Sofía Ramos

Melatonin blocks the activation of estrogen receptor for DNA binding

The present study shows that melatonin prevents, within the first cell cycle, the estradiol‐induced growth of synchronized MCF7 breast cancer cells. By using nuclear extracts of these cells, we first examined the binding of estradiol–estrogen receptor complexes to estrogen‐responsive elements and found that the addition of estradiol to whole cells activates the binding of the estrogen receptor to DNA whereas melatonin blocks this interaction. By contrast, melatonin neither affects the binding of estradiol to its receptor nor the receptor nuclear localization. Moreover, we also show that addition of estradiol to nuclear extracts stimulates the binding of estrogen receptor to DNA, but this activation is also prevented by melatonin. The inhibitory effect caused by melatonin is saturable at nanomolar concentrations and does not appear to be mediated by RZR nuclear receptors. The effect is also specific, since indol derivatives do not cause significant inhibition. Furthermore, we provide evidence that melatonin does not interact with the estrogen receptor in the absence of estradiol. Together, these results demonstrate that melatonin interferes with the activation of estrogen receptor by estradiol. The effect of melatonin suggests the presence of a receptor that, upon melatonin addition, destabilizes the binding of the estradiol–estrogen receptor complex to the estrogen responsive element.—Rato, A. G., Pedrero, J. G., Martínez, M. A., del Rio, B., Lazo, P. S., Ramos, S. Melatonin blocks the activation of estrogen receptor for DNA binding. FASEB J. 13, 857–868 (1999)

The Mechanism of Intracellular Acidification Induced by Glucose in Saccharomyces cerevisiae

Addition of glucose or fructose to cells of Saccharomyces cerevisiae adapted to grow in the absence of glucose induced an acidification of the intracellular medium. This acidification appeared to be due to the phosphorylation of the sugar since: (i) glucose analogues which are not efficiently phosphorylated did not induce internal acidification; (ii) glucose addition did not cause internal acidification in a mutant deficient in all the three sugar-phosphorylating enzymes; (iii) fructose did not affect the intracellular pH in a double mutant having only glucokinase activity; (iv) glucose was as effective as fructose in inducing the internal pH drop in a mutant deficient in phosphoglucose isomerase activity; and (v) in strains deficient in two of the three sugar-phosphorylating activities, there was a good correlation between the specific glucose- or fructose-phosphorylating activity of cell extracts and the sugar-induced internal acidification. In addition, in whole cells any of the three yeast sugar kinases were capable of mediating the internal acidification described. Glucose-induced internal acidification was observed even when yeast cells were suspended in growth medium and in cells suspended in buffer containing K+, which supports the possible signalling function of the glucose-induced internal acidification. Evaluation of internal pH by following fluorescence changes of fluorescein-loaded cells indicated that the change in intracellular pH occurred immediately after addition of sugar. The apparent Km for glucose in this process was 2 mM. Changes in both the internal and external pH were determined and it was found that the internal acidification induced by glucose was followed by a partial alkalinization coincident with the initiation of H+ efflux. This reversal of acidification could be due to the activity of the H+-ATPase, since it was inhibited by diethylstilboestrol. Coincidence between internal alkalinization and the H+ efflux was also observed after addition of ethanol.

[74] The use of flow dialysis for determinations of ΔpH and active transport

Publisher Summary This chapter discusses that pH gradients (ΔpH) in biological systems that are not amenable to a direct electrophysiological approach can be estimated by measuring the distribution of certain weak acids or bases. Generally, if the internal pH is more alkaline than that of the medium, accumulation of weak acids is observed; conversely, accumulation of weak bases is observed if the internal pH is relatively acidic. The principle on which this approach is based stipulates that the undissociated species of the weak acid or base be freely permeant, whereas the dissociated, ionic species cannot traverse the membrane. Thus, if the internal compartment of the system under consideration is more alkaline than the external medium, the equilibrium between the undissociated and dissociated species of the weak acid will be displaced toward the ionic form, which will accumulate within that compartment. The chapter is concerned with a specific technique that have found to be particularly useful for measuring the distribution of weak organic acids into plasma membrane vesicles isolated from the enteric bacteria Escherichia coli and Salmonella typhimurium. The technique provides a notably accurate, reproducible method for measuring the active transport of a variety of solutes.

Amplification of ERBB oncogenes in squamous cell carcinomas of the head and neck

The activation of ERBB oncogenes has been described in various human tumours, including squamous cell carcinomas of the head and neck (SCCHN), and, in some of them, it has been correlated with a poor prognosis. Tissue samples from 59 patients with SCCHN were studied. After DNA extraction, the ERBB1, ERBB2 and ERBB3 copy number in tumour samples was estimated with the polymerase chain reaction (PCR) method. The PCR products were analysed by agarose gel electrophoresis and quantified by image analysis techniques. 9 (15%) cases presented with ERBB1 amplification, which was correlated with lymph node involvement (P = 0.04), poorly differentiated tumours (P = 0.03) and a hypopharyngeal primary site (P = 0.035). No correlation among amplification status, recurrence, metastases and survival was observed, although this may be due to the small number of patients in the amplified group. None of the 59 cases presented amplification of ERBB2 and ERBB3 oncogenes.

Variability of Genetic Alterations in Different Sites of Head and Neck Cancer

Objective Tumors arising from different sites of the head and neck area have different clinical behavior. However, most of the studies on genetic alterations in head and neck squamous cell carcinomas do not make a distinction between the sites within this area. The objective of this study is to compare the genetic alterations in three different sites of the head and neck (larynx, oropharynx, and hypopharynx).

Primary effects of yeast killer toxin

Abstract Killer toxin from Saccharomyces cerevisiae binds to sensitive cells immediately after addition to the cells. However, 50% mortality was obtained only after 40 minutes. Although it is thought that a lag phase is required for the killer to exert its action, we report experiments showing that the killer starts affecting the cell immediately after binding. Thus, shortly after addition the toxin was able to inhibit the transport of L-|;3H|; leucine as well as that of protons which are cotransported with this amino-acid or with histidine. Moreover, killer toxin inhibited the pumping of protons to the medium by cells which were actively metabolizing glucose. These effects were a function of the concentration of toxin used. The results suggest that killer toxin acts by affecting the electrochemical proton gradient across the plasma membrane of yeast.

Molecular cloning of a mouse homologue for the Drosophila splicing regulator Tra2.

We report the identification of a mouse cDNA, SIG41, encoding a protein of 288 amino acids that is 45% identical (58% similar) to the Drosophila splicing regulator Tra2. SIG41 cDNA contains four polyadenylation signals whose alternative use gives rise to four types of transcripts (2.1, 2.0, 1.5, and 1.4 kb) in mouse cells. Northern analysis and RT‐PCR assays showed that SIG41 mRNA is present in virtually all the cell lines and tissues studied, with remarkable levels of expression in uterus and brain tissues. Differential stability of the SIG41 mRNAs was detected in mouse macrophage cells.

Polyinosinic acid induces TNF and NO production as well as NF-κB and AP-1 transcriptional activation in the monocytemacrophage cell line RAW 264.7

Abstract.Objective: This study evaluates the poly inosinic acid (poly I)-induced activation in the murine monocytemacrophage cell line RAW 264.7, which led to an inflammatory phenotype.Material: RAW 264.7, and WEHI 164 cell lines were used.Results: The activation process is characterized by the acquisition of a mature macrophage morphology and the production of inflammatory mediators tumor necrosis factor (TNF) and nitric oxide (NO). The activation by poly I has distinctive features. Thus, poly I induced an increase in nuclear factor κB (NF-κB) transcriptional activity due to a long-term degradation of inhibitory NF-κB (IκB) β while lipopolysaccharide (LPS) induced the degradation of both IκBα and IκBβ. Poly I also induced an increase in activator protein 1 (AP-1) transcriptional activity, possibly due to the activation of the mitogen activated protein kinases (MAPKs) ERK, Jun N terminal kinase (JNK) and p38. Dextran sulphate (DS) efficiently inhibited the activation induced by poly I including the production of the inflammatory mediators. Dextran sulphate also inhibited AP-1 and NF-κB transcriptional activities in poly I-stimulated cells. RAW 264.7 cells express macrophage scavenger receptor 1 (Msr1) type I and Msr1 type II that are differently up-regulated upon treatment with poly I.Conclusions: The results presented demonstrate that the well-known blocker of scavenger receptors poly I activates macrophages to produce TNF and NO, triggering specific signal transduction pathways.

Measurement of cytotoxicity by propidium iodide staining of target cell DNA: Application to the quantification of murine TNF-α

A rapid and sensitive method is described for the determination of murine tumor necrosis factor (TNF-alpha), which can be performed in microtiter plates using a fluorescence plate scanner. The method is based on the binding of propidium iodide (PI), a membrane-impermeant dye, to nucleic acids of WEHI 164 cells, whose plasma membrane permeable due to TNF-alpha-induced cell damage. The analytical range for the proposed method is 0.3-200 pg/ml of TNF-alpha after 5 h of incubation. The optimal number of target cells was found to be 4-5X10(4)/well. The variability obtained for the PI assay was 7.6%; lower than that obtained with a commonly employed method in which MTT is used to determine cell viability (11.3%). Thus, the PI assay appears to be a reliable and reproducible method for the determination of biologically active TNF-alpha. The assay can be performed in a few hours and has the advantage over the current MTT and 51Cr-released assays that kinetic studies of TNF-alpha toxicity are possible since it permits multiple, sequenced measurements of cell viability during the incubation of the sample. The method can also be used for the determination of human TNF-alpha.

Genetic alterations in squamous cell carcinomas of the hypopharynx with correlations to clinicopathological features

The objective of this study is to describe the molecular alterations in carcinomas in one specific location of the head and neck, the hypopharynx. Thirty-seven hypopharyngeal squamous cell carcinomas were studied. The DNA from tumour and healthy tissue was evaluated for amplification of the 11q13 region and of the MYC and ERBB1 oncogenes, for integration of the Human Papillomavirus (HPV), and for loss of heterozygosity (LOH) at p53 and NAT2 loci. The most common alteration was the amplification of the 11q13 region (78% of the cases), followed by LOH at p53 locus (70%). MYC amplification was found in 19% of the cases, ERBB1 amplification in 29%, LOH at NAT2 locus in 25%, and integration of the HPV in 29%. 11q13 amplification was related with nodal metastases and higher tumour recurrence rates. These findings confirm that 11q13 amplification is one of the most frequent genetic alterations in hypopharyngeal squamous cell carcinomas, and that it may have prognostic significance in these tumours.