Effect of storage time on gene expression data acquired from unfrozen archived newborn blood spots.

Unfrozen archived newborn blood spots (NBS) have been shown to retain sufficient messenger RNA (mRNA) for gene expression profiling. However, the effect of storage time at ambient temperature for NBS samples in relation to the quality of gene expression data is relatively unknown. Here, we evaluated mRNA expression from quantitative real-time PCR (qRT-PCR) and microarray data obtained from NBS samples stored at ambient temperature to determine the effect of storage time on the quality of gene expression. These data were generated in a previous case-control study examining NBS in 53 children with cerebral palsy (CP) and 53 matched controls. NBS sample storage period ranged from 3 to 16years at ambient temperature. We found persistently low RNA integrity numbers (RIN=2.3±0.71) and 28S/18S rRNA ratios (~0) across NBS samples for all storage periods. In both qRT-PCR and microarray data, the expression of three common housekeeping genes-beta cytoskeletal actin (ACTB), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and peptidylprolyl isomerase A (PPIA)-decreased with increased storage time. Median values of each microarray probe intensity at log2 scale also decreased over time. After eight years of storage, probe intensity values were largely reduced to background intensity levels. Of 21,500 genes tested, 89% significantly decreased in signal intensity, with 13,551, 10,730, and 9925 genes detected within 5years, > 5 to <10years, and >10years of storage, respectively. We also examined the expression of two gender-specific genes (X inactivation-specific transcript, XIST and lysine-specific demethylase 5D, KDM5D) and seven gene sets representing the inflammatory, hypoxic, coagulative, and thyroidal pathways hypothesized to be related to CP risk to determine the effect of storage time on the detection of these biologically relevant genes. We found the gender-specific genes and CP-related gene sets detectable in all storage periods, but exhibited differential expression (between male vs. female or CP vs. control) only within the first six years of storage. We concluded that gene expression data quality deteriorates in unfrozen archived NBS over time and that differential gene expression profiling and analysis is recommended for those NBS samples collected and stored within six years at ambient temperature.

Chromosomal amplification of leucine-rich repeat kinase-2 (LRRK2) is required for oncogenic MET signaling in papillary renal and thyroid carcinomas.

The receptor tyrosine kinase MET is frequently amplified in human tumors, resulting in high cell surface densities and constitutive activation even in the absence of growth factor stimulation by its endogenous ligand, hepatocyte growth factor (HGF). We sought to identify mechanisms of signaling crosstalk that promote MET activation by searching for kinases that are coordinately dysregulated with wild-type MET in human tumors. Our bioinformatic analysis identified leucine-rich repeat kinase-2 (LRRK2), which is amplified and overexpressed in papillary renal and thyroid carcinomas. Down-regulation of LRRK2 in cultured tumor cells compromises MET activation and selectively reduces downstream MET signaling to mTOR and STAT3. Loss of these critical mitogenic pathways induces cell cycle arrest and cell death due to loss of ATP production, indicating that MET and LRRK2 cooperate to promote efficient tumor cell growth and survival in these cancers.

Identification and mechanism of 10-carbon fatty acid as modulating ligand of peroxisome proliferator-activated receptors.

Peroxisome proliferator-activated receptors (PPARα, -ß/δ, and -γ) are a subfamily of nuclear receptors that plays key roles in glucose and lipid metabolism. PPARγ is the molecular target of the thiazolidinedione class of antidiabetic drugs that has many side effects. PPARγ is also activated by long chain unsaturated or oxidized/nitrated fatty acids, but its relationship with the medium chain fatty acids remains unclear even though the medium chain triglyceride oils have been used to control weight gain and glycemic index. Here, we show that decanoic acid (DA), a 10-carbon fatty acid and a major component of medium chain triglyceride oils, is a direct ligand of PPARγ. DA binds and partially activates PPARγ without leading to adipogenesis. Crystal structure reveals that DA occupies a novel binding site and only partially stabilizes the AF-2 helix. DA also binds weakly to PPARα and PPARß/δ. Treatments with DA and its triglyceride form improve glucose sensitivity and lipid profiles without weight gain in diabetic mice. Together, these results suggest that DA is a modulating ligand for PPARs, and the structure can aid in designing better and safer PPARγ-based drugs.

Pericyte coverage of differentiated vessels inside tumor vasculature is an independent unfavorable prognostic factor for patients with clear cell renal cell carcinoma.

BACKGROUND: The objective of this study was to evaluate the effect of pericyte coverage (PC) of differentiated tumor microvessels on the prognosis of patients with clear cell renal cell carcinoma (CCRCC). METHODS: Samples from 2 cohorts of patients with CCRCC (101 Asian patients and 524 US patients) were prepared using 2 different histologic approaches: routine sectioning versus tissue microarray. Then, the samples were immunohistochemically doubled-stained for a pericyte marker (alpha smooth muscle actin [α-SMA]) and a differentiated vessel marker (cluster of differentiation 34 [CD34]), followed by multispectral image capturing and computerized image analyses to quantify the microvessel density (MVD) and the PC of differentiated vessels. The correlations of PC and the MVD:PC ratio with clinicopathologic characteristics were analyzed. RESULTS: There was an inverse correlation between differentiated MVD and PC. Higher PC correlated with more aggressive clinicopathologic characteristics of CCRCC in both cohorts, including more advanced T-classification, higher pathologic grades, and the occurrence of tumor necrosis. The MVD:PC ratio was an independent favorable prognostic factor for overall and recurrence-free survival in the Asian cohort and for recurrence-free survival in the US cohort. PC also was an independent prognostic factor, with higher PC predicting a poorer outcome. The combination of PC and MVD was better at distinguishing the outcome of patients with CCRCC. PC combined with differentiated MVD or with the MVD:PC ratio provided additional, independent prognostic information to the Leibovich risk model, and that information was used to generate improved risk models. CONCLUSIONS: The authors consistently observed that higher PC was correlated with more aggressive clinicopathologic characteristics. PC was an independent unfavorable prognostic factor. The authors concluded that pericytes should be considered for therapeutic targeting.

Gene expression in archived newborn blood spots distinguishes infants who will later develop cerebral palsy from matched controls.

BACKGROUND: Gene expression in archived newborn blood spots remaining from newborn screening may reflect pathophysiological disturbances useful in understanding the etiology of cerebral palsy (CP). METHODS: We quantified the expression of gene sets representing four physiological pathways hypothesized to contribute to CP in archived unfrozen residual newborn blood spot specimens from 53 children with CP and 53 age-, gender-, and gestational age-matched controls. We selected four empirical and three canonical gene sets representing the inflammatory, hypoxic, coagulative, and thyroidal pathways and examined mRNA expression using an 8 × 60,000 oligonucleotide microarray. The log2 fold change of gene expression between matched cases and controls was analyzed using the generally applicable gene set enrichment method. RESULTS: The empirical inflammatory and empirical hypoxic gene sets were significantly downregulated in term-born CP cases (n = 33) as compared with matched controls (P = 0.0007 and 0.0009, respectively), whereas both gene sets were significantly upregulated (P =0.0055 and 0.0223, respectively) in preterm-born CP cases (n = 20). The empirical thyroidal gene set was significantly upregulated in preterm-born CP cases (P = 0.0023). CONCLUSION: The newborn blood spot transcriptome can serve as a platform for investigating distinctive gene expression patterns in children who later develop CP.

The adaptor protein Tks5/Fish is required for podosome formation and function, and for the protease-driven invasion of cancer cells.

Tks5/Fish is a scaffolding protein with five SH3 domains and one PX domain. In Src-transformed cells, Tks5/Fish localizes to podosomes, discrete protrusions of the ventral membrane. We generated Src-transformed cells with reduced Tks5/Fish levels. They no longer formed podosomes, did not degrade gelatin, and were poorly invasive. We detected Tks5/Fish expression in podosomes in invasive cancer cells, as well as in human breast cancer and melanoma samples. Tks5/Fish expression was also required for protease-driven matrigel invasion in human cancer cells. Finally, coexpression of Tks5/Fish and Src in epithelial cells resulted in the appearance of podosomes. Thus, Tks5/Fish appears to be required for podosome formation, for degradation of the extracellular matrix, and for invasion of some cancer cells.

Mycoplasmosis in ferrets.

We report an outbreak of severe respiratory disease associated with a novel Mycoplasma species in ferrets. During 2009-2012, a respiratory disease characterized by nonproductive coughing affected ≈8,000 ferrets, 6-8 weeks of age, which had been imported from a breeding facility in Canada. Almost 95% became ill, but almost none died. Treatments temporarily decreased all clinical signs except cough. Postmortem examinations of euthanized ferrets revealed bronchointerstitial pneumonia with prominent hyperplasia of bronchiole-associated lymphoid tissue. Immunohistochemical analysis with polyclonal antibody against Mycoplasma bovis demonstrated intense staining along the bronchiolar brush border. Bronchoalveolar lavage samples from 12 affected ferrets yielded fast-growing, glucose-fermenting mycoplasmas. Nucleic acid sequence analysis of PCR-derived amplicons from portions of the 16S rDNA and RNA polymerase B genes failed to identify the mycoplasmas but showed that they were most similar to M. molare and M. lagogenitalium. These findings indicate a causal association between the novel Mycoplasma species and the newly recognized pulmonary disease.

Genomic characterization of explant tumorgraft models derived from fresh patient tumor tissue.

BACKGROUND: There is resurgence within drug and biomarker development communities for the use of primary tumorgraft models as improved predictors of patient tumor response to novel therapeutic strategies. Despite perceived advantages over cell line derived xenograft models, there is limited data comparing the genotype and phenotype of tumorgrafts to the donor patient tumor, limiting the determination of molecular relevance of the tumorgraft model. This report directly compares the genomic characteristics of patient tumors and the derived tumorgraft models, including gene expression, and oncogenic mutation status. METHODS: Fresh tumor tissues from 182 cancer patients were implanted subcutaneously into immune-compromised mice for the development of primary patient tumorgraft models. Histological assessment was performed on both patient tumors and the resulting tumorgraft models. Somatic mutations in key oncogenes and gene expression levels of resulting tumorgrafts were compared to the matched patient tumors using the OncoCarta (Sequenom, San Diego, CA) and human gene microarray (Affymetrix, Santa Clara, CA) platforms respectively. The genomic stability of the established tumorgrafts was assessed across serial in vivo generations in a representative subset of models. The genomes of patient tumors that formed tumorgrafts were compared to those that did not to identify the possible molecular basis to successful engraftment or rejection. RESULTS: Fresh tumor tissues from 182 cancer patients were implanted into immune-compromised mice with forty-nine tumorgraft models that have been successfully established, exhibiting strong histological and genomic fidelity to the originating patient tumors. Comparison of the transcriptomes and oncogenic mutations between the tumorgrafts and the matched patient tumors were found to be stable across four tumorgraft generations. Not only did the various tumors retain the differentiation pattern, but supporting stromal elements were preserved. Those genes down-regulated specifically in tumorgrafts were enriched in biological pathways involved in host immune response, consistent with the immune deficiency status of the host. Patient tumors that successfully formed tumorgrafts were enriched for cell signaling, cell cycle, and cytoskeleton pathways and exhibited evidence of reduced immunogenicity. CONCLUSIONS: The preservation of the patient's tumor genomic profile and tumor microenvironment supports the view that primary patient tumorgrafts provide a relevant model to support the translation of new therapeutic strategies and personalized medicine approaches in oncology.

VACM-1/cul5 expression in vascular tissue in vivo is induced by water deprivation and its expression in vitro regulates aquaporin-1 concentrations.

VACM-1, a cul5 gene product, when overexpressed in vitro, has an antiproliferative effect. In vivo, VACM-1/cul5 is present in tissues involved in the regulation of water balance. Neither proteins targeted for VACM-1/cul5-specific degradation nor factors that may regulate its expression in those tissues have been studied. To identify genes that may be misregulated by VACM-1 cDNA, we performed microarray analysis. Our results indicate that in cos-1 cells transfected with VACM-1 cDNA, mRNA levels for several genes, including AQP1, were decreased when compared to the control group. Our results also indicate that in cos-1 cells transfected with VACM-1 cDNA, endogenous AQP1 protein was decreased about 6-fold when compared to the controls. To test the hypothesis that VACM-1/cul5 may be regulated by conditions that compromise water homeostasis in vivo, we determined if 24 h of water deprivation affects VACM-1/cul5 levels or the effect of VACM-1/cul5 on AQP1. VACM-1 mRNA and protein levels were significantly higher in rat mesenteric arteries, skeletal muscle and the heart ventricle but not in the heart atrium from 24-h water-deprived rats when compared to the controls. Interestingly, 24 h of water deprivation increased modification of VACM-1 by an ubiquitin-like protein, Nedd8, essential for cullin-dependent E3 ligase activity. Although water deprivation did not significantly change AQP1 levels in the mesenteric arteries, AQP1 protein concentrations were inversely correlated with the ratio of the VACM-1 to Nedd8-modified VACM-1. These results suggest that VACM-1/cul5 may regulate endothelial AQP1 concentration both in vivo and in vitro.

Evaluation of sex-specific gene expression in archived dried blood spots (DBS).

Screening newborns for treatable serious conditions is mandated in all US states and many other countries. After screening, Guthrie cards with residual blood (whole spots or portions of spots) are typically stored at ambient temperature in many facilities. The potential of archived dried blood spots (DBS) for at-birth molecular studies in epidemiological and clinical research is substantial. However, it is also challenging as analytes from DBS may be degraded due to preparation and storage conditions. We previously reported an improved assay for obtaining global RNA gene expression from blood spots. Here, we evaluated sex-specific gene expression and its preservation in DBS using oligonucleotide microarray technology. We found X inactivation-specific transcript (XIST), lysine-specific demethylase 5D (KDM5D) (also known as selected cDNA on Y, homolog of mouse (SMCY)), uncharacterized LOC729444 (LOC729444), and testis-specific transcript, Y-linked 21 (TTTY21) to be differentially-expressed by sex of the newborn. Our finding that trait-specific RNA gene expression is preserved in unfrozen DBS, demonstrates the technical feasibility of performing molecular genetic profiling using such samples. With millions of DBS potentially available for research, we see new opportunities in using newborn molecular gene expression to better understand molecular pathogenesis of perinatal diseases.

Host response transcriptional profiling reveals extracellular components and ABC (ATP-binding cassette) transporters gene enrichment in typhoid fever-infected Nigerian children.

BACKGROUND: Salmonella enterica serovar Typhi (S. Typhi) is a human-specific pathogen that causes typhoid fever, and remains a global health problem especially in developing countries. Its pathogenesis is complex and host response is poorly understood. In Africa, typhoid fever can be a major cause of morbidity in young infected children. The onset of the illness is insidious and clinical diagnosis is often unreliable. Gold standard blood culture diagnostic services are limited, thus rapid, sensitive, and affordable diagnostic test is essential in poor-resourced clinical settings. Routine typhoid fever vaccination is highly recommended but currently licensed vaccines provide only 55-75% protection. Recent epidemiological studies also show the rapid emergence of multi-drug resistant S. Typhi strains. High-throughput molecular technologies, such as microarrays, can dissect the molecular mechanisms of host responses which are S. Typhi-specific to provide a comprehensive genomic component of immunological responses and suggest new insights for diagnosis and treatment. METHODS: Global transcriptional profiles of S. Typhi-infected young Nigerian children were obtained from their peripheral blood and compared with that of other bacteremic infections using Agilent gene expression microarrays. The host-response profiles of the same patients in acute vs. convalescent phases were also determined. The top 96-100 differentially-expressed genes were identified and four genes were validated by quantitative real-time PCR. Gene clusters were obtained and functional pathways were predicted by DAVID (Database for Annotation, Visualization and Integrated Discovery). RESULTS: Transcriptional profiles from S. Typhi-infected children could be distinguished from those of other bacteremic infections. Enriched gene clusters included genes associated with extracellular peptides/components such as lipocalin (LCN2) and systemic immune response which is atypical in bacterial invasion. Distinct gene expression profiles can also be obtained from acute vs. convalescent phase during typhoid fever infection. We found novel down-regulation of ABC (ATP-binding cassette) transporters genes such as ABCA7, ABCC5, and ABCD4 and ATPase activity as the highest enriched pathway. CONCLUSIONS: We identified unique extracellular components and ABC transporters gene enrichments in typhoid fever-infected Nigerian children, which have never been reported. These enriched gene clusters may represent novel targeted pathways to improve diagnostic, prognostic, therapeutic and next-generation vaccine strategies for typhoid fever in Africa.

Acquiring genome-wide gene expression profiles in Guthrie card blood spots using microarrays.

Standard Guthrie cards have been widely used to collect blood samples from essentially all USA and Japanese neonates for newborn screening programs. Thus, archival blood spot samples are a unique and comprehensive resource for molecular pathology studies. However, the challenge in using these samples is the presumed low quantity and degraded quality of nucleic acids that can be isolated from these samples, particularly the RNA. Here, we report a new assay using Agilent 4x44K microarrays for acquiring genome-wide gene expression profiles from blood spots on Guthrie cards. Due to the small amount of RNA obtained from each sample, major modifications, such as concentrating and amplifying the RNA and using a different labeling procedure, were performed. Approximately 9000 expressed genes can be detected after normalization of data, an increment of 260% in detection power compared with previously reported cDNA microarrays made in-house with standard procedures. The correlation coefficients in technical and biological replicates were 0.92 and 0.85, respectively, confirming the reproducibility of this study. This new and comprehensive assay will add value to the utility of archival Guthrie cards (e.g. neonatal blood spot cards) and open new opportunities to molecular epidemiology, pathology, genomic, and diagnostic studies of perinatal diseases.

Molecular subtype analysis determines the association of advanced breast cancer in Egypt with favorable biology.

BACKGROUND: Prognostic markers and molecular breast cancer subtypes reflect underlying biological tumor behavior and are important for patient management. Compared to Western countries, women in North Africa are less likely to be prognosticated and treated based on well-characterized markers such as the estrogen receptor (ER), progesterone receptor (PR) and Her2. We conducted this study to determine the prevalence of breast cancer molecular subtypes in the North African country of Egypt as a measure of underlying biological characteristics driving tumor manifestations. METHODS: To determine molecular subtypes we characterized over 200 tumor specimens obtained from Egypt by performing ER, PR, Her2, CK5/6, EGFR and Ki67 immunohistochemistry. RESULTS: Our study demonstrated that the Luminal A subtype, associated with favorable prognosis, was found in nearly 45% of cases examined. However, the basal-like subtype, associated with poor prognosis, was found in 11% of cases. These findings are in sharp contrast to other parts of Africa in which the basal-like subtype is over-represented. CONCLUSIONS: Egyptians appear to have favorable underlying biology, albeit having advanced disease at diagnosis. These data suggest that Egyptians would largely profit from early detection of their disease. Intervention at the public health level, including education on the benefits of early detection is necessary and would likely have tremendous impact on breast cancer outcome in Egypt.

Serum adiponectin, resistin, and circulating soluble receptor for advanced glycation end products in colectomy patients.

AIM: Surgical trauma and associated complications are frequently related to physiological stress during colectomy. This study evaluated the response of adiponectin, resistin, and circulating soluble receptor for advanced glycation end products (sRAGE) in colectomy patients with or without an enhanced recovery protocol. METHOD: Serum samples were collected from 44 colectomy patients at 3 timframes. The surgical procedures were laparoscopic (LAP), hand-assisted laparoscopic (HALS), or open colectomy (OPEN). Adiponectin, resistin, and sRAGE levels were determined by ELISA. Repeated measures ANOVA was applied and P values < 0.05 were considered significant. RESULTS: A total of 132 (44 × 3) sera were used for analysis. Levels of adiponectin was significantly decreased between PREOP and POD3 (P < 0.001). Conversely, concentrations of resistin significantly increased from PREOP to POD1 and returned to baseline value by POD3 (P < 0.001). Serum sRAGE levels were significantly higher in LAP in comparison with HALS (P = 0.004) and OPEN (P < 0.001). sRAGE levels were significantly higher in sera of patients that underwent ERP (P < 0.001). CONCLUSIONS: Serum adiponectin, resistin, and sRAGE have the potential to develop into a panel of stress markers. Higher sRAGE levels in sera of LAP and ERP patients may be indicative of a protective and syngeristic role for colectomy recovery.

LRP5 and LRP6 are not required for protective antigen-mediated internalization or lethality of anthrax lethal toxin.

Anthrax toxin (AnTx) plays a key role in the pathogenesis of anthrax. AnTx is composed of three proteins: protective antigen (PA), edema factor, and lethal factor (LF). PA is not toxic but serves to bind cells and translocate the toxic edema factor or LF moieties to the cytosol. Recently, the low-density lipoprotein receptor-related protein LRP6 has been reported to mediate internalization and lethality of AnTx. Based on its similarity to LRP6, we hypothesized that LRP5 may also play a role in cellular uptake of AnTx. We assayed PA-dependent uptake of anthrax LF or a cytotoxic LF fusion protein (FP59) in cells and mice harboring targeted deletions of Lrp5 or Lrp6. Unexpectedly, we observed that uptake was unaltered in the presence or absence of either Lrp5 or Lrp6 expression. Moreover, we observed efficient PA-mediated uptake into anthrax toxin receptor (ANTXR)-deficient Chinese hamster ovary cells (PR230) that had been stably engineered to express either human ANTXR1 or human ANTXR2 in the presence or absence of siRNA specific for LRP5 or LRP6. Our results demonstrate that neither LRP5 nor LRP6 is necessary for PA-mediated internalization or lethality of anthrax lethal toxin.

Inactivation of Apc in the mouse prostate causes prostate carcinoma.

Alterations of the Wnt/beta-catenin signaling pathway are positively associated with the development and progression of human cancer, including carcinoma of the prostate. To determine the role of activated Wnt/beta-catenin signaling in mouse prostate carcinogenesis, we created a mouse prostate tumor model using probasin-Cre-mediated deletion of Apc. Prostate tumors induced by the deletion of Apc have elevated levels of beta-catenin protein and are highly proliferative. Tumor formation is fully penetrant and follows a consistent pattern of progression. Hyperplasia is observed as early as 4.5 weeks of age, and adenocarcinoma is observed by 7 months. Continued tumor growth usually necessitated sacrifice between 12 and 15 months of age. Despite the high proliferation rate, we have not observed metastasis of these tumors to the lymph nodes or other organs. Surgical castration of 6-week-old mice inhibited tumor formation, and castration of mice with more advanced tumors resulted in the partial regression of specific prostate glands. However, significant areas of carcinoma remained 2 months postcastration, suggesting that tumors induced by Apc loss of function are capable of growth under conditions of androgen depletion. We conclude that the prostate-specific deletion of Apc and the increased expression of beta-catenin associated with prostate carcinoma suggests a role for beta-catenin in prostate cancer and offers an appropriate animal model to investigate the interaction of Wnt signaling with other genetic and epigenetic signals in prostate carcinogenesis.

Mitogen-activated protein kinase kinase signaling promotes growth and vascularization of fibrosarcoma.

We hypothesized that signaling through multiple mitogen-activated protein kinase (MAPK) kinase (MKK) pathways is essential for the growth and vascularization of soft-tissue sarcomas, which are malignant tumors derived from mesenchymal tissues. We tested this using HT-1080, NCI, and Shac fibrosarcoma-derived cell lines and anthrax lethal toxin (LeTx), a bacterial toxin that inactivates MKKs. Western blots confirmed that LeTx treatment reduced the levels of phosphorylated extracellular signal-regulated kinase and p38 MAPK in vitro. Although short treatments with LeTx only modestly affected cell proliferation, sustained treatment markedly reduced cell numbers. LeTx also substantially inhibited the extracellular release of angioproliferative factors including vascular endothelial growth factor, interleukin-8, and basic fibroblast growth factor. Similar results were obtained with cell lines derived from malignant fibrous histiocytomas, leiomyosarcomas, and liposarcomas. In vivo, LeTx decreased MAPK activity and blocked fibrosarcoma growth. Growth inhibition correlated with decreased cellular proliferation and extensive necrosis, and it was accompanied by a decrease in tumor mean vessel density as well as a reduction in serum expression of angioproliferative cytokines. Vital imaging using high-resolution ultrasound enhanced with contrast microbubbles revealed that the effects of LeTx on tumor perfusion were remarkably rapid (<24 h) and resulted in a marked reduction of perfusion within the tumor but not in nontumor tissues. These results are consistent with our initial hypothesis and lead us to propose that MKK inhibition by LeTx is a broadly effective strategy for targeting neovascularization in fibrosarcomas and other similar proliferative lesions.

A highly invasive human glioblastoma pre-clinical model for testing therapeutics.

Animal models greatly facilitate understanding of cancer and importantly, serve pre-clinically for evaluating potential anti-cancer therapies. We developed an invasive orthotopic human glioblastoma multiforme (GBM) mouse model that enables real-time tumor ultrasound imaging and pre-clinical evaluation of anti-neoplastic drugs such as 17-(allylamino)-17-demethoxy geldanamycin (17AAG). Clinically, GBM metastasis rarely happen, but unexpectedly most human GBM tumor cell lines intrinsically possess metastatic potential. We used an experimental lung metastasis assay (ELM) to enrich for metastatic cells and three of four commonly used GBM lines were highly metastatic after repeated ELM selection (M2). These GBM-M2 lines grew more aggressively orthotopically and all showed dramatic multifold increases in IL6, IL8, MCP-1 and GM-CSF expression, cytokines and factors that are associated with GBM and poor prognosis. DBM2 cells, which were derived from the DBTRG-05MG cell line were used to test the efficacy of 17AAG for treatment of intracranial tumors. The DMB2 orthotopic xenografts form highly invasive tumors with areas of central necrosis, vascular hyperplasia and intracranial dissemination. In addition, the orthotopic tumors caused osteolysis and the skull opening correlated to the tumor size, permitting the use of real-time ultrasound imaging to evaluate antitumor drug activity. We show that 17AAG significantly inhibits DBM2 tumor growth with significant drug responses in subcutaneous, lung and orthotopic tumor locations. This model has multiple unique features for investigating the pathobiology of intracranial tumor growth and for monitoring systemic and intracranial responses to antitumor agents.

The parafibromin tumor suppressor protein is part of a human Paf1 complex.

Parafibromin, the product of the HRPT2 (hyperparathyroidism-jaw tumor syndrome 2) tumor suppressor gene, is the human homologue of yeast Cdc73, part of the yeast RNA polymerase II/Paf1 complex known to be important for histone modification and connections to posttranscriptional events. By purifying cellular parafibromin and characterizing its associated proteins, we have identified a human counterpart to the yeast Paf1 complex including homologs of Leo1, Paf1, and Ctr9. Like the yeast complex, the parafibromin complex associates with the nonphosphorylated and Ser2 and Ser5 phosphorylated forms of the RNA polymerase II large subunit. Immunofluorescence experiments show that parafibromin is a nuclear protein. In addition, cotransfection data suggest that parafibromin can interact with a histone methyltransferase complex that methylates histone H3 on lysine 4. Some mutant forms of parafibromin lack association with hPaf1 complex members and with the histone methyltransferase complex, suggesting that disruption of these complexes may correlate with the oncogenic process.

Anthrax lethal toxin inhibits growth of and vascular endothelial growth factor release from endothelial cells expressing the human herpes virus 8 viral G protein coupled receptor.

PURPOSE: In this study, we tested the hypothesis that inhibition of mitogen-activated protein kinase kinases (MKK) inhibits tumor growth by acting on angiogenic signaling and by extension may form the basis of an effective strategy for treatment of Kaposi's sarcoma. EXPERIMENTAL DESIGN: Murine endothelial cells expressing the human herpes virus 8 G protein-coupled receptor (vGPCR-SVEC) were treated with anthrax lethal toxin (LeTx). LeTx is a binary toxin ordinarily secreted by Bacillus anthracis and is composed of two proteins: protective antigen (the binding moiety) and lethal factor (the active moiety). Lethal factor is a protease that cleaves and inactivates MKKs. RESULTS: In vitro, treatment of vGPCR-SVEC with LeTx inhibited MKK signaling, moderately inhibited cell proliferation, and blocked the ability of these cells to form colonies in soft agar. Treatment with LeTx also blocked the ability of these cells to release several angioproliferative cytokines, notably vascular endothelial growth factor (VEGF). In contrast, inhibition of mitogen-activated protein kinase/extracellular signal-regulated kinase 1/2 with U0126 caused a substantial inhibition of proliferation but only modestly inhibited VEGF release. In xenograft models, i.v. injection of LeTx caused reduced tumor growth characterized immunohistochemically by inhibition of MKK signaling, decreased rates of proliferation, and reduced levels of VEGF and VEGF receptor 2, with a corresponding decrease in vascular density. CONCLUSIONS: These data support a role for MKK signaling in tumor growth and vascularization and are consistent with the hypothesis that inhibition of MKK signaling by LeTx or a similar agent may be an effective strategy for the treatment of Kaposi's sarcoma as well as other vascular tumors.