Inorganic polyphosphate as an energy source in tumorigenesis.

Cancer cells have high demands for energy to maintain their exceedingly proliferative growth. However, the mechanism of energy expenditure in cancer is not well understood. We hypothesize that cancer cells might utilize energy-rich inorganic polyphosphate (polyP), as energetic reserve. PolyP is comprised of orthophosphates linked by phosphoanhydride bonds, as in ATP. Here, we show that polyP is highly abundant in several types of cancer cells, including brain tumor-initiating cells (BTICs), i.e., stem-like cells derived from a mouse brain tumor model that we have previously described. The polymer is avidly consumed during starvation of the BTICs. Depletion of ATP by inhibiting glycolysis and mitochondrial ATP-synthase (OXPHOS) further decreases the levels of polyP and alters morphology of the cells. Moreover, enzymatic hydrolysis of the polymer impairs the viability of cancer cells and significantly deprives ATP stores. These results suggest that polyP might be utilized as a source of phosphate energy in cancer. While the role of polyP as an energy source is established for bacteria, this finding is the first demonstration that polyP may play a similar role in the metabolism of cancer cells.

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.

Characterization of brain tumor initiating cells isolated from an animal model of CNS primitive neuroectodermal tumors.

CNS Primitive Neuroectodermal tumors (CNS-PNETs) are members of the embryonal family of malignant childhood brain tumors, which remain refractory to current therapeutic treatments. Current paradigm of brain tumorigenesis implicates brain tumor-initiating cells (BTIC) in the onset of tumorigenesis and tumor maintenance. However, despite their significance, there is currently no comprehensive characterization of CNS-PNETs BTICs. Recently, we described an animal model of CNS-PNET generated by orthotopic transplantation of human Radial Glial (RG) cells - the progenitor cells for adult neural stem cells (NSC) - into NOD-SCID mice brain and proposed that BTICs may play a role in the maintenance of these tumors. Here we report the characterization of BTIC lines derived from this CNS-PNET animal model. BTIC's orthotopic transplantation generated highly aggressive tumors also characterized as CNS-PNETs. The BTICs have the hallmarks of NSCs as they demonstrate self-renewing capacity and have the ability to differentiate into astrocytes and early migrating neurons. Moreover, the cells demonstrate aberrant accumulation of wild type tumor-suppressor protein p53, indicating its functional inactivation, highly up-regulated levels of onco-protein cMYC and the BTIC marker OCT3/4, along with metabolic switch to glycolysis - suggesting that these changes occurred in the early stages of tumorigenesis. Furthermore, based on RNA- and DNA-seq data, the BTICs did not acquire any transcriptome-changing genomic alterations indicating that the onset of tumorigenesis may be epigenetically driven. The study of these BTIC self-renewing cells in our model may enable uncovering the molecular alterations that are responsible for the onset and maintenance of the malignant PNET phenotype.

Stabilization of HIF-1α and HIF-2α, up-regulation of MYCC and accumulation of stabilized p53 constitute hallmarks of CNS-PNET animal model.

Recently, we described a new animal model of CNS primitive neuroectodermal tumors (CNS-PNET), which was generated by orthotopic transplantation of human Radial Glial (RG) cells into NOD-SCID mice's brain sub-ventricular zone. In the current study we conducted comprehensive RNA-Seq analyses to gain insights on the mechanisms underlying tumorigenesis in this mouse model of CNS-PNET. Here we show that the RNA-Seq profiles derived from these tumors cluster with those reported for patients' PNETs. Moreover, we found that (i) stabilization of HIF-1α and HIF-2α, which are involved in mediation of the hypoxic responses in the majority of cell types, (ii) up-regulation of MYCC, a key onco-protein whose dysregulation occurs in ~70% of human tumors, and (iii) accumulation of stabilized p53, which is commonly altered in human cancers, constitute hallmarks of our tumor model, and might represent the basis for CNS-PNET tumorigenesis in this model. We discuss the possibility that these three events might be interconnected. These results indicate that our model may prove invaluable to uncover the molecular events leading to MYCC and TP53 alterations, which would be of broader interest considering their relevance to many human malignancies. Lastly, this mouse model might prove useful for drug screening targeting MYCC and related members of its protein interaction network.

The P4 Health Spectrum - A Predictive, Preventive, Personalized and Participatory Continuum for Promoting Healthspan.

Chronic diseases (i.e., noncommunicable diseases), mainly cardiovascular disease, cancer, respiratory diseases and type-2-diabetes, are now the leading cause of death, disability and diminished quality of life on the planet. Moreover, these diseases are also a major financial burden worldwide, significantly impacting the economy of many countries. Healthcare systems and medicine have progressively improved upon the ability to address infectious diseases and react to adverse health events through both surgical interventions and pharmacology; we have become efficient in delivering reactive care (i.e., initiating interventions once an individual is on the verge of or has actually suffered a negative health event). However, with slowly progressing and often 'silent' chronic diseases now being the main cause of illness, healthcare and medicine must evolve into a proactive system, moving away from a merely reactive approach to care. Minimal interactions among the specialists and limited information to the general practitioner and to the individual receiving care lead to a fragmented health approach, non-concerted prescriptions, a scattered follow-up and a suboptimal cost-effectiveness ratio. A new approach in medicine that is predictive, preventive, personalized and participatory, which we label here as "P4" holds great promise to reduce the burden of chronic diseases by harnessing technology and an increasingly better understanding of environment-biology interactions, evidence-based interventions and the underlying mechanisms of chronic diseases. In this concept paper, we propose a 'P4 Health Continuum' model as a framework to promote and facilitate multi-stakeholder collaboration with an orchestrated common language and an integrated care model to increase the healthspan.

Expression of miR-18a and miR-210 in Normal Breast Tissue as Candidate Biomarkers of Breast Cancer Risk.

miRNAs are noncoding RNAs with abnormal expression in breast cancer; their expression in high-risk benign breast tissue may relate to breast cancer risk. We examined miRNA profiles in contralateral unaffected breasts (CUB) of patients with breast cancer and validated resulting candidates in two additional sample sets. Expression profiles of 754 mature miRNAs were examined using TaqMan Low Density Arrays in 30 breast cancer samples [15 estrogen receptor (ER)-positive and 15 ER-negative] and paired CUBs and 15 reduction mammoplasty controls. Pairwise comparisons identified miRNAs with significantly differential expression. Seven candidate miRNAs were examined using qRT-PCR in a second CUB sample set (40 cases, 20 ER+, 20 ER-) and 20 reduction mammoplasty controls. Further validation was performed in 80 benign breast biopsy (BBB) samples; 40 from cases who subsequently developed breast cancer and 40 from controls who did not. Logistic regression, using tertiles of miRNA expression, was used to discriminate cases from controls. Seven miRNAs were differentially expressed in tumors and CUBs versus reduction mammoplasty samples. Among them, miR-18a and miR-210 were validated in the second CUB set, showing significantly higher expression in tumor and CUBs than in reduction mammoplasty controls. The expression of miR-18a and miR-210 was also significantly higher in BBB cases than in BBB controls. When both miR-18a and miR-210 were expressed in the upper tertiles in BBB, OR for subsequent cancer was 3.20, P = 0.023. miR-18a and miR-210 are expressed at higher levels in CUBs of patients with breast cancer, and in BBB prior to cancer development, and are therefore candidate breast cancer risk biomarkers. Cancer Prev Res; 10(1); 89-97. ©2016 AACR.

Extensive miRNA expression analysis in craniopharyngiomas.

PURPOSE: Craniopharyngiomas are benign tumors of the sellar or parasellar regions. They arise from the remnants of Rathke's pouch and are considered a "developmental disease." microRNAs are short non-coding RNAs that play a key regulatory role in the control of expression of entire gene networks. We performed an extensive analysis of miRNAs in craniopharyngiomas aiming to identify a miRNA expression signature that might aid in the prognosis of disease progression and outcome. METHODS: Thirty-seven craniopharyngioma samples from twenty-three patients, ten age-matched controls from autopsy, and ten infant controls from the developing pituitary from autopsy were evaluated for the expression of 754 miRNAs using TaqMan® Low Density Arrays (TLDAs) v2.0 (Applied Biosystems, Foster City, CA). RESULTS: Among the most differentially expressed miRNAs, downregulation of miR-132 appears to be a marker of aggressiveness and also plays a role in epithelial-mesenchymal transition. CONCLUSIONS: This is the first time that an extensive study of miRNA expression has been performed in craniopharyngiomas. Further research needs to be performed to investigate the potential role of miR-132 in the development and progression of craniopharyngiomas, and its value as a prognostic marker of aggressiveness.

MicroRNA-10a Regulation of Proinflammatory Mediators: An Important Component of Untreated Juvenile Dermatomyositis.

OBJECTIVE: To identify differentially expressed microRNA (miRNA) in muscle biopsies (MBx) from 15 untreated children with juvenile dermatomyositis (JDM) compared with 5 controls. METHODS: Following MBx miRNA profiling, differentially expressed miRNA and their protein targets were validated by quantitative real-time PCR (qRT-PCR) and immunological assay. The association of miRNA-10a and miRNA-10b with clinical data was evaluated, including Disease Activity Score (DAS), von Willebrand factor antigen (vWF:Ag), nailfold capillary end row loops, duration of untreated disease, and tumor necrosis factor (TNF)-α-308A allele. RESULTS: In JDM, 16/362 miRNA were significantly differentially expressed [false discovery rate (FDR) < 0.05]. Among these, miRNA-10a was the most downregulated miRNA in both FDR and ranking of fold change: miRNA-10a = -2.27-fold, miRNA-10b = -1.80-fold. Decreased miRNA-10a and miRNA-10b expressions were confirmed using q RT-PCR: -4.16 and -2.59 fold, respectively. The qRT-PCR documented that decreased miRNA-10a expression was related to increased vascular cell adhesion molecule 1 in 13 of these JDM cases (correlation -0.67, p = 0.012), unlike miRNA-10b data (not significant). Concurrent JDM plasma contained increased levels of interleukin (IL) 6 (p = 0.0363), IL-8 (p = 0.0005), TNF-α (p = 0.0011), and monocyte chemoattractant proteins 1 (p = 0.0139). Decreased miRNA-10a, but not miRNA-10b, was associated with the TNF-α-308A allele (p = 0.015). In the 15 JDM, a trend of association of miRNA-10a (but not miRNA-10b) with vWF:Ag and DAS was observed. CONCLUSION: MiRNA-10a downregulation is an important element in untreated JDM muscle pathophysiology. We speculate that muscle miRNA expression in adult dermatomyositis differs from muscle miRNA expression in untreated childhood JDM.

A mouse model of human primitive neuroectodermal tumors resulting from microenvironmentally-driven malignant transformation of orthotopically transplanted radial glial cells.

There is growing evidence and a consensus in the field that most pediatric brain tumors originate from stem cells, of which radial glial cells constitute a subtype. Here we show that orthotopic transplantation of human radial glial (RG) cells to the subventricular zone of the 3rd ventricle--but not to other transplantation sites--of the brain in immunocompromised NOD-SCID mice, gives rise to tumors that have the hallmarks of CNS primitive neuroectodermal tumors (PNETs). The resulting mouse model strikingly recapitulates the phenotype of PNETs. Importantly, the observed tumorigenic transformation was accompanied by aspects of an epithelial to mesenchymal transition (EMT)-like process. It is also noteworthy that the tumors are highly invasive, and that they effectively recruit mouse endothelial cells for angiogenesis. These results are significant for several reasons. First, they show that malignant transformation of radial glial cells can occur in the absence of specific mutations or inherited genomic alterations. Second, they demonstrate that the same radial glial cells may either give rise to brain tumors or differentiate normally depending upon the microenvironment of the specific region of the brain to which the cells are transplanted. In addition to providing a prospect for drug screening and development of new therapeutic strategies, the resulting mouse model of PNETs offers an unprecedented opportunity to identify the cancer driving molecular alterations and the microenvironmental factors that are responsible for committing otherwise normal radial glial cells to a malignant phenotype.

Human rhinovirus infection causes different DNA methylation changes in nasal epithelial cells from healthy and asthmatic subjects.

BACKGROUND: Mechanisms underlying the development of virus-induced asthma exacerbations remain unclear. To investigate if epigenetic mechanisms could be involved in virus-induced asthma exacerbations, we undertook DNA methylation profiling in asthmatic and healthy nasal epithelial cells (NECs) during Human Rhinovirus (HRV) infection in vitro. METHODS: Global and loci-specific methylation profiles were determined via Alu element and Infinium Human Methylation 450 K microarray, respectively. Principal components analysis identified the genomic loci influenced the most by disease-status and infection. Real-time PCR and pyrosequencing were used to confirm gene expression and DNA methylation, respectively. RESULTS: HRV infection significantly increased global DNA methylation in cells from asthmatic subjects only (43.6% to 44.1%, p = 0.04). Microarray analysis revealed 389 differentially methylated loci either based on disease status, or caused by virus infection. There were disease-associated DNA methylation patterns that were not affected by HRV infection as well as HRV-induced DNA methylation changes that were unique to each group. A common methylation locus stood out in response to HRV infection in both groups, where the small nucleolar RNA, H/ACA box 12 (SNORA12) is located. Further analysis indicated that a relationship existed between SNORA12 DNA methylation and gene expression in response to HRV infection. CONCLUSIONS: We describe for the first time that Human rhinovirus infection causes DNA methylation changes in airway epithelial cells that differ between asthmatic and healthy subjects. These epigenetic differences may possibly explain the mechanism by which respiratory viruses cause asthma exacerbations.

Onset of rosette formation during spontaneous neural differentiation of hESC and hiPSC colonies.

In vitro neural differentiation of human embryonic stem cells (hESCs) is an advantageous system for studying early neural development. The process of early neural differentiation in hESCs begins by initiation of primitive neuroectoderm, which is manifested by rosette formation, with consecutive differentiation into neural progenitors and early glial-like cells. In this study, we examined the involvement of early neural markers - OTX2, PAX6, Sox1, Nestin, NR2F1, NR2F2, and IRX2 - in the onset of rosette formation, during spontaneous neural differentiation of hESC and human induced pluripotent stem cell (hiPSC) colonies. This is in contrast to the conventional way of studying rosette formation, which involves induction of neuronal differentiation and the utilization of embryoid bodies. Here we show that OTX2 is highly expressed at the onset of rosette formation, when rosettes comprise no more than 3-5 cells, and that its expression precedes that of established markers of early neuronal differentiation. Importantly, the rise of OTX2 expression in these cells coincides with the down-regulation of the pluripotency marker OCT4. Lastly, we show that cells derived from rosettes that emerge during spontaneous differentiation of hESCs or hiPSCs are capable of differentiating into dopaminergic neurons in vitro, and into mature-appearing pyramidal and serotonergic neurons weeks after being injected into the motor cortex of NOD-SCID mice.

Differentially expressed miRNAs in retinoblastoma.

MicroRNAs (miRNAs) are short non-coding RNA transcripts that have the ability to regulate the expression of target genes, and have been shown to influence the development of various tumors. The purpose of our study is to identify aberrantly expressed miRNAs in retinoblastoma for the discovery of potential therapeutic targets for this disease, and to gain a greater understanding of the mechanisms driving retinoblastoma progression. We report 41 differentially expressed miRNAs (p<0.05) in 12 retinoblastomas as compared to three normal human retinae. Of these miRNAs, many are newly identified as being differentially expressed in retinoblastoma. Further, we report the validations of five of the most downregulated miRNAs in primary human retinoblastomas (p<0.05), human retinoblastoma cell lines, and mouse retinoblastoma cell lines. This serves as the largest and most comprehensive retinoblastoma miRNA analysis to date with corresponding clinical and pathological characteristics. This is an essential step in the discovery of miRNAs associated with retinoblastoma progression, and in the identification of potential therapeutic targets for this disease.

ANALYSIS OF ALEXANDRIUM TAMARENSE (DINOPHYCEAE) GENES REVEALS THE COMPLEX EVOLUTIONARY HISTORY OF A MICROBIAL EUKARYOTE().

Microbial eukaryotes may extinguish much of their nuclear phylogenetic history due to endosymbiotic/horizontal gene transfer (E/HGT). We studied E/HGT in 32,110 contigs of expressed sequence tags (ESTs) from the dinoflagellate Alexandrium tamarense (Dinophyceae) using a conservative phylogenomic approach. The vast majority of predicted proteins (86.4%) in this alga are novel or dinoflagellate-specific. We searched for putative homologs of these predicted proteins against a taxonomically broadly sampled protein database that includes all currently available data from algae and protists and reconstructed a phylogeny from each of the putative homologous protein sets. Of the 2,523 resulting phylogenies, 14-17% are potentially impacted by E/HGT involving both prokaryote and eukaryote lineages, with 2-4% showing clear evidence of reticulate evolution. The complex evolutionary histories of the remaining proteins, many of which may also have been affected by E/HGT, cannot be interpreted using our approach with currently available gene data. We present empirical evidence of reticulate genome evolution that combined with inadequate or highly complex phylogenetic signal in many proteins may impede genome-wide approaches to infer the tree of microbial eukaryotes.

Histone deacetylase 1 deficiency impairs differentiation and electrophysiological properties of cardiomyocytes derived from induced pluripotent cells.

Epigenetic and chromatin modifications play particularly important roles in embryonic and induced pluripotent stem cells (ESCs and iPSCs) allowing for the cells to both differentiate and dedifferentiate back to a pluripotent state. We analyzed how the loss of a key chromatin-modifying enzyme, histone deacetylase 1 (HDAC1), affects early and cardiovascular differentiation of both ESCs and iPSCs. We also investigated potential differences between these two cell types when differentiation is induced. Our data indicate an essential role for HDAC1 in deacetylating regulatory regions of key pluripotency-associated genes during early differentiation. Although HDAC1 functions primarily as a HDAC, its loss also affects DNA methylation in ESCs and iPSCs both during pluripotency and differentiation. We show that HDAC1 plays a crucial, nonredundant role in cardiomyocyte differentiation and maturation. Our data also elucidate important differences between ESCs and iPSCs, when levels of this enzyme are reduced, that affect their ability to differentiate into functional cardiomyocytes. As varying levels of chromatin-modifying enzymes are likely to exist in patient-derived iPSCs, understanding the molecular circuitry of these enzymes in ESCs and iPSCs is critical for their potential use in cardiovascular therapeutic applications

Putative multifunctional signature of lung metastases in dedifferentiated chondrosarcoma.

Chondrosarcomas are among the most malignant skeletal tumors. Dedifferentiated chondrosarcoma is a highly aggressive subtype of chondrosarcoma, with lung metastases developing within a few months of diagnosis in 90% of patients. In this paper we performed comparative analyses of the transcriptomes of five individual metastatic lung lesions that were surgically resected from a patient with dedifferentiated chondrosarcoma. We document for the first time a high heterogeneity of gene expression profiles among the individual lung metastases. Moreover, we reveal a signature of "multifunctional" genes that are expressed in all metastatic lung lesions. Also, for the first time, we document the occurrence of massive macrophage infiltration in dedifferentiated chondrosarcoma lung metastases.

Efficacy of vincristine administered via convection-enhanced delivery in a rodent brainstem tumor model documented by bioluminescence imaging.

PURPOSE: Brain stem gliomas account for 20% of childhood brain tumors. Presently, there is no effective treatment for these tumors, and the prognosis remains poor. One reason for this is that chemotherapeutic drugs cannot cross the blood-brain barrier. In this study, we used a rodent brainstem tumor model, monitored both qualitatively and quantitatively, to examine the effectiveness of vincristine (VCR) administered via convection-enhanced delivery (CED). METHODS: C6 rat glioblastoma cells, transduced with an oncoretroviral plasmid containing a luciferase coding sequence, were inoculated into Fischer 344 rat brainstems. Tumor growth was monitored by bioluminescence intensity (BLI), and tumor volume was calculated from serial histopathologic sections. Therapeutic efficacy of VCR delivered via CED was assessed. Intravenous (I.V.) and intraperitoneal (I.P.) drug administration were used as a comparison for CED efficacy. RESULTS: BLI monitoring revealed progressive tumor growth in inoculated rats. Symptoms caused by tumor burden were evident 16-18 days after inoculation. BLI correlated quantitatively with tumor volume (r(2) = 0.9413), established by histopathological analysis of tumor growth within the pons. VCR administered through CED was more effective than I.V. or I.P. administration in reducing tumor size and increasing survival times. TUNEL assay results suggest that VCR induced glioblastoma cell apoptosis. CONCLUSIONS: VCR administered by CED was effective in reducing tumors and prolonging survival time.

TGF-ß mediated DNA methylation in prostate cancer.

Almost all tumors harbor a defective negative feedback loop of signaling by transforming growth factor-ß (TGF-ß). Epigenetic mechanisms of gene regulation, including DNA methylation, are fundamental to normal cellular function and also play a major role in carcinogenesis. Recent evidence demonstrated that TGF-ß signaling mediates cancer development and progression. Many key events in TGF-ß signaling in cancer included auto-induction of TGF-ß1 and increased expression of DNA methyltransferases (DNMTs), suggesting that DNA methylation plays a significant role in cancer development and progression. In this review, we performed an extensive survey of the literature linking TGF-ß signaling to DNA methylation in prostate cancer. It appeared that almost all DNA methylated genes detected in prostate cancer are directly or indirectly related to TGF-ß signaling. This knowledge has provided a basis for our future directions of prostate cancer research and strategies for prevention and therapy for prostate cancer.

Swarm rat chondrosarcoma cells as an in vivo model: lung colonization and effects of tissue environment on tumor growth.

Swarm rat chondrosarcoma cells have been used extensively for biochemical studies of extra-cellular matrix metabolism in cartilage. However, these cells also possess tumor-like behavior in vivo and are useful in investigation of chondrosarcoma biology. the current study was designed to develop a metastatic model using swarm rat chondrosarcoma cells, and to assess the effect of tissue-environment on tumor behavior in vivo. Tumors were implanted subcutaneously or into bone, and animals were assessed radiographically and microscopically for tumor growth and metastasis. The subcutaneous tumor grew to an average mass of 35 g, while tumor implanted into bone grew 75 mg. Transplantation of the cells into the bone led to extensive bone remodeling with invasion of the medullary cavity and destruction of the bone cortex. Light microscopy demonstrated no significant differences in the number of mitoses, cellular atypia or extracellular matrix staining between the two sites of tumor implantation. Interestingly, lung colonization was observed in none of the animals in the subcutaneous tumor injection group, while tumors colonized the lungs in 95% of the rats with tumor injected into bone. Analysis of cDNA libraries from subcutaneous and bone-transplanted tumors demonstrated a complex and diverse array of expressed transcripts, and there were significant differences in gene expression between tumors at different sites. The results of this study suggest swarm rat chondrosarcoma is a model that resembles human chondrosarcoma mimicking its ability to infiltrate and remodel local bone and to colonize the lungs. Furthermore, the interaction between host-tissue and tumor cells plays a major role in the tumor behavior in this model. Identifying these interactions will lead to further understanding of chondrosarcoma and contribute to therapeutic targets in the future.

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.

Genetic and epigenetic variations contributed by Alu retrotransposition.

BACKGROUND: De novo retrotransposition of Alu elements has been recognized as a major driver for insertion polymorphisms in human populations. In this study, we exploited Alu-anchored bisulfite PCR libraries to identify evolutionarily recent Alu element insertions, and to investigate their genetic and epigenetic variation. RESULTS: A total of 327 putatively recent Alu insertions were identified, altogether represented by 1,762 sequence reads. Nearly all such de novo retrotransposition events (316/327) were novel. Forty-seven out of forty-nine randomly selected events, corresponding to nineteen genomic loci, were sequence-verified. Alu element insertions remained hemizygous in one or more individuals in sixteen of the nineteen genomic loci. The Alu elements were found to be enriched for young Alu families with characteristic sequence features, such as the presence of a longer poly(A) tail. In addition, we documented the occurrence of a duplication of the AT-rich target site in their immediate flanking sequences, a hallmark of retrotransposition. Furthermore, we found the sequence motif (TT/AAAA) that is recognized by the ORF2P protein encoded by LINE-1 in their 5'-flanking regions, consistent with the fact that Alu retrotransposition is facilitated by LINE-1 elements. While most of these Alu elements were heavily methylated, we identified an Alu localized 1.5 kb downstream of TOMM5 that exhibited a completely unmethylated left arm. Interestingly, we observed differential methylation of its immediate 5' and 3' flanking CpG dinucleotides, in concordance with the unmethylated and methylated statuses of its internal 5' and 3' sequences, respectively. Importantly, TOMM5's CpG island and the 3 Alu repeats and 1 MIR element localized upstream of this newly inserted Alu were also found to be unmethylated. Methylation analyses of two additional genomic loci revealed no methylation differences in CpG dinucleotides flanking the Alu insertion sites in the two homologous chromosomes, irrespective of the presence or absence of the insertion. CONCLUSIONS: We anticipate that the combination of methodologies utilized in this study, which included repeat-anchored bisulfite PCR sequencing and the computational analysis pipeline herein reported, will prove invaluable for the generation of genetic and epigenetic variation maps.