Mirk/Dyrk1B Kinase Inhibitors in Targeted Cancer Therapy.

During the last years, there has been an increased effort in the discovery of selective and potent kinase inhibitors for targeted cancer therapy. Kinase inhibitors exhibit less toxicity compared to conventional chemotherapy, and several have entered the market. Mirk/Dyrk1B kinase is a promising pharmacological target in cancer since it is overexpressed in many tumors, and its overexpression is correlated with patients' poor prognosis. Mirk/Dyrk1B acts as a negative cell cycle regulator, maintaining the survival of quiescent cancer cells and conferring their resistance to chemotherapies. Many studies have demonstrated the valuable therapeutic effect of Mirk/Dyrk1B inhibitors in cancer cell lines, mouse xenografts, and patient-derived 3D-organoids, providing a perspective for entering clinical trials. Since the majority of Mirk/Dyrk1B inhibitors target the highly conserved ATP-binding site, they exhibit off-target effects with other kinases, especially with the highly similar Dyrk1A. In this review, apart from summarizing the data establishing Dyrk1B as a therapeutic target in cancer, we highlight the most potent Mirk/Dyrk1B inhibitors recently reported. We also discuss the limitations and perspectives for the structure-based design of Mirk/Dyrk1B potent and highly selective inhibitors based on the accumulated structural data of Dyrk1A and the recent crystal structure of Dyrk1B with AZ191 inhibitor.

A miR-124-mediated post-transcriptional mechanism controlling the cell fate switch of astrocytes to induced neurons.

The microRNA (miRNA) miR-124 has been employed supplementary to neurogenic transcription factors (TFs) and other miRNAs to enhance direct neurogenic conversion. The aim of this study was to investigate whether miR-124 is sufficient to drive direct reprogramming of astrocytes to induced neurons (iNs) on its own and elucidate its independent mechanism of reprogramming action. Our data show that miR-124 is a potent driver of the reprogramming switch of astrocytes toward an immature neuronal fate by directly targeting the RNA-binding protein Zfp36L1 implicated in ARE-mediated mRNA decay and subsequently derepressing Zfp36L1 neurogenic interactome. To this end, miR-124 contribution in iNs' production largely recapitulates endogenous neurogenesis pathways, being further enhanced upon addition of the neurogenic compound ISX9, which greatly improves iNs' differentiation and functional maturation. Importantly, miR-124 is potent in guiding direct conversion of reactive astrocytes to immature iNs in vivo following cortical trauma, while ISX9 supplementation confers a survival advantage to newly produced iNs.

MicroRNA-934 is a novel primate-specific small non-coding RNA with neurogenic function during early development.

Integrating differential RNA and miRNA expression during neuronal lineage induction of human embryonic stem cells we identified miR-934, a primate-specific miRNA that displays a stage-specific expression pattern during progenitor expansion and early neuron generation. We demonstrate the biological relevance of this finding by comparison with data from early to mid-gestation human cortical tissue. Further we find that miR-934 directly controls progenitor to neuroblast transition and impacts on neurite growth of newborn neurons. In agreement, miR-934 targets are involved in progenitor proliferation and neuronal differentiation whilst miR-934 inhibition results in profound global transcriptome changes associated with neurogenesis, axonogenesis, neuronal migration and neurotransmission. Interestingly, miR-934 inhibition affects the expression of genes associated with the subplate zone, a transient compartment most prominent in primates that emerges during early corticogenesis. Our data suggest that mir-934 is a novel regulator of early human neurogenesis with potential implications for a species-specific evolutionary role in brain function.

Minibrain-related kinase/dual-specificity tyrosine-regulated kinase 1B implication in stem/cancer stem cells biology.

Dual-specificity tyrosine phosphorylation-regulated kinase 1B (DYRK1B), also known as minibrain-related kinase (MIRK) is one of the best functionally studied members of the DYRK kinase family. DYRKs comprise a family of protein kinases that are emerging modulators of signal transduction pathways, cell proliferation and differentiation, survival, and cell motility. DYRKs were found to participate in several signaling pathways critical for development and cell homeostasis. In this review, we focus on the DYRK1B protein kinase from a functional point of view concerning the signaling pathways through which DYRK1B exerts its cell type-dependent function in a positive or negative manner, in development and human diseases. In particular, we focus on the physiological role of DYRK1B in behavior of stem cells in myogenesis, adipogenesis, spermatogenesis and neurogenesis, as well as in its pathological implication in cancer and metabolic syndrome. Thus, understanding of the molecular mechanisms that regulate signaling pathways is of high importance. Recent studies have identified a close regulatory connection between DYRK1B and the hedgehog (HH) signaling pathway. Here, we aim to bring together what is known about the functional integration and cross-talk between DYRK1B and several signaling pathways, such as HH, RAS and PI3K/mTOR/AKT, as well as how this might affect cellular and molecular processes in development, physiology, and pathology. Thus, this review summarizes the major known functions of DYRK1B kinase, as well as the mechanisms by which DYRK1B exerts its functions in development and human diseases focusing on the homeostasis of stem and cancer stem cells.

Cend1, a Story with Many Tales: From Regulation of Cell Cycle Progression/Exit of Neural Stem Cells to Brain Structure and Function.

Neural stem/precursor cells (NPCs) generate the large variety of neuronal phenotypes comprising the adult brain. The high diversity and complexity of this organ have its origin in embryonic life, during which NPCs undergo symmetric and asymmetric divisions and then exit the cell cycle and differentiate to acquire neuronal identities. During these processes, coordinated regulation of cell cycle progression/exit and differentiation is essential for generation of the appropriate number of neurons and formation of the correct structural and functional neuronal circuits in the adult brain. Cend1 is a neuronal lineage-specific modulator involved in synchronization of cell cycle exit and differentiation of neuronal precursors. It is expressed all along the neuronal lineage, from neural stem/progenitor cells to mature neurons, and is associated with the dynamics of neuron-generating divisions. Functional studies showed that Cend1 has a critical role during neurogenesis in promoting cell cycle exit and neuronal differentiation. Mechanistically, Cend1 acts via the p53-dependent/Cyclin D1/pRb signaling pathway as well as via a p53-independent route involving a tripartite interaction with RanBPM and Dyrk1B. Upon Cend1 function, Notch1 signaling is suppressed and proneural genes such as Mash1 and Neurogenins 1/2 are induced. Due to its neurogenic activity, Cend1 is a promising candidate therapeutic gene for brain repair, while the Cend1 minimal promoter is a valuable tool for neuron-specific gene delivery in the CNS. Mice with Cend1 genetic ablation display increased NPC proliferation, decreased migration, and higher levels of apoptosis during development. As a result, they show in the adult brain deficits in a range of motor and nonmotor behaviors arising from irregularities in cerebellar cortex lamination and impaired Purkinje cell differentiation as well as a paucity in GABAergic interneurons of the cerebral cortex, hippocampus, and amygdala. Taken together, these studies highlight the necessity for Cend1 expression in the formation of a structurally and functionally normal brain.

Expression of Mammalian BM88/CEND1 in Drosophila Affects Nervous System Development by Interfering with Precursor Cell Formation.

We used Drosophila melanogaster as an experimental model to express mouse and pig BM88/CEND1 (cell cycle exit and neuronal differentiation 1) in order to investigate its potential functional effects on Drosophila neurogenesis. BM88/CEND1 is a neuron-specific protein whose function is implicated in triggering cells to exit from the cell cycle and differentiate towards a neuronal phenotype. Transgenic flies expressing either mouse or pig BM88/CEND1 in the nervous system had severe neuronal phenotypes with variable expressivity at various stages of embryonic development. In early embryonic stage 10, BM88/CEND1 expression led to an increase in the neural-specific antigenicity of neuroectoderm at the expense of precursor cells [neuroblasts (Nbs) and ganglion mother cells (GMCs)] including the defective formation and differentiation of the MP2 precursors, whereas at later stages (12-15), protein accumulation induced gross morphological defects primarily in the CNS accompanied by a reduction of Nb and GMC markers. Furthermore, the neuronal precursor cells of embryos expressing BM88/CEND1 failed to carry out proper cell-cycle progression as revealed by the disorganized expression patterns of specific cell-cycle markers. BM88/CEND1 accumulation in the Drosophila eye affected normal eye disc development by disrupting the ommatidia. Finally, we demonstrated that expression of BM88/CEND1 modified/reduced the levels of activated MAP kinase indicating a functional effect of BM88/CEND1 on the MAPK signaling pathway. Our findings suggest that the expression of mammalian BM88/CEND1 in Drosophila exerts specific functional effects associated with neuronal precursor cell formation during embryonic neurogenesis and proper eye disc development. This study also validates the use of Drosophila as a powerful model system in which to investigate gene function and the underlying molecular mechanisms.

A novel regulatory role of RGS4 in STAT5B activation, neurite outgrowth and neuronal differentiation.

The Regulator of G protein Signalling 4 (RGS4) is a multitask protein that interacts with and negatively modulates opioid receptor signalling. Previously, we showed that the δ-opioid receptor (δ-OR) forms a multiprotein signalling complex consisting of Gi/Go proteins and the Signal Transducer and Activator of Transcription 5B (STAT5B) that leads to neuronal differentiation and neurite outgrowth upon δ-ΟR activation. Here, we investigated whether RGS4 could participate in signalling pathways to regulate neurotropic events. We demonstrate that RGS4 interacts directly with STAT5B independently of δ-ΟR presence both in vitro and in living cells. This interaction involves the N-terminal portion of RGS4 and the DNA-binding SH3 domain of STAT5B. Expression of RGS4 in HEK293 cells expressing δ-OR and/or erythropoietin receptor results in inhibition of [D-Ser2, Leu5, Thr6]-enkephalin (DSLET)-and erythropoietin-dependent STAT5B phosphorylation and subsequent transcriptional activation. DSLET-dependent neurite outgrowth of neuroblastoma cells is also blocked by RGS4 expression, whereas primary cortical cultures of RGS4 knockout mice (RGS4-/-) exhibit enhanced neuronal sprouting after δ-OR activation. Additional studies in adult brain extracts from RGS4-/- mice revealed increased levels of p-STAT5B. Finally, neuronal progenitor cultures from RGS4-/- mice exhibit enhanced proliferation with concomitant increases in the mRNA levels of the anti-apoptotic STAT5B target genes bcl2 and bcl-xl. These observations suggest that RGS4 is implicated in opioid dependent neuronal differentiation and neurite outgrowth via a "non-canonical" signaling pathway regulating STAT5B-directed responses.

Hepatitis C virus modulates lipid regulatory factor Angiopoietin-like 3 gene expression by repressing HNF-1α activity.

BACKGROUND & AIMS: HCV relies on host lipid metabolism to complete its life cycle and HCV core is crucial to this interaction. Liver secreted ANGPTL-3 is an LXR- and HNF-1α-regulated protein, which plays a key role in lipid metabolism by increasing plasma lipids via inhibition of lipase enzymes. Here we aimed to investigate the modulation of ANGPTL-3 by HCV core and identify the molecular mechanisms involved. METHODS: qRT-PCR and ELISA were used to assess ANGPTL-3 mRNA and protein levels in HCV patients, the JFH-1 infectious system and liver cell lines. Transfections, chromatin immunoprecipitation and immunofluorescence delineated parts of the molecular mechanisms implicated in the core-mediated regulation of ANGPTL-3 gene expression. RESULTS: ANGPTL-3 gene expression was decreased in HCV-infected patients and the JFH-1 infectious system. mRNA and promoter activity levels were down-regulated by core. The response was lost when an HNF-1α element in ANGPTL-3 promoter was mutated, while loss of HNF-1α DNA binding to this site was recorded in the presence of HCV core. HNF-1α mRNA and protein levels were not altered by core. However, trafficking between nucleus and cytoplasm was observed and then blocked by an inhibitor of the HNF-1α-specific kinase Mirk/Dyrk1B. Transactivation of LXR/RXR signalling could not restore core-mediated down-regulation of ANGPTL-3 promoter activity. CONCLUSIONS: ANGPTL-3 is negatively regulated by HCV in vivo and in vitro. HCV core represses ANGPTL-3 expression through loss of HNF-1α binding activity and blockage of LXR/RXR transactivation. The putative ensuing increase in serum lipid clearance and uptake by the liver may sustain HCV virus replication and persistence.

Functional Interactions between BM88/Cend1, Ran-binding protein M and Dyrk1B kinase affect cyclin D1 levels and cell cycle progression/exit in mouse neuroblastoma cells.

BM88/Cend1 is a neuronal-lineage specific modulator with a pivotal role in coordination of cell cycle exit and differentiation of neuronal precursors. In the current study we identified the signal transduction scaffolding protein Ran-binding protein M (RanBPM) as a BM88/Cend1 binding partner and showed that BM88/Cend1, RanBPM and the dual specificity tyrosine-phosphorylation regulated kinase 1B (Dyrk1B) are expressed in mouse brain as well as in cultured embryonic cortical neurons while RanBPM can form complexes with either of the two other proteins. To elucidate a potential mechanism involving BM88/Cend1, RanBPM and Dyrk1B in cell cycle progression/exit, we transiently co-expressed these proteins in mouse neuroblastoma Neuro 2a cells. We found that the BM88/Cend1-dependent or Dyrk1B-dependent down-regulation of cyclin D1 is reversed following their functional interaction with RanBPM. More specifically, functional interaction of RanBPM with either BM88/Cend1 or Dyrk1B stabilizes cyclin D1 in the nucleus and promotes 5-bromo-2'-deoxyuridine (BrdU) incorporation as a measure of enhanced cell proliferation. However, the RanBPM-dependent Dyrk1B cytosolic retention and degradation is reverted in the presence of Cend1 resulting in cyclin D1 destabilization. Co-expression of RanBPM with either BM88/Cend1 or Dyrk1B also had a negative effect on Neuro 2a cell differentiation. Our results suggest that functional interactions between BM88/Cend1, RanBPM and Dyrk1B affect the balance between cellular proliferation and differentiation in Neuro 2a cells and indicate that a potentially similar mechanism may influence cell cycle progression/exit and differentiation of neuronal precursors.

δ-opioid receptor activation leads to neurite outgrowth and neuronal differentiation via a STAT5B-Gαi/o pathway.

It remains unclear how opioid receptors (δ, µ, κ) are implicated in mechanisms controlling differentiation, cell proliferation, and survival. Opioid receptors are coupled to Gi/Go proteins and recent findings have shown that opioid receptors can form a multicomponent signaling complex, consisting of members of G protein and the signal transducer and activator of transcription (STAT)5B. We thus wondered whether activation of the opioid receptors could direct differentiation and neurite outgrowth through a molecular pathway involving STAT5B and other signaling intermediates. We demonstrate that prolonged δ-opioid receptor (δ-OR) activation with opioid agonists induces STAT5B phosphorylation in Neuro-2A cells. Moreover, [D-Ser2, Leu5, Thr6]-enkephalin-activation of δ-OR triggers neurite outgrowth and neuronal survival; these effects are blocked by the selective antagonist naltrindole, by treatment with pertussis toxin, and after expression of a dominant negative mutant of STAT5B (DN-STAT5B), suggesting that the signaling pathway participating in this mechanism involves Gi/o proteins and p-STAT5B. Additional studies have shown that while [D-Ser(2) , Leu(5) , Thr(6) ]-enkephalin exposure of neuroblastoma cells induces a marked increase in the differentiation marker proteins, ßIII-tubulin (Tuj-1), synaptophysin, and neural cell adhesion molecule, over-expression of the DN-STAT5B attenuated significantly their expression levels. Taken together, our findings demonstrate that δ-OR activation leads to a number of neurotropic events via a Gαi/o-linked and STAT5B-dependent manner. We propose a novel signalling pathway for δ-opioid receptor (δ-ΟR)-mediated neurotropic events. STAT5B interacts with the δ-ΟR and upon prolonged receptor activation phosphorylates STAT5B in a Gi/Go dependent manner leading to increased neuronal survival, neurite outgrowth and differentiation. These findings contribute to a better understanding of the molecular and cellular events following δ-OR activation and suggest a possible neuroprotective role opioids could exert.

Soluble forms of the cell adhesion molecule L1 produced by insect and baculovirus-transduced mammalian cells enhance Schwann cell motility.

For biotechnological applications, insect cell lines are primarily known as hosts for the baculovirus expression system that is capable to direct synthesis of high levels of recombinant proteins through use of powerful viral promoters. Here, we demonstrate the implementation of two alternative approaches based on the baculovirus system for production of a mammalian recombinant glycoprotein, comprising the extracellular part of the cell adhesion molecule L1, with potential important therapeutic applications in nervous system repair. In the first approach, the extracellular part of L1 bearing a myc tag is produced in permanently transformed insect cell lines and purified by affinity chromatography. In the second approach, recombinant baculoviruses that express L1-Fc chimeric protein, derived from fusion of the extracellular part of L1 with the Fc part of human IgG1, under the control of a mammalian promoter are used to infect mammalian HEK293 and primary Schwann cells. Both the extracellular part of L1 bearing a myc tag accumulating in the supernatants of insect cultures as well as L1-Fc secreted by transduced HEK293 or Schwann cells are capable of increasing the motility of Schwann cells with similar efficiency in a gap bridging bioassay. In addition, baculovirus-transduced Schwann cells show enhanced motility when grafted on organotypic cultures of neonatal brain slices while they retain their ability to myelinate CNS axons. This proof-of-concept that the migratory properties of myelin-forming cells can be modulated by recombinant protein produced in insect culture as well as by means of baculovirus-mediated adhesion molecule expression in mammalian cells may have beneficial applications in the field of CNS therapies.

Lentivirus-mediated expression of insulin-like growth factor-I promotes neural stem/precursor cell proliferation and enhances their potential to generate neurons.

Strategies to enhance neural stem/precursor cell (NPC) capacity to yield multipotential, proliferative, and migrating pools of cells that can efficiently differentiate into neurons could be crucial for structural repair after neurodegenerative damage. Here, we have generated a lentiviral vector for expression of insulin-like growth factor-I (IGF-1) and investigated the impact of IGF-1 transduction on the properties of cultured NPCs (IGF-1-NPCs). Under proliferative conditions, IGF-1 transduction promoted cell cycle progression via cyclin D1 up-regulation and Akt phosphorylation. Remarkably upon differentiation-inducing conditions, IGF-1-NPCs cease to proliferate and differentiate to a greater extent into neurons with significantly longer neurites, at the expense of astrocytes. Moreover, using live imaging we provide evidence that IGF-1 transduction enhances the motility and tissue penetration of grafted NPCs in cultured cortical slices. These results illustrate the important consequence of IGF-1 transduction in regulating NPC functions and offer a potential strategy to enhance the prospective repair potential of NPCs.

Leishmania donovani Ran-GTPase interacts at the nuclear rim with linker histone H1.

Ran-GTPase regulates multiple cellular processes such as nucleocytoplasmic transport, mitotic spindle assembly, nuclear envelope assembly, cell-cycle progression and the mitotic checkpoint. The leishmanial Ran protein, in contrast with its mammalian counterpart which is predominately nucleoplasmic, is localized at the nuclear rim. The aim of the present study was to characterize the LdRan (Leishmania donovani Ran) orthologue with an emphasis on the Ran-histone association. LdRan was found to be developmentally regulated, expressed 3-fold less in the amastigote stage. LdRan overexpression caused a growth defect linked to a delayed S-phase progression in promastigotes as for its mammalian counterpart. We report for the first time that Ran interacts with a linker histone, histone H1, in vitro and that the two proteins co-localize at the parasite nuclear rim. Interaction of Ran with core histones H3 and H4, creating in metazoans a chromosomal Ran-GTP gradient important for mitotic spindle assembly, is speculative in Leishmania spp., not only because this parasite undergoes a closed mitosis, but also because the main localization of LdRan is different from that of core histone H3. Interaction of Ran with the leishmanial linker histone H1 (LeishH1) suggests that this association maybe involved in modulation of pathways other than those documented for the metazoan Ran-core histone association.

BM88/Cend1 expression levels are critical for proliferation and differentiation of subventricular zone-derived neural precursor cells.

Neural stem cells remain in two areas of the adult mammalian brain, the subventricular zone (SVZ) and the dentate gyrus of the hippocampus. Ongoing neurogenesis via the SVZ-rostral migratory stream pathway maintains neuronal replacement in the olfactory bulb (OB) throughout life. The mechanisms determining how neurogenesis is restricted to only a few regions in the adult, in contrast to its more widespread location during embryogenesis, largely depend on controlling the balance between precursor cell proliferation and differentiation. BM88/Cend1 is a neuronal lineage-specific regulator implicated in cell cycle exit and differentiation of precursor cells in the embryonic neural tube. Here we investigated its role in postnatal neurogenesis. Study of in vivo BM88/Cend1 distribution revealed that it is expressed in low levels in neuronal precursors of the adult SVZ and in high levels in postmitotic OB interneurons. To assess the functional significance of BM88/Cend1 in neuronal lineage progression postnatally, we challenged its expression levels by gain- and loss-of-function approaches using lentiviral gene transfer in SVZ-derived neurospheres. We found that BM88/Cend1 overexpression decreases proliferation and favors neuronal differentiation, whereas its downregulation using new-generation RNA interference vectors yields an opposite phenotype. Our results demonstrate that BM88/Cend1 participates in cell cycle control and neuronal differentiation mechanisms during neonatal SVZ neurogenesis and becomes crucial for the transition from neuroblasts to mature neurons when reaching high levels.

A critical role for HSP90 in cancer cell invasion involves interaction with the extracellular domain of HER-2.

HSP90 is a ubiquitously expressed molecular chaperone that controls the folding, assembly, intracellular disposition, and proteolytic turnover of many proteins, most of which are involved in signal transduction processes. Recently, a surface form of HSP90 has been identified and associated with cell migration events. In this paper, we explore the interaction of surface HSP90 with HER-2, a receptor-like glycoprotein and member of the ErbB family of receptor tyrosine kinases that play central roles in cellular proliferation, differentiation, and migration as well as in cancer progress. The involvement of HSP90 in the regulation of HER-2 has been attributed so far to receptor stabilization via interaction with its cytoplasmic kinase domain. Here we present evidence, using glutathione S-transferase pull-down and transfection assays, for a novel interaction between surface HSP90 and the extracellular domain of HER-2. Specific disruption of this interaction using mAb 4C5, a function-blocking monoclonal antibody against HSP90, inhibits cell invasion accompanied by altered actin dynamics in human breast cancer cells under ligand stimulation conditions with heregulin. Additionally, disruption of surface HSP90/HER-2 interaction leads to inhibition of heregulin-induced HER-2-HER-3 heterodimer formation, reduced HER-2 phosphorylation, and impaired downstream kinase signaling. Interestingly, this disruption does not affect HER-2 internalization. Our data suggest that surface HSP90 is involved in heregulin-induced HER-2 activation and signaling, leading to cytoskeletal rearrangement, essential for cell invasion.

BM88 is a dual function molecule inducing cell cycle exit and neuronal differentiation of neuroblastoma cells via cyclin D1 down-regulation and retinoblastoma protein hypophosphorylation.

Control of cell cycle progression/exit and differentiation of neuronal precursors is of paramount importance during brain development. BM88 is a neuronal protein associated with terminal neuron-generating divisions in vivo and is implicated in mechanisms underlying neuronal differentiation. Here we have used mouse neuroblastoma Neuro 2a cells as an in vitro model of neuronal differentiation to dissect the functional properties of BM88 by implementing gain- and loss-of-function approaches. We demonstrate that stably transfected cells overexpressing BM88 acquire a neuronal phenotype in the absence of external stimuli, as judged by enhanced expression of neuronal markers and neurite outgrowth-inducing signaling molecules. In addition, cell cycle measurements involving cell growth assays, BrdUrd incorporation, and fluorescence-activated cell sorting analysis revealed that the BM88-transfected cells have a prolonged G(1) phase, most probably corresponding to cell cycle exit at the G(0) restriction point, as compared with controls. BM88 overexpression also results in increased levels of the cell cycle regulatory protein p53, and accumulation of the hypophosphorylated form of the retinoblastoma protein leading to cell cycle arrest, with concomitant decreased levels and, in many cells, cytoplasmic localization of cyclin D1. Conversely, BM88 gene silencing using RNA interference experiments resulted in acceleration of cell proliferation accompanied by impairment of retinoic acid-induced neuronal differentiation of Neuro 2a cells. Taken together, our results suggest that BM88 plays an essential role in regulating cell cycle exit and differentiation of Neuro 2a cells toward a neuronal phenotype and further support its involvement in the proliferation/differentiation transition of neural stem/progenitor cells during embryonic development.

Baculovirus-mediated gene delivery into Mammalian cells does not alter their transcriptional and differentiating potential but is accompanied by early viral gene expression.

Gene delivery to neural cells is central to the development of transplantation therapies for neurological diseases. In this study, we used a baculovirus derived from the domesticated silk moth, Bombyx mori, as vector for transducing a human cell line (HEK293) and primary cultures of rat Schwann cells. Under optimal conditions of infection with a recombinant baculovirus containing the reporter green fluorescent protein gene under mammalian promoter control, the infected cells express the transgene with high efficiency. Toxicity assays and transcriptome analyses suggest that baculovirus infection is not cytotoxic and does not induce differential transcriptional responses in HEK293 cells. Infected Schwann cells retain their characteristic morphological and molecular phenotype as determined by immunocytochemistry for the marker proteins S-100, glial fibrillary acidic protein, and p75 nerve growth factor receptor. Moreover, baculovirus-infected Schwann cells are capable of differentiating in vitro and express the P0 myelination marker. However, transcripts for several immediate-early viral genes also accumulate in readily detectable levels in the transduced cells. This transcriptional activity raises concerns regarding the long-term safety of baculovirus vectors for gene therapy applications. Potential approaches for overcoming the identified problem are discussed.