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Biomedical subjects

I E Alexander

Publications and source records attributed to I E Alexander.

8 recordsLinked to original sources
PubMed1992

Expression and regulation of retinoic acid receptors in human breast cancer cells.

Retinoic acid is known to inhibit mammary carcinogenesis in rodents and to inhibit proliferation and steroid hormone receptor gene expression in human breast cancer cells. Since these effects are likely to be mediated by nuclear retinoic acid receptors (RARs) the present study was initiated to determine the expression and regulation of RARs in human breast cancer cell lines. Differential cellular gene expression of the RARs was determined by Northern blot analysis of total RNA prepared from 5 ER+ and 6 ER- cell lines. RAR alpha was detected as mRNA species of 2.7 and 3.4 kilobases in all cell lines and the level of gene expression was greater in ER+ cell lines (P less than 0.001). RAR beta mRNA (3.7 kilobases) was detected in seven of the eleven lines tested and was expressed most commonly in ER- cell lines. RAR gamma mRNA was expressed in all cell lines as a transcript of 3.4 kilobases at levels that were similar in both ER+ and ER- cell lines. Retinoic acid failed to regulate the expression of the RAR alpha and RAR gamma genes. The effect of steroid hormones on RAR alpha and RAR gamma mRNA levels was also examined. In four PR+ cell lines (T-47D, BT 474, MCF-7M, and MDA-MB-361), progestins markedly decreased RAR alpha mRNA levels. The progestin effect on RAR alpha levels in T-47D cells was detectable at concentrations of 0.05 nM and was maximal at 1 nM 16 alpha-ethyl-21-hydroxy-19-nor-4-pregnene-3,20-dione ORG 2058, whereas dihydrotestosterone and dexamethasone were without effect. RAR alpha and RAR gamma mRNA levels were rapidly decreased by progestin, and the effect was maximal 3-6 h after ORG 2058 treatment. However, the mRNA loss was transient, and recovery of RAR alpha and RAR gamma mRNA levels was noted 12-24 h after retinoic acid treatment. Although RAR gamma mRNA returned to control levels by 24 h, the decrease in RAR alpha mRNA was maintained at around 50% control until at least 48 h. In summary, RAR alpha and RAR gamma were expressed in all human breast cancer cell lines and were regulated by progestins in the PR+ T-47D cell line. The previously reported ability of retinoic acid to down-regulate PR mRNA and the present demonstration that progestins down-regulate RAR alpha and RAR gamma mRNA suggest that mutual regulation may be a mechanism through which PR and the RARs interact in human breast cancer cells.

Breast Neoplasms↗
PubMed1990

Steroid hormone receptor gene expression in human breast cancer cells: inverse relationship between oestrogen and glucocorticoid receptor messenger RNA levels.

The relative expression in human breast cancer cells of messenger ribonucleic acids (mRNA) encoding different steroid hormone receptors is unknown. Accordingly, mRNA levels in total RNA extracted from 13 human breast cancer cell lines were measured by Northern analysis employing complementary DNA probes for the human oestrogen (ER), progesterone (PR), androgen (AR), vitamin D3 (VDR) and glucocorticoid receptors (GR). The 7 ER+ lines expressed a single 6.4 kilobases (kb) ER mRNA. Interestingly, low concentrations of ER mRNA were detected in the ER- cell lines, MDA-MB-330 and BT 20. PR mRNA, predominantly a 13.5 kb species, was expressed in the 6 lines known to be ER+, PR+ by radioligand binding; however, one ER+ cell line, MDA-MB-134, failed to express PR mRNA. A 10.5 kb AR mRNA was expressed at significantly higher levels in ER+ than ER- cell lines. All cell lines expressed a single 4.6 kb mRNA for VDR and a single 7.4 kb mRNA for GR. ER and PR mRNA levels were positively correlated (p = 0.011) and each was positively correlated with androgen receptor (AR) mRNA levels (p less than or equal to 0.009). ER, PR and AR mRNAs were negatively associated with GR levels (p less than or equal to 0.012), while ER and AR mRNA levels were negatively correlated with mRNA for the epidermal growth factor receptor. In contrast, levels of VDR mRNA were unrelated to the concentration of any other steroid receptor mRNA. Our data demonstrate the coordinate expression of ER, PR and AR genes, and an inverse relationship between sex steroid hormone receptor and GR gene expression in human breast cancer cell lines.

Blotting, Northern↗
PubMed1990

Progestin regulation of estrogen receptor messenger RNA in human breast cancer cells.

Progestin antagonism of estrogen action is thought to be due, at least in part, to progestin down-regulation of the estrogen receptor (ER). The molecular mechanisms subserving this effect, and the functional consequences in terms of target cell sensitivity to estrogens, are poorly understood. The present study was undertaken to address these issues with particular emphasis on progestin regulation of ER gene expression at the mRNA level. The T-47D human breast cancer cell line was treated with the synthetic progestin, ORG 2058, and the resultant changes in ER mRNA and ER levels determined by Northern analysis and radioligand binding, respectively. Treatment of T-47D cells with ORG 2058 resulted in rapid down-regulation of ER mRNA levels to a nadir of 35-40% of control by 6 h. This fall in ER mRNA levels was accompanied by a slower but more sustained fall in ER binding to a nadir of 20% of control at 24 h. Between 12 and 24 h ER mRNA levels recovered partially while ER ligand binding continued to fall. At 48 h both ER mRNA and ER concentrations remained depressed, although the latter to a greater extent. ER mRNA half-life was determined by [3H]uridine incorporation to be approximately 60 min and was unaffected by progestin treatment during the early rapid phase of ER mRNA down-regulation. These data demonstrate that progestins cause rapid down-regulation of the ER mRNA and suggest that during the early rapid phase of this effect, reduced transcription of the ER gene rather than altered ER mRNA half-life mediate this effect.(ABSTRACT TRUNCATED AT 250 WORDS)

Breast Neoplasms↗
PubMed1990

Inverse relationship between estrogen receptor and epidermal growth factor receptor mRNA levels in human breast cancer cell lines.

Epidermal growth factor receptors (EGF-R) are present in a number of human breast cancer cell lines and tumor biopsies. Furthermore, it has been suggested that EGF-R levels are higher in estrogen receptor negative (ER-) than in ER+ human breast tumors and that EGF-R status may be a prognostic indicator in breast cancer. The present study was undertaken to establish whether there is a quantitative relationship between EGF-R and ER mRNA concentrations in a series of 10 well-characterized human breast cancer cell lines. All cell lines expressed detectable quantities of EGF-R mRNA by Northern analysis but the relative abundance of EGF-R mRNA varied more than 50-fold. Two transcripts corresponding to the 10.5- and 5.8-kb mRNAs described in other cell types were present but in different relative proportions in different cell lines. When these lines were divided into an ER+ and an ER- group based on their ability to bind estradiol, ER- cell lines were shown to express significantly higher concentrations of EGF-R mRNA than did ER+ cell lines (p less than 0.005). Furthermore, linear-regression analysis revealed a significant inverse relationship between ER and EGF-R mRNA concentrations both within the group of 10 human breast cancer cell lines as a whole (r = 0.66) and within the 6 functionally ER + lines (r = 0.77). This demonstration of a significant (p less than 0.005) inverse relationship between the concentrations of ER and EGF-R mRNAs in ER + cell lines raises the possibility of reciprocal regulation of the expression of these genes in human breast cancer.

Blotting, Northern↗
PubMed1989

Progestin inhibition of progesterone receptor gene expression in human breast cancer cells.

The present study was designed to investigate whether inhibition of progesterone receptor (PR) gene transcription and/or regulation of PR mRNA half-life were involved in the progestin-mediated decrease of PR in T-47D human breast cancer cells. Cells were treated with the progestin ORG 2058 and PR mRNA measured by Northern blot analysis of total RNA. A major PR mRNA around 13.5 kilobases and minor species around the 28S ribosomal RNA subunit were decreased upon ORG 2058 treatment. The decrease was not detectable until 2-3 h after treatment and was the same at all ORG 2058 concentrations (1-100 nM) tested. The decrease in PR mRNA was unaffected by actinomycin D in the first 3 h but was inhibited thereafter. There was a partial recovery of PR mRNA levels 24 h after ORG 2058 exposure. Immunoblot analysis showed that immunoreactive PR decreased in parallel with PR mRNA. The rate of protein loss in the first 12 h after progestin treatment was related to the ORG 2058 concentration used. Nuclear run-on experiments showed that ORG 2058 caused a decrease of up to 70% in the transcription rate of the PR gene. The half-life of PR mRNA was shown to be 2-2.5 h by [3H]uridine incorporation, which was much shorter than estimates obtained using actinomycin D, and was unaffected by ORG 2058. In summary, these data have shown that the mechanism by which progestins decrease the concentration of PR includes inhibition of transcription of the PR gene.

Blotting, Northern↗
PubMed1988

Genetic instability and the development of steroid hormone insensitivity in cultured T 47D human breast cancer cells.

The estrogen receptor (ER)-positive human breast cancer cell line T 47D exhibited genetic instability under cell culture conditions which maintained almost continuous exponential growth. This resulted in the spontaneous generation of three ER-positive sublines with a range of DNA ploidies and distinctive phenotypes. One of these sublines, T 47D-5, exhibited resistance to the growth-inhibitory effects of the synthetic nonsteroidal antiestrogen tamoxifen and the synthetic progestin ORG 2058, in marked contrast to "wild type" T 47D cells (designated T 47D-7 in this study). T 47D-5 cells were cloned by limiting dilution and 11 clonal cell lines were tested for sensitivity to tamoxifen. Although all clones of T 47D-5 were significantly less sensitive than T 47D-7 cells, a spectrum of sensitivities was observed. Three clones, T 47D-5-13, T 47D-5-21, and T 47D-5-23, were further characterized by measuring the concentrations of receptors for estrogen, progesterone, growth hormone, and epidermal growth factor and responses to estradiol, tamoxifen, and progestin, in terms of both induction of specific proteins and effects on cellular proliferation. Although the T 47D-5 subline and clone T 47D-5-23 were insensitive to both the growth-stimulatory effects of estradiol and the inhibitory effects of tamoxifen, this was not related to the concentration of ER or its ability to induce progesterone receptor. Estrogen receptor levels were similar in resistant and sensitive clones of T 47D-5 [70,000-81,000 sites/cell] and were 2.5-fold greater than in the sensitive T 47D-7 line [32,600 +/- 5,000 (SEM) sites/cell]. Northern blots showed no difference in the size of ER mRNA transcripts between sensitive and resistant clones. Estradiol treatment increased progesterone receptor (PR) levels in all cell lines but the magnitude and sensitivity of this response were unrelated to growth responses indicating a divergence in estrogenic control of cellular proliferation and specific protein synthesis within these clones. T 47D-5, T 47D-5-13, T 47D-5-21, and T 47D-5-23 were all insensitive to the growth-inhibitory effects of ORG 2058. The progestin was also unable to increase lactogenic and epidermal growth factor receptor concentrations in these four lines in contrast to the response in T 47D-7 cells. The insensitivity to progestin in the T 47D-5 subline and its three clonal cell lines could be accounted for, in part, by a 75-80% reduction in PR levels when compared with T 47D-7 cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Breast Neoplasms↗
PubMed1979

Death anxiety, dissent, and competence.

A total of 64 male undergraduates were administered a multistage interview which was structured to assess (a) their level of overtly expressed death anxiety, (b) covert (GSR) arousal to death stimuli (c) self-perceived competence, and (d) agreement with or dissent from life threatening national policies. The analyses that followed were concerned with examining the relationships among these variables. In previous studies of this kind it had been typically found that (1) self-perceived competence and magnitude of expressed death concern are inversely related and (2) overt expressions of death concern and covert physiological arousal to death cues are inversely related. Psychodynamic formulations centering on the ego-defensive nature of inhibited expressions of death anxiety have been cited to explain these past data. The current investigation proposed that the magnitude of expressed death concern would bear an inverse relationship to both felt competence and covert death arousal only when the level of overt concern was not contingent upon the individual's attitudes concerning the imminence of real life threatening circumstances in the environment. The rationale behind these predictions inheres in the notion that the neurotic components of strongly expressed death anxiety derive from its lack of anchoring in "real" external threats. Conversely, the expression of low death fear can only be regarded as "defensive" when real threats are perceived and acknowledged. The obtained results strongly support this rationale and the discussion centers on the impact of social conditions on psychodynamic processes.

Achievement↗