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Biomedical subjects

R L Sutherland

Publications and source records attributed to R L Sutherland.

At least 145 records · Page 8Linked to original sources
PubMed1986

Definition of two distinct mechanisms of action of antiestrogens on human breast cancer cell proliferation using hydroxytriphenylethylenes with high affinity for the estrogen receptor.

Treatment of MCF 7 human breast cancer cells with three high affinity hydroxylated antiestrogens (Kd for the estrogen receptor = 0.11-0.45 nM) resulted in biphasic inhibition of cell growth. Administration of 0.1-1.0 nM of each drug caused a concentration-dependent decrease in cell number to a maximum of 30-40% of control but no further change was observed as the drug concentration was increased to 1 microM. Between 1.0 and 10 microM, however, a further concentration-dependent decrease in cell proliferation was observed. Among these compounds relative potencies paralleled their affinities for estrogen receptor in the 0.1-10 nM range but at micromolar concentrations this relationship did not hold. It is concluded that antiestrogens inhibit cell proliferation by two distinct mechanisms one of which involves the estrogen receptor and the other a mechanism yet to be defined. The parallel changes in cell cycle kinetic parameters accompanying growth inhibition in both concentration ranges i.e. accumulation of cells in the G1 phase at the expense of S phase cells, suggests that both mechanisms may converge on common pathways critical to cell cycle progression.

Binding, Competitive↗
PubMed1986

Microsomal binding sites for antioestrogens in rat liver. Properties and detergent solubilization.

The properties of an antioestrogen binding site (AEBS), which has high affinity and specificity for nonsteroidal antioestrogens and structurally related compounds, have been studied in rat liver microsomes. When subcellular organelles were separated on Percoll density gradients the distribution of the AEBS paralleled that of NADPH-cytochrome c reductase, indicating that the AEBS is associated with the endoplasmic reticulum. Saturation analysis showed that [3H]tamoxifen was bound to a single class of saturable binding sites in liver microsomes with a KD of 0.9 +/- 0.1 nM at 0 degrees C. The equilibrium KD was not significantly different at 22 degrees C. The KD calculated from the association and dissociation rate constants for [3H]tamoxifen binding at 0 degrees C and 22 degrees C was compatible with the KD measured at equilibrium. Ligand specificity studies using tamoxifen analogues showed qualitatively similar structure-affinity relationships for the AEBS from both rat liver and the MCF 7 breast cancer cell line. In general structural modifications caused correspondingly greater changes in affinity for rat liver AEBS than for MCF 7 AEBS. The AEBS was solubilized from microsomal membranes with sodium cholate. This was the only detergent of nine tested that solubilized the site in high yield without loss of activity. Solubilization using cholate was more effective in the presence of 1 M-NaCl. In the solubilized state there was an apparent loss of [3H]tamoxifen binding activity which could be restored by dilution of the detergent. Gel filtration indicated an Mr of 440,000-490,000 for the AEBS-cholate complex. These studies demonstrate that rat liver contains high concentrations of a microsomal AEBS which has similar properties and specificity to the AEBS previously described in human breast cancer cells. This site can be solubilized by sodium cholate to supply material suitable for further purification.

Animals↗
PubMed1986

Substituted-vinyl hydroxytriarylethylenes, 1-[4-[2-(diethylamino) ethoxy]phenyl]-1-(4-hydroxyphenyl)-2-phenylethylenes: synthesis and effects on MCF 7 breast cancer cell proliferation.

A series of triarylethylene compounds related to 4-hydroxyclomiphene (2) in which the vinyl Cl substituent was replaced by ethyl (5), Br (6), H (7), CN (8), or NO2 (9) substituents were synthesized to facilitate studies of the molecular actions of synthetic nonsteroidal antiestrogens. The relative binding affinities of these compounds for the estrogen receptor (ER) and the antiestrogen binding site (AEBS) in MCF 7 human mammary carcinoma cells were measured and correlated with the effects of these drugs on cell proliferation kinetics. Affinities for ER and AEBS were highly correlated, illustrating that vinyl substituents influence binding to ER and AEBS in a parallel manner. All compounds except 7 had biphasic effects on cell proliferation kinetics, indicating the presence of at least two distinct mechanisms by which hydroxytriarylethylenes inhibit breast cancer cell proliferation. In the concentration range 10(-10)-10(-8) M, cell proliferation was inhibited by 60-70%, these effects were estrogen-reversible, and the degree of growth inhibition was in the order Cl greater than Et greater than Br greater than NO2 greater than CN greater than H, which paralleled the order of affinities for ER. There was no further inhibition of cell growth between 10(-8) and 10(-6) M, but at concentrations greater than 10(-6) M there was a further dose-dependent decrease in cell growth mediated by mechanisms yet to be defined but apparently distinct from ER-mediated events. In both concentration ranges, growth inhibition was accompanied by accumulation of cells in the G1 phase of the cell cycle. These data, obtained with a novel series of hydroxytriarylethylenes, have enabled clear definition of two distinct mechanisms of growth inhibition by triarylethylene antiestrogens. They also indicate that among the vinyl substitutions examined to date the Cl substituent yields the most active molecule both in terms of affinity for ER and AEBS and potency as a growth inhibitory agent.

Antineoplastic Agents↗
PubMed1986

Modulation of lactogenic receptors by progestins in cultured human breast cancer cells.

Progesterone receptors (PgR) are present in many breast cancers, but few specific actions of progestins in breast cancer cells have been reported. We now report that progestins specifically modulate lactogenic receptor expression in cultured T-47D and MCF-7 human mammary carcinoma cells. When T-47D cells were preincubated for 24 h with 1 nM medroxyprogesterone acetate (17 alpha-acetoxy-6 alpha-methyl-4-pregnene-3,20-dione), specific binding of [125I]human GH ([125I]hGH) and [125I]human PRL was increased to 205 +/- 22% (+/- SE) (P less than 0.01) and 175 +/- 32% (P less than 0.05), respectively, of that in control cultures. There was no significant effect on cell number and no significant enhancement of specific binding of [125I]porcine insulin, [125I]salmon calcitonin, [125I]human transferrin, or [3H] Concanavalin A. Lactogenic receptor number was increased from 6,490 +/- 500 (n = 12) to 13,180 +/- 3,270 (n = 7; P less than 0.01) sites/cell, with no significant change in affinity for hGH. Progesterone, which is readily metabolized by these cells, was less potent than the synthetic progestins (medroxyprogesterone acetate, R 5020 (17 alpha, 21-dimethyl-19-norpregn-4,9-diene-3,20-dione), and ORG 2058 (16 alpha-ethyl-21-hydroxy-19-norpregn-4-en-3,20-dione), but physiological concentrations of progesterone (1 nM) significantly enhanced specific binding of [125I]hGH to 153 +/- 23% of the control value (n = 6; P less than 0.05). Physiological concentrations of androgens, estrogens, and glucocorticoids had no significant effect. MCF-7 cells were considerably less sensitive to these effects of progestins than T-47D cells, probably due to the lower PgR concentration in MCF-7 cells. These observations, which indicate that lactogenic receptor expression is controlled, at least in part, by progestins in these mammary carcinoma cell lines, may have important implications in the management of human breast cancer, where high levels of this receptor may reflect a functional PgR and a highly hormone-dependent phenotype.

Breast Neoplasms↗
PubMed1986

Progestin regulation of epidermal growth factor receptor in human mammary carcinoma cells.

Epidermal growth factor (EGF) receptors are present in human breast cancer and probably mediate the effects of EGF and the autocrine effects of alpha-transforming growth factors, produced by breast cancer cells. Steroid hormones influence the growth of some human cancers, and both direct and indirect effects on cell proliferation have been proposed. One potential indirect effect of steroids would be to augment sensitivity to other endocrine and autocrine factors by up-regulation of their receptors. We therefore investigated the effects of various steroids on EGF receptor expression in T-47D, MCF-7, and BT 20 human mammary carcinoma cells in culture. Preincubation of T-47D cells for 24 h with a series of androgens, estrogens, glucocorticoids, and progestins resulted in a significant enhancement of specific 125I-EGF binding in the presence of progestins only. Increased binding of EGF was associated with neither a change in cell number nor changes in the specific binding of concanavalin A, insulin, or calcitonin but was accompanied by an increase in lactogenic receptor expression. When assayed at 20 degrees C, increased EGF binding was due to an increase in receptor number (33,380 +/- 7,410 sites/cell in control cultures; 67,460 +/- 20,330 sites/cell in cultures treated with 1 nM medroxyprogesterone acetate for 24 h; P less than 0.05) without a change in receptor affinity. Two- to 3-fold increases in receptor number were also apparent when binding was measured at 4 degrees C, indicating that the effect was due to an increase in expression of receptor at the cell surface rather than progestin effects on internalization and degradation. These data illustrate that the expression of EGF receptor in some breast cancer cells is regulated in part by mechanisms mediated via the progesterone receptor, since the effect was confined to progestins, potency among a series of progestins was correlated with their affinities for progesterone receptor, and sensitivity among the three cell lines studied was related to the presence and concentration of cellular progesterone receptor.

Androgens↗
PubMed1985

Regulation of growth hormone and epidermal growth factor receptors by progestins in breast cancer cells.

A 24 hr incubation of T-47D human breast cancer cells with R5020, a synthetic progestin, resulted in a 200-250% increase in the specific binding of human growth hormone (hGH) and epidermal growth factor (EGF) by these cells. This effect was specific for progestins in that similar responses were observed with progesterone, medroxyprogesterone acetate and ORG 2058 but no significant increases in hGH or EGF binding were observed in cells incubated with testosterone, estradiol or hydrocortisone. Increased binding was due to an increase in the concentration of receptors (hGH, control = 6,490 +/- 500, progestin treated = 13,180 +/- 3,270 sites/cell; EGF, control = 33,380 +/- 7,410, progestin treated = 67,460 +/- 20,330 sites/cell) while the affinity constants for the hormone-receptor interactions were unchanged by progestin treatment. The specific binding of insulin, calcitonin, transferrin and concanavalin A was unaffected by these treatments. It is concluded that expression of hGH and EGF receptors in this breast cancer cell line is regulated by progestins.

Breast Neoplasms↗
PubMed1985

Cell cycle effects of iron depletion on T-47D human breast cancer cells.

T-47D human breast cancer cells grown in culture medium containing low concentrations of fetal calf serum (FCS) proliferated very slowly, with an accumulation of cells in the G2 phase of the cell cycle, increased polyploid cells, and increased expression of transferrin receptors. Cell proliferation was stimulated by the addition of human transferrin or ammonium ferric citrate to the medium. Growth inhibition and accumulation of G2-phase cells could also be produced in T-47D cells grown in medium containing 10% FCS by the addition of the iron chelator, desferrioxamine. It is concluded that cellular deprivation of iron and/or transferrin is the major cause of reduced proliferation rates and G2-phase arrest which accompany the culture of these cells in medium supplemented with low concentrations of FCS.

Animals↗
PubMed1985

Differential effects of tamoxifen and analogs with nonbasic side chains on cell proliferation in vitro.

Structural analogs of the nonsteroidal antiestrogen tamoxifen, in which the basic dimethylaminoethoxy side chain was either absent or replaced with a variety of nonbasic side chains, were examined for their ability to inhibit the proliferation of a hormonally responsive cell line, MCF 7 human breast cancer. The degree of inhibition was compared with relative binding affinities for the estrogen receptor (RE) and a microsomal antiestrogen binding site (AEBS). All modifications resulted in loss of detectable affinity for AEBS. Replacement of the basic side chain of tamoxifen with a series of nonbasic side chains reduced affinity for RE by 78-93% except in the case of 1-(4-(1,2-diphenylbut-1-enyl)phenyl)-2,3-butanediol (ICI 145-680) where affinity was unchanged. When the basic side chain of tamoxifen was replaced by a hydroxyl group, to form the estrogenic analog ICI 141389 (Metabolite E), affinity for RE was reduced by 39%. ICI 141389 was a very weak inhibitor of MCF 7 cell growth, showing no significant growth inhibition at concentrations less than 10 microM. Despite the fact that ICI 145680 and tamoxifen had identical affinities for RE, ICI 145680 was a significantly weaker growth inhibitor than tamoxifen over the concentration range studied, i.e. 0.1-20 microM. Differences in potency were greatest at concentrations greater than 7.5 microM where the effects were not reversed by estradiol and where cytotoxicity played a major role in the decrease in cell numbers induced by tamoxifen. Like tamoxifen, ICI 145680 demonstrated both estrogen-reversible (at concentrations between 0.5-7.5 microM) and estrogen-irreversible (10-20 microM) inhibition of MCF 7 cell proliferation which was associated with a concentration-dependent accumulation of cells in the G0/G1 phase of the cell cycle. In contrast to tamoxifen, however, ICI 145680 appeared not to possess cytotoxic activity. Whereas ICI 145680 was without effect on proliferation of the RE negative human breast cancer cell line, MDA-MB-330, at doses less than 20 microM, tamoxifen inhibited growth at concentrations greater than 5 microM, but with changes in cell cycle kinetic parameters that were markedly different from those seen in RE positive cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Breast Neoplasms↗
PubMed1985

Differential sensitivity of human breast cancer cell lines to the growth-inhibitory effects of tamoxifen.

In eight estrogen receptor (ER)-positive breast cancer cell lines (including three sublines of MCF-7) and five ER-negative breast lines, the action of the nonsteroidal antiestrogen, tamoxifen, was studied, and the concentrations of ER and antiestrogen binding site were measured. The concentration of antiestrogen binding site was significantly [P less than 0.005] greater in ER-positive cells [236,600 +/- 29,900 (SE) sites/cell] than in ER-negative cell lines [66,600 +/- 16,800 sites/cell]. In ER-positive cell lines, a cell cycle phase-specific growth-inhibitory effect, 20% inhibitory dose less than 0.1 to 1.0 microM, was seen which was shown for some representative cell lines to be estrogen reversible. Within this group of cell lines, the degree of tamoxifen-induced inhibition of growth correlated with control population doubling time, but not ER or antiestrogen binding site concentration. The changes in cell cycle kinetic parameters characteristic of all ER-positive lines were a decrease in percentage of S-phase cells and a corresponding increase in percentage of G0-G1 cells. In all cell lines, 5 to 12.5 microM tamoxifen caused cytotoxicity, and this was shown to be estrogen-irreversible in 3 representative cell lines; moreover, estradiol synergistically enhanced the cytotoxic effects of tamoxifen under some experimental conditions. The cell cycle effects of tamoxifen in three ER-negative cell lines (Hs0578T, MDA-MB-231, MDA-MB-330) were decreased proportions of G0-G1 cells with an increase in percentages of S and G2+M cells. These results implied that the mechanism of tamoxifen cytotoxicity may differ in ER-positive and ER-negative breast cancer cells. However, although the ER-negative BT-20 line was much less sensitive to tamoxifen than were the ER-positive cells, growth inhibition and cytotoxicity in this line were associated with a slight decrease in percentage of S-phase cells. These results confirm that ER-positive breast cancer cells are more sensitive (4- to greater than 75-fold) than ER-negative breast cells to the growth-inhibitory effects of tamoxifen and demonstrate that, in all ER-positive cells, growth inhibition and cytotoxicity are accompanied by characteristic changes in cell cycle kinetic parameters. In contrast, different mechanisms may be involved in the effects of tamoxifen on different ER-negative cell lines.

Breast Neoplasms↗
PubMed1984

High affinity specific antiestrogen binding sites are concentrated in rough microsomal membranes of rat liver.

Saturation and competitive binding analyses demonstrated the presence of a high affinity (KD = 0.92 nM), specific antiestrogen binding site (AEBS) in rat liver microsomes and at least 75% of total liver AEBS was recovered in this fraction. When microsomes were further separated into smooth and rough fractions, AEBS was concentrated in the latter. Subsequent dissociation of ribosomes from the rough membranes revealed that AEBS was associated with the membrane and not the ribosomal fraction. Antiestrogen binding activity could not be extracted from membranes with 1 M KCl or 0.5 M acetic acid but could be solubilized with sodium cholate. These data indicate that AEBS is an integral membrane component of the rough microsomal fraction of rat liver.

Animals↗
PubMed1984

Microsomal binding sites for nonsteroidal anti-estrogens in MCF 7 human mammary carcinoma cells. Demonstration of high affinity and narrow specificity for basic ether derivatives of triphenylethylene.

In the presence of estradiol, at a concentration sufficient to saturate the estrogen receptor, the antiestrogenic and anti-tumor agent tamoxifen was bound to a high affinity (KD = 0.97 +/- 0.15 nM at 4 degrees C) saturable binding site (141,300 +/- 20,100 sites/cell) in MCF 7 human mammary carcinoma cells. The distribution of this site between cytosol, mitochondrial, microsomal (heavy and light), and nuclear fractions paralleled that of NADPH-cytochrome c reductase, an enzyme marker for endoplasmic reticulum. The interaction between tamoxifen and the high affinity site was influenced by changes in pH, ionic strength, and temperature. The kinetic rate constants k+1 and k-1 showed strong temperature dependence, but KD was unaffected by changes in temperature. Competition studies employing analogs of the anit-estrogens tamoxifen, clomiphene, and CI 628 revealed narrow specificity for triphenylethylene derivatives with basic ether side chains.

Breast Neoplasms↗
PubMed1984

Tamoxifen stimulation of human breast cancer cell proliferation in vitro: a possible model for tamoxifen tumour flare.

When T-47D human breast cancer cells were grown in medium supplemented with foetal calf serum (FCS) which had been depleted of endogenous oestradiol (E2) by dextran-charcoal treatment (CS-FCS) tamoxifen and E2 had biphasic effects on cellular proliferation. At lower concentrations E2 and, to a lesser extent, tamoxifen stimulated cell proliferation, with maximal effect at 10(-9) M, and with corresponding increases in the % S phase cells; this was not seen with medium containing untreated FCS. At higher doses tamoxifen (1-1.5 X 10(-5) M) and E2 (1.5 X 10(-5) M) in the presence of CS-FCS inhibited cell proliferation. These observations may explain features of the clinical phenomenon of tamoxifen-induced tumour flare: tamoxifen may temporarily exert a weak oestrogen agonist activity on the tumour as its concentration gradually increases after initiation of treatment; further, patients with tamoxifen-induced tumour flare often experience a remission with continued administration of the drug, presumably as the intratumour concentration of tamoxifen continues to accumulate to an inhibitory level.

Breast Neoplasms↗
PubMed1984

Growth hormone binding to cultured human breast cancer cells.

The role of pituitary hormones in the pathogenesis of human breast cancer is unclear, although hypophysectomy is of therapeutic benefit in some patients with advanced breast cancer. Agents that lower serum PRL are of little value in the treatment of breast cancer, suggesting that other pituitary hormones may be important in the control of the growth of human breast cancer in vivo. Since human (h) GH is lactogenic in rodents, we investigated the binding of [125I]hGH and [125I]hPRL to the cultured human breast cancer cell lines T-47D and MCF-7. Both [125I]hGH and [125I]hPRL bound to a saturable binding site with high affinity (Ka = 0.94-1.70 X 10(9) M-1) and low capacity (4140-6560 sites/cells) in the two cell types. hGH and hPRL were mutually competitive, indicating that both hormones bound to the same receptor site. After binding of [125I]hGH to cell monolayers, the hormone was rapidly internalized in a time-, temperature-, and energy-dependent fashion. Lysosomotropic agents inhibited degradation of [125I]hGH and enhanced specific binding. Preincubation of MCF-7 cells with either hGH or hPRL resulted in loss of hGH/hPRL-binding sites, although hGH was consistently more potent in inducing down-regulation of the receptor. On the basis of these observations we suggest that hGH is a potent ligand for the lactogenic receptor in human breast cancer cells in vitro and may be important in the pathogenesis, growth, and metastasis of human breast cancer.

Aged↗
PubMed1984

Correlation of lactogenic receptor concentration in human breast cancer with estrogen receptor concentration.

The presence of receptors for lactogenic hormones in human breast cancer tissue has been documented previously, but the relationship between the expression of these receptors and estrogen receptor (ER) status has not been adequately studied. In this report, the specificity of 125I-human growth hormone (HGH) binding in both cultured human breast cancer cell lines and tumor biopsies was studied to establish that HGH was a suitable ligand for investigating lactogenic receptor concentration in these tissues. In addition, the relationship between specific binding of 125I-HGH and ER concentration in human breast cancer was investigated. Specific 125I-HGH binding to 14 breast cancer cell lines in long term culture and to membrane preparations (microsomal and plasma membrane fractions) from 31 breast cancer biopsy specimens was examined. Human prolactin and HGH were approximately equipotent in inhibiting binding of 125I-HGH to both cultured breast cancer cell lines and to membrane preparations from breast cancer biopsy specimens. Competitive inhibition experiments using lactogenic and somatogenic hormones established that the specificity of 125I-HGH binding to breast cancer biopsy material was similar to that of cultured breast cancer cell lines and similar to that reported for subprimate lactogenic receptors. Saturable, high-affinity (Ka = 0.53 to 2.33 nM-1), low-capacity (330 to 6560 sites/cell) growth hormone binding sites were found on each of the ER-positive cell lines, whereas no specific 125I-HGH binding to ER-negative cell monolayers was detected. When all cell lines were considered, a significant linear correlation (r = 0.745, p less than 0.001) between ER and lactogenic receptor concentrations was found. Significant specific 125I-HGH binding, greater than 1% of the total radioactivity added, was detected in 20 of 31 (65%) breast tumor biopsy specimens. The mean affinity and capacity of the lactogenic receptor as measured in 8 separate membrane preparations were Ka = 0.52 +/- 0.09 (S.E.) nM-1 and 255 +/- 85 fmol/mg protein. Membrane preparations from ER-negative tumors (less than 3 fmol ER/mg cytosol protein) bound significantly less 125I-HGH than did membrane preparations from ER-positive tumor biopsies (1.22 +/- 0.44 versus 3.21 +/- 0.56%, p less than 0.05). A significant linear correlation between specifically bound 125I-HGH and ER concentration (r = 0.412, p less than 0.02) was demonstrated in the 31 breast cancer biopsy specimens studied.(ABSTRACT TRUNCATED AT 400 WORDS)

Binding, Competitive↗
PubMed1984

A new human breast carcinoma cell line (PMC42) with stem cell characteristics. III. Hormone receptor status and responsiveness.

PMC42 is a new human breast carcinoma cell line. In this report the content of estrogen, progesterone, and glucocorticoid receptors in PMC42 has been determined. The estrogen receptor content (1,750 cytoplasmic sites and 350 nuclear sites) was lower than that described for MCF7 and T47D. The cells would not proliferate in serum-free medium without the addition of beta-estradiol (optimum concentration 10(-8) M) or progesterone (5 X 10(-8) M). The addition of both hormones induced a more than additive increase in proliferation (P less than .005, n = 18). Similarly, addition of insulin or hydrocortisone induced proliferation; however, in this case, the effect of the hormones together was only additive. The addition of tamoxifen (10(-6) M) led to a significant decrease in cell numbers and inhibited the stimulatory effects of 10(-8) M beta-estradiol.

Breast Neoplasms↗
PubMed1984

Factors affecting the sensitivity of T-47D human breast cancer cells to tamoxifen.

Cell proliferation kinetics during growth of an estrogen receptor-positive human breast carcinoma cell line, T-47D, was defined, and some factors which modify its response to tamoxifen were investigated in vitro. T-47D cells were estrogen responsive when grown in charcoal-stripped fetal calf serum, but the addition of 17 beta-estradiol did not fully restore the growth rate to that observed in the same concentration of fetal calf serum. Tamoxifen had both a low-dose, estrogen-reversible, growth-inhibitory effect and a high-dose, estrogen-irreversible, growth-inhibitory and cytotoxic effect on T-47D cells. Tamoxifen-induced growth inhibition was associated with a decrease in the percentage of S-phase cells and, to a lesser extent, G2-M-phase cells and an increase in G0-G1-phase cells. Plateau-phase cells were considerably less sensitive than were exponentially growing cells, and this was accompanied by a fall in unoccupied estrogen receptor content from 4407 +/- 655 (S.E.) sites/cell in exponentially growing cultures to 1420 +/- 315 sites/cell in plateau-phase cultures. T-47D cells were more sensitive to tamoxifen cytostasis when grown in fetal calf serum rather than charcoal-stripped fetal calf serum. However, with both types of growth medium, the sensitivity to tamoxifen was inversely related to the serum concentration, e.g., the 50%-inhibitory dose concentration increased 75-fold as the fetal calf serum concentration was increased from 0.25 to 10%. Addition of insulin to the culture medium had no effect on the growth rate, estrogen receptor content, or tamoxifen sensitivity of T-47D cells. These results illustrate that the conditions under which cells are cultured markedly affect their sensitivity to tamoxifen and highlight the need to specify these conditions when reporting effects of this drug.

Breast Neoplasms↗