Kinetic alterations in estrogen receptors associated with estrogen receptor processing in human breast cancer cells

After nuclear translocation of estrogen receptors in MCF-7 human breast cancer cells, a processing takes place resulting in a 30-70% decline in the number of estradiol-binding sites measured in nuclear extracts. We have investigated the mechanism of estrogen receptor processing and obtained evidence that multiple events are involved. We confirm, as others have shown previously, that processing involves a decrease in the amount of estradiol binding in MCF-7 cells. In addition, evidence is provided for the generation of a rapidly dissociating population of estradiol-binding sites as an early event in processing. There is a single, slowly dissociating population of estrogen binding sites when MCF-7 cells are exposed to estradiol in the presence of actinomycin D, an inhibitor of receptor processing. One hour after the addition of sufficient estradiol to induce receptor processing, an additional, more rapidly dissociating population of estrogen binding sites is detected. When cells are exposed to estradiol and ethidium bromide, a drug which shares many actions with actinomycin D, but does not inhibit receptor processing, the rapidly dissociating population of estradiol-binding sites is again observed. Significantly, the loss of estradiol-binding sites from MCF-7 cells associated with processing between 1 and 6 h of estradiol exposure, occurs exclusively from the rapidly dissociating population of sites. Whole cell equilibrium-binding assays were performed with MCF-7 cells after 30 min or 5 h of estradiol exposure to determine whether the detected changes in estradiol dissociation reflected affinity changes in a subpopulation of estrogen-binding sites. Although the number of sites detected per cell varied with the assay method employed, binding to a single saturable class of higher affinity sites is always observed. High affinity estradiol-binding sites were reduced by 45% after a 5-h incubation with estradiol in both assay methods. The loss of estradiol binding during processing may therefore be explained by the conversion of certain high affinity estrogen receptors to a rapidly dissociating form which then fails to rebind hormone, or undergoes subsequent reactions that destroy hormone binding activity. Additionally, after 6 h of exposure to estradiol, the remaining receptor-bound estradiol dissociates from intact cells with a rate increased by 50% over that seen from the slow dissociating receptors present at 1 h.(ABSTRACT TRUNCATED AT 400 WORDS)     

Activation of growth factor secretion in tumorigenic states of breast cancer induced by 17 beta-estradiol or v-Ha-ras oncogene.

The MCF-7 human breast cancer cell line responds to estrogen stimulation in vitro by increased secretion of growth factors and proliferation and in vivo by tumor formation in the nude mouse. To test a possible role of growth factor secretion in expression of the tumorigenic phenotype, we stably transfected MCF-7 cells with the v-Ha-ras oncogene to produce the MCF-7ras cell line. The MCF-7ras cell line was tumorigenic in the absence of estrogens and secreted 3- to 5-fold elevated levels of a high molecular weight form of a type alpha transforming growth factor-like growth factor, type beta transforming growth factor, and insulin-like growth factor I. MCF-7ras cells, in contrast to MCF-7, were less sensitive to further growth stimulation by estrogen, type alpha transforming growth factor, and insulin-like growth factor I and showed little change in receptor levels for these hormones. Conditioned medium from MCF-7ras cells as well as two of its component growth factors (insulin-like growth factor I and type alpha transforming growth factor) replaced estrogen in stimulating MCF-7 colony formation in vitro. A coordinate increase in growth factor secretion by human breast cancer may contribute to its escape from estrogen dependence.     

The effect of estradiol on in vivo tumorigenesis is modulated by the human epidermal growth factor receptor 2/phosphatidylinositol 3-kinase/Akt1 pathway

To determine whether the epidermal growth factor receptor 2 (ErbB2) and Akt1 can alter the in vivo growth of MCF-7 cells, parental cells or cells stably transfected with constitutively active Akt1 (myr-Akt1) or dominant-negative Akt1 mutants (K179M-Akt1 and R25C-Akt1) were implanted into athymic nude mice. Tumor growth was monitored in the presence or absence of the antiestrogen tamoxifen and the selective ErbB2 inhibitor, AG825. MCF-7 [parental or empty vector transfected, cytomegalovirus (CMV)] and myr-Akt1 cells formed tumors upon estradiol supplementation after 20-30 d (59-, 29-, and 17-fold increase in tumor volume, respectively). Tamoxifen and AG825 blocked the estradiol effect by 93 and 96% in MCF-7 xenografts, 88 and 81% in CMV xenografts, and 91% in myr-Akt1 xenografts. Furthermore, AG825 suppressed the growth of established tumors in CMV and myr-Akt1 inoculated animals by 68 and 75%, respectively, as compared with continued estrogen supplementation, suggesting a role for ErbB2. When K179M-Akt1 or R25C-Akt1 cells were injected into ovariectomized animals, tumor growth was reduced upon estradiol treatment by 95% and 98%, respectively, supporting a role for Akt1. In contrast to ovariectomized animals, in intact animals, myr-Akt1 cells could establish tumors without estradiol priming after 40-50 d (20-fold increase in tumor volume). Loss of Akt1 phosphorylation was associated with tumor growth inhibition. Immunohistochemical assays showed that in tumors from parental and CMV xenografts, estradiol decreased estrogen receptor-alpha expression and induced progesterone receptor expression and Akt phosphorylation, effects that were inhibited by tamoxifen, AG825, and R25C-Akt1 by 89, 82, and 77% for progesterone receptor expression and 48, 66, and 73% for pAkt expression, respectively. Cumulatively, our results suggest that Akt1 and ErbB2 are involved in in vivo tumorigenesis and modulation of estrogen receptor-alpha expression and activity.     

An autostereoscopic 3D display can improve visualization of 3D models from intracranial MR angiography

Purpose  

An autostereoscopic display with image quality comparable to ordinary 2D displays has recently been developed. The purpose of our study was to evaluate whether the visualization of static 3D models from intracranial time-of-flight (TOF) MR angiography (MRA) was improved by this display.     

Impact of age on the prognostic value of body mass index in ST-Elevation myocardial infarction

Background and AimNo data are so far available on the impact of age and obesity in ST-elevation myocardial infarction (STEMI) submitted to percutaneous coronary intervention (PCI).Methods and ResultsWe assessed the impact of age on the prognostic value of body mass index (BMI) in 1268 consecutive STEMI patients admitted to our Intensive Cardiac Care Unit (ICCU). BMI categories were as follows: 37 “lean” patients (37/1268, 2.9%), 403 “normal” patients (403/1268, 31.8%), 656 “overweight” patients (656/1268, 51.7%), 172 “obese” patients (172/1268, 13.6%). Among patients aged <75 years, as BMI increased, the number of males and diabetic patients significantly increased (p < 0.001 and p = 0.004, respectively). Among STEMI patients aged ≥75 years, lean patients showed a higher in-ICCU mortality in respect to the other BMI categories but this did not reach statistical significance. BMI was an independent predictor of In-ICCU mortality in the whole population (lean vs. “normal”: OR 3.47, 95%CI 1.08–11.14, p = 0.036) and it was associated with long term mortality only in patients <75 years since lean and overweight patients showed lower survival rate (lean vs. “normal”: HR 9.25, 95%CI 3.09–27.63, p < 0.001; overweight vs. “normal”: OR 2.10; 95%CI 1.04–4.23, p = 0.039).ConclusionsIn our series, underweight is associated with the highest mortality across all age subgroups, while only in patients <75 years, overweight patients showed increased in-hospital mortality rate and a poorer long term survival rate. According to our data, the “so called obesity paradox” should be probably age-contextualized.     

Correlation of constitutive activation of raf-1 with morphological transformation and abrogation of tyrosine phosphorylation of distinct sets of proteins in human squamous carcinoma cells

We and others have reported an association between raf-1 protein serine/threonine kinase activity and transformation of mammalian cells. Because constitutive tyrosine phosphorylation of specific polypeptides is, in general, indicative of the transformed state of cells, we investigated the effect of activation of raf-1 on phosphotyrosine-containing proteins in a human head and neck squamous cell carcinoma-derived cell line, PCI-06A. raf-1 expression and activity were modulated in PCI-06A cells by means of stable DNA transfection of either the entire coding domain of the human c-raf-1 cDNA (in the sense or antisense orientation) of a fragment of c-raf-1 cDNA coding for the kinase domain of raf-1. Our data showed that constitutive activation of raf-1 correlated with morphological transformation, whereas the inhibition of raf-1 expression and activity had no detectable effect on cell morphology as compared with the untransfected cells. Immunoprecipitation of whole-cell lysates with anti-phosphotyrosine antibody followed by anti-phosphotyrosine immunol lotting revealed four phosphotyrosine-containing proteins of approximately 129, 120, 110, and 63 kDa (I–IV, respectively) in the antisense c-raf-1 cDNA-transfected cells showing relatively diminished raf-1 activity but not in the transfectants expressing activated raf. We concluded that tyrosine phosphorylation of I–IV proteins is abrogated in PCI-06A squamous carcinoma cells transformed with constitutively active raf-1 protein kinase. Mol.Carcinog. 18:1–6, 1997. © 1997 Wiley-Liss, Inc.     

Systemic delivery of RafsiRNA using cationic cardiolipin liposomes silences Raf-1 expression and inhibits tumor growth in xenograft model of human prostate cancer

Raf-1, a protein serine-threonine kinase, plays a critical role in mitogen-activated protein kinase kinase (MKK/MEK)- mitogen-activated protein kinase (extracellular signal-regulated kinase) (MAPK/ERK) pathways. We show here that systemically delivered novel cationic cardiolipin liposomes (NeoPhectin-AT) containing a small interfering RNA (siRNA) against Raf-1 silence the expression of Raf-1 in tumor tissues and inhibit tumor growth in xenograft model of human prostate cancer. The knockdown of Raf-1 expression by siRNA is also associated with down-regulation of cyclin D1 expression in vivo.     

Impact of hypertension on short- and long-term prognoses in patients with ST elevation myocardial infarction and without previously known diabetes

Hypertension is well established as a risk factor for the development of atherosclerosis. Data on the impact of hypertension in patients with ST elevation myocardial infarction are so far inconsistent, and are mainly related to studies performed in the thrombolytic era. We assessed the impact of hypertension over the short and long term in 560 patients with ST elevation myocardial infarction (STEMI) and without previously known diabetes, all of whom were submitted to mechanical revascularization and consecutively admitted to our Intensive Cardiac Care Unit. Hypertensive patients were older (p?<?0.001), more frequently male (0.005), and they showed a reduced eGFR (p?<?0.001). Smoking was more frequent in nonhypertensive patients (p?<?0.001), while the incidence of three-vessel coronary artery disease was higher in hypertensive patients (p?=?0.003). No difference in the in-hospital mortality rates for the two subgroups was detected. At follow-up (median 32.5?months, 25th–75th percentile 16.9–47.3?months), Kaplan–Meier survival analysis detected no differences in mortality between hypertensive and nonhypertensive patients (log rank χ ² 0.38, p?=?0.538). According to our data, obtained from a large series of consecutive STEMI patients without previously known diabetes, all of whom were submitted to primary PCI, a history of hypertension does not affect mortality over either the short or the long term. Moreover, hypertensive patients showed an altered glucose response to stress, as indicated by higher admission glucose values, poorer in-hospital glucose control, and a higher incidence of acute insulin resistance (as indicated by the HOMA index). Hypertensive patients therefore appear to warrant careful metabolic management during their hospital courses.