Miguel Saceda

Role of insulin-like growth factor-I in regulating estrogen receptor-α gene expression

The role of insulin‐like growth factor‐I (IGF‐I) in regulating estrogen receptor‐α (ER‐α) gene expression and activity was investigated in the human breast cancer cell line MCF‐7. Treatment of cells with 40 ng/ml IGF‐I resulted in a 60% decrease in ER‐α protein concentration by 3 h, and the amount of ER‐α remained suppressed for 24 h. A multiple‐dose ligand‐binding assay demonstrated that the decrease in ER‐α protein corresponded to a similar decrease of 50% in estradiol‐binding sites with no effect on the binding affinity of ER‐α. The dissociation constant of the estradiol‐ER‐α complex in the absence of IGF‐I (Kd = 3 × 10−10 ± 0.5 × 10−10 M) was similar to the dissociation constant in the presence of IGF‐I (Kd = 6 × 10−10 ± 0.3 × 10−10 M). The decrease in ER‐α protein concentration was paralleled by an 80% decrease in the steady‐state amount of ER‐α mRNA by 3 h. The IGF‐I induced decrease in ER‐α mRNA was due to the inhibition of ER‐α gene transcription. When an 128‐base pair ER‐α‐promoter‐CAT construct was transfected into MCF‐7 cells, treatment with IGF‐I resulted in a 40% decrease in CAT activity. In contrast to the effects on ER‐α, treatment with IGF‐I induced two endogenous estrogen‐regulated genes, progesterone receptor and pS2, by 4‐ and twofold, respectively. The pure antiestrogen ICI‐164,384 blocked this induction, suggesting that ER‐α mediates the effects of IGF‐I. Transient co‐transfections of wild‐type ER‐α and an estrogen response element‐CAT reporter into COS‐1 cells demonstrated that IGF‐I increased reporter gene activity. This effect was also blocked by ICI 164,384. Protein kinase A and phosphatidylinositol 3‐kinase inhibitors blocked the IGF‐I effects on ER‐α expression and activity, suggesting that these kinases may be involved in the cross‐talk between the IGF‐I and ER‐α pathways. J. Cell. Biochem. 76:605–614, 2000.

Estradiol regulates estrogen receptor mRNA stability.

Previous studies suggest that post-transcriptional events play an important role in estrogen-induced loss of estrogen receptor expression. The present study shows that treatment of MCF-7 cells with estradiol resulted in a six-fold decrease in estrogen receptor mRNA half-life from 4 h in control cells to 40 min in estradiol treated cells. To determine the role of protein synthesis in the regulation of estrogen receptor mRNA stability, several translational inhibitors were utilized. Pactamycin and puromycin, which prevent ribosome association with mRNA, inhibited the effect of estradiol on receptor mRNA stability, whereas cycloheximide, which has no effect on ribosome association with mRNA, had no effect on estradiol regulation of estrogen receptor mRNA stability. In control cells, the total cellular content of estrogen receptor mRNA was associated with high molecular weight polyribosomes. Treatment with estradiol resulted in a 70% decrease in estrogen receptor mRNA associated with polyribosomes but had no effect on the polyribosome distribution of estrogen receptor mRNA. In an in vitro degradation assay, polyribosomes isolated from estradiol-treated cells degraded ER mRNA faster than polyribosomes isolated from control cells. The nuclease activity associated with the polysome fraction appeared to be Mg2+ independent and inhibited by RNasin. Freeze-thawing and heating at 90 degrees C for 10 min resulted in the loss of nuclease activity. These studies suggest that an estrogen-regulated nuclease activity associated with ribosomes alters the stability of estrogen receptor mRNA.

Regulation of estrogen receptor‐α gene expression by 1,25‐dihydroxyvitamin D in MCF‐7 cells

This report describes an investigation of the role of 1,25‐dihydroxyvitamin D (VD3) in the regulation of estrogen receptor‐α (ER) in the ER‐positive breast cancer cell line, MCF‐7. Treatment of cells with 10 nM VD3 resulted in a 50% decline in the concentration of ER protein at 24 h. Scatchard analysis showed a corresponding decrease in the number of estradiol binding sites and no alteration in the binding affinity of estradiol for the ER (Kd = 0.08 nM in VD3‐treated cells compared with Kd = 0.07 nM in control cells). Vitamin D treatment also caused a 50% decrease in the steady state amount of ER mRNA, which was maximal by 18 h. In vitro transcription run‐on experiments demonstrated a decrease of approximately 60% in transcription of the estrogen receptor gene. Transient transfections using an ER promoter‐CAT construct also demonstrated a 40% decrease in CAT activity after VD3 treatment. Sequence analysis identified a potential vitamin D response element (nVDRE) within the ER promoter. When this element was mutated, the ability of VD3 to block transcription from the ER promoter was lost. When the nVDRE was placed upstream of a heterologous promoter, nVDRE‐SV40‐CAT, treatment with VD3 resulted in a 50% decrease in CAT activity. Interestingly, co‐transfection of either the ER promoter‐CAT or the nVDRE‐SV40‐CAT construct and a vitamin D receptor expression vector into COS‐1 or CV‐1 cells showed an approximately 4‐fold increase in CAT activity after VD3 treatment. Taken together these data suggest that VD3 inhibition of ER gene transcription is mediated through a nVDRE in the ER promoter. Inhibition appears to be cell specific. J. Cell. Biochem. 75:640–651, 1999.

The Role of Transforming Growth Factor-β in the Regulation of Estrogen Receptor Expression in the MCF-7 Breast Cancer Cell Line1

The role of transforming growth factor-β1 (TGFβ1) in the regulation of estrogen receptor (ER) expression in MCF-7 cells was investigated. After treatment of the cells with 100 pm TGFβ1, ER protein declined by about 30% at 6 h from a concentration of 413.5 fmol/mg protein in control cells to 289.5 fmol/mg protein in treated cells. The concentration of receptor remained suppressed for 24 h. Scatchard analysis demonstrated that the decrease in ER protein corresponded to a decrease in estradiol-binding sites, with no effect on the binding affinity of the ER. The dissociation constant of the estradiol-ER complex was 0.117 nm in TGFβ1-treated cells compared to 0.155 nm in control cells. Treatment with TGFβ1 did not influence the half-life of the ER. In TGFβ1-treated cells, as well as in control cells, the half-life of the receptor was approximately 4 h. In contrast to the effect on ER concentration, TGFβ1 treatment resulted in a greater decrease in the steady state level of ER messenger RNA (∼75%) at 6 h. By 24...

Regulation of estrogen receptor expression

One of the most prevalent of all cancers, breast cancer, is characterized by hormonal control of its growth. In cell culture, the control of proliferation and differentiation of human breast cancer cells involves steroid hormone, peptide hormones, and growth factors [1-5] however, epidemiological and clinical findings suggest that the major stimulus for growth of breast cancer is estrogen. Epidemiological studies show that endocrine status is an important factor in the prediction of risk. These risk factors include sex, age, age at menarche and menopause, age at first pregnancy, dietary factors, and family history of breast cancer [6]. In addition to epidemiological studies, clinical observations that support the role of estrogen in the growth of breast cancer are the marked decrease in tumor growth following ovariectomy and remission following treatment with antiestrogens and progestins [6]. Because breast cancer is characterized by hormonal control of its growth; the estrogen receptor (ER) is used to predict those patients who benefit from hormonal therapy. Although the presence of estrogen receptor is employed to predict the hormone dependency of a tumor, the response to endocrine therapy is not perfect. Significant levels of estrogen receptor have been detected in more than 60% of human breast cancers, however, only twothirds of these ER-positive tumors respond to endocrine therapy [7-10]. In addition, 5-10% of the ER-negative patients respond to endocrine therapy [11, 12]. To increase the prognostic value of the ER, the presence of an estrogen regulated protein such as the progesterone receptor (PR) is also measured. In normal tissue, progesterone receptor expression is regulated by estrogen [13, 14]. Although the presence of progesterone receptor improves the predictability of hormone dependency of a tumor; the improvement is not great. Retrospective studies show that only 70% of PR-positive and 25-30% of PR-negative tumors respond to hormone therapy. The reasons for the discrepancies between the level of receptors and their predictive value are not clear but may be attributed to the ability of ER to bind to nuclei in the absence of ligand [15], to the ability of ER to bind ligand but not to bind to nuclei, or to the synthesis of estrogen-regulated proteins in the absence of a functional ER pathway. Mutants of the estrogen receptor have also been identified which include base pair insertions, transitions, deletions, and alternate spliced forms of the receptor [16-22]. Additional assays, such as binding of the ER to an estrogen responsive element (ERE) or mutational analysis of ER by the polymerase chain reaction, may be necessary to accurately predict tumor response to endocrine therapy.

Regulation of Estrogen Receptor Expression in Breast Cancer

One of the most prevalent of cancers, breast cancer, is characterized by hormonal control of its growth. Expression of the estrogen receptor (ER) in MCF-7 breast cancer cells appears to be a complex process involving multiple steps subject to hormonal regulation by estrogen. Treatment of MCF-7 cells with estradiol results in the suppression of estrogen receptor protein. By 6 hours, the receptor protein declined by about 60% from a level of approximately 3.6 to 1.2 fmol/micrograms DNA and remained suppressed for 24-48 hours. Similar results were obtained with an estrogen receptor binding assay. Estrogen treatment also resulted in a decrease of receptor mRNA to approximately 10% of control values by 6 hours. Estrogen receptor remained at the suppressed level for up to 48 hours. Transcription run-on experiments demonstrated a transient decrease of about 90% in receptor gene transcription after 1 hour. By 3-6 hours transcription increased approximately 2-fold and remained elevated for at least 48 hours. These data suggest that estrogen suppresses ER mRNA by inhibition of ER gene transcription at early times and by a post-transcriptional effect on receptor mRNA at later times. To determine whether post-transcriptional regulation of ER gene expression is mediated by an ER-dependent mechanism independent of protein synthesis, we used the competitive estrogen antagonist, 4-hydroxytamoxifen, and the inhibitor of protein synthesis, cycloheximide, to study the regulation of ER mRNA by estradiol. 4-Hydroxytamoxifen had no effect on the steady-state level of receptor mRNA and effectively blocked the suppression of ER mRNA by estradiol. The metabolic inhibitor, cycloheximide, was unable to prevent the estrogen induced decrease in ER mRNA. These data provide evidence that the post-transcriptional suppression of ER expression through estradiol is mediated through the ER independent of protein synthesis.

Regulation of breast cancer cells by hormones and growth factors: effects on proliferation and basement membrane invasiveness.

The current understanding of the regulation of breast cancer cell proliferation and invasiveness by hormones and growth factors is reviewed. It has been shown that polypeptide growth factors are involved in hormone-independent breast cancer, and are sometimes oestrogen-regulated in hormone-responsive models. Basement-membrane invasiveness, relating to the metastatic potential of these cells, is also stimulated by oestrogen in hormone-dependent models, elevated in hormone-independent models, and is growth factor sensitive. Further understanding of the differential effects of growth factors on breast cancer cell proliferation and invasiveness should facilitate better therapeutic exploitation of regulation at this level.

Cell Death and Cancer, Novel Therapeutic Strategies

Life and death are essential parts of the natural cycle of all multicellular organisms. In metazoans, somatic cells divide normally during the process known as mitosis. Cell proliferation is tightly controlled, according to the organism needs. An increase in the number of cells takes place during growth and when one of these cells finishes its physiological function or detects DNA or cell damage, it undergoes a physiological process known as apoptosis that induces its own death. In humans about a hundred thousand cells are formed every second through mitosis, while a similar number is destroyed by apoptosis [1]. This dynamic balance between proliferation and cell death is known as homeostasis. If altered, different pathologic processes such as carcinogenesis can take place. Besides its role in embryonic development, homeostasis maintenance and aging, apoptosis is also a defence mechanism by which infected, injured or mutated cells as a result of irradiation or chemotherapeutic drugs are eliminated. This type of cell death involves the activation of an evolutionary conserved and tightly regulated intracellular machinery that requires energy consumption [2]. An important feature of apoptosis is that the cell is eliminated without triggering an immune response, avoiding thus tissue damage [3].