scholarly journals Unoccupied binding sites for oestrogen in nuclei of a breast tumour cell line (MCF-7)

1981 ◽  
Vol 200 (3) ◽  
pp. 515-520 ◽  
Author(s):  
A Geier ◽  
M Haimsohn ◽  
Z Malik ◽  
B Lunenfeld
Keyword(s):  
Nuclear Receptor ◽  
Fold Increase ◽  
Breast Tumour ◽  
Considerable Change ◽  
Tumour Cell Line ◽  
Nuclear Fraction ◽  
Oestrogen Receptors ◽  
Triton X ◽  
Mcf 7 ◽  
In Cells

1. A method to measure both occupied and unoccupied oestrogen receptors directly in the crude nuclear fraction of the MCF-7 cells was developed. The receptors had high affinity for oestradiol (Kd approx. 0.7 nM) and binding specificity characteristics of oestrogen receptors. 2. A substantial amount of the unoccupied receptors were found in the crude nuclear fraction. 3. Several experiments excluded the possibility that the unoccupied nuclear receptor might be a cytoplasmic contaminant. (a) Multiple extractions with Tris buffer released about 75% of the total receptor content, leaving the rest unextractable in the crude nuclear fraction. (b) Nuclei purified by centrifugation through 1.8M-sucrose and treatment with 0.7% Triton X-100, or by centrifugation through 50% glycerol with 0.1% Triton X-100 contained similar amounts of unoccupied receptors to that found in the crude nuclear fraction. (c) In cells cultured during 5 days after preconfluency a 3-fold increase in the amount of unoccupied cytoplasmic receptors occurred, whereas the amount of unoccupied nuclear receptors did not change significantly and conversely in cells exposed to increasing concentrations of oestradiol the unoccupied cytoplasmic receptor was continuously depleted but no considerable change in the unoccupied nuclear receptor was found.

scholarly journals Coregulatory protein–orphan nuclear receptor interactions in the human adrenal cortex

2005 ◽  
Vol 186 (1) ◽  
pp. 33-42 ◽  
Author(s):  
Sinead N Kelly ◽  
T Joseph McKenna ◽  
Leonie S Young
Keyword(s):  
Transcriptional Regulation ◽  
Adrenal Cortex ◽  
Nuclear Receptor ◽  
Tumour Cell Line ◽  
Zona Fasciculata ◽  
Orphan Nuclear Receptor ◽  
Response Element ◽  
Coregulatory Proteins ◽  
Coregulatory Protein ◽  
In Cells

The capacity of the adrenal to produce steroids is controlled in part through the transcriptional regulation of steroid enzymes. The orphan nuclear receptor steroidogenic factor 1 (SF-1) is central to the transcriptional regulation of all steroid hydroxylase enzymes, whereas nur77 can preferentially regulate steroid enzyme genes relevant to cortisol production. We hypothesised that, in the presence of secretagogues, SF-1 and nur77 may differentially interact with coregulatory proteins in the human adrenal cortex. Both coregulatory proteins, steroid receptor coactivator (SRC-1) and silencing mediator for retinoid and thyroid hormones (SMRT), were found to be expressed in the zona fasciculata and reticularis in the human adrenal cortex, but were largely absent from the zona glomerulosa. Both coregulatory proteins were colocalised with SF-1 and nur77. In the H295R adrenal tumour cell line, SF-1 and nur77 transcripts were increased in cells in the presence of forskolin, whereas nur77 mRNA was also induced with angiotensin II (AII). The coactivator SRC-1 mRNA was increased in the presence of both forskolin and AII. Forskolin induced recruitment of SRC-1 to the SF-1 response element and induced SRC-1–SF-1 interactions, whereas AII increased recruitment of SRC-1 to the nur77 response element and induced SRC-1–nur77 interactions. The corepressor SMRT interacted with SF-1 in the presence of AII and with nur77 in cells treated with forskolin. Orphan nuclear receptor–coregulatory protein interactions may have consequences for the regulation of key steroidogenic enzymes in the human adrenal cortex.

Tumour cell growth in culture: dependence on arginine

2004 ◽  
Vol 107 (4) ◽  
pp. 371-379 ◽  
Author(s):  
Giuseppe CASO ◽  
Margaret A. McNURLAN ◽  
Nelson D. McMILLAN ◽  
Oleg EREMIN ◽  
Peter J. GARLICK
Keyword(s):  
Protein Synthesis ◽  
Amino Acid ◽  
Tumour Cell ◽  
Urea Cycle ◽  
Breast Tumour ◽  
Cell Protein ◽  
Tumour Cell Line ◽  
Intracellular Enzyme ◽  
Polyamine Synthesis ◽  
Mcf 7

The amino acid arginine has been shown to affect the growth of several tumours, although the mechanisms of its action are not clear. In the present study, using a human breast tumour cell line (MCF-7), we investigated the arginine requirements of tumour cells for optimal protein synthesis and growth, and the metabolic pathway responsible for the arginine-dependent growth. The results showed that MCF-7 cells are highly dependent on arginine for growth and that the requirement for arginine is much higher than for an indispensable amino acid, leucine, indicating that arginine is needed for pathways other than protein synthesis. In arginine-free cultures, growth could be completely restored by the urea cycle intermediate citrulline. However, arginine could not be replaced by the urea cycle intermediate and the direct precursor for polyamine synthesis, ornithine, or by the polyamine putrescine, suggesting that the high dependence on arginine is not due to a requirement for polyamine synthesis. Moreover, inhibition of NOS [NO (nitric oxide) synthase] did not affect cell protein synthesis and growth, and the arginine analogue and substrate for NOS, homoarginine, could not replace arginine, implying that the conversion of arginine into NO is not involved in the growth-promoting effects of arginine. The major determinant for the high dependence of MCF-7 cells for arginine was found to be the irreversible conversion of this amino acid into ornithine by the intracellular enzyme arginase. The conversion into ornithine caused a progressive depletion of arginine from the culture medium, which ultimately inhibited cell protein synthesis and halted growth. Intracellular arginase activity may be the major factor determining the requirement for arginine of all cells in culture.

scholarly journals Developmental changes in the content of oestrogen receptors in the hypothalamus of the female rat

1979 ◽  
Vol 184 (2) ◽  
pp. 465-468 ◽  
Author(s):  
J O White ◽  
C Hall ◽  
L Lim
Keyword(s):  
Nuclear Receptors ◽  
Early Development ◽  
Nuclear Receptor ◽  
Sexual Differentiation ◽  
Fold Increase ◽  
Developmental Changes ◽  
Female Rat ◽  
Oestrogen Receptors

Hypothalamic cytosol and nuclear oestrogen receptors are present at birth. A 2-fold increase in cytoplasmic receptor content occurs by the second week, whereas the first significant and equivalent increase in nuclear receptor occurs in the fourth week. The latter reflects reported increases in oestradiol availability thought to lead to complete feminine sexual differentiation. The presence of nuclear receptors in the newborn suggests a requirement for oestrogenic stimulation in early development.

scholarly journals Nucleo-cytoplasmic relationships of oestrogen receptors in rat liver during the oestrous cycle and in response to administered natural and synthetic oestrogen

1980 ◽  
Vol 190 (1) ◽  
pp. 17-25 ◽  
Author(s):  
W Marr ◽  
J O White ◽  
M G Elder ◽  
L Lim
Keyword(s):  
Nuclear Receptors ◽  
Nuclear Receptor ◽  
Rat Liver ◽  
Fold Increase ◽  
Oestrous Cycle ◽  
Physiological Changes ◽  
Oestrogen Receptors ◽  
High Affinity ◽  
Cyclic Changes ◽  
Similar Affinity

Oestrogen receptors were measured in the cytosolic and purified nuclear fractions of rat liver. Both cytosolic and nuclear receptors bind oestrogen with high affinity (Kd = 1.47 and 2.28 nM respectively) and specificity similar to that of receptors in order oestrogen-target tissues such as the uterus. During the 4-day oestrous cycle the receptor content and distribution between cytosol and nucleus did not vary; in particular, the content of nuclear receptor did not appear to fluctuate in concert with known cyclic changes in the concentration of plasma oestrogen. Injection of 50 micrograms of oestradiol-17 beta or 10 micrograms of ethynyloestradiol resulted in a 4–6-fold increase in the nuclear receptor content, with a concomitant decrease in the unoccupied-receptor content of cytosol 1 h after injection. The nuclear receptors present after injection bind oestrogens with similar affinity (Kd = 2.78 nM) and specificity to receptors present in uninjected animals. The administration of lower doses of either oestrogen was less effective in producing increases in nuclear receptor content. Hence there is apparently substantial translocation of receptor to the nucleus in response to hyperphysiological doses of oestrogen, but not to the physiological changes in plasma oestrogen concentrations during the oestrous cycle. The response to exogenous oestrogens is discussed in relation to the clinical use of synthetic oestrogens and progestogens.

scholarly journals DBC1 (Deleted in Breast Cancer 1) modulates the stability and function of the nuclear receptor Rev-erbα

2013 ◽  
Vol 451 (3) ◽  
pp. 453-461 ◽  
Author(s):  
Claudia C. S. Chini ◽  
Carlos Escande ◽  
Veronica Nin ◽  
Eduardo N. Chini
Keyword(s):  
Breast Cancer ◽  
Nuclear Receptor ◽  
Clock Genes ◽  
Mrna Levels ◽  
Protein Levels ◽  
The Stability ◽  
And Function ◽  
Interfering Rna ◽  
In Cells

The nuclear receptor Rev-erbα has been implicated as a major regulator of the circadian clock and integrates circadian rhythm and metabolism. Rev-erbα controls circadian oscillations of several clock genes and Rev-erbα protein degradation is important for maintenance of the circadian oscillations and also for adipocyte differentiation. Elucidating the mechanisms that regulate Rev-erbα stability is essential for our understanding of these processes. In the present paper, we report that the protein DBC1 (Deleted in Breast Cancer 1) is a novel regulator of Rev-erbα. Rev-erbα and DBC1 interact in cells and in vivo, and DBC1 modulates the Rev-erbα repressor function. Depletion of DBC1 by siRNA (small interfering RNA) in cells or in DBC1-KO (knockout) mice produced a marked decrease in Rev-erbα protein levels, but not in mRNA levels. In contrast, DBC1 overexpression significantly enhanced Rev-erbα protein stability by preventing its ubiquitination and degradation. The regulation of Rev-erbα protein levels and function by DBC1 depends on both the N-terminal and C-terminal domains of DBC1. More importantly, in cells depleted of DBC1, there was a dramatic decrease in circadian oscillations of both Rev-erbα and BMAL1. In summary, our data identify DBC1 as an important regulator of the circadian receptor Rev-erbα and proposes that Rev-erbα could be involved in mediating some of the physiological effects of DBC1.

Cholinephosphotransferase activities in microsomes and neuronal nuclei isolated from immature rabbit cerebral cortex: the use of endogenously generated diacylglycerols as substrate

1982 ◽  
Vol 60 (7) ◽  
pp. 724-733 ◽  
Author(s):  
R. Roy Baker ◽  
Huu-Yi Chang
Keyword(s):  
Phospholipase C ◽  
Microsomal Fraction ◽  
Neuronal Development ◽  
Nuclear Fraction ◽  
Neuronal Nuclei ◽  
Nuclear Membranes ◽  
Choline Phosphotransferase ◽  
Rabbit Cerebral Cortex ◽  
Low Levels ◽  
Triton X

A neuronal nuclear fraction (N1) and a microsomal fraction (P3) were isolated from homogenates of cerebral cortices of 15-day-old rabbits. A nuclear envelope fraction (E) was prepared from N1. To assay cholinephosphotransferase, diacylglycerols were first generated in the membranes of these subfractions using a phospholipase C (Bacillus cereus) preincubation. With levels of endogenous diacylglycerols producing maximal specific cholinephosphotransferase activities, an activity ratio of 1:1:5 was found for N1, P3, and E, respectively. An independent neuronal nuclear cholinephosphotransferase, concentrated in nuclear membranes, is indicated. With regard to changes in pH and concentrations of MgCl2 and CDP-choline, N1 and P3 activities responded in a similar manner. However, in contrast to P3, N1 activities were much more profoundly inhibited at low levels of Triton X-100 (0.01–0.02 w/v%) and N1 showed quite significant levels of cholinephosphotransferase activity in the absence of a phospholipase C preincubation. Choline phosphotransferase in N1 and P3 showed Km values for CDP-choline (0.028 and 0.031 mM, respectively) which were much lower than corresponding literature values determined using exogenous diacylglycerols as substrates for this enzyme. The presence of cholinephosphotransferase in neuronal nuclear membranes reflects a rather exceptional nuclear autonomy. This may be related to a need to maintain nuclear phospholipid in the absence of a well-developed endoplasmic reticulum at early stages of neuronal development or to synthesize phospholipid in response to functions unique to the nucleus.

Development of orally bioavailable prodrugs of fulvestrant for the treatment of metastatic/advanced breast cancer.

2021 ◽  
Vol 39 (15_suppl) ◽  
pp. e13027-e13027
Author(s):  
Parva Purohit ◽  
Pathik Brahmkshatriya ◽  
Vishalgiri Goswami
Keyword(s):  
Breast Cancer ◽  
Oral Delivery ◽  
Tumor Volume ◽  
Estrogen Therapy ◽  
Xenograft Model ◽  
Fold Increase ◽  
Tumor Growth Inhibition ◽  
Pharmacokinetic Studies ◽  
Hormone Receptor Positive ◽  
Mcf 7

e13027 Background: Fulvestrant, a potent, selective estrogen receptor degrader, is a primary drug of choice for treating advanced metastatic hormone receptor-positive breast cancer in postmenopausal women following anti-estrogen therapy. However, the existing therapy limits to inconvenient intramuscular injections due to low solubility, weak permeation, high metabolism, and poor pharmacokinetics profile. Additionally, it takes 30 days to reach maximal steady-state plasma concentration, limiting clinical efficacy. To overcome these issues, we modulated physicochemical properties of fulvestrant, enabling its oral delivery to improve bioavailability. Methods: Structurally diverse pro-moieties were appended on fulvestrant to improve solubility and ADME profile. Thermodynamic solubility, plasma/liver microsomal stability, and Caco-2 permeability studies were performed to identify lead molecules. Pharmacokinetic studies were performed for selected molecules in mice. Antitumor activity of once-daily oral dose of three molecules was evaluated in female nude mice using the MCF-7 xenograft model. The efficacy of lead molecules was compared with subcutaneously administered faslodex in terms of percentage tumor growth inhibition. Results: Several prodrugs of fulvestrant were synthesized and evaluated for their intrinsic properties suitable for increasing bioavailability of fulvestrant. Remarkable improvements (̃500 to 2000-fold increase) were achieved in solubility and permeability. The PoC established an increase in systemic plasma exposure of fulvestrant upon oral administration of prodrugs in mice with enhanced bioavailability (1.5-8.7-fold) as compared to fulvestrant given subcutaneously (Table). Herewith, we report the identification of KSHN001022, KSHN001075, and KSHN001126, the prodrugs of fulvestrant, which showed enhanced efficacy with better tumor volume reduction (̃48-88% regression in tumor volume) as compared to that of fulvestrant (78%) in the estrogen-dependent MCF-7 xenograft model. Conclusions: KSHN001 lead candidates demonstrated significantly higher bioavailability, hence, provides a novel strategy to deliver fulvestrant orally to pursue the potential benefits in patients with advanced metastatic disease.[Table: see text]

beta-Carotene is Accumulated, Metabolized, and Possibly Converted to Retinol in Human Breast Carcinoma Cells (MCF-7)

2004 ◽  
Vol 74 (3) ◽  
pp. 171-177 ◽  
Author(s):  
Torres ◽  
Borojevic ◽  
Trugo
Keyword(s):  
Breast Carcinoma ◽  
Beta Carotene ◽  
Human Breast Carcinoma ◽  
Breast Carcinoma Cell Line ◽  
Human Breast ◽  
Cell Extracts ◽  
Human Breast Carcinoma Cells ◽  
Dose Dependent ◽  
Mcf 7 ◽  
In Cells

The aims of the present study were to investigate the uptake, accumulation, and metabolism of beta-carotene by the human breast carcinoma cell line MCF-7. Beta-carotene uptake was time- and dose-dependent, and independent of cell polarity. Beta-carotene accumulation in cells was linear as a function of its concentration in medium (1.3–4.1 mumol/L). It was accompanied by increasing amounts of retinol, which accumulated in cells following a sigmoid pattern, and by other four putative metabolites. Beta-apocarotenals, epoxides, endoperoxides, retinal, retinoic acid, and retinyl esters were not detected in cell extracts. Beta-carotene and its metabolites did not induce alterations in cell morphology or subcellular localization of epithelial mucins. Beta-carotene and retinol were released from cells that had previously accumulated beta-carotene, and were further incubated in beta-carotene- and retinol-free medium, but intracellular retinol content remained constant whereas b-carotene decreased. In conclusion, beta-carotene added to culture medium in physiological concentrations (1–6 mumol/L) is taken up and metabolized in MCF-7 cells, and is possibly converted to retinol.

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