Clonally Selected Lines After CRISPR-Cas Editing Are Not Isogenic.

The CRISPR-Cas9 system has enabled researchers to precisely modify/edit the sequence of a genome. A typical editing experiment consists of two steps: (1) editing cultured cells; (2) cell cloning and selection of clones with and without intended edit, presumed to be isogenic. The application of CRISPR-Cas9 system may result in off-target edits, whereas cloning will reveal culture-acquired mutations. We analyzed the extent of the former and the latter by whole genome sequencing in three experiments involving separate genomic loci and conducted by three independent laboratories. In all experiments we hardly found any off-target edits, whereas detecting hundreds to thousands of single nucleotide mutations unique to each clone after relatively short culture of 10-20 passages. Notably, clones also differed in copy number alterations (CNAs) that were several kb to several mb in size and represented the largest source of genomic divergence among clones. We suggest that screening of clones for mutations and CNAs acquired in culture is a necessary step to allow correct interpretation of DNA editing experiments. Furthermore, since culture associated mutations are inevitable, we propose that experiments involving derivation of clonal lines should compare a mix of multiple unedited lines and a mix of multiple edited lines.

Intrapulmonary administration of purified NEIL2 abrogates NF-κB-mediated inflammation.

Aberrant or constitutive activation of nuclear factor kappa B (NF-κB) contributes to various human inflammatory diseases and malignancies via the upregulation of genes involved in cell proliferation, survival, angiogenesis, inflammation, and metastasis. Thus, inhibition of NF-κB signaling has potential for therapeutic applications in cancer and inflammatory diseases. We reported previously that Nei-like DNA glycosylase 2 (NEIL2), a mammalian DNA glycosylase, is involved in the preferential repair of oxidized DNA bases from the transcriptionally active sequences via the transcription-coupled base excision repair pathway. We have further shown that Neil2-null mice are highly sensitive to tumor necrosis factor α (TNFα)- and lipopolysaccharide-induced inflammation. Both TNFα and lipopolysaccharide are potent activators of NF-κB. However, the underlying mechanism of NEIL2's role in the NF-κB-mediated inflammation remains elusive. Here, we have documented a noncanonical function of NEIL2 and demonstrated that the expression of genes, such as Cxcl1, Cxcl2, Cxcl10, Il6, and Tnfα, involved in inflammation and immune cell migration was significantly higher in both mock- and TNFα-treated Neil2-null mice compared with that in the WT mice. NEIL2 blocks NF-κB's binding to target gene promoters by directly interacting with the Rel homology region of RelA and represses proinflammatory gene expression as determined by co-immunoprecipitation, chromatin immunoprecipitation, and electrophoretic mobility-shift assays. Remarkably, intrapulmonary administration of purified NEIL2 via a noninvasive nasal route significantly abrogated binding of NF-κB to cognate DNA, leading to decreased expression of proinflammatory genes and neutrophil recruitment in Neil2-null as well as WT mouse lungs. Our findings thus highlight the potential of NEIL2 as a biologic for inflammation-associated human diseases.

Genomic and Phenotypic Characterization of a Broad Panel of Patient-Derived Xenografts Reflects the Diversity of Glioblastoma.

PURPOSE: Glioblastoma is the most frequent and lethal primary brain tumor. Development of novel therapies relies on the availability of relevant preclinical models. We have established a panel of 96 glioblastoma patient-derived xenografts (PDX) and undertaken its genomic and phenotypic characterization. EXPERIMENTAL DESIGN: PDXs were established from glioblastoma, IDH-wildtype (n = 93), glioblastoma, IDH-mutant (n = 2), diffuse midline glioma, H3 K27M-mutant (n = 1), and both primary (n = 60) and recurrent (n = 34) tumors. Tumor growth rates, histopathology, and treatment response were characterized. Integrated molecular profiling was performed by whole-exome sequencing (WES, n = 83), RNA-sequencing (n = 68), and genome-wide methylation profiling (n = 76). WES data from 24 patient tumors was compared with derivative models. RESULTS: PDXs recapitulate many key phenotypic and molecular features of patient tumors. Orthotopic PDXs show characteristic tumor morphology and invasion patterns, but largely lack microvascular proliferation and necrosis. PDXs capture common and rare molecular drivers, including alterations of TERT, EGFR, PTEN, TP53, BRAF, and IDH1, most at frequencies comparable with human glioblastoma. However, PDGFRA amplification was absent. RNA-sequencing and genome-wide methylation profiling demonstrated broad representation of glioblastoma molecular subtypes. MGMT promoter methylation correlated with increased survival in response to temozolomide. WES of 24 matched patient tumors showed preservation of most genetic driver alterations, including EGFR amplification. However, in four patient-PDX pairs, driver alterations were gained or lost on engraftment, consistent with clonal selection. CONCLUSIONS: Our PDX panel captures the molecular heterogeneity of glioblastoma and recapitulates many salient genetic and phenotypic features. All models and genomic data are openly available to investigators.

Improved Drug Delivery to Brain Metastases by Peptide-Mediated Permeabilization of the Blood-Brain Barrier.

Patients with melanoma have a high risk of developing brain metastasis, which is associated with a dismal prognosis. During early stages of metastasis development, the blood-brain barrier (BBB) is likely intact, which inhibits sufficient drug delivery into the metastatic lesions. We investigated the ability of the peptide, K16ApoE, to permeabilize the BBB for improved treatment with targeted therapies preclinically. Dynamic contrast enhanced MRI (DCE-MRI) was carried out on NOD/SCID mice to study the therapeutic window of peptide-mediated BBB permeabilization. Further, both in vivo and in vitro assays were used to determine K16ApoE toxicity and to obtain mechanistic insight into its action on the BBB. The therapeutic impact of K16ApoE on metastases was evaluated combined with the mitogen-activated protein kinase pathway inhibitor dabrafenib, targeting BRAF mutated melanoma cells, which is otherwise known not to cross the intact BBB. Our results from the DCE-MRI experiments showed effective K16ApoE-mediated BBB permeabilization lasting for up to 1 hour. Mechanistic studies showed a dose-dependent effect of K16ApoE caused by induction of endocytosis. At concentrations above IC50, the peptide additionally showed nonspecific disturbances on plasma membranes. Combined treatment with K16ApoE and dabrafenib reduced the brain metastatic burden in mice and increased animal survival, and PET/CT showed that the peptide also facilitated the delivery of compounds with molecular weights as large as 150 kDa into the brain. To conclude, we demonstrate a transient permeabilization of the BBB, caused by K16ApoE, that facilitates enhanced drug delivery into the brain. This improves the efficacy of drugs that otherwise do not cross the intact BBB.

Cationic carrier peptide enhances cerebrovascular targeting of nanoparticles in Alzheimer's disease brain.

Accumulation of amyloid beta (Aß) peptides in the cerebral vasculature, referred to as cerebral amyloid angiopathy (CAA), is widely observed in Alzheimer's disease (AD) brain and was shown to accelerate cognitive decline. There is no effective method for detecting cerebrovascular amyloid (CVA) and treat CAA. The targeted nanoparticles developed in this study effectively migrated from the blood flow to the vascular endothelium as determined by using quartz crystal microbalance with dissipation monitoring (QCM-D) technology. We also improved the stability, and blood-brain barrier (BBB) transcytosis of targeted nanoparticles by coating them with a cationic BBB penetrating peptide (K16ApoE). The K16ApoE-Targeted nanoparticles demonstrated specific targeting of vasculotropic DutchAß40 peptide accumulated in the cerebral vasculature. Moreover, K16ApoE-Targeted nanoparticles demonstrated significantly greater uptake into brain and provided specific MRI contrast to detect brain amyloid plaques.

Adult infiltrating gliomas with WHO 2016 integrated diagnosis: additional prognostic roles of ATRX and TERT.

The "integrated diagnosis" for infiltrating gliomas in the 2016 revised World Health Organization (WHO) classification of tumors of the central nervous system requires assessment of the tumor for IDH mutations and 1p/19q codeletion. Since TERT promoter mutations and ATRX alterations have been shown to be associated with prognosis, we analyzed whether these tumor markers provide additional prognostic information within each of the five WHO 2016 categories. We used data for 1206 patients from the UCSF Adult Glioma Study, the Mayo Clinic and The Cancer Genome Atlas (TCGA) with infiltrative glioma, grades II-IV for whom tumor status for IDH, 1p/19q codeletion, ATRX, and TERT had been determined. All cases were assigned to one of 5 groups following the WHO 2016 diagnostic criteria based on their morphologic features, and IDH and 1p/19q codeletion status. These groups are: (1) Oligodendroglioma, IDH-mutant and 1p/19q-codeleted; (2) Astrocytoma, IDH-mutant; (3) Glioblastoma, IDH-mutant; (4) Glioblastoma, IDH-wildtype; and (5) Astrocytoma, IDH-wildtype. Within each group, we used univariate and multivariate Cox proportional hazards models to assess associations of overall survival with patient age at diagnosis, grade, and ATRX alteration status and/or TERT promoter mutation status. Among Group 1 IDH-mutant 1p/19q-codeleted oligodendrogliomas, the TERT-WT group had significantly worse overall survival than the TERT-MUT group (HR: 2.72, 95% CI 1.05-7.04, p = 0.04). In both Group 2, IDH-mutant astrocytomas and Group 3, IDH-mutant glioblastomas, neither TERT mutations nor ATRX alterations were significantly associated with survival. Among Group 4, IDH-wildtype glioblastomas, ATRX alterations were associated with favorable outcomes (HR: 0.36, 95% CI 0.17-0.81, p = 0.01). Among Group 5, IDH-wildtype astrocytomas, the TERT-WT group had significantly better overall survival than the TERT-MUT group (HR: 0.48, 95% CI 0.27-0.87), p = 0.02). Thus, we present evidence that in certain WHO 2016 diagnostic groups, testing for TERT promoter mutations or ATRX alterations may provide additional useful prognostic information.

2-Methoxyestradiol-Mediated Induction of Frzb Contributes to Cell Death and Autophagy in MG63 Osteosarcoma Cells.

Osteosarcoma is a bone tumor that mainly affects children and adolescents. Although its pathogenesis is still not fully understood, activation of Wnt signaling has been implicated in the development and metastasis of osteosarcoma. In this report, we have investigated the effect of the anti-tumor compound, 2-methoxyestradiol (2-ME) on Wnt antagonist frizzled-related protein b (Frzb), also known as secreted frizzled-related protein (sFRP)3 in human osteosarcoma (MG63) cells. Our results show that 2-ME treatment induces Frzb gene promoter activity, and increases Frzb mRNA and protein levels in osteosarcoma cells. In addition, 2-ME treatment regulates downstream Wnt signaling, increasing the cytoplasmic levels of ß-catenin, and blocking ß-catenin-mediated Wnt activation in osteosarcoma cells. 2-ME-mediated induction of Frzb protein expression is specific to osteosarcoma cells, as it does not affect Frzb expression in normal primary human osteoblasts. Furthermore, 2-ME-induced apoptosis and autophagy are blocked in osteosarcoma cells transfected with Frzb siRNAs. Taken together, these studies demonstrate that Frzb protein plays an important role in 2-ME-mediated anti-tumor mechanisms in osteosarcoma cells. J. Cell. Biochem. 118: 1497-1504, 2017. © 2016 Wiley Periodicals, Inc.

Delineation of MGMT Hypermethylation as a Biomarker for Veliparib-Mediated Temozolomide-Sensitizing Therapy of Glioblastoma.

BACKGROUND: Sensitizing effects of poly-ADP-ribose polymerase inhibitors have been studied in several preclinical models, but a clear understanding of predictive biomarkers is lacking. In this study, in vivo efficacy of veliparib combined with temozolomide (TMZ) was evaluated in a large panel of glioblastoma multiforme (GBM) patient-derived xenografts (PDX) and potential biomarkers were analyzed. METHODS: The efficacy of TMZ alone vs TMZ/veliparib was compared in a panel of 28 GBM PDX lines grown as orthotopic xenografts (8-10 mice per group); all tests of statistical significance were two-sided. DNA damage was analyzed by γH2AX immunostaining and promoter methylation of DNA repair gene O6-methylguanine-DNA-methyltransferase (MGMT) by Clinical Laboratory Improvement Amendments-approved methylation-specific polymerase chain reaction. RESULTS: The combination of TMZ/veliparib statistically significantly extended survival of GBM models (P < .05 by log-rank) compared with TMZ alone in five of 20 MGMT-hypermethylated lines (average extension in median survival = 87 days, range = 20-150 days), while the combination was ineffective in six MGMT-unmethylated lines. In the MGMT promoter-hypermethylated GBM12 line (median survival with TMZ+veliparib = 189 days, 95% confidence interval [CI] = 59 to 289 days, vs TMZ alone = 98 days, 95% CI = 49 to 210 days, P = .04), the profound TMZ-sensitizing effect of veliparib was lost when MGMT was overexpressed (median survival with TMZ+veliparib = 36 days, 95% CI = 28 to 38 days, vs TMZ alone = 35 days, 95% CI = 32 to 37 days, P = .87), and a similar association was observed in two nearly isogenic GBM28 sublines with an intact vs deleted MGMT locus. In comparing DNA damage signaling after dosing with veliparib/TMZ or TMZ alone, increased phosphorylation of damage-responsive proteins (KAP1, Chk1, Chk2, and H2AX) was observed only in MGMT promoter-hypermethylated lines. CONCLUSION: Veliparib statistically significantly enhances (P < .001) the efficacy of TMZ in tumors with MGMT promoter hypermethylation. Based on these data, MGMT promoter hypermethylation is being used as an eligibility criterion for A071102 (NCT02152982), the phase II/III clinical trial evaluating TMZ/veliparib combination in patients with GBM.

Glioma Groups Based on 1p/19q, IDH, and TERT Promoter Mutations in Tumors.

BACKGROUND: The prediction of clinical behavior, response to therapy, and outcome of infiltrative glioma is challenging. On the basis of previous studies of tumor biology, we defined five glioma molecular groups with the use of three alterations: mutations in the TERT promoter, mutations in IDH, and codeletion of chromosome arms 1p and 19q (1p/19q codeletion). We tested the hypothesis that within groups based on these features, tumors would have similar clinical variables, acquired somatic alterations, and germline variants. METHODS: We scored tumors as negative or positive for each of these markers in 1087 gliomas and compared acquired alterations and patient characteristics among the five primary molecular groups. Using 11,590 controls, we assessed associations between these groups and known glioma germline variants. RESULTS: Among 615 grade II or III gliomas, 29% had all three alterations (i.e., were triple-positive), 5% had TERT and IDH mutations, 45% had only IDH mutations, 7% were triple-negative, and 10% had only TERT mutations; 5% had other combinations. Among 472 grade IV gliomas, less than 1% were triple-positive, 2% had TERT and IDH mutations, 7% had only IDH mutations, 17% were triple-negative, and 74% had only TERT mutations. The mean age at diagnosis was lowest (37 years) among patients who had gliomas with only IDH mutations and was highest (59 years) among patients who had gliomas with only TERT mutations. The molecular groups were independently associated with overall survival among patients with grade II or III gliomas but not among patients with grade IV gliomas. The molecular groups were associated with specific germline variants. CONCLUSIONS: Gliomas were classified into five principal groups on the basis of three tumor markers. The groups had different ages at onset, overall survival, and associations with germline variants, which implies that they are characterized by distinct mechanisms of pathogenesis. (Funded by the National Institutes of Health and others.).

Peptide carrier-mediated non-covalent delivery of unmodified cisplatin, methotrexate and other agents via intravenous route to the brain.

BACKGROUND: Rapid pre-clinical evaluation of chemotherapeutic agents against brain cancers and other neurological disorders remains largely unattained due to the presence of the blood-brain barrier (BBB), which limits transport of most therapeutic compounds to the brain. A synthetic peptide carrier, K16ApoE, was previously developed that enabled transport of target proteins to the brain by mimicking a ligand-receptor system. The peptide carrier was found to generate transient BBB permeability, which was utilized for non-covalent delivery of cisplatin, methotrexate and other compounds to the brain. APPROACH: Brain delivery of the chemotherapeutics and other agents was achieved either by injecting the carrier peptide and the drugs separately or as a mixture, to the femoral vein. A modification of the method comprised injection of K16ApoE pre-mixed with cetuximab, followed by injection of a 'small-molecule' drug. PRINCIPAL FINDINGS: Seven-of-seven different small molecules were successfully delivered to the brain via K16ApoE. Depending on the method, brain uptake with K16ApoE was 0.72-1.1% for cisplatin and 0.58-0.92% for methotrexate (34-50-fold and 54-92 fold greater for cisplatin and methotrexate, respectively, with K16ApoE than without). Visually intense brain-uptake of Evans Blue, Light Green SF and Crocein scarlet was also achieved. Direct intracranial injection of EB show locally restricted distribution of the dye in the brain, whereas K16ApoE-mediated intravenous injection of EB resulted in the distribution of the dye throughout the brain. Experiments with insulin suggest that ligand-receptor signaling intrinsic to the BBB provides a natural means for passive transport of some molecules across the BBB. SIGNIFICANCE: The results suggest that the carrier peptide can non-covalently transport various chemotherapeutic agents to the brain. Thus, the method offers an avenue for pre-clinical evaluation of various small and large therapeutic molecules against brain tumors and other neurological disorders.

Effective intravenous therapy for neurodegenerative disease with a therapeutic enzyme and a peptide that mediates delivery to the brain.

The blood-brain barrier (BBB) presents a major challenge to effective treatment of neurological disorders, including lysosomal storage diseases (LSDs), which frequently present with life-shortening and untreatable neurodegeneration. There is considerable interest in methods for intravenous delivery of lysosomal proteins across the BBB but for the most part, levels achievable in the brain of mouse models are modest and increased lifespan remains to be demonstrated. In this study, we have investigated delivery across the BBB using a mouse model of late-infantile neuronal ceroid lipofuscinosis (LINCL), a neurodegenerative LSD caused by loss of tripeptidyl peptidase I (TPP1). We have achieved supraphysiological levels of TPP1 throughout the brain of LINCL mice by intravenous (IV) coadministration of recombinant TPP1 with a 36-residue peptide that contains polylysine and a low-density lipoprotein receptor binding sequence from apolipoprotein E. Importantly, IV administration of TPP1 with the peptide significantly reduces brain lysosomal storage, increases lifespan and improves neurological function. This simple "mix and inject" method is immediately applicable towards evaluation of enzyme replacement therapy to the brain in preclinical models and further exploration of its clinical potential is warranted.

A carrier for non-covalent delivery of functional beta-galactosidase and antibodies against amyloid plaques and IgM to the brain.

BACKGROUND: Therapeutic intervention of numerous brain-associated disorders currently remains unrealized due to serious limitations imposed by the blood-brain-barrier (BBB). The BBB generally allows transport of small molecules, typically <600 daltons with high octanol/water partition coefficients, but denies passage to most larger molecules. However, some receptors present on the BBB allow passage of cognate proteins to the brain. Utilizing such receptor-ligand systems, several investigators have developed methods for delivering proteins to the brain, a critical requirement of which involves covalent linking of the target protein to a carrier entity. Such covalent modifications involve extensive preparative and post-preparative chemistry that poses daunting limitations in the context of delivery to any organ. Here, we report creation of a 36-amino acid peptide transporter, which can transport a protein to the brain after routine intravenous injection of the transporter-protein mixture. No covalent linkage of the protein with the transporter is necessary. APPROACH: A peptide transporter comprising sixteen lysine residues and 20 amino acids corresponding to the LDLR-binding domain of apolipoprotein E (ApoE) was synthesized. Transport of beta-galactosidase, IgG, IgM, and antibodies against amyloid plques to the brain upon iv injection of the protein-transporter mixture was evaluated through staining for enzyme activity or micro single photon emission tomography (micro-SPECT) or immunostaining. Effect of the transporter on the integrity of the BBB was also investigated. PRINCIPAL FINDINGS: The transporter enabled delivery to the mouse brain of functional beta-galactosidase, human IgG and IgM, and two antibodies that labeled brain-associated amyloid beta plaques in a mouse model of Alzheimer's disease. SIGNIFICANCE: The results suggest the transporter is able to transport most or all proteins to the brain without the need for chemically linking the transporter to a protein. Thus, the approach offers an avenue for rapid clinical evaluation of numerous candidate drugs against neurological diseases including cancer. (299 words).

Sleeping beauty-mediated somatic mutagenesis implicates CSF1 in the formation of high-grade astrocytomas.

The Sleeping Beauty (SB) transposon system has been used as an insertional mutagenesis tool to identify novel cancer genes. To identify glioma-associated genes, we evaluated tumor formation in the brain tissue from 117 transgenic mice that had undergone constitutive SB-mediated transposition. Upon analysis, 21 samples (18%) contained neoplastic tissue with features of high-grade astrocytomas. These tumors expressed glial markers and were histologically similar to human glioma. Genomic DNA from SB-induced astrocytoma tissue was extracted and transposon insertion sites were identified. Insertions in the growth factor gene Csf1 were found in 13 of the 21 tumors (62%), clustered in introns 5 and 8. Using reverse transcription-PCR, we documented increased Csf1 RNAs in tumor versus adjacent normal tissue, with the identification of transposon-terminated Csf1 mRNAs in astrocytomas with SB insertions in intron 8. Analysis of human glioblastomas revealed increased levels of Csf1 RNA and protein. Together, these results indicate that SB-insertional mutagenesis can identify high-grade astrocytoma-associated genes and they imply an important role for CSF1 in the development of these tumors.

Preferential expression of the secreted and membrane forms of tumor endothelial marker 7 transcripts in osteosarcoma.

BACKGROUND: High expression of tumor endothelial marker 7 (TEM7) is correlated with osteogenic sarcoma (OS) metastasis and poor survival of patients. The TEM7 gene produces four alternatively spliced transcripts with distinct functional domains; the expression pattern of these transcripts in OS is unknown. MATERIALS AND METHODS: mRNA expression was assessed in 5 OS cell lines, 7 normal bone, and 9 OS tumor specimens by reverse transcriptase polymerase chain reaction. RESULTS: All OS cell lines, 6/9 tumors but none of the bone specimens expressed mRNA of TEM7 secreted forms 1 and 2. A total of 3/5 OS cell lines, 8/9 of tumors and 4/7 of bone specimens expressed mRNA of the TEM7 intracellular form. One out of 5 cell lines, 2/7 tumors and none of the bone specimens expressed mRNA of the TEM7 membrane form. The secreted forms had 20-fold higher expression in metastatic (LM7) compared to non-metastatic (SAOS-2) cells. CONCLUSION: The mRNA of secreted and the membrane forms of TEM7 are preferentially expressed in OS.

Osteoblastic and osteolytic human osteosarcomas can be studied with a new xenograft mouse model producing spontaneous metastases.

There is no animal model that reflects the histological and radiographical heterogeneity of osteosarcoma. We assessed seven osteosarcoma cell lines for their potential to develop orthotopic tumors and lung metastasis in SCID mice. Whereas radiologically, 143B developed osteolytic tumors, SaOS-LM7 developed osteoblastic primary tumors. The mineralization status was confirmed by assessing the alkaline phosphatase activity and the microarray expression profile. We herein report a xenograft orthotopic osteosarcoma mouse model to assess osteoblastic and osteolytic lesions, which may contribute in the search for new diagnostic and therapeutic approaches.

Cellular uptake of gold nanoparticles directly cross-linked with carrier peptides by osteosarcoma cells.

Nanoparticles have been extensively used for a variety of biomedical applications and there is a growing need for highly specific and efficient delivery of the nanoparticles into target cells and subcellular location. We attempted to accomplish this goal by modifying gold particles with peptide motif's that are known to deliver a 'cargo' into chosen cellular location specifically, we intended to deliver nanogold particles into cells through chemical cross-linking with different peptides known to carry protein into cells. Our results suggest that specific sequence of such 'carrier peptides' can efficiently deliver gold nanoparticles into cells when chemically cross-linked with the metal particles.

2-methoxyestradiol-induced cell death in osteosarcoma cells is preceded by cell cycle arrest.

2-Methoxyestradiol (2-ME), a naturally occurring mammalian metabolite of 17beta-Estradiol (E2), induces cell death in osteosarcoma cells. To further understand the molecular mechanisms of action, we have investigated cell cycle progression in 2-ME-treated human osteosarcoma (MG63, SaOS-2 and LM7 [corrected]) cells. At 5 microM, 2-ME induced growth arrest by inducing a block in cell cycle; 2-ME-treatment resulted in 2-fold increases in G1 phase cells and a decrease in S phase cells in MG63 and SaOS-2 osteosarcoma cell lines, compared to the appropriate vehicle controls. 2-ME-treatment induced a threefold increase in the G2 phase in LM7 [corrected] osteosarcoma cells. The results demonstrated steroid specificity, as the tumorigenic metabolite, 16alpha-hydroxyestradiol (16-OHE), did not have any effect on cell cycle progression in osteosarcoma cells. The cell cycle arrest coincided with an increase in expression of the cell cycle markers p21, p27 and p53 proteins in 2-ME-treated osteosarcoma cells. Also, MG63 cells, transiently transfected with cDNA for a 'loss of function mutant' RNA-dependent protein kinase (PKR) protein, were resistant to 2-ME-induced cell cycle arrest. These results suggest that 2-ME works in concert with factors regulating cell cycle progression, and cell cycle arrest precedes cell death in 2-ME-treated osteosarcoma cells.

Cathepsins and osteosarcoma: Expression analysis identifies cathepsin K as an indicator of metastasis.

Osteosarcoma is the most frequent malignant bone tumor with a poor survival rate for patients with metastasis. Previous studies have shown that beside other proteases, distinct sets of cathepsins are involved in the process of metastasis of different tumors. In this study we investigated the expression of cathepsin proteases in human osteosarcoma metastasis. First, the mRNA expression of 14 human cathepsins was studied in SAOS-2 osteosarcoma cells and the highly metastatic LM5 and LM7 sublines by reverse transcriptase (RT)-polymerase chain reaction (PCR). The expression of cathepsin D, K, and L mRNA was found upregulated and that of cathepsin F, H, and V downregulated in the highly metastatic LM5 and LM7 cells. A subgroup of the cathepsin proteases was further studied at the protein level by Western blot analysis of cell extracts. The expression of cathepsin B and H was decreased and that of cathepsin D, K, and L was increased in the highly metastatic cell lines as compared to the SAOS-2 cell line. Diagnostic relevance of cathepsin K expression in osteosarcoma was revealed upon correlation of survival and metastasis with immunohistochemical cathepsin K staining of biopsies collected from 92 patients prior to chemotherapy. Patients with metastatic high-grade osteosarcoma and low cathepsin K expression at diagnosis had a better prognosis than those with high expression. Thus, it appears that cathepsin K expression is of predictive prognostic value for patients with high-grade tumors and metastasis at diagnosis.

Evidence of genetic locus heterogeneity for familial bicuspid aortic valve.

OBJECTIVE: We sought to determine if the gene responsible for bicuspid aortic valve (BAV) in an extended family corresponds to previously reported loci for inherited forms of the disorder. BACKGROUND: Loci at 15q25.1-26 and 9q34 have been reported to be associated with cardiovascular abnormalities involving BAV. METHODS: Linkage analysis was performed on DNA collected from a large multigenerational family in which BAV disease segregates in an autosomal dominant manner, using microsatellite markers from the regions previously reported to segregate with the phenotype. RESULTS: Lod scores were determined for genetic markers near the NOTCH1 gene and for a locus on chromosome 15q25.1-26 previously reported as being linked to BAV. The lod scores were negative or less than 1.0 for all markers tested. CONCLUSIONS: There is no evidence of linkage of BAV in our pedigree to either the NOTCH1 gene or to the chromosome 15 locus. The disorder in this family appears to be caused by a gene at a novel locus.

High expression of tumor endothelial marker 7 is associated with metastasis and poor survival of patients with osteogenic sarcoma.

Our objective is to identify genes regulating metastasis of osteogenic sarcoma (OGS) since metastasis is the primary cause of mortality among patients with OGS. To identify such genes, we first created a database of differentially expressed genes between six low-grade and six high-grade OGS tumors, and between a normal immortalized osteoblast cell line (FOB) and four commercially available OGS-derived cell lines. We specifically searched for surface proteins over-expressed in high-grade OGS, since we hypothesize that tumor-cell specific surface markers are key to metastasis. A gene encoding Tumor Endothelial Marker7 (TEM7) was selected as a candidate for further study. TEM7 expression pattern was assessed by RT-PCR, Western blotting and immunostaining. TEM7 mRNA was abundantly expressed in SAOS cells (derived from high-grade OGS), but not in FOB or MG63 cells (derived from low-grade OGS). Virtually no expression of TEM7 protein was observed in FOB cells but abundant expression was noted in SAOS and TE85 cells. Employing immunostaining of 92 human OGS specimens (50 high-grade and 42 low-grade) collected before chemotherapy show 97% (37 of 38) of high-grade OGS specimens with metastasis have high TEM7 staining. Further, we found that elevated expression of TEM7 correlated with poor survival (p<0.04) of affected patients. Inhibiting TEM7 function by siRNA inhibited invasion and migration of OGS cells with metastatic potential. Our results suggest TEM7 expression level closely parallels histology-based prognostication of OGS metastasis and, therefore, it is a therapeutic target. This is the first demonstration of a link between TEM7 and cancer metastasis.