Alternative splicing in lung cancer

Abstract: Alterations in alternative splicing affect essential biologic processes and are the basis for a number of pathologic conditions, including cancer. In this review we will summarize the evidence supporting the relevance of alternative splicing in lung cancer. An example that illustrates this relevance is the altered balance between Bcl-xL and Bcl-xS, two splice variants of the apoptosis regulator Bcl-x. Splice modifications in cancer-related genes can be associated with modifications either in cis-acting splicing regulatory sequences or in trans-acting splicing factors. In fact, lung tumors show abnormal expression of splicing regulators such as ASF/SF2 or some members of the heterogeneous nuclear ribonucleoprotein family. The potential significance of alternative splicing as a target for lung cancer diagnosis or treatment will also be discussed

Breakthroughs in lung cancer management

In the present issue of Anales del Sistema Sanitario de Navarra, Rico et al summarize the progress in key areas of the radiotherapeutic strategies to fight against advanced lung cancer1. Progress in radiotherapy strategies for lung cancer management has been remarkable in the last decade. We will devote some comments in the present editorial to these achievements. Nevertheless, radiotherapy (RT) has been only one of the many areas that have benefited from successful basic, translational and clinical research in lung cancer during the last decade. In the following paragraphs, we will very briefly mention some of these breakthroughs and seminal achievements that have improved the survival prospects of lung cancer patients in the decade from 2010 to 2020

The regenerative nidi of the locust midgut as a model to study epithelial cell differentiation from stem cells

A better knowledge of the regulatory mechanisms involved in stem cell proliferation and/or differentiation could reveal new methods for the treatment of some diseases. Most previous studies in the field of stem cell biology have been carried out on cultured isolated cells. In the case of adult tissue stem cells, mesenchymal bone marrow derived cells have been most widely studied, while the undifferentiated stem cells present in the epithelial tissues are less known. In order to advance further our understanding of epithelial tissue stem cells, new in vivo models are required. The present study focuses on the dynamics of a new and simple model of intestinal epithelial regeneration found in the midgut of the migratory locust, Locusta migratoria (Linnaeus 1758). The locust midgut consists of three cell types: columnar cells, endocrine cells and undifferentiated regenerative clustered cells. The undifferentiated epithelial midgut cells give rise to two other cell types and are located in a nest of regenerative cells known as regenerative niche. We have performed single and continuous bromodeoxyuridine (BrdU) administration experiments to study regeneration niches and their cellular dynamics. Immunocytochemistry and immunofluorescence techniques were used to detect the incorporation of BrdU into regenerative niches and the presence of FMRFamide-like immunoreactivity, as a marker for endocrine cell differentiation. Some isolated label retaining cells (LRC) were observed at the niche base 10-15 days after the final BrdU administration. We propose that these cells are the stem cells of this epithelial tissue. We also calculated the length of the cell cycle phases for a subpopulation of transit amplifying cells within the regenerative niche: G1, 2.5+/-0.5 h; S, 5.5+/-0.5 h; G2, 0.75+/-0.25 h; M, 2.5+/-0.5 h. These amplifying cells will give rise to the columnar epithelial non-endocrine lineage. The differentiation of an endocrine cell from a niche stem cell occurs less frequently and thus leads to a lower proportion of endocrine cells as compared with epithelial columnar digestive cells (up to three endocrine cells per niche). Endocrine cell commitment seems to occur very early in the differentiation process within the niche, as double-labelled BrdU and FMRF endocrine cells have never been found. The only exception is the endocrine cells located in the ampullar region of the midgut, some of which show double immunostaining after long-term chronic BrdU injection. In summary, we have characterized a new and simple animal model of epithelial stem cell regeneration that may be useful for understanding the complex biological process that drives tissue renewal from undifferentiated and uncommitted progenitor cells

Adrenomedullin prevents apoptosis in prostate cancer cells

The 52-aminoacid peptide adrenomedullin (AM) is expressed in the normal and malignant prostate. We have previously shown that prostate cancer cells produce and secrete AM, which acts as an autocrine growth inhibitory factor. We have evaluated in the present study the role of AM in prostate cancer cell apoptosis, induced either by serum deprivation or treatment with the chemotherapeutic agent etoposide (which acts as an inhibitor of topoisomerase II). For this purpose we over-expressed AM in PC-3, DU 145 and LNCaP cells, which were transfected with an expression vector carrying AM. We also treated the parental cell lines with synthetic AM in normal culture conditions and in conditions of induced-apoptosis. After serum removal, AM prevented apoptosis in DU 145 and PC-3 cells, but not in LNCaP cells. When treated with etoposide, AM prevented apoptosis in PC-3 and LNCaP cells, but not in DU 145 cells. Cell cycle analysis demonstrated a significant decrease in the percentage of AM-overexpressing PC-3 cells in the subG0/G1 phase after treatment with etoposide, as compared to the percentage of mock-transfected PC-3 treated cells. Western blot showed that protein levels of phosphorylated ERK1/2 increased in parental PC-3 cells after treatment with etoposide. In PC-3 cells overexpressing AM, phosphorylated ERK1/2 basal levels were lower than basal levels of parental PC-3 cells, and treatment with etoposide did not result in such an increase. Etoposide produced a significant increase in cleaved PARP in parental PC-3 cells. However, PC-3 clones overexpressing AM that were treated with etoposide only showed a mild increase in fragmented PARP. The ratio Bcl-2/Bax was reduced in parental or mock-transfected PC-3 cells after treatment with etoposide. On the contrary, this ratio was not reduced in PC-3 clones with AM overexpression that were treated with etoposide. All these data demonstrate that AM plays a protective role against induced apoptosis in prostate cancer cells. These results may have important implications in prostate cancer resistance to chemotherapeutic agents

Effects of food nutrient content, insect age and stage in the feeding cycle on the FMRFamide immunoreactivity of diffuse endocrine cells in the locust gut

We have studied the influence of variations in dietary protein and digestible carbohydrate content, of insect age and of time during the feeding cycle on the endocrine cells of the ampullar region of the midgut in the African migratory locust Locusta migratoria L. Morphometric analysis of FMRFamide-like immunoreactivity was used as an indirect measure of the amount of FMRFamide-related peptides (FaRPs) stored in the gut endocrine cells. There was a highly significant correlation between FaRP content and the nutritional quality of the food, measured relative to the concentrations and ratio of protein to digestible carbohydrate in a nutritionally optimal diet. The direction of the relationship between FaRP content and diet quality varied with age during the fifth stadium. On day 1, FaRP levels increased with the nutritional quality of the food, while on day 4 the opposite relationship was observed. Release of peptide was triggered by the onset of a meal during ad libitum feeding, with cell FaRP levels returning to premeal values within 15 min of the meal ending. The results also suggested that cell contents were released during food deprivation beyond the normal intermeal interval. Locusts switched for a single meal during ad libitum feeding on day 4 from a low- to a high-carbohydrate food did not respond by reducing endocrine cell FaRP content. Our results show a relationship between the diffuse gut endocrine system and feeding and nutrition in locusts. The ampullar endocrine cells are in three-way contact with the midgut luminal contents, with the primary urine from the Malpighian tubules and with the haemolymph. They are thus ideally positioned to play an integrative receptor-secretory function in the regulation of a variety of post-ingestive processes, such as enzyme secretion, absorption, gut motility or nutrient metabolism

A new type of arthropod photoreceptor

A new type of photoreceptor for the phylum Arthropoda, found in the class Collembola (Arthropoda, Hexapoda) is reported. This new light-sensitive structure consists of a pair of interocular vesicles present in the genus Vesicephalus Richards, 1964 and is anatomically related to the cluster of ommatidia. The absence of a lens, the presence of a rabdome in the upper part of the vesicle and the reflection and refraction of light by a hemolymph bubble with incidence to the rhabdomeric structure are the main traits of this new photoreceptor

Adrenomedullin expression in a rat model of acute lung injury induced by hypoxia and LPS

Adrenomedullin (ADM) is upregulated independently by hypoxia and LPS, two key factors in the pathogenesis of acute lung injury (ALI). This study evaluates the expression of ADM in ALI using experimental models combining both stimuli: an in vivo model of rats treated with LPS and acute normobaric hypoxia (9% O2) and an in vitro model of rat lung cell lines cultured with LPS and exposed to hypoxia (1% O2). ADM expression was analyzed by in situ hybridization, Northern blot, Western blot, and RIA analyses. In the rat lung, combination of hypoxia and LPS treatments overcomes ADM induction occurring after each treatment alone. With in situ techniques, the synergistic effect of both stimuli mainly correlates with ADM expression in inflammatory cells within blood vessels and, to a lesser extent, to cells in the lung parenchyma and bronchiolar epithelial cells. In the in vitro model, hypoxia and hypoxia LPS treatments caused a similar strong induction of ADM expression and secretion in epithelial and endothelial cell lines. In alveolar macrophages, however, LPS-induced ADM expression and secretion were further increased by the concomitant exposure to hypoxia, thus paralleling the in vivo response. In conclusion, ADM expression is highly induced in a variety of key lung cell types in this rat model of ALI by combination of hypoxia and LPS, suggesting an essential role for this mediator in this syndrom

Effects of acute hypoxia and lipopolysaccharide on nitric oxide synthase-2 expression in acute lung injury

The potential role of nitric oxide synthase-2 (NOS2) in acute lung injury (ALI) has gained increasing attention. This study evaluates the effects of hypoxia, an important feature of ALI, on NOS2 expression in a rat model of ALI caused by exposure to hypoxia and LPS. Exposure to hypoxia alone had no effect on the expression of NOS2 in rat lungs. LPS treatment resulted in a significant increase in NOS2 in the lungs, which was further enhanced by concomitant exposure to hypoxia. Immunohistochemical analysis and in situ hybridization showed no changes in the expression of NOS2 in lung resident cells under any conditions. The increase in NOS2 levels is mainly due to the influx of NOS2-expressing inflammatory cells. By morphologic analysis, these inflammatory cells were identified as neutrophils, lymphocytes, and monocytes. In vitro experiments of lung epithelial and endothelial cell lines showed no detectable expression of NOS2 with any of the treatments. In a macrophage cell line, LPS-induced NOS2 expression was not affected by the concomitant exposure to hypoxia. In conclusion, LPS increases NOS2 expression in rat lungs through the recruitment of NOS2-producing leukocytes. Simultaneous exposure to LPS and hypoxia results in a greater influx of inflammatory cells that further enhances NOS2 expression

Adrenomedullin inhibits prostate cancer cell proliferation through a cAMP-independent autocrine mechanism

Adrenomedullin (AM) is a multifunctional peptide expressed in the normal and malignant prostate, and in prostate cancer cells. To elucidate the potential role of AM in prostate cancer, we have transfected the human AM gene into PC-3, DU 145, and LNCaP prostate cancer cells. Northern blot, Western blot, and radioimmunoassay techniques confirmed an increase in the synthesis and secretion of the 6kDa mature peptide, in the AM-transfected clones. Proliferation and cell cycle assays demonstrated that AM overexpression inhibited cell proliferation in PC-3 and LNCaP cells through a G0/G1 cell cycle arrest, but not in DU 145 cells. In vivo growth assays also confirmed that, at least in PC-3, AM produced a very significant reduction of tumor volume. In addition, the three cell lines expressed the CL/RCP/RAMP-2 receptor complex by RT-PCR, which suggests that AM peptide acts through an autocrine loop in prostate cancer cells. Although cAMP elevation is the most common pathway involved in AM signalling, stimulation of PC-3, DU 145, and LNCaP with synthetic AM did not increase intracellular cAMP. However, short-term stimulation of PC-3 cells with synthetic AM increased ERK1/2 activation. On the contrary, long-term stimulation, or AM overexpression, caused a reduction in the basal activation of ERK1/2. In summary, our results demonstrate that AM (either overexpressed or exogenously added) causes an inhibition of prostate cancer cell growth. This inhibition does not depend on changes in intracellular cAMP levels, but may be related to ERK1/2 activation

Proadrenomedullin NH(2)-terminal 20 peptide (PAMP) and adrenomedullin bind to teratocarcinoma cells.

Proadrenomedullin NH(2-)terminal 20 peptide (PAMP) and adrenomedullin (ADM) bind to teratocarcinoma cells. The effects of PAMP and ADM on teratocarcinoma cells were investigated. (125)I-PAMP bound to PA1 cells with moderate affinity (K(d) = 110 nM) to a single class of sites (B(max) = 110 000/cell). Specific (125)I-PAMP binding was inhibited by PAMP (IC(50) of 100 nM) but not ADM, calcitonin gene-related peptide (CGRP), or amylin. Specific (125)I-ADM binding was inhibited with high affinity by ADM, CGRP, and CGRP(8-37) (IC(50) values of 10, 10, and 15 nM respectively) but not PAMP or amylin. ADM elevated cAMP (ED(50) value of 100 nM), whereas PAMP had no effect on basal cAMP but inhibited the increase in cAMP caused by 10 nM ADM. Also, the increase in cAMP caused by ADM was inhibited CGRP(8-37), suggesting that ADM is binding to CGRP receptors. ADM (100 nM) stimulated transiently c-fos mRNA, whereas PAMP (1000 nM) had little effect; however, PAMP inhibited the increase in c-fos mRNA caused by ADM. ADM stimulated [(3)H]thymidine uptake into PA1 cells, whereas PAMP inhibited the increase in thymidine uptake caused by ADM. These results indicate that ADM and PAMP are both biologically active in teratocarcinoma cells