High MYC mRNA expression is more clinically relevant than MYC DNA amplification in triple-negative breast cancer

DNA abnormalities are used in inclusion criteria of clinical trials for treatments with specific targeted molecules. MYC is one of the most powerful oncogenes and is known to be associated with triple-negative breast cancer (TNBC). Its DNA amplification is often part of the targeted DNA-sequencing panels under the assumption of reflecting upregulated signaling. However, it remains unclear if MYC DNA amplification is a surrogate of its upregulated signaling. Thus, we investigated the difference between MYC DNA amplification and mRNA high expression in TNBCs utilizing publicly available cohorts. MYC DNA amplified tumors were found to have various mRNA expression levels, suggesting that MYC DNA amplification does not always result in elevated MYC mRNA expression. Compared to other subtypes, both MYC DNA amplification and mRNA high expression were more frequent in the TNBCs. MYC mRNA high expression, but not DNA amplification, was significantly associated with worse overall survival in the TNBCs. The TNBCs with MYC mRNA high expression enriched MYC target genes, cell cycle related genes, and WNT/β-catenin gene sets, whereas none of them were enriched in MYC DNA amplified TNBCs. In conclusion, MYC mRNA high expression, but not DNA amplification, reflects not only its upregulated signaling pathway, but also clinical significance in TNBCs

Increased expression of aggrecan and biglycan mRNA in Achilles tendinopathy

To determine the expression of mRNA encoding the proteoglycans aggrecan, versican, biglycan and decorin in mid-tendon samples of chronic painful Achilles tendinopathy and ruptured Achilles tendons, compared with normal tendons. Total RNA isolated from frozen tendon samples (14 normal, 13 painful, 14 ruptured) was assayed by relative quantitative reverse transcription polymerase chain reaction for aggrecan, versican, biglycan and decorin mRNA, normalized using 18S rRNA. Differences between sample groups were tested by univariate analysis of variance with age as co-variate. In normal tendon samples expression of each of the proteoglycan mRNA decreased with increasing age. Decorin mRNA was the most highly-expressed of the proteoglycan mRNA, while versican mRNA expression was higher (3.8-fold) than that of aggrecan. In painful tendinopathy both aggrecan and biglycan mRNA expression increased (more than 10-fold and 5-fold, respectively) compared with normal tendon samples, but levels of versican and decorin mRNA were not significantly changed. In ruptured tendons the levels of aggrecan, biglycan and versican mRNA were not changed compared with normal tendon samples, but decorin mRNA decreased markedly. Increased aggrecan and biglycan mRNA expression in painful tendinopathy resembles the pattern in fibrocartilaginous regions of tendon, and may reflect an altered mechanical environment at the site of the lesion. Increased aggrecan mRNA expression may underlie the increase in glycosaminoglycan observed in painful tendinopathy

The onset of labor alters corticotropin-releasing hormone type 1 receptor variant expression in human myometrium : putative role of interleukin-1ß

CRH targets the human myometrium during pregnancy. The efficiency of CRH actions is determined by expression of functional receptors (CRH-R), which are dynamically regulated. Studies in myometrial tissue biopsies using quantitative RT-PCR demonstrated that the onset of labor, term or preterm, is associated with a significant 2- to 3-fold increase in CRH-R1 mRNA levels. Detailed analysis of myometrial CRH-R1 mRNA variants showed a decline of the pro-CRH-R1 mRNA encoding the CRH-R1ß variant during labor and increased mRNA levels of CRH-R1d mRNA. Studies in myometrial cells identified IL-1ß as an important regulator of myometrial CRH-R1 gene expression because prolonged treatment of myometrial cells with IL-1ß (1 ng/ml) for 18 h induced expression of CRH-R1 mRNA levels by 1.5- to 2-fold but significantly attenuated CRH-R1ß mRNA expression by 70%. In contrast, IL-1ß had no effect on CRH-R1d mRNA expression. Studies using specific inhibitors suggest that ERK1/2, p38 MAPK, and downstream nuclear translocation of nuclear factor-B mediate IL-1ß effects on myometrial CRH-R1 gene. However, the increased CRH-R1 mRNA expression was associated with a dampening of the receptor efficacy to activate the adenylyl cyclase/cAMP signaling cascade. Thus, our findings suggest that IL-1ß is an important regulator of CRH-R1 expression and functional activity, and this interaction might play a role in the transition of the uterus from quiescence to active contractions necessary for the onset of parturition

Ciprofloxacin enhances the stimulation of matrix metalloproteinase 3 expression by interleukin-1beta in human tendon-derived cells

To determine whether the fluoroquinolone antibiotic ciprofloxacin, which can cause tendon pain and rupture in a proportion of treated patients, affects the expression of matrix metalloproteinases (MMPs) in human tendon-derived cells in culture. Cell cultures were derived from 6 separate tendon explants, and were incubated in 6-well culture plates for 2 periods of 48 hours each, with ciprofloxacin (or DMSO in controls) and interleukin-1ß (IL-1ß), alone and in combination. Samples of supernatant medium from the second 48-hour incubation were assayed for MMPs 1, 2, and 3 by Western blotting. RNA was extracted from the cells and assayed for MMP messenger RNA (mRNA) by semiquantitative reverse transcription–polymerase chain reaction, with normalization for GAPDH mRNA. Unstimulated tendon cells expressed low or undetectable levels of MMP-1 and MMP-3, and substantial levels of MMP-2. IL-1ß induced a substantial output of both MMP-1 and MMP-3 into cell supernatants, reflecting increases (typically 100-fold) in MMP mRNA, but had only minor effects on MMP-2 expression. Ciprofloxacin had no detectable effect on MMP output in unstimulated cells. Preincubation with ciprofloxacin potentiated IL-1ß–stimulated MMP-3 output, reflecting a similar effect on MMP-3 mRNA expression. Ciprofloxacin also potentiated IL-1ß–stimulated MMP-1 mRNA expression, but did not potentiate the output of MMP-1, and had no significant effects on MMP-2 mRNA expression or output. Ciprofloxacin can selectively enhance MMP expression in tendon-derived cells. Such effects might compromise tendon microstructure and integrity

In vivo effect of dried chicory root (Cichorium intybus L.) on xenobiotica metabolising cytochrome P450 enzymes in porcine liver

Cytochrome P450 (CYP) enzymes are widely studied for their involvement in metabolism of drugs and endogenous compounds. In porcine liver, CYP1A2,2Aand 2E1 are important for the metabolism of skatole.Feeding chicory roots to pigs is known to decrease the skatole concentration in plasma and fat. In the present study we investigated the effect of chicory on CYP mRNA and protein expression, as well as their activity. Male pigs were feed dried chicory root for 16 days before liver samples were collected. By the use of RT-PCR and Western blotting we showed that the mRNA and protein expression of CYP1A2 and 2A were increased in chicory fed pigs. The mRNA expression of CYP2E1 was increased, while there was no effect on protein expression. Activity of CYP1A2 and 2A were increased in chicory feed pigs; this was not the case for CYP2E1 activity. In conclusion; oral administration of chicory root for 16 days to pigs increased the mRNA expression of CYP1A2, 2A and 2E1; and the protein expression of CYP1A2 and 2A. The activities of CYP1A2 and 2A were increased

Characterization of serotonin transporter expression in human T lymphocytes

Serotonin transporter (SERT) expression has been demonstrated in human lymphocytes, including B lymphocytes, NK cells and other immune cells. However, discussion remains on whether human T lymphocytes express SERT. Given the potentially important role of serotonin in lymphocyte activation and proliferation, we investigated SERT expression in purified human T lymphocytes both in resting and activated state. Blood samples were collected from 9 healthy volunteers. PBMCs were isolated using Ficoll density centrifugation and T lymphocytes were further purified with magnetic activated cell sorting. T cells were either processed for mRNA and protein isolation immediately, or activated using anti-CD3/CD28 coated magnetic beads and allowed to proliferate for 72h at 37°C and 5% CO2. SERT mRNA expression was measured using droplet digital PCR to allow for increased sensitivity in comparison with qRT-PCR. SERT protein was detected on western blot. SERT expression was detected both on mRNA and protein level, although expression levels were very low. On mRNA level, SERT was expressed in both resting and activated cells. On the protein level however, only activated cells displayed SERT expression. This observation might point to a ‘translational readiness’ were resting T lymphocytes already produce SERT mRNA, but translation is only induced after activation of the cell

Versican splice variant messenger RNA expression in normal human Achilles tendon and tendinopathies

Versican is the principal large proteoglycan expressed in mid-tendon, but its role in tendon pathology is unknown. Our objective was to define the expression of versican isoform splice variant messenger ribonucleic acid (mRNA) in normal Achilles tendons, in chronic painful tendinopathy and in ruptured tendons. Total RNA isolated from frozen tendon samples (normal n = 14; chronic painful tendinopathy n = 10; ruptured n = 8) was assayed by relative quantitative reverse transcriptase polymerase chain reaction (RT-PCR) for total versican, versican variants V0, V1, V2, V3 and type I collagen a1 mRNA, normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Differences between sample groups were tested by Wilcoxon statistics. Painful and ruptured tendons showed a significant decrease (median 2-fold) in the expression of versican mRNA, in contrast to an increased expression (median 8-fold) of type I collagen a1 mRNA in painful tendons. Versican splice variants V0 and V1 mRNA were readily detected in normal samples, V3 levels were substantially lower, and V2 levels were more variable. Each of V1, V2 and V3 mRNA showed significant decreases in expression in painful and ruptured tendons, but V0 was not significantly changed. Changes in versican expression relative to that of collagen, and alterations in the balance of versican splice variants, may contribute to changes in matrix structure and function in tendinopathies

Distribution of α7 Nicotinic Acetylcholine Receptor Subunit mRNA in the Developing Mouse.

Homomeric α7 nicotinic acetylcholine receptors (nAChRs) are abundantly expressed in the central and peripheral nervous system (CNS and PNS, respectively), and spinal cord. In addition, expression and functional responses have been reported in non-neuronal tissue. In the nervous system, α7 nAChR subunit expression appears early during embryonic development and is often transiently upregulated, but little is known about their prenatal expression outside of the nervous system. For understanding potential short-term and long-term effects of gestational nicotine exposure, it is important to know the temporal and spatial expression of α7 nAChRs throughout the body. To that end, we studied the expression of α7 nAChR subunit mRNA using highly sensitive isotopic in situ hybridization in embryonic and neonatal whole-body mouse sections starting at gestational day 13. The results revealed expression of α7 mRNA as early as embryonic day 13 in the PNS, including dorsal root ganglia, parasympathetic and sympathetic ganglia, with the strongest expression in the superior cervical ganglion, and low to moderate levels were detected in brain and spinal cord, respectively, which rapidly increased in intensity with embryonic age. In addition, robust α7 mRNA expression was detected in the adrenal medulla, and low to moderate expression in selected peripheral tissues during embryonic development, potentially related to cells derived from the neural crest. Little or no mRNA expression was detected in thymus or spleen, sites of immune cell maturation. The results suggest that prenatal nicotine exposure could potentially affect the nervous system with limited effects in non-neural tissues

Expression of plasma prekallikrein mRNA in human nonhepatic tissues and cell lineages suggests special local functions of the enzyme

At present it is generally accepted that plasma prekallikrein (PPK) is synthesized in the liver and secreted into the bloodstream. Surprisingly, it has recently been shown that PPK mRNA is present also in RNA from the kidney, adrenal gland and placenta. In spite of its novelty and possible important physiological implications this finding has been neglected. Here we report that PPK mRNA is expressed also in the human brain, heart, lung, trachea, endothelial cells and leukocytes as well as in a variety of fibroblast and epithelial cell lines. Expression of PPK mRNA in fibroblasts, endothelial cells and leukocytes suggests that PPK mRNA detected in RNA preparations from whole tissue may originate solely from these ubiquitously occurring cells. However, PPK mRNA expression in various epithelial cell lines demonstrates that tissue-specific cells also transcribe the PPK gene. The presence of PPK mRNA in nonhepatic tissues and cells indicates that they have the capacity to synthesize the protein. The physiological role of PPK synthesized in extrahepatic tissue is unknown. It may participate in local actions within tissues as well as contributing to the PPK pool in blood plasma. Cultured cells will provide a valuable model for exploring the physiological significance of extrahepatic PPK expression

Differential regulation of anti-inflammatory genes by p38 MAP kinase and MAP kinase kinase 6.

BackgroundConventional p38α inhibitors have limited efficacy in rheumatoid arthritis, possibly because p38 blockade suppresses the counter-regulatory mechanisms that limit inflammation. In contrast, targeting the upstream MAP kinase kinases, MKK3 and MKK6, partially maintains p38-mediated anti-inflammatory responses in bone marrow-derived macrophages (BMDM). In this study, we explored the mechanisms that preserve anti-inflammatory gene expression by evaluating differential regulation of IL-10 and p38-dependent anti-inflammatory genes in MKK3-/-, MKK6-/-, and p38 inhibitor-treated wildtype cells.MethodsBMDM from wild type (WT), MKK3-/-, and MKK6-/- mice were pre-treated with p38 inhibitor SB203580 (SB), JNK inhibitor SP600125 (SP), and/or ERK inhibitor PD98059 (PD) and stimulated with LPS. Supernatant protein levels were measured by multiplex bead immunoassay. mRNA expression was determined by qPCR and protein expression by Western blot analysis. De novo IL-10 mRNA synthesis was quantified in cells treated with ethynyl-uridine and LPS followed by reverse transcription and qPCR. mRNA half-life was measured in LPS-treated cells that were then incubated with actinomycin D ± SB203580.ResultsPre-treatment of WT BMDM with p38 inhibitor significantly reduced IL-10 production in the three groups, while ERK and JNK inhibitors had minimal effects. IL-10 production was significantly decreased in MKK3-/- BMDM compared with either WT or MKK6-/- cells. IL-10 mRNA expression was modestly reduced in MKK3-/- BMDM but was preserved in MKK6-/- cells compared with WT. De novo IL-10 mRNA synthesis was inhibited in MKK3-/- and p38 inhibitor pre-treated cells, but not MKK6-/- cells compared with WT. IL-10 mRNA half-life was markedly reduced in p38 inhibitor-treated WT cells while MKK-deficiency had minimal effect. DUSP1 mRNA levels were preserved in MKK-deficient cells but not in p38 inhibitor-treated WT cells. Tristetraprolin mRNA and protein levels were reduced in p38 inhibitor-treated WT cells compared with MKK6-/- cells.ConclusionUnlike p38-inhibition, the absence of MKK6 mostly preserves IL-10 and TTP protein expression in BMDM. MKK6-deficiency also spares DUSP1 and IL-1RA, which are key negative regulators of the inflammatory response. Together, these data suggest that MKK6 is a potential therapeutic target in RA