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C J Ormandy

Publications and source records attributed to C J Ormandy.

28 records · Page 2Linked to original sources
PubMed1995

Differential expression of oestrogen regulated genes in breast cancer.

Pathological endpoints such as tumour size, lymph node status and vascular invasion remain the most useful guides in selecting treatment strategies for breast cancer. There is a need, however, to further investigate the molecular mechanisms that determine the properties of an individual tumour e.g., hormone responsiveness and probability of metastasis. While numerous prognostic factors have now been identified few have contributed to defining clinical response to therapy. Oestrogen-regulated genes are likely to be important since they not only define a functional oestrogen receptor, but alterations in their expression might provide insights into the mechanisms involved in tumour progression and loss of endocrine sensitivity. Recently an oestrogen responsive gene, pLIV1, has been isolated and shown to be expressed in ER+ disease where it appears to predict nodal involvement. The present paper describes aspects of its regulation and discusses the potential role of this and other genes in the development of endocrine resistance.

Breast Neoplasms↗
PubMed1993

Estradiol induction of retinoic acid receptors in human breast cancer cells.

Retinoic acid inhibits proliferation and steroid receptor gene expression in human breast cancer cell lines. Retinoic acid receptors (RAR)alpha, -beta, and -gamma are expressed in these cells and the expression of RAR alpha is significantly greater in estrogen receptor (ER)-positive cells. This study was undertaken to determine whether the same relationship between RAR alpha and ER gene expression was present in human breast cancers and to explore the possibility that the higher level of RAR alpha in ER-positive cells was due to estrogen regulation of RAR alpha gene expression. RAR alpha and ER mRNA expression were determined by Northern blot analysis in 116 primary breast tumors; 94 (81%) tumors were ER-positive and of these 87 (93%) were also RAR alpha-positive. The coexpression of ER and RAR alpha was statistically significant (P = 0.0052 by chi 2 contingency analysis). There was also a positive correlation (by linear regression analysis) between the levels of expression of ER and RAR alpha mRNA (r2 = 0.251, P = 0.0001), which confirmed the relationship previously documented in breast cancer cell lines and suggested that RAR alpha expression may be modulated in breast cancer in vivo by estrogens acting via the ER. The ability of estradiol to regulate RAR alpha gene expression was examined in vitro using T-47D cells which had been rendered sensitive to estrogen by repeated passage in steroid-depleted medium. Estradiol increased RAR alpha gene expression, but not that of RAR beta or RAR gamma, in a concentration-dependent manner, with the effect being maximal at 10(-10) M and less marked at higher concentrations. The effect was rapid, being detectable 1 h after and maximal 6 h after treatment with 10(-10) M estradiol. Co-treatment of cells with estradiol and antiestrogens (tamoxifen or ICI 164384, 4 x 10(-7) M for 6 h) inhibited the estradiol induction of RAR alpha gene expression, demonstrating that the effect was ER mediated. The estradiol sensitivity of the effect was underscored by the demonstration that addition of untreated serum to cells growing under steroid-depleted conditions was sufficient to induce maximal RAR alpha gene expression. This effect was totally abolished by addition of ICI 164384. In summary, the demonstration that estradiol increased RAR alpha mRNA levels in breast cancer cells supports the hypothesis that the correlation between RAR alpha and ER gene expression in breast tumors and breast cancer cell lines is due to estradiol augmentation of RAR alpha gene expression.

Breast Neoplasms↗
PubMed1993

Regulation of prolactin receptor expression by the tumour promoting phorbol ester 12-O-tetradecanoylphorbol-13-acetate in human breast cancer cells.

In both the normal and malignant human breast, cellular sensitivity to the proliferative and differentiative activities of the lactogenic hormones is conferred by expression of the prolactin receptor (PRLR). The PRLR is regulated by steroid hormones; however, recent findings have suggested that PRLR may also be regulated by protein kinase C. To examine this possibility we have studied the effect of various modulators of PKC activity on PRLR binding activity and gene expression in five PRLR positive human breast cancer cell lines. Treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA), a tumour promoter and modulator of PKC activity, decreased PRLR binding activity in all cell lines examined. In MCF-7 cells, 10 nM TPA caused a 70% loss of PRLR mRNA after 12 h, paralleled 3 h later by a comparable loss of cell surface PRLR. Mezerein, a non-phorbol ester modulator of PKC activity and 1,2-dioctanoyl-sn-glycerol, a permeant analogue of the endogenous activator of PKC, also reduced PRLR binding activity and gene expression in a time- and concentration-dependent manner. Cycloheximide failed to abrogate the TPA-induced decline in PRLR mRNA levels, indicating that this process was not dependent upon continuing protein synthesis. No change in the stability of PRLR mRNA was observed during 24 h of TPA treatment and TPA reduced the rate of PRLR gene transcription within 3 h of treatment. These results demonstrate that modulators of PKC activity reduce PRLR binding activity and gene expression, implicating this signal transduction pathway in PRLR regulation.

Breast Neoplasms↗
PubMed1992

Androgen regulation of prolactin-receptor gene expression in MCF-7 and MDA-MB-453 human breast cancer cells.

Lactogenic hormones which bind to the PRLR are likely to be growth-stimulatory in human breast-cancer cells. Oestrogen and progesterone control cellular expression of the PRLR; however, elevated androgen levels in some breast-cancer patients raised the possibility that androgens may also influence breast-cancer sensitivity to lactogenic hormones. This study investigated whether androgens could affect expression of the PRLR in the MCF-7 breast-cancer cell line. PRLR binding activity was increased approximately 2-fold by treatment for 24 hr with 10 nM R1881, TEST, DHT, MPA and ORG 2058. Northern analysis indicated that DHT also increased the level of PRLR mRNA. The antiprogesterone, RU 38486, displaced tritiated ORG 2058 binding but not tritiated DHT binding to MCF-7 cells; it completely antagonized ORG 2058 and partially antagonized R1881 induction of the PRLR, but had no effect on induction by DHT. The anti-androgen, RU 23908, displaced tritiated DHT binding but not tritiated ORG 2058 binding, and antagonized DHT and R1881 induction of PRLR but not induction of the PRLR by ORG 2058. These data indicated that ORG 2058 acting via the PR and DHT acting via the AR were able to induce PRLR expression in MCF-7 cells. In MDA-MB-453 cells, which express the AR but not the ER or PR, DHT and R1881 increased PRLR binding to 150% of control values at 0.1 nM. ORG 2058 was ineffective, demonstrating androgen induction of PRLR in the absence of PR and ER. These data indicate that PRLR can be regulated by androgens in MCF-7 and MDA-MB-453 human breast-cancer cells.

Androgens↗
PubMed1992

Coordinate regulation of oestrogen and prolactin receptor expression by sodium butyrate in human breast cancer cells.

Prolactin receptor and oestrogen receptor are co-ordinately expressed in human breast cancer cell lines and in human breast tumour biopsies, leading to the suggestion that the expression of these receptors may be coupled. To examine this hypothesis, T-47D and MCF-7 human breast cancer cells were treated with sodium butyrate, a known modulator of oestrogen receptor levels, and the changes in oestrogen and prolactin receptor mRNA and binding activity were measured. In both cell lines treatment with 0.3-10 mM sodium butyrate resulted in a parallel decrease in prolactin and oestrogen receptor mRNA levels and binding activity. In T-47D cells, where the effect was transient, mRNA levels of both receptors recovered in parallel. These data indicated that oestrogen receptor and prolactin receptor gene expression is modulated in parallel by sodium butyrate and supported the hypothesis that the expression of these two receptors is coupled.

Breast Neoplasms↗
PubMed1992

The effect of progestins on prolactin receptor gene transcription in human breast cancer cells.

The sex steroid hormone progesterone modulates the developmental and lactogenic activity of prolactin in the mammary gland. Regulation of the level of prolactin receptor (PRLR) provides one possible mechanism by which this may occur, prompting this investigation of the molecular mechanisms involved in progestin regulation of prolactin receptor levels. Treatment of T-47D and MCF-7 human breast cancer cells with 10 nM of the synthetic progestin ORG 2058 for 24 hr resulted in an increase in all four PRLR mRNA transcripts detected. The effect of ORG 2058 was shown in T-47D cells to be time- and concentration-dependent and resulted in an approximate two-fold increase in PRLR mRNA after 24 hr of treatment with 10 nM or 100 nM ORG 2058. Nuclear run-on assays indicated that ORG 2058 increased the rate of T-47D PRLR gene transcription at all times between 1 hr and 28 hr of treatment. The protein synthesis inhibitors cycloheximide and puromycin abrogated the induction of PRLR gene transcription at 1 hr and 2 hr, which demonstrated that on-going protein synthesis was required for the ORG 2058 effect and suggested that progestins may exert some transcriptional effects via the induction of an intermediary protein. These experiments demonstrated that progestin induced a transcriptionally based increase in PRLR gene expression and provided a mechanism by which progesterone may modulate the mitogenic activity of prolactin during mammary gland development.

Autoradiography↗
PubMed1992

Transcriptional regulation of prolactin receptor gene expression by sodium butyrate in MCF-7 human breast cancer cells.

The prolactin receptor (PRLR) mediates the diverse effects of prolactin, which in the mammary gland include the development of lobuloalveolar structures and increased tumor cell proliferation. Treatment of mammary carcinoma cells with the differentiating agent sodium butyrate (NaB) is known to reduce PRLR binding activity and PRLR gene expression, however the mechanism which mediates these changes is unknown, prompting this investigation. Using MCF-7 human breast cancer cells, assay of the rate of PRLR gene transcription by the nuclear run-on technique indicated that 3 mM NaB reduced PRLR gene transcription by 50% after 3 h of treatment and that this effect was maintained for at least 24 h. The protein synthesis inhibitor cycloheximide failed to abrogate this effect, which indicated that NaB did not require continuing protein synthesis to reduce the rate of PRLR transcription. Measurement of PRLR mRNA stability, using Northern blot analysis at various times after the inhibition of transcription with actinomycin D, showed that NaB treatment did not alter PRLR mRNA half-life. These results indicate that NaB inhibits PRLR gene expression by a transcriptional mechanism that does not require continuing protein synthesis.

Blotting, Northern↗
PubMed1990

Solubilization and characterization of a lactogenic receptor from human placental chorion membranes.

Prolactin has a wide range of actions, including osmoregulation and the control of mammary gland development and lactation. These effects are mediated through a high-affinity cell surface receptor, which has been well characterized in a number of animal tissues. The molecular characteristics of the human receptor are unknown, however. The present studies were initiated, therefore, to determine the binding and molecular characteristics of the lactogenic receptor of human placental chorion membranes. Subcellular fractionation studies showed that the bulk of the receptor sedimented in the microsomal fraction at 45,000gav. Endogenous ligand was dissociated from the receptor with 3.5 M MgCl2 or 0.05 M acetate buffer (pH 4.8) with preservation of binding activity. The microsomal receptor bound human growth hormone (hGH), human prolactin (hPRL), ovine prolactin (oPRL), and human placental lactogen (hPL) but not non-primate growth hormones, indicating a narrow specificity for lactogenic hormones. The binding was only partially reversible in agreement with the known binding kinetics of animal lactogenic receptors. The receptor was solubilized with 45% yield from the microsomes using 16 mM 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulphonate (CHAPS) detergent-250 mM NaCl, and the binding activity was fully restored by a two-fold dilution in the binding reaction to reveal a KD of 0.8 nM for hGH and a binding capacity of 200 fmol of specifically bound hGH per mg of microsomal protein. Gel filtration chromatography indicated the minimum molecular weight of the ligand-receptor complex was approximately 60,000 daltons, and sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) of covalently cross-linked 125I-hGH-receptor complexes revealed a molecular size of 58,000 daltons. When account was taken of the contribution of the ligand, a molecular weight of 36,000 for the receptor's binding domain was obtained. These data indicate that the chorion lactogenic receptor has very similar binding and molecular characteristics to the lactogenic receptors from other mammalian species. Chorion membranes are thus a convenient source of material for the further purification and characterization of the human lactogenic receptor.

Cell Fractionation↗
PubMed1988

Human growth hormone binds to lactogenic receptors in bovine, ovine and rat adrenals.

The distribution of 125I radioactivity in the liver, kidneys, adrenals and serum of male rats was measured 10 minutes after an intravenous bolus of 125I-labelled human growth hormone (hGH) was administered in the presence or absence of a large excess of ovine growth hormone or ovine prolactin. The hGH binding sites in the adrenals had displacement properties characteristic of lactogenic receptors, whereas those in the liver had displacement properties characteristic of somatogenic receptors. Bovine and ovine adrenal microsomal membrane fractions contained high affinity (Ka = 1.4-3.3 nM-1) binding sites for hGH which showed ligand specificity typical of lactogenic receptors. It is concluded that the hGH binding site in the adrenal gland is a classical lactogenic receptor and that this tissue is a convenient and rich (42.6 +/- 6.4 fmol hGH specifically bound/mg protein) source of receptor suitable for further characterization.

Adrenal Glands↗