Imidazopyridine-Based Fatty Acid Synthase Inhibitors That Show Anti-HCV Activity and in Vivo Target Modulation.

Potent imidazopyridine-based inhibitors of fatty acid synthase (FASN) are described. The compounds are shown to have antiviral (HCV replicon) activities that track with their biochemical activities. The most potent analogue (compound 19) also inhibits rat FASN and inhibits de novo palmitate synthesis in vitro (cell-based) as well as in vivo.

ß-catenin/Wnt signaling controls progenitor fate in the developing and regenerating zebrafish retina.

BACKGROUND: The zebrafish retina maintains two populations of stem cells: first, the germinal zone or ciliary marginal zone (CMZ) contains multipotent retinal progenitors that add cells to the retinal periphery as the fish continue to grow; second, radial glia (Müller cells) occasionally divide asymmetrically to generate committed progenitors that differentiate into rod photoreceptors, which are added interstitially throughout the retina with growth. Retinal injury stimulates Müller glia to dedifferentiate, re-enter the cell cycle, and generate multipotent retinal progenitors similar to those in the CMZ to replace missing neurons. The specific signals that maintain these two distinct populations of endogenous retinal stem cells are not understood. RESULTS: We used genetic and pharmacological manipulation of the ß-catenin/Wnt signaling pathway to show that it is required to maintain proliferation in the CMZ and that hyperstimulation of ß-catenin/Wnt signaling inhibits normal retinal differentiation and expands the population of proliferative retinal progenitors. To test whether similar effects occur during regeneration, we developed a method for making rapid, selective photoreceptor ablations in larval zebrafish with intense light. We found that dephosphorylated ß-catenin accumulates in Müller glia as they re-enter the cell cycle following injury, but not in Müller glia that remain quiescent. Activation of Wnt signaling is required for regenerative proliferation, and hyperstimulation results in loss of Müller glia from the INL as all proliferative cells move into the ONL. CONCLUSIONS: ß-catenin/Wnt signaling is thus required for the maintenance of retinal progenitors during both initial development and lesion-induced regeneration, and is sufficient to prevent differentiation of those progenitors and maintain them in a proliferative state. This suggests that the ß-catenin/Wnt cascade is part of the shared molecular circuitry that maintains retinal stem cells for both homeostatic growth and epimorphic regeneration.

TRP channel regulates EGFR signaling in hair morphogenesis and skin barrier formation.

A plethora of growth factors regulate keratinocyte proliferation and differentiation that control hair morphogenesis and skin barrier formation. Wavy hair phenotypes in mice result from naturally occurring loss-of-function mutations in the genes for TGF-alpha and EGFR. Conversely, excessive activities of TGF-alpha/EGFR result in hairless phenotypes and skin cancers. Unexpectedly, we found that mice lacking the Trpv3 gene also exhibit wavy hair coat and curly whiskers. Here we show that keratinocyte TRPV3, a member of the transient receptor potential (TRP) family of Ca(2+)-permeant channels, forms a signaling complex with TGF-alpha/EGFR. Activation of EGFR leads to increased TRPV3 channel activity, which in turn stimulates TGF-alpha release. TRPV3 is also required for the formation of the skin barrier by regulating the activities of transglutaminases, a family of Ca(2+)-dependent crosslinking enzymes essential for keratinocyte cornification. Our results show that a TRP channel plays a role in regulating growth factor signaling by direct complex formation.

Cytosine deaminase/5-fluorocytosine exposure induces bystander and radiosensitization effects in hypoxic glioblastoma cells in vitro.

PURPOSE: Treatment of glioblastoma (GBM) is limited by therapeutic ratio; therefore, successful therapy must be specifically cytotoxic to cancer cells. Hypoxic cells are ubiquitous in GBM, and resistant to radiation and chemotherapy, and, thus, are logical targets for gene therapy. In this study, we investigated whether cytosine deaminase (CD)/5-fluorocytosine (5-FC) enzyme/prodrug treatment induced a bystander effect (BE) and/or radiosensitization in hypoxic GBM cells. METHODS AND MATERIALS: We stably transfected cells with a gene construct consisting of the SV40 minimal promoter, nine copies of a hypoxia-responsive element, and the yeast CD gene. During hypoxia, a hypoxia-responsive element regulates expression of the CD gene and facilitates the conversion of 5-FC to 5-fluorouracil, a highly toxic antimetabolite. We used colony-forming efficiency (CFE) and immunofluorescence assays to assess for BE in co-cultures of CD-expressing clone cells and parent, pNeo- or green fluorescent protein-stably transfected GBM cells. We also investigated the radiosensitivity of CD clone cells treated with 5-FC under hypoxic conditions, and we used flow cytometry to investigate treatment-induced cell cycle changes. RESULTS: Both a large BE and radiosensitization occurred in GBM cells under hypoxic conditions. The magnitude of the BE depended on the number of transfected cells producing CD, the functionality of the CD, the administered concentration of 5-FC, and the sensitivity of cell type to 5-fluorouracil. CONCLUSION: Hypoxia-inducible CD/5-FC therapy in combination with radiation therapy shows both a pronounced BE and a radiosensitizing effect under hypoxic conditions.

Response of intracerebral human glioblastoma xenografts to multifraction radiation exposures.

PURPOSE: We investigated the effects of fractionated radiation treatments on the life spans of athymic rats bearing intracerebral brain tumors. METHODS AND MATERIALS: U-251 MG or U-87 MG human glioblastoma cells were implanted into the brains of athymic rats, and the resulting tumors were irradiated once daily with various doses of ionizing radiation for 5 consecutive days or for 10 days with a 2-day break after Day 5. RESULTS: Five daily doses of 1 and 1.5 Gy, and 10 doses of 0.75 and 1 Gy, cured some U-251 MG tumors. However, five daily doses of 0.5 Gy increased the survival time of animals bearing U-251 MG tumors 5 days without curing any animals of their tumors. Ten doses of 0.3 Gy given over 2 weeks extended the lifespan of the host animals 9 days without curing any animals. For U-87 MG tumors, 5 daily doses of 3 Gy produced an increased lifespan of 8 days without curing any animals, and 10 doses of 1 Gy prolonged lifespan 5.5 days without curing any animals. The differences in extension of life span between the 5- and 10-fraction protocols were minor for either tumor type. CONCLUSION: The finding that the U-251 MG tumors are more sensitive than U-87 MG tumors, despite the fact that U-251 MG tumors contain many more hypoxic cells than U-87 MG tumors, suggests the intrinsic cellular radiosensitivities of these cell lines are more important than hypoxia in determining their in vivo radiosensitivities.

Chromosome transfer experiments link regions on chromosome 7 to radiation resistance in human glioblastoma multiforme.

Glioblastoma multiforme (GM) is the most lethal form of brain tumor, with a median survival of approximately 1 year. Treatment options are limited. Radiation therapy is a common form of treatment, but many tumors are resistant. In earlier studies, we found that gain of chromosome 7 is associated with radiation resistance in human primary GM. In this study, we extend that result to a model system in which we transferred chromosome 7 to recipient cells and confirmed radiation resistance as a function of chromosome 7 gain. We identified three candidate regions on chromosome 7 that conferred radiation resistance in our model system.

Hypoxia-induced BAX overexpression and radiation killing of hypoxic glioblastoma cells.

One major challenge in treating glioblastoma multiforme (GBM) has been the presence of radiation-resistant hypoxic cells. The pro-apoptosis protein BAX has been reported to be a possible radiation sensitizer of cancer cells; however, to our knowledge, no studies have reported on the effects of BAX on radiation sensitivity under hypoxic conditions. Therefore, in this study, we specifically overexpressed murine Bax in hypoxic cells in an attempt to enhance radiation-induced cell killing. We have previously stably transfected U-251 MG and U-87 MG human GBM cells with constructs containing murine Bax under the regulation of nine copies of hypoxia-responsive elements (HREs). During hypoxia, the transcriptional complex hypoxia-inducible factor 1 (HIF1) forms and binds to HRE; this binding facilitates the transcription of downstream genes. In the experiments reported here, two protocols were used. In the first protocol, parent and clone cells were exposed to graded doses of X rays under hypoxic conditions, left hypoxic for 0, 4, 16 or 24 h, and then assayed for clonogenic cell survival. In the second protocol, cells were incubated under hypoxic conditions for 20 h, irradiated with graded doses under hypoxia, then left in hypoxic conditions for 4 h before being assayed for clonogenic cell survival. Western blots showed that we had successfully increased Bax expression in both U-251 MG and U-87 MG Bax clone cells after 16 h of hypoxic incubation, yet dose-response curves showed no difference in radiation-induced cell killing between control non-Bax-expressing pNeo clone cells and the U-251 MG Bax clone cells using either protocol. In U-87 MG cells, the first protocol showed no difference in radiation response between control pNeo clone cells and the Bax clone, similar to the results obtained in U-251 cells. However, the second protocol revealed that Bax overexpression did render these cells more sensitive to radiation under hypoxic conditions. Therefore, we conclude that whether Bax is a radiation enhancer under hypoxia not only is cell line-dependent but also depends on when the Bax overexpression occurs.

Functionality of hypoxia-induced BAX expression in a human glioblastoma xenograft model.

The effectiveness of radiation therapy for human brain tumors is limited by the presence of radiation-resistant hypoxic cells. In order to improve patient outcomes, therapeutic methods that increase hypoxic cell killing must be developed. To investigate the possibility of using the hypoxic tumor microenvironment itself as a target for gene therapy, we stably transfected U-251 MG human glioblastoma cells with constructs containing the suicide gene Bax under the regulation of a nine-copy concatemer of hypoxia responsive elements (HREs). Previously, we demonstrated that the expression of BAX protein under anoxic conditions in transfected U-251 MG clones leads to increased cell killing in vitro. Our recent studies revealed that HIF-1alpha induction under anoxic conditions occurs prior to the increase in BAX expression, thereby implicating HIF-1 induction as the basis of BAX upregulation. To test the effect of BAX-mediated cell killing in vivo, we implanted five stably transfected clones subcutaneously into the flanks of athymic mice. Compared to nontransfected controls, tumor growth in four of five clones was significantly retarded. Histopathological analysis demonstrated decreased hypoxic fractions and increased amounts of apoptosis in clone-derived tumors. These results suggest that the tumor microenvironment is sufficiently hypoxic to trigger HRE-mediated cell killing via the BAX apoptotic pathway.

Development of a hypoxia-inducible cytosine deaminase expression vector for gene-directed prodrug cancer therapy.

One important feature of human solid tumors is the presence of a hypoxic microenvironment. Under hypoxia, genes that contain a hypoxia-response element (HRE) can be activated by the binding of hypoxia-inducible factor-1. To reach the goal of selectively killing tumor cells in a hypoxic microenvironment using a gene therapy approach, we developed a cytosine deaminase (CD) gene construct (pH9YCD2) that contains an HRE gene enhancer. CD is an enzyme that catalyzes the conversion of noncytotoxic 5-fluorocytosine (5-FC) to the cytotoxic and radiosensitizing drug 5-fluorouracil (5-FU). Yeast CD was cloned into an SV40 promoter-based mammalian expression vector, and an HRE enhancer was inserted in front of the promoter. Human glioblastoma U-87 MG cells were transfected with pH9YCD2. Western blots revealed that CD was strongly expressed under hypoxic conditions (0.3-1% O2), whereas only minor CD expression was seen under normoxic conditions. To confirm that the expressed CD enzyme retains catalytic activity, we performed a 5-FC/5-FU-conversion assay in which 5-FC was incubated with the lysates of pH9YCD2-transfected cells. The percentage of conversion from 5-FC to 5-FU was 63% under hypoxia versus 13% under normoxia. In vitro, cell viability and colony-forming efficiency assays demonstrated that the gene construct was able to significantly kill glioblastoma cells in a hypoxia-dependent manner. In addition, 5-FC treatment of hypoxic pH9YCD2-transfected cells produced a marked bystander effect, which could be a distinct advantage for gene therapy. If this construct exhibits antitumor efficacy in vivo, it may have promise as an antitumor agent in humans.

Antitumor effects of specific telomerase inhibitor GRN163 in human glioblastoma xenografts.

Telomerase is a ribonucleoprotein complex that elongates telomeric DNA and appears to play an important role in cellular immortalization of cancers. Because telomerase is expressed in the vast majority of malignant gliomas but not in normal brain tissues, it is a logical target for gliomaspecific therapy. The telomerase inhibitor GRN163, a 13-mer oligonucleotide N3'-->P5' thio-phosphoramidate (Geron Corporation, Menlo Park, Calif.), is complementary to the template region of the human telomerase RNA subunit hTR. When athymic mice bearing U-251 MG human brain tumor xenografts in their flanks were treated intratumorally with GRN163, a significant growth delay in tumor size was observed (P < 0.01 in all groups) as compared to the tumor size in mice receiving a mismatched oligonucleotide or the carrier alone. We also investigated biodistribution of the drug in vivo in an intracerebral rat brain-tumor model. Fluorescein-labeled GRN163 was loaded into an osmotic minipump and infused directly into U-251 MG brain tumors over 7 days. Examination of the brains revealed that GRN163 was present in tumor cells at all time points studied. When GRN163 was infused into intracerebral U-251 MG tumors shortly after their implantation, it prevented their establishment and growth. Lastly, when rats with larger intracerebral tumors were treated with the inhibitor, GRN163 increased animal survival times. Our results demonstrate that the antitelomerase agent GRN163 inhibits growth of glioblastoma in vivo, exhibits favorable intracerebral tumor uptake properties, and prevents the growth of intracerebral tumors. These findings support further development of this compound as a potential anticancer agent.

Growth of human glioblastomas as xenografts in the brains of athymic rats.

BACKGROUND: We have developed xenografts of human glioblastoma (GBM) and established the baseline growth parameters and histopathological features of these tumors. MATERIALS-METHODS: Cells from 4 different human GBM cell lines were injected into the right caudate-putamen of brain in athymic rats. We measured tumor weights and the estimated survival time of each rat. RESULTS-CONCLUSION: U-251 MG and U-87 MG cells produced solid intracerebral tumors with a 100% tumor take rate, while SF-767 and SF-126 cells did not grow in the brains of athymic rats. Under the conditions employed, U-87 MG tumors grew faster than U-251 MG tumors, but both types of tumors exhibited reproducible growth characteristics from animal to animal. There was heterogeneity in the growth characteristics and histologies between the 2 tumor types, indicating that these tumor models might be useful for simulating some of the heterogeneity that occurs between GBM in humans.

Abnormalities of SNARE mechanism proteins in anterior frontal cortex in severe mental illness.

A fundamental molecular component of neural connectivity is the SNARE (SNAP receptor) protein complex, which consists of three proteins, syntaxin, SNAP-25 and VAMP. Under appropriate conditions, the SNARE complex can be formed in vitro. To investigate the hypothesis that dysregulation of SNARE proteins or their interactions could be abnormal in severe mental disorders, the three SNARE proteins and the complex were studied in post-mortem anterior frontal cortex homogenates. An ELISA was used to quantify SNARE protein immunoreactivities in cortical homogenates from four groups: patients with schizophrenia who died of causes other than suicide (n = 6), patients with schizophrenia and suicide (n = 7), patients with depression and suicide (n = 11), and controls (n = 11). Differences between groups in patterns of SNARE protein immuno-reactivities were demonstrated [Wilks' Lambda F(9,68) = 3.57, P = 0.001]. Protein-by-protein analyses indicated a significant reduction in SNAP-25 immunoreactivity in the schizophrenia non-suicide group [28% decrease relative to controls, F(3,31) = 6.45, P = 0.002, Student-Newman-Keuls test, P < 0.01]. The intercorrelations between SNARE protein and synaptophysin immunoreactivities were high in controls, but lower in the other groups, further indicating disturbances in relationships between these proteins. The extent of SNARE complex formation in vitro was studied using immuno-blotting. Significant differences related to group membership were observed for the SNARE complexes identified by SNAP-25 [Wilks' Lambda F(3,31) = 4.76, P = 0.008] and by syntaxin immunostaining [Wilks' Lambda F(3,31) = 9.16, P = 0.0002]. In both groups with suicide as a cause of death, relatively more SNAP-25 and syntaxin was present in the heterotrimeric SNARE complex than in other molecular forms. These abnormalities in the SNARE complex could represent a molecular substrate for abnormalities of neural connectivity in severe mental disorders.