Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.1.3.1 (alkaline phosphatase)
47,916 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A combination of immunocytochemistry and microdensitometry has been used to localize and quantify the expression of the proto-oncogene c-myc within chondrocytes of the proximal growth plates of rat and chick long bones. Although the c-myc protein was localized in all chondrocytes of the growth plate of both species the most intense staining was restricted to the proliferating and differentiating chondrocytes. These were identified by their ability to synthesize DNA (bromodeoxyuridine positive) and the presence of alkaline phosphatase activity, respectively. Species differences did exist with the c-myc concentration of the chick proliferating and differentiating chondrocytes being higher (128% and 240%, respectively) than the respective chondrocytes of the rat. The higher c-myc concentration in the chick proliferating chondrocytes paralleled the differences in the bromodeoxyuridine labelling index between the two species. In the rat, the concentration of c-myc protein present in the differentiating chondrocytes was 74% higher than in the respective proliferating chondrocytes, while in the chick it was 146% higher. The data not only provides further evidence for a role of the c-myc protein in cell proliferation but also suggests involvement of this protein in chondrocyte differentiation and/or hypertrophy.
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PMID:The proto-oncogene c-myc is involved in cell differentiation as well as cell proliferation: studies on growth plate chondrocytes in situ. 161 15

The N-myc and c-myc genes encode closely related nuclear phosphoproteins. We found that the N-myc protein from human tumor cell lines appears as four closely migrating polypeptide bands (p58 to p64) in sodium dodecyl sulfate-polyacrylamide gels. This and the recent finding that the c-myc protein is synthesized from two translational initiation sites located in the first and second exons of the gene (S. R. Hann, M. W. King, D. L. Bentley, C. W. Anderson, and R. N. Eisenman, Cell 52:185-195, 1988) prompted us to study the molecular basis of the N-myc protein heterogeneity. Dephosphorylation by alkaline phosphatase reduced the four polypeptide bands to a doublet with an electrophoretic mobility corresponding to the two faster-migrating N-myc polypeptides (p58 and p60). When expressed transiently in COS cells, an N-myc deletion construct lacking the first exon produced polypeptides similar to the wild-type N-myc protein, indicating that the first exon of the N-myc gene is noncoding. Furthermore, mutants deleted of up to two thirds of C-terminal coding domains still retained the capacity to produce a doublet of polypeptides, suggesting distinct amino termini for the two N-myc polypeptides. The amino-terminal primary structure of the N-myc protein was studied by site-specific point mutagenesis of the 5' end of the long open reading frame and by N-terminal radiosequencing of the two polypeptides. Our results show that the N-myc polypeptides are initiated from two alternative in-phase AUG codons located 24 base pairs apart at the 5' end of the second exon. Both of these polypeptides are phosphorylated and localized to the nucleus even when expressed separately. Interestingly, DNA rearrangements activating the c-myc gene are often found in the 1.7-kilobase-pair region between the two c-myc translational initiation sites and correlate with the loss of the longer c-myc polypeptide. Thus the close spacing of the two N-myc initiation codons could explain the relative resistance of the N-myc gene to similar modes of oncogenic activation.
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PMID:Two N-myc polypeptides with distinct amino termini encoded by the second and third exons of the gene. 265 99

The c-myc and N-myc nuclear oncoproteins are implicated in the genesis and maintenance of the transformed phenotype in several types of neoplastic disease, and the c-myc protein is involved in the progression of normal cells through the cell cycle. We have designed and developed sensitive and quantitative ELISAs for these proteins. Myc proteins are captured from cell lysates by an antibody directed against a peptide sequence substantially conserved in all known myc proteins; the captured proteins are recognised by a specific anti-c-myc or anti-N-myc monoclonal antibody conjugated to alkaline phosphatase; bound alkaline phosphatase is measured with an extremely sensitive cycling enzyme system that generates a coloured end-product. The c-myc assay is calibrated using bacterially expressed human c-myc protein. We have used this assay to estimate the number of c-myc molecules in a range of normal and transformed cells of human, murine, and feline origin; to monitor increases in c-myc expression when quiescent cells are stimulated with growth factors; and to follow the decrease in c-myc protein levels when HL60 promyelocytic leukaemia cells are induced to differentiate with dimethylsulphoxide or phorbol esters.
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PMID:A sensitive and quantitative enzyme-linked immunosorbence assay for the c-myc and N-myc oncoproteins. 333 75

Chondrocytes isolated from the proliferative and differentiating zones of 3-wk-old chick growth plates were cultured in the presence of 10% fetal bovine serum (FBS) and ascorbic acid for up to 21 d in a high cell density culture within Eppendorf tubes. The proliferative, differentiating, and calcification properties of the chondrocytes were examined by immunolocalization and by enzyme histochemical and biochemical methods. The cells maintained a chondrocyte phenotype throughout culture: they were round in shape and synthesized both collagen type II and proteoglycans. The expression of a hypertrophic phenotype was evident by Day 3 of culture and from this time onwards characteristics of terminal differentiation were observed. The cells were positive for both alkaline phosphatase (ALP) activity and c-myc protein and the surrounding matrix stained strongly for collagen type X. Small foci of mineralization associated with individual chondrocytes were first evident by Day 6 and more widespread areas of mineralization occupying large areas of matrix were present by Day 15. Mineralization occurred without the addition of exogenous phosphate to the medium. This culture system displays characteristics that are similar in both morphological and developmental terms to that of chick chondrocyte differentiation and calcification in vivo and therefore offers an excellent in vitro model for endochondral ossification.
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PMID:Differentiation and mineralization in chick chondrocytes maintained in a high cell density culture: a model for endochondral ossification. 754 Sep 18

Cholesteatoma epithelium is characterised by a keratinocyte dysregulation with a hyperproliferative growth and altered differentiation. In a variety of cells c-myc oncogene was found to be highly linked to the control of growth and differentiation. The expression of c-myc was studied in cholesteatoma epithelium using a monoclonal antibody directed against the 67 kD c-myc protein product and the alkaline phosphatase-anti-alkaline phosphatase-method. Furthermore for quantitative analysis we used a computer aided analysing system. In contrast to normal skin the keratinocytes of suprabasal layers showed a nuclear staining in cholesteatoma epithelium. The incidence of nuclear stainings of epithelial cells in cholesteatoma was significantly increased. Simultaneously, cytoplasmatic staining was observed in both skin and cholesteatoma with a stronger reactivity in the latter. Our findings suggest a participation of the c-myc oncogene in the reported dysregulation of cholesteatoma epithelium.
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PMID:[Immunohistochemical detection of c-myc proto-oncogene products in middle ear cholesteatoma]. 766 77

Cholesteatoma epithelium is characterized by a dysregulation with a hyperproliferative growth and altered differentiation. In a variety of cells c-myc oncogene was found to be highly linked to the control of growth and differentiation. Expression of c-myc was studied in cholesteatoma epithelium using a monoclonal antibody directed against the 67 kDa c-myc protein product and the alkaline phosphatase-antialkaline phosphatase method. For quantitative analysis a computer-linked analyzing system was used. In contrast to normal skin, keratinocytes of basal and suprabasal layers showed nuclear staining in cholesteatoma epithelium. The extent of nuclear staining of epithelial cells in the cholesteatomas studied was significantly increased. Concurrent cytoplasmic staining was observed in both skin and cholesteatoma, but with a stronger reactivity in the latter. These findings suggest participation of the c-myc oncogene in cholesteatoma epithelium.
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PMID:Immunohistochemical demonstration of c-myc oncogene product in middle ear cholesteatoma. 867 57

Fermentation of dietary fiber within the colonic lumen yields short chain fatty acids (SCFA) such as butyrate, which may modulate colonic mucosal biology and inhibit the development of a malignant phenotype. However, different fibers yield varying proportions of various SCFA. We studied the effects of the three most common SCFA, acetate, butyrate, and propionate, on the proliferation, adhesion, and motility of the human intestinal Caco-2 cell line, as well as the effects of these SCFA on alkaline phosphatase and dipeptidyl dipeptidase specific activity (common laboratory markers of differentiation). In addition, we examined the modulation of c-myc protein and the tyrosine phosphorylation of cellular proteins by these SCFA in order to determine whether the variations in the potency of these three SCFA for phenotypic change extended to variations in effects on intracellular signaling and protooncogene expression. All three SCFA tended to slow proliferation, promote brush border enzyme activity, and inhibit both adhesion to and motility across a type I collagen matrix substrate. However, we observed substantial differences in the potency of these three SCFA with regard to these effects. In particular, butyrate was uniformly more potent than an equimolar concentration of acetate whereas equimolar propionate achieved comparable effects with regard to proliferation and brush border enzyme activity but was intermediate between butyrate and acetate with regard to modulation of cell-matrix interactions. Similarly, the SCFA downregulated c-myc protein levels and modulated the phosphorylation of several intracellular tyrosine phosphoproteins, but the effects of the three SCFA varied substantially for these parameters. These results suggest that the common short chain fatty acids are not equipotent in their effects on human Caco-2 colon cancer cell biology. Such differences in potency could contribute to the observed differences in effects of different dietary fibers in vivo.
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PMID:Differential modulation of human (Caco-2) colon cancer cell line phenotype by short chain fatty acids. 952 Oct 97

Short chain fatty acids may protect colonic mucosa against neoplastic transformation by modulating colonocyte phenotype, DNA synthesis, and c-myc levels. To test this hypothesis, nonmalignant and malignant human colonocytes were isolated from surgical specimens and treated with 10 mM acetate, propionate, or butyrate. Markers of cellular phenotype, DNA synthesis, and c-myc protein levels were assayed by alkaline phosphatase and dipeptidyl dipeptidase IV activities, [3H]thymidine labeling, and western blotting, respectively. Butyrate, in particular, exerted discordant effects on alkaline phosphatase (P < 0.05), and c-myc levels (P < 0.05, N > or = 6) in nonmalignant and malignant human colonocytes. DPDD was unaffected by any of the short chain fatty acids tested. [3H]Thymidine labeling was differentially stimulated by short chain fatty acids in both cell types and greater DNA synthesis rates were observed in malignant colonocytes (P < 0.005, N = 16). These data suggest that in vitro, butyrate, in particular, may differentially modulate phenotype, DNA synthesis, and c-myc in nonmalignant and malignant human colonocytes.
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PMID:Short chain fatty acids differentially modulate cellular phenotype and c-myc protein levels in primary human nonmalignant and malignant colonocytes. 1127 Aug

Non-transformed, rat intestinal epithelial cells (IEC-6), and human intestinal colonic carcinoma cells (CACO-2) have both been used to study processes of epithelial cell differentiation. However, only CACO-2 cells have been described as spontaneously expressing phenotypic changes of differentiation in culture. We report here that when IEC-6 cells are grown in post-confluent culture, they develop structural changes similar to those seen in cells induced to differentiate by culture on Englebreth-Holm-Swarm (EHS) extracellular matrix proteins. Correlated with this morphological change is loss of nuclear localization of c-myc protein and development of cell surface alkaline phosphatase (ALP) enzymatic activity. Messenger RNAs for liver and intestinal isoforms of ALP were expressed in both pre- and post-confluent cells. Inhibition of ALP activity in post-confluent cells by levamisole indicated the expressed ALP activity to be of the liver isoform. We suggest the expression of ALP activity, which occurs concomitantly with morphological alterations in post-confluent IEC-6 cells, represents increased expression and localization to the cell surface of the liver isoform of ALP. Cultured IEC-6 cells may provide a non-transformed, in vitro alternative to CACO-2 cells for study of epithelial cell differentiation.
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PMID:Altered morphology in cultured rat intestinal epithelial IEC-6 cells is associated with alkaline phosphatase expression. 1258 29

DNA-dependent protein kinase (DNA-PK) has been intensively investigated for its roles in the nonhomologous end-joining (NHEJ) pathway of DNA double-strand break repair and maintenance of genomic stability. Its catalytic subunit, DNA-PKcs, a serine/threonine protein kinase, has recently been reported to be overexpressed in various human cancers, but its significance is unclear. In our study, we synthesized 3 small interfering RNA (siRNA) oligonucleotides, which separately target the translation initiation region, catalytic motif and a sequence between the scid-mutation region and the FATC motif of DNA-PKcs; 3 stable cell lines were generated from HeLa cells transfected with these siRNA constructs, respectively. All 3 siRNAs resulted in remarkable depression on DNA-PKcs expression in HeLa cells, and led to an increased sensitivity to 2 or 4 Gy of gamma-ray as well as 5 or 10 J/m(2) of ultraviolet (UV) irradiation. The siRNA targeting the catalytic motif of DNA-PKcs exhibited the greatest efficiency of radiosensitization. We demonstrated that c-myc protein level was suppressed more than 80% by siRNA-mediated silencing of DNA-PKcs. Using an E-box enhancer (c-myc binding element) driving a secreted alkaline phosphatase (SEAP) reporter strategy, we further found that the transcriptional activity of c-myc was extremely suppressed by silencing DNA-PKcs. The highest suppression effect on c-myc expression was observed in the cells transfected with the siRNA targeting the catalytic motif of DNA-PKcs. Moreover, a similar suppression on c-myc expression and activity was also detected in HeLa cells treated with wortmannin, a phosphatidylinositol (PI)-3 kinase inhibitor. However, silencing DNA-PKcs did not change the level of c-myc mRNA. We have further identified the interaction between DNA-PKcs and c-myc protein. Together, our results imply that DNA-PKcs activity is necessary or contributory to the expression of c-myc protein. Targeting DNA-PKcs is an attractive anticancer strategy, which can achieve through at least two mechanistic pathways: (i) sensitizing cancer cells to radiotherapy or chemotherapy of DNA-damaging agents and (ii) downregulation of c-myc protein.
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PMID:Downregulation of c-myc protein by siRNA-mediated silencing of DNA-PKcs in HeLa cells. 1592 10


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