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)

Recent studies have demonstrated the presence of estrogen receptor (ER) in both normal human osteoblast-like and osteoblast-like osteosarcoma cells. The number of ER in cultured osteoblastic cells is very low (200-500 sites/cell). This has complicated characterization of the biological role of estrogens in bone cells. To study the responsiveness of bone cells to estrogens, we established osteoblast-like cell lines expressing higher ER levels. ROS 17/2.8, an osteoblastic cell line, was stably transfected with the cDNA encoding for the mouse ER. After a selection period, positive clones were isolated and evaluated for the presence of ER by both Northern blot analysis and ligand binding assays. Using these techniques, we detected a significant increase in the level of both ER transcript and binding compared to that in wild-type cells. The levels of expressed ER protein were similar to those reported in normal human osteoblast-like cells in primary culture (approximately 2000 sites/cell). To test whether the exogenously inserted ER was responsive, both wild-type and ER stably transfected cells were transiently transfected with a reporter construct containing an estrogen-responsive element linked to a truncated thymidine kinase promoter and a chloramphenicol acetyltransferase (CAT) reporter gene. Exposure of the cells to increased concentrations of estradiol induced a slight increase in CAT activity in wild-type cells (approximately 1.5-fold) at maximal stimulation; however, it provoked a clear concentration-dependent increase in CAT activity in the ER stably transfected cells, with a maximal stimulation of approximately 10-fold. This event was receptor mediated, since ICI 164,384, an ER antagonist, blocked the enhancement of estradiol-induced CAT activity, and it was specific, since other steroid hormones did not stimulate CAT activity. Finally, we evaluated the ability of ER to modulate an endogenous estrogen-responsive gene by measuring the activity of the enzyme alkaline phosphatase. In addition, diethylstilbestrol, a synthetic estrogen agonist, increased the activity of both the CAT reporter gene and the endogenous alkaline phosphatase enzyme. In summary, we have established osteoblast-like cells expressing high levels of an exogenously inserted ER, which has characteristics similar to those of the endogenous ER in terms of its Kd. Finally, the exogenous ER regulates both exogenously inserted construct (VITERECAT) and endogenous properties of the cells (enzymatic activity and proliferation).
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PMID:Estrogens modulate the responsiveness of osteoblast-like cells (ROS 17/2.8) stably transfected with estrogen receptor. 157 85

The authors immunohistochemically assessed the presence of estrogen receptor (ER) in formalin-fixed, paraffin-embedded tissue sections of 68 breast carcinomas by an automated method using Pronase (CalBiochem, La Jolla, CA) predigestion and alkaline phosphatase detection (Method 1). These results were compared with those obtained by an automated peroxidase-antiperoxidase method with DNAse pretreatment of fixed embedded sections (Method 2), with ER immunostain on frozen sections (Method 3), and with biochemical results (dextran-coated charcoal cytosolic [DCC] assay). Compared with the DCC assay, Methods 1, 2, and 3 gave sensitivities of 54%, 25%, and 89%, respectively. The sensitivity for Method 1 was increased to 74% in those cases with DCC results showing greater than 50 fmol/mg protein. These findings indicate that ER immunohistochemical studies on formalin-fixed paraffin-embedded tissues (as assayed by Method 1) provide useful clinical information when the results are positive. A negative result, especially if surrounding normal elements are not positive, may indicate no receptors, receptor levels less than 50 fmol/mg protein, or improper tissue preservation. In the absence of fresh tissue for ER assay by DCC assay or of frozen sections for immunostaining, and with an understanding of its limitations, this method may be useful.
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PMID:Automated immunohistochemical estrogen receptor in fixed embedded breast carcinomas. 170 91

Monoclonal antibody to human estrogen receptor (ER) provides a useful immunohistochemical tool for the evaluation of ER content in breast carcinoma, but visual interpretation is subjective. Computer-assisted image analysis has proved effective in immunohistochemical quantitation of ER in fresh tumor imprints and cryostat sections. We examined the usefulness of this technique in 5-microns-thick formalin-fixed paraffin-embedded tissue sections of 66 cases of primary breast carcinoma previously assayed by dextran-coated charcoal (DCC) analysis. Immunohistochemistry was automated and performed on a Code-on slide stainer (Instrumentation Laboratories, Lexington, MA) using Pronase predigestion, a monoclonal antibody (ER-ICA; Abbott, Chicago, IL), and a biotin-labeled secondary antibody. Detection was achieved with an avidin-alkaline phosphatase conjugate and nitroblue tetrazolium (NBT) bromochloroindoyl phosphate (BCIP) substrate. The immunohistochemical ER staining was analyzed visually and with the CAS/200 image analyzer (Elmhurst, IL). The visual semiquantitative histologic scores (HSCORE), the automated quantitative assays including the percentage of positive nuclear areas (PNA), and the quantitative immunocytochemical scores (QIC SCORE = PNA x % of positive stain/10) were compared with the corresponding DCC results. Linear correlations were demonstrated between all immunohistochemical assays and the logarithm of DCC, the strongest correlation seen with PNA (r = 0.91). Threshold points for positive HSCORE, QIC SCORE, and PNA assays were extrapolated using DCC as the reference. ER immunodetection by PNA as compared with visual examination alone was enhanced by 18% (up to 88%) in sensitivity and 34% (up to 94%) in specificity, and the DCC concordance rate increased by 26% (up to 91%). A comparative chart extrapolating DCC from PNA was thus established.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Image analysis for quantitation of estrogen receptor in formalin-fixed paraffin-embedded sections of breast carcinoma. 170 2

Paraffin embedded sections of 64 breast carcinomas were stained immunohistochemically using a commercially available monoclonal antibody to estrogen receptor. To improve the sensitivity of the staining, the authors used a Pronase enzyme pretreatment, biotinylated antibody to rat IgG as secondary antibody, streptavidin-alkaline phosphatase as tertiary reagent and fast red as chromogen. When compared to the results of estrogen receptor enzyme immunoassay, this method yielded an 85.9% concordance rate, 86.2% specificity and 85.7% sensitivity. When compared to estrogen receptor immunocytochemistry(ER-ICA) in frozen section and considering the inherent advantages of immunohistochemical staining over biochemical assay, the major advantages of this method are good morphology, suitability for retrospective study and reduced cost of staining due to dilution of expensive primary antibody. Thus, this method offers an alternative to ER assay using fresh tissue and should provide additional valuable information about estrogen receptor.
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PMID:Demonstration of estrogen receptor by immunohistochemical staining in paraffin sections of breast carcinoma. 194 14

Recent studies suggest that, estriol, like estradiol, is biosynthetically esterified with fatty acids. We have synthesized the stearate estriol, at C-16 alpha, C-17 beta and the diester, C-16 alpha,17 beta and tested these D-ring esters for their estrogenic action both in vivo and in vitro, comparing them to estradiol, estriol and estradiol-17-stearate. None of the estriol esters bind to the estrogen receptor. They are only weakly estrogenic in a microtiter plate estrogen bioassay: stimulation of alkaline phosphatase in the Ishikawa endometrial cells. However, both estriol monoesters are extremely potent estrogens when injected subcutaneously (in aqueous alcohol) into ovariectomized mice. Compared to the free steroids, they produced a dramatically increased uterine weight with a greatly prolonged duration of stimulation. The 16 alpha,17 beta-diester also induced a protracted uterotrophic response, but the stimulation of uterine weight was comparatively low. Since the esters of estradiol and estriol do not bind to the estrogen receptor, their estrogenic signal must be generated through the action of esterase enzymes. These naturally occurring esters have the potential of being extremely useful pharmacological agents for long-lived estrogenic stimulation.
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PMID:Estrogenic action of estriol fatty acid esters. 203 55

Further therapeutical investigations are necessary to obtain a satisfactory survival rate of patients by improving the conventional methods of the treatment of osteosarcoma. Our working hypothesis is that an estrogenic hormonal influence is available for the effective treatment of osteosarcoma. An attempt was made to clarify this idea using experimentally induced hamster osteosarcoma. Male and female Syrian golden hamsters were used. Small amounts of minced tumor pieces were transplanted into hamsters subcutaneously. The levels of the circulating estradiol (E2) and alkaline phosphatase (ALPase) in blood were determined after the transplantation. An estrogen receptor (ER) on tumor cells was also demonstrated. Hamsters with an increased level of serum E2 were likely to have a smaller size of primary tumors and a smaller number of nodes in the lung metastasis. These tumor cells were successfully shown to be positive for ER stain. This suggests that E2 treatment may possibly control the osteosarcoma-condition.
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PMID:[Analyses regarding estrogen and estrogen receptors in an experimental osteosarcoma]. 205 36

A retrospective chart review was conducted of women with stage III breast cancer seen at the Princess Margaret Hospital between January 1977 and December 1980. Three hundred and sixty-nine patients were available for analysis. These cases were evaluated to determine the prognostic factors of patients presenting with this stage of the disease using a recursive partitioning technique, RECPAM, and a Cox regression model. A non-mathematical description of RECPAM is presented and the advantages of RECPAM over Cox analysis are discussed. The results identify primary tumour size, axillary node involvement, internal mammary node involvement, and estrogen receptor status as the most important prognostic variables. RECPAM identified 3 prognostic groups and simultaneously provided rules based on the prognostic variables to assign patients to poor, intermediate, or good prognosis categories. Patients with estrogen receptor negative tumours, or those with axillary node involvement, primary tumours greater than 5 cm, and serum alkaline phosphatase greater than 60 IU/L, or those with internal mammary node involvement, no skin changes, and serum alkaline phosphatase greater than 60 IU/L, define a group with a poor prognosis. Patients with estrogen receptor positive tumours, no axillary node involvements, and primary tumours greater than 5 cm, or estrogen receptor positive tumours, axillary node involvement, primary tumours greater than 5 cm, but serum alkaline phosphatase less than or equal to 60 U/L, have an intermediate prognosis. The good prognosis group consists of those patients with estrogen receptor positive tumours who have either skin changes or primary tumours less than or equal to 5 cm. The effect of loco-regional and systemic therapy was assessed and there was no association between treatment assignment and prognostic group. On the basis of this RECPAM analysis, we have defined important prognostic variables to be used in the design of clinical trials, and three major patient subgroups which can be used in routine oncologic practice as a guide to patient management.
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PMID:RECPAM analysis of prognostic factors in patients with stage III breast cancer. 208 74

A monoclonal antibody (anti-estrogen receptor [ER] from the ER-immunocytochemical assay [ICA] [Abbott Laboratories, Berkshire, England]), which binds to epitopes of the estrogen receptor in surgical biopsy specimens of breast carcinomas, was used in an improved alkaline phosphatase anti-alkaline phosphatase (APAAP) technique for in-situ receptor detection. This method was compared with a standard radioligand binding biochemical assay in assessing the ER status of 48 human breast carcinomas. The monoclonal antibody allowed detection of ER in the presence of estrogens or anti-estrogens and avoided problems of nonspecific intrinsic reactions, due to endogenous steroid hormones. The immunohistologic results (positive and negative) correlated well with the radioligand binding results (rs = 0.760; P less than 0.001) and allowed the additional assessment of cell morphology and tumor heterogeneity in the cryostat sections. The APAAP method provides diagnostic rapidity (approximately 4 hours) combined with sensitivity as compared with the radioligand assay (an approximately 2-day assay). The vivid red color associated with APAAP staining also was a distinct advantage compared with the brown reaction product of ER-ICA in interpreting stained sections. The results of this study indicate that the APAAP technique is an improved method for detection of ER and is applicable in routine hospital practice, thereby contributing to the immediate clinical management of patients with breast tumors.
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PMID:Detection of estrogen receptor proteins in breast tumors using an improved APAAP immunohistochemical technique. 246 Feb 11

We have studied the cytosolic estrogen receptor in uterus of rats after castration and diabetes induction with Streptozotocin, and the relationship of estradiol (E2) binding with phosphatase activities. Ovariectomy (OVX) and diabetes produced a significant reduction in Type I and II binding sites, but did not affect the equilibrium dissociation constants. The reduction of receptor levels was partially reversed by homogenization and incubation with 20 mM molybdate (MoO4) and also by chronic treatment with E2. Considering the possibility that the increase in E2 binding in the presence of MoO=4 was due to phosphatase inhibition, the activities of these enzymes hypothetically involved in receptor dephosphorylation and inactivation were determined in uterine homogenate and cytosol from intact, OVX, and diabetic rats with and without E2 treatment. Chronic OVX and diabetes induced stimulation of alkaline, acid and neutral phosphatase activities. On the contrary, the increment of estrogenic receptors due to E2 treatment was not correlated with changes in phosphatase activity. It is possible that this effect was due to the protection of the receptor or to the induction of receptor synthesis by E2. Molybdate inhibited acid and neutral phosphatases and increased alkaline phosphatase, which suggest that neutral and acid phosphatases are identical and that they were responsible for the receptor inactivation. However, it is unclear at present the relationship between the increment of alkaline phosphatase and the reduction of receptors.
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PMID:Estradiol binding and phosphatase activity in the uterus after castration and chronic diabetes: effect of molybdate and estrogen replacement. 300 2

An immunohistochemical technique using a commercially available monoclonal antibody to the estrogen receptor in formalin-fixed breast tumors is described. The author's technique is compared to three other recently published techniques in 27 case studies. The authors' method using pronase enzyme pretreatment and alkaline phosphatase as the third antibody yielded the best results. Comparison with the standard dextran-coated charcoal cytosolic assay results from the cases selected yielded a 100% sensitivity and 89% specificity for the authors' technique. The advantages of the immunohistochemical method over the biochemical assay are discussed and clinical implications are suggested. A step-by-step procedure for the authors' technique follows the text.
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PMID:Demonstration of estrogen receptors by monoclonal antibody in formalin-fixed breast tumors. 327 63


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