Immunological detection of the oestradiol receptor protein in cell lines derived from the lymphatic system and the haematopoietic system: variability of specific hormone binding in vitro

1992 ◽  
Vol 134 (3) ◽  
pp. 397-404 ◽  
Author(s):  
F. Jakob ◽  
H. P. Tony ◽  
D. Schneider ◽  
H. H. Thole

ABSTRACT Extracts of human MCF 7 mammary carcinoma cells, the human lymphoblastoid cell lines AEH 1 and IM 9, T-cell derived CCRF cells, HL 60 myeloic leukaemia cells and murine myeloma cells SP 0 and NS I were analysed for immunoreactivity with polyclonal goat antibodies raised against homogeneous preparations of C-terminal fragments (32 kDa) of porcine uterine oestradiol receptor (ER). Whole cells and low speed cytosols were analysed for specific oestradiol-binding activity. ERs were enriched from cell extracts by either fractionated ethanol precipitation (0–25% (v/v) ethanol) and/or microscale-immunoaffinity chromatography. Immunoreactive proteins of identical molecular weight (approximately 65 kDa) were detected in all cell lines examined. Whole cell binding assays showed specific oestradiol-binding activity in MCF 7, IM 9 and CCRF cells. Borderline binding was found in HL 60 myeloid cells. No specific binding could be detected in AEH 1, NS I and SP 0 cells. Identical results were obtained using agarelectrophoresis after dextran-coated charcoal treatment. Immunoaffinity purified ERs from MCF 7, AEH 1 and HL 60 cells were subjected to limited proteolysis, where identical tryptic fragments were generated. In conclusion, we have confirmed by immunological methods that ERs are expressed in a variety of cell lines derived from the immune system and the haematopoietic system. The lack of specific hormone binding or very low-affinity hormone binding in some of the cells examined may be due to post-translational events or point mutations. Journal of Endocrinology (1992) 134, 397–404

2019 ◽  
Vol 18 (9) ◽  
pp. 1295-1302 ◽  
Author(s):  
Sajjad Ahmadpour ◽  
Zohreh Noaparast ◽  
Seyed Mohammad Abedi ◽  
Seyed Jalal Hosseinimehr

Background: Breast cancer is a malignant disease with high mortality rate among women in the world. It is necessary to diagnose breast cancer at the early stage before it metastasizes in patients. Objective: The aim of this study is the evaluation of 99mTc-(tricine)-HYNIC-Lys-FROP for breast tumor imaging. Method: Lys-FROP peptide was labeled with 99mTc using HYNIC as chelator and tricine as co-ligand. Specific binding of this radiolabeled peptide on breast cancerous cell was assessed in different cell lines as well as in tumor-bearing mice. Results: HYNIC-Lys-FROP peptide was labeled with 99mTc at radiochemical purity more than 99%. It was observed high stability in normal saline and serum about 95%. The highest cellular uptake was observed in MCF-7 breast tumor cells treated with 99mTc-(tricine)-HYNIC-Lys-FROP as compared to other cell lines (lung, ovarian, T47D breast cancer cell lines). Biodistribution results in female MCF-7 tumor-bearing mice showed the relatively high tumor uptake and tumor-muscle ratio as 3.82 ± 0.66 after 15 min post-injection of 99mTc-(tricine)- HYNIC-Lys-FROP. Tumor uptake was reduced in mice that were co-injected with excess of unlabeled peptide to be 0.91 ± 0.08. Conclusion: Findings showed this radiolabeled peptide is a promising candidate for tumor targeting and molecular imaging of breast cancer.


1990 ◽  
Vol 1 (6) ◽  
pp. 487-498 ◽  
Author(s):  
D J Liska ◽  
J L Slack ◽  
P Bornstein

The first intron of the human alpha 1(I) collagen gene contains a positive, orientation-dependent cis-acting sequence located between bases +292 and +670. Transient transfection experiments indicate that this sequence is functional in both primary chicken tendon fibroblasts and in a human fibroblast-like cell line derived from SV40-transformed marrow stromal cells. DNase I footprint, methylation interference, and mobility shift analyses provide evidence for a sequence-specific binding activity and show that the region of binding corresponds to a 29-base-pair sequence that is also present in the rat alpha 1(I) collagen intron. This conserved sequence contains an AP1 consensus motif. Sequence-specific binding activity is present in nuclear extracts from HeLa and fibroblast cell lines but not in extracts from two lymphoid cell lines. Mutation of the AP1 consensus sequence indicates that this motif is required for function of the cis-acting element. These data indicate that transcriptional modulation of the alpha 1(I) collagen gene involves an interaction between an intronic AP1-containing sequence and its cognate transcription factors.


1989 ◽  
Vol 92 (2) ◽  
pp. 291-301
Author(s):  
A. M. PARISSENTI ◽  
M. B. COUKELL

Using ion-exchange chromatography, we have identified and isolated two forms of a cyclic GMP-specific binding activity in filter-broken cell extracts of Dictyostelium discoideum. Upon addition of excess cold ligand, one form (S-type) released bound 3H-labelled cyclic GMP very slowly (t½ ≈ 68 min), while the other form (F-type) released the cyclic GMP in <1 min. After photoaffinity labelling with 32P-labelled cyclic GMP, both forms revealed a major 160x103Mr band (and a few bands of lower molecular weight) on autoradiograms of sodium dodecyl sulphate-polyacrylamide gels. Addition of 500 mM-NaCl to S-type activity converted the activity to a fast-dissociating form indistinguishable from F-type, and this conversion was reversed by dialysis. Salt treatment or dialysis had no appreciable effect on the association/dissociation kinetics of F-type activity. When crude S-type activity was heated (to destroy cyclic GMP binding) and then added to F-type activity, the latter activity acquired slow-dissociating properties identical to S-type. This result suggested that the cells possess a ‘factor’ that can dramatically alter binding properties of this cyclic GMP-binding protein. Crude preparations of this factor were by boiling or proteases, but were sensitive to RNase A. Further studies revealed that acids (in particular, DNA) could effectively mimic the factor in its ability to modulate the binding kinetics of the cyclic GMP-binding activity.


1975 ◽  
Vol 149 (1) ◽  
pp. 123-132 ◽  
Author(s):  
K W Cheng

A simple method for preparing plasma membranes from bovine testes is described. Bovine testicular receptor has a high affinity and specificity for 125I-labelled human FSH (follicle-stimulating hormone). The specific binding of 125I-labelled human FSH to the plasma membranes is a saturable process with respect to the amounts of receptor protein and FSH added. The association and dissociation of 125I-labelled human FSH are time- and temperature-dependent, and the binding of labelled human FSH to bovine testicular receptor is strong and not readily reversible. Scatchard [Ann. N.Y. Acad. Sci. (1949) 51, 660-672] analysis indicates a dissociation constant, Kd, of 9.8 ×10-11M, and 5.9 × 10-14mol of binding sites/mg of membrane protein. The testicular membrane receptor is heat-labile. Preheating at 40°C for 15 min destroyed 30% of the binding activity. Specific binding is pH-dependent, with an optimum between pH 7.0 and 7.5. Brief exposure to extremes of pH caused irreversible damage to the receptors. The ionic strength of the incubation medium markedly affects the association of 125I-labelled human FSH with its testicular receptor. Various cations at concentrations of 0.1M inhibit almost completely the binding of 125I-labelled human FSH. Nuclectides and steroid hormones at concentrations of 1mM and 5 μg/ml respectively have no effect on the binding of FSH to its receptor. Incubation of membranes with and chymotrypsin resulted in an almost complete loss of binding activity, suggesting that protein moieties are essential for the binding of 125I-labelled human FSH. Binding of 125I-labelled human FSH to bovine testicular receptor does not result in destruction or degradation of the hormone.


1991 ◽  
Vol 66 (03) ◽  
pp. 310-314 ◽  
Author(s):  
David C Sane ◽  
Tammy L Moser ◽  
Charles S Greenberg

SummaryVitronectin (VN) stabilizes plasminogen activator inhibitor type 1 (PAI-1) activity and prevents the fibrin(ogen)-induced acceleration of plasminogen activation by t-PA. These antifibrinolytic activities as well as other functions are mediated by the glycosaminoglycan (GAG) binding domain of VN. Since the GAG binding region is rich in arginyl and lysyl residues, it is a potential target for enzymes such as plasmin. In this paper, the dose and time-dependent proteolysis of VN by plasmin is demonstrated. The addition of urokinase or streptokinase (200 units/ml) to plasma also produced proteolysis of VN. With minimal proteolysis, the 75 kDa band was degraded to a 62-65 kDa form of VN. This minimal proteolysis destroyed the binding of [3H]-heparin to VN and reversed the neutralization of heparin by VN.Thus, the plasmin-mediated proteolysis of the GAG binding activity of VN could destroy the antifibrinolytic activity of VN during physiologic conditions and during thrombolytic therapy. Furthermore, other functions of VN in complement and coagulation systems that are mediated by the GAG binding domain may be destroyed by plasmin proteolysis.


2018 ◽  
Vol 8 (3) ◽  
pp. 159 ◽  
Author(s):  
Meghan Fragis ◽  
Abdulmonem I. Murayyan ◽  
Suresh Neethirajan

Background: Breast cancer is the most commonly diagnosed cancer and the second leading cause of cancer deaths among Canadian women. Cancer management through changes in lifestyle, such as increased intake of foods rich in dietary flavonoids, have been shown to decrease the risk associated with breast, liver, colorectal, and upper-digestive cancers in epidemiologic studies. Onions are high in flavonoid content and one of the most common vegetables. Additionally, onions are used in most Canadian cuisines.Methods: We investigated the effect of five prominent Ontario grown onion (Stanley, Ruby Ring, LaSalle, Fortress, and Safrane) extracts on two subtypes of breast cancer cell lines: a triple negative breast cancer line MDA-MB-231 and an ER+ breast cancer line MCF-7.Results: These onion extracts elicited strong anti-proliferative, anti-migratory, and cytotoxic activities on both the cancer cell lines. Flavonoids present in these onion extracts induced apoptosis, cell cycle arrest in the G2/M phase, and a reduction in mitochondrial membrane potential at dose-dependent concentrations. Onion extracts were more effective against MDA-MB-231 compared to the MCF-7 cell line. Conclusion: In this study, we investigated the extracts synthesized from Ontario-grown onion varieties in inducing anti-migratory, cytostatic, and cytotoxic activities in two sub-types of human breast cancer cell lines. Anti-tumor activity of these extracts depends upon the varietal and can be formulated into nutraceuticals and functional foods for the wellbeing of cancer patients. Overall, the results suggest that onion extracts are a good source of flavonoids with anti-cancerous properties.Keywords: onion extracts; flavonoids; anti-proliferative; breast cancer; cytotoxic activity


2020 ◽  
Vol 17 ◽  
Author(s):  
Tarek Faris ◽  
Gamaleldin I. Harisa ◽  
Fars K. Alanazi ◽  
Mohamed M. Badran ◽  
Afraa Mohammad Alotaibi ◽  
...  

Aim: This study aimed to explore an affordable technique for the fabrication of Chitosan Nanoshuttles (CSNS) at the ultrafine nanoscale less than 100 nm with improved physicochemical properties, and cytotoxicity on the MCF-7 cell line. Background: Despite several studies reported that the antitumor effect of CS and CSNS could achieve intracellular compartment target ability, no enough available about this issue and further studies are required to address this assumption. Objectives: The objective of the current study was to investigate the potential processing variables for the production of ultrafine CSNS (> 100 nm) using Box-Benhken Design factorial design (BBD). This was achieved through a study of the effects of processing factors, such as CS concentration, CS/TPP ratio, and pH of the CS solution, on PS, PDI, and ZP. Moreover, the obtained CSNS was evaluated for physicochemical characteristics, morphology Also, hemocompatibility, and cytotoxicity using Red Blood Cells (RBCs) and MCF-7 cell lines were investigated. Methods: Box-Benhken Design factorial design (BBD) was used in the analysis of different selected variables. The effects of CS concentration, sodium tripolyphosphate (TPP) ratio, and pH on particle size, Polydispersity Index (PDI), and Zeta Potential (ZP) were measured. Subsequently, the prepared CS nanoshuttles were exposed to stability studies, physicochemical characterization, hemocompatibility, and cytotoxicity using red blood cells and MCF-7 cell lines as surrogate models for in vivo study. Result: The present results revealed that the optimized CSNS have ultrafine nanosize, (78.3±0.22 nm), homogenous with PDI (0.131±0.11), and ZP (31.9±0.25 mV). Moreover, CSNS have a spherical shape, amorphous in structure, and physically stable. Also, CSNS has biological safety as indicated by a gentle effect on red blood cell hemolysis, besides, the obtained nanoshuttles decrease MCF-7 viability. Conclusion: The present findings concluded that the developed ultrafine CSNS has unique properties with enhanced cytotoxicity. thus promising for use in intracellular organelles drug delivery.


2020 ◽  
Vol 17 (5) ◽  
pp. 563-573 ◽  
Author(s):  
Chandrakant Dhondiram Pawar ◽  
Dattatraya Navnath Pansare ◽  
Devanand Baburao Shinde

Background: Thiophene ring forms important building block in medicinal chemistry. Literature reveals that thiophene ring in combination with different groups shows different activity. By keeping these things in mind we have designed and synthesized a new series of amide and sulfonamide coupled thiophene. A series of novel substituted 3-sulfamoylbenzo[b]thiophene-4- carboxamide molecules containing sulfonamide and amide group were designed, synthesized and used for anti-proliferative activity study. Methods: The final compounds 16-36 were synthesized by using series of reactions comprising sulfonation, sulfonamide coupling, hydrolysis and peptide coupling. The yields of compounds 16- 36 are in the range of 90-98%. The structures of the synthesized compounds were elucidated and confirmed by 1H NMR, 13C NMR, LCMS and the purity was checked through HPLC analysis. The compounds were further tested for their in vitro anticancer activity against a series of cell lines A549, HeLa, MCF-7 and Du-145. Results: The intermediates 8-13, 15 and final compounds 16-36 were synthesized in good yields. The synthesized compounds were further tested for their anticancer activity and most of compounds showed moderate to good anticancer activity against all four cell lines. Conclusion: We have synthesized 21 compounds and were screened for anticancer activity against MCF-7, HeLa, A-549 and Du-145 cancer cell lines. Most of the compounds were active for tested cell lines with IC50 value in the range of 1.81 to 9.73 μM. The compounds 18, 19, 21, 25, 30, 31 and 33 are most active in cell line data with IC50 value in the range of 1.81 to 2.52 μM.


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