Targeting CD133 improves chemotherapeutic efficacy of recurrent pediatric pilocytic astrocytoma following prolonged chemotherapy.

BACKGROUND: Pilocytic astrocytomas (PAs) are the most common pediatric central nervous system neoplasms. In the majority of cases these tumors are benign and receive favorable prognosis following gross total surgical resection. In patients with progressive or symptomatic tumors, aggressive surgical resection is generally not feasible, thus radiation or chemotherapy are accepted initial or adjuvant interventions. Due to serious long-lasting side-effects, radiation is limited in young children; therefore, chemotherapy is widely practiced as an adjuvant treatment for these patients. However, chemotherapy can promote the emergence of multidrug resistant tumor cells that are more malignant than those of the original tumor. CD133, a putative stem cell marker in normal tissue and malignant brain tumors, enhances multidrug resistant gene 1 (MDR1) expression following chemotherapy in adult malignant glioblastomas. This study examines the relationship between CD133 and MDR1 in pediatric PAs exposed to chemotherapy, with the goal of identifying therapeutic targets that manifest as a result of chemotherapy. METHODS: Slides were obtained for 15 recurrent PAs, seven of which had received chemotherapy prior to surgical treatment for the recurrent tumor. These samples, as well as primary tumor tissue slides from the same patients were used to investigate CD133 and MDR1 expression via immunofluorescence. Archived frozen tissue samples from the same patients were used to examine CD133, MDR1 and PI3K-Akt-NF-κB signaling mediators, via western blot. Two drug resistant pediatric PA cell lines Res186 and Res199 were also used to evaluate the role of CD133 on cell response to cytotoxic therapy. RESULTS: CD133 and MDR1 were co-expressed and their expression was elevated in recurrent PAs from patients that had received chemotherapy, compared to patients that had not received chemotherapy. PI3K-Akt-NF-κB signaling mediator expression was also elevated in recurrent, chemotherapy-treated PA. Suppressing CD133 expression with siCD133 decreased levels of PI3K-Akt-NF-κB signaling mediators and MDR1, while increasing cell chemosensitivity, as indicated by quantification of apoptotic cells following chemotherapy. CONCLUSIONS: CD133 contributes to multidrug resistance by regulating MDR1 levels via the PI3K-Akt-NF-κB signal pathway not only in adult glioblastomas, but also in pediatric PAs. Targeting CD133, adjuvant to conventional chemotherapy may improve outcomes for children with recurrent PA.

Folate Receptor Alpha Upregulates Oct4, Sox2 and Klf4 and Downregulates miR-138 and miR-let-7 in Cranial Neural Crest Cells.

Prenatal folic acid (FA) supplementation prevents neural tube defects. Folate receptor alpha (FRα) is critical for embryonic development, including neural crest (NC) development. Previously we showed that FRα translocates to the nucleus in response to FA, where it acts as a transcription factor. In this study, we examined if FA through interaction with FRα regulates stem cell characteristics of cranial neural crest cells (CNCCs)-critical for normal development. We hypothesized that FRα upregulates coding genes and simultaneously downregulates non-coding miRNA which targets coding genes in CNCCs. Quantitative RT-PCR and chromatin immunoprecipitation showed that FRα upregulates Oct4, Sox2, and Klf4 by binding to their cis-regulator elements-5' enhancer/promoters defined by H3K27Ac and p300 occupancy. FA via FRα downregulates miRNAs, miR-138 and miR-let-7, which target Oct4 and Trim71 (an Oct4 downstream effector), respectively. Co-immunoprecipitation data suggests that FRα interacts with the Drosha-DGCR8 complex to affect pre-miRNA processing. Transfecting anti-miR-138 or anti-miR-let-7 into non-proliferating neural crest cells (NCCs) derived from Splotch (Sp-/- ), restored their proliferation potential. In summary, these results suggest a novel pleiotropic role of FRα: (a) direct activation of Oct4, Sox2, and Klf4 genes; and (b) repression of biogenesis of miRNAs that target these genes or their effector molecules. Stem Cells 2016;34:2721-2732.

A critical role of noggin in developing folate-nonresponsive NTD in Fkbp8 -/- embryos.

PURPOSE: Maternal folate intake has reduced the incidence of human neural tube defects by 60-70 %. However, 30-40 % of cases remain nonresponsive to folate intake. The main purpose of this study was to understand the molecular mechanism of folate nonresponsiveness in a mouse model of neural tube defect. METHODS: We used a folate-nonresponsive Fkbp8 knockout mouse model to elucidate the molecular mechanism(s) of folate nonresponsiveness. Neurospheres were grown from neural stem cells isolated from the lumbar neural tube of E9.5 Fkbp8 (-/-) and wild-type embryos. Immunostaining was used to determine the protein levels of oligodendrocyte transcription factor 2 (Olig2), Nkx6.1, class III beta-tubulin (TuJ1), O4, glial fibrillary acidic protein (GFAP), histone H3 Lys27 trimethylation (H3K27me3), ubiquitously transcribed tetratricopeptide repeat (UTX), and Msx2, and quantitative real-time (RT)-PCR was used to determine the message levels of Olig2, Nkx6.1, Msx2, and noggin in neural stem cells differentiated in the presence and absence of folic acid. RESULTS: Fkbp8 (-/-)-derived neural stem cells showed (i) increased noggin expression; (ii) decreased Msx2 expression; (iii) premature differentiation--neurogenesis, oligodendrogenesis (Olig2 expression), and gliogenesis (GFAP expression); and (iv) increased UTX expression and decreased H3K27me3 polycomb modification. Exogenous folic acid did not reverse these markers. CONCLUSIONS: Folate nonresponsiveness could be attributed in part to increased noggin expression in Fkbp8 (-/-) embryos, resulting in decreased Msx2 expression. Folate treatment further increases Olig2 and noggin expression, thereby exacerbating ventralization.

Convection-enhanced delivery of nanodiamond drug delivery platforms for intracranial tumor treatment.

This study examined a novel drug delivery system for treatment of malignant brain gliomas: DOX complexed with nanodiamonds (ND-Dox), and administered via convection-enhanced delivery (CED). Drug retention and toxicity were examined in glioma cell lines, and distribution, retention and toxicity were examined in normal rat parenchyma. Efficacy was assessed in a bioluminescence rodent tumor model. NDs markedly enhanced DOX uptake and retention in glioma cells. ND-Dox delivered via CED extended DOX retention and localized DOX toxicity in normal rodent parenchyma, and was significantly more efficient at killing tumor cells than uncomplexed DOX. Outcomes from this work suggest that CED of ND-Dox is a promising approach for brain tumor treatment. FROM THE CLINICAL EDITOR: In this paper, nanodiamonds were utilized to enhance delivery of DOX in a preclinical glioma model using a convection-enhanced delivery method, demonstrating remarkably enhanced efficacy.

WDR82-Mediated H3K4me3 Is Associated with Tumor Proliferation and Therapeutic Efficacy in Pediatric High-Grade Gliomas.

Pediatric high-grade gliomas (pHGGs) are common malignant brain tumors without effective treatment and poor patient survival. Abnormal posttranslational modification at the histone H3 tail plays critical roles in tumor cell malignancy. We have previously shown that the trimethylation of lysine 4 at histone H3 (H3K4me3) plays a significant role in pediatric ependymoma malignancy and is associated with tumor therapeutic sensitivity. Here, we show that H3K4me3 and its methyltransferase WDR82 are elevated in pHGGs. A reduction in H3K4me3 by downregulating WDR82 decreases H3K4me3 promoter occupancy and the expression of genes associated with stem cell features, cell proliferation, the cell cycle, and DNA damage repair. A reduction in WDR82-mediated H3K4me3 increases the response of pediatric glioma cells to chemotherapy. These findings suggest that WDR82-mediated H3K4me3 is an important determinant of pediatric glioma malignancy and therapeutic response. This highlights the need for a more thorough understanding of the potential of WDR82 as an epigenetic target to increase therapeutic efficacy and improve the prognosis for children with malignant gliomas.

Efficacy of interstitial continuous vincristine infusion in a bioluminescent rodent intracranial tumor model.

Interstitial chemotherapeutic drug infusion can bypass the blood-brain barrier, and provide high regional drug concentrations without systemic exposure. However, toxicity and efficacy for drugs administered via interstitial continuous (i.c.) infusion have not been characterized. In the current study, vincristine (VIN) was infused into the right frontal lobes of healthy Fisher 344 rats at 30, 45, 60, and 120 µg/ml over a period of 7 days at 1 µl/h, using an Alzet osmotic pump to evaluate toxicity. C6 rat glioblastoma cells transduced with a luciferase gene were inoculated into the right frontal lobe of a second group of rats. VIN was administered to tumor bearing rats via i.c. infusion 7 days later and tumor growth was monitored by bioluminescence intensity (BLI) to assess VIN efficacy, intravenous (i.v.) drug administration was used as a comparison drug delivery method. The results suggested that VIN toxicity is dose-dependent. Efficacy studies showed increased BLI, which correlates with histopathological tumor size, in saline-infused and i.v.-treated tumor-bearing rats. These rats survived an average of 28 ± 0.85 days and 33 ± 1.38 days, respectively. Both groups had large tumors at the time of death. Animals treated with VIN via i.c. infusion survived until day 90, the observation endpoint for this study. This was significantly longer than average survival times in the previous two groups. These results demonstrate that VIN via i.c. infusion is effective in reducing C6 glioblastoma tumors and prolonging rodent survival time compared to i.v. injection and suggest that chemotherapeutic drug administration via i.c. infusion may be a promising strategy for treating malignant brain tumors.

Folic acid remodels chromatin on Hes1 and Neurog2 promoters during caudal neural tube development.

The mechanism(s) behind folate rescue of neural tube closure are not well understood. In this study we show that maternal intake of folate prior to conception reverses the proliferation potential of neural crest stem cells in homozygous Splotch embryos (Sp(-/-)) via epigenetic mechanisms. It is also shown that the pattern of differentiation seen in these cells is similar to wild-type (WT). Cells from open caudal neural tubes of Sp(-/-) embryos exhibit increased H3K27 methylation and decreased expression of KDM6B possibly due to up-regulation of KDM6B targeting micro-RNAs such as miR-138, miR-148a, miR-185, and miR-339-5p. In our model, folate reversed these epigenetic marks in folate-rescued Sp(-/-) embryos. Using tissue from caudal neural tubes of murine embryos we also examined H3K27me2 and KDM6B association with Hes1 and Neurog2 promoters at embryonic day E10.5, the proliferative stage, and E12.5, when neural differentiation begins. In Sp(-/-) embryos compared with WT, levels of H3K27me2 associated with the Hes1 promoter were increased at E10.5, and levels associated with the Neurog2 promoter were increased at E12.5. KDM6B association with Hes1 and Neurog2 promoters was inversely related to H3K27me2 levels. These epigenetic changes were reversed in folate-rescued Sp(-/-) embryos. Thus, one of the mechanisms by which folate may rescue the Sp(-/-) phenotype is by increasing the expression of KDM6B, which in turn decreases H3K27 methylation marks on Hes1 and Neurog2 promoters thereby affecting gene transcription.

Advanced Pediatric Diffuse Pontine Glioma Murine Models Pave the Way towards Precision Medicine.

Diffuse intrinsic pontine gliomas (DIPGs) account for ~15% of pediatric brain tumors, which invariably present with poor survival regardless of treatment mode. Several seminal studies have revealed that 80% of DIPGs harbor H3K27M mutation coded by HIST1H3B, HIST1H3C and H3F3A genes. The H3K27M mutation has broad effects on gene expression and is considered a tumor driver. Determination of the effects of H3K27M on posttranslational histone modifications and gene regulations in DIPG is critical for identifying effective therapeutic targets. Advanced animal models play critical roles in translating these cutting-edge findings into clinical trial development. Here, we review current molecular research progress associated with DIPG. We also summarize DIPG animal models, highlighting novel genomic engineered mouse models (GEMMs) and innovative humanized DIPG mouse models. These models will pave the way towards personalized precision medicine for the treatment of DIPGs.

Bone Morphogenetic Protein 4 Targeting Glioma Stem-Like Cells for Malignant Glioma Treatment: Latest Advances and Implications for Clinical Application.

Malignant gliomas are heterogeneous neoplasms. Glioma stem-like cells (GSCs) are undifferentiated and self-renewing cells that develop and maintain these tumors. These cells are the main population that resist current therapies. Genomic and epigenomic analyses has identified various molecular subtypes. Bone morphogenetic protein 4 (BMP4) reduces the number of GSCs through differentiation and induction of apoptosis, thus increasing therapeutic sensitivity. However, the short half-life of BMP4 impedes its clinical application. We previously reviewed BMP4 signaling in central nervous system development and glioma tumorigenesis and its potential as a treatment target in human gliomas. Recent advances in understanding both adult and pediatric malignant gliomas highlight critical roles of BMP4 signaling pathways in the regulation of tumor biology, and indicates its potential as a therapeutic molecule. Furthermore, significant progress has been made on synthesizing BMP4 biocompatible delivery materials, which can bind to and markedly extend BMP4 half-life. Here, we review current research associated with BMP4 in brain tumors, with an emphasis on pediatric malignant gliomas. We also summarize BMP4 delivery strategies, highlighting biocompatible BMP4 binding peptide amphiphile nanostructures as promising novel delivery platforms for treatment of these devastating tumors.

Key basic helix-loop-helix transcription factor genes Hes1 and Ngn2 are regulated by Pax3 during mouse embryonic development.

Pax3 is expressed early during embryonic development in spatially restricted domains including limb muscle, neural crest, and neural tube. Pax3 functions at the nodal point in melanocyte stem cell differentiation, cardiogenesis and neurogenesis. Additionally Pax3 has been implicated in migration and differentiation of precursor cell populations. Currently there are questions about how Pax3 regulates these diverse functions. In this study we found that in the absence of functional Pax3, as in Splotch embryos, the neural crest cells undergo premature neurogenesis, as evidenced by increased Brn3a positive staining in neural tube explants, in comparison with wild-type. Premature neurogenesis in the absence of functional Pax3 may be due to a change in the regulation of basic helix-loop-helix transcription factors implicated in proliferation and differentiation. Using promoter-luciferase activity measurements in transient co-transfection experiments and electro-mobility shift assays, we show that Pax3 regulates Hairy and enhancer of split homolog-1 (Hes1) and Neurogenin2 (Ngn2) by directly binding to their promoters. Chromatin immunoprecipitation assays confirmed that Pax3 bound to cis-regulatory elements within Hes1 and Ngn2 promoters. These observations suggest that Pax3 regulates Hes1 and Ngn2 and imply that it may couple migration with neural stem cell maintenance and neurogenesis.

Interstitial continuous infusion therapy in a malignant glioma model in rats.

PURPOSE: Local direct delivery of chemotherapeutic agents for the treatment of brain tumors is an area of focus in the development of new therapeutic paradigms. These techniques need improvement, especially in terms of drug retention in brain tissue. MATERIALS AND METHODS: In this study, we used a rat glioma model to examine carboplatin distribution, as measured by platinum penetration, after delivery via interstitial continuous (i.c.) infusion. We also examined rat survival times in response to carboplatin and oxaliplatin. I.C. infusion, a modified version of convection-enhanced delivery (CED) for local drug delivery, uses low volume (1 microl per hour) continuous infusion directly into the tumor. RESULTS: I.C. infusion produced a nearly 360-fold higher concentration of platinum in tumor tissue and significantly prolonged rodent survival time compared to intraperitoneal (i.p.) infusion. CONCLUSIONS: We showed i.c. infusion allows for circumvention of the blood-brain barrier, focused drug distribution, and sustained drug delivery. This method could be a promising strategy for treating brain tumors.

Global Reduction of H3K4me3 Improves Chemotherapeutic Efficacy for Pediatric Ependymomas.

BACKGROUND: Ependymomas (EPNs) are the third most common brain tumor in children. These tumors are resistant to available chemotherapeutic treatments, therefore new effective targeted therapeutics must be identified. Increasing evidence shows epigenetic alterations including histone posttranslational modifications (PTMs), are associated with malignancy, chemotherapeutic resistance and prognosis for pediatric EPNs. In this study we examined histone PTMs in EPNs and identified potential targets to improve chemotherapeutic efficacy. METHODS: Global histone H3 lysine 4 trimethylation (H3K4me3) levels were detected in pediatric EPN tumor samples with immunohistochemistry and immunoblots. Candidate genes conferring therapeutic resistance were profiled in pediatric EPN tumor samples with micro-array. Promoter H3K4me3 was examined for two candidate genes, CCND1 and ERBB2, with chromatin-immunoprecipitation coupled with real-time PCR (ChIP-PCR). These methods and MTS assay were used to verify a relationship between H3K4me3 levels and CCND1 and ERBB2, and to investigate cell viability in response to chemotherapeutic drugs in primary cultured pediatric EPN cells. RESULTS: H3K4me3 levels positively correlate with WHO grade malignancy in pediatric EPNs and are associated with progression free survival in patients with posterior fossa group A EPNs (PF-EPN-A). Reduction of H3K4me3 by silencing its methyltransferase SETD1A, in primary cultured EPN cells increased cell response to chemotherapy. CONCLUSIONS: Our results support the development of a novel treatment that targets H3K4me3 to increase chemotherapeutic efficacy in pediatric PF-EPN-A tumors.

Therapeutic Potential for Bone Morphogenetic Protein 4 in Human Malignant Glioma.

Human glioma, in particular, malignant forms such as glioblastoma exhibit dismal survival rates despite advances in treatment strategies. A population of glioma cells with stem-like features, glioma cancer stem-like cells (GCSCs), contribute to renewal and maintenance of the tumor cell population and appear responsible for chemotherapeutic and radiation resistance. Bone morphogenetic protein 4 (BMP4), drives differentiation of GCSCs and thus improves therapeutic efficacy. Based on this observation it is imperative that the clinical merits of BMP4 in treating human gliomas should be addressed. This article reviews BMP4 signaling in central nervous system development and in glioma tumorigenesis, and the potential of this molecule as a treatment target in human gliomas. Further work needs to be done to determine if distinct lineages of GCSCs, associated with different glioma sub-classifications, proneural, neural, classical and mesenchymal, differ in responsiveness to BMP4 treatment. Additionally, interaction among BMP4 and cell matrix, tumor-vascular molecules and microglial immune cells also needs to be investigated, as this will enhance our knowledge about the role of BMP4 in human glioma and lead to the identification and/or development of novel therapeutic approaches that improve treatment outcomes of these devastating tumors.

Amniotic fluid and serum biomarkers from women with neural tube defect-affected pregnancies: a case study for myelomeningocele and anencephaly: clinical article.

OBJECT: The authors sought to identify novel biomarkers for early detection of neural tube defects (NTDs) in human fetuses. METHODS: Amniotic fluid and serum were drawn from women in the second trimester of pregnancy. The study group included 2 women pregnant with normal fetuses and 4 with fetuses displaying myelomeningocele (n = 1), anencephaly (n = 1), holoprosencephaly (n = 1), or encephalocele (n = 1). Amniotic fluid stem cells (AFSCs) were isolated and cultured. The cells were immunostained for the stem cell markers Oct4, CD133, and Sox2; the epigenetic biomarkers H3K4me2, H3K4me3, H3K27me2, H3K27me3, H3K9Ac, and H3K18Ac; and the histone modifiers KDM6B (a histone H3K27 demethylase) and Gcn5 (a histone acetyltransferase). The levels of 2 markers for neural tube development, bone morphogenetic protein-4 (BMP4) and sonic hedgehog (Shh), were measured in amniotic fluid and serum using an enzyme-linked immunosorbent assay. RESULTS: The AFSCs from the woman pregnant with a fetus affected by myelomeningocele had higher levels of H3K4me2, H3K4me3, H3K27me2, and H3K27me3 and lower levels of KDM6B than the AFSCs from the women with healthy fetuses. The levels of H3K9ac, H3K18ac, and Gcn5 were also decreased in the woman with the fetus exhibiting myelomeningocele. In AFSCs from the woman carrying an anencephalic fetus, levels of H3K27me3, along with those of H3K9Ac, H3K18ac, and Gcn5, were increased, while that of KDM6B was decreased. Compared with the normal controls, the levels of BMP4 in amniotic fluid and serum from the woman with a fetus with myelomeningocele were increased, whereas levels of Shh were increased in the woman pregnant with a fetus displaying anencephaly. CONCLUSIONS: The levels of epigenetic marks, such as H3K4me, H3K27me3, H3K9Ac, and H3K18A, in cultured AFSCs in combination with levels of key developmental proteins, such as BMP4 and Shh, are potential biomarkers for early detection and identification of NTDs in amniotic fluid and maternal serum.

Fetal neural tube stem cells from Pax3 mutant mice proliferate, differentiate, and form synaptic connections when stimulated with folic acid.

Although maternal intake of folic acid (FA) prevents neural tube defects in 70% of the population, the exact mechanism of prevention has not been elucidated. We hypothesized that FA affects neural stem cell (NSC) proliferation and differentiation. This hypothesis was examined in a folate-responsive spina bifida mouse model, Splotch (Sp(-/-)), which has a homozygous loss-of-function mutation in the Pax3 gene. Neurospheres were generated with NSCs from the lower lumbar neural tube of E10.5 wild-type (WT) and Sp(-/-) embryos, in the presence and absence of FA. In the absence of FA, the number of neurospheres generated from Sp(-/-) embryos compared with WT was minimal (P<0.05). Addition of FA to Sp(-/-) cultures increased the expression of a Pax3 downstream target, fgfr4, and rescued NSC proliferative potential, as demonstrated by a significant increase in neurosphere formation (P<0.01). To ascertain if FA affected cell differentiation, FA-stimulated Sp(-/-) neurospheres were allowed to differentiate in the continued presence or absence of FA. Neurospheres from both conditions expressed multi-potent stem cell characteristics and the same differentiation potential as WT. Further, multiple neurospheres from both WT and FA-stimulated Sp(-/-) cell cultures formed extensive synaptic connections. On the whole, FA-mediated rescue of neural tube defects in Sp(-/-) embryos promotes NSC proliferation at an early embryonic stage. FA-stimulated Sp(-/-) neurospheres differentiate and form synaptic connections, comparable to WT.

Nuclear localization of folate receptor alpha: a new role as a transcription factor.

Folic acid (FA) has traditionally been associated with prevention of neural tube defects; more recent work suggests that it may also be involved in in the prevention of adult onset diseases. As the role of FA in human health and disease expands, it also becomes more critical to understand the mechanisms behind FA action. In this work we examined the hypothesis that folate receptor alpha (FRα) acts as a transcription factor. FRα is a GPI-anchored protein and a component of the caveolae fraction. The work described here shows that FRα translocates to the nucleus, where it binds to cis-regulatory elements at promoter regions of Fgfr4 and Hes1, and regulates their expression. The FRα recognition domain mapped to AT rich regions on the promoters. Until this time FRα has only been considered as a folate transporter, these studies describe a novel role for FRα as a transcription factor.

Epigenetic regulation of sensory neurogenesis in the dorsal root ganglion cell line ND7 by folic acid.

The epigenetic mechanism of folic acid (FA) action on dorsal root ganglion (DRG) cell proliferation and sensory neuron differentiation is not well understood. In this study, the ND7 cell line, derived from DRG cells, was used to elucidate this mechanism. In ND7 cells differentiated with dbcAMP and NGF, Hes1 and Pax3 levels decreased, whereas Neurog2 levels showed a modest increase. Chromatin immunoprecipitation (ChIP) assays examining epigenetic marks at the Hes1 promoter showed that FA favored increased H3K9 and H3K19 acetylation and decreased H3K27 methylation. Hence, FA plays a positive role in cell proliferation. In differentiated ND7 cells, H3K27 methylation decreased, whereas H3K9 and H3K18 acetylation increased at the Neurog2 promoter. FA did not favor this phenotypic outcome. Additionally, in differentiated ND7 Neurog2 associated with the NeuroD1 promoter, FA decreased this association. The results suggest that the switch from proliferation to sensory neuron differentiation in DRG cells is regulated by alterations in epigenetic marks, H3K9/18 acetylation and H3K27 methylation, at Hes1 and Neurog2 promoters, as well as by Neurog2 association with NeuroD1 promoter. FA although positive for proliferation, does not appear to play a role in differentiation.

Role of Pax3 acetylation in the regulation of Hes1 and Neurog2.

Pax3 plays a role in regulating Hes1 and Neurog2 activity and thereby stem cell maintenance and neurogenesis. A mechanism for Pax3 regulation of these two opposing events, during caudal neural tube development, is examined in this study. Pax3 acetylation on C-terminal lysine residues K437 and K475 may be critical for proper regulation of Hes1 and Neurog2. Removal of these lysine residues increased Hes1 but decreased Neurog2 promoter activity. SIRT1 deacetylase may be a key component in regulating Pax3 acetylation. Chromatin immunoprecipitation assays showed that SIRT1 is associated with Hes1 and Neurog2 promoters during murine embryonic caudal neural tube development at E9.5, but not at E12.5. Overexpression of SIRT1 decreased Pax3 acetylation, Neurog2 and Brn3a positive staining. Conversely, siRNA-mediated silencing of SIRT1 increased these factors. These studies suggest that Pax3 acetylation results in decreased Hes1 and increased Neurog2 activity, thereby promoting sensory neuron differentiation.

Transcriptional regulation by Pax3 and TGFbeta2 signaling: a potential gene regulatory network in neural crest development.

Pax3 regulates neural crest cell migration and is critical during neural crest development. TGFbs modify neural crest cell migration and differentiation. TGFbeta2 nullizygous embryos (TGFbeta2(-/-)Pax3(+/+)) display open neural tube and bifid spine, whereas in wild type embryos, the neural tube is closed. In previous work, we have demonstrated that Pax3 regulates TGFbeta2 by directly binding to cis-regulatory elements on its promoter. In this study, we found that the TGFbeta2 nullizygous phenotype can be reversed to the wild type phenotype by down-regulating one allele of Pax3, as in TGFbeta2(-/-)Pax3(+/-) embryos obtained through breeding TGFb2(+/-)Pax3(+/-) mice. The data in this paper suggest that Pax3 and TGFbeta2 interact in a coordinated gene regulatory network, linked by common downstream effector genes, to bring about this phenotypic reversal. Downstream effectors may include Hes1, Ngn2 and Sox9, as well as other genes involved in neuronal differentiation.

Longitudinal assessment of regional directed delivery in a rodent malignant glioma model.

OBJECT: Direct delivery of chemotherapeutic agents for the treatment of brain tumors is an area of focus in the development of therapeutic paradigms because this method of delivery circumvents the blood-brain barrier without causing adverse systemic side effects. Few studies have investigated longitudinal tumor response to this type of therapy. In this study, the authors examined the time course of tumor response to direct delivery of a chemotherapeutic agent in a rodent malignant glioma model. METHODS: To visualize tumor response to chemotherapy, the authors used bioluminescence imaging in a rodent model. Rat 9L gliosarcoma cells expressing a luciferase gene were inoculated into adult male rat striata. Ten days following surgery the animals were randomly divided into 4 groups. Groups 1 and 2 received 20 and 40 microl carboplatin (1 mg/ml), respectively, via convection-enhanced delivery (CED); Group 3 received 60 mg/kg carboplatin intraperitoneally; and Group 4 received no treatment. Tumor growth was correlated with luminescence levels twice weekly. RESULTS: Differential growth curves were observed for the 4 groups. Systemically treated rats showed decreasing photon flux emission at 15.0 + or - 4.7 days; rats treated with 20- or 40-microl CED showed decreased emissions at 4.0 + or - 2.0 and 3.2 + or - 1.3 days after treatment, respectively. Histopathologically, 6 of 12 CED-treated animals exhibited no residual tumor at the end point of the study. CONCLUSIONS: Direct and systemic delivery of carboplatin was examined to determine how the method of drug delivery affects tumor growth. The present report is one of the first in vivo studies to examine the time course of tumor response to direct drug delivery. The results indicate that direct drug delivery may be a promising option for treating gliomas.