Reevaluation of the proposed autocrine proliferative function of prolactin in breast cancer

The pituitary hormone prolactin (PRL) has been implicated in tumourigenesis. Expression of PRL and its receptor (PRLR) was reported in human breast epithelium and breast cancer cells. It was suggested that PRL may act as an autocrine/paracrine growth factor. Here, we addressed the role of locally synthesised PRL in breast cancer. We analysed the expression of PRL in human breast cancer tumours using qPCR analysis and in situ hybridization (ISH). PRL mRNA expression was very low or undetectable in the majority of samples in three cDNA arrays representing samples from 144 breast cancer patients and in 13 of 14 breast cancer cell lines when analysed by qPCR. In accordance, PRL expression did not reach detectable levels in any of the 19 human breast carcinomas or 5 cell lines, which were analysed using a validated ISH protocol. Two T47D-derived breast cancer cell lines were stably transfected with PRL-expressing constructs. Conditioned medium from the T47D/PRL clones promoted proliferation of lactogen-dependent Nb2 cells and control T47D cells. Surprisingly, the PRL-producing clones themselves displayed a lower proliferation rate as compared to the control cells. Their PRLR protein level was reduced and the cells were no longer responsive to exogenous recombinant PRL. Taken together, these data strongly indicate that autocrine PRL signalling is unlikely to be a general mechanism promoting tumour growth in breast cancer patients. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s10549-013-2731-7) contains supplementary material, which is available to authorized users

Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)1.

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field

Parvalbumin expression during developmental differentiation of the rat ovary

Parvalbumin (PV) is a high-affinity Ca2+-binding protein which is expressed in a limited number of vertebrates tissues and restricted to a few distinct cell types. It has been shown by biochemical methods to be present in the adult rat ovary, but cellular localizations or developmental appearance have not been described until now. This study describes the presence of PV and its transcripts, analyzed by immunohistochemistry and in situ hybridization, respectively, during the postnatal development of the rat ovary: 13 developmental stages between day 1 and day 83 were examined. In ovaries 1-16 days old, neither PV mRNA nor PV was observed in any cell of the ovary. By contrast, starting from day 18 postpartum, both PV mRNA and PV were detected in low amounts, simultaneously with the onset of differentiation of secondary interstitial gland cells in the ovarian interfollicular stroma. PV and its transcripts were primarily detected in conspicuous patches of interstitial gland tissue and in the differentiated thecal cells around the large follicles, and PV appeared to be fully expressed 33 days after birth. The parallel time courses of PV mRNA and PV accumulation during developmental differentiation, and the appearance of a steroid-producing cellular phenotype as well as the strict cellular colocalization of these two features, strongly suggest involvement of PV in the steroid metabolism of these cells, as earlier proposed for the Leydig cells of the testis. According to this hypothesis, we also show that suppression of gonadotrophic hormone production by hypophysectomy of adult rats totally suppresses PV production in parallel with the disappearance of the morphological features typical of steroid-producing cells in the remaining interstitial tissue of the ovary.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

Properties of the co-chaperone protein p23 erroneously attributed to ALG-2 (apoptosis linked gene 2)

AbstractA commercial antibody (clone 22) directed against the apoptosis-linked gene 2 (alg2, pdcd6) encoded protein has been used by several groups. Up-regulated expression of the antigen was observed in primary tumours and in metastatic tissue and also during rat brain ischemia. Furthermore, antigen down-regulation was found in human atherosclerotic plaques. Recently, we found that the clone 22 antibody does not recognise ALG-2. In the present study the antigen of the clone 22 antibody was identified as the heat shock protein 90 (HSP90) co-chaperone protein p23, identical to the cytosolic prostaglandin E2 synthase, by immunoprecipitation followed by tryptic in-gel digests and mass spectrometry of the purified peptides. Moreover, the heterogeneous ribonuclear protein A2/B1 was found to be a part of the p23 co-immunoprecipitated protein complex