Immortalized mouse embryo fibroblasts are resistant to miR-290-induced senescence regardless of p53 status

Immortalized mouse embryo fibroblasts are resistant to miR-290-induced senescence regardless of p53 status. Physiol Genomics 43: 1153-1159, 2011. First published August 16, 2011; doi:10.1152/physiolgenomics.00064.2011.-The prosenescence role of miR-290 and nocodazole has been documented in primary mouse embryo fibroblasts (MEF), while it is not clear whether immortal murine fibroblasts are still responsive to these senescence inducing stimuli. To establish this point, immortal murine fibroblasts with functional (NIH3T3) or nonfunctional p53 (I-MEF) and low levels of miR-290 were tested for their capability to undergo senescence after exposure to either nocodazole or miR-290. Our results clearly indicate that nocodazole induces senescence only in NIH3T3 cells with a functional p53 but not in I-MEF lacking a functional p53. miR-290 overexpression is unable to address any of the tested immortalized clones toward senescence, regardless of the p53 status, suggesting that the prosenescence role of miR-290 is specific for primary but not for immortal murine fibroblasts. Moreover our findings suggest that the mere downregulation of a potential tumor suppressor miRNA in a given cell type does not necessarily imply that it behaves as a tumor suppressor

The analysis of the expression pattern of the intracellular and extracellular miRNAs in prostate tumor cell lines exposed to cytotoxic drugs.

It has been recently discovered that microRNAs are stably expressed in body fluids of several organisms and that the expression patterns in the plasma/serum of cancer patients could represent a diagnostic/predictive tool of the disease. Extracellular miRNAs have been also found in the growth medium of cells in culture and this prompted us to verify whether prostate tumor cell lines release miRNAs specific of prostate tumor patients and whether anticancer drugs affect the expression patterns of the extracellular miRNAs. First of all we determined the expression of either prostate specific (PS-miRNA) or non prostate specific (NPS-miRNA) miRNAs in the growth medium of PC3 and DU-145 cells (prostate metastatic tumor cell lines) in comparison to PNT1A (immortalized prostate cell line). We found that the levels of both the intracellular and extracellular PS-miRNAs were higher in PC3 and DU-145 tumor cell lines in comparison to the immortalized cell line PNT1A and that a positive correlation exists between the intracellular and the extracellular levels suggesting that the release of miRNA in the growth medium may be a manner to maintain the intracellular miRNAs at physiological level. Thereafter, we investigated whether the release of miRNAs is affected in cells upon their exposure to a cytotoxic drug. To address the point, PC-3 and DU-145 cells were exposed to a cytotoxic concentration of either fludarabine (10 μg/mL) or taxotere (30 nM) for 48 hours. At the end of the treatment both the intracellular and extracellular PS-miRNAs and NPS-miRNAs were quantified. Data showed that i) those miRNAs that were up regulated in cells surviving to either fludarabine or taxotere were not released and that ii) the up regulated miRNAs found in fludarabine-or in taxotere-surviving cells were not the same. Overall data indicate for the first time a possible involvement of miRNAs in the survival/resistance of tumor cells to cytotoxic drugs and suggest that the expression pattern of the intracellular and extracellular miRNAs could be an useful tool to identify in tumor cell lines miRNAs responsible of survival/resistance to cytotoxic drugs and in plasma/serum of cancer patients the efficacy of an anticancer treatment

The RNA Activator ds-p21 Potentiates the Cytotoxicity Induced by Fludarabine in Dohh2 Cells

Recently, it has been reported that, in several tumor cell lines, short double-stranded RNAs tailored for promoter regions of specific genes are able to activate their transcription. Such molecules (named RNA activators) act opposite to other double-stranded RNA molecules (named RNA inhibitors) in that the overexpression instead of underexpression of a given gene is triggered. In Dohh2 non-Hodgkin lymphoma cells, the transcriptional repressor BCL6, which negatively controls both p53 and p21, is overexpressed, so that the cells can escape the check point governed by p53 and proliferate. The aim of this work was to investigate whether the RNA activator p21 can represent a tool to circumvent the transcriptional control of BCL6 and induce the blockage of cell proliferation in Dohh2 non-Hodgkin lymphoma cells. For that, Dohh2 cells were transfected with either a control RNA activator (ds-NC) or an RNA activator specific for human p21 promoter (ds-p21). At various time points after transfection, the cells were collected and p21 was measured. Dohh2 cells transfected with ds-p21 showed a slight but significant overexpression of p21 at both mRNA and protein levels. Nonetheless, cell proliferation, cell cycle, and apoptosis were not significantly modified. In contrast, the exposure of Dohh2 cells transfected with ds-p21 to fludarabine potentiates the cytotoxicity of the drug, suggesting the RNA activator p21 complements the fludarabine-dependent cell death pathways

Clinical applications of TSPO PET for glioma imaging: current evidence and future perspective a systematic review

Our aim was to provide a comprehensive overview of the existing literature concerning the clinical applications of positron emission computed tomography (PET) with radiopharmaceuticals targeting the translocator protein (TSPO) in gliomas. A literature search for studies about TSPO PET in the last 10 years (from 2013 to February 2023) was carried out on PubMed, Scopus, and Web of Science using the following keywords: "PET" AND "Gliomas" AND "TSPO". The Critical Appraisal Skills Program checklist for diagnostic test studies was used for testing the quality of selected papers. Ten articles were selected, encompassing 314 glioma patients submitted to PET/CT (9/10) or PET/MRI (1/10) with TSPO ligands. Among the various available TSPO tracers, the most frequently used was the third-generation ligand, [F-18]-GE-180. TSPO PET results were useful to identify anaplastic transformation in gliomas and for the prognostic stratification of patients bearing homogeneous genetic alterations. When compared to amino-acid PET, TSPO PET with [F-18]-GE-180 presented superior image quality and provided larger and only partially overlapping PET-based volumes. Although biased by some issues (i.e., small sample size, most of the studies coming from the same country), preliminary applications of TSPO PET were encouraging. Further studies are needed to define implications in clinical practice and shape the role of TSPO PET for patients' selection for potential TSPO-targeted molecular therapies

Immortalization of MEF is characterized by the deregulation of specific miRNAs with potential tumor suppressor activity

The life span (Hayflick limit) of primary mouse embryo fibroblasts (MEF) in culture is variable but it is still unclear if the escape of the Hayflick limit is also variable. To address this point MEF were expanded every fifteen days (6T15) instead of every three days (6T3) until they became immortal. With this protocol MEF lifespan was extended and immortalization accordingly delayed. By testing a panel of genes (p19ARF, p16, p21) and miRNAs (miR-20a, miR-21, miR-28, miR-290) related to primary MEF senescence, a switch of p21 from up to down regulation, the down regulation of specific miRNAs as well as a massive shift from diploidy to hyperdiploidy were observed in coincidence with the resumption of cell proliferation. Collectively, these data indicate that the inactivation of genes and miRNAs, important in controlling cell proliferation, might be determinant for the escape from the Hayflick limit. In support of this hypothesis was the finding that some of the down regulated miRNAs transfected in immortalized MEF inhibited cell proliferation thus displaying a tumor suppressor-like activity

The miRNA Pull Out Assay as a Method to Validate the miR-28-5p Targets Identified in Other Tumor Contexts in Prostate Cancer

miR-28-5p is an intragenic miRNA which is underexpressed in several tumor types showing a tumor suppressor (TS) activity. Routinely, the known miR-28-5p targets are validated in specific tumor contexts but it is unclear whether these targets are also being regulated in other tumor types. To this end, we adopted the miRNA pull out assay to capture the miR-28-5p targets in DU-145 prostate cancer (PCa) cells. Firstly, we demonstrated that miR-28-5p acts as a TS-miRNA in PCa, affecting cell proliferation, survival, and apoptosis. Secondly, we evaluated the enrichment of the 10 validated miR-28-5p targets in the pull out sample. We showed that E2F6, TEX-261, MAPK1, MPL, N4BP1, and RAP1B but not BAG1, OTUB1, MAD2L1, and p21 were significantly enriched, suggesting that not all the miR-28-5p targets are regulated by this miRNA in PCa. We then verified whether the miR-28-5p-interacting targets were regulated by this miRNA. We selected E2F6, the most enriched target in the pull out sample, and demonstrated that miR-28-5p downregulated E2F6 at the protein level suggesting that our approach was effective. In general terms, these findings support the miRNA pull out assay as a useful method to identify context-specific miRNA targets

The zebrafish/tumor xenograft angiogenesis assay as a tool for screening anti-angiogenic miRNAs

The zebrafish/tumor xenograft angiogenesis assay is used to approach tumor angiogenesis, a pivotal step in cancer progression and target for anti-tumor therapies. Here, we evaluated whether the assay could allow the identification of microRNAs having an anti-angiogenic potential. For that, we transfected DU-145 prostate cancer cells with four microRNAs (miR-125a, miR-320, miR-487b, miR-492) responsive to both anti- and pro-angiogenic stimuli applied to human umbilical vein endothelial cells. After transfection, DU-145 cells were injected close to the developing subintestinal vessels of transgenic Tg(Kdrl:eGFP)s843 zebrafish embryos that express green fluorescent protein under the control of Kdrl promoter. At 72 h post-fertilization, we observed that green fluorescent protein–positive neo-vessels infiltrated the graft of DU-145 transfected with miR-125a, miR-320, and miR-487b. Vice versa, neo-vessel formation and tumor cell infiltration were inhibited when DU-145 cells transfected with miR-492 were used. These results indicated that the zebrafish/tumor xenograft assay was adequate to identify microRNAs able to suppress the release of angiogenic growth factors by angiogenic tumor cells

Visualization of coronary arteries and coronary stents by low dose 320-slice multi-detector computed tomography in a patient with atrial fibrillation

Abstract Cardiac multi-detector computed tomography (MDCT) is widely used in the diagnosis of coronary disease. However, the predictive value of this technique is limited in the presence of atrial fibrillation and coronary stents. Here we present a case showing the ability of the new 320-slice MDCT to assess coronary anatomy in a patient with atrial fibrillation and coronary stents