Antiestrogens and selective estrogen receptor modulators as multifunctional medicines. 1. Receptor interactions.

The action of androgens is essential for the development of benign prostatic hyperplasia and carcinoma of the prostate. The androgen receptor is a ligand-dependent nuclear transcription factor. The transcriptional activation domain of the androgen receptor gene contains a polymorphic CAG repeat sequence. A shorter CAG repeat sequence within the normal range has been reported to be associated with increased risk of prostate cancer and symptomatic benign prostatic hyperplasia. Here, we examine the in vitro transcriptional activity of the androgen receptor (AR) with different numbers of CAG repeats within the normal range in a number of different cell lines of prostatic (LNCaP, PC3) and non-prostatic (COS-1, MCF7) origin. We utilize a luciferase reporter driven by the rat probasin promoter (-286/+28) containing two androgen receptor binding sites. Transcriptional activation of the androgen responsive reporter was observed to be greater with the AR containing 15 vs 31 CAG repeats in COS-1 cells (123.2+/-16.6 vs 78.2+/-10.9, P value 0.01) and the well differentiated prostate cancer cell line LNCaP (103.4+/-17.7 vs 81.4+/-7.7, P value 0.045). No difference was observed in the poorly differentiated prostate cancer cell line, PC3 (106.9+/-21.9 vs 109. 6+/-21.4, P>0.5) or the breast cancer cell line MCF7 (120.4+/-39.4 vs 103.1+/-23.1, P value >0.5). Dose-response experiments with varying quantities of ligand (0.01, 0.1, 1 and 10 nM dihydrotestosterone) or AR cDNA did not demonstrate significant differences in transactivation of the androgen responsive reporter in PC3 cells by the different AR constructs. This suggests that the lack of influence of CAG number in this prostatic cell line is not related to dose of ligand or quantity of androgen receptor. Western immunoblot analysis of androgen receptor protein in transiently transfected COS-1 cells did not demonstrate a difference in the expression of the androgen receptor protein with different numbers of CAG repeats following incubation in the presence or absence of androgen. Gel shift assay did not demonstrate increased DNA binding by androgen receptor with a shorter CAG repeat sequence. These experiments using a relatively androgen- and prostate-specific reporter provide evidence for an inverse relationship between androgen receptor transcriptional activity and the number of CAG repeats in the transcriptional activation domain. The effect of CAG repeat number was cell specific suggesting the involvement of accessory factors expressed differentially between different cell lines.

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Effect of the androgen receptor CAG repeat polymorphism on transcriptional activity: specificity in prostate and non-prostate cell lines.

Tricyclic antidepressants, such as amitriptyline (Elavil), and the nontricyclic agent, fluoxetine (Prozac), bind to growth-regulatory intracellular histamine receptors, associated with anti-estrogen binding sites in microsomes and nuclei. The prototype anti-estrogen binding site/intracellular histamine receptor ligand, N,N-diethyl-2-[4-(phenylmethyl)phenoxy]ethanamine HCl, inhibits normal cell proliferation in vitro but stimulates tumor growth in vivo. Because of their structural similarity to N,N-diethyl-2-[4-(phenylmethyl)phenoxy]ethanamine HCl, we carried out studies to determine whether amitriptyline and fluoxetine stimulate tumor growth and/or development in rodents at concentrations relevant to the treatment of human depression (equivalent human dose range, approximately 100-150 mg/day for amitriptyline and approximately 20-80 mg/day for fluoxetine). All experiments were performed blinded. In studies of growth stimulation of transplantable syngeneic tumors, groups of mice were inoculated s.c. with C-3 fibrosarcoma cells or given i.v. or s.c. injections of B16f10 melanoma cells, followed 24 h later by daily i.p. injections of saline, amitriptyline, or fluoxetine. Tumor latency (fibrosarcoma), aggregate tumor weight (s.c. injected melanoma), or time to death from pulmonary metastasis (i.v. injected melanoma) was determined; drug-induced stimulation of DNA synthesis in C-3 fibrosarcoma cells in vitro was correlated with tumor growth acceleration in vivo. In a mammary carcinogenesis model, the effects of chronic saline, amitriptyline, or fluoxetine administration on the rate and frequency of development of mammary tumors in rats fed dimethylbenzanthracene (DMBA) were compared. Eight of 20 amitriptyline- or fluoxetine-treated mice developed fibrosarcoma tumors by day 5, as compared to none of 20 saline controls (P less than 0.002). Similarly, 20 of 21 DMBA-treated rats receiving the antidepressant drugs developed 33 mammary tumors by week 15 as compared to 5 tumors in 4 of 7 DMBA-treated rats receiving saline (P less than 0.001). For both models, tumor latency decreased 30-40% and, in the DMBA model, tumor frequency increased greater than 2-fold in the antidepressant-treated rats as compared to controls. Stimulation of fibrosarcoma growth in vivo correlated with a corresponding bell-shaped drug-induced increase in DNA synthesis in vitro. While the median time to death from pulmonary metastases did not differ among groups given i.v. injections of melanoma cells, a significant (P less than 0.01) stimulation of growth of s.c. injected melanoma was observed in mice receiving the antidepressants.(ABSTRACT TRUNCATED AT 400 WORDS)

/pdf/stimulation-of-malignant-growth-in-rodents-by-antidepressant-ksed1wd6uz.pdf

Stimulation of malignant growth in rodents by antidepressant drugs at clinically relevant doses.

Do you know the sex of your cells? Not a question that is frequently heard around the lab bench, yet thanks to recent research is probably one that should be asked. It is self-evident that cervical epithelial cells would be derived from female tissue and prostate cells from a male subject (exemplified by HeLa and LnCaP, respectively), yet beyond these obvious examples, it would be true to say that the sex of cell lines derived from non-reproductive tissue, such as lung, intestine, kidney, for example, is given minimal if any thought. After all, what possible impact could the presence of a Y chromosome have on the biochemistry and cell biology of tissues such as the exocrine pancreatic acini? Intriguingly, recent evidence has suggested that far from being irrelevant, genes expressed on the sex chromosomes can have a marked impact on the biology of such diverse tissues as neurons and renal cells. It is also policy of AJP-Cell Physiology that the source of all cells utilized (species, sex, etc.) should be clearly indicated when submitting an article for publication, an instruction that is rarely followed (http://www.the-aps.org/mm/Publications/Info-For-Authors/Composition). In this review we discuss recent data arguing that the sex of cells being used in experiments can impact the cell's biology, and we provide a table outlining the sex of cell lines that have appeared in AJP-Cell Physiology over the past decade.

Do you know the sex of your cells

Reductive elimination of the 2′-hydroxyl group from ribonucleotides to yield 2′-deoxyribonucleotides, the monomeric precursors of DNA, requires an uncommon type of enzyme catalysis in which the transition metals, manganese, iron, or cobalt, and free radical intermediates cooperate In the group of deoxyadenosylcobalamin (coenzyme B 12)-dependent ribonucleotide reductases the coenzyme supplies a transient radical pair of deoxyadenosyl· and cob(II)alamin whereas in the nonheme-iron group of enzymes a protein subunit carries a stable tyrosyl radical coordinated to a binuclear iron(III) complex; the manganese-dependent enzymes are less precisely known The radicals are thought to function in hydrogen transfer from cysteine SH to the ribonucleotide substrate, and the metal complexes are apparently needed to generate and stabilize the radicals In aerobic organisms oxygen also plays a critical role in these processes and hence in DNA synthesis and cell proliferation In addition to the transition metals, Mg2+ or Ca2+ are required by several ribonucleotide reductases for structural integrity However the most potent inhibitors of deoxy-ribonucleotide biosynthesis (of potential interest in chemotherapy) are not metal chelators but radical scavengers Cell cycle arrest and cell death produced by simple chemicals like N-hydroxyurea and hydroxamates can be traced back to their reaction with ribonucleotide reductase Evidence is accumulating that independent enzymes of deoxyribonucleotide and DNA synthesis are functionally coupled in a novel type of supramolecular structure

The ribonucleotide reductases — A unique group of metalloenzymes essential for cell proliferation

A diphenylmethane derivative specific for the antiestrogen binding site found in rat liver microsomes.

The estrogen receptor (ER)-positive human breast cancer cell line MCF-7 was incubated continuously in the presence of pharmacological concentrations of diethylstilbestrol (DES) in an attempt to correlate receptor status with DES sensitivity. It was consistently observed that cytotoxicity occurred at DES concentrations greater than 5 X 10(-6)M; however, a small percentage of cells, both from the wild-type MCF-7 line and from subclones derived in soft agar from single MCF-7 cells, survived, with altered morphology, up to 4 months of continuous exposure to DES concentrations ranging from 5 X 10(-6) to 1 X 10(-4)M. Characterization of seven regenerated surviving cell populations suggested that they remained ER positive; no evidence could be found for a block in the pathway of hormonal activation, as determined by progesterone receptor induction, to explain the ability of these cells to survive DES. Three regenerated cell populations were reexposed to DES. Two remained as sensitive to growth inhibition as untreated parent cells from which they were derived; however, one of these, designated MCF-7(35-1), was found to have autonomously high progesterone receptor (463 +/- 94 fmol/mg of cytosol; Kd = 1.8 +/- 0.2 X 10(-9)M) which was not significantly stimulated by the addition of 1 X 10(-8)M 17beta-estradiol for 72 hr. The third population, designated MCF-7(35-3), which survived initial exposure to 5 X 10(-5)M DES for 109 days and which remained ER positive (27 +/- 3 fmol/mg of cytosol; Kd = 0.8 +/- 0.2 X 10(-10)M) and progesterone receptor inducible, demonstrated significantly decreased sensitivity (p = 0.025) on reexposure to DES; conversely, significantly increased sensitivity (p less than 0.03) to the antiestrogen tamoxifen was observed. The mechanisms by which some MCF-7 cells survive prolonged exposure to DES are not certain; the data suggest that there is no clear relationship between ER status and sensitivity to DES and that there is no way of predicting the ultimate status of cells surviving DES treatment.

https://cancerres.aacrjournals.org/content/canres/43/6/2831.full.pdf

Receptor status and subsequent sensitivity of subclones of MCF-7 human breast cancer cells surviving exposure to diethylstilbestrol.

The use of (a) N,N-dialkyl-2-[(4-phenylmethyl)phenoxy]ethanamine and N-morpholino-2-[(4-phenylmethyl)phenoxy]ethanamine compounds and their salts, as anticancer agents, is described. These compounds bind significantly to the anti-estrogen binding sites but only poorly to the estrogen receptor sites, and are cytotoxic to cancer cells. Compositions including mixtures of these ethanamine compounds (a) with (b) therapeutically active anticancer compounds such as tamoxifen, have been found particularly beneficial.

Aminoalkyl ethers of phenols as anticancer agents for the breast and colon

A degradation product of fenitrothion, 3-methyl-4-nitrophenol, is an inhibitor of mammalian ribonucleotide reductase.

This invention is concerned with N,N-dialkyl-2-[(4-phenylmethyl)phenoxy]ethanamine and N-morpholino-2-[(4-phenylmethyl)phenoxylethanamine and their salts, and the production of these compounds. These compounds bind singificantly to the anti-estrogen binding sites but only poorly to the estrogen receptor sites, and are cytotoxic to breast cancer cells in vitro.

Mark W. Hermonat

Papers

A diphenylmethane derivative specific for the antiestrogen binding site found in rat liver microsomes.

Receptor status and subsequent sensitivity of subclones of MCF-7 human breast cancer cells surviving exposure to diethylstilbestrol.

Aminoalkyl ethers of phenols as anticancer agents for the breast and colon

A degradation product of fenitrothion, 3-methyl-4-nitrophenol, is an inhibitor of mammalian ribonucleotide reductase.

Novel cytotoxic aminoalkyl phenol ethers