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

A simple procedure to calibrate soil parameters for slope stability modelling: the Langhe (1994) case study

Shallow landslides triggered by rainfall represent common geotechnical hazards in Italy. In this context, the hundreds of landslides which occurred in the Piedmont Langhe area in November 1994, still identified among the most extensive areal event in the last 30 years in Italy, are investigated. Exploiting data from the surveys of Campus et al. (1998), here we calibrate simple soil water and mechanical properties (i.e., the saturated permeability and cohesion values) to overcome the large uncertainty affecting the determination of parameters to be used as inputs for physically based soil slip models. This work aims to contribute to the development of reliable soil data inventory that may be of direct interest in slope stability modelling, following Vannocci et al. (2020). The analysis was conducted on a small number of cases selected from a sample of 238 observed landslides, to which geometries and geotechnical features were attributed from a regional database. The calibration was performed using a simple hydrological model, i.e. that of Rosso et al. (2006), since it allows a reasonable check on the sensitivity of soil parameter values to the instability conditions. For saturated conductivity, a variation range was obtained, whose upper limit referred to the so-called bucket model. Assuming that part of the rainfall contributes to surface runoff, a lower permeability value was derived using the proportional flow method ψ, consistently with the real dynamics of the processes. Soil cohesion was calibrated by mechanical analysis based on the infinite slope theory, by targeting the Safety Factor SF to assume the value 0.99. When comparing locally calibrated parameters and the reference ones found in the database some differences arise; in particular, in several cases, based on calibrated values, SFs quite lower than 1 were derived. It must be pointed out that the calibration procedure allowed us to characterize shallow soils, made up of remolded and often vegetated soil, while the regional dataset provide information on undisturbed soil samples, typically collected at depths greater than those of interest. The possibility of getting reliable soil parameters to be used in physically based modelling of shallow landslides is a complex task. Here we use a calibration method to obtain meaningful saturated hydraulic conductivity and cohesion values, compatible with the observed instabilities. The implications of the differences found between the calibrated parameters and those published in the regional dataset will lay the foundations for subsequent investigations, as this analysis will be part of the research framework of the RETURN Extended Partnership Project

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

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

The miR-28-5p Targetome Discovery Identified SREBF2 as One of the Mediators of the miR-28-5p Tumor Suppressor Activity in Prostate Cancer Cells

miR-28-5p is downregulated in some tumor tissues in which it has been demonstrated to have tumor suppressor (TS) activity. Here, we demonstrate that miR-28-5p acts as a TS in prostate cancer (PCa) cells affecting cell proliferation/survival, as well as migration and invasion. Using the miRNA pull out assay and next generation sequencing, we collected the complete repertoire of miR-28-5p targets, obtaining a data set (miR-28-5p targetome) of 191 mRNAs. Filtering the targetome with TargetScan 7, PITA and RNA22, we found that 61% of the transcripts had miR-28-5p binding sites. To assign a functional value to the captured transcripts, we grouped the miR-28-5p targets into gene families with annotated function and showed that six transcripts belong to the transcription factor category. Among them we selected SREBF2, a gene with an important role in PCa. We validated miR-28-5p/SREBF2 interaction, demonstrating that SREBF2 inhibition affects almost all the tumor processes altered by miR-28-5p re-expression, suggesting that SREBF2 is an important mediator of miR-28-5p TS activity. Our findings support the identification of the targetome of cancer-related miRNAs as a tool to discover genes and pathways fundamental for tumor development, and potential new targets for anti-tumor therapy

Factors influencing choice of chemotherapy in metastatic colorectal cancer (mCRC)

Management of metastatic colorectal cancer requires a multimodal approach and must be performed by an experienced, multidisciplinary expert team. The optimal choice of the individual treatment modality, according to disease localization and extent, tumor biology, and patient clinical characteristics, will be one that can maintain quality of life and long-term survival, and even cure selected patients. This review is an overview of the different therapeutic approaches available in metastatic colorectal cancer, for the purpose of defining personalized therapeutic algorithms according to tumor biology and patient clinical features