Effect of prolactin and actinomycin D on gene expression in murine AML12 and Hepa 1-6 hepatocytes
ABSTRACT: Transcriptional profiling of murine hepatocyte gene expression following exposure to prolactin or actinomycin D Prolactin or actinomycin D-treated versus serum-free control cells: AML12 and Hepa 1-6. Biological replicates: 2 per cell line per treatment protocol.
Project description:Primary hyperparathyroidism is a common endocrine disorder frequently affecting postmenopausal women. In this study we have investigated expression of the prolactin receptor (PRLr) in a panel of 37 sporadic parathyroid tumours, as well as functionality in vitro in cultured parathyroid tumour cells. High levels of the prolactin receptor gene (PRLR) transcripts were demonstrated in parathyroid tissues as compared to other reference tissues and breast cancer cells. PRLr products of 60/70 kDa were highly expressed in all parathyroid tumours. In addition varying levels of the 80 kDa PRLr isoform, with known proliferative activity, were demonstrated. In parathyroid tumours PRLr immunoreactivity was observed in cytoplasm in all cases and in addition in the plasma membrane (n = 12) or enlarged lysosomes (n = 4). In normal parathyroid rim PRLr was expressed in cytoplasm and granulae. In in vitro studies of short-term cultured human parathyroid tumour cells prolactin stimulation was associated with transcriptional changes in JAK/STAT, RIG-I like receptor and type II interferon signaling pathways as documented by gene expression profiling. Moreover, PRLR gene expression in parathyroid tumors was significantly inversely correlated with plasma total Ca2+ levels. In conclusion, the prolactin receptor was found highly abundant in human parathyroid gland, parathyroid tumours, correlated with patient Ca2+ levels and functionally responsive to physiological levels of prolactin. These findings suggest a role for the prolactin receptor in human parathyroid adenomas. Expression profiling was done in parathyroid adenomas subjected to prolactin treatment in culture. In addition, corresponding paraffin sections were obtained for verification of PRLr expression by immunohistochemistry. 200 mg/L prolactin (recombinant human prolactin, Cat. No. JM-4687-50, MBL Woburn, MA) was added to 1× 10^6 attached parathyroid tumour cells. Cells were harvested using RNAlater (QIAGEN) and homogenized with QIAshredder for RNA extraction after 3 h and 24 h in culture, respectively. Negative controls were collected in parallel with each case at the same time points. RNA was extracted using QIA Cube, and quality assessed with Bioanalyser and Nanodrop. Expression array profiling and data analysis was done at the KI core facility Bioinformatics and Expression Analysis (BEA, Novum, Huddinge) using the Affymetrix platform and the TITAN ST 1.1 array. A total of 16 samples were analysed including 4 parathyroid adenomas cultured for 3 h or 24 h in the presence of prolactin plus control samples cultured in parallel without prolactin.
Project description:Aim: PROLACTIN (PRL), normally produced by the pituitary gland, acts through its receptor (PRL-R) to initiate a signalling cascade to the genome. PRL can also be produced by cancer cells and become oncogenic by stimulating its receptor following an autocrine or paracrine route. Here we investigated the oncogenic activities of PRL in lung cancer. Results: PRL is ectopically activated in a subset of very aggressive lung tumours, associated with a rapid fatal outcome, in our cohort of 293 lung tumour patients as well as in an external independent series of patients. An investigation of the molecular basis of PRL adverse effects surprisingly showed an absence of PRL-R expression in the vast majority of PRL-expressing lung tumours. Additionally, a detailed analysis of the ectopically expressed PRL transcripts in lung tumours and cell lines revealed systematic alterations of its first exons encoding the signal peptide. Finally, the transcriptomes of two PRL expressing lung cancer cell lines, with or without silencing of PRL, showed that PRL directly or indirectly sustains the expression of a group of genes that are frequently up-regulated in a variety of unrelated cancers. Interestingly, the gene signature repressed by PRL ectopic expression in lung tumour cells is also specifically up-regulated by HDAC inhibitor treatment or HDAC1/2/3 knock-down. Innovation and conclusion: Altogether, this work sheds lights on the poorly understood impact of the recently-shown large-scale out-of-context gene activity in cancer and also suggests that PRL-expressing aggressive lung cancers could be particularly responsive to a HDAC inhibitor-based treatment. Total RNA was extracted from two small cell lung carcinoma (SCLC) cell lines expressing PRL (H146 and h524: si control) or not (siPRL) and the differentially expressed genes. Five replicates were analysed for each of the four conditions.
Project description:The hormone prolactin is implicated in the pathogenesis of breast cancer, and a subset of prolactin-induced gene expression is mediated by HDAC6 activity. The NSAID, bufexamac, is a fairly specific inhibitor of HDAC6 and HDAC10. We used bufexamac in combination with prolactin treatment in MCF7 cells to determine differential expression under these conditions. Overall design: MCF7 cells were serum starved for 24 hours prior to treatment with bufexamac and/or prolactin, and subsequent RNA extraction.
Project description:Analysis of the extent to which inter-individual variation in mRNA decay contributes to inter-individual variation in gene expression levels in humans. The study examines properties of genome-wide decay rates and the relationship between mRNA decay and gene expression across genes, across individuals, and finally across genotype classes. 70 human lymphoblastoid cell lines were treated with Actinomycin D to arrest transcription. Transcript abundance was measured at 5 timepoints: before transcription (0 hours) and after transcription (0.5 hours, 1 hour, 2 hours, and 4 hours). Data was then used to estimate gene-specific decay rates genome-wide.
Project description:Expression data from treatment of actinomycin D (2.5uM) and triptolide (500 nM) on MCF7 cells for 2, 4 and 6 hours. We used microarrays to explore the mechanism of triptolide action. Overall design: MCF7 cells were treated with actinomycin D or triptolide for 2, 4 and 6 hours, RNA was extracted and hybridization on Affymetrix microarrays. Data were normalized by RMA.