Publications by authors named "Antonio Sorrentino"

29 Publications

  • Page 1 of 1

Novel insights into the disease transcriptome of human diabetic glomeruli and tubulointerstitium.

Nephrol Dial Transplant 2020 12;35(12):2059-2072

Department of Clinical Science, Intervention and Technology, Division of Renal Medicine, Karolinska Institutet, Stockholm, Sweden.

Background: Diabetic nephropathy (DN) is the most common cause of end-stage renal disease, affecting ∼30% of the rapidly growing diabetic population, and strongly associated with cardiovascular risk. Despite this, the molecular mechanisms of disease remain unknown.

Methods: RNA sequencing (RNAseq) was performed on paired, micro-dissected glomerular and tubulointerstitial tissue from patients diagnosed with DN [n = 19, 15 males, median (range) age: 61 (30-85) years, chronic kidney disease stages 1-4] and living kidney donors [n = 20, 12 males, median (range) age: 56 (30-70) years].

Results: Principal component analysis showed a clear separation between glomeruli and tubulointerstitium transcriptomes. Differential expression analysis identified 1550 and 4530 differentially expressed genes, respectively (adjusted P < 0.01). Gene ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analyses highlighted activation of inflammation and extracellular matrix (ECM) organization pathways in glomeruli, and immune and apoptosis pathways in tubulointerstitium of DN patients. Specific gene modules were associated with renal function in weighted gene co-expression network analysis. Increased messengerRNA (mRNA) expression of renal damage markers lipocalin 2 (LCN) and hepatitis A virus cellular receptor1 (HAVCR1) in the tubulointerstitial fraction was observed alongside higher urinary concentrations of the corresponding proteins neutrophil gelatinase-associated lipocalin (NGAL) and kidney injury molecule-1 (KIM-1) in DN patients.

Conclusions: Here we present the first RNAseq experiment performed on paired glomerular and tubulointerstitial samples from DN patients. We show that prominent disease-specific changes occur in both compartments, including relevant cellular processes such as reorganization of ECM and inflammation (glomeruli) as well as apoptosis (tubulointerstitium). The results emphasize the potential of utilizing high-throughput transcriptomics to decipher disease pathways and treatment targets in this high-risk patient population.
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http://dx.doi.org/10.1093/ndt/gfaa121DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7716805PMC
December 2020

CAMK1D Triggers Immune Resistance of Human Tumor Cells Refractory to Anti-PD-L1 Treatment.

Cancer Immunol Res 2020 09 14;8(9):1163-1179. Epub 2020 Jul 14.

Regensburg Center for Interventional Immunology (RCI), University Regensburg, Regensburg, Germany.

The success of cancer immunotherapy is limited by resistance to immune checkpoint blockade. We therefore conducted a genetic screen to identify genes that mediated resistance against CTLs in anti-PD-L1 treatment-refractory human tumors. Using PD-L1-positive multiple myeloma cells cocultured with tumor-reactive bone marrow-infiltrating CTL as a model, we identified calcium/calmodulin-dependent protein kinase 1D (CAMK1D) as a key modulator of tumor-intrinsic immune resistance. CAMK1D was coexpressed with PD-L1 in anti-PD-L1/PD-1 treatment-refractory cancer types and correlated with poor prognosis in these tumors. CAMK1D was activated by CTL through Fas-receptor stimulation, which led to CAMK1D binding to and phosphorylating caspase-3, -6, and -7, inhibiting their activation and function. Consistently, CAMK1D mediated immune resistance of murine colorectal cancer cells The pharmacologic inhibition of CAMK1D, on the other hand, restored the sensitivity toward Fas-ligand treatment in multiple myeloma and uveal melanoma cells Thus, rapid inhibition of the terminal apoptotic cascade by CAMK1D expressed in anti-PD-L1-refractory tumors via T-cell recognition may have contributed to tumor immune resistance.
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http://dx.doi.org/10.1158/2326-6066.CIR-19-0608DOI Listing
September 2020

A Guide to Concentration Alternating Frequency Response Analysis of Fuel Cells.

J Vis Exp 2019 12 11(154). Epub 2019 Dec 11.

Max Planck Institute for Dynamics of Complex Technical Systems;

An experimental setup capable of generating a periodic concentration input perturbation of oxygen was used to perform concentration-alternating frequency response analysis (cFRA) on proton-exchange membrane (PEM) fuel cells. During cFRA experiments, the modulated concentration feed was sent to the cathode of the cell at different frequencies. The electric response, which can be cell potential or current depending on the control applied on the cell, was registered in order to formulate a frequency response transfer function. Unlike traditional electrochemical impedance spectroscopy (EIS), the novel cFRA methodology makes it possible to separate the contribution of different mass transport phenomena from the kinetic charge transfer processes in the frequency response spectra of the cell. Moreover, cFRA is able to differentiate between varying humidification states of the cathode. In this protocol, the focus is on the detailed description of the procedure to perform cFRA experiments. The most critical steps of the measurements and future improvements to the technique are discussed.
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http://dx.doi.org/10.3791/60129DOI Listing
December 2019

The Role of BRAF-Targeted Therapy for Advanced Melanoma in the Immunotherapy Era.

Curr Oncol Rep 2019 07 29;21(9):76. Epub 2019 Jul 29.

Unit of Melanoma, Cancer Immunotherapy and Development Therapeutics, Istituto Nazionale Tumori IRCCS Fondazione G. Pascale, Via Mariano Semmola snc, Naples, Italy.

Purpose Of Review: The treatment of advanced melanoma has changed dramatically in recent years with several new drugs having been approved for the treatment of melanoma since 2011. This review aims to evaluate the role of BRAF-targeted therapy for advanced melanoma in the immunotherapy era.

Recent Findings: Currently, in patients with BRAF wild-type advanced melanoma, anti-PD-1 (nivolumab or pembrolizumab) is the main treatment. The combination of nivolumab and ipilimumab (anti-CTLA-4) is also an important option for these patients, resulting in a better outcome, but with less favorable toxicity profile. In patients with BRAF mutations, three regimens of BRAF plus MEK inhibitors are now approved (vemurafenib plus cobimetinib, dabrafenib plus trametinib, and encorafenib plus binimetinib), which achieve rapid antitumor responses and a significant survival benefit. In these patients, as well as in BRAF wild-type patients, immunotherapy can be also effective and is regularly used. Immunotherapy and targeted therapy have become the new standards of care, substantially improving survival rates. However, many questions still remain unanswered, such as what is the best first- and second-line treatment and the best treatment sequence. New combinations of drugs, targeted therapy combined with immunotherapy, and sequencing approaches are now underway in many ongoing clinical trials.
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http://dx.doi.org/10.1007/s11912-019-0827-xDOI Listing
July 2019

An alternative to plaster cast treatment in a pediatric trauma center using the CAD/CAM technology to manufacture customized three-dimensional-printed orthoses in a totally hospital context: a feasibility study.

J Pediatr Orthop B 2019 May;28(3):248-255

Santobono-Pausilipon Foundation, Naples.

The aim of this study is to implement the clinical use of the three-dimensional (3D) design and printing technology in pediatric pathologies requiring immobilization. We describe the manufacturing process of the 3D device in place of the plaster cast usually applied to a child 48/72 h after the access to the Trauma Center Traumatology Hub. This procedure had already been performed at Level II, Trauma Center, Campania Region, Orthopaedic Division of Santobono Children's Hospital, Naples, Italy. The operative phase was performed by two 3D printers and a scanner in the bioengineering laboratory of the hospital's outpatient area. The phase of software elaboration requires close cooperation among physicians and engineers. We decided to use a model with a double-shell design and holes varying in width to ensure complete ventilation and lightness of the device. We chose to treat nondisplaced metaphyseal distal fractures of the radius in 18 patients enrolled from January 2017 to November 2017. The flow chart includes clinical and radiological examinations of every enrolled child, collecting information required by the program and its elaboration by bioengineers, and then transfer of the results to 3D printers. The child, immobilized by a temporary splint, wore his 3D device after 12/24 h. Then, he underwent serial check-ups in which the effectiveness and appropriateness of the treatment were clinically monitored and evaluated using subjective scales: visual analogue scale and patient-rated wrist evaluation. All the fractures consolidated both radiologically and clinically after the treatment, with no complications reported. Only one partial breakage of the device happened because of an accidental fall. The statistical analysis of the visual analogue scale and patient-rated wrist evaluation data shows that children's activities of everyday life improved during the immobilization thanks to this treatment. This first study shows that using a 3D device instead of a traditional plaster cast can be an effective alternative approach in the treatment of pediatric nondisplaced metaphyseal distal radius fractures, with high overall patient satisfaction. We believe that 3D technology could be extended to the treatment of more complex fractures; this will be the subject of our second study.
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http://dx.doi.org/10.1097/BPB.0000000000000589DOI Listing
May 2019

Increased PD-L1 expression in radioresistant HNSCC cell lines after irradiation affects cell proliferation due to inactivation of GSK-3beta.

Oncotarget 2019 Jan 15;10(5):573-583. Epub 2019 Jan 15.

Department of Oral and Maxillofacial Surgery, University Hospital Regensburg, Regensburg, Germany.

At present, targeting PD-1/PD-L1 axis for immune checkpoint inhibition has improved treatment of various tumor entities, including head and neck squamous cell carcinoma (HNSCC). However, one part of the patient cohort still shows little improvement or even hyperprogression. We established three radioresistant (RR) and three radiosensitive (RS) HNSCC cell lines. RR cells showed prolonged survival as well as delayed and diminished apoptosis after irradiation with vimentin expression but no E-cadherin expression, whereas RS cell lines died early and exhibited early apoptosis after irradiation and high vimentin expression. Here, we present results demonstrating differential basal PD-L1 gene and protein expression in RR and RS HNSCC cell lines. Moreover, we observed a radiation dose dependent increase of total PD-L1 protein expression in RR cell lines up to 96h after irradiation compared to non-irradiated (non-IRR) cells. We found a significant GSK-3beta phosphorylation, resulting in an inactivation, after irradiation of RR cell lines. Co-immunoprecipitation experiments revealed decreased interaction of GSK-3beta with PD-L1 in non-IRR compared to irradiated (IRR) RR cells leading to PD-L1 stabilization in RR cells. PD-L1 knockdown in RR cells showed a strong decrease in cell survival. In summary, our results suggest an irradiation dependent increase in basal PD-L1 expression in RR HNSCC cell lines via GSK-3beta inactivation.
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http://dx.doi.org/10.18632/oncotarget.26542DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6355177PMC
January 2019

Cell engineering with microfluidic squeezing preserves functionality of primary immune cells in vivo.

Proc Natl Acad Sci U S A 2018 11 31;115(46):E10907-E10914. Epub 2018 Oct 31.

SQZ Biotechnologies, Watertown, MA 02472.

The translational potential of cell-based therapies is often limited by complications related to effectively engineering and manufacturing functional cells. While the use of electroporation is widespread, the impact of electroporation on cell state and function has yet to be fully characterized. Here, we use a genome-wide approach to study optimized electroporation treatment and identify striking disruptions in the expression profiles of key functional transcripts of human T cells. These genetic disruptions result in concomitant perturbation of cytokine secretion including a 648-fold increase in IL-2 secretion ( < 0.01) and a 30-fold increase in IFN-γ secretion ( < 0.05). Ultimately, the effects at the transcript and protein level resulted in functional deficiencies in vivo, with electroporated T cells failing to demonstrate sustained antigen-specific effector responses when subjected to immunological challenge. In contrast, cells subjected to a mechanical membrane disruption-based delivery mechanism, cell squeezing, had minimal aberrant transcriptional responses [0% of filtered genes misregulated, false discovery rate (FDR) q < 0.1] relative to electroporation (17% of genes misregulated, FDR q < 0.1) and showed undiminished effector responses, homing capabilities, and therapeutic potential in vivo. In a direct comparison of functionality, T cells edited for PD-1 via electroporation failed to distinguish from untreated controls in a therapeutic tumor model, while T cells edited with similar efficiency via cell squeezing demonstrated the expected tumor-killing advantage. This work demonstrates that the delivery mechanism used to insert biomolecules affects functionality and warrants further study.
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http://dx.doi.org/10.1073/pnas.1809671115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6243275PMC
November 2018

Effect of miR-204&211 and RUNX2 control on the fate of human mesenchymal stromal cells.

Regen Med Res 2017 5;5. Epub 2017 Dec 5.

Department of Oncology and Molecular Medicine, Istituto Superiore di Sanità, Rome, Italy - Sbarro Institute for Cancer Research and Molecular Medicine & Center of Biotechnology, College of Science and Technology, Temple University, Philadelphia, Pennsylvania.

MiR-204 and 211 enforced expression in murine mesenchymal stromal cells (MSCs) has been shown to induce adipogenesis and impair osteogenesis, through RUNX2 down-modulation. This mechanism has been suggested to play a role in osteoporosis associated with obesity. However, two further fundamental MSC functions, chondrogenesis and hematopoietic supporting activity, have not yet been explored. To this end, we transduced, by a lenti-viral vector, miR-204 and 211 in a model primary human MSC line, opportunely chosen among our MSC collection for displaying all properties of canonical bone marrow MSCs, except adipogenesis. Enforced expression of miR-204&211 in these cells, rescued adipogenesis, and inhibited osteogenesis, as previously reported in murine MSCs, but, surprisingly, also damaged cartilage formation and hematopoietic supporting activity, which were never explored before. RUNX2 has been previously indicated as the target of miR-204&211, whose down modulation is responsible for the switch from osteogenesis to adipogenesis. However, the additional disruption of chondrogenesis and hematopoietic supporting activity, which we report here, might depend on diverse miR-204&211 targets. To investigate this hypothesis, permanent RUNX2 knock-down was performed. Sh-RUNX2 fully reproduced the phenotypes induced by miR-204&211, confirming that RUNX2 down modulation is the major event leading to the reported functional modification on our MSCs. It seems thus apparent that RUNX2, a recognized master gene for osteogenesis, might rule all four MSC commitment and differentiation processes. Hence, the formerly reported role of miR204&211 and RUNX2 in osteoporosis and obesity, coupled with our novel observation showing inhibition of cartilage differentiation and hematopoietic support, strikingly resemble the clinical traits of metabolic syndrome, where osteoarthritis, osteoporosis, anaemia and obesity occur together. Our observations, corroborating and extending previous observations, suggest that miR-204&211-RUNX2 axis in human MSCs is possibly involved in the pathogenesis of this rapidly growing disease in industrialized countries, for possible therapeutic intervention to regenerate former homeostasis.
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http://dx.doi.org/10.1051/rmr/170004DOI Listing
December 2017

Differential expression of plasma miRNAs in patients with unprovoked venous thromboembolism and healthy control individuals.

Thromb Res 2015 Sep 15;136(3):566-72. Epub 2015 Jul 15.

K. G. Jebsen - Thrombosis Research and Expertise Center (TREC), Department of Clinical Medicine, UiT - The Arctic University of Norway, Tromsø, Norway; Division of Internal Medicine, University Hospital of North Norway, Tromsø, Norway.

Background: Venous thromboembolism (VTE) remains the third most common cardiovascular disease with a vague pathogenesis. Circulating miRNAs are small regulatory RNAs found in plasma, serum and other body fluids in an apparently stable form. Although circulating miRNAs, a novel family of regulatory molecules, emerge as a promising class of biomarkers in many cardiovascular diseases and malignancies, knowledge on plasma miRNA levels in VTE remains sparse.

Aims: The present work was conducted as a pilot study in order to estimate the plasma levels of miRNAs in patients with unprovoked VTE and to assess miRNAs as potential novel biomarkers of VTE.

Methods: Twenty patients with a history of unprovoked VTE 1-5 years prior to inclusion in the study and twenty age- and sex-matched healthy control participants were enrolled in a case-control study (Tromsø IV). Plasma levels of 742 miRNAs were assessed after RNA extraction and reverse transcription. Profiling of miRNA was conducted on the Universal RT microRNA PCR Human panels I and II (Exiqon, Denmark). For normalization of the data, the average of the assays detected in all samples (n=40 samples) was applied.

Results: Ninety-seven miRNAs were detected throughout all samples. Of these, miR-10b-5p, -320a, -320b, -424-5p, and -423-5p were upregulated, whereas miR-103a-3p, -191-5p, -301a-3p, and 199b-3p were downregulated in plasmas of VTE patients versus controls (P≤0.05). These miRNAs were confined to the extracellular vesicles-depleted plasma fraction, and yielded clear clustering distinguishing samples from the VTE and control groups.

Conclusions: The results of this pilot study indicate that plasma miRNAs profiling can provide novel biomarkers of unprovoked VTE.
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http://dx.doi.org/10.1016/j.thromres.2015.07.005DOI Listing
September 2015

Activity-Based Protein Profiling of Oncogene-Driven Changes in Metabolism Reveals Broad Dysregulation of PAFAH1B2 and 1B3 in Cancer.

ACS Chem Biol 2015 Jul 7;10(7):1624-30. Epub 2015 May 7.

†Program in Metabolic Biology, Department of Nutritional Sciences and Toxicology, University of California, Berkeley, 127 Morgan Hall, Berkeley, California 94720, United States.

Targeting dysregulated metabolic pathways is a promising therapeutic strategy for eradicating cancer. Understanding how frequently altered oncogenes regulate metabolic enzyme targets would be useful in identifying both broad-spectrum and targeted metabolic therapies for cancer. Here, we used activity-based protein profiling to identify serine hydrolase activities that were consistently upregulated by various human oncogenes. Through this profiling effort, we found oncogenic regulatory mechanisms for several cancer-relevant serine hydrolases and discovered that platelet activating factor acetylhydrolase 1B2 and 1B3 (PAFAH1B2 and PAFAH1B3) activities were consistently upregulated by several oncogenes, alongside previously discovered cancer-relevant hydrolases fatty acid synthase and monoacylglycerol lipase. While we previously showed that PAFAH1B2 and 1B3 were important in breast cancer, our most recent profiling studies have revealed that these enzymes may be dysregulated broadly across many types of cancers. Here, we find that pharmacological blockade of both enzymes impairs cancer pathogenicity across multiple different types of cancer cells, including breast, ovarian, melanoma, and prostate cancer. We also show that pharmacological blockade of PAFAH1B2 and 1B3 causes unique changes in lipid metabolism, including heightened levels of tumor-suppressing lipids. Our results reveal oncogenic regulatory mechanisms of several cancer-relevant serine hydrolases using activity-based protein profiling, and we show that PAFAH1B2 and 1B3 are important in maintaining cancer pathogenicity across a wide spectrum of cancer types.
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http://dx.doi.org/10.1021/acschembio.5b00053DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4702252PMC
July 2015

A high-throughput RNAi screen for detection of immune-checkpoint molecules that mediate tumor resistance to cytotoxic T lymphocytes.

EMBO Mol Med 2015 Apr;7(4):450-63

Division of Translational Immunology, German Cancer Research Center (DKFZ), Heidelberg, Germany

The success of T cell-based cancer immunotherapy is limited by tumor's resistance against killing by cytotoxic T lymphocytes (CTLs). Tumor-immune resistance is mediated by cell surface ligands that engage immune-inhibitory receptors on T cells. These ligands represent potent targets for therapeutic inhibition. So far, only few immune-suppressive ligands have been identified. We here describe a rapid high-throughput siRNA-based screening approach that allows a comprehensive identification of ligands on human cancer cells that inhibit CTL-mediated tumor cell killing. We exemplarily demonstrate that CCR9, which is expressed in many cancers, exerts strong immune-regulatory effects on T cell responses in multiple tumors. Unlike PDL1, which inhibits TCR signaling, CCR9 regulates STAT signaling in T cells, resulting in reduced T-helper-1 cytokine secretion and reduced cytotoxic capacity. Moreover, inhibition of CCR9 expression on tumor cells facilitated immunotherapy of human tumors by tumor-specific T cells in vivo. Taken together, this method allows a rapid and comprehensive determination of immune-modulatory genes in human tumors which, as an entity, represent the 'immune modulatome' of cancer.
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http://dx.doi.org/10.15252/emmm.201404414DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4403046PMC
April 2015

Linking tumor mutations to drug responses via a quantitative chemical-genetic interaction map.

Cancer Discov 2015 02 12;5(2):154-67. Epub 2014 Dec 12.

University of California, San Francisco, San Francisco, California.

Unlabelled: There is an urgent need in oncology to link molecular aberrations in tumors with therapeutics that can be administered in a personalized fashion. One approach identifies synthetic-lethal genetic interactions or dependencies that cancer cells acquire in the presence of specific mutations. Using engineered isogenic cells, we generated a systematic and quantitative chemical-genetic interaction map that charts the influence of 51 aberrant cancer genes on 90 drug responses. The dataset strongly predicts drug responses found in cancer cell line collections, indicating that isogenic cells can model complex cellular contexts. Applying this dataset to triple-negative breast cancer, we report clinically actionable interactions with the MYC oncogene, including resistance to AKT-PI3K pathway inhibitors and an unexpected sensitivity to dasatinib through LYN inhibition in a synthetic lethal manner, providing new drug and biomarker pairs for clinical investigation. This scalable approach enables the prediction of drug responses from patient data and can accelerate the development of new genotype-directed therapies.

Significance: Determining how the plethora of genomic abnormalities that exist within a given tumor cell affects drug responses remains a major challenge in oncology. Here, we develop a new mapping approach to connect cancer genotypes to drug responses using engineered isogenic cell lines and demonstrate how the resulting dataset can guide clinical interrogation.
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http://dx.doi.org/10.1158/2159-8290.CD-14-0552DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4407699PMC
February 2015

Inositol phosphate recycling regulates glycolytic and lipid metabolism that drives cancer aggressiveness.

ACS Chem Biol 2014 Jun 28;9(6):1340-50. Epub 2014 Apr 28.

Program in Metabolic Biology, Department of Nutritional Sciences and Toxicology, University of California, Berkeley , Berkeley, California 94720, United States.

Cancer cells possess fundamentally altered metabolism that supports their pathogenic features, which includes a heightened reliance on aerobic glycolysis to provide precursors for synthesis of biomass. We show here that inositol polyphosphate phosphatase 1 (INPP1) is highly expressed in aggressive human cancer cells and primary high-grade human tumors. Inactivation of INPP1 leads to a reduction in glycolytic intermediates that feed into the synthesis of the oncogenic signaling lipid lysophosphatidic acid (LPA), which in turn impairs LPA signaling and further attenuates glycolytic metabolism in a feed-forward mechanism to impair cancer cell motility, invasiveness, and tumorigenicity. Taken together these findings reveal a novel mode of glycolytic control in cancer cells that can serve to promote key oncogenic lipid signaling pathways that drive cancer pathogenicity.
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http://dx.doi.org/10.1021/cb5001907DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4076040PMC
June 2014

Management of dermatologic toxicities associated with monoclonal antibody epidermal growth factor receptor inhibitors: A case review.

J Pharmacol Pharmacother 2013 Dec;4(Suppl 1):S78-85

Department of Science of Health, School of Medicine, University of Catanzaro, Catanzaro, Italy ; Pharmacovigilance's Centre Calabria Region, University Hospital Mater Domini, Catanzaro, Italy.

Introduction: The epidermal growth factor receptor inhibitors (EGFRIs), cetuximab and panitumumab, represent an effective treatment option for patients affected by metastatic colorectal cancer (mCRC); furthermore, they are relatively devoid of systemic toxicities, which are commonly observed with standard cytotoxic chemotherapy. However, the majority of patients treated with these monoclonal antibodies (mAbs), will experience dermatologic toxicities, most notably the papulopustular skin rash, which can impact quality-of-life and affect adherence to therapy. This paper reviews the most recent practices in the management of skin rash related to anti-epidermal growth factor receptor (EGFR) mAbs, cetuximab and panitumumab, in the treatment of mCRC.

Materials And Methods: We reviewed relevant literature regarding dermatologic toxicities associated with anti-EGFR mAbs in order to give important indications about prevention and reactive treatment of skin rash.

Results: Two case reports were presented to show how skin rash could hamper mAb EGFRIs use in clinical practice, underscoring the need of implementing a comprehensive management strategy of skin toxicity in order to promote patients' compliance with anti-EGFR therapy and maintain quality-of-life. Based on randomized data, recent guidelines established by the Multinational Association for Supportive Care in Cancer Skin Toxicity Study Group suggest that prophylactic use of oral doxycycline or minocycline reduces the risk and severity of skin rash, improving clinical outcomes.

Conclusions: At the start of treatment with cetuximab and panitumumab, the proper patient education about the skin rash associated with these mAbs and the implementation of a pre-emptive, comprehensive skin toxicity program significantly contribute to improve adherence to therapy, optimize anti-EGFR therapy and maintain quality-of-life.
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http://dx.doi.org/10.4103/0976-500X.120966DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3853676PMC
December 2013

Synergy between enzastaurin doxorubicin in inducing melanoma apoptosis.

Pigment Cell Melanoma Res 2013 Nov 7;26(6):900-11. Epub 2013 Aug 7.

Dipartimento di Medicina Molecolare e Biotecnologie Mediche, Federico II University, Napoli, Italy.

Melanoma is resistant to most standard chemotherapeutics. We analysed the combined effect of doxorubicin and enzastaurin on cell death of four melanoma cell lines, namely G361, SK-MEL3, A375 and SAN. Enzastaurin IC50 was calculated by measure of growth inhibition with MTS assay and corresponded to 2 μM; the half maximal cytotoxicity of doxorubicin was obtained at 3 μM dose. Evaluation of combination index showed synergism (CI > 1) or additive effect (CI = 1) with all melanoma cell lines, with enzastaurin doses ≥0.6 μM and doxorubicin doses ≥1 μM. Combination of the two drugs resulted in increase in caspase 3 and 8 activation, in comparison with activation by single agents. Caspase 8 activation was impaired by TNFR-1 blocking. Our results show doxorubicin-stimulated production of TNFα, whereas enzastaurin-stimulated TNFR-1 expression on plasma membrane. The effect on TNFR-1 appeared to be mediated by PKCζ inhibition. Taken together, our findings suggest that enzastaurin increases doxorubicin-induced apoptosis of melanoma by a mechanism involving, at least in part, activation of the TNF-α signal.
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http://dx.doi.org/10.1111/pcmr.12144DOI Listing
November 2013

Circulating microRNAs in hematological diseases: principles, challenges, and perspectives.

Blood 2013 Jun 2;121(25):4977-84. Epub 2013 Apr 2.

Department of Internal Medicine III, University Hospital of Ulm, Ulm, Germany.

The complex microRNA (miRNA) network plays an important role in the regulation of cellular processes such as development, differentiation, and apoptosis. Recently, the presence of cell-free miRNAs that circulate in body fluids was discovered. The ability of these circulating miRNAs to mirror physiological and pathophysiological conditions as well as their high stability in stored patient samples underlines the potential of these molecules to serve as biomarkers for various diseases. In this review, we describe recent findings in miRNA-mediated cell-to-cell communication and the functions of circulating miRNAs in the field of hematology. Furthermore, we discuss current approaches to design biomarker studies with circulating miRNAs. This article critically reviews the novel field of circulating miRNAs and highlights their suitability for clinical and basic research in addition to their potential as a novel class of biomarkers.
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http://dx.doi.org/10.1182/blood-2013-01-480079DOI Listing
June 2013

Identity and ranking of colonic mesenchymal stromal cells.

J Cell Physiol 2012 Sep;227(9):3291-300

Department of Hematology, Oncology and Molecular Medicine, Istituto Superiore di Sanità, Rome, Italy.

Although ongoing clinical trials utilize systemic administration of bone-marrow mesenchymal stromal cells (BM-MSCs) in Crohn's disease (CD), nothing is known about the presence and the function of mesenchymal stromal cells (MSCs) in the normal human bowel. MSCs are bone marrow (BM) multipotent cells supporting hematopoiesis with the potential to differentiate into multiple skeletal phenotypes. A recently identified new marker, CD146, allowing to prospectively isolate MSCs from BM, renders also possible their identification in different tissues. In order to elucidate the presence and functional role of MSCs in human bowel we analyzed normal adult colon sections and isolated MSCs from them. In colon (C) sections, resident MSCs form a net enveloping crypts in lamina propria, coinciding with structural myofibroblasts or interstitial stromal cells. Nine sub-clonal CD146(+) MSC lines were derived and characterized from colon biopsies, in addition to MSC lines from five other human tissues. In spite of a phenotype qualitative identity between the BM- and C-MSC populations, they were discriminated and categorized. Similarities between C-MSC and BM-MSCs are represented by: Osteogenic differentiation, hematopoietic supporting activity, immune-modulation, and surface-antigen qualitative expression. The differences between these populations are: C-MSCs mean intensity expression is lower for CD13, CD29, and CD49c surface-antigens, proliferative rate faster, life-span shorter, chondrogenic differentiation rare, and adipogenic differentiation completely blocked. Briefly, BM-MSCs, deserve the rank of progenitors, whereas C-MSCs belong to the restricted precursor hierarchy. The presence and functional role of MSCs in human colon provide a rationale for BM-MSC replacement therapy in CD, where resident bowel MSCs might be exhausted or diverted from their physiological functions.
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http://dx.doi.org/10.1002/jcp.24027DOI Listing
September 2012

Differential effect of atorvastatin and tacrolimus on proliferation of vascular smooth muscle and endothelial cells.

Am J Physiol Heart Circ Physiol 2012 Jan 4;302(1):H135-42. Epub 2011 Nov 4.

Invasive Cardiology Unit, Pineta Grande Hospital, Castelvolturno, Italy.

Although considered promising for use in drug-eluting stents (DES), tacrolimus failed clinically. Tacrolimus inhibits growth factor production but can also act as a growth factor on vascular smooth muscle cells (VSMC). This unexpected proliferative stimulus could reverse the beneficial effects of the drug on restenosis. We hypothesized that tacrolimus' association with statins, which lower cholesterol and impair cell proliferation, could restore tacrolimus' beneficial effect by abrogating the aberrant proliferative stimulus. Additionally, since maintenance of endothelial function represents a challenge for new-generation DES, we investigated the combined effect of tacrolimus and atorvastatin on endothelial cells. Human VSMC and umbilical vein endothelial cells (HUVEC) were incubated with 100 nM tacrolimus and increasing doses of atorvastatin (0-3.0 μM). Atorvastatin plus tacrolimus dose-dependently inhibited VSMC proliferation. The percentage of cells incorporating 5-bromo-2'-deoxyuridine (BrdU) in their DNA was 49 ± 14% under basal conditions, 62 ± 15% (P = 0.01) with tacrolimus, 40 ± 22% with 3 μM atorvastatin, and 30 ± 7% (P < 0.05) with 3 μM atorvastatin plus tacrolimus. Atorvastatin downregulated β-catenin, Erk1 and Erk2, and cyclin B in tacrolimus-stimulated VSMC. In contrast, atorvastatin plus tacrolimus did not affect proliferation of endothelial cells. The percentage of HUVEC incorporating BrdU in their DNA was 47 ± 8% under basal conditions, 58 ± 6% (P = 0.01) with tacrolimus, 45 ± 4% with 3 μM atorvastatin, and 49 ± 1% with 3 μM atorvastatin plus tacrolimus. Both agents stimulated endoglin production by HUVEC. Taken together, these results suggest that, when combined with tacrolimus, atorvastatin exerts a dose-dependent antiproliferative effect on VSMC. In contrast, atorvastatin acts in concert with tacrolimus in HUVEC to stimulate production of endoglin, a factor that has an important role in endothelial repair. Our study supports the conclusion that prevention of postcoronary in-stent restenosis and late thrombosis may benefit of concomitant association of tacrolimus and high doses of atorvastatin.
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http://dx.doi.org/10.1152/ajpheart.00490.2011DOI Listing
January 2012

Regulatory circuitries coordinated by transcription factors and microRNAs at the cornerstone of hematopoietic stem cell self-renewal and differentiation.

Curr Stem Cell Res Ther 2011 Jun;6(2):142-61

Department of Histology and Medical Embryology, University La Sapienza, 00161 Rome, Italy.

In mammals, hematopoiesis is the continuous formation of all blood cell types from a limited pool of hematopoietic stem cells (HSCs) residing in specialized niches in the bone marrow (BM). Hierarchical specification of hematopoietic lineages, as well as stem cell kinetics, are dynamic processes influenced by an intricate network of soluble growth factors and membrane-anchored signals orchestrated by the microenvironment (extrinsic signals), coupled with cell-autonomous changes in gene expression (intrinsic signals). At the molecular level, during the early steps of hematopoietic differentiation from the HSC, the chromatin progressively becomes more accessible at genes poised for expression, rapidly followed by an increased expression of lineage-associated genes with concomitant repression of alternative-lineage genes, resulting in commitment and differentiation. These events are established by the coordinated action of transcription factors (TFs), chromatin remodeling factors and microRNAs (miRNAs). In this review we describe the combinatorial molecular circuitries managed by TFs and miRNAs underlying HSC emergence, maintenance, and lineage development.
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http://dx.doi.org/10.2174/157488811795495431DOI Listing
June 2011

FK506 binding proteins as targets in anticancer therapy.

Anticancer Agents Med Chem 2010 Nov;10(9):651-6

Department of Biochemistry and Medical Biotechnology, University Federico II, Naples, Italy.

FK506 binding proteins (FKBPs) are the intracellular ligands of FK506 and rapamycin, two natural compounds with powerful and clinically efficient immunosuppressive activity. In recent decades, a relevant role for immunosuppressants as anticancer agents has emerged. Especially, rapamycin and its derivatives are used, with successful results, across a variety of tumors. Of note, rapamycin and FK506 bind to FKBP12, and the resulting complexes interfere with distinct intracellular signaling pathways driven, respectively, by the mammalian target of rapamycin and calcineurin phosphatase. These pathways are related to T-cell activation and growth. Hyperactivation of the mammalian target of rapamycin (mTOR), particularly in cancers that have lost the tumor suppressor gene PTEN, plays an important pathogenetic role in tumor transformation and growth. The signaling pathway involving calcineurin and nuclear factors of activated T-lymphocytes is also involved in the pathogenesis of different cancer types and in tumor metastasis, providing a rationale for use of FK506 in anticancer therapy. Recent studies have focused on FKBPs in apoptosis regulation: Targeting of FKBP12 promotes apoptosis in chronic lymphocytic leukemia, FKBP38 knockdown sensitizes hepatoma cells to apoptosis, and FKBP51 silencing overcomes resistance to apoptosis in acute lymphoblastic leukemia, prostate cancer, melanoma, and glioma. Interestingly, derivatives of FK506 that have the same FKBP12-binding properties as FK506 but lack functional immunosuppressant activity, exert the same apoptotic effect as FK506 in chronic lymphocytic leukemia.These findings suggest that a direct FKBP inhibition represents a further mechanism of immunosuppressants.' anticancer activity. In this review, we focus on the role of FKBP members in apoptosis control and summarize the data on the antitumor effect of selective targeting of FKBP.
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http://dx.doi.org/10.2174/187152010794479816DOI Listing
November 2010

Regulation of monocytopoiesis by microRNAs.

Methods Mol Biol 2010 ;667:165-76

Department of Hematology, Oncology and Molecular Medicine, Istituto Superiore di Sanità, Rome, Italy.

MicroRNAs (miRNAs or miRs) are approximately 22 nt single-stranded noncoding RNAs that control gene expression in eukaryotes. miRNAs play an essential role in all basic cellular processes including cell development, proliferation, differentiation, and apoptosis. Importantly, miRNAs regulate hematopoietic progenitor cells differentiation toward the different hematopoietic lineages. This occurs through the regulation of key factors involved in hematopoiesis (e.g., transcription factors, growth factor receptors). We, hereby, describe how to investigate the role of miRNAs in monocytopoiesis.
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http://dx.doi.org/10.1007/978-1-60761-811-9_11DOI Listing
January 2011

NFI-A directs the fate of hematopoietic progenitors to the erythroid or granulocytic lineage and controls beta-globin and G-CSF receptor expression.

Blood 2009 Aug 19;114(9):1753-63. Epub 2009 Jun 19.

Department of Histology and Medical Embryology, University La Sapienza, Rome, Italy.

It is generally conceded that selective combinations of transcription factors determine hematopoietic lineage commitment and differentiation. Here we show that in normal human hematopoiesis the transcription factor nuclear factor I-A (NFI-A) exhibits a marked lineage-specific expression pattern: it is upmodulated in the erythroid (E) lineage while fully suppressed in the granulopoietic (G) series. In unilineage E culture of hematopoietic progenitor cells (HPCs), NFI-A overexpression or knockdown accelerates or blocks erythropoiesis, respectively: notably, NFI-A overexpression restores E differentiation in the presence of low or minimal erythropoietin stimulus. Conversely, NFI-A ectopic expression in unilineage G culture induces a sharp inhibition of granulopoiesis. Finally, in bilineage E + G culture, NFI-A overexpression or suppression drives HPCs into the E or G differentiation pathways, respectively. These NFI-A actions are mediated, at least in part, by a dual and opposite transcriptional action: direct binding and activation or repression of the promoters of the beta-globin and G-CSF receptor gene, respectively. Altogether, these results indicate that, in early hematopoiesis, the NFI-A expression level acts as a novel factor channeling HPCs into either the E or G lineage.
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http://dx.doi.org/10.1182/blood-2008-12-196196DOI Listing
August 2009

MicroRNA 223-dependent expression of LMO2 regulates normal erythropoiesis.

Haematologica 2009 Apr 10;94(4):479-86. Epub 2009 Mar 10.

Dept. of Hematology, Oncology and Molecular Medicine, Istituto Superiore di Sanità, Viale Regina Elena 299, 00161 Rome, Italy.

Background: MicroRNAs are small non-coding RNAs that regulate gene expression through mRNA degradation or translational inhibition. MicroRNAs are emerging as key regulators of normal hematopoiesis and hematologic malignancies. Several miRNAs are differentially expressed during hematopoiesis and their specific expression regulates key functional proteins involved in hematopoietic lineage differentiation. This study focused on the functional role of microRNA-223 (miR-223) on erythroid differentiation.

Design And Methods: Purified cord blood CD34+ hematopoietic progenitor cells were grown in strictly controlled conditions in the presence of saturating dosage of erythropoietin to selectively induce erythroid differentiation. The effects of enforced expression of miR-223 in unilin-eage erythroid cultures were evaluated in liquid phase culture experiments and clonogenic studies.

Results: In unilineage erythroid culture of cord blood CD34+ hematopoietic progenitor cells miR-223 is down-regulated, whereas LMO2, an essential protein for erythroid differentiation, is up-regulated. Functional studies showed that enforced expression of miR-223 reduces the mRNA and protein levels of LMO2, by binding to LMO2 3' UTR, and impairs differentiation of erythroid cells. Accordingly, knockdown of LMO2 by short interfering RNA mimics the action of miR-223. Furthermore, hematopoietic progenitor cells transduced with miR-223 showed a significant reduction of their erythroid clonogenic capacity, suggesting that downmodulation of this miRNA is required for erythroid progenitor recruitment and commitment.

Conclusions: These results show that the decline of miR-223 is an important event for erythroid differentiation that leads to the expansion of erythroblast cells at least partially mediated by unblocking LMO2 protein expression.
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http://dx.doi.org/10.3324/haematol.2008.002345DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2663611PMC
April 2009

Role of microRNAs in haemopoiesis, heart hypertrophy and cancer.

Biochem Soc Trans 2008 Dec;36(Pt 6):1206-10

Department of Hematology, Oncology and Molecular Medicine, Istituto Superiore di Sanità, Rome, Italy.

miRNAs (microRNAs) are important regulatory molecules that control gene expression in all eukaryotes. miRNAs play an essential role in basic cellular activities such as proliferation, differentiation, morphogenesis and apoptosis. In haemopoiesis, several miRNA-based pathways have been identified. Importantly, miRNA mutations or mis-expression correlate with various human diseases. In cancer, deregulated miRNAs can function as tumour suppressors or oncogenes. The present review focuses on the recent literature concerning the role of miRNAs in three different research areas: haematology, cardiology and oncology, with particular focus on the results obtained by our group.
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http://dx.doi.org/10.1042/BST0361206DOI Listing
December 2008

Role of microRNAs in drug-resistant ovarian cancer cells.

Gynecol Oncol 2008 Dec 26;111(3):478-86. Epub 2008 Sep 26.

Department of Hematology, Istituto Superiore di Sanità, Rome, Italy.

Objectives: Chemotherapy is the preferred therapeutic approach for the therapy of advanced ovarian cancer, but a successful long-term treatment is prevented by the development of drug resistance. Recent works have underlined the involvement of non-coding RNAs, microRNAs (miRNAs) in cancer development, with several conjectures regarding their possible involvement in the evolution of drug resistance. This work was aimed to identify selected microRNAs involved in the development of chemoresistance in ovarian cancer.

Methods: High-throughput analysis of the miRNA profile in a panel of paclitaxel- (A2780TAX, A2780TC1 and A2780TC3) and cisplatin-resistant (A2780CIS) cells was assessed using a microarray platform and subsequent validation with qPCR and Northern blots. Downstream target validation was performed for miR-130a and the target M-CSF.]

Results: Six miRNAs (let-7e, miR-30c, miR-125b, miR-130a and miR-335) were always diversely expressed in all the resistant cell lines. Let-7e was upregulated in A2780TAX cells, while it was downregulated in the other resistant cell lines. The opposite phenomenon was obtained for miR-125b, which was downregulated in A2780TAX and upregulated in the other cell lines. The miR-30c, miR-130a and miR-335 were downregulated in all the resistant cell lines, thereby suggesting a direct involvement in the development of chemoresistance. Finally downstream target validation was proven for the miR-130a, whose downregulation was linked to the translational activation of the M-CSF gene, a known resistance factor for ovarian cancer.

Conclusions: Our results indicate that ovarian cancer drug resistance is associated with a distinct miRNA fingerprint, and miRNA microarrays could represent a prognostic tool to monitor the chemotherapy outcome.
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http://dx.doi.org/10.1016/j.ygyno.2008.08.017DOI Listing
December 2008

The mesenchymal stromal cell contribution to homeostasis.

J Cell Physiol 2008 Nov;217(2):296-300

Department of Hematology, Oncology, and Molecular Medicine, Istituto Superiore di Sanità, Rome, Italy.

Adult mesenchymal stromal cells (MSCs) are undifferentiated multi-potent cells predominantly residing in the bone marrow (BM), but also present with similar but not identical features in many other tissues such as blood, placenta, dental pulp, and adipose tissue. MSCs have the potential to differentiate into multiple skeletal phenotypes like osteoblasts, chondrocytes, adipocytes, stromal cells, fibroblasts, and possibly tendons. MSCs differentiation potential, ex vivo expansion capacity, nurturing and immunomodulatory proficiencies oriented these versatile cells in several areas of ongoing clinical applications. However, the absence of MSC-specific markers for isolation and characterization together with the lack of a comprehensive view of the molecular pathways governing their particular biological properties, remains a primary obstacle to their research and application. In this review we discuss some areas of growing interest in MSCs biology: their contribution to the hematopoietic stem cell (HSC) niche, to regenerative medicine, their role in cancer and in therapy as delivery tools and their micro-RNA (miRNA) signatures. Despite rapid progress in the MSC field, it is generally thought that only a fraction of their full potential has been realized thus far.
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http://dx.doi.org/10.1002/jcp.21521DOI Listing
November 2008

Isolation and characterization of CD146+ multipotent mesenchymal stromal cells.

Exp Hematol 2008 Aug 27;36(8):1035-46. Epub 2008 May 27.

Department of Hematology, Oncology, and Molecular Medicine, Istituto Superiore di Sanità, Rome, Italy.

Mesenchymal stromal cells (MSCs) represent a bone marrow (BM) population, classically defined by five functional properties: extensive proliferation, ability to differentiate into osteoblasts, chondrocytes, adipocytes, and stromal cells-supporting hematopoiesis. However, research progress in this area has been hampered by lack of suitable markers and standardized procedures for MSC isolation. We have isolated a CD146(+) multipotent MSC population from 20 human BM donors displaying the phenotype of self-renewing osteoprogenitors; an extensive 12-week proliferation; and the ability to differentiate in osteoblasts, chondrocytes, adipocytes, and stromal cells supporting hematopoiesis. Furthermore, the CD146(+) MSCs secrete a complex combination of growth factors (GFs) controlling hematopoietic stem cells (HSCs) function, while providing a >2-log increase in the long-term culture (LTC) colony output in 8-week LTC over conventional assays. The hematopoietic stromal function exhibited by the MSCs was further characterized by manipulating LTCs with the chemical inhibitors Imatinib or SU-5416, targeting two GF receptors (GFRs), KIT or VEGFR2/1, respectively. Both treatments similarly impaired LTC colony output, indicating key roles for these two GF/GFR interactions to support LTC-initiating cell activity. CD146(+) MSCs may thus represent a tool to explore the MSC-HSC cross-talk in an in vitro surrogate model for HSC "niches," and for regenerative therapy studies. In addition, the MSC microRNA (miRNA) expression profile was analyzed by microarrays in both basic conditions and chondrogenic differentiation. Our analysis revealed that several miRNAs are modulated during chondrogenesis, and many of their putative targets are genes involved in chondrogenic differentiation.
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http://dx.doi.org/10.1016/j.exphem.2008.03.004DOI Listing
August 2008

Telomerase inhibition by stable RNA interference impairs tumor growth and angiogenesis in glioblastoma xenografts.

Int J Cancer 2006 May;118(9):2158-67

Institute of Neurosurgery, Catholic University School of Medicine, Rome, Italy.

Telomerase is highly expressed in advanced stages of most cancers where it allows the clonal expansion of transformed cells by counteracting telomere erosion. Telomerase may also contribute to tumor progression through still undefined cell growth-promoting functions. Here, we inhibited telomerase activity in 2 human glioblastoma (GBM) cell lines, TB10 and U87MG, by targeting the catalytic subunit, hTERT, via stable RNA interference (RNAi). Although the reduction in telomerase activity had no effect on GBM cell growth in vitro, the development of tumors in subcutaneously and intracranially grafted nude mice was significantly inhibited by antitelomerase RNAi. The in vivo effect was observed within a relatively small number of population doublings, suggesting that telomerase inhibition may hinder cancer cell growth in vivo prior to a substantial shortening of telomere length. Tumor xenografts that arose from telomerase-inhibited GBM cells also showed a less-malignant phenotype due both to the absence of massive necrosis and to reduced angiogenesis.
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http://dx.doi.org/10.1002/ijc.21613DOI Listing
May 2006

MicroRNAs 221 and 222 inhibit normal erythropoiesis and erythroleukemic cell growth via kit receptor down-modulation.

Proc Natl Acad Sci U S A 2005 Dec 5;102(50):18081-6. Epub 2005 Dec 5.

Department of Hematology, Oncology, and Molecular Medicine, Istituto Superiore di Sanità, Rome, Italy.

MicroRNAs (miRs) are small noncoding RNAs that regulate gene expression primarily through translational repression. In erythropoietic (E) culture of cord blood CD34+ progenitor cells, the level of miR 221 and 222 is gradually and sharply down-modulated. Hypothetically, this decline could promote erythropoiesis by unblocking expression of key functional proteins. Indeed, (i) bioinformatic analysis suggested that miR 221 and 222 target the 3' UTR of kit mRNA; (ii) the luciferase assay confirmed that both miRs directly interact with the kit mRNA target site; and (iii) in E culture undergoing exponential cell growth, miR down-modulation is inversely related to increasing kit protein expression, whereas the kit mRNA level is relatively stable. Functional studies show that treatment of CD34+ progenitors with miR 221 and 222, via oligonucleotide transfection or lentiviral vector infection, causes impaired proliferation and accelerated differentiation of E cells, coupled with down-modulation of kit protein: this phenomenon, observed in E culture releasing endogenous kit ligand, is magnified in E culture supplemented with kit ligand. Furthermore, transplantation experiments in NOD-SCID mice reveal that miR 221 and 222 treatment of CD34+ cells impairs their engraftment capacity and stem cell activity. Finally, miR 221 and 222 gene transfer impairs proliferation of the kit+ TF-1 erythroleukemic cell line. Altogether, our studies indicate that the decline of miR 221 and 222 during exponential E growth unblocks kit protein production at mRNA level, thus leading to expansion of early erythroblasts. Furthermore, the results on kit+ erythroleukemic cells suggest a potential role of these miRs in cancer therapy.
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http://dx.doi.org/10.1073/pnas.0506216102DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1312381PMC
December 2005