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)

Immortalized rat proximal tubule cell (IRPTC) lines should be useful for investigation of proximal tubule (PT) regulation and function but previously have been unavailable. We now report the establishment and characterization of an immortalized transformed, temperature-sensitive IRPTC cell line containing renin-angiotensin system (RAS) components. Primary PT cells prepared from male Wistar rats (4-5 wk old) after collagenase digestion, sieving, and Percoll gradient were cultured on collagen-coated T-75 flasks in Dulbecco's modified Eagle's medium containing 5% fetal calf serum. Subconfluent PT cells were transfected with the temperature-sensitive SV40 mutant viruses (tsA SV40) by direct exposure. After 7-8 wk, several clones were obtained, from which one has been characterized and designated as line 3-2. This cell line appears stable up to 45 passages. Clonal cells transformed with this virus exhibit a transformed phenotype at a permissive temperature of 34 degrees C and grow in multiple layers. When the cells are subsequently placed at a nonpermissive temperature of 41 degrees C, they return to morphology similar to that of untransformed cells of the same lineage. At either 34 degrees C or 41 degrees C, this cell line expresses a variety of PT markers including alkaline phosphatase, cytokeratin, carbonic anhydrase, and glucose transporter isoform 2 (GLUT2), while not expressing factor VIII. Uniquely, these cells also appear to express PT proteins gp330 and CHIP28, markers which are usually lost in cultured cells. Furthermore, the cell line expresses protein and mRNA components of RAS, including angiotensinogen, angiotensin converting enzyme, and renin. The IRPTC cell line expresses few angiotensin II (ANG II) receptors at 34 degrees C, the permissive temperature. However, at the nonpermissive temperature, 41 degrees C, IRPTC expresses ANG II receptor (dissociation constant of 0.7 nM; maximum binding capacity of 265 fmol/mg protein). ANG II (10(-8) M) induced a transient rise in cytoplasmic Ca2+ concentration, which was nearly abolished with losartan but not PD-123319, suggesting this finding is AT1 receptor mediated. This cell line should provide an excellent model of PT and should make it possible to study the cell and molecular biology of the RAS, as well as other regulatory systems of the PT.
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PMID:Temperature-sensitive SV40 immortalized rat proximal tubule cell line has functional renin-angiotensin system. 790 Aug 43

Mouse blastocyst-derived embryonic stem (ES) cells are multipotent cells that can be used in vitro as models of differentiation and in vivo can contribute to all embryonic tissues including the germ line. The culture of ES cells requires a source of leukemia inhibitory factor (LIF), often provided by culture with a mouse fibroblast (STO) feeder layer, buffalo rat liver cell-conditioned media (BRL-CM), or the addition of recombinant LIF. To date, all of the ES cell culture systems use mammalian sources of LIF. We found that mouse ES cells can be maintained for over 10 passages in an undifferentiated state with media conditioned by a chicken liver cell line (LMH-CM) or on a feeder layer made with primary chicken embryonic fibroblasts (CEF). These ES cells can undergo both spontaneous and induced differentiation, which is associated with the disappearance or reduction of the expression of alkaline phosphatase and SSEA-1, similar to that observed for ES cells cultured with BRL-CM or STO feeder layers. The ES cells cultured in LMH-CM did not express cytokeratin Endo-A antigen recognized by TROMA-1, but their differentiated progeny did express this antigen. In contrast to LMH-CM, Endo-A was expressed in ES cells cultured on CEF feeder layers and in differentiated progeny. These results indicate that avian cells can produce a LIF-like cytokine that is active in inhibiting the differentiation of mouse ES cells. This could provide a biological end point for the isolation and characterization of avian LIF.
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PMID:Use of avian cytokines in mammalian embryonic stem cell culture. 793 84

We describe the development and application of a sensitive high-resolution fluorescence alkaline phosphatase (APase)-Fast Red immunocytochemical (ICC) staining method in combination with fluorescence in situ hybridization (ISH) and bromodeoxyuridine (BrdU) detection. The high fluorescence intensity, accurate localization, and advantageous slow-fading properties make the APase-Fast Red reaction a valuable tool for detection of antigens or specific DNA probes in biological cell preparations. Since the enzyme precipitate proved to be resistant to enzymatic pre-treatment steps and stable during the entire ISH procedure, APase-Fast Red immunostaining could be combined with subsequent visualization of DNA target sequences by fluorescence ISH. The lung cancer cell lines NCI-H82 and EPLC 65 were used as a model system for simultaneous detection of cell proteins, such as the neural cell adhesion molecule (N-CAM), cytokeratin filaments, lamin or the Ki67 antigen (Ki67-Ag), and centromere-specific DNA probes for human chromosomes 1, 7, or 17. In addition, the combined ICC/ISH procedure could be extended with the immunodetection of BrdU incorporated by tumor cells in S-phase. As a consequence, a combined ICC/ISH/BrdU detection procedure is now available that enables analysis of relatively complex tumor populations on the basis of different ICC and genetic markers as well as proliferative activity.
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PMID:Combined immunocytochemistry and fluorescence in situ hybridization for simultaneous tricolor detection of cell cycle, genomic, and phenotypic parameters of tumor cells. 801 80

Short-term cultures of purified murine trophoblast were used to investigate the potential trophic effects of a number of cytokines. Both granulocyte-macrophage colony stimulating factor (GM-CSF) and colony stimulating factor-1 (CSF-1) increased [3H]thymidine (TdR) uptake (3-8 times control values) by trophoblast harvested from placentae on day 12 or 14 of pregnancy. In contrast, interleukin-3 (IL-3) had only a mild stimulatory effect ([3H]TdR uptake 1.5 times control), and IL-2 did not alter the level of DNA synthesis in these cells. Immunocytochemical analysis confirmed that the cells engaged in DNA synthesis were cytokeratin-positive trophoblast cells and revealed that these cells predominantly bore markers (alkaline phosphatase, transferrin receptors) characteristic of trophoblast cells from the placental labyrinth. The increased DNA synthesis observed after exposure to GM-CSF or CSF-1 was not associated with a change in the proportion of nuclei involved in synthesis, nor did it result in significantly increased trophoblast cell numbers in the cultures. These findings suggest that DNA-synthesizing trophoblast cells were not proliferating, but were more likely engaged in endoreduplicative cycles leading to the formation of terminally differentiated trophoblast giant cells. These results caution against the presumption of proliferation when measuring [3H]thymidine incorporation by placental or trophoblast cells in standard in vitro cultures. In addition, taken together with the reports of high levels of CSF-1 in the pregnant uterus and the expression of the CSF-1 receptor on placental trophoblast cells, they suggest that the hemopoietic cytokines may play a role in the differentiation and/or function of trophoblast cells in the developing murine placenta.
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PMID:GM-CSF and CSF-1 stimulate DNA synthesis but not cell proliferation in short-term cultures of mid-gestation murine trophoblast. 804 Aug 36

Studies assessing mechanisms of proximal tubular cell (PTC) physiology and pathophysiology increasingly utilize cell culture systems to avoid the complexity of whole organ/whole animal experiments. However, no well-differentiated PTC line derived from adult human kidney currently exists. Therefore, the goal of this research was to establish such a line by transduction with human papilloma virus (HPV 16) E6/E7 genes. A primary PTC culture from normal adult human renal cortex was exposed to a recombinant retrovirus containing the HPV 16 E6/E7 genes, resulting in a cell line designated HK-2 (human kidney-2) which has grown continuously in serum free media for more than one year. HK-2 cell growth is epidermal growth factor dependent and the cells retain a phenotype indicative of well-differentiated PTCs (positive for alkaline phosphatase, gamma glutamyltranspeptidase, leucine aminopeptidase, acid phosphatase, cytokeratin, alpha 3 beta 1 integrin, fibronectin; negative for factor VIII-related antigen, 6.19 antigen and CALLA endopeptidase). Furthermore, HK-2 cells retain functional characteristics of proximal tubular epithelium (Na+ dependent/phlorizin sensitive sugar transport; adenylate cyclase responsiveness to parathyroid, but not to antidiuretic, hormone). The E6/E7 genes are present in the HK-2 genome, as determined by PCR. To assess its potential usefulness as a tool for studying injury and repair, HK-2 cells were exposed to a toxic concentration of H2O2 +/- iron chelation (deferoxamine) or hydroxyl radical scavenger (Na benzoate) therapy. Only the former blocked H2O2 cytotoxicity, reproducing results previously obtained with freshly isolated rat proximal tubular segments. In conclusion, an immortalized adult human PTC line has been established by transduction with HPV 16 E6/E7 genes. It appears to be well-differentiated on the basis of its histochemical, immune cytochemical, and functional characteristics, and it can reproduce experimental results obtained with freshly isolated PTCs. Thus, this new PTC line could have substantial research application.
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PMID:HK-2: an immortalized proximal tubule epithelial cell line from normal adult human kidney. 812 21

To determine whether the poorly differentiated AMOC-2 human ovarian adenocarcinoma cell line was capable of undergoing differentiation, AMOC-2 cells were exposed to 2 mM sodium butyrate, 2.5% dimethylsulfoxide, or 4 mM dibutyryl cyclic adenosine monophosphate (dibutyryl cAMP) for 6 days. These treatments resulted in growth inhibition, a reduction in clonogenicity and an increase in cellular glycogen content. Significant increases in heat stable alkaline phosphatase activity also occurred after exposure to sodium butyrate. In addition, a thorn-like microfilament structure observed in untreated cells was diminished concomitantly with morphological changes that included flattening, enlargement and extended cytoplasmic processes after exposure to sodium butyrate or dibutyryl cAMP. Furthermore, treatment with sodium butyrate increased the intracellular concentrations of beta-tubulin, vimentin, neurofilaments (M(r) 210,000) and cytokeratin (M(r) 56,000-58,000). These changes were completely reversed after removal of the inducing agent. The findings suggest that treatment of AMOC-2 cells with sodium butyrate induced a more differentiated phenotype, although terminal differentiation was not achieved.
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PMID:Effects of sodium butyrate, dimethylsulfoxide and dibutyryl cAMP on the poorly differentiated ovarian adenocarcinoma cell line AMOC-2. 830 43

A small subpopulation of pulmonary epithelial cells (PE) proliferates in low-density primary culture of alveolar type II cells and forms colonies of cells that could be passaged for several generations and that in some respects maintain a differentiated phenotype of the alveolar type II cells. At this time it is not known if these cells are some form of progenitor epithelial cells or type II cells that are not fully differentiated in vitro. The proliferation of the PE cells was dependent on serum, alveolar macrophage-conditioned medium, and insulin being included in the culture medium. Under these conditions, approximately 0.5-1.0% of the seeded cells that adhered to the culture dishes were capable of forming colonies. Efficiency of colony formation increased to 5-10% in subsequent passages. PE cells maintained a high level (> 40%) of saturated phosphatidylcholine (PC) as a percentage of total PC throughout the culture period (> 28 days). However, the saturated PC content was not constant throughout the long-term culture period and the subsequent passages (41.3% at 29 days and 37.3% in the 3rd passage). These cells also contained numerous lamellar bodies and were able to bind the Maclura pomifera lectin. PE cells also expressed cytokeratin No. 19, as well as alkaline phosphatase activity, both possible markers for differentiated type II cells. However, PE cell synthesized low levels of Pg (approximately 2%), were squamous, and tended to form multiple strata, unlike the cuboidal type II cells in vivo. The cells did not exhibit immunocytochemically demonstrable surfactant-associated protein A (SP-A). Additional factors and culture requirements may be necessary for complete maturation of cultured PE cells. This was demonstrated by culturing PE cells on EHS matrix. Aggregates of cells surrounding a central lumen were formed after a few hours in culture and were maintained for 20 days. The cells contained lamellar bodies and some intercellular junctions. PE cells can be regarded as a highly selected subpopulation of pulmonary epithelial cells that concomitantly maintain proliferation and aspects of differentiated alveolar type II cells in long-term culture.
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PMID:Pulmonary epithelial cell proliferation in primary culture of alveolar type II cells. 846 60

We developed methodology to isolate and culture rat alveolar Type II cells under conditions that preserved their proliferative capacity, and applied lipofection to introduce an immortalizing gene into the cells. Briefly, the alveolar Type II cells were isolated from male F344 rats using airway perfusion with a pronase solution followed by incubation for 30 min at 37 degrees C. Cells obtained by pronase digestion were predominantly epithelial in morphology and were positive for Papanicolaou and alkaline phosphatase staining. These cells could be maintained on an extracellular matrix of fibronectin and Type IV collagen in a low serum, insulin-supplemented Ham's F12 growth medium for four to five passages. Rat alveolar epithelial cells obtained by this method were transformed with the SV40-T antigen gene and two immortalized cell lines (RLE-6T and RLE-6TN) were obtained. The RLE-6T line exhibits positive nuclear immunostaining for the SV40-T antigen and the RLE-6TN line does not. PCR analysis of genomic DNA from the RLE-6T and RLE-6TN cells demonstrated the T-antigen gene was present only in the RLE-6T line indicating the RLE-6TN line is likely derived from a spontaneous transformant. After more than 50 population doublings, the RLE-6T cells stained positive for cytokeratin, possessed alkaline phosphatase activity, and contained lipid-containing inclusion bodies (phosphine 3R staining); all characteristics of alveolar Type II cells. The RLE-6TN cells exhibited similar characteristics except they did not express alkaline phosphatase activity. Early passage RLE-6T and 6TN cells showed a near diploid chromosome number.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Establishment of immortalized alveolar type II epithelial cell lines from adult rats. 852

During a systematic, immunocytochemical screening of 40 human cutaneous melanomas (30 primary and 10 metastatic) for immunophenotype (IP) heterogeneity, we employed a library of 20 well characterized, commercially available mono- and polyclonal antibodies. The use of the sensitive, indirect, four to six step immunoperoxidase or alkaline phosphatase conjugated streptavidin-biotin antigen detection technique provided excellent results. The immunocytochemically most characteristic IP for primary cutaneous melanoma, as detected by us was: HMB45+, S-100+, CEA+, vimentin+, cytokeratin 19+, p53+, Rbgene+, nm23+, HLA-DR+, HL.A-DP+, c-erbB3/HER-3+/-, cytokeratin 10/13+/-, HLA-DQ-, cytokeratin 5/8-, EMA-, c-myc-, and actin-. During melanoma progression, a tendency toward poor differentiation (dedifferentiation) and an increase in c-myc expression have both been observed, the latter downregulating HLA-A,B,C expression and consequently diminishing the possibility of melanoma cell Iysis by powerful CD8+, cytotoxic T lymphocytes (CTL) or other cytotoxic cells which requires HLA class I antigens. The development of the metastatic potential in melanomas caused an increase in CEA expression, eliminated the presence of nm23, and prompted the appearance of actin among the intermediate filaments, composing the cytoskeleton of these malignant tumor cells. The most characteristic IP for MMs, identified by this study was HMB45+, S-100+, CEA+, EMA+, vimentin+, HLA-DR+, HLA-DP+, cytokeratin 19+, actin-, c-erbB3/HER-3+, p53+, cytokeratin 10/13+/-, c-myc+/-, c-erbB2/HER-2+/-, HLA-DQ-, cytokeratin 5/8-, Rb gene-, nm23-. It has been observed that adhesion molecules and integrins play a significant role in the complex process of melanoma metastasis and thus we propose a blocking of these de novo expressed molecules with the appropriate antibodies as a form of immunotherapy of PMs and early stages of MMs.
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PMID:Immunophenotypically varied cell subpopulations in primary and metastatic human melanomas. Monoclonal antibodies for diagnosis, detection of neoplastic progression and receptor directed immunotherapy. 861 65

Minimal residual disease in patients with operable esophageal cancer is frequently missed by current noninvasive tumor staging. Here we applied an immunocytochemical cytokeratin assay that allows identification of individual esophageal carcinoma cells disseminated to bone marrow. Prior to therapy, bone marrow was aspirated from the upper iliac crest of 71 patients with esophageal cancer at various disease stages as well as an age-matched control group of 20 noncarcinoma patients. Tumor cells in cytologic bone marrow preparations were detected with monoclonal antibodies (mAbs) CK2, KL1, and A45-B/B3 to epithelial cytokeratins (CKs) using the alkaline phosphatase antialkaline phosphatase method. CK-positive cells were found in 14 (36.8%) of 38 cancer patients treated with curative intent and 16 (48.5%) of 33 patients with extended disease. The overall frequency of these cells was 1 per 4 x 10(5) to 82 per 4 x 10(5) mononuclear cells with no significant differences between patients at different tumor stages. After a short median follow-up of 9.5 months (3-24 months), 7 of 11 patients who underwent complete surgical resection but had tumor cells in bone marrow presented with tumor relapse compared to 2 of 19 corresponding patients without such cells (p < 0.01). It was concluded that although bone marrow is not a preferential site of overt metastasis of esophageal cancer, the frequent occurrence of isolated tumor cells at this distant site indicates that hematogenous dissemination of viable malignant cells occurs early in tumor progression.
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PMID:Disseminated epithelial tumor cells in bone marrow of patients with esophageal cancer: detection and prognostic significance. 866 32


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