bluuebunny/crossref_metadata_2025 huggingface dataset:
Molecular Basis for G2 Arrest Induced by 2′-C-Cyano-2′-Deoxy-1-β-d-Arabino-Pentofuranosylcytosine and Consequences of Checkpoint Abrogation
Authors: Liu Xiaojun , Guo Ying , Li Yexiong , Jiang Yingjun , Chubb Sherri , Azuma Atsushi , Huang Peng , Matsuda Akira , Hittelman Walter , Plunkett William
Published: September 2005

2′-C-cyano-2′-deoxy-1-β-d-arabino-pentofuranosylcytosine (CNDAC) is a nucleoside analogue with a novel mechanism of action that is currently being evaluated in clinical trials. Incorporation of CNDAC triphosphate into DNA and extension during replication leads to single-strand breaks directly caused by β-elimination. These breaks, or the lesions that arise from further processing, cause cells to arrest in G2. The purpose of this investigation was to define the molecular basis for G2 checkpoint activation and to delineate the sequelae of its abrogation. Cell lines derived from diverse human tissues underwent G2 arrest after CNDAC treatment, suggesting a common mechanism of response to the damage created. CNDAC-induced G2 arrest was instituted by activation of the Chk1-Cdc25C-Cdk1/cyclin B checkpoint pathway. Neither Chk2, p38, nor p53 was required for checkpoint activation. Inhibition of Chk1 kinase with 7-hydroxystaurosporine (UCN-01) abrogated the checkpoint pathway as indicated by dephosphorylation of checkpoint proteins and progression of cells through mitosis and into G1. Cell death was first evident in hematologic cell lines after G1 entry. As indicated by histone H2AX phosphorylation, DNA damage initiated by CNDAC incorporation was transformed into double-strand breaks when ML-1 cells arrested in G2. Some breaks were manifested as chromosomal aberrations when the G2 checkpoint of CNDAC-arrested cells was abrogated by UCN-01 but also in a minor population of cells that escaped to mitosis during treatment with CNDAC alone. These findings provide a mechanistic rationale for the design of new strategies, combining CNDAC with inhibitors of cell cycle checkpoint regulation in the therapy of hematologic malignancies.
Characterization of a Proapoptotic Antiganglioside GM2 Monoclonal Antibody and Evaluation of Its Therapeutic Effect on Melanoma and Small Cell Lung Carcinoma Xenografts
Authors: Retter Marc W. , Johnson Jeffrey C. , Peckham David W. , Bannink Jeannette E. , Bangur Chaitanya S. , Dresser Karen , Cai Feng , Foy Teresa M. , Fanger Neil A. , Fanger Gary R. , Woda Bruce , Rock Kenneth L.
Published: September 2005

Monoclonal antibodies have begun to show great clinical promise for the treatment of cancer. Antibodies that can directly affect a tumor cell's growth and/or survival are of particular interest for immunotherapy. Previously, we described monoclonal antibody DMF10.62.3 that had antiproliferative and proapoptotic effects when it bound an antigen of unknown identity on tumor cells in vitro. In this report, we determined that DMF10.62.3 and a clonally related antibody DMF10.167.4 recognize the ganglioside GM2. These antibodies react with a GM2 epitope that is expressed on a large number of tumor cell lines, including human melanoma and small cell lung carcinoma, but not on normal primary lines or most normal tissues. Interestingly, this pattern of cellular reactivity is distinct from that reported for other previously described GM2 antibodies, a difference that is presumably due to DMF10.167.4's binding to a unique GM2-associated epitope. Additional characterization of DMF10.167.4 revealed that this antibody was able to induce apoptosis and/or block cellular proliferation when cultured in vitro with the human Jurkat T lymphoma, CHL-1 melanoma, and SBC-3 small cell lung carcinoma lines. In vivo, DMF10.167.4 antibody was well tolerated in mice and did not detectably bind to or damage normal tissues. However, this antibody was able to prevent murine E710.2.3 lymphoma, human CHL-1 melanoma, and SBC-3 small cell lung carcinoma lines from establishing tumors in vivo and blocked progression of established CHL-1 and SBC-3 tumors in vivo. Therefore, monoclonal antibody DMF10.167.4 has immunotherapeutic potential.
RAD001 (Everolimus) Improves the Efficacy of Replicating Adenoviruses that Target Colon Cancer
Authors: Homicsko Krisztian , Lukashev Alexander , Iggo Richard D.
Published: September 2005

Selectively replicating adenoviruses have the potential to cure cancer but have shown little efficacy in clinical trials. We have tested the ability of the mTOR kinase inhibitor RAD001 (everolimus) to enhance the response of xenografts to an oncolytic adenovirus. The virus has Tcf sites inserted in the early viral promoters and replicates selectively in cells with activation of the Wnt signaling pathway. To enhance tumor cell infection, an integrin targeting peptide (CDCRGDCFC) was inserted into the fiber gene of the virus. RAD001 combines three useful properties: it inhibits tumor cell growth directly, blocks angiogenesis, and suppresses the immune response. RAD001 does not block viral protein expression, DNA replication, or cytopathic effect in tumor cells in vitro. After 6 weeks of daily RAD001 treatment, ongoing viral DNA replication could be detected in tumor xenografts, showing that RAD001 does not inhibit virus replication in vivo. I.v. injection of virus alone produced a small delay in xenograft growth, whereas combination therapy substantially prolonged the survival of the mice. We suggest that collapsing the tumor vasculature after the initial infection traps the virus and facilitates local spread within the tumor. Unlike conventional drugs, which require continued access to the tumor through the vascular system, oncolytic viruses are in principle less sensitive to late reductions in perfusion because they are produced locally within the tumor.
Somatic Mosaicism and Cancer: A Micro-Genetic Examination into the Role of the Androgen Receptor Gene in Prostate Cancer
Authors: Alvarado Carlos , Beitel Lenore K. , Sircar Kanishka , Aprikian Armen , Trifiro Mark , Gottlieb Bruce
Published: September 2005

Recent evidence has shown that the androgen receptor (AR) plays a major role in all prostate cancer stages, including both androgen-dependent and -independent tumors. A large number of studies have examined the possible effects of a functional polymorphism in the AR gene, a variable-length CAG repeat, on the development of prostate cancer, but the results to date have been inconclusive. We have considered the fact that the tissue heterogeneity present in almost all prostate cancer tumors has rarely been regarded as an indicator of AR genetic heterogeneity. To determine if genetic heterogeneity exists and is a significant event in prostate cancer development, we have examined prostate cancer tumors for somatic shortening of the AR gene CAG repeat. All 72 laser capture microdissected samples from archival prostate cancer tissues, as well as samples from freshly prepared prostate cancer tissues, showed some genetic heterogeneity (somatic mosaicism) for AR CAG repeat length. Cancerous tissues showed a much greater degree of genetic heterogeneity than adjacent benign tissues, as well as a very significant shortening of their CAG repeat lengths. However, CAG repeat length heterogeneity was not observed in normal prostate tissues. It is hypothesized that somatic mosaicism of the AR CAG repeat in prostate cancer tumors may be found to be an important genetic event in precancerous tissue, which may subsequently lead to the development of prostate cancer.
A New Three-Dimensional Ultrasound Microimaging Technology for Preclinical Studies Using a Transgenic Prostate Cancer Mouse Model
Authors: Wirtzfeld Lauren A. , Wu Guojun , Bygrave Michael , Yamasaki Yasuto , Sakai Hideki , Moussa Madeleine , Izawa Jonathan I. , Downey Dónal B. , Greenberg Norman M. , Fenster Aaron , Xuan Jim W. , Lacefield James C.
Published: September 2005

Prostate cancer is the most common cancer in adult men in North America. Preclinical studies of prostate cancer employ genetically engineered mouse models, because prostate cancer does not occur naturally in rodents. Widespread application of these models has been limited because autopsy was the only reliable method to evaluate treatment efficacy in longitudinal studies. This article reports the first use of three-dimensional ultrasound microimaging for measuring tumor progression in a genetically engineered mouse model, the 94–amino acid prostate secretory protein gene-directed transgenic prostate cancer model. Qualitative comparisons of three-dimensional ultrasound images with serial histology sections of prostate tumors show the ability of ultrasound to accurately depict the size and shape of malignant masses in live mice. Ultrasound imaging identified tumors ranging from 2.4 to 14 mm maximum diameter. The correlation coefficient of tumor diameter measurements done in vivo with three-dimensional ultrasound and at autopsy was 0.998. Prospective tumor detection sensitivity and specificity were both >90% when diagnoses were based on repeated ultrasound examinations done on separate days. Representative exponential growth curves constructed via longitudinal ultrasound imaging indicated volume doubling times of 5 and 13 days for two prostate tumors. Compared with other microimaging and molecular imaging modalities, the application of three-dimensional ultrasound imaging to prostate cancer in mice showed advantages, such as high spatial resolution and contrast in soft tissue, fast and uncomplicated protocols, and portable and economical equipment that will likely enable ultrasound to become a new microimaging modality for mouse preclinical trial studies.
Caspase-Mediated p65 Cleavage Promotes TRAIL-Induced Apoptosis
Authors: Kim Hun Sik , Chang Inik , Kim Ja Young , Choi Kyung-Hee , Lee Myung-Shik
Published: September 2005

Tumor necrosis factor (TNF)–related apoptosis-inducing ligand (TRAIL) is cytotoxic to a wide variety of transformed cells, but not to most normal cells, implying potential therapeutic value against advanced cancer. However, signal transduction in TRAIL-mediated apoptosis is not clearly understood compared with other TNF family members. Specifically, it is not yet understood how TRAIL controls nuclear factor κB (NF-κB) activation and overcomes its antiapoptotic effect. We explored the regulation of NF-κB activity by TRAIL and its role in apoptosis. TRAIL combined with IκBα-“superrepressor” induced potent apoptosis of SK-Hep1 hepatoma cells at low concentrations of TRAIL that do not independently induce apoptosis. Apoptosis by high concentrations of TRAIL was not affected by IκBα-superrepressor. Although TRAIL alone did not induce NF-κB activity, TRAIL combined with z-VAD significantly increased NF-κB activation. Analysis of the NF-κB activation pathway indicated that TRAIL unexpectedly induced cleavage of p65 at Asp97, which was blocked by z-VAD, accounting for all of these findings. p65 expression abrogated apoptosis and increased NF-κB activity in TRAIL-treated cells. Cleavage-resistant p65D97A further increased NF-κB activity in TRAIL-treated cells, whereas the COOH-terminal p65 fragment acted as a dominant-negative inhibitor. XIAP levels were increased by TRAIL in combination with z-VAD, whereas XIAP levels were decreased by TRAIL alone. Cleavage of p65 was also detected after FRO thyroid cancer cells were treated with TRAIL. These results suggest that TRAIL induces NF-κB activation, but simultaneously abrogates NF-κB activation by cleaving p65, and thereby inhibits the induction of antiapoptotic proteins such as XIAP, which contributes to the strong apoptotic activity of TRAIL compared with other TNF family members.
Angiopoietin 1 Is Mitogenic for Cultured Endothelial Cells
Authors: Kanda Shigeru , Miyata Yasuyoshi , Mochizuki Yasushi , Matsuyama Toshifumi , Kanetake Hiroshi
Published: September 2005

The angiopoietin (Ang)/Tie2 system is implicated in blood vessel formation and maturation. However, the mitogenic effects of angiopoietins remain to be elucidated. Here, we show that Ang1 is mitogenic for cultured endothelial cells. Ang1 dose-dependently induced the proliferation and increased the labeling index of a murine brain capillary endothelial cell line, IBE cells. Ang1 also increased the labeling index of human umbilical vein endothelial cells (HUVEC). Ang1 up-regulated the expression of cyclin D1 in both of these cells. Ang1 activated mitogen-activated protein kinase (MAPK) and phosphoinositide 3-kinase (PI3K) in IBE cells and HUVECs. Activated PI3K was associated with c-Fes protein tyrosine kinase in these cells, but not with Tie2. p70 S6 kinase (p70 S6K) was activated by Ang1-treatment, although this activation was blocked by a PI3K inhibitor, LY294002. Simultaneous treatment of cells with PD98059 (MAPK/extracellular regulated kinase kinase inhibitor) and rapamycin (mTOR inhibitor) completely blocked Ang1-induced mitogenic activity for IBE cells and HUVECs. Although Ang2 at high concentration weakly activated Tie2 and p70 S6K, it failed to activate Ras and MAPK, or to induce cell proliferation. Taken together, these findings indicate that Ang1 exerts mitogenic activity on endothelial cells, which requires activation of both MAPK and p70 S6K.
The Myc Target Gene JPO1/CDCA7 Is Frequently Overexpressed in Human Tumors and Has Limited Transforming Activity In vivo
Authors: Osthus Rebecca C. , Karim Baktiar , Prescott Julia E. , Smith B. Douglas , McDevitt Michael , Huso David L. , Dang Chi V.
Published: September 2005

MYC is frequently overexpressed in human cancers, but the downstream events contributing to tumorigenesis remain incompletely understood. MYC encodes an oncogenic transcription factor, of which target genes presumably contribute to cellular transformation. Although Myc regulates about 15% of genes and combinations of target genes are likely required for tumorigenesis, we studied in depth the expression of the Myc target gene, JPO1/CDCA7, in human cancers and its ability to provoke tumorigenesis in transgenic mice. JPO1/CDCA7 is frequently overexpressed in human cancers, and in particular, its expression is highly elevated in chronic myelogenous leukemia blast crisis as compared with the chronic phase. In murine lymphoid tissues, ectopic human JPO1/CDCA7 expression resulted in a 2-fold increased risk of lymphoid malignancies at 1 year. The transgene, which was driven by the H2-K promoter, exhibited leaky expression in nonlymphoid tissues such as kidney. We observed a significant increased incidence of transgenic animal solid tumors, which were not seen in littermate controls. These observations suggest that JPO1/CDCA7 may contribute to Myc-mediated tumorigenesis.
Human T-Cell Leukemia Virus Type I at Age 25: A Progress Report
Authors: Matsuoka Masao , Jeang Kuan-Teh
Published: September 2005

It has been 25 years since the discovery of human T-cell leukemia virus type I (HTLV-I) and its role in adult T-cell leukemia. Here, in brief, we review the current state of our understanding of HTLV-I epidemiology, viral biology, pathogenesis, and treatment. We discuss how HTLV-I may transform cells through destabilization of cellular genomic integrity and induction of cellular tolerance for chromosomal errors.
A Mouse Model for the Carney Complex Tumor Syndrome Develops Neoplasia in Cyclic AMP–Responsive Tissues
Authors: Kirschner Lawrence S. , Kusewitt Donna F. , Matyakhina Ludmila , Towns William H. , Carney J. Aidan , Westphal Heiner , Stratakis Constantine A.
Published: September 2005

Carney complex is an autosomal dominant neoplasia syndrome characterized by spotty skin pigmentation, myxomatosis, endocrine tumors, and schwannomas. This condition may be caused by inactivating mutations in PRKAR1A, the gene encoding the type 1A regulatory subunit of protein kinase A. To better understand the mechanism by which PRKAR1A mutations cause disease, we have developed conventional and conditional null alleles for Prkar1a in the mouse. Prkar1a+/− mice developed nonpigmented schwannomas and fibro-osseous bone lesions beginning at ∼6 months of age. Although genotype-specific cardiac and adrenal lesions were not seen, benign and malignant thyroid neoplasias were observed in older mice. This spectrum of tumors overlaps that seen in Carney complex patients, confirming the validity of this mouse model. Genetic analysis indicated that allelic loss occurred in a subset of tumor cells, suggesting that complete loss of Prkar1a plays a key role in tumorigenesis. Similarly, tissue-specific ablation of Prkar1a from a subset of facial neural crest cells caused the formation of schwannomas with divergent differentiation. These observations confirm the identity of PRKAR1A as a tumor suppressor gene with specific importance to cyclic AMP–responsive tissues and suggest that these mice may be valuable tools not only for understanding endocrine tumorigenesis but also for understanding inherited predispositions for schwannoma formation.
A Novel Human tRNA-Dihydrouridine Synthase Involved in Pulmonary Carcinogenesis
Authors: Kato Tatsuya , Daigo Yataro , Hayama Satoshi , Ishikawa Nobuhisa , Yamabuki Takumi , Ito Tomoo , Miyamoto Masaki , Kondo Satoshi , Nakamura Yusuke
Published: September 2005

An increased level of dihydrouridine in tRNAPhe was found in human malignant tissues nearly three decades ago, but its biological significance in carcinogenesis has remained unclear. Through analysis of genome-wide gene-expression profiles among non–small cell lung carcinomas (NSCLC), we identified overexpression of a novel human gene, termed hDUS2, encoding a protein that shared structural features with tRNA-dihydrouridine synthases (DUS). The deduced 493-amino-acid sequence showed 39% homology to the dihydrouridine synthase 2 enzyme (Dus2) of Saccharomycescerevisiae and contained a conserved double-strand RNA-binding motif (DSRM). We found that hDUS2 protein had tRNA-DUS activity and that it physically interacted with EPRS, a glutamyl-prolyl tRNA synthetase, and was likely to enhance translational efficiencies. A small interfering RNA against hDUS2 transfected into NSCLC cells suppressed expression of the gene, reduced the amount of dihydrouridine in tRNA molecules, and suppressed growth. Immunohistochemical analysis showed significant association between higher levels of hDUS2 in tumors and poorer prognosis of lung cancer patients. Our data imply that up-regulation of hDUS2 is a relatively common feature of pulmonary carcinogenesis and that selective suppression of hDUS2 enzyme activity and/or inhibition of formation of the hDUS2-tRNA synthetase complex could be a promising therapeutic strategy for treatment of many lung cancers.
Proteasome Inhibitor MG132 Induces Death Receptor 5 through CCAAT/Enhancer-Binding Protein Homologous Protein
Authors: Yoshida Tatsushi , Shiraishi Takumi , Nakata Susumu , Horinaka Mano , Wakada Miki , Mizutani Yoichi , Miki Tsuneharu , Sakai Toshiyuki
Published: September 2005

Combined treatment with a proteasome inhibitor and tumor necrosis factor–related apoptosis-inducing ligand (TRAIL) is a promising strategy for cancer therapy. Proteasome inhibitors induce the expression of death receptor 5 (DR5), a receptor for TRAIL, and sensitize cancer cells to TRAIL-induced apoptosis; however, the molecular mechanism of DR5 up-regulation has not been elucidated. In this study, we report that CCAAT/enhancer-binding protein homologous protein (CHOP) is a regulator of DR5 induction by proteasome inhibitor MG132. MG132 induced DR5 expression at a protein and mRNA level in prostate cancer DU145 cells. Furthermore, MG132 increased DR5 promoter activity. Using a series of deletion mutant plasmids containing DR5 promoters of various sizes, we found that MG132 stimulated the promoter activity via the region of −289 to −253. This region contained a CHOP-binding site. Site-directed mutation of the site abrogated the promoter activity enhanced by MG132. An electrophoretic mobility shift assay showed that CHOP directly bound to the MG132-responsive site on the DR5 promoter. Expression of the CHOP protein was increased with MG132 along with DR5 up-regulation. Furthermore, CHOP small interfering RNA attenuated the DR5 up-regulation due to MG132. These results indicate that the proteasome inhibitor MG132 induces DR5 expression through CHOP up-regulation.
8-Oxoguanine Formation Induced by Chronic UVB Exposure Makes Ogg1 Knockout Mice Susceptible to Skin Carcinogenesis
Authors: Kunisada Makoto , Sakumi Kunihiko , Tominaga Yohei , Budiyanto Arief , Ueda Masato , Ichihashi Masamitsu , Nakabeppu Yusaku , Nishigori Chikako
Published: September 2005

8-Oxoguanine is one of the oxidative DNA damages that can result in stable mutations. The Ogg1 gene encodes the repair enzyme 8-oxoguanine-DNA glycosylase, which removes the oxidized base from DNA. In this study, we investigated the role of 8-oxoguanine in skin carcinogenesis induced by UVB irradiation using Ogg1 knockout mice (C57Bl/6J background). We examined the effect of UVB irradiation on the formation of 8-oxoguanine in epidermal cells using immunostaining and found that the level of 8-oxoguanine in Ogg1 knockout mice 24 hours after UVB irradiation remained high compared with that in wild-type and heterozygous mice. To verify the effect of chronic UVB irradiation on 8-oxoguanine formations in epidermal cells, we irradiated wild-type, heterozygous, and Ogg1 knockout mice with UVB at a dose of 2.5 kJ/m2 thrice a week for 40 weeks. We found that the mean number of tumors in Ogg1 knockout mice was 3.71, which was significantly more than in wild-type and heterozygous mice, being 1.71 and 2.28, respectively. The rate of developing malignant tumors in Ogg1 knockout mice was also significantly higher (88.5%; squamous cell carcinomas, 73.1%; sarcomas, 15.4%) than in wild-type mice (50.0%; squamous cell carcinomas, 41.7%; sarcomas, 8.3%). Moreover, the age of onset of developing skin tumors in Ogg1 knockout mice was earlier than in the other types of mice. These results clearly indicate that oxidative DNA damage induced by sunlight plays an important role in the development of skin cancers.
Nestin-Linked Green Fluorescent Protein Transgenic Nude Mouse for Imaging Human Tumor Angiogenesis
Authors: Amoh Yasuyuki , Yang Meng , Li Lingna , Reynoso Jose , Bouvet Michael , Moossa Abdool R. , Katsuoka Kensei , Hoffman Robert M.
Published: September 2005

We report here a novel transgenic nude mouse for the visualization of human tumor angiogenesis. We have recently shown that the neural stem cell marker nestin is expressed in hair follicle stem cells and blood vessel networks in the skin of C57/B6 transgenic mice with nestin regulatory element–driven green fluorescent protein (ND-GFP). Others have shown ND-GFP is expressed in the brain, pancreas, and testes in these mice. In the present study, the nestin ND-GFP gene was crossed into nude mice on the C57/B6 background to obtain ND-GFP nude mice. ND-GFP was expressed in the brain, spinal cord, pancreas, stomach, esophagus, heart, lung, blood vessels of glomeruli, blood vessels of skeletal muscle, testes, hair follicles, and blood vessel network in the skin of ND-GFP nude mice. Human lung cancer, pancreatic cancer, and colon cancer cell lines as well as a murine melanoma cell line and breast cancer tumor cell line expressing red fluorescent protein were implanted orthotopically, and a red fluorescent protein–expressing human fibrosarcoma was implanted s.c. in the ND-GFP nude mice. These tumors grew extensively in the ND-GFP mice. ND-GFP was highly expressed in proliferating endothelial cells and nascent blood vessels in the growing tumors, visualized by dual-color fluorescence imaging. Results of immunohistochemical staining showed that CD31 was expressed in the ND-GFP–expressing nascent blood vessels. The ND-GFP transgenic nude mouse model enables the visualization of nascent angiogenesis in human and mouse tumor progression. These results suggest that this model is useful for the imaging of the angiogenesis of human as well as rodent tumors and visualization of the efficacy of angiogenetic inhibitors.
Human LATS1 Is a Mitotic Exit Network Kinase
Authors: Bothos John , Tuttle Robyn L. , Ottey Michelle , Luca Francis C. , Halazonetis Thanos D.
Published: September 2005

The kinase LATS/WARTS is a tumor suppressor protein conserved in evolution, but its function at the molecular level is not well understood. We report here that human LATS1 interacts with MOB1A, a protein whose homologue in budding yeast associates with kinases involved in mitotic exit. This suggested that LATS1 may be a component of the previously uncharacterized mitotic exit network in higher eukaryotes. Indeed, moderate overexpression of human LATS1 in cells exposed to microtubule poisons facilitated mitotic exit, and this activity required MOB1A. Reciprocally, small interfering RNA–mediated suppression of LATS1 or MOB1A prolonged telophase, but had no effect on the length of the earlier phases of mitosis. A role of LATS1 in mitotic exit may explain its previously described abilities to induce G2 arrest and promote cytokinesis.
Intrapulmonary IFN-β Gene Therapy Using an Adenoviral Vector Is Highly Effective in a Murine Orthotopic Model of Bronchogenic Adenocarcinoma of the Lung
Authors: Wilderman Michael J. , Sun Jing , Jassar Arminder S. , Kapoor Veena , Khan Mohamed , Vachani Anil , Suzuki Eiji , Kinniry Paul A. , Sterman Daniel H. , Kaiser Larry R. , Albelda Steven M.
Published: September 2005

Given previous work showing that an adenoviral vector expressing IFN-β (Ad.IFNβ) was highly effective in eradicating i.p. mesothelioma tumors, the antitumor efficacy of this agent was evaluated in an orthotopic model of bronchogenic adenocarcinoma of the lung. These transgenic mice have a conditionally expressed, oncogenic K-rasG12D allele that can be activated by intratracheal administration of an adenovirus expressing Cre recombinase (Ad.Cre). K-rasG12D mutant mice were given Ad.Cre intranasally to activate the oncogene. Mice were then given 109 plaque-forming units of a control vector (Ad.LacZ) or Ad.IFNβ intranasally 3 and 4 weeks later, a time when lung tumors had been established. Cells derived from K-ras-mutated lung tumors were also grown in the flanks of mice to study mechanisms of therapeutic responses. In two separate experiments, untreated tumor-bearing mice all died by day 57 (median survival, 49 days). Ad.LacZ-treated mice all died by day 71 (median survival, 65 days). In contrast, 90% to 100% of mice treated with Ad.IFNβ were long-term survivors (>120 days; P < 0.001). In addition, immunity to re-challenge with tumor cells was induced. In vitro and flank tumor studies showed that Ad.IFNβ induced direct tumor cell killing and that depleting natural killer or CD8+ T cells, but not CD4+ T cells, with antibodies attenuated the effect of Ad.IFNβ. These studies, showing remarkable antitumor activity in this orthotopic lung cancer model, provide strong preclinical support for a trial of Ad.IFNβ to treat human non–small cell lung cancer.
Kinase-Inactive Glycogen Synthase Kinase 3β Promotes Wnt Signaling and Mammary Tumorigenesis
Authors: Farago Marganit , Dominguez Isabel , Landesman-Bollag Esther , Xu Xin , Rosner Andrea , Cardiff Robert D. , Seldin David C.
Published: September 2005

Recent studies have implicated ectopic activation of the Wnt pathway in many human cancers, including breast cancer. β-catenin is a critical coactivator in this signaling pathway and is regulated in a complex fashion by phosphorylation, degradation, and nuclear translocation. Glycogen synthase kinase 3β (GSK3β) phosphorylation of the NH2-terminal domain of β-catenin targets it for ubiquitination and proteosomal degradation. We hypothesized that expression of kinase-inactive GSK3β (KI-GSK3β) in mammary glands would function in a dominant-negative fashion by antagonizing the endogenous activity of GSK3β and promoting breast cancer development. Consistent with this, we find that KI-GSK3β stabilizes β-catenin expression, catalyzes its localization to the nucleus, and up-regulates the downstream target gene, cyclin D1, in vitro. In vivo, transgenic mice overexpressing the KI-GSK3β under the control of the mouse mammary tumor virus-long terminal repeat develop mammary tumors with overexpression of β-catenin and cyclin D1. Thus, antagonism of GSK3β activity is oncogenic in the mammary epithelium; mutation or pharmacologic down-regulation of GSK3β could promote mammary tumors.
Therapeutic Index by Combination of Adriamycin and Docetaxel Depends on Dosing Time in Mice
Authors: Tabuchi Mayumi , To Hideto , Sakaguchi Hiromi , Goto Naho , Takeuchi Ayako , Higuchi Shun , Ohdo Shigehiro
Published: September 2005

Although the combination of adriamycin and docetaxel showed a better cure rate against metastatic breast cancer, severe myelosuppression and cardiotoxicity were dose-limiting factors. The purpose of this study was to establish a suitable dosing schedule, based on a chronopharmacologic approach, to relieve severe adverse effects. In experiment 1, adriamycin or docetaxel was injected i.p. at 2, 6, 10, 14, 18, or 22 hours after light onset (HALO) to estimate toxicities. In experiment 2, the dosing time dependency of toxicity and pharmacokinetics were assessed in the combination of adriamycin and docetaxel. In addition, G2-M phase in myelocyte cells was determined in nontreated mice. Adverse effects caused by adriamycin were shown to be the worst at 2 HALO and the best at 14 HALO. On the other hand, docetaxel-induced adverse effects were more severe at 14 HALO than at 2 HALO. In the combination study, the D(2)-A(14) group, in which docetaxel was administered at 2 HALO followed by adriamycin at 14 HALO, showed the most toxicity relief of all the treated groups. In the pharmacokinetic study, the dosing time dependency of toxicities was not related to the daily variation of pharmacokinetics of adriamycin and docetaxel. A significant 24-hour rhythm of G2-M phase distribution was found in myelocyte cells of nontreated mice. The daily variation of leukopenia caused by docetaxel corresponded to the 24-hour rhythm of G2-M phase distribution. These findings reveal that the therapeutic index of the combined chemotherapy can be improved by administering adriamycin and docetaxel at the time when the most adverse effects are relieved in each drug.
Tumor Dormancy and MYC Inactivation: Pushing Cancer to the Brink of Normalcy
Authors: Shachaf Catherine M. , Felsher Dean W.
Published: September 2005

Upon MYC inactivation, tumors variously undergo proliferative arrest, cellular differentiation, and apoptosis and in some cases, apparently permanently revoking tumorigenesis. In liver tumor cells, we recently showed that MYC inactivation uncovers stem cell properties and triggers differentiation, but in this case, their neoplastic properties are restorable by MYC reactivation. Thus, whereas oncogene inactivation can push cancer to the brink of normalcy, some cells retain the latent capacity to turn cancerous again, arguing that they may exist in a state of tumor dormancy.
Identification of a Novel Prostate Tumor Target, Mindin/RG-1, for Antibody-Based Radiotherapy of Prostate Cancer
Authors: Parry Renate , Schneider Doug , Hudson Debra , Parkes Debbie , Xuan Jian-Ai , Newton Alicia , Toy Pam , Lin Rick , Harkins Rick , Alicke Bruno , Biroc Sandra , Kretschmer Peter J. , Halks-Miller Meredith , Klocker Helmut , Zhu Ying , Larsen Brent , Cobb Ronald R. , Bringmann Peter , Roth Georg , Lewis Jason S. , Dinter Harald , Parry Gordon
Published: September 2005

Gene expression analysis showed that a human mindin homologue, mindin/RG-1, is expressed selectively in prostate tissues and that its expression level is elevated in some prostate tumors. Mindin/RG-1 protein expression is maintained in >80% of prostate cancers metastatic to bone or lymph nodes as well as in locally recurrent tumors in androgen-unresponsive patients. In contrast, mindin/RG-1 expression in other normal tissues is significantly lower than that seen in the prostate. A fully human antibody, 19G9, was generated against mindin/RG-1 protein and was shown to accumulate at high abundance in LNCaP tumor xenografts. Conjugates of this antibody with the chelator CHX-A″-DTPA were generated and radiolabeled with either 111In, 90Y, or 86Y. Small animal positron emission tomography imaging with the 86Y-radiolabeled conjugate showed very specific accumulation of the antibody in LNCaP tumor xenografts with clear tumor delineation apparent at 4 hours. The therapeutic efficacy of [90Y]-CHX-A″-DTPA-19G9 was evaluated in mice bearing LNCaP xenografts. A dose-finding study identified a nontoxic therapeutic dose to be ∼75 μCi. Significant antitumor effects were seen with a single administration of radiolabeled antibody to animals bearing 200 to 400 mm3 tumors. Inhibition of tumor growth was observed in all treated animals over a 49-day period. At 49 days posttreatment, slow tumor growth recurred but this could be prevented for an additional 40-day period by a second administration of a 75 μCi dose at day 49. We conclude that [90Y]-CHX-A″-DTPA-19G9 is a novel antibody conjugate that has considerable promise for therapy of metastatic prostate cancer in androgen-unresponsive patients.
Immortal ALT+ Human Cells Do Not Require Telomerase Reverse Transcriptase for Malignant Transformation
Authors: Sun Beicheng , Chen Meizhen , Hawks Christina L. , Hornsby Peter J.
Published: September 2005

Many human cancer cells lack telomerase activity but nevertheless maintain telomeres via a process termed “alternative lengthening of telomeres” (ALT). Despite being immortal and having a telomere maintenance mechanism, ALT+ human fibroblasts require telomerase reverse transcriptase (hTERT) for tumor formation in immunodeficient mice when tested by s.c. injection. Here we show that three ALT+ human SV40-immortalized fibroblast cell lines require only oncogenic RasV12G to be converted to a fully tumorigenic state. When cells were implanted beneath the kidney capsule of immunodeficient mice, they invaded the kidney and neighboring organs and metastasized to the lungs. RasV12G-expressing ALT+ cells remained completely telomerase negative. Introduction of hTERT conferred strong telomerase activity but did not appreciably change the malignant properties of the cells. However, when cells were tested by s.c. injection, RasV12G-transduced ALT+ cells did not form tumors, and in this site, hTERT was required for tumorigenicity. These data show that when the s.c. injection method is used as an assay for tumorigenicity, hTERT may be artifactually scored as an oncogene; the subrenal capsule assay shows that ALT, as a telomere maintenance mechanism, is equivalent to hTERT in neoplastic transformation of human cells by oncogenes.
Control of Genomic Instability and Epithelial Tumor Development by the p53-Fbxw7/Cdc4 Pathway
Authors: Perez-Losada Jesus , Mao Jian-Hua , Balmain Allan
Published: September 2005

Mouse models of cancer have provided novel insights into the timing of p53 loss during tumorigenesis. We have recently identified Fbxw7/Cdc4 as a downstream target of p53 loss that controls genomic instability and tumor development in epithelial tumors. Although p53-deficient mice primarily develop lymphomas and sarcomas, the additional loss of one copy of the Fbxw7 gene drives tumor development in a range of epithelial tissues. These data highlight the importance of genetic instability at the chromosome level in the development of common cancer types, and further illustrate the value of mouse models in identifying causal genetic events in epithelial tumor formation.
Bone Morphogenetic Protein-6 Promotes Osteoblastic Prostate Cancer Bone Metastases through a Dual Mechanism
Authors: Dai Jinlu , Keller Jill , Zhang Jian , Lu Yi , Yao Zhi , Keller Evan T.
Published: September 2005

Prostate cancer frequently metastasizes to bone where it forms osteoblastic lesions through unknown mechanisms. Bone morphogenetic proteins (BMP) are mediators of skeletal formation. Prostate cancer produces a variety of BMPs, including BMP-6. We tested the hypothesis that BMP-6 contributes to prostate cancer–induced osteosclerosis at bone metastatic sites. Prostate cancer cells and clinical tissues produced BMP-6 that increased with aggressiveness of the tumor. Prostate cancer-conditioned medium induced SMAD phosphorylation in the preosteoblast MC3T3 cells, and phosphorylation was diminished by anti–BMP-6 antibody. Prostate cancer-conditioned medium induced mineralization of MC3T3 cells, which was blocked by both the BMP inhibitor noggin and anti–BMP-6. Human fetal bones were implanted in severe combined immunodeficient mice and after 4 weeks, LuCaP 23.1 prostate cancer cells were injected both s.c. and into the bone implants. Anti–BMP-6 or isotype antibody administration was then initiated. Anti–BMP-6 reduced LuCaP 23.1–induced osteoblastic activity, but had no effect on its osteolytic activity. This was associated with increased osteoblast numbers and osteoblast activity based on bone histomorphometric evaluation. As endothelin-1 has been implicated in bone metastases, we measured serum endothelin-1 levels but found they were not different among the treatment groups. In addition to decreased bone production, anti–BMP-6 reduced intraosseous, but not s.c., tumor size. We found that BMP-2, BMP-4, BMP-6, and BMP-7 had no direct effect on prostate cancer cell growth, but BMP-2 and BMP-6 increased the in vitro invasive ability of prostate cancer cell. These data show that prostate cancer promotes osteoblastic activity through BMP-6 and that, in addition to its bone effects, suggest that BMPs promote the ability of the prostate cancer cells to invade the bone microenvironment.
Marrying Immunotherapy with Chemotherapy: Why Say IDO?
Authors: Muller Alexander J. , Prendergast George C.
Published: September 2005

Activation of the tryptophan catabolizing enzyme indoleamine 2,3-dioxygenase (IDO) in cancer cells facilitates immune escape. A recent study now shows how small-molecule inhibitors of IDO can be used to leverage the efficacy of traditional chemotherapeutic drugs that are used to treat cancer in the clinic. By promoting antitumor immune responses in combination with cytotoxic chemotherapy, IDO inhibitors may offer a drug-based strategy to more effectively attack systemic cancer.
Transfection of Keratin 18 Gene in Human Breast Cancer Cells Causes Induction of Adhesion Proteins and Dramatic Regression of Malignancy In vitro and In vivo
Authors: Bühler Helmut , Schaller Gerhard
Published: September 2005

This study shows that high keratin 18 (K18) expression in tumor cells is associated with reduced invasiveness in vitro and lack of tumorigenicity in nude mice. We previously showed that high K18 expression correlated with a good prognosis and that reducing K18 expression increased the aggressiveness of established breast cancer cell lines. To confirm these observations, we transfected the human K18 gene into the human breast cancer cell line MDA-MB-231 and isolated a stable overexpressing clone. The forced K18 expression was associated with a complete loss of the previously strong vimentin expression in the parent cell line, induction of the K18 dimerization partner K8, and up-regulation of adhesion proteins. These changes were accompanied by a dramatic reduction in the aggressiveness of the K18 transfectants in vitro and in vivo. We conclude that forced reexpression of K18 causes at least partial redifferentiation of the tumor cell, followed by a corresponding regression of malignant phenotype.
Pim-1 Kinase Stability Is Regulated by Heat Shock Proteins and the Ubiquitin-Proteasome Pathway
Authors: Petersen Shay Kate , Wang Zeping , Xing Pei-xiang , McKenzie Ian F.C. , Magnuson Nancy S.
Published: September 2005

Elevated expression of the serine/threonine kinase Pim-1 increases the incidence of lymphomas in Pim-1 transgenic mice and has also been found to occur in some human cancers. Pim-1 acts as a cell survival factor and may prevent apoptosis in malignant cells. It was therefore of interest to understand to what extent maintenance and degradation of Pim-1 protein is affected by heat shock proteins (Hsp) and the ubiquitin-proteasome pathway in K562 and BV173 human leukemic cells. The half-life of Pim-1 protein in these cells was found to increase from 1.7 to 3.1 hours when induced by heat shock or by treating the cells with the proteasome inhibitor PS-341 (bortezomib). The Hsp90 inhibitor geldanamycin prevented the stabilization of Pim-1 by heat shock. Using immunoprecipitation, it was determined that Pim-1 is targeted for degradation by ubiquitin and that Hsp70 is associated with Pim-1 under these circumstances. Conversely, Hsp90 was found to protect Pim-1 from proteasomal degradation. A luminescence-based kinase assay showed that Pim-1 kinase bound to Hsp70 or Hsp90 remains active, emphasizing the importance of its overall cellular levels. This study shows how Pim-1 levels can be modulated in cells through degradation and stabilization.
Molecular Consequences of Silencing Mutant K-ras in Pancreatic Cancer Cells: Justification for K-ras–Directed Therapy
Authors: Fleming Jason B. , Shen Guo-Liang , Holloway Shane E. , Davis Mishel , Brekken Rolf A.
Published: September 2005

Mutation of the K-ras gene is an early event in the development of pancreatic adenocarcinoma and, therefore, RNA interference (RNAi) directed toward mutant K-ras could represent a novel therapy. In this study, we examine the phenotypic and molecular consequences of exposure of pancreatic tumor cells to mutant-specific K-ras small interfering RNA. Specific reduction of activated K-ras via RNAi in Panc-1 and MiaPaca-2 cells resulted in cellular changes consistent with a reduced capacity to form malignant tumors. These changes occur through distinct mechanisms but likely reflect an addiction of each cell line to oncogene stimulation. Both cell lines show reduced proliferation after K-ras RNAi, but only MiaPaca-2 cells showed increased apoptosis. Both cell lines showed reduced migration after K-ras knockdown, but changes in integrin levels were not consistent between the cell lines. Both cell lines showed alteration of the level of GLUT-1, a metabolism-associated gene that is downstream of c-myc, with Panc-1 cells demonstrating decreased GLUT-1 levels, whereas MiaPaca-2 cells showed increased levels of expression after K-ras knockdown. Furthermore, after K-ras RNAi, there was a reduction in angiogenic potential of both Panc-1 and MiaPaca-2 cells. Panc-1 cells increased the level of expression of thrombospondin-1, an endogenous inhibitor of angiogenesis, whereas MiaPaca-2 cells decreased the production of vascular endothelial growth factor, a primary stimulant of angiogenesis in pancreatic tumors. We have found that silencing mutant K-ras through RNAi results in alteration of tumor cell behavior in vitro and suggests that targeting mutant K-ras specifically might be effective against pancreatic cancer in vivo.
Vitamin K Epoxide Reductase: A Protein Involved in Angiogenesis
Authors: Wang Yibo , Zhen Yisong , Shi Yi , Chen Jingzhou , Zhang Channa , Wang Xiaojian , Yang Xu , Zheng Yi , Liu Yuqing , Hui Rutai
Published: September 2005

Vitamin K epoxide reductase (VKOR) is a newly identified protein which has been reported to convert the epoxide of vitamin K back to vitamin K, a cofactor essential for the posttranslational γ-carboxylation of several blood coagulation factors. We found that the gene is expressed ubiquitously including vascular endothelial cells, smooth muscle cells, fibroblasts and cardiomyocytes, and is overexpressed in 11 tumor tissues on microarray. Stable transfection of VKOR cDNA into tumor cell line A549 and H7402 did not promote the cell proliferation. These results promoted us to hypothesize that VKOR may also be involved in angiogenesis. To test this hypothesis, the expression of VKOR was studied in different vascular cells in developmental and pathologic heart tissues. The effects of overexpression and suppressing expression of VKOR on endothelial cell proliferation, migration, adhesion, and tubular network formation were explored. We found that VKOR expression in arteries was prominent in vascular endothelial cells and was high in the ventricular aneurysm tissue of human heart and human fetal heart. In vitro studies showed that overexpression of VKOR slightly but significantly stimulated human umbilical vein endothelial cell proliferation (by 120%), migration (by 118%), adhesion (by 117%), as well as tubular network formation. Antisense to VKOR gene inhibited the proliferation (by 67%), migration (by 64%), adhesion (by 50%), and tubular network formation. Our findings support the impact of VKOR in the process of angiogenesis; hence, the molecule may have a potential application in cardiovascular disease and cancer therapy.
Pim Family Kinases Enhance Tumor Growth of Prostate Cancer Cells
Authors: Chen Wei Wei , Chan Daniel C. , Donald Carlton , Lilly Michael B. , Kraft Andrew S.
Published: September 2005

Recent analyses indicate that the expression of the Pim-1 protein kinase is elevated in biopsies of prostate tumors. To identify the mechanism by which the Pim kinases may affect the growth of prostate tumors, we expressed Pim-1, Pim-2, or a kinase-dead Pim-2 protein in human PC3 prostate cancer cells. On implantation of the transfectants in nude mice, the growth of the cells expressing Pim-1 or Pim-2 was significantly faster than the growth of the control cells transfected with the neomycin-resistant gene or the kinase-dead Pim-2 protein. When grown in medium, the doubling time of the Pim-1 and Pim-2 transfectants was faster (0.75 days) than that of the control cells (1.28 days). We, therefore, examined the ability of Pim to control the phosphorylation of proteins that regulate protein synthesis. On growth factor starvation or rapamycin treatment, the Pim-1 and Pim-2 transfectants maintained their ability to phosphorylate 4E-BP1 and S6 kinase, although this phosphorylation did not occur in the control-transfected PC3 cells. We have found that the cellular levels of c-Myc were elevated in the Pim-1 and Pim-2 transfectants under these conditions. The Pim-1 and Pim-2 transfectants have lower levels of serine/threonine protein phosphatase 2A (PP2A) activity and the α- and β-subunit B56γ of the PP2A phosphatase do not coimmunoprecipitate in these cells. Thus, the effects of Pim on PP2A activity may mediate the levels of c-Myc and the phosphorylation of proteins needed for increased protein synthesis. Both of these changes could have a significant impact on tumor growth.
Crk-Associated Substrate Tyrosine Phosphorylation Sites Are Critical for Invasion and Metastasis of Src-Transformed Cells
Authors: Brábek Jan , Constancio Sábata S. , Siesser Priscila Fonseca , Shin Nah-Young , Pozzi Ambra , Hanks Steven K.
Published: September 2005

Crk-associated substrate (CAS, p130Cas) is a major tyrosine phosphorylated protein in cells transformed by v-crk and v-src oncogenes. We recently reported that reexpression of CAS in CAS-deficient mouse embryo fibroblasts transformed by oncogenic Src promoted an invasive phenotype associated with enhanced cell migration through Matrigel, organization of actin into large podosome ring and belt structures, activation of matrix metalloproteinase-2, and elevated tyrosine phosphorylation of the focal adhesion proteins FAK and paxillin. We have now extended these studies to examine the mechanism by which CAS achieves these changes and to evaluate the potential role for CAS in promoting in vivo tumor growth and metastasis. Whereas the presence or absence of CAS did not alter the primary growth of subcutaneous-injected Src-transformed mouse embryo fibroblasts, CAS expression was required to promote lung metastasis following removal of the primary tumor. The substrate domain YxxP tyrosines, the major sites of CAS phosphorylation by Src that mediate interactions with Crk, were found to be critical for promoting both invasive and metastatic properties of the cells. The ability of CAS to promote Matrigel invasion, formation of large podosome structures, and tyrosine phosphorylation of Src substrates, including FAK, paxillin, and cortactin, was also strictly dependent on the YxxP tyrosines. In contrast, matrix metalloproteinase-2 activation was most dependent on the CAS SH3 domain, whereas the substrate domain YxxP sites also contributed to this property. Thus multiple CAS-mediated signaling events are implicated in promoting invasive and metastatic properties of Src-transformed cells.