Other articles by Daniel Lindner on PubMed

• The Interferon (IFN)-induced GTPase, MGBP-2. Role in IFN-gamma-induced Murine Fibroblast Proliferation The Journal of Biological Chemistry. Feb, 2002  |   Pubmed ID: 11726669 To investigate the function of mGBP-2, a member of the interferon (IFN)-induced guanylate-binding protein family of GTPases, NIH 3T3 fibroblasts were generated that constitutively expressed mGBP-2. mGBP-2 induced a faster growth rate, with the highest expressing clones showing approximately a 50% reduction in doubling time. mGBP-2-expressing cells also grew to higher density and exhibited partial loss of contact growth inhibition, as evidenced by the formation of foci in post-confluent cultures. In addition, mGBP-2-expressing cells showed decreased dependence on serum-derived growth factors. However, they did not lose the requirement for anchorage-dependent growth. Finally, NIH 3T3 cells expressing mGBP-2 formed tumors in athymic mice. An mGBP-2 protein carrying a point mutation (S52N) that reduced GTP binding failed to produce these phenotypes when expressed at the same levels as wild type. The additional finding that IFN-gamma treatment of NIH 3T3 cells resulted in an increase in proliferation similar to that observed for mGBP-2 in the absence of other IFN-induced proteins suggests that mGBP-2 may indeed be important for these growth changes.
• Inositol Hexakisphosphate Kinase 2 Sensitizes Ovarian Carcinoma Cells to Multiple Cancer Therapeutics Oncogene. Mar, 2002  |   Pubmed ID: 11896621 We recently identified inositol hexakisphosphate kinase 2 (IP6K2) as a positive regulator of apoptosis. Overexpression of IP6K2 enhances apoptosis induced by interferon-beta (IFN-beta) and cytotoxic agents in NIH-OVCAR-3 ovarian carcinoma cells. In this study, we contrast and compare IFN-beta and radiation-induced death, and show that IP6K2 expression sensitizes tumor cells. Unirradiated NIH-OVCAR-3 cells transfected with IP6K2 formed fewer colonies compared to unirradiated vector-expressing cells. IP6K2 overexpression caused increased radiosensitivity, evidenced by decreased colony forming units (CFU). Both IFN-beta and radiation induced caspase 8. IFN-beta, but not gamma-irradiation, induced TRAIL in NIH-OVCAR-3 cells. Gamma irradiation, but not IFN-beta, induced DR4 mRNA. Apoptotic effects of IFN-beta or gamma-irradiation were blocked by expression of a dominant negative mutant death receptor 5 (DR5Delta) or by Bcl-2. Caspase-8 mRNA induction was more pronounced in IP6K2-expressing cells compared to vector-expressing cells. These data suggest that overexpression of IP6K2 enhances sensitivity of some ovarian carcinomas to radiation and IFN-beta. IP6K2 may function to enhance the expression and/or function of caspase 8 and DR4 following cell injury. Both IFN-beta and gamma-irradiation induce apoptosis through the extrinsic, receptor-mediated pathway, IFN-beta through TRAIL, radiation through DR4, and both through caspase 8. The function of both death inducers is positively regulated by IP6K2.
• Mutational Analysis of Human Thioredoxin Reductase 1. Effects on P53-mediated Gene Expression and Interferon and Retinoic Acid-induced Cell Death The Journal of Biological Chemistry. Jun, 2002  |   Pubmed ID: 11953436 The interferon (IFN)-beta and all-trans-retinoic acid combination suppresses tumor growth by inducing apoptosis in several tumor cell lines. A genetic technique permitted the isolation of human thioredoxin reductase (TR) as a critical regulator of IFN/all-trans-retinoic acid-induced cell death. Our recent studies have shown that TR1:thioredoxin 1-regulated cell death is effected in part through the activation of p53-dependent responses. To understand its death regulatory function, we have performed a mutational analysis of TR. Human TR1 has three major structural domains, the FAD binding domain, the NADPH binding domain, and an interface domain (ID). Here, we show that the deletion of the C-terminal interface domain results in a constitutive activation of TR-dependent death responses and promotes p53-dependent gene expression. TR mutant without the ID still retains its dependence on thioredoxin for promoting these responses. Thus, our data suggest that TR-ID acts as a regulatory domain.
• MEKK1 Plays a Critical Role in Activating the Transcription Factor C/EBP-beta-dependent Gene Expression in Response to IFN-gamma Proceedings of the National Academy of Sciences of the United States of America. Jun, 2002  |   Pubmed ID: 12048245 IFN-gamma induces a number of genes to up-regulate cellular responses by using specific transcription factors and the cognate elements. We recently discovered that CCAAT/enhancer-binding protein-beta (C/EBP-beta) induces gene transcription through an IFN-response element called gamma-IFN-activated transcriptional element (GATE). Using mutant cells, chemical inhibitors, and specific dominant negative inhibitors, we show that induction of GATE-driven gene expression depends on MEK1 (mitogen-activated protein kinase kinase/extracellular signal-regulated protein kinase kinase) and ERKs (extracellular signal-regulated protein kinases) but is independent of Raf-1. Interestingly in cells lacking the MEKK1 gene or expressing the dominant negative MEKK1, ERK activation, and GATE dependent gene expression is inhibited. A dominant negative MEKK1 blocks C/EBP-beta-driven gene expression stimulated by IFN-gamma. These studies describe an IFN-gamma-stimulated pathway that involves MEKK1-MEK1-ERK1/2 kinases to regulate C/EBP-beta-dependent gene expression.
• Effects of Interferon Beta on Transcobalamin II-receptor Expression and Antitumor Activity of Nitrosylcobalamin Journal of the National Cancer Institute. Jul, 2002  |   Pubmed ID: 12096086 The ubiquitous plasma membrane transcobalamin II receptor (TC II-R) mediates uptake of cobalamin (Cbl; vitamin B12), an essential micronutrient. Tumors often require more Cbl than normal tissue, and increased Cbl uptake may result from increased TC II-R expression. To examine whether Cbl could therefore be used as a carrier molecule to target a chemotherapy drug, we tested an analogue of Cbl with nitric oxide as a ligand, nitrosylcobalamin (NO-Cbl). Because interferon beta (IFN-beta) has antitumor effects and increases expression of some membrane receptors, we examined whether it may enhance the effects of NO-Cbl.
• Interferons As Antiangiogenic Agents Current Oncology Reports. Nov, 2002  |   Pubmed ID: 12354364 Interferons (IFNs), pleiotropic cytokines that regulate antiviral, antitumor, apoptotic, and cellular immune responses, were the first endogenous antiangiogenic regulators identified. In a species-specific manner, IFNs inhibit secretion of such angiogenic factors as basic fibroblast growth factor from tumor cells. The antiangiogenic activity of IFNs is enhanced when they are combined with other antiangiogenic agents, such as tamoxifen and thalidomide.
• Thioredoxin Reductase Plays a Critical Role in IFN Retinoid-mediated Tumor-growth Control in Vivo Clinical Cancer Research : an Official Journal of the American Association for Cancer Research. Oct, 2002  |   Pubmed ID: 12374691 The IFN and retinoic acid (RA) combination suppresses cell growth by inducing apoptosis in the cultured tumor cells. Using a genetic technique, we have isolated several "genes associated with retinoid-IFN-induced mortality" (GRIM) that participate in this death pathway. One such gene, GRIM-12, encodes the redox enzyme thioredoxin reductase (TR). Antisense-mediated inhibition of TR abrogates cell death. To test the in vivo relevance of TR for growth suppression, we have conducted the following study. A wild-type TR or a catalytically defective mutant were expressed in MCF-7 breast carcinoma cells and transplanted into athymic nude mice. These mice were treated with IFN-beta and all-trans RA combination. Tumors expressing the vector or wild-type TR were readily suppressed by the IFN/RA combination. In contrast, the tumors bearing a mutant TR were resistant to regression. We further show that markers of apoptosis are stimulated in the regressing tumors. These studies show a prominent role for TR in tumor-growth suppression.
• Anticancer Activity of Sodium Stibogluconate in Synergy with IFNs Journal of Immunology (Baltimore, Md. : 1950). Nov, 2002  |   Pubmed ID: 12421984 Cancer cell resistance limits the efficacy of IFNs. In this study, we show that sodium stibogluconate (SSG) and IFN-alpha synergized to overcome IFN-alpha resistance in various human cancer cell lines in culture and eradicated IFN-alpha-refractory WM9 human melanoma tumors in nude mice with no obvious toxicity. SSG enhanced IFN-alpha-induced Stat1 tyrosine phosphorylation, inactivated intracellular SHP-1 and SHP-2 that negatively regulate IFN signaling, and induced cellular protein tyrosine phosphorylation in cancer cell lines. These effects are consistent with inactivation of phosphatases as the basis of SSG anticancer activity. Characterization of SSG by chromatography revealed that only selective compounds in SSG were effective protein tyrosine phosphatase inhibitors. These observations suggest the potential of SSG as a clinically usable protein tyrosine phosphatase inhibitor in cancer treatment and provide insights for developing phosphatase-targeted therapeutics.
• Pentamidine is an Inhibitor of PRL Phosphatases with Anticancer Activity Molecular Cancer Therapeutics. Dec, 2002  |   Pubmed ID: 12516958 The PRL family oncogenic phosphatases are attractive targets for developing inhibitors as anticancer therapeutics given their potentially pathogenic role in human malignancies. Herein we demonstrate that pentamidine, an anti-protozoa drug with an unknown mechanism of action, is an inhibitor of PRLs with anticancer potential. Pentamidine at its therapeutic doses inhibited recombinant PRL phosphatases in vitro and inactivated ectopically expressed PRLs in NIH3T3 transfectants with an effective duration more than 24 h after a pulse cell treatment. The drug had in vitro growth-inhibitory activity against human cancer cell lines that express the endogenous PRLs. Pentamidine at a tolerable dose markedly inhibited the growth of WM9 human melanoma tumors in nude mice coincident with the induction of tumor cell necrosis and is capable of inactivating ectopically expressed PRL-2 in the cancer cells. These observations suggest the potential of pentamidine in anticancer therapies and may provide a basis for developing novel PTPase-targeted therapeutics.
• IFN-alpha2b and Thalidomide Synergistically Inhibit Tumor-induced Angiogenesis Journal of Interferon & Cytokine Research : the Official Journal of the International Society for Interferon and Cytokine Research. Jan, 2003  |   Pubmed ID: 12639293 Angiogenesis is an absolute requirement for tumor growth and metastasis. The purpose of this study was to evaluate the antiangiogenic activity of interferon-alpha2b (IFN-alpha2b) and thalidomide, as single agents and in combination. The murine dermis model was used to assess tumor-induced angiogenesis in nude mice. Human ACHN (renal), NIH-OVCAR-3 (ovarian), LNCaP (prostate), and SK-Mel-1 (melanoma) tumor cells were inoculated intradermally into the flanks of nude mice. IFN-alpha2b and thalidomide, administered daily, were effective inhibitors of angiogenesis induced by all four tumor types. The combination of IFN-alpha2b and thalidomide caused a synergistic decrease in mean vessel count in tumors that were resistant to the antiproliferative effects of IFN-alpha2b and thalidomide in vitro. This enhanced suppression of angiogenesis translated into synergistic antitumor activity in a xenograft model. Pegylated IFN-alpha (PEG-IFN-alpha2b) (10(6) U) administered once in 10 days was as effective as daily IFN-alpha2b treatment (10(6) U x 10 days). IFN-alpha2b and thalidomide have potentiated antiangiogenic activity when used in combination. A single dose of PEG-IFN-alpha2b (10(6) U) was as effective at suppressing vessel growth as an equivalent dose of IFN-alpha2b given daily for 10 days.
• Suppression of NF-kappa B Survival Signaling by Nitrosylcobalamin Sensitizes Neoplasms to the Anti-tumor Effects of Apo2L/TRAIL The Journal of Biological Chemistry. Oct, 2003  |   Pubmed ID: 12881518 We have previously demonstrated the anti-tumor activity of nitrosylcobalamin (NO-Cbl), an analog of vitamin B12 that delivers nitric oxide (NO) and increases the expression of tumor necrosis factor-related apoptosis-inducing ligand (Apo2L/TRAIL) and its receptors in human tumors. The specific aim of this study was to examine whether NO-Cbl could sensitize drug-resistant melanomas to Apo2L/TRAIL. Antiproliferative effects of NO-Cbl and Apo2L/TRAIL were assessed in malignant melanomas and non-tumorigenic melanocyte and fibroblast cell lines. Athymic nude mice bearing human melanoma A375 xenografts were treated with NO-Cbl and Apo2L/TRAIL. Apoptosis was measured by TUNEL and confirmed by examining levels and activity of key mediators of apoptosis. The activation status of NF-kappa B was established by assaying DNA binding, luciferase reporter activity, the phosphorylation status of I kappa B alpha, and in vitro IKK activity. NO-Cbl sensitized Apo2L/TRAIL-resistant melanoma cell lines to growth inhibition by Apo2L/TRAIL but had minimal effect on normal cell lines. NO-Cbl and Apo2L/TRAIL exerted synergistic anti-tumor activity against A375 xenografts. Treatment with NO-Cbl followed by Apo2L/TRAIL induced apoptosis in Apo2L/TRAIL-resistant tumor cells, characterized by cleavage of caspase-3, caspase-8, and PARP. NO-Cbl inhibited IKK activation, characterized by decreased phosphorylation of I kappa B alpha and inhibition of NF-kappa B DNA binding activity. NO-Cbl suppressed Apo2L/TRAIL- and TNF-alpha-mediated activation of a transfected NF-kappa B-driven luciferase reporter. XIAP, an inhibitor of apoptosis, was inactivated by NO-Cbl. NO-Cbl treatment rendered Apo2L/TRAIL-resistant malignancies sensitive to the anti-tumor effects of Apo2L/TRAIL in vitro and in vivo. The use of NO-Cbl and Apo2L/TRAIL capitalizes on the tumor-specific properties of both agents and represents a promising anti-cancer combination.
• Synergistic Activation of Innate Immunity by Double-stranded RNA and CpG DNA Promotes Enhanced Antitumor Activity Cancer Research. Aug, 2004  |   Pubmed ID: 15313929 Double-stranded RNA (dsRNA) and unmethylated CpG sequences in DNA are pathogen-associated molecular patterns of viruses and bacteria that activate innate immunity. To examine whether dsRNA and CpG DNA could combine to provide enhanced stimulation of innate immune cells, murine macrophages were stimulated with poly-rI:rC (pIC), a dsRNA analog, and CpG-containing oligodeoxynucleotides (CpG-ODN). Combined treatments demonstrated synergy in nitric oxide, interleukin (IL)-12, tumor necrosis factor alpha, and IL-6 production. Studies using neutralizing antibodies for type I interferons (IFNs), IFN-alpha and IFN-beta, indicated that nitric oxide synthase synergism is mediated by paracrine/autocrine effects of IFN-beta. In contrast, enhanced cytokine production occurred independent of type I IFN and was maintained in macrophages from IFN-alpha/beta receptor knockout mice. Cotransfection of human Toll-like receptors 3 and 9 (receptors for dsRNA and CpG DNA, respectively) into 293T cells supported synergistic activation of an IL-8 promoter reporter construct by pIC, indicating interaction of the signaling pathways in driving the synergy response. In vivo stimulation of mice with pIC and CpG-ODN demonstrated synergy for serum IL-6 and IL-12p40 levels that correlated with an enhanced antitumor effect against established B16-F10 experimental pulmonary metastases. Treatment of tumor-bearing mice with pIC and CpG-ODN in combination resulted in enhanced nitric oxide synthase expression in lung tissue and enhanced up-regulation of class I major histocompatibility complex on splenic dendritic cells relative to treatments with either agent alone. In conclusion, the combined detection of viral pathogen-associated molecular patterns, i.e., dsRNA and CpG DNA, may mimic definitive viral recognition, resulting in an enhanced innate immune response that could be used for tumor vaccination or immunotherapy.
• Phase 2 Trial of Interferon-beta As Second-line Treatment of Ovarian Cancer, Fallopian Tube Cancer, or Primary Carcinoma of the Peritoneum Oncology. 2004  |   Pubmed ID: 15331919 The protocol was designed to examine the biological effects and clinical activity of interferon-beta in patients with platinum/taxane-resistant ovarian cancer.
• Apo2L/TRAIL Induction and Nuclear Translocation of Inositol Hexakisphosphate Kinase 2 During IFN-beta-induced Apoptosis in Ovarian Carcinoma The Biochemical Journal. Jan, 2005  |   Pubmed ID: 15634191 Previously, we have reported that overexpression of IHPK2 (inositol hexakisphosphate kinase 2) sensitized NIH-OVCAR-3 ovarian carcinoma cell lines to the growth-suppressive and apoptotic effects of IFN-beta (interferon-beta) treatment and gamma-irradiation. In the present study, we demonstrate that Apo2L/TRAIL (Apo2L/tumour-necrosis-factor-related apoptosis-inducing ligand) is a critical mediator of IFN-induced apoptosis in these cells. Compared with IFN-alpha2, IFN-beta is a more potent inducer of Apo2L/TRAIL and IHPK2 activity. Overexpression of IHPK2 converts IFN-alpha2-resistant cells into cells that readily undergo apoptosis in response to IFN-alpha2. In untreated cells transfected with IHPK2-eGFP (where eGFP stands for enhanced green fluorescent protein), the fusion protein is localized to the cytoplasm and perinuclear region. After treatment with IFN-beta, IHPK2-eGFP translocated to the nucleus. In cells transfected with mutant IHPK2-NLS-eGFP (where NLS stands for nuclear localization sequence), containing point mutations in the NLS, the fusion protein remained trapped in the cytoplasm, even after IFN-beta treatment. Cells expressing mutant NLS mutation were more resistant to IFN-beta. The IC50 value of IHPK2-expressing cells was 2-3-fold lower than vector control. The IC50 value of NLS-mutant-expressing cells was 3-fold higher than vector control. Blocking antibodies to Apo2L/TRAIL or transfection with a dominant negative Apo2L/TRAIL receptor (DR5Delta) inhibited the antiproliferative effects of IFN-beta. Thus overexpression of IHPK2 enhanced apoptotic effects of IFN-beta, and expression of the NLS mutant conferred resistance to IFN-beta. Apo2L/TRAIL expression and nuclear localization of IHPK2 are both required for the induction of apoptosis by IFN-beta in ovarian carcinoma.
• Sodium Stibogluconate Interacts with IL-2 in Anti-Renca Tumor Action Via a T Cell-dependent Mechanism in Connection with Induction of Tumor-infiltrating Macrophages Journal of Immunology (Baltimore, Md. : 1950). Nov, 2005  |   Pubmed ID: 16272361 IL-2 therapy results in 10-20% response rates in advanced renal cell carcinoma (RCC) via activating immune cells, in which the protein tyrosine phosphatase Src homology 2 domain-containing phosphatase 1 (SHP-1) is a key negative regulator. Based on finding that sodium stibogluconate (SSG) inhibited SHP-1, the anti-RCC potential and action mechanism of SSG and SSG/IL-2 in combination were investigated in a murine renal cancer model (Renca). Despite its failure to inhibit Renca cell proliferation in cultures, SSG induced 61% growth inhibition of Renca tumors in BALB/c mice coincident with an increase (2-fold) in tumor-infiltrating macrophages (Mphi). A combination of SSG and IL-2 was more effective in inhibiting tumor growth (91%) and inducing tumor-infiltrating Mphi (4-fold), whereas IL-2 alone had little effect. Mphi increases were also detected in the spleens of mice treated with SSG (3-fold) or SSG/IL-2 in combination (6-fold), suggesting a systemic Mphi expansion similar to those in SHP-deficient mice. T cell involvement in the anti-Renca tumor action of the combination was suggested by the observations that the treatment induced spleen IFN-gamma T cells in BALB/c mice, but failed to inhibit Renca tumor growth in athymic nude mice and that SSG treatment of T cells in vitro increased production of IFN-gamma capable of activating tumoricidal Mphi. The SSG and SSG/IL-2 combination treatments were tolerated in the mice. These results together demonstrate an anti-Renca tumor activity of SSG that was enhanced in combination with IL-2 and functions via a T cell-dependent mechanism with increased IFN-gamma production and expansion/activation of Mphi. Our findings suggest that SSG might improve anti-RCC efficacy of IL-2 therapy by enhancing antitumor immunity.
• N-terminally PEGylated Human Interferon-beta-1a with Improved Pharmacokinetic Properties and in Vivo Efficacy in a Melanoma Angiogenesis Model Bioconjugate Chemistry. Jan-Feb, 2006  |   Pubmed ID: 16417267 PEGylation of IFN-alpha has been used successfully to improve the pharmacokinetic properties and efficacy of the drug. To prepare a PEGylated form of human interferon-beta-1a (IFN-beta-1a) suitable for testing in vivo, we have synthesized 20 kDa mPEG-O-2-methylpropionaldehyde and used it to modify the N-terminal alpha-amino group of the cytokine. The PEGylated protein retained approximately 50% of the activity of the unmodified protein and had significantly improved pharmacokinetic properties following intravenous administration in rats. The clearance and volume of distribution at steady state were reduced approximately 30-fold and approximately 4-fold, respectively, resulting in a significant increase in systemic exposure as determined by the area under the curve. The elimination half-life of the PEGylated protein was approximately 13-fold greater than for the unmodified protein. The unmodified and PEGylated proteins were tested for their ability to inhibit the formation of radially oriented blood vessels entering the periphery of human SK-MEL-1 melanoma tumors in athymic nude homozygous (nu/nu) mice. In a single dose comparison study, administration of 1 x 10(6) units of unmodified IFN-beta-1a resulted in a 29% reduction in vessel number, while 1 x 10(6) units of PEGylated IFN-beta-1a resulted in a 58% reduction. Both treatments resulted in statistically significant reductions in mean vessel number as compared to the vehicle (control)-treated mice, with the PEGylated IFN-beta-1a-treated mice showing a statistically significantly greater reduction in mean vessel number as compared to the unmodified IFN-beta-1a-treated mice. In a multiple versus single dose comparison study, daily administration of 1 x 10(6) units of unmodified IFN-beta-1a for 9 days resulted in a 51% reduction in vessel number, while a single dose of 1 x 10(6) units of the PEGylated protein resulted in a 66% reduction. Both treatments resulted in statistically significant reductions in mean vessel number as compared to the vehicle-treated mice, with the PEGylated IFN-beta-1a-treated mice showing a statistically significantly greater reduction in mean vessel number as compared to the unmodified IFN-beta-1a-treated mice. Therefore, the improved pharmacokinetic properties of the modified protein translated into improved efficacy. Since unmodified IFN-beta is used for the treatment of multiple sclerosis and hepatitis C virus infection, a PEGylated form of the protein such as 20 kDa mPEG-O-2-methylpropionaldehyde-modified IFN-beta-1a may serve as a useful adjunct for the treatment of these diseases. In addition, the antiangiogenic effects of PEGylated IFN-beta-1a may be harnessed for the treatment of certain cancers, either as a sole agent or in combination with other antitumor drugs.
• IFN-alpha1,8 Inhibits Tumor-induced Angiogenesis in Murine Angiosarcomas Journal of Interferon & Cytokine Research : the Official Journal of the International Society for Interferon and Cytokine Research. May, 2006  |   Pubmed ID: 16689662 Interferon-alpha (IFN-alpha) has proved effective in the treatment of hemangiomas, hemangioblastomas, and Kaposi's sarcoma. To investigate the ability of IFNs to inhibit angiosarcoma, we used two transformed murine endothelial cell lines that form angiosarcomas in vivo. SVR and MS1-VEGF cell lines express oncogenic H-ras or vascular endothelial growth factor (VEGF), respectively. IFN-alpha1,8, which is active against murine and human cells, inhibited SVR and MS1-VEGF proliferation in vitro by 40% at 10(3) U/mL (p = 0.028). In vivo, IFN-alpha1,8 inhibited SVR tumor volume by 71% (p = 0.047) and MS1-VEGF volume by 79% (p = 0.003). Tumor-induced angiogenesis was decreased in SVR tumors by 52% (p = 0.005) and in MS1-VEGF tumors by 58% (p = 0.001). Sera from IFN-alpha1,8-treated mice bearing either SVR or MS1-VEGF tumors demonstrated a 5-fold increase in IP-10/CXCL10 (p = 0.001), an IFN-induced antiangiogenic protein. Both recombinant IP-10 and IFN-alpha1,8 inhibited human umbilical vein endothelial cell (HUVEC) vessel formation in the fibrin gel assay, a three-dimensional culture model of angiogenesis, by 56% at 25 ng/mL and 50% at 1.2 ng/mL, respectively (p < 0.001). An IP-10 blocking antibody restored vessel formation to 80% of untreated controls (p = 0.001). Given the magnitude of the in vivo response, these data suggested that the antitumor effects of IFN-alpha1,8 were likely mediated through angiogenesis inhibition rather than solely by direct inhibition of tumor cell proliferation.
• Overcoming Resistance to Interferon-induced Apoptosis of Renal Carcinoma and Melanoma Cells by DNA Demethylation Journal of Clinical Oncology : Official Journal of the American Society of Clinical Oncology. Aug, 2006  |   Pubmed ID: 16801630 Epigenetic editing of gene expression by aberrant methylation of DNA may help tumor cells escape attack from the innate and acquired immune systems. Resistance to antiproliferative effects and apoptosis induction by interferons (IFNs) was postulated to result from silencing of IFN response genes by promoter hypermethylation. Treatment of human ACHN renal cell carcinoma (RCC) and A375 melanoma cells with the DNA demethylating nucleoside analog 5-AZA-2'-deoxycytidine (5-AZA-dC) synergistically augmented antiproliferative effects of IFN- alpha (alpha) 2 and IFN-beta (beta). Either 5-AZA-dC or an antisense to DNA methyltransferase 1 (DNMT1) overcame resistance to apoptosis induction by IFNs with up to 85% apoptotic cells resulting from the combinations. No similar potentiation occurred in normal kidney epithelial cells. IFN response genes were augmented more than 10 times in expression by 5-AZA-dC. Demethylation by 5-AZA-dC of the promoter of the prototypic, apoptosis-associated IFN response gene XAF1 was confirmed by methylation-specific polymerase chain reaction. siRNA to XAF1 inhibited IFN-induced apoptosis; conversely, overexpression of XAF1 overcame resistance to apoptosis induction by IFN-beta. As occurred with apoptosis-resistant melanoma cells in vitro, tumor growth inhibition in the nude mouse of human A375 melanoma xenografts resulted from treatment with 5-AZA-dC in combination with IFN-beta, an effect not resulting from either single agent. The importance of epigenetic remodeling of expression of immune-modifying genes in tumor cells was further suggested by identifying reactivation of the cancer-testis antigens MAGE and RAGE in ACHN cells after DNMT1 depletion. Thus, inhibitors of DNMT1 may have clinical relevance for immune modulation by augmentation of cytokine effects and/or expression of tumor-associated antigens.
• Nitrosylcobalamin Promotes Cell Death Via S Nitrosylation of Apo2L/TRAIL Receptor DR4 Molecular and Cellular Biology. Aug, 2006  |   Pubmed ID: 16847314 We have previously demonstrated that nitrosylcobalamin (NO-Cbl), an analogue of vitamin B12 that delivers nitric oxide (NO), had potent antiproliferative activity against several human cancer cell lines. NO-Cbl induced apoptosis via a death receptor/caspase-8 pathway. In this study, we demonstrate that a functional Apo2L/TRAIL receptor was necessary for the induction of cell death by NO-Cbl. Furthermore, the Apo2L/TRAIL death receptor DR4 (TRAIL R1) was S nitrosylated following NO-Cbl treatment. Human melanoma (A375), renal carcinoma (ACHN), and ovarian carcinoma (NIH-OVCAR-3) cells were treated with NO-Cbl and subjected to the biotin switch assay; S-nitrosylated DR4 was detected in all three cell lines. NO-Cbl treatment did not cause S nitrosylation of DR5. The seven cysteine residues located in the cytoplasmic domain of DR4 were individually point mutated to alanines. NIH-OVCAR-3 cells expressing the DR4 C336A mutation lacked S nitrosylation following NO-Cbl treatment. Overexpression of wild-type DR4 sensitized cells to growth inhibition by NO-Cbl. Cells expressing the DR4 C336A mutant were more resistant to NO-Cbl and Apo2L/TRAIL than were the other six C-A mutations or wild-type cells. The C336A mutant also displayed blunted caspase-8 enzymatic activity following NO-Cbl treatment compared to the other mutants. Thus, DR4 residue C336 becomes S nitrosylated and promotes apoptosis following NO-Cbl treatment.
• Species Diversity of Polyporoid and Corticioid Fungi in Northern Hardwood Forests with Differing Management Histories Mycologia. Mar-Apr, 2006  |   Pubmed ID: 16894965 Effects of forest management on fungal diversity were investigated by sampling fruit bodies of polyporoid and corticioid fungi in forest stands that have different management histories. Fruit bodies were sampled in 15 northern hardwood stands in northern Wisconsin and the upper peninsula of Michigan. Sampling was conducted in five old-growth stands, five uneven-age stands, three even-age unthinned stands and two even-age thinned stands. Plots 100 m x 60 m were established and 3000 m2 within each plot was sampled during the summers of 1996 and 1997. A total of 255 polyporoid and corticioid morphological species were identified, 46 (18%) of which could not be assigned to a described species. Species accumulation curves for sites and management classes differed from straight lines, although variability from year to year suggests that more than 2 y of sampling are needed to characterize annual variation. Mean species richness and diversity index values did not vary significantly by management class, although mean richness on large diameter wood (> or = 15 cm diam) varied with moderate significance. Richness values on small diameter debris varied significantly by year, indicating that a large part of year-to-year variability in total species richness is due to small diameter debris. Ten species had abundance levels that varied by management class. Two of these species. Changes in the diversity and species composition of the wood-inhabiting fungal community could have significant implications for the diversity, health and productivity of forest ecosystems.
• Human Telomerase RNA Degradation by 2'-5'-linked Oligoadenylate Antisense Chimeras in a Cell-free System, Cultured Tumor Cells, and Murine Xenograft Models Oligonucleotides. 2006  |   Pubmed ID: 16978086 Ribonuclease L (RNase L) is a latent single-stranded RNA-directed endoribonuclease that is activated on binding to short 2'-5'-linked oligoadenylates (2-5A), a feature that has led to its use in antisense therapeutic strategies. By attaching a 2-5A moiety to the 5' terminus of standard antisense oligonucleotides, it is possible to activate RNase L and guide it to specific RNAs for degradation. These 2-5A antisense chimeras have been used successfully to target a variety of cellular and viral RNAs. Telomerase is a nuclear ribonucleoprotein complex that elongates telomeric DNA and contributes to cellular immortalization. Telomerase is composed of a protein catalytic subunit and an RNA (hTR or TERC) component, both of which are critical for holoenzyme activity. We describe the characterization of 2-5A antisense chimeras targeting the hTR component of telomerase (2-5A antihTR). Newly designed 2-5A anti-hTR molecules were assayed for their abilities to selectively degrade hTR in a cell-free system. Of the five chimeras tested, one (RBI011) degraded hTR by 97%, and two others (RBI013 and RBI009) were also found to be highly active (73-76% degradation). The ability of transfected RBI011, and its homolog RBI254, to degrade hTR in cultured tumor cells was assessed by real-time RT-PCR. In these studies, RBI011 and RBI254 effectively degraded hTR in a variety of hTR-positive tumor cell lines. The hTR degradation studies were extended to growth assays to determine whether hTR ablation affected tumor cell viability or proliferation. RBI254 treatment resulted in reduced tumor cell viability over the course of 4-day growth assays, effects that were augmented by cotreatment with interferon-beta. To extend these results to an in vivo system, nude mice were implanted subcutaneously or orthotopically with hTR-positive prostate tumors and treated with RBI254. RBI254-treated mice exhibited enhanced tumor cell apoptosis and reduced tumor volume as compared with controls. These findings demonstrated the effectiveness of highly active forms of 2-5A antisense against hTR, and also highlight the usefulness of the cell-free system in predicting chimera efficacy before to inception of cell-based and in vivo studies.
• Short Communication: Phase I Clinical and Gene Modulatory Evaluation of Tamoxifen and IFN-alpha2b Journal of Interferon & Cytokine Research : the Official Journal of the International Society for Interferon and Cytokine Research. Nov, 2006  |   Pubmed ID: 17115898 Preclinical studies had determined that tamoxifen and interferon-alpha2b (IFN-alpha2b) synergistically inhibited growth of both estrogen-receptor positive and negative murine tumor xenografts and had combined antiangiogenic effects and that tamoxifen potentiated IFN-stimulated gene (ISG) expression. A phase I trial in 26 patients was conducted using the combination to define tolerance and potentiation of ISG expression. IFN- alpha2b at a dose of 3 x 10(6) units/m(2) daily was given subcutaneously (s.c.), and tamoxifen was initiated as a loading dose of 150 mg/m(2) and then 60 mg/m(2) twice daily on day 8. At this initial dose, reduction of dose of IFN- alpha2b was required in 4 of 11 patients, primarily because of fatigue. Another group of patients was treated with an identical tamoxifen dose but with IFN-alpha2b reduced to 2 x 10(6)/m(2) U; this was better tolerated. As the projected serum tamoxifen level to reduplicate preclinical effects was 300 mg/m(2), dose escalation in a third cohort was undertaken; it had to be discontinued secondary to grade III or IV toxicity in 2 of 2 patients. Increases in products of transcriptionally regulated ISGs, beta (2)-microglobulin, neopterin, and ISG15 were assessed. All ISGs increased after IFN-alpha2b, but only ISG15 had a further significant rise after initiation of tamoxifen. Because at doses not limited by unacceptable toxicities, no marked potentiation of ISGs by tamoxifen could be identified, clinical evaluation of the combination was terminated.
• Efficacy of SSG and SSG/IFNalpha2 Against Human Prostate Cancer Xenograft Tumors in Mice: a Role for Direct Growth Inhibition in SSG Anti-tumor Action Cancer Chemotherapy and Pharmacology. Aug, 2007  |   Pubmed ID: 17103170 Pre-clinical activity of SSG against melanoma and renal cancer has been identified recently although the drug's mechanism of action and activity against tumors of additional histological-types remain undefined.
• Effect of Inositol Hexakisphosphate Kinase 2 on Transforming Growth Factor Beta-activated Kinase 1 and NF-kappaB Activation The Journal of Biological Chemistry. May, 2007  |   Pubmed ID: 17379600 We previously showed that inositol hexakisphosphate kinase 2 (IHPK2) functions as a growth-suppressive and apoptosis-enhancing kinase during cell stress. Overexpression of IHPK2 sensitized ovarian carcinoma cell lines to the growth-suppressive and apoptotic effects of interferon beta (IFN-beta), IFN-alpha2, and gamma-irradiation. Expression of a kinase-dead mutant abrogated 50% of the apoptosis induced by IFN-beta. Because the kinase-dead mutant retained significant response to cell stressors, we hypothesized that a portion of the death-promoting function of IHPK2 was independent of its kinase activity. We now demonstrate that IHPK2 binds to tumor necrosis factor (TNF) receptor-associated factor (TRAF) 2 and interferes with phosphorylation of transforming growth factor beta-activated kinase 1 (TAK1), thereby inhibiting NF-kappaB signaling. IHPK2 contains two sites required for TRAF2 binding, Ser-347 and Ser-359. Compared with wild type IHPK2-transfected cells, cells expressing S347A and S359A mutations displayed 3.5-fold greater TAK1 activation following TNF-alpha. This mutant demonstrated a 6-10-fold increase in NF-kappaB DNA binding following TNF-alpha compared with wild type IHPK2-expressing cells in which NF-kappaB DNA binding was inhibited. Cells transfected with wild type IHPK2 or IHPK2 mutants that lacked S347A and S359A mutations displayed enhanced terminal deoxynucleotidyltransferase-mediated dUTP nick end-labeling staining following TNF-alpha. We believe that IHPK2-TRAF2 binding leads to attenuation of TAK1- and NF-kappaB-mediated signaling and is partially responsible for the apoptotic activity of IHPK2.
• Arthromyces and Blastosporella, Two New Genera of Conidia-producing Lyophylloid Agarics (Agaricales, Basidiomycota) from the Neotropics Mycological Research. May, 2007  |   Pubmed ID: 17572336 Two new genera encompassing three new species of lyophylloid agarics that produce conidia on the basidiomata are described. Arthromyces is a genus comprised of two very different arthrospore-producing mushroom species found in the Greater Antilles and Central America. Blastosporella is a monotypic genus with spherical balls of blastospores covering the pileus surface with age and is known from Hispaniola and Colombia. A key to the species of Arthromyces is included.
• FRA-1 Proto-oncogene Induces Lung Epithelial Cell Invasion and Anchorage-independent Growth in Vitro, but is Insufficient to Promote Tumor Growth in Vivo Cancer Research. Jul, 2007  |   Pubmed ID: 17616677 FRA-1 forms activator protein-1 complexes in association with members of the JUN family and drives gene transcription. FRA-1 has been implicated in the development of airway squamous metaplasia and is frequently overexpressed in squamous cell carcinomas of the esophagus and stomach. We and others have shown a high level of persistent induction of FRA-1 by lung carcinogens, such as cigarette smoke and asbestos, in pulmonary epithelial cells. However, the exact roles of FRA-1 in regulating lung epithelial cell growth and invasion are poorly understood. To examine this aspect, we have stably overexpressed FRA-1 in human type-II-like alveolar malignant cell line (A549) and a nonmalignant bronchial epithelial cell line (BEAS-2B). FRA-1 greatly enhanced the rate of proliferation, motility, and invasion of A549 and BEAS-2B cells. In athymic nude mice, FRA-1, but not the control vector, rapidly enhanced tumor formation and metastasis by A549 cells. In contrast, FRA-1 failed to promote tumor formation by BEAS-2B. We suggest that FRA-1 can promote motility, invasion, and anchorage-independent growth of lung epithelial cells in vitro, but is insufficient for tumor formation.
• Tumor-suppressive Activity of the Cell Death Activator GRIM-19 on a Constitutively Active Signal Transducer and Activator of Transcription 3 Cancer Research. Jul, 2007  |   Pubmed ID: 17616678 Signal transducers and activators of transcription 3 (STAT3) was originally identified as a transcription factor that mediates cytokine-induced responses. In these pathways, Janus-activated kinase (JAK)-induced transient tyrosine phosphorylation of STAT3 promotes gene expression in response to a number of cytokines, which is inhibited by feedback mechanisms. A number of studies have shown that STAT3 is constitutively activated in human cancer cells, leading to cell proliferation. It is unclear, apart from a chronic tyrosyl phosphorylation of STAT3, what mechanisms contribute to the STAT3 deregulation in tumors. Earlier, we have isolated a novel growth inhibitory gene product, gene associated with retinoid-IFN-induced mortality 19 (GRIM-19), using a genetic approach. GRIM-19 is an IFN/retinoic acid-regulated growth suppressor. Subsequent analyses have shown that GRIM-19 binds to STAT3 and prevents interleukin-6-induced transcription of cellular genes. However, its effects on a constitutively active STAT3 and cellular transformation are unknown. In this study, we show that GRIM-19 suppresses constitutive STAT3-induced cellular transformation in vitro and in vivo by down-regulating the expression of a number of cellular genes involved in cell proliferation and apoptosis.
• Ovarian Cancer G Protein-coupled Receptor 1, a New Metastasis Suppressor Gene in Prostate Cancer Journal of the National Cancer Institute. Sep, 2007  |   Pubmed ID: 17728215 Metastasis is a process by which tumors spread from primary organs to other sites in the body and is the major cause of death for cancer patients. The ovarian cancer G protein-coupled receptor 1 (OGR1) gene has been shown to be expressed at lower levels in metastatic compared with primary prostate cancer tissues.
• Tumor Suppressive Protein Gene Associated with Retinoid-interferon-induced Mortality (GRIM)-19 Inhibits Src-induced Oncogenic Transformation at Multiple Levels The American Journal of Pathology. Oct, 2007  |   Pubmed ID: 17823279 Interferons (IFNs) inhibit the growth of infectious pathogens and tumor development. Although IFNs are potent tumor suppressors, they modestly inhibit the growth of some human solid tumors. Their weak activity against such tumors is augmented by co-treatment with differentiation-inducing agents such as retinoids. Previous studies from our laboratory identified a novel gene product, gene associated with retinoid-interferon-induced mortality (GRIM)-19, as an IFN/all-trans retinoic acid-induced growth suppressor. However, the mechanisms of its growth suppressive actions are unclear. The src-family of tyrosine kinases is important regulators of various cell growth responses. Mutational activation of src causes cellular transformation by altering transcription and cytoskeletal properties. In this study, we show that GRIM-19 suppresses src-induced cellular transformation in vitro and in vivo by down-regulating the expression of a number of signal transducer and activator of transcription-3 (STAT3)-dependent cellular genes. In addition, GRIM-19 inhibited the src-induced cell motility and metastasis by suppressing the tyrosyl phosphorylation of focal adhesion kinase, paxillin, E-cadherin, and gamma-catenin. Effects of GRIM-19 on src-induced cellular transformation are reversible in the presence of specific short hairpin RNA, indicating its direct effect on transformation. GRIM-19-mediated inhibition of the src-induced tyrosyl phosphorylation of cellular proteins, such as focal adhesion kinase and paxillin, seems to occur independently of the STAT3 protein. GRIM-19 had no significant effect on the cellular transformation induced by other oncogenes such as myc and Ha-ras. Thus, GRIM-19 not only blocks src-induced gene expression through STAT3 but also the activation of cell adhesion molecules.
• Nitrosylcobalamin Potentiates the Anti-neoplastic Effects of Chemotherapeutic Agents Via Suppression of Survival Signaling PloS One. 2007  |   Pubmed ID: 18074035 Nitrosylcobalamin (NO-Cbl) is a chemotherapeutic pro-drug derived from vitamin B12 that preferentially delivers nitric oxide (NO) to tumor cells, based upon increased receptor expression. NO-Cbl induces Apo2L/TRAIL-mediated apoptosis and inhibits survival signaling in a variety of malignant cell lines. Chemotherapeutic agents often simultaneously induce an apoptotic signal and activation of NF-kappaB, which has the undesired effect of promoting cell survival. The specific aims of this study were to 1) measure the anti-tumor effects of NO-Cbl alone and in combination with conventional chemotherapeutic agents, and to 2) examine the mechanism of action of NO-Cbl as a single agent and in combination therapy.
• The Role and Target Potential of Protein Tyrosine Phosphatases in Cancer Current Oncology Reports. Mar, 2008  |   Pubmed ID: 18377824 Protein tyrosine phosphatases (PTPases) are attractive targets for developing novel cancer therapeutics. Activated via gain-of-function point mutations or overexpression, several PTPases have been identified as critical oncogenic molecules in human malignancies that may be targeted with small chemical inhibitors as a therapeutic strategy. Tumor suppressor PTPases have also been discovered as contributing factors in cancer development that may be targeted via intervention of downstream signaling events for therapeutic purposes. In addition, PTPases have been identified as key negative regulators of cytokines or immune cells. Targeting these negative PTPases may improve the efficacy of cytokine therapy and immunotherapy, which currently have modest response rates and limited survival benefit. Inhibitors of selective PTPases have demonstrated significant preclinical antitumor activity, leading to early-phase clinical trials. Further research and development could lead to PTPase-targeted cancer therapeutics in the near future.
• Molecular Phylogeny of Laetiporus and Other Brown Rot Polypore Genera in North America Mycologia. May-Jun, 2008  |   Pubmed ID: 18751549 Phylogenetic relationships were investigated among North American species of Laetiporus, Leptoporus, Phaeolus, Pycnoporellus and Wolfiporia using ITS, nuclear large subunit and mitochondrial small subunit rDNA sequences. Members of these genera have poroid hymenophores, simple septate hyphae and cause brown rots in a variety of substrates. Analyses indicate that Laetiporus and Wolfiporia are not monophyletic. All North American Laetiporus species formed a well supported monophyletic group (the "core Laetiporus clade" or Laetiporus s.s.) with the exception of L. persicinus, which showed little affinity for any genus for which sequence data are available. Based on data from GenBank, the southern hemisphere species L. portentosus also fell well outside the core Laetiporus clade. Wolfiporia dilatohypha was found to represent a sister group to the core Laetiporus clade. Isolates of Phaeolus, Pycnoporellus and members of the core Laetiporus clade all fell within the Antrodia clade of polypores, while Leptoporus mollis and Laetiporus portentosus fell within the phlebioid clade of polypores. Wolfiporia cocos isolates also fell in the Antrodia clade, in contrast to previous studies that placed W. cocos in the core polyporoid clade. ITS analyses resolved eight clades within Laetiporus s.s., three of which might represent undescribed species. A combined analysis using the three DNA regions resolved five major clades within Laetiporus s.s.: a clade containing conifer-inhabiting species ("Conifericola clade"), a clade containing L. cincinnatus ("Cincinnatus clade"), a clade containing L. sulphureus s.s. isolates with yellow pores ("Sulphureus clade I"), a clade containing L. sulphureus s.s. isolates with white pores ("Sulphureus clade II") and a clade containing L. gilbertsonii and unidentified isolates from the Caribbean ("Gilbertsonii clade"). Although there is strong support for groups within the core Laetiporus clade, relationships among these groups remain poorly resolved.
• Ectomycorrhizal Characterization of an American Chestnut (Castanea Dentata)-dominated Community in Western Wisconsin Mycorrhiza. Dec, 2008  |   Pubmed ID: 18807258 Circa 1900, a farmer from the eastern US planted 11 American chestnut (Castanea dentata) seeds on a newly established farm near West Salem in western Wisconsin. These trees were very successful, producing a large stand of over 6,000 trees. Since this area is well outside the natural range of chestnut, these trees remained free from chestnut blight until 1987. In the West Salem stand, chestnuts are the dominant species of a mixed forest community, reminiscent of the chestnut-oak ecosystems of pre-1900 Appalachia. To identify putative mycorrhizal associates of chestnut in this unique forest, our approach was twofold: (1) an extensive fruiting body survey was conducted for four seasons that yielded approximately 100 putative mycorrhizal species and (2) a belowground molecular approach was used to generate DNA sequences of the internal transcribed spacer region from ectomycorrhizae. Unexpectedly, chestnut did not appear to be the dominant underground ectomycorrhizal-forming plant species. This study highlights the need to identify the plant host species when conducting belowground molecular-based surveys and provides preliminary identification of ectomycorrhizal fungi associated with a disjunct stand of American chestnut.
• Identification of Interferon-beta-stimulated Genes That Inhibit Angiogenesis in Vitro Journal of Interferon & Cytokine Research : the Official Journal of the International Society for Interferon and Cytokine Research. Dec, 2008  |   Pubmed ID: 18937547 Interferons (IFNs) have proven antitumor activity against a variety of human malignancies, which may result, at least in part, from inhibition of angiogenesis. The objective of this study was to identify IFN-stimulated genes (ISGs) that played a role in mediation of angiogenic inhibition. IFN-beta was a more potent antiangiogenic agent compared to IFN-alpha2b (80% versus 20%, respectively) and suggests that IFNs inhibited angiogenesis by preventing endothelial cell differentiation, and not by direct antiproliferative effects. To identify ISGs that were key inhibitors of angiogenesis, we utilized an in vitro fibrin gel angiogenic assay which closely recapitulated the in vivo processes of angiogenesis. DNA microarray analysis of IFN-beta-treated endothelial cells in the fibrin gel assay identified 11 ISGs that were induced >10-fold during angiogenesis inhibition. Recombinant IP-10 inhibited angiogenesis in a dose-dependent fashion, but was a less effective inhibitor compared to IFN-beta, suggesting that additional ISGs are involved in inhibiting angiogenesis. ISG20 was upregulated by microarray analysis, but did not inhibit angiogenesis when overexpressed in human umbilical vein endothelial cells (HUVECs). However, a dominant negative mutant of ISG20 inhibited angiogenesis by 43%. Results suggest that IFN-induced angiogenic inhibition was likely mediated by multiple ISGs; our novel finding is that decreased exonuclease activity in HUVECs associated with expression of the ISG20 ExoII mutant inhibited angiogenesis.
• Effects of Cloning and Root-tip Size on Observations of Fungal ITS Sequences from Picea Glauca Roots Mycologia. Jan-Feb, 2009  |   Pubmed ID: 19271678 To better understand the effects of cloning on observations of fungal ITS sequences from Picea glauca (white spruce) roots two techniques were compared: (i) direct sequencing of fungal ITS regions from individual root tips without cloning and (ii) cloning and sequencing of fungal ITS regions from individual root tips. Effect of root tip size was investigated by selecting 20 small root tips (SRT, 1.0-2.0 mm long) and 20 large root tips (LRT, 5.0-6.0 mm long). DNA was isolated from each tip and PCR-amplified with fungal-specific primers. PCR reactions were divided into two portions, one of which was sequenced directly and one of which was cloned first followed by sequencing of 12 random clones. With direct sequencing all 20 SRT produced an identifiable sequence, while only 13 of 20 LRT (65%) yielded an identifiable sequence. With cloning and sequencing all 40 tips produced identifiable fungal ITS sequences regardless of size. Failure of direct sequencing in LRT was associated with the presence of multispecies assemblages. Cloning identified 18 taxa overall while direct sequencing identified four. Cloning was not affected by tip size and identified more taxa relative to direct sequencing, although cost and probability of observing lab-based contaminants (e.g., airborne or reagent-based) were higher. We suggest that standardized controls be run whenever clones are sequenced from environmental samples, including positive controls derived from pure cultures and negative controls that cover the entire extraction, amplification and cloning process. Additional studies on larger root segments and bulked samples are needed to determine whether cloning can detect fungi accurately and cost-effectively in complex environmental samples.
• ADAMTS9 is a Cell-autonomously Acting, Anti-angiogenic Metalloprotease Expressed by Microvascular Endothelial Cells The American Journal of Pathology. Mar, 2010  |   Pubmed ID: 20093484 The metalloprotease ADAMTS9 participates in melanoblast development and is a tumor suppressor in esophageal and nasopharyngeal cancer. ADAMTS9 null mice die before gastrulation, but, ADAMTS9+/- mice were initially thought to be normal. However, when congenic with the C57Bl/6 strain, 80% of ADAMTS9+/- mice developed spontaneous corneal neovascularization. beta-Galactosidase staining enabled by a lacZ cassette targeted to the ADAMTS9 locus showed that capillary endothelial cells (ECs) in embryonic and adult tissues and in capillaries growing into heterotopic tumors expressed ADAMTS9. Heterotopic B.16-F10 melanomas elicited greater vascular induction in ADAMTS9+/- mice than in wild-type littermates, suggesting a potential inhibitory role in tumor angiogenesis. Treatment of cultured human microvascular ECs with ADAMTS9 small-interfering RNA resulted in enhanced filopodial extension, decreased cell adhesion, increased cell migration, and enhanced formation of tube-like structures on Matrigel. Conversely, overexpression of catalytically active, but not inactive, ADAMTS9 in ECs led to fewer tube-like structures, demonstrating that the proteolytic activity of ADAMTS9 was essential. However, unlike the related metalloprotease ADAMTS1, which exerts anti-angiogenic effects by cleavage of thrombospondins and sequestration of vascular endothelial growth factor165, ADAMTS9 neither cleaved thrombospondins 1 and 2, nor bound vascular endothelial growth factor165. Taken together, these data identify ADAMTS9 as a novel, constitutive, endogenous angiogenesis inhibitor that operates cell-autonomously in ECs via molecular mechanisms that are distinct from those used by ADAMTS1.
• Novel SHP-1 Inhibitors Tyrosine Phosphatase Inhibitor-1 and Analogs with Preclinical Anti-tumor Activities As Tolerated Oral Agents Journal of Immunology (Baltimore, Md. : 1950). Jun, 2010  |   Pubmed ID: 20421638 Src homology region 2 domain-containing phosphatase 1 (SHP-1) has been implicated as a potential cancer therapeutic target by its negative regulation of immune cell activation and the activity of the SHP-1 inhibitor sodium stibogluconate that induced IFN-gamma(+) cells for anti-tumor action. To develop more potent SHP-1-targeted anti-cancer agents, inhibitory leads were identified from a library of 34,000 drug-like compounds. Among the leads and active at low nM for recombinant SHP-1, tyrosine phosphatase inhibitor-1 (TPI-1) selectively increased SHP-1 phospho-substrates (pLck-pY394, pZap70, and pSlp76) in Jurkat T cells but had little effects on pERK1/2 or pLck-pY505 regulated by phosphatases SHP-2 or CD45, respectively. TPI-1 induced mouse splenic-IFN-gamma(+) cells in vitro, approximately 58-fold more effective than sodium stibogluconate, and increased mouse splenic-pLck-pY394 and -IFN-gamma(+) cells in vivo. TPI-1 also induced IFN-gamma(+) cells in human peripheral blood in vitro. Significantly, TPI-1 inhibited ( approximately 83%, p < 0.002) the growth of B16 melanoma tumors in mice at a tolerated oral dose in a T cell-dependent manner but had little effects on B16 cell growth in culture. TPI-1 also inhibited B16 tumor growth and prolonged tumor mice survival as a tolerated s.c. agent. TPI-1 analogs were identified with improved activities in IFN-gamma(+) cell induction and in anti-tumor actions. In particular, analog TPI-1a4 as a tolerated oral agent completely inhibited the growth of K1735 melanoma tumors and was more effective than the parental lead against MC-26 colon cancer tumors in mice. These results designate TPI-1 and the analogs as novel SHP-1 inhibitors with anti-tumor activity likely via an immune mechanism, supporting SHP-1 as a novel target for cancer treatment.
• Identification of a Structural Motif in the Tumor-suppressive Protein GRIM-19 Required for Its Antitumor Activity The American Journal of Pathology. Aug, 2010  |   Pubmed ID: 20595633 We have previously isolated GRIM-19, a novel growth suppressor, using a genetic method. GRIM-19 ablates cell growth by inhibiting the transcription factor signal transducer and activator of transcription 3 (STAT3). Up-regulation of STAT3 and growth promotion were observed in a number of human tumors. Although the tumor-suppressive actions of GRIM-19 are known, the structural elements required for its antitumor actions are not understood. Mutational and protein sequence analyses identified a motif in the N terminus of GRIM-19 that exhibited similarity to certain RNA viral proteins. We show that disruption of specific amino acids within this motif cripples the antitumor actions of GRIM-19. These mutants fail to interact with STAT3 efficiently and consequently do not inhibit growth-promoting gene expression. More importantly, we show that a clinically observed mutation in the N terminus of GRIM-19 also weakened its interaction with STAT3 and antitumor action. Together, these studies identify a major role for the N terminus of GRIM-19 in mediating its tumor-suppressive actions.
• Relationships Among North American and Japanese Laetiporus Isolates Inferred from Molecular Phylogenetics and Single-spore Incompatibility Reactions Mycologia. Jul-Aug, 2010  |   Pubmed ID: 20648757 Relationships were investigated among North American and Japanese isolates of Laetiporus using phylogenetic analysis of ITS sequences and single-spore isolate incompatibility. Single-spore isolate pairings revealed no significant compatibility between North American and Japanese isolates. ITS analysis revealed 12 clades within the core Laetiporus clade, seven of which are known to occur in North America (including Hawaii and the Caribbean), three in Japan, two in South America, three in Europe and one in South Africa. The identity of L. sulphureus s.s. has yet to be determined and could be either L. "sulphureus" (clade C), which appears to be restricted to Europe and occurs on angiosperms and gymnospersm, or L. "sulphureus" (clade E), which is found in Europe, North America and South America exclusively on angiosperms. Three clades, one from the Caribbean, one from Hawaii and one from South Africa, have yet to be named formally. Of the three Laetiporus species found in Japan two have been named recently (L. cremeiporus and L. montanus) and one has been epitypified (L. versisporus). The single-spore incompatibility and ITS data support recognition of the three Japanese taxa as distinct biological and evolutionary species.
• Identification and Characterization of GRIM-1, a Cell-death-associated Gene Product Journal of Cell Science. Aug, 2010  |   Pubmed ID: 20663920 Using a genome-wide technical knockout, we isolated a newly identified set of GRIM (genes associated with retinoid-interferon-induced mortality) genes; GRIM genes mediate IFN- and retinoic-acid (RA)-induced cell death. Here, we describe the isolation and characterization of one such gene, GRIM-1. Three proteins, with identical C-termini, were produced from the GRIM-1 open reading frame when this gene was transcribed and translated in vitro. These protein isoforms, designated GRIM-1alpha, GRIM-1beta and GRIM-1gamma, differentially suppressed growth via apoptosis in various cell lines. We also show that a caspase-dependent mechanism generates the proapoptotic GRIM-1 isoforms. Lastly, GRIM-1 isoforms differentially blocked maturation of 18S ribosomal RNA, consistent with their respective growth-suppressive ability. Together, these studies identified a novel protein involved in growth suppression and cell death.
• Tyrosine Phosphatase Inhibitor-3 Sensitizes Melanoma and Colon Cancer to Biotherapeutics and Chemotherapeutics Molecular Cancer Therapeutics. Aug, 2010  |   Pubmed ID: 20682647 Drug resistance is a major obstacle in cancer treatments and diminishes the clinical efficacy of biological, cytotoxic, or targeted therapeutics. Being an antiapoptotic mediator of chemoresistance in breast and lung cancer cells, MKP1 phosphatase might be targeted for overcoming chemoresistance and improving therapeutic efficacy. In this work, tyrosine phosphatase inhibitor-3 (TPI-3) was identified as a novel small molecule inhibitor of MKP1 and was capable of sensitizing tumors to bio- and chemotherapeutics in mice as a tolerated oral agent. Effective against recombinant MKP1, TPI-3 selectively increased MKP1 phosphosubstrates in Jurkat cells and induced cell death via apoptosis at nanomolar concentrations. TPI-3 also increased MKP1 phosphosubstrates in WM9 human melanoma cells and synergized with biotherapeutic IFN alpha 2b in the growth inhibition of melanoma cells in vitro (combination index,
• DNA-based Detection of the Fungal Pathogen Geomyces Destructans in Soils from Bat Hibernacula Mycologia. Mar-Apr, 2011  |   Pubmed ID: 20952799 White-nose syndrome (WNS) is an emerging disease causing unprecedented morbidity and mortality among bats in eastern North America. The disease is characterized by cutaneous infection of hibernating bats by the psychrophilic fungus Geomyces destructans. Detection of G. destructans in environments occupied by bats will be critical for WNS surveillance, management and characterization of the fungal lifecycle. We initiated an rRNA gene region-based molecular survey to characterize the distribution of G. destructans in soil samples collected from bat hibernacula in the eastern United States with an existing PCR test. Although this test did not specifically detect G. destructans in soil samples based on a presence/absence metric, it did favor amplification of DNA from putative Geomyces species. Cloning and sequencing of PCR products amplified from 24 soil samples revealed 74 unique sequence variants representing 12 clades. Clones with exact sequence matches to G. destructans were identified in three of 19 soil samples from hibernacula in states where WNS is known to occur. Geomyces destructans was not identified in an additional five samples collected outside the region where WNS has been documented. This study highlights the diversity of putative Geomyces spp. in soil from bat hibernacula and indicates that further research is needed to better define the taxonomy of this genus and to develop enhanced diagnostic tests for rapid and specific detection of G. destructans in environmental samples.
• Intragenomic Variation in the ITS RDNA Region Obscures Phylogenetic Relationships and Inflates Estimates of Operational Taxonomic Units in Genus Laetiporus Mycologia. Jul-Aug, 2011  |   Pubmed ID: 21289107 Regions of rDNA are commonly used to infer phylogenetic relationships among fungal species and as DNA barcodes for identification. These regions occur in large tandem arrays, and concerted evolution is believed to reduce intragenomic variation among copies within these arrays, although some variation still might exist. Phylogenetic studies typically use consensus sequencing, which effectively conceals most intragenomic variation, but cloned sequences containing intragenomic variation are becoming prevalent in DNA databases. To understand effects of using cloned rDNA sequences in phylogenetic analyses we amplified and cloned the ITS region from pure cultures of six Laetiporus species and one Wolfiporia species (Basidiomycota, Polyporales). An average of 66 clones were selected randomly and sequenced from 21 cultures, producing a total of 1399 interpretable sequences. Significant variation (≥ 5% variation in sequence similarity) was observed among ITS copies within six cultures from three species clades (L. cincinnatus, L. sp. clade J, and Wolfiporia dilatohypha) and phylogenetic analyses with the cloned sequences produced different trees relative to analyses with consensus sequences. Cloned sequences from L. cincinnatus fell into more than one species clade and numerous cloned L. cincinnatus sequences fell into entirely new clades, which if analyzed on their own most likely would be recognized as "undescribed" or "novel" taxa. The use of a 95% cut off for defining operational taxonomic units (OTUs) produced seven Laetiporus OTUs with consensus ITS sequences and 20 OTUs with cloned ITS sequences. The use of cloned rDNA sequences might be problematic in fungal phylogenetic analyses, as well as in fungal bar-coding initiatives and efforts to detect fungal pathogens in environmental samples.
• Noncytotoxic Differentiation Treatment of Renal Cell Cancer Cancer Research. Feb, 2011  |   Pubmed ID: 21303982 Current drug therapy for metastatic renal cell cancer (RCC) results in temporary disease control but not cure, necessitating continued investigation into alternative mechanistic approaches. Drugs that inhibit chromatin-modifying enzymes involved in transcription repression (chromatin-relaxing drugs) could have a role, by inducing apoptosis and/or through differentiation pathways. At low doses, the cytosine analogue decitabine (DAC) can be used to deplete DNA methyl-transferase 1 (DNMT1), modify chromatin, and alter differentiation without causing apoptosis (cytotoxicity). Noncytotoxic regimens of DAC were evaluated for in vitro and in vivo efficacy against RCC cell lines, including a p53-mutated RCC cell line developed from a patient with treatment-refractory metastatic RCC. The cell division-permissive mechanism of action-absence of early apoptosis or DNA damage, increase in expression of HNF4α (hepatocyte nuclear factor 4α), a key driver associated with the mesenchymal to epithelial transition, decrease in mesenchymal marker expression, increase in epithelial marker expression, and late increase in cyclin-dependent kinase inhibitor CDKN1B (p27) protein-was consistent with differentiation-mediated cell-cycle exit. In vivo blood counts and animal weights were consistent with minimal toxicity of therapy. The distinctive mechanism of action of a dose and schedule of DAC designed for noncytotoxic depletion of DNMT1 suggests a potential role in treating RCC.
• Identification of an MRNP Complex Regulating Tumorigenesis at the Translational Elongation Step Molecular Cell. Feb, 2011  |   Pubmed ID: 21329880 Transcript-selective translational regulation of epithelial-mesenchymal transition (EMT) by transforming growth factor-β (TGF-β) is directed by the hnRNP E1-containing TGF-β-activated-translational (BAT) mRNP complex. Herein, eukaryotic elongation factor-1 A1 (eEF1A1) is identified as an integral component of the BAT complex. Translational silencing of Dab2 and ILEI, two EMT transcripts, is mediated by the binding of hnRNP E1 and eEF1A1 to their 3'UTR BAT element, whereby hnRNP E1 stalls translational elongation by inhibiting the release of eEF1A1 from the ribosomal A site. TGF-β-mediated hnRNP E1 phosphorylation, through Akt2, disrupts the BAT complex, thereby restoring translation of target EMT transcripts. Attenuation of hnRNP E1 expression in two noninvasive breast epithelial cells (NMuMG and MCF-7) not only induced EMT but also enabled cells to form metastatic lesions in vivo. Thus, translational regulation by TGF-β at the elongation stage represents a critical checkpoint coordinating the expression of EMT transcripts required during development and in tumorigenesis and metastatic progression.
• MR Molecular Imaging of Prostate Cancer with a Peptide-targeted Contrast Agent in a Mouse Orthotopic Prostate Cancer Model Pharmaceutical Research. Apr, 2012  |   Pubmed ID: 22139536 To study the effectiveness of a peptide targeted nanoglobular Gd-DOTA complexes for MR molecular imaging of prostate cancer in a mouse orthotopic PC-3 prostate cancer model.
• Armillaria Altimontana, a New Species from the Western Interior of North America Mycologia. Sep-Oct, 2012  |   Pubmed ID: 22505437 Armillaria altimontana, previously considered North American biological species (NABS) X, is described as new. To date, it appears that A. altimontana prefers higher-elevation, mesic sites within the dry, conifer forest zone of western interior North America. This species has been found on hardwoods and conifers and is associated most commonly with Abies-dominated forest types in southern British Columbia, Washington, Oregon, Idaho and northern California. Partial elongation factor 1-alpha (tef1) sequences were generated from six isolates of A. altimontana originating from three locations in northern Idaho. Phylogenetic analyses of all 10 North American Armillaria species were carried out with maximum parsimony and maximum likelihood. Results indicate that isolates of A. altimontana formed a monophyletic group and clustered with A. calvescens, A. cepistipes, A. gallica and A. nabsnona, which is in agreement with recent phylogenetic studies of Armillaria.
• Synthesis and Evaluation of a Peptide Targeted Small Molecular Gd-DOTA Monoamide Conjugate for MR Molecular Imaging of Prostate Cancer Bioconjugate Chemistry. Aug, 2012  |   Pubmed ID: 22812444 Tumor extracellular matrix has an abundance of cancer related proteins that can be used as biomarkers for cancer molecular imaging. Innovative design and development of safe and effective targeted contrast agents to these biomarkers would allow effective MR cancer molecular imaging with high spatial resolution. In this study, we synthesized a low molecular weight CLT1 peptide targeted Gd(III) chelate CLT1-dL-(Gd-DOTA)(4) specific to clotted plasma proteins in tumor stroma for cancer MR molecular imaging. CLT1-dL-(Gd-DOTA)(4) was synthesized by conjugating four Gd-DOTA monoamide chelates to a CLT1 peptide via generation 1 lysine dendrimer. The T(1) relaxivity of CLT1-dL-(Gd-DOTA)(4) was 40.4 mM(-1) s(-1) per molecule (10.1 mM(-1) s(-1) per Gd) at 37 °C and 1.5 T. Fluorescence imaging showed high binding specificity of CLT1 to orthotopic PC3 prostate tumor in mice. The contrast agent resulted in improved tumor contrast enhancement in male athymic nude mice bearing orthotopic PC3 prostate tumor xenograft at a dose of 0.03 mmol Gd/kg. The peptide targeted MRI contrast agent is promising for high-resolution MR molecular imaging of prostate tumor.
• A Small Molecule Agonist of EphA2 Receptor Tyrosine Kinase Inhibits Tumor Cell Migration in Vitro and Prostate Cancer Metastasis in Vivo PloS One. 2012  |   Pubmed ID: 22916121 During tumor progression, EphA2 receptor can gain ligand-independent pro-oncogenic functions due to Akt activation and reduced ephrin-A ligand engagement. The effects can be reversed by ligand stimulation, which triggers the intrinsic tumor suppressive signaling pathways of EphA2 including inhibition of PI3/Akt and Ras/ERK pathways. These observations argue for development of small molecule agonists for EphA2 as potential tumor intervention agents. Through virtual screening and cell-based assays, we report here the identification and characterization of doxazosin as a novel small molecule agonist for EphA2 and EphA4, but not for other Eph receptors tested. NMR studies revealed extensive contacts of doxazosin with EphA2/A4, recapitulating both hydrophobic and electrostatic interactions recently found in the EphA2/ephrin-A1 complex. Clinically used as an α1-adrenoreceptor antagonist (Cardura®) for treating hypertension and benign prostate hyperplasia, doxazosin activated EphA2 independent of α1-adrenoreceptor. Similar to ephrin-A1, doxazosin inhibited Akt and ERK kinase activities in an EphA2-dependent manner. Treatment with doxazosin triggered EphA2 receptor internalization, and suppressed haptotactic and chemotactic migration of prostate cancer, breast cancer, and glioma cells. Moreover, in an orthotopic xenograft model, doxazosin reduced distal metastasis of human prostate cancer cells and prolonged survival in recipient mice. To our knowledge, doxazosin is the first small molecule agonist of a receptor tyrosine kinase that is capable of inhibiting malignant behaviors in vitro and in vivo.
• Bat White-nose Syndrome: a Real-time TaqMan Polymerase Chain Reaction Test Targeting the Intergenic Spacer Region of Geomyces Destructans Mycologia. Mar-Apr, 2013  |   Pubmed ID: 22962349 The fungus Geomyces destructans is the causative agent of white-nose syndrome (WNS), a disease that has killed millions of North American hibernating bats. We describe a real-time TaqMan PCR test that detects DNA from G. destructans by targeting a portion of the multicopy intergenic spacer region of the rRNA gene complex. The test is highly sensitive, consistently detecting as little as 3.3 fg genomic DNA from G. destructans. The real-time PCR test specifically amplified genomic DNA from G. destructans but did not amplify target sequence from 54 closely related fungal isolates (including 43 Geomyces spp. isolates) associated with bats. The test was qualified further by analyzing DNA extracted from 91 bat wing skin samples, and PCR results matched histopathology findings. These data indicate the real-time TaqMan PCR method described herein is a sensitive, specific and rapid test to detect DNA from G. destructans and provides a valuable tool for WNS diagnostics and research.
• A Culture-based Survey of Fungi in Soil from Bat Hibernacula in the Eastern United States and Its Implications for Detection of Geomyces Destructans, the Causal Agent of Bat White-nose Syndrome Mycologia. Mar-Apr, 2013  |   Pubmed ID: 23074174 The recent emergence of white-nose syndrome (WNS), a fungal disease causing unprecedented mortality among hibernating bats of eastern North America, has revealed a knowledge gap regarding fungal communities associated with bats and their hibernacula. We used culture-based techniques to investigate the diversity of fungi in soil samples collected from 24 bat hibernacula in the eastern United States. Ribosomal RNA regions (internal transcribed spacer and partial intergenic spacer) were sequenced to preliminarily characterize isolates. Geomyces species were one of the most abundant and diverse groups cultured, representing approximately 33% of all isolates. Geomyces destructans was isolated from soil samples from three hibernacula in states where WNS is known to occur, and many of the other cultured Geomyces isolates likely represent undescribed taxa. Further characterization of the diversity of fungi that occur in hibernacula both will facilitate an improved understanding of the ecology of G. destructans within this complex fungal community and provide an opportunity to identify characteristics that differentiate G. destructans from non-pathogenic relatives.
• Thrombospondin-1 Expression in Melanoma is Blocked by Methylation and Targeted Reversal by 5-Aza-deoxycytidine Suppresses Angiogenesis Matrix Biology : Journal of the International Society for Matrix Biology. Mar, 2013  |   Pubmed ID: 23202046 Reversibility of aberrant methylation via pharmacological means is an attractive target for therapies through epigenetic reprogramming. To establish that pharmacologic reversal of methylation could result in functional inhibition of angiogenesis, we undertook in vitro and in vivo studies of thrombospondin-1 (TSP1), a known inhibitor of angiogenesis. TSP1 is methylated in several malignancies, and can inhibit angiogenesis in melanoma xenografts. We analyzed effects of 5-Aza-deoxycytidine (5-Aza-dC) on melanoma cells in vitro to confirm reversal of promoter hypermethylation and restoration of TSP1 expression. We then investigated the effects of TSP1 expression on new blood vessel formation and tumor growth in vivo. Finally, to determine potential for clinical translation, the methylation status of TSP1 promoter regions of nevi and melanoma tissues was investigated.
• Distribution and Environmental Persistence of the Causative Agent of White-nose Syndrome, Geomyces Destructans, in Bat Hibernacula of the Eastern United States Applied and Environmental Microbiology. Feb, 2013  |   Pubmed ID: 23241985 White-nose syndrome (WNS) is an emerging disease of hibernating bats caused by the recently described fungus Geomyces destructans. First isolated in 2008, the origins of this fungus in North America and its ability to persist in the environment remain undefined. To investigate the correlation between manifestation of WNS and distribution of G. destructans in the United States, we analyzed sediment samples collected from 55 bat hibernacula (caves and mines) both within and outside the known range of WNS using a newly developed real-time PCR assay. Geomyces destructans was detected in 17 of 21 sites within the known range of WNS at the time when the samples were collected; the fungus was not found in 28 sites beyond the known range of the disease at the time when environmental samples were collected. These data indicate that the distribution of G. destructans is correlated with disease in hibernating bats and support the hypothesis that the fungus is likely an exotic species in North America. Additionally, we examined whether G. destructans persists in infested bat hibernacula when bats are absent. Sediment samples were collected from 14 WNS-positive hibernacula, and the samples were screened for viable fungus by using a culture technique. Viable G. destructans was cultivated from 7 of the 14 sites sampled during late summer, when bats were no longer in hibernation, suggesting that the fungus can persist in the environment in the absence of bat hosts for long periods of time.
• Tumor-derived Mutations in the Gene Associated with Retinoid Interferon-induced Mortality (GRIM-19) Disrupt Its Anti-signal Transducer and Activator of Transcription 3 (STAT3) Activity and Promote Oncogenesis The Journal of Biological Chemistry. Mar, 2013  |   Pubmed ID: 23386605 The signal transducer and activator of transcription 3 (STAT3) protein is critical for multiple cytokine and growth factor-induced biological responses in vivo. Its transcriptional activity is controlled by a transient phosphorylation of a critical tyrosine. Constitutive activation of STAT3 imparts resistance to apoptosis, promotes cell proliferation, and induces de novo micro-angiogenesis, three of the six cardinal hallmarks of a typical cancer cell. Earlier we reported the isolation of GRIM-19 as a growth suppressor using a genome-wide expression knockdown strategy. GRIM-19 binds to STAT3 and suppresses its transcriptional activity. To understand the pathological relevance of GRIM-19, we screened a set of primary head and neck tumors and identified three somatic mutations in GRIM-19. Wild-type GRIM-19 suppressed cellular transformation by a constitutively active form of STAT3, whereas tumor-derived mutants L71P, L91P and A95T significantly lost their ability to associate with STAT3, block gene expression, and suppress cellular transformation and tumor growth in vivo. Additionally, these mutants lost their capacity to prevent metastasis. These mutations define a mechanism by which STAT3 activity is deregulated in certain human head and neck tumors.
• The Investigational Aurora Kinase A Inhibitor MLN8237 Induces Defects in Cell Viability and Cell-cycle Progression in Malignant Bladder Cancer Cells in Vitro and in Vivo Clinical Cancer Research : an Official Journal of the American Association for Cancer Research. Apr, 2013  |   Pubmed ID: 23403633 Despite more than 70,000 new cases of bladder cancer in the United States annually, patients with advanced disease have a poor prognosis due to limited treatment modalities. We evaluated Aurora kinase A, identified as an upregulated candidate molecule in bladder cancer, as a potential therapeutic target.
• Synthesis and Evaluation of a Targeted Nanoglobular Dual-Modal Imaging Agent for MR Imaging and Image-Guided Surgery of Prostate Cancer Pharmaceutical Research. Mar, 2013  |   Pubmed ID: 23471641 PURPOSE: To synthesize and evaluate a peptide targeted nanoglobular dual modal imaging agent specific to a cancer biomarker in tumor stroma for MRI and fluorescence visualization of prostate tumor in image-guided surgery. METHODS: A peptide (CGLIIQKNEC, CLT1) targeted generation 2 nanoglobular (polylysine dendrimer with a silsesquioxane core) dual modal imaging agent, CLT1-G2-(Gd-DOTA-MA)-Cy5, was synthesized by stepwise conjugation of Gd-DOTA-MA, Cy5 and peptide to the dendrimer. Contrast enhanced MR imaging of the targeted dual imaging agent was evaluated on a Bruker 7T animal scanner with male athymic nude mice bearing orthotopic PC3-GFP prostate tumor. Fluorescence tumor imaging of the agent was carried out on a Maestro fluorescence imaging system. RESULTS: The targeted agent CLT1-G2-(Gd-DOTA-MA)-Cy5 produced greater contrast enhancement in the tumor tissue than the control agent KAREC-G2-(Gd-DOTA-MA)-Cy5 at a dose of 30 μmol-Gd/kg in the MR images of the tumor bearing mice. Signal-to-noise ratio (SNR) of CLT1-G2-(Gd-DOTA-MA)-Cy5 in the tumor tissue was approximately 2 fold of that of the control agent in the first 15 min post-injection. The targeted agent also resulted in bright fluorescence signals in the tumor tissue. CONCLUSION: The CLT1 peptide targeted nanoglobular dual-imaging agent CLT1-G2-(Gd-DOTA-MA)-Cy5 has a potential for MRI and fluorescence visualization of prostate tumor.
• The Cardioprotective Protein Apolipoprotein A1 Promotes Potent Anti-tumorigenic Effects The Journal of Biological Chemistry. Jul, 2013  |   Pubmed ID: 23720750 Here, we show that apolipoprotein A1 (apoA1), the major protein component of high density lipoprotein (HDL), through both innate and adaptive immune processes, potently suppresses tumor growth and metastasis in multiple animal tumor models, including the aggressive B16F10L murine malignant melanoma model. Mice expressing the human apoA1 transgene (A1Tg) exhibited increased infiltration of CD11b(+) F4/80(+) macrophages with M1, anti-tumor phenotype, reduced tumor burden and metastasis, and enhanced survival. In contrast, apoA1-deficient (A1KO) mice showed markedly heightened tumor growth and reduced survival. Injection of human apoA1 into A1KO mice inoculated with tumor cells remarkably reduced both tumor growth and metastasis, enhanced survival, and promoted regression of both tumor and metastasis burden when administered following palpable tumor formation and metastasis development. Studies with apolipoprotein A2 revealed the anti-cancer therapeutic effect was specific to apoA1. In vitro studies ruled out substantial direct suppressive effects by apoA1 or HDL on tumor cells. Animal models defective in different aspects of immunity revealed both innate and adaptive arms of immunity contribute to complete apoA1 anti-tumor activity. This study reveals a potent immunomodulatory role for apoA1 in the tumor microenvironment, altering tumor-associated macrophages from a pro-tumor M2 to an anti-tumor M1 phenotype. Use of apoA1 to redirect in vivo elicited tumor-infiltrating macrophages toward tumor rejection may hold benefit as a potential cancer therapeutic.
• Employing 454 Amplicon Pyrosequencing to Reveal Intragenomic Divergence in the Internal Transcribed Spacer RDNA Region in Fungi Ecology and Evolution. Jun, 2013  |   Pubmed ID: 23789083 The rDNA internal transcribed spacer (ITS) region has been accepted as a DNA barcoding marker for fungi and is widely used in phylogenetic studies; however, intragenomic ITS variability has been observed in a broad range of taxa, including prokaryotes, plants, animals, and fungi, and this variability has the potential to inflate species richness estimates in molecular investigations of environmental samples. In this study 454 amplicon pyrosequencing of the ITS1 region was applied to 99 phylogenetically diverse axenic single-spore cultures of fungi (Dikarya: Ascomycota and Basidiomycota) to investigate levels of intragenomic variation. Three species (one Basidiomycota and two Ascomycota), in addition to a positive control species known to contain ITS paralogs, displayed levels of molecular variation indicative of intragenomic variation; taxon inflation due to presumed intragenomic variation was ≈9%. Intragenomic variability in the ITS region appears to be widespread but relatively rare in fungi (≈3-5% of species investigated in this study), suggesting this problem may have minor impacts on species richness estimates relative to PCR and/or pyrosequencing errors. Our results indicate that 454 amplicon pyrosequencing represents a powerful tool for investigating levels of ITS intragenomic variability across taxa, which may be valuable for better understanding the fundamental mechanisms underlying concerted evolution of repetitive DNA regions.
• A Phylogenetic Overview of the Antrodia Clade (Basidiomycota, Polyporales) Mycologia. Aug, 2013  |   Pubmed ID: 23935025 Phylogenetic relationships among members of the antrodia clade were investigated using molecular data from two nuclear ribosomal DNA regions, LSU and ITS. A total of 123 species representing 26 genera producing a brown rot were included in the present study. Three DNA datasets (combined LSU-ITS dataset, LSU dataset, ITS dataset) comprising sequences of 449 isolates were evaluated using three different phylogenetic analyses (Maximum Likelihood, Maximum Parsimony, Bayesian inference). We present a phylogenetic overview of the five main groups recovered: the fibroporia, laetiporus, postia, laricifomes and core antrodia groups. Not all of the main groups received strong support in the analyses, requiring further research. We were able to identify a number of well-supported clades within the main groups.