Andrea V. Leisewitz

PPAR γ activators induce growth arrest and process extension in B12 oligodendrocyte‐like cells and terminal differentiation of cultured oligodendrocytes

Peroxisome proliferator‐activated receptors (PPARs) are key transcription factors in the control of lipid homeostasis and cell differentiation, but little is known about their function in oligodendrocytes, the major lipid‐synthesizing cells in the central nervous system (CNS). Using the B12 oligodendrocyte‐like cell line and rat spinal cord‐derived oligodendrocytes, we evaluated the importance of PPARγ in the maturation process of these cells. B12 cells express all PPAR isoforms (α, β/δ, and γ), as assessed by RT‐PCR, Western‐blot, and transactivation assays. B12 cells respond specifically to PPARγ agonists by arresting cell proliferation and extending cell processes, events that are blocked by the PPARγ antagonist GW9662. In addition, alkyl‐dihydroxyacetone phosphate synthase (ADAPS), a key peroxisomal enzyme involved in the synthesis of myelin‐rich lipid plasmalogens, is increased in PPARγ agonist‐treated B12 cells. In contrast with B12 cells, both immature and mature isolated spinal cord oligodendrocytes presented a high and similar expression level of ADAPS, as assessed by immunocytochemistry. However, as in B12 cells, isolated spinal cord oligodendrocytes were also found to respond specifically to PPARγ agonists with a four‐fold increase in the number of mature cells. Our data suggest a relevant role for PPARγ in oligodendrocyte lipid metabolism and differentiation.

Targeting Serous Epithelial Ovarian Cancer with Designer Zinc Finger Transcription Factors

Background: There is a need for novel targeted therapies for metastatic ovarian cancers. Results: We reactivated the tumor suppressor Maspin in ovarian carcinoma cells by delivering tumor-specific nanoparticles encapsulating a chemically modified ATF-mRNA. Conclusion: LPR nanoparticles encapsulating the ATF mRNA inhibited ovarian cancer tumor growth in a mouse model. Significance: We report the first non-viral delivery of an ATF in vivo and the discovery of novel anti-metastatic targets for ovarian cancer. Ovarian cancer is the leading cause of death among gynecological malignancies. It is detected at late stages when the disease is spread through the abdominal cavity in a condition known as peritoneal carcinomatosis. Thus, there is an urgent need to develop novel therapeutic interventions to target advanced stages of ovarian cancer. Mammary serine protease inhibitor (Maspin) represents an important metastasis suppressor initially identified in breast cancer. Herein we have generated a sequence-specific zinc finger artificial transcription factor (ATF) to up-regulate the Maspin promoter in aggressive ovarian cancer cell lines and to interrogate the therapeutic potential of Maspin in ovarian cancer. We found that although Maspin was expressed in some primary ovarian tumors, the promoter was epigenetically silenced in cell lines derived from ascites. Transduction of the ATF in MOVCAR 5009 cells derived from ascitic cultures of a TgMISIIR-TAg mouse model of ovarian cancer resulted in tumor cell growth inhibition, impaired cell invasion, and severe disruption of actin cytoskeleton. Systemic delivery of lipid-protamine-RNA nanoparticles encapsulating a chemically modified ATF mRNA resulted in inhibition of ovarian cancer cell growth in nude mice accompanied with Maspin re-expression in the treated tumors. Gene expression microarrays of ATF-transduced cells revealed an exceptional specificity for the Maspin promoter. These analyses identified novel targets co-regulated with Maspin in human short-term cultures derived from ascites, such as TSPAN12, that could mediate the anti-metastatic phenotype of the ATF. Our work outlined the first targeted, non-viral delivery of ATFs into tumors with potential clinical applications for metastatic ovarian cancers.

Bone marrow stromal cells modulate mouse ent1 activity and protect leukemia cells from cytarabine induced apoptosis

Background Despite a high response rate to chemotherapy, the majority of patients with acute myeloid leukemia (AML) are destined to relapse due to residual disease in the bone marrow (BM). The tumor microenvironment is increasingly being recognized as a critical factor in mediating cancer cell survival and drug resistance. In this study, we propose to identify mechanisms involved in the chemoprotection conferred by the BM stroma to leukemia cells. Methods Using a leukemia mouse model and a human leukemia cell line, we studied the interaction of leukemia cells with the BM microenvironment. We evaluated in vivo and in vitro leukemia cell chemoprotection to different cytotoxic agents mediated by the BM stroma. Leukemia cell apoptosis was assessed by flow cytometry and western blotting. The activity of the equilibrative nucleoside transporter 1 (ENT1), responsible for cytarabine cell incorporation, was investigated by measuring transport and intracellular accumulation of 3H-adenosine. Results Leukemia cell mobilization from the bone marrow into peripheral blood in vivo using a CXCR4 inhibitor induced chemo-sensitization of leukemia cells to cytarabine, which translated into a prolonged survival advantage in our mouse leukemia model. In vitro, the BM stromal cells secreted a soluble factor that mediated significant chemoprotection to leukemia cells from cytarabine induced apoptosis. Furthermore, the BM stromal cell supernatant induced a 50% reduction of the ENT1 activity in leukemia cells, reducing the incorporation of cytarabine. No protection was observed when radiation or other cytotoxic agents such as etoposide, cisplatin and 5-fluorouracil were used. Conclusion The BM stroma secretes a soluble factor that significantly protects leukemia cells from cytarabine-induced apoptosis and blocks ENT1 activity. Strategies that modify the chemo-protective effects mediated by the BM microenvironment may enhance the benefit of conventional chemotherapy for patients with AML.

A PPARs cross-talk concertedly commits C6 glioma cells to oligodendrocytes and induces enzymes involved in myelin synthesis.

Peroxisome proliferator activated receptors (PPARs, α, β/δ, γ) control lipid homeostasis and differentiation in various tissues and tumor cells. PPARβ and PPARγ increase oligodendrocyte maturation in glial mixed populations and spinal cord oligodendrocytes, respectively, and PPARβ is known to modulate the activity of other PPARs. To assess a possible interaction between PPARs in glial cell differentiation we used the undifferentiated C6 glioma cell line as model. These cells express all three PPARs, but only PPARγ shows transcriptional activity in agonist‐based reporter gene assay. Agonist‐activated PPARγ up‐regulates oligodendrocyte markers, down‐regulates an astrocyte marker, and increases alkyl‐dihydroxyacetone phosphate synthase, enzyme involved in the synthesis of myelin‐rich plasmalogens. Similar effects are induced in PPARγ overexpressing cells, which in addition show PPARβ up‐regulation. PPARβ or PPARα agonists show no effect. Nevertheless, PPARβ overexpression up‐regulates PPARγ and commits C6 cells to oligodendrocytes; effect that is abrogated by a PPARγ antagonist or PPARγ interference RNA. Moreover, PPARβ overexpression also induces PPARα and its target genes, including acyl‐CoA oxidase, enzyme involved in very long chain fatty acid recycling, and in the synthesis of myelin components such as docosahexaenoic acid. These results indicate for the first time, that PPARs concertedly cooperate in C6 glioma cell differentiation to oligodendrocytes. Further, they suggest that active PPARβ might be essential for increasing oligodendrocyte distinctive markers and enzymes required for myelin synthesis in C6 glioma cells through up‐regulation of PPARγ and PPARα. J. Cell. Physiol. 217: 367–376, 2008.

Waking up dormant tumor suppressor genes with zinc fingers, TALEs and the CRISPR/dCas9 system

The aberrant epigenetic silencing of tumor suppressor genes (TSGs) plays a major role during carcinogenesis and regaining these dormant functions by engineering of sequence-specific epigenome editing tools offers a unique opportunity for targeted therapies. However, effectively normalizing the expression and regaining tumor suppressive functions of silenced TSGs by artificial transcription factors (ATFs) still remains a major challenge. Herein we describe novel combinatorial strategies for the potent reactivation of two class II TSGs, MASPIN and REPRIMO, in cell lines with varying epigenetic states, using the CRISPR/dCas9 associated system linked to a panel of effector domains (VP64, p300, VPR and SAM complex), as well as with protein-based ATFs, Zinc Fingers and TALEs. We found that co-delivery of multiple effector domains using a combination of CRISPR/dCas9 and TALEs or SAM complex maximized activation in highly methylated promoters. In particular, CRISPR/dCas9 VPR with SAM upregulated MASPIN mRNA (22,145-fold change) in H157 lung cancer cells, with accompanying re-expression of MASPIN protein, which led to a concomitant inhibition of cell proliferation and induction of apoptotic cell death. Consistently, CRISPR/dCas9 VP64 with SAM upregulated REPRIMO (680-fold change), which led to phenotypic reprogramming in AGS gastric cancer cells. Altogether, our results outlined novel sequence-specific, combinatorial epigenome editing approaches to reactivate highly methylated TSGs as a promising therapy for cancer and other diseases.

PPARα and PPARγ regulate the nucleoside transporter hENT1.

Human equilibrative nucleoside transporter 1 (hENT1) is an important determinant for nucleoside analog based chemotherapy success. Preliminary data suggest hENT1 regulation by PPARs. Using A2780 cells, we investigated the role of PPARs on hENT1 expression and activity. PPARα and PPARγ agonists, Wy14,643 and RGZ, increased hENT1 expression, but only PPARα activation or overexpression resulted in higher hENT1 transport activity. On the other hand, promoter analysis showed two putative PPRE in hENT1 promoter and luciferase-coupled promoter constructs were generated and analyzed. Our results suggest that PPARα-but not PPARγ-mediated expression regulation of hENT1 is PPRE-dependent. In conclusion, PPARα and PPARγ activation modulate hENT1 expression.

The CCL2/CCR2 Axis Affects Transmigration and Proliferation but Not Resistance to Chemotherapy of Acute Myeloid Leukemia Cells

Acute myeloid leukemia (AML) has a high mortality rate despite chemotherapy and transplantation. Both CXCR4/SDF-1 and VLA-4/VCAM1 axes are involved in leukemia protection but little is known about the role of CCL2/CCR2 in AML biology and protection against chemotherapy. We measured CCR2 expression in AML cell lines and primary AML cells by flow cytometry (FCM), real time PCR (RT-PCR) and western blot (WB). CCL2 production was quantified by solid phase ELISA in peripheral blood (PB) and bone marrow (BM) serum. We measured chemotaxis in a transwell system with different concentrations of CCL2/CCR2 blockers; cell cycle with BrDU and propidium iodide and proliferation with yellow tetrazolium MTT. We determined synergy in in vitro cell apoptosis combining chemotherapy and CCL2/CCR2 blockade. Finally, we performed chemoprotection studies in an in vivo mouse model. Of 35 patients, 23 (65%) expressed CCR2 by FCM in PB. Two cell lines expressed high levels of CCR2 (THP-1 and murine AML). RT-PCR and WB confirmed CCR2 production. CCL2 solid phase ELISA showed significantly lower levels of CCL2 in PB and BM compared to normal controls. Chemotaxis experiments confirmed a dose-dependent migration in AML primary cells expressing CCR2 and THP-1 cells. A significant inhibition of transmigration was seen after CCL2/CCR2 blockade. Proliferation of CCR2+ AML cell lines was slightly increased (1.4-fold) after axis stimulation. We observed a non-significant increase in phase S THP-1 cells exposed to CCL2 and a concomitant decrease of cells in G1. The chemotherapy studies did not show a protective effect of CCL2 on cytarabine-induced apoptosis or synergy with chemotherapy after CCL2/CCR2 blockade both in vitro and in vivo. In conclusion, CCL2/CCR2 axis is expressed in the majority of monocytoid AML blasts. The axis is involved in cell trafficking and proliferation but no in vitro and in vivo chemotherapy protective effect was seen.

Reusing and composing models of cell fate regulation of human bone precursor cells

In order to treat osteoporosis and other bone mass disorders it is necessary to understand the regulatory processes that control the cell fate decisions responsible for going from bone precursor cells to bone tissue. Many processes interact to regulate cell division, differentiation and apoptosis. There are models for these basic processes, but not for their interactions. In this work we use the theory of switched systems, reuse and composition of validated models to describe the cell fate decisions leading to bone and fat formation. We describe the differentiation of osteo-adipo progenitor cells by composing its model with differentiation stimuli. We use the activation of the Wnt pathway as stimulus to osteoblast lineage, including regulation of cell division and apoptosis. This model is our first step to simulate physiological responses in silico to treatments for bone mass disorders.

Su1991 Reactivation of Reprimo by an Artificial Transcription Factor in Gastric Cancer Cell Lines

Introduction: Gastric cancer (GC) is the second cause of cancer-related death worldwide and the leading one in Latin America. It has been proposed that the silencing of Reprimo (RPRM), by epigenetic mechanisms, is a putative biomarker of GC. RPRM is a p53 dependent G2 arrest mediator and is considered to be tumor suppressor gene involved in the pathogenesis of numerous cancers such as gastric, colon, pituitary, among others. We propose that the reactivation of RPRM expression, by means of an activator Artificial Transcription Factor (ATF), modifies the proliferation rate of GC cell lines in vitro. Methods: An activator ATF for RPRM was designed using the method proposed by zincfingertools.org. Thus, an ATF with a unique domain of six zinc-finger capable of binding specifically to 18 consecutive base pairs, located in the promoter region of RPRM, was engineered. This ATF was then inserted into a modified pcDNA3.1+ vector coupled with a potent transcription activator domain (VP64). A luciferase reporter assay was performed using a pGL3 vector (Promega), linked to a 634 base pairs region of the promoter region of RPRM, in order to evaluate the efficiency and specificity of the synthetized ATF. Finally, the ATF was transiently transfected with lipofectamine2000 (Invitrogen). Subsequently, RPRM mRNA expression was evaluated by a RT-qPCR and proliferation rate was evaluated by a MTS assay. The experiments were conducted on AGS and MKN28 GC cell lines. Results: The ATF designed was able to significantly increase the luciferase activity vs controls, both in MKN28 and AGS cell lines. Accordingly, RPRM mRNA was upregulated in the presence of the ATF. These findings were associated with a lower proliferation rate in both cell lines. Since the AGS cell line is highly methylated in the promoter region of RPRM, it might explain a significantly lower response to the presence of ATF, in comparison with that of MKN28. Discussion: Our work suggests that a RPRM specific ATF could be used as a novel tool to reactivate this epigenetically silenced tumor suppressor gene. Moreover, the upregulation of RPRM modifies one of the hallmarks of cancer pathways by reducing the proliferation rate of cancerous cell lines in vitro. Grant Support: Fondecyt #1111014 and Fondef #D09I1137 from the Government of Chile to AHC.