I am passionate about translational research and how single cell technologies could open up new avenues for better and more accurate predictive models. Currently, I am focus on integrating single cell RNA and protein expression data to develop models to predict patient at risk for Antigen Loss relapse after CAR T cells immunotherapy.
Instructor, Pediatrics - Hematology & Oncology
Honors & Awards
ASH Scholar Award, American Society of Hematology (2022 - 2024)
Best Abstracts Award, Transplantation & Cellular Therapy Meetings of ASTCT and CIBMTR (2022)
Outstanding Oral Presentation, 12th Annual Pediatrics Research Retreat (2021)
Abstract selected for 6th Annual Immuno-Oncology Young Investigators’ Forum, Immuno-Oncology Young Investigators’ Forum (2020)
ASH Abstract Achievement Award for the 2019 ASH Annual Meeting, American Society of Hematology (2019)
Charles B. Carrington Memorial Award for Outstanding Poster Presentation, Stanford Pathology Annual Research Retreat. (2017)
Boards, Advisory Committees, Professional Organizations
Member, Society for ImmunoTherapy of Cancer (SITC) (2020 - Present)
Member, American Society of Hematology (ASH) (2018 - Present)
Member, American Association for Cancer Research (AACR) (2018 - Present)
Ph.D., National University of Rosario. Argentina. (2015)
B.Sc., National University of Rosario. Argentina. (2009)
Current Research and Scholarly Interests
CD19-directed chimeric antigen receptor T cell (CAR-T) therapy has shown impressive results in children and adults with relapsed or refractory (r/r) B-ALL or non-Hodgkin lymphoma. However, 30 – 70% of initial responders will eventually relapse with CD19 antigen loss (CD19Neg). The development of tools to accurately predict which patients are at risk for CD19Neg relapse would guide treatment decisions regarding alternative therapies.
The goal of my research is to develop a model to predict which patients are at risk for CD19Neg relapse.
I hypothesize that resistant tumor cells, and their features associated with CD19Neg relapse, are present before CAR-T administration and can be detected and used to build a model to predict which patients are at risk for CD19Neg relapse. To that end, I used a combination of single cell mass cytometry (CyTOF) and simultaneous whole transcriptome analysis and antibodies sequencing (WTA-AbSeq) data from B-ALL patient samples collected before or after CD19-directed CAR-T administration. Through these analyses I expect to identify and deeply characterize those tumor populations present before CAR-T administration responsible for CD19Neg relapse.
Dasatinib overcomes glucocorticoid resistance in B-cell acute lymphoblastic leukemia.
2023; 14 (1): 2935
Resistance to glucocorticoids (GC) is associated with an increased risk of relapse in B-cell progenitor acute lymphoblastic leukemia (BCP-ALL). Performing transcriptomic and single-cell proteomic studies in healthy B-cell progenitors, we herein identify coordination between the glucocorticoid receptor pathway with B-cell developmental pathways. Healthy pro-B cells most highly express the glucocorticoid receptor, and this developmental expression is conserved in primary BCP-ALL cells from patients at diagnosis and relapse. In-vitro and in vivo glucocorticoid treatment of primary BCP-ALL cells demonstrate that the interplay between B-cell development and the glucocorticoid pathways is crucial for GC resistance in leukemic cells. Gene set enrichment analysis in BCP-ALL cell lines surviving GC treatment show enrichment of B cell receptor signaling pathways. In addition, primary BCP-ALL cells surviving GC treatment in vitro and in vivo demonstrate a late pre-B cell phenotype with activation of PI3K/mTOR and CREB signaling. Dasatinib, a multi-kinase inhibitor, most effectively targets this active signaling in GC-resistant cells, and when combined with glucocorticoids, results in increased cell death in vitro and decreased leukemic burden and prolonged survival in an in vivo xenograft model. Targeting the active signaling through the addition of dasatinib may represent a therapeutic approach to overcome GC resistance in BCP-ALL.
View details for DOI 10.1038/s41467-023-38456-y
View details for PubMedID 37217509
Single-Cell Proteomic Analysis Defines Metabolic Heterogeneity in Response to Venetoclax in AML
AMER SOC HEMATOLOGY. 2022: 1028-1029
View details for DOI 10.1182/blood-2022-171009
View details for Web of Science ID 000893223201014
IKAROS MEDIATES ANTIGEN ESCAPE FOLLOWING CD19 CAR T CELL THERAPY IN R/R B-ALL
View details for Web of Science ID 000859203900105
CytofIn enables integrated analysis of public mass cytometry datasets using generalized anchors.
2022; 13 (1): 934
The increasing use of mass cytometry for analyzing clinical samples offers the possibility to perform comparative analyses across public datasets. However, challenges in batch normalization and data integration limit the comparison of datasets not intended to be analyzed together. Here, we present a data integration strategy, CytofIn, using generalized anchors to integrate mass cytometry datasets from the public domain. We show that low-variance controls, such as healthy samples and stable channels, are inherently homogeneous, robust against stimulation, and can serve as generalized anchors for batch correction. Single-cell quantification comparing mass cytometry data from 989 leukemia files pre- and post normalization with CytofIn demonstrates effective batch correction while recapitulating the gold-standard bead normalization. CytofIn integration of public cancer datasets enabled the comparison of immune features across histologies and treatments. We demonstrate the ability to integrate public datasets without necessitating identical control samples or bead standards for fast and robust analysis using CytofIn.
View details for DOI 10.1038/s41467-022-28484-5
View details for PubMedID 35177627
Chromatin Content Capture Reveals Acute Leukaemia Oncogenic Vulnerability Point in Human B Cell Development
AMER SOC HEMATOLOGY. 2021
View details for DOI 10.1182/blood-2021-153114
View details for Web of Science ID 000736398802212
Ikaros Mediates Antigen Escape Following CD19 CAR T Cell Therapy in r/r B-ALL
AMER SOC HEMATOLOGY. 2021
View details for DOI 10.1182/blood-2021-151002
View details for Web of Science ID 000736398802154
Glucocorticoid-Resistant B-Cell Acute Lymphoblastic Leukemic Cells Can be Targeted By BCR-Signaling Inhibition
AMER SOC HEMATOLOGY. 2021
View details for DOI 10.1182/blood-2021-150356
View details for Web of Science ID 000736398802158
In-depth triacylglycerol profiling using MS3 Q-Trap mass spectrometry.
Analytica chimica acta
2021; 1184: 339023
Total triacylglycerol (TAG) level is a key clinical marker of metabolic and cardiovascular diseases. However, the roles of individual TAGs have not been thoroughly explored in part due to their extreme structural complexity. We present a targeted mass spectrometry-based method combining multiple reaction monitoring (MRM) and multiple stage mass spectrometry (MS3) for the comprehensive qualitative and semiquantitative profiling of TAGs. This method referred as TriP-MS3 - triacylglycerol profiling using MS3 - screens for more than 6,700 TAG species in a fully automated fashion. TriP-MS3 demonstrated excellent reproducibility (median interday CV0.15) and linearity (median R2=0.978) and detected 285 individual TAG species in human plasma. The semiquantitative accuracy of the method was validated by comparison with a state-of-the-art reverse phase liquid chromatography (RPLC)-MS (R2=0.83), which is the most commonly used approach for TAGs profiling. Finally, we demonstrate the utility and the versatility of the method by characterizing the effects of a fatty acid desaturase inhibitor on TAG profiles invitro and by profiling TAGs in Caenorhabditis elegans.
View details for DOI 10.1016/j.aca.2021.339023
View details for PubMedID 34625255
A Cancer Biologist's Primer on Machine Learning Applications in High-Dimensional Cytometry.
Cytometry. Part A : the journal of the International Society for Analytical Cytology
The application of machine learning and artificial intelligence to high-dimensional cytometry data sets has increasingly become a staple of bioinformatic data analysis over the past decade. This is especially true in the field of cancer biology, where protocols for collecting multiparameter single-cell data in a high-throughput fashion are rapidly developed. As the use of machine learning methodology in cytometry becomes increasingly common, there is a need for cancer biologists to understand the basic theory and applications of a variety of algorithmic tools for analyzing and interpreting cytometry data. We introduce the reader to several keystone machine learning-based analytic approaches with an emphasis on defining key terms and introducing a conceptual framework for making translational or clinically relevant discoveries. The target audience consists of cancer cell biologists and physician-scientists interested in applying these tools to their own data, but who may have limited training in bioinformatics. © 2020 International Society for Advancement of Cytometry.
View details for DOI 10.1002/cyto.a.24158
View details for PubMedID 32602650
Expansion of Bone Precursors through Jun as a Novel Treatment for Osteoporosis-Associated Fractures.
Stem cell reports
Osteoporosis and osteoporotic fractures lead to decreased life quality and high healthcare costs. Current treatments prevent losses in bone mass and fractures to some extent but have side effects. Therefore, better therapies are needed. This study investigated whether the transcriptionfactor Jun has a specific pro-osteogenic potency and whether modulating Jun could serve as a novel treatment for osteoporosis-associated fractures. We demonstrate that ectopically transplanted whole bones and distinct osteoprogenitors increase bone formation. Perinatal Jun induction disturbs growth plate architecture, causing a striking phenotype with shortened and thickened bones. Molecularly, Jun induces hedgehog signaling in skeletal stem cells. Therapeutically, Jun accelerates bone growth and healing in a drilling-defect model. Altogether, these results demonstrate that Jun drives bone formation by expanding osteoprogenitor populations and forcing them into the bone fate, providing a rationale for future clinical applications.
View details for DOI 10.1016/j.stemcr.2020.02.009
View details for PubMedID 32197115
Activation of JUN in fibroblasts promotes pro-fibrotic programme and modulates protective immunity.
2020; 11 (1): 2795
The transcription factor JUN is highly expressed in pulmonary fibrosis. Its induction in mice drives lung fibrosis, which is abrogated by administration of anti-CD47. Here, we use high-dimensional mass cytometry to profile protein expression and secretome of cells from patients with pulmonary fibrosis. We show that JUN is activated in fibrotic fibroblasts that expressed increased CD47 and PD-L1. Using ATAC-seq and ChIP-seq, we found that activation of JUN rendered promoters and enhancers of CD47 and PD-L1 accessible. We further detect increased IL-6 that amplified JUN-mediated CD47 enhancer activity and protein expression. Using an in vivo mouse model of fibrosis, we found two distinct mechanisms by which blocking IL-6, CD47 and PD-L1 reversed fibrosis, by increasing phagocytosis of profibrotic fibroblasts and by eliminating suppressive effects on adaptive immunity. Our results identify specific immune mechanisms that promote fibrosis and suggest a therapeutic approach that could be used alongside conventional anti-fibrotics for pulmonary fibrosis.
View details for DOI 10.1038/s41467-020-16466-4
View details for PubMedID 32493933
Prediction of Patients at Risk of CD19(Neg) Relapse Following CD19-Directed CAR T Cell Therapy in B Cell Precursor Acute Lymphoblastic Leukemia
AMER SOC HEMATOLOGY. 2019
View details for DOI 10.1182/blood-2019-128883
View details for Web of Science ID 000518218500504
Preconditioning of Rat Bone Marrow-Derived Mesenchymal Stromal Cells with Toll-Like Receptor Agonists.
Stem cells international
2019; 2019: 7692973
Bone marrow-derived mesenchymal stromal cells (BM-MSCs) are dynamic cells that can sense the environment, adapting their regulatory functions to different conditions. Accordingly, the therapeutic potential of BM-MSCs can be modulated by preconditioning strategies aimed at modifying their paracrine action. Although rat BM-MSCs (rBM-MSCs) have been widely tested in preclinical research, most preconditioning studies have employed human and mouse BM-MSCs. Herein, we investigated whether rBM-MSCs modify their phenotype and paracrine functions in response to Toll-like receptor (TLR) agonists. The data showed that rBM-MSCs expressed TLR3, TLR4, and MDA5 mRNA and were able to internalize polyinosinic-polycytidylic acid (Poly(I:C)), a TLR3/MDA5 agonist. rBM-MSCs were then stimulated with Poly(I:C) or with lipopolysaccharide (LPS, a TLR4 agonist) for 1 h and were grown under normal culture conditions. LPS or Poly(I:C) stimulation did not affect the viability or the morphology of rBM-MSCs and did not modify the expression pattern of key cell surface markers. Poly(I:C) did not induce statistically significant changes in the release of several inflammatory mediators and VEGF by rBM-MSCs, although it tended to increase IL-6 and MCP-1 secretion, whereas LPS increased the release of IL-6, MCP-1, and VEGF, three factors that were constitutively secreted by unstimulated cells. The neurotrophic activity of the conditioned medium from unstimulated and LPS-preconditioned rBM-MSCs was investigated using dorsal root ganglion explants, showing that soluble factors produced by unstimulated and LPS-preconditioned rBM-MSCs can stimulate neurite outgrowth similarly, in a VEGF-dependent manner. LPS-preconditioned cells, however, were slightly more efficient in increasing the number of regrowing axons in a model of sciatic nerve transection in rats. In conclusion, LPS preconditioning boosted the production of constitutively secreted factors by rBM-MSCs, without changing their mesenchymal identity, an effect that requires further investigation in exploratory preclinical studies.
View details for DOI 10.1155/2019/7692973
View details for PubMedID 31531025
View details for PubMedCentralID PMC6721436
Generation of patient-specific pluripotent induced stem cell line UFRJi007-A from a Brazilian familial amyotrophic lateral sclerosis patient.
Stem cell research
2019; 39: 101490
Induced pluripotent stem cell (iPSC) line were generated from erythroblasts of a Brazilian patient with familiar form of amyotrophic lateral sclerosis (ALS). NGS analysis demonstrated that patient carried a mutation in SOD1 gene, as well as a deletion in FUS gene. CytoTune™-iPS 2.0 Sendai Reprogramming Kit (containing the reprogramming factors OCT3/4, KLF4, SOX2 and cMYC) was used to generate the cell lines. The iPSCs express pluripotency markers, have normal karyotype and differentiated spontaneously in the three germ layers. The expression of Sendai virus was lost in all iPSC lines after 15 passages.
View details for DOI 10.1016/j.scr.2019.101490
View details for PubMedID 31301488
Generation of four patient-specific pluripotent induced stem cell lines from two Brazilian patients with amyotrophic lateral sclerosis and two healthy subjects.
Stem cell research
2019; 37: 101448
Induced pluripotent stem cell (iPSC) lines were generated from erythroblasts of two patients with amyotrophic lateral sclerosis (ALS) and two healthy individuals. One familial and one sporadic ALS patients were used, both with genetic alterations in VAPB gene. CytoTune™-iPS 2.0 Sendai Reprogramming Kit (containing the reprogramming factors OCT3/4, KLF4, SOX2 and cMYC) was used to generate the iPSC cell lines. The four iPSCs express pluripotency markers, have normal karyotype and differentiated spontaneously in the three germ layers. The expression of Sendai virus was lost in all iPSC lines after 15 passages.
View details for DOI 10.1016/j.scr.2019.101448
View details for PubMedID 31077962
Direct targeting of the mouse optic nerve for therapeutic delivery
JOURNAL OF NEUROSCIENCE METHODS
2019; 313: 1–5
View details for DOI 10.1016/j.jneumeth.2018.10.038
View details for Web of Science ID 000458596100001
KDM2B regulates choline kinase expression and neuronal differentiation of neuroblastoma cells.
2019; 14 (1): e0210207
The process of neuronal differentiation is associated with neurite elongation and membrane biogenesis, and phosphatidylcholine (PtdCho) is the major membrane phospholipid in mammalian cells. During neuroblast differentiation, the transcription of two genes involved in PtdCho biosynthesis are stimulated: Chka gene for choline kinase (CK) alpha isoform and Pcyt1a gene for CTP:phosphocholine cytidylyltransferase (CCT) alpha isoform. Here we show that CKα is essential for neuronal differentiation. In addition, we demonstrated that KDM2B regulates CKα expression and, as a consequence, neuronal differentiation. This factor is up-regulated in the course of the neuroblasts proliferative and undifferentiated state and down-regulated during differentiation induced by retinoic acid (RA). During proliferation, KDM2B binds to the Box2 located in the Chka promoter repressing its transcription. Interestingly, KDM2B knockdown enhances the levels of CKα expression in neuroblast cells and induces neuronal differentiation even in the absence of RA. These results suggest that KDM2B is required for the appropriate regulation of CKα during neuronal differentiation and to the maintaining of the undifferentiated stage of neuroblast cells.
View details for DOI 10.1371/journal.pone.0210207
View details for PubMedID 30629659
View details for PubMedCentralID PMC6328129
High-efficiency CRISPR induction of t(9;11) chromosomal translocations and acute leukemias in human blood stem cells.
2019; 3 (19): 2825–35
Chromosomal rearrangements involving the mixed lineage leukemia (MLL) gene, also known as KMT2A, are often observed in human leukemias and are generally associated with a poor prognosis. To model these leukemias, we applied clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 gene editing to induce MLL chromosomal rearrangements in human hematopoietic stem and progenitor cells purified from umbilical cord blood. Electroporation of ribonucleoprotein complexes containing chemically modified synthetic single guide RNAs and purified Cas9 protein induced translocations between chromosomes 9 and 11 [t(9;11)] at an efficiency >1%. Transplantation of gene-edited cells into immune-compromised mice rapidly induced acute leukemias of different lineages and often with multiclonal origins dictated by the duration of in vitro culture prior to transplantation. Breakpoint junction sequences served as biomarkers to monitor clonal selection and progression in culture and in vivo. High-dimensional cell surface and intracellular protein analysis by mass cytometry (CyTOF) revealed that gene-edited leukemias recapitulated disease-specific protein expression observed in human patients and showed that MLL-rearranged (MLLr) mixed phenotype acute leukemias (MPALs) were more similar to acute myeloid leukemias (AMLs) than to acute lymphoblastic leukemias (ALLs). Therefore, highly efficient generation of MLL chromosomal translocations in primary human blood stem cells using CRISPR/Cas9 reliably models human acute MLLr leukemia and provides an experimental platform for basic and translational studies of leukemia biology and therapeutics.
View details for DOI 10.1182/bloodadvances.2019000450
View details for PubMedID 31582391
Direct targeting of the mouse optic nerve for therapeutic delivery.
Journal of neuroscience methods
BACKGROUND: Animal models of optic nerve injury are often used to study central nervous system (CNS) degeneration and regeneration, and targeting the optic nerve is a powerful approach for axon-protective or remyelination therapy. However, the experimental delivery of drugs or cells to the optic nerve is rarely performed because injections into this structure are difficult in small animals, especially in mice.NEW METHOD: We investigated and developed methods to deliver drugs or cells to the mouse optic nerve through 3 different routes: a) intraorbital, b) through the optic foramen and c) transcranial.RESULTS: The methods targeted different parts of the mouse optic nerve: intraorbital proximal (intraorbital), intracranial middle (optic-foramen) or intracranial distal (transcranial) portion.COMPARISON WITH EXISTING METHODS: Most existing methods target the optic nerve indirectly. For instance, intravitreally delivered cells often cannot cross the inner limiting membrane to reach retinal neurons and optic nerve axons. Systemic delivery, eye drops and intraventricular injections do not always successfully target the optic nerve. Intraorbital and transcranial injections into the optic nerve or chiasm have been performed but these methods have not been well described. We approached the optic nerve with more selective and precise targeting than existing methods.CONCLUSIONS: We successfully targeted the murine optic nerve intraorbitally, through the optic foramen, and transcranially. Of all methods, the injection through the optic foramen is likely the most innovative and fastest. These methods offer additional approaches for therapeutic intervention to be used by those studying white matter damage and axonal regeneration in the CNS.
View details for PubMedID 30389488
Stem cell therapy for treatment of ischemic optic neuropathy
ASSOC RESEARCH VISION OPHTHALMOLOGY INC. 2018
View details for Web of Science ID 000442912501176
Therapeutic effects of sphingosine kinase inhibitor N,N-dimethylsphingosine (DMS) in experimental chronic Chagas disease cardiomyopathy.
2017; 7 (1): 6171
Chagas disease cardiomyopathy is a parasite-driven inflammatory disease to which there are no effective treatments. Here we evaluated the therapeutic potential of N,N-dimethylsphingosine(DMS), which blocks the production of sphingosine-1-phosphate(S1P), a mediator of cellular events during inflammatory responses, in a model of chronic Chagas disease cardiomyopathy. DMS-treated, Trypanosoma cruzi-infected mice had a marked reduction of cardiac inflammation, fibrosis and galectin-3 expression when compared to controls. Serum concentrations of galectin-3, IFNγ and TNFα, as well as cardiac gene expression of inflammatory mediators were reduced after DMS treatment. The gene expression of M1 marker, iNOS, was decreased, while the M2 marker, arginase1, was increased. DMS-treated mice showed an improvement in exercise capacity. Moreover, DMS caused a reduction in parasite load in vivo. DMS inhibited the activation of lymphocytes, and reduced cytokines and NO production in activated macrophage cultures in vitro, while increasing IL-1β production. Analysis by qRT-PCR array showed that DMS treatment modulated inflammasome activation induced by T. cruzi on macrophages. Altogether, our results demonstrate that DMS, through anti-parasitic and immunomodulatory actions, can be beneficial in the treatment of chronic phase of T. cruzi infection and suggest that S1P-activated processes as possible therapeutic targets for the treatment of Chagas disease cardiomyopathy.
View details for DOI 10.1038/s41598-017-06275-z
View details for PubMedID 28733584
View details for PubMedCentralID PMC5522404
Lysophosphatidylcholine Drives Neuroblast Cell Fate.
2016; 53 (9): 6316-6331
Neuronal differentiation plays a key role during embryogenesis. However, based on the capacity of neuronal stem cells to either generate or regenerate neurons and because differentiation stops aberrant neuroblasts proliferation, neuronal differentiation is crucial during neuropathological conditions. Although phosphatidylcholine (PtdCho) has been proposed as an important molecule for neurite growth and neuronal regeneration, the identity of the molecular target has remained elusive. This study originally describes that lysophosphatidylcholine (LPtdCho), either exogenously supplied or generated by the imbalance of PtdCho metabolism through the enzymatic action of cytosolic phospholipase A2, acts as a neurotrophic-like factor. We demonstrated that LPtdCho induces neuronal differentiation by activation of the small G protein Ras followed by the Raf/MEK/ERK signaling pathway. Accordingly, LPtdCho redirects neuroblasts gene expression leading to the generation of functional mature neurons expressing βIII-tubulin and having increased acetylcholinesterase activity and membrane biosynthesis required for neuritogenesis. These findings provide mechanistic details of the role of cytidine-5-diphosphocholine (CDP-choline) and PtdCho as neuroprotectors. Furthermore, as LPtdCho recapitulates the effect of the therapeutic agent retinoic acid, these results open new avenues for drug discovery for the treatment of neuropathological conditions.
View details for DOI 10.1007/s12035-015-9528-0
View details for PubMedID 26567110
G-quadruplexes as novel cis-elements controlling transcription during embryonic development.
Nucleic acids research
2016; 44 (9): 4163-73
G-quadruplexes are dynamic structures folded in G-rich single-stranded DNA regions. These structures have been recognized as a potential nucleic acid based mechanism for regulating multiple cellular processes such as replication, transcription and genomic maintenance. So far, their transcriptional role in vivo during vertebrate embryonic development has not yet been addressed. Here, we performed an in silico search to find conserved putative G-quadruplex sequences (PQSs) within proximal promoter regions of human, mouse and zebrafish developmental genes. Among the PQSs able to fold in vitro as G-quadruplex, those present in nog3, col2a1 and fzd5 promoters were selected for further studies. In cellulo studies revealed that the selected G-quadruplexes affected the transcription of luciferase controlled by the SV40 nonrelated promoter. G-quadruplex disruption in vivo by microinjection in zebrafish embryos of either small ligands or DNA oligonucleotides complementary to the selected PQSs resulted in lower transcription of the targeted genes. Moreover, zebrafish embryos and larvae phenotypes caused by the presence of complementary oligonucleotides fully resembled those ones reported for nog3, col2a1 and fzd5 morphants. To our knowledge, this is the first work revealing in vivo the role of conserved G-quadruplexes in the embryonic development, one of the most regulated processes of the vertebrates biology.
View details for DOI 10.1093/nar/gkw011
View details for PubMedID 26773060
View details for PubMedCentralID PMC4872077
Coordinated induction of GST and MRP2 by cAMP in Caco-2 cells: Role of protein kinase A signaling pathway and toxicological relevance.
Toxicology and applied pharmacology
2015; 287 (2): 178-190
The cAMP pathway is a universal signaling pathway regulating many cellular processes including metabolic routes, growth and differentiation. However, its effects on xenobiotic biotransformation and transport systems are poorly characterized. The effect of cAMP on expression and activity of GST and MRP2 was evaluated in Caco-2 cells, a model of intestinal epithelium. Cells incubated with the cAMP permeable analog dibutyryl cyclic AMP (db-cAMP: 1,10,100 μM) for 48 h exhibited a dose-response increase in GST class α and MRP2 protein expression. Incubation with forskolin, an activator of adenylyl cyclase, confirmed the association between intracellular cAMP and upregulation of MRP2. Consistent with increased expression of GSTα and MRP2, db-cAMP enhanced their activities, as well as cytoprotection against the common substrate 1-chloro-2,4-dinitrobenzene. Pretreatment with protein kinase A (PKA) inhibitors totally abolished upregulation of MRP2 and GSTα induced by db-cAMP. In silico analysis together with experiments consisting of treatment with db-cAMP of Caco-2 cells transfected with a reporter construct containing CRE and AP-1 sites evidenced participation of these sites in MRP2 upregulation. Further studies involving the transcription factors CREB and AP-1 (c-JUN, c-FOS and ATF2) demonstrated increased levels of total c-JUN and phosphorylation of c-JUN and ATF2 by db-cAMP, which were suppressed by a PKA inhibitor. Co-immunoprecipitation and ChIP assay studies demonstrated that db-cAMP increased c-JUN/ATF2 interaction, with further recruitment to the region of the MRP2 promoter containing CRE and AP-1 sites. We conclude that cAMP induces GSTα and MRP2 expression and activity in Caco-2 cells via the PKA pathway, thus regulating detoxification of specific xenobiotics.
View details for DOI 10.1016/j.taap.2015.06.003
View details for PubMedID 26049102
Choline kinase alpha expression during RA-induced neuronal differentiation: role of C/EBPβ.
Biochimica et biophysica acta
2014; 1841 (4): 544-51
Neuronal differentiation is a complex process characterized by a halt in proliferation and extension of neurites from the cell body. This process is accompanied by changes in gene expression that mediate the redirection leading to neurite formation and function. Acceleration of membrane phospholipids synthesis is associated with neurite elongation, and phosphatidylcholine (PtdCho) is the major membrane phospholipid in mammalian cells. The transcription of two genes in particular encoding key enzymes in the CDP-choline pathway for PtdCho biosynthesis are stimulated; the Chka gene for choline kinase (CK) alpha isoform and the Pcyt1a gene for the CTP:phosphocholine cytidylyltransferase (CCT) alpha isoform. We report that the stimulation of CKα expression during retinoic acid (RA) induced differentiation depends on a promoter region that contains two CCAAT/Enhancer-binding Protein-β (C/EBPβ) sites. We demonstrate that during neuronal differentiation of Neuro-2a cells, RA induces Chka expression by a mechanism that involves ERK1/2 activation which triggers C/EBPβ expression. Elevated levels of C/EBPβ bind to the Chka proximal promoter (Box1) inducing CKα expression. In addition we identified a downstream sequence named Box2 which together with Box1 is required for the promoter to reach the full induction. This is the first elucidation of the mechanism by which the expression of Chka is coordinately regulated during neuronal differentiation.
View details for DOI 10.1016/j.bbalip.2014.01.007
View details for PubMedID 24440820
Role of phosphatidylcholine during neuronal differentiation.
2011; 63 (9): 714-20
Neuronal differentiation is characterized by neuritogenesis and neurite outgrowth, processes, which are critically dependent on membrane biosynthesis, and therefore, on the expression and regulation of enzymes involved in phospholipid biosynthesis. During the last decade a great effort was made to clarify where membrane lipids are synthesized, how the newly synthesized membrane components reach the membrane and are inserted during neuritogenesis and to elucidate the mechanism by which the supply of new membrane components is coordinated with the demand for growth. Phosphatidylcholine is the principal and essential component for mammalian membranes. This review updates the mechanism by which phosphatidylcholine biosynthesis takes place and how it is coordinately regulated during neuronal differentiation.
View details for DOI 10.1002/iub.521
View details for PubMedID 21818839