissue were prepared and decalcified for one week at 4C in a 10% solution of (tetrasodium salt of ethylendiamintetraacetate (EDTA), as a neutral solution adjusted to pH 7.0 with 5M citric acid. After rinsing in disstilled water, blocks were sagittally sectioned at 18,u in a cryostat with a sliding microtome. Serial sections were fixed (10% neutral formalin, 30 min) and stained with hematoxylin and eosin. The substrate solution for acid phosphatase consisted of 5 mg of naphthol AS-TR phosphate, dissolved in 0.1 ml of N-N dimethylformamide with 30 ml of 0.1M acetate buffer at pH 5.8 and added to 30 mg of fast red violet LB.* In sections incubated 1 to 1.5 hours at 37C, dark purple precipitation indicated enzyme-positive sites. The substrate solution for alkaline phosphatase consisted of 5 mg of naphthol AS-MX phosphate, dissolved in 0.1 ml of NCN dimethylformamide and 30 ml of Clark and Lub's buffer (K. KAWAKATSU and M. MORI, Cancer Res 23:539545, 1963) at pH 9.2 and added to 30 mg of fast red violet LB. After 30 to 50 minutes at 20C, enzyme-positive sites appeared red on a yellowbrown background. In normal tissues, moderate amounts of acid and alkaline phosphatases were observed in the periodontal ligaments. Alveolar bone surface and

The effects of aldehyde fixation on acid phosphatase activity in tissue blocks.

The effect of various aldehydes on phosphatase(s) of rat liver and kidney hydrolyzing the monophosphates of β-glycerol, phenol, p-nitrophenol and naphthol AS-TR at pH 5.0 was determined. Biochemical data were correlated with histochemical staining results. On the basis of enzyme recovery after fixation, the aldehydes could be divided into 3 broad groups: (1) well over 50 per cent after hydroxyadipaldehyde and glyoxal; (2) near 50 per cent after formaldehyde and methacrolein; and (3) well below 50 per cent after crotonaldehyde, glutaraldehyde, and acrolein. Acid phosphatase(s) hydrolyzing all 4 substrates showed a similar differential response to fixation by 4 aldehydes, but individual recovery values with naphthol AS-TR phosphate were distinctly lower. Tissues immersed in the first aldehyde group were soft, showed poor morphologic preservation, and a large percentage of the activity was in a soluble state; these features were reflected in the histochemical demonstration of acid phosphatase by an intense staining reaction which was judged to be unsatisfactory. The reverse held true for tissues fixed in aldehydes of the third group, using glutaraldehyde in the staining tests Evidence is presented suggesting that acid phosphatase staining results after formaldehyde fixation is dependent in part on the duration of fixation.

APPLICATION OF THE STEREOSPECIFIC INHIBITOR L-PHENYLALANINE TO THE ENZYMORPHOLOGY OF INTESTINAL ALKALINE PHOSPHATASE.

The intestine-specific inhibitor of alkaline phosphatase, l-phenylalanine, has been employed in the study of alkaline phosphatase in rat intestine. Five substrates were used, β-glycerophosphate, o-carboxyphenylphosphate, α-naphthyl acid phosphate, naphthol AS-BI phosphate and naphthol AS-TR phosphate. The hydrolytic rate and specific l-phenylalanine inhibition was most marked in the case of the first three substrates. When formol-Ca fixed, gum-sucrose treated sections of rat intestine were incubated in these substrates in the presence separately of 0.05 Md- and l-phenylalanine and Gomori's calcium-cobalt method was employed, a striking inhibition by the l-isomer was regularly found. Azo dye methods were applied under different conditions of time, temperature and cation concentration in some instances. The contrast was marked but not dramatic in the case of α-naphthyl acid phosphate, and it was evident but not marked in the case of naphthol AS phosphate substrates. These findings have been discussed. The alkaline phosphatase of intestinal epithelial cells (human) grown in tissue culture exhibited great sensitivity to l-phenylalanine. Rat kidney and leukocyte alkaline phosphatase were relatively insensitive under the same conditions. Morphologically, the l-phenylalanine-sensitive alkaline phosphatase in rat intestine is largely confined to the striated border of the epithelial cells. In animals maintained on a high fat diet, azo dye methods demonstrated the presence of fine granules in the region of the Golgi apparatus and terminal web in the presence of d-phenylalanine. These reactions were absent in companion sections incubated in l-phenylalanine-containing substrate solutions. The possibility of comparing the morphology of a tissue in which both test and control solutions are identical except for the configuration of one component may contribute to a more precise interpretation of the results obtained in the test section.