bluuebunny/crossref_metadata_2025 huggingface dataset:
Hormone-Induced Chromosomal Instability in p53-Null Mammary Epithelium
Authors: Pati Debananda , Haddad Bassem R. , Haegele Albert , Thompson Henry , Kittrell Frances S. , Shepard Anne , Montagna Cristina , Zhang Nenggang , Ge Gouqing , Otta Subhendu Kumar , McCarthy Maureen , Ullrich Robert L. , Medina Daniel
Published: September 2005

The absence of p53 function increases risk for spontaneous tumorigenesis in the mammary gland. Hormonal stimulation enhances tumor risk in p53-null mammary epithelial cells as well as the incidence of aneuploidy. Aneuploidy appears in normal p53-null mammary epithelial cells within 5 weeks of hormone stimulation. Experiments reported herein assessed a possible mechanism of hormone-induced aneuploidy. Hormones increased DNA synthesis equally between wild-type (WT) and p53-null mammary epithelial cells. There were two distinct responses in p53-null cells to hormone exposure. First, Western blot analysis demonstrated that the levels of two proteins involved in regulating sister chromatid separation and the spindle checkpoint, Mad2 and separase (ESPL1) were increased in null compared with WT cells. In contrast, the levels of securin and Rad21 proteins were not increased in hormone-stimulated p53-null compared with WT cells. ESPL1 RNA was also increased in p53-null mouse mammary cells in vivo by 18 h of hormone stimulation and in human breast MCF7 cells in monolayer culture by 8 h of hormone stimulation. Furthermore, both promoters contained p53 and steroid hormone response elements. Mad2 protein was increased as a consequence of the absence of p53 function. The increase in Mad2 protein was observed also at the cellular level by immunohistochemistry. Second, hormones increased gene amplication in the distal arm of chromosome 2, as shown by comparative genomic hybridization. These results support the hypothesis that hormone stimulation acts to increase aneuploidy by several mechanisms. First, by increasing mitogenesis in the absence of the p53 checkpoint in G2, hormones allow the accumulation of cells that have experienced chromosome missegregation. Second, the absolute rate of chromosome missegregation may be increased by alterations in the levels of two proteins, separase and Mad2, which are important for maintaining chromosomal segregation and the normal spindle checkpoint during mitosis.
Cyr61 Is Overexpressed in Gliomas and Involved in Integrin-Linked Kinase-Mediated Akt and β-Catenin-TCF/Lef Signaling Pathways
Authors: Xie Dong , Yin Dong , Tong Xiangjun , O’Kelly James , Mori Akio , Miller Carl , Black Keith , Gui Dorina , Said Johathan W. , Koeffler H. Phillip
Published: September 2005

Cyr61 is a member of the CCN family of growth factors; these proteins are secreted and can act as ligands of distinct integrins. We show that Cyr61 can enhance tumorigenicity of glioma cells acting through activated integrin-linked kinase (ILK) to stimulate β-catenin-TCF/Lef and Akt signaling pathways. Overexpression of Cyr61 occurred in highly tumorigenic glioma cell lines and in 68% of the most malignant glioblastoma multiforme brain tumors. Forced expression of Cyr61 in U343 glioma cells accelerated their growth in liquid culture, enhanced their anchorage-independent proliferation in soft agar, and significantly increased their ability to form large, vascularized tumors in nude mice. Overexpression of Cyr61 in the U343 cells led to the up-regulation of distinct integrins, including β1 and ανβ3, which have been shown to interact with Cyr61 and ILK. The activity of ILK was increased dramatically in these cells. Overexpression of Cyr61 also resulted in the phosphorylation of glycogen synthase kinase-3β and accumulation and nuclear translocation of β-catenin, leading to activation of the β-catenin-TCF/Lef-1 signaling pathway. Furthermore, forced expression of Cyr61 in the glioma cells activated phosphatidylinositol 3′-kinase pathway, resulting in prominent phosphorylation of Akt and the antiapoptotic protein Bad. Cyr61 appears to stimulate several signaling pathways in the development of gliomas.
A Mutant High-Density Lipoprotein Receptor Inhibits Proliferation of Human Breast Cancer Cells
Authors: Cao Wen M. , Murao Koji , Imachi Hitomi , Yu Xiao , Abe Hiroshi , Yamauchi Akira , Niimi Michio , Miyauchi Akira , Wong Norman C. W. , Ishida Toshihiko
Published: September 2005

High-density lipoprotein (HDL) stimulates the growth of many types of cells, including those of breast cancer. High levels of HDL are associated with an increased risk of breast cancer development. A scavenger receptor of the B class (SR-BI)/human homolog of SR-BI, CD36, and LIMPII analogous-1 (CLA-1) facilitates the cellular uptake of cholesterol from HDL and thus augments cell growth. Furthermore, HDL is also believed to have antiapoptotic effects on various cell types, and this feature adds to its ability to promote cell growth. These collaborative roles of HDL and CLA-1 prompted us to assess the function of these components on human breast cancer cells. In this study, we created a mutant CLA-1 (mCLA) that lacked the COOH-terminal tail to determine its potential role in breast cancer cell growth. Expression of mCLA inhibited the proliferation of breast cancer cell line MCF-7. This inhibitory action of mCLA required the transcriptional factor activator protein-1 (AP-1), and the mutant receptor also affected the antiapoptotic features of HDL. The effect of HDL on AP-1 activation and [3H]thymidine incorporation was abrogated by wortmannin, a specific inhibitor of phosphoinositide 3-kinase. Furthermore, the dominant negative mutant of Akt abolished the ability of HDL to activate AP-1. These findings raise the possibility that the inhibitors of the effects of HDL may be of therapeutic value for breast cancer.
Nickel Compounds Act through Phosphatidylinositol-3-kinase/Akt-Dependent, p70S6k-Independent Pathway to Induce Hypoxia Inducible Factor Transactivation and Cap43 Expression in Mouse Epidermal Cl41 Cells
Authors: Li Jingxia , Davidson Gerard , Huang Yi , Jiang Bing-Hua , Shi Xianglin , Costa Max , Huang Chuanshu
Published: September 2005

Nickel compounds are a somewhat unique class of carcinogens. Previous studies have demonstrated that NiCl2 exposure leads to marked induction of hypoxia inducible factor 1 (HIF-1) in human osteosarcoma and BALB/c 3T3 cells, a transcription factor that has been considered to play an important role in tumor promotion and progression. However, the signal transduction pathways leading to HIF-1 induction are not well understood. The present study indicated that exposure of mouse epidermal Cl41 cells to either Ni3S2 or NiCl2 resulted in activation of phosphatidylinositol 3-kinase (PI-3K), Akt, and p70 S6 kinase (p70S6k). Inhibition of PI-3K, Akt, and p70S6k by overexpression of a dominant-negative mutant of PI-3K (Δp85) impaired nickel-induced HIF-1 transactivation. Furthermore, an overexpression of the dominant-negative Akt mutant (Akt-T308A/S473A) blocked nickel-induced Akt phosphorylation and HIF-1 transactivation, whereas inhibition of p70S6k activation by pretreatment of cells with rapamycin did not show significant inhibitory effects on HIF-1 transactivation induced by nickel compounds. Consistent with HIF-1 transactivation, inhibition of the PI-3K/Akt pathway by either overexpression of Δp85 or Akt-T308A/S473A caused dramatic inhibition of Cap43 protein expression induced by nickel compounds, whereas pretreatment of cells with rapamycin did not exhibit inhibition of Cap43 induction. These results demonstrated that nickel compounds induce HIF-1 transactivation and Cap43 protein expression through a PI-3K/Akt-dependent and p70S6k-independent pathway. This study should help us understand the signal transduction pathways involved in the carcinogenic effects of nickel compounds.
A Constitutively Active Dioxin/Aryl Hydrocarbon Receptor Promotes Hepatocarcinogenesis in Mice
Authors: Moennikes Oliver , Loeppen Sandra , Buchmann Albrecht , Andersson Patrik , Ittrich Carina , Poellinger Lorenz , Schwarz Michael
Published: September 2005

The dioxin/aryl hydrocarbon receptor (AhR) functions as a ligand-activated transcription factor regulating transcription of a battery of genes encoding enzymes involved in drug metabolism. Known ligands include polycyclic aromatic hydrocarbons, certain polychlorinated biphenyls, and the polyhalogenated dioxins including 2,3,7,8-tetrachlorodibenzo-p-dioxin. Both polyhalogenated biphenyls and 2,3,7,8-tetrachlorodibenzo-p-dioxin are potent promoters of rodent hepatocarcinogenesis in two-stage initiation-promotion experiments. Although several lines of evidence indicate the involvement of the AhR in toxic effects mediated by polyhalogenated biphenyls and dioxins, its involvement in tumor promotion has not been unequivocally proven. In the present study, a transgenic mouse line expressing a constitutively active AhR (CA-AhR) has been used to investigate the role of the AhR in hepatocarcinogenesis. Male AhR wild-type and CA-AhR-transgenic B6C3F1-mice were treated with a single injection of the hepatocarcinogen N-nitrosodiethylamine at 6 weeks of age and were subsequently kept untreated on control diet. Thirty five weeks after carcinogen treatment, mice were sacrificed, and the prevalence and multiplicity of liver tumors were determined. Whereas only 1 small liver tumor was observed in 15 AhR-wild-type mice, 19 tumors (two >1 cm in diameter) were present in 18 CA-AhR-transgenic mice. This result demonstrates the oncogenic potential of the activated AhR and implicates an important role of the receptor in promotion of hepatocarcinogenesis. A microarray-based gene expression-profiling analysis revealed down-regulation in the liver of CA-AhR-transgenic mice of a cluster of genes encoding heat shock proteins, including GRP78/BiP, Herp1, Hsp90, DnaJ (Hsp40) homologue B1, and Hsp105, which are important for protein folding and quality control.
Prostate Pathology of Genetically Engineered Mice: Definitions and Classification. The Consensus Report from the Bar Harbor Meeting of the Mouse Models of Human Cancer Consortium Prostate Pathology Committee
Authors: Shappell Scott B. , Thomas George V. , Roberts Richard L. , Herbert Ron , Ittmann Michael M. , Rubin Mark A. , Humphrey Peter A. , Sundberg John P. , Rozengurt Nora , Barrios Roberto , Ward Jerrold M. , Cardiff Robert D.
Published: September 2005

The Pathological Classification of Prostate Lesions in Genetically Engineered Mice (GEM) is the result of a directive from the National Cancer Institute Mouse Models of Human Cancer Consortium Prostate Steering Committee to provide a hierarchical taxonomy of disorders of the mouse prostate to facilitate classification of existing and newly created mouse models and the translation to human prostate pathology. The proposed Bar Harbor Classification system is the culmination of three meetings and workshops attended by various members of the Prostate Pathology Committee of the Mouse Models of Human Cancer Consortium. A 2-day Pathology Workshop was held at The Jackson Laboratory in Bar Harbor, Maine, in October 2001, in which study sets of 93 slides from 22 GEM models were provided to individual panel members. The comparison of mouse and human prostate anatomy and disease demonstrates significant differences and considerable similarities that bear on the interpretation of the origin and natural history of their diseases. The recommended classification of mouse prostate pathology is hierarchical, and includes developmental, inflammatory, benign proliferative, and neoplastic disorders. Among the neoplastic disorders, preinvasive, microinvasive, and poorly differentiated neoplasms received the most attention. Specific criteria were recommended and will be discussed. Transitions between neoplastic states were of particular concern. Preinvasive neoplasias of the mouse prostate were recognized as focal, atypical, and progressive lesions. These lesions were designated as mouse prostatic intraepithelial neoplasia (mPIN). Some atypical lesions were identified in mouse models without evidence of progression to malignancy. The panel recommended that mPIN lesions not be given histological grades, but that mPIN be further classified as to the absence or presence of documented associated progression to invasive carcinoma. Criteria for recognizing microinvasion, for classification of invasive gland-forming adenocarcinomas, and for characterizing poorly differentiated tumors, including neuroendocrine carcinomas, were developed and are discussed. The uniform application of defined terminology is essential for correlating results between different laboratories and models. It is recommended that investigators use the Bar Harbor Classification system when characterizing new GEM models or when conducting experimental interventions that may alter the phenotype or natural history of lesion progression in existing models.
p14ARF Expression Increases Dihydrofolate Reductase Degradation and Paradoxically Results in Resistance to Folate Antagonists in Cells with Nonfunctional p53
Authors: Magro Pellegrino G. , Russo Angelo J. , Li Wei-Wei , Banerjee Debabrata , Bertino Joseph R.
Published: September 2005

The p14ARF protein, the product of an alternate reading frame of the INK4A/ARF locus on human chromosome 9p21, disrupts the ability of MDM2 to target p53 for proteosomal degradation and causes an increase in steady-state p53 levels, leading to a G1 and G2 arrest of cells in the cell cycle. Although much is known about the function of p14ARF in the p53 pathway, not as much is known about its function in human tumor growth and chemosensitivity independently of up-regulation of p53 protein levels. To learn more about its effect on cellular proliferation and chemoresistance independent of p53 up-regulation, human HT-1080 fibrosarcoma cells null for p14ARF and harboring a defective p53 pathway were stably transfected with p14ARF cDNA under the tight control of a doxycycline-inducible promoter. Induction of p14ARF caused a decrease in cell proliferation rate and colony formation and a marked decrease in the level of dihydrofolate reductase (DHFR) protein. The effect of p14ARF on DHFR protein levels was specific, because thymidylate kinase and thymidylate synthase protein levels were not decreased nor were p53 or p21WAF1 protein levels increased. The decrease in DHFR protein was abolished when the cells were treated with the proteasome inhibitor MG132, demonstrating that p14ARF augments proteasomal degradation of the protein. Surprisingly, induction of p14ARF increased resistance to the folate antagonists methotrexate, trimetrexate, and raltitrexed. Depletion of thymidine in the medium reversed this resistance, indicating that p14ARF induction increases the reliance of these cells on thymidine salvage.
Endothelial P-Selectin as a Target of Heparin Action in Experimental Melanoma Lung Metastasis
Authors: Ludwig Ralf J. , Boehme Beatrice , Podda Maurizio , Henschler Reinhard , Jager Elke , Tandi Christa , Boehncke Wolf-Henning , Zollner Thomas M. , Kaufmann Roland , Gille Jens
Published: September 2005

Spontaneous and experimental metastasis can be effectively inhibited by the widely used anticoagulant heparin in different tumor models. At the cellular level, many of the antimetastatic effects of heparin in vivo are due to its action on P-selectin-mediated binding. Whereas previous attention has focused on P-selectin-dependent tumor-cell–platelet interactions in blood-borne metastasis, we sought to address the potential contribution of endothelial P-selectin expression to adhesive events between the microvasculature and melanoma cells in vivo. Transplantation of bone marrow from P-selectin-deficient into wild-type mice conveyed inhibition of ex-perimental melanoma metastasis. However, the extent to which bone marrow-conferred lack of platelet P-selectin expression attenuated melanoma lung metastasis was significantly less than that seen in P-selectin-deficient mice, suggesting that endothelial P-selectin expression may additionally contribute to formation of hematogenous metastases. This assumption was supported by our intravital microscopy studies, in which a significant proportion of melanoma cells were capable of directly interacting with postcapillary venules of the murine ear in a P-selectin-dependent manner. Heparin not only inhibits P-selectin-mediated melanoma cell rolling but also attenuates melanoma metastasis formation in vivo, further supporting the concept that endothelial P-selectin expression may represent an additional target of heparin action in experimental melanoma lung metastasis.
Transdominant ΔTAp73 Isoforms Are Frequently Up-regulated in Ovarian Cancer. Evidence for Their Role as Epigenetic p53 Inhibitors
in Vivo
Authors: Concin Nicole , Becker Kirsten , Slade Neda , Erster Susan , Mueller-Holzner Elizabeth , Ulmer Hanno , Daxenbichler Guenter , Zeimet Alain , Zeillinger Robert , Marth Christian , Moll Ute M.
Published: September 2005

Despite strong homology, the roles of TP53 and TP73 in tumorigenesis seem to be fundamentally different. In contrast to TP53, tumor-associated overexpression of TP73 in many different cancers, combined with virtual absence of inactivating mutations and lack of a cancer phenotype in the TP73 null mouse are inconsistent with a suppressor function but instead support an oncogenic function. The discovery of NH2-terminally truncated p73 isoforms, collectively called ΔTAp73, is now the focus of intense interest because they act as potent transdominant inihibitors of wild-type p53 and transactivation-competent TAp73. Therefore, establishing deregulated ΔTAp73 expression in tumors could be the crucial link to decipher which of the two opposing roles of this bipolar gene is the biologically relevant one. This study is the largest to date and encompasses 100 ovarian carcinomas with complete expression profile of all NH2-terminal isoforms, discriminating between TAp73 and ΔTAp73 (ΔNp73, ΔN′p73, Ex2p73, and Ex2/3p73) by isoform-specific real-time reverse transcription-PCR. We find that the set of NH2-terminal p73 isoforms distinguishes ovarian cancer patients from healthy controls and thus is a molecular marker for this diagnosis. Ovarian cancers strongly and almost universally overexpress ΔN′p73 compared with normal tissues (95% of cancers). About one-third of tumors also exhibit concomitant up-regulation of the antagonistic TAp73, whereas only a small subgroup of tumors overexpress ΔNp73. Thus, deregulation of the E2F1-responsive P1 promoter, rather than the alternate P2 promoter, is mainly responsible for the production of transdominant p53/TAp73 antagonists in ovarian cancer. Tumor stage, grade, presence of metastases, p53 status, and residual disease after resection are significant prognostic markers for overall and recurrence-free survival. A trend is found for better overall survival in patients with low expression of ΔN′p73/ΔNp73, compared with patients with high expression. A strong correlation between deregulated ΔTAp73 and p53 status exists. p53 wild-type cancers exhibit significantly higher deregulation of ΔN′p73, ΔNp73, and Ex2/3p73 than p53 mutant cancers. This data strongly supports the hypothesis that overexpression of transdominant p73 isoforms can function as epigenetic inhibitors of p53 in vivo, thereby alleviating selection pressure for p53 mutations in tumors.
In Vivo
Imaging of HIV Protease Activity in Amplicon Vector-transduced Gliomas
Authors: Shah Khalid , Tung Ching-Hsuan , Chang Chung-Hsun , Slootweg Eric , O’Loughlin Terence , Breakefield Xandra O. , Weissleder Ralph
Published: September 2005

In vivo imaging of endogenously expressed mammalian proteases has been useful for the detection of cancer and preneoplastic lesions, for staging of inflammatory and autoimmune diseases, and for testing the efficacy of novel protease inhibitors. Here we report on the synthesis of a novel imaging probe that is specific for HIV-1 protease (PR). The probe was designed to be biocompatible, i.v. injectable, and detectable by fluorescence imaging. Human Gli36 glioblastoma cells infected with an human simplex virus amplicon vector expressing HIV-1PR showed specific fluorescence activation, an effect that could be inhibited by the HIV-1PR inhibitor, indinavir. The transfer of the HIV-1PR marker gene could be detected in vivo after intratumoral delivery of the human simplex virus-amplicon vector. These results are the first proof of principle that viral proteases can directly be imaged in vivo. These findings may be directly applicable in using viral protease expression as a transgene marker in tumor therapy and may have implications in testing the efficacy of HIV-1PR inhibitors in vivo.
Specific Histone Patterns and Acetylase/Deacetylase Activity at the Breakpoint-Cluster Region of the Human
MLL
Gene
Authors: Khobta Andriy , Carlo-Stella Carmelo , Capranico Giovanni
Published: September 2005

The MLL gene breakpoint-cluster region (BCR) is a known hot-spot for chromosomal translocations in human leukemias. We mapped core histone modifications and histone H1 along the MLL gene in Jurkat cells and human CD34+ progenitor blood cells by chromatin immunoprecipitation. Within the BCR, we found specific histone patterns that were different from other genomic regions and a histone H1-free fragment at the telomeric end. Core histone acetylase/deacetylase activities were also found within the BCR. In the studied cell models, chromatin components at the MLL BCR suggest an asymmetric organization that may influence early molecular events eventually leading to chromosomal translocations.
Expression of
POT1
is Associated with Tumor Stage and Telomere Length in Gastric Carcinoma
Authors: Kondo Tomohiro , Oue Naohide , Yoshida Kazuhiro , Mitani Yoshitsugu , Naka Kazuhito , Nakayama Hirofumi , Yasui Wataru
Published: September 2005

Pot1, a telomere end-binding protein in fission yeast and human, is proposed not only to cap telomeres but also to recruit telomerase to the ends of chromosomes. No study has been performed regarding Pot1 expression status in human cancers. Thus, we examined POT1 mRNA expression in 51 gastric cancer (GC) tissues and evaluated telomere length and 3′ telomeric overhang signals in 20 of the 51 GC tissues. Quantitative reverse transcription-PCR analysis showed that POT1 expression levels in the tumor relative to those in nonneoplastic mucosa (T/N ratio) were significantly higher in stage III/IV tumors than in stage I/II tumors (P = 0.005). Down-regulation of POT1 (T/n < 0.5) was observed more frequently in stage I/II GC (52.4%, 11 of 21) than in stage III/IV GC (23.3%, 7 of 30; P = 0.033), whereas up-regulation of POT1 (T/n > 2.0) was observed more frequently in stage III/IV GC (33.3%, 10 of 30) than in stage I/II GC (9.5%, 2 of 21; P = 0.048). POT1 expression levels showed decreased in accordance with telomere shortening (r = 0.713, P = 0.002). In-gel hybridization analysis showed that 3′ telomeric overhang signals decreased in accordance with decreases in POT1 expression levels (r = 0.696, P = 0.002) and telomere shortening (r = 0.570, P = 0.013). Reduced POT1 expression was observed in GC cell lines with telomeres shortened by treatment with azidothymidine. In addition, inhibition of Pot1 by antisense oligonucleotides led to telomere shortening as well as inhibition of telomerase activity in GC cells. Moreover, inhibition of Pot1 decreased 3′ overhang signals and increased the frequency of anaphase bridge (P = 0.0005). These data suggest that Pot1 may play an important role in regulation of telomere length and that inhibition of Pot1 may induce telomere dysfunction. Moreover, changes in POT1 expression levels may be associated with stomach carcinogenesis and GC progression.
Expression of the Insulin-Like Growth Factor I Receptor and Urokinase Plasminogen Activator in Breast Cancer Is Associated with Poor Survival
Authors: Nielsen Torsten O. , Andrews Heather N. , Cheang Maggie , Kucab Jill E. , Hsu Forrest D. , Ragaz Joseph , Gilks C. Blake , Makretsov Nikita , Bajdik Chris D. , Brookes Christy , Neckers Leonard M. , Evdokimova Valentina , Huntsman David G. , Dunn Sandra E.
Published: September 2005

Urokinase plasminogen activator (uPA) expression in breast cancer is associated with relapse and a reduction in disease-specific survival. Thus, efforts are under way to identify uPA inhibitors. By screening a chemical library of >1000 compounds, 17-allyaminogeldanamycin (17AAG) was identified as a potent inhibitor of uPA by the National Cancer Institute and is now in Phase I clinical trials. At this time, it remains unclear how 17AAG blocks uPA; one possibility is through disruption of the insulin-like growth factor I receptor (IGF-IR) pathway. This would be consistent with studies from our laboratory showing that activation of IGF-IR results in the induction of uPA protein. In the study described herein, we observed that IGF-IR and uPA were highly expressed in 87 and 55% of breast cancer by screening tumor tissue microarrays representing 930 cases. A significant proportion (52.1% = 354 of 680 cases, P < 0.0001) of the patients had tumors expressing both proteins. uPA alone (P = 0.033) or in combination with IGF-IR (P = 0.0104) was indicative of decreased disease-specific survival. Next, we demonstrated that treating MDA-MB-231 cells with increasing concentrations of 17AAG resulted in IGF-IR degradation (IC50 = 1.0 μm) and blocked signal transduction through the Akt and mitogen-activated protein kinase pathways. Finally, we found that 17AAG had a robust inhibitory effect on the production of uPA mRNAand protein in the presence of IGF-I. Thus, our study raises the possibility that 17AAG could prove to be an effective therapeutic agent for a large number of breast cancer patients by inhibiting the IGF-IR and ultimately uPA.
Molecular Classification and Survival Prediction in Human Gliomas Based on Proteome Analysis
Authors: Iwadate Yasuo , Sakaida Tsukasa , Hiwasa Takaki , Nagai Yuichiro , Ishikura Hiroshi , Takiguchi Masaki , Yamaura Akira
Published: September 2005

The biological features of gliomas, which are characterized by highly heterogeneous biological aggressiveness even in the same histological category, would be precisely described by global gene expression data at the protein level. We investigated whether proteome analysis based on two-dimensional gel electrophoresis and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry can identify differences in protein expression between high- and low-grade glioma tissues. Proteome profiling patterns were compared in 85 tissue samples: 52 glioblastoma multiforme, 13 anaplastic astrocytomas, 10 atrocytomas, and 10 normal brain tissues. We could completely distinguish the normal brain tissues from glioma tissues by cluster analysis based on the proteome profiling patterns. Proteome-based clustering significantly correlated with the patient survival, and we could identify a biologically distinct subset of astrocytomas with aggressive nature. Discriminant analysis extracted a set of 37 proteins differentially expressed based on histological grading. Among them, many of the proteins that were increased in high-grade gliomas were categorized as signal transduction proteins, including small G-proteins. Immunohistochemical analysis confirmed the expression of identified proteins in glioma tissues. The present study shows that proteome analysis is useful to develop a novel system for the prediction of biological aggressiveness of gliomas. The proteins identified here could be novel biomarkers for survival prediction and rational targets for antiglioma therapy.
Adenoviral Gene Transfer of the Human Inducible Nitric Oxide Synthase Gene Enhances the Radiation Response of Human Colorectal Cancer Associated with Alterations in Tumor Vascularity
Authors: Wang Zifa , Cook Tracy , Alber Sean , Liu Kaihong , Kovesdi Imre , Watkins Simon K. , Vodovotz Yoram , Billiar Timothy R. , Blumberg David
Published: September 2005

Nitric oxide is a potent radiosensitizer of tumors, but its use clinically is limited by serious side effects when administered systemically. We have demonstrated previously that gene transfer of the inducible nitric oxide synthase gene (iNOS) into colorectal cancer cells enhances radiation-induced apoptosis in vitro. The objectives of this study were to further characterize the effects of iNOS gene transfer on the radiosensitivity of human colorectal cancer cells in vitro and tumors grown in athymic nude mice. Adenoviral gene transfer of iNOS (AdiNOS) into human colorectal cancer cell lines (HCT-116 and SNU-1040 cells) significantly enhanced the effects of radiation with sensitizing enhancement ratios (0.1) of 1.65 and 1.6, respectively. The radiation enhancement induced by iNOS was associated with increased iNOS expression and nitric oxide production and prevented by L-NIO, an enzymatic inhibitor of iNOS. AdiNOS treatment of HCT-116 tumors combined with radiation (2 Gy × three fractions) led to a 3.4-fold greater (P < 0.005) tumor growth delay compared with radiation (RT) alone. AdiNOS plus RT also caused significant (P < 0.01) tumor regression with 63% of tumors regressing compared with only 6% of tumors treated with RT. AdiNOS plus RT significantly (P ≤ 0.001) increased the percentage of apoptotic cells (22 ± 4%) compared with either tumors treated with control vector plus RT (9 ± 1%), AdiNOS alone (9 ± 3%), or no treatment (2 ± 1%). These radiosensitizing effects of AdiNOS occurred at low infection efficiency (4% of tumor infected), indicating a significant bystander effect.
Human Telomerase Reverse Transcriptase-Transduced Human Cytotoxic T Cells Suppress the Growth of Human Melanoma in Immunodeficient Mice
Authors: Verra Natascha C. V. , Jorritsma Annelies , Weijer Kees , Ruizendaal Janneke J. , Voordouw Arie , Weder Pauline , Hooijberg Erik , Schumacher Ton N. M. , Haanen John B. A. G. , Spits Hergen , Luiten Rosalie M.
Published: September 2005

Immunotherapy of melanoma by adoptive transfer of tumor-reactive T lymphocytes aims at increasing the number of activated effectors at the tumor site that can mediate tumor regression. The limited life span of human T lymphocytes, however, hampers obtaining sufficient cells for adoptive transfer therapy. We have shown previously that the life span of human T cells can be greatly extended by transduction with the human telomerase reverse transcriptase (hTERT) gene, without altering antigen specificity or effector function. We developed a murine model to evaluate the efficacy of hTERT-transduced human CTLs with antitumor reactivity to eradicate autologous tumor cells in vivo. We transplanted the human melanoma cell line melAKR or melAKR-Flu, transduced with a retrovirus encoding the influenza virus/HLA-A2 epitope, in RAG-2−/− IL-2Rγ −/− double knockout mice. Adoptive transfer of the hTERT-transduced influenza virus-specific CTL clone INFA24 or clone INFA13 inhibited the growth of melAKR-Flu tumors in vivo and not of the parental melAKR melanoma cells. Furthermore, the hTERT-transduced CTL clone INFA13 inhibited tumor growth to the same extent in vivo as the untransduced CTL clone, as determined by in vivo imaging of luciferase gene-transduced melAKR-Flu tumors, indicating that hTERT did not affect the in vivo function of CTL. These results demonstrate that hTERT-transduced human CTLs are capable of mediating antitumor activity in vivo in an antigen-specific manner. hTERT-transduced MART-1-specific CTL clones AKR4D8 and AKR103 inhibited the growth of syngeneic melAKR tumors in vivo. Strikingly, melAKR-Flu cells were equally killed by the MART-1-specific CTL clones and influenza virus-specific CTL clones in vitro, but only influenza-specific CTLs were able to mediate tumor regression in vivo. The influenza-specific CTL clones were found to produce higher levels of IFNγ on tumor cell recognition than the MART-1-specific CTL clones, which may result from the higher functional avidity of the influenza virus-specific CTL clones. Also, melAKR-Flu tumors were growing faster than melAKR tumors, which may have surpassed the relatively modest antitumor effect of the MART-1-specific CTL, as compared with the influenza virus-specific CTL. Taken together, the adoptive transfer model described here shows that hTERT-transduced T cells are functional in vivo, and allows us to evaluate the balance between functional activity of the CTL and tumor growth rate in vivo, which determines the efficacy of CTLs to eradicate tumors in adoptive transfer therapy.
The Wnt Antagonist
sFRP1
in Colorectal Tumorigenesis
Authors: Caldwell Germaine M. , Jones Carolyn , Gensberg Karl , Jan Shamem , Hardy Robert G. , Byrd Philip , Chughtai Shaheen , Wallis Yvonne , Matthews Glenn M. , Morton Dion G.
Published: September 2005

Regions of the short arm of chromosome 8 are deleted frequently in a range of solid tumors, indicating that tumor suppressor genes reside at these loci. In this study, we have examined the properties of the Wnt signaling antagonist secreted frizzled-related protein (sFRP) 1 as a candidate for this role at c8p11.2. An initial survey of 10 colorectal tumors, selected by the presence of isolated short deletions of the 8p11.2 region, identified three chain-terminating mutations, all within the first exon, which encodes the cysteine-rich domain. None of these tumors exhibited microsatellite instability, indicating intact mismatch repair gene function. The preserved sFRP1 alleles in the remaining seven tumors each contained a polymorphic three-base insertion in the signal sequence, but in a broader study, no association was found between this and the development of colorectal cancer. Epigenetic inhibition of sFRP1 transcription was investigated, and increased methylation of the promotor region was demonstrated in an additional cohort of 51 locally advanced colorectal cancers. Hypermethylation was identified in 40 of 49 (82%) cancers and in only 11 of 36 (30%) matched normal mucosal samples (P < 0.001). Semiquantitative analysis, by real-time PCR, of mRNA expression in 37 of the same cohort of 51 cancers revealed that sFRP1 mRNA expression was down-regulated in 28 (76%) cases compared with matched normal large bowel mucosa. The 3′ end of the sFRP1 mRNA also was found to be alternatively spliced, compared with the prototype liver and lung forms, in the colon and a number of other tissues, yielding an extended COOH terminus, which may influence its activity in a tissue-specific manner. The inactivation and down-regulation of sFRP1 observed are consistent with it acting as a tumor suppressor gene in colorectal carcinogenesis. Because β-catenin is constitutively active in the majority of colorectal tumors, it is unlikely that sFRP1 can act in the canonical Wnt response pathway. Therefore, we propose that the reduced activity or absence of sFRP1 allows the transduction of noncanonical Wnt signals, which contribute to tumor progression.
Collagenase Increases the Transcapillary Pressure Gradient and Improves the Uptake and Distribution of Monoclonal Antibodies in Human Osteosarcoma Xenografts
Authors: Eikenes Live , Bruland Øyvind S. , Brekken Christian , Davies Catharina de Lange
Published: September 2005

Cancer therapy based on tumor-selective macromolecules may fail due to the elevated interstitial fluid pressure (IFP) that reduces the transvascular and interstitial convection in solid tumors. Modulation of the tumor extracellular matrix (ECM) may reduce IFP and enhance transvascular filtration and interstitial transport of macromolecules. We therefore measured the effect of the ECM-degrading enzyme collagenase on IFP and microvascular pressure (MVP) in human osteosarcoma xenografts using the wick-in-needle and micropipette methods, respectively. The tumor uptake and distribution of a systemically administered osteosarcoma-associated monoclonal antibody (TP-3) after i.v. injection of collagenase were analyzed using confocal laser scanning microscopy. Collagenase (0.1%) reduced both IFP (45%) and MVP (60%), but the kinetics of the recoveries differed, because MVP had recovered by the time IFP reached its minimum level. Thus, collagenase increased the transcapillary pressure gradient, inducing a 2-fold increase in the tumor uptake and improving the distribution of the monoclonal antibody, which was localized further into the tumor. To study the mechanism of the reduction in MVP, mean arterial blood pressure was measured and found not to be affected by the collagenase treatment. The reduction in MVP was rather due to reduced vascular resistance because microvascular-associated collagen was totally or partially disintegrated. Although collagenase may favor metastasis and thus not be clinically relevant, this study shows proof of principle that degradation of the ECM leads to a favorable change in the transvascular pressure gradient, thereby increasing antibody penetration and binding to tumor cells.
Imatinib Mesylate Resistance Through BCR-ABL Independence in Chronic Myelogenous Leukemia
Authors: Donato Nicholas J. , Wu Ji Y. , Stapley Jonathan , Lin Hui , Arlinghaus Ralph , Aggarwal Bharat , Shishodin Shishir , Albitar Maher , Hayes Kimberly , Kantarjian Hagop , Talpaz Moshe
Published: September 2005

Imatinib mesylate (IM) binds to the BCR-ABL protein, inhibiting its kinase activity and effectively controlling diseases driven by this kinase. IM resistance has been associated with kinase mutations or increased BCR-ABL expression. However, disease progression may be mediated by other mechanisms that render tumor cells independent of BCR-ABL. To demonstrate this potential, IM-resistant cells were found in chronic myelogenous leukemia patients with continuous BCR-ABL gene expression but undetectable BCR-ABL protein expression. These cells were unresponsive to IM and acquired BCR-ABL-independent signaling characteristics. IM resistance in some patients may be mediated through loss of kinase target dependence.
Effect Of Human Natural Killer and γδ T Cells on the Growth of Human Autologous Melanoma Xenografts in SCID Mice
Authors: Lozupone Francesco , Pende Daniela , Burgio Vito Lelio , Castelli Chiara , Spada Massimo , Venditti Massimo , Luciani Francesca , Lugini Luana , Federici Cristina , Ramoni Carlo , Rivoltini Licia , Parmiani Giorgio , Belardelli Filippo , Rivera Paola , Marcenaro Stefania , Moretta Lorenzo , Fais Stefano
Published: September 2005

Natural killer (NK) cells were first identified for their ability to kill tumor cells of different origin in vitro. Similarly, γδ T lymphocytes display strong cytotoxic activity against various tumor cell lines. However, the ability of both the NK and γδ cells to mediate natural immune response against human malignant tumors in vivo is still poorly defined. Severe combined immunodeficient (SCID) mice have been successfully engrafted with human tumors. In this study, the antitumor effect of local as well as of systemic treatments based on NK cells or Vδ1 or Vδ2 γ/δ T lymphocytes against autologous melanoma cells was investigated in vivo. The results show that all three of the populations were effective in preventing growth of autologous human melanomas when both tumor and lymphoid cells were s.c. inoculated at the same site. However, when lymphoid cells were infused i.v., only NK cells and Vδ1 γ/δ T lymphocytes could either prevent or inhibit the s.c. growth of autologous melanoma. Accordingly, both NK cells and Vδ1 γδ T lymphocytes could be detected at the s.c. tumor site. In contrast, Vδ2 γδ T lymphocytes were only detectable in the spleen of the SCID mice. Moreover, NK cells maintained their inhibitory effect on tumor growth even after discontinuation of the treatment. Indeed they were present at the tumor site for a longer period.

These data support the possibility to exploit NK cells and Vδ1 γδ T lymphocytes in tumor immunotherapy. Moreover, our study emphasizes the usefulness of human tumor/SCID mouse models for preclinical evaluation of immunotherapy protocols against human tumors.
A Mimic of Tumor Rejection Antigen-Associated Carbohydrates Mediates an Antitumor Cellular Response
Authors: Monzavi-Karbassi Behjatolah , Luo Ping , Jousheghany Fariba , Torres-Quiñones Marta , Cunto-Amesty Gina , Artaud Cecile , Kieber-Emmons Thomas
Published: September 2005

Tumor-associated carbohydrate antigens are typically perceived as inadequate targets for generating tumor-specific cellular responses. Lectin profile reactivity and crystallographic studies demonstrate that MHC class I molecules can present to the immune system posttranslationally modified cytosolic peptides carrying O-β-linked N-acetylglucosamine (GlcNAc). Here we report that a peptide surrogate of GlcNAc can facilitate an in vivo tumor-specific cellular response to established Meth A tumors that display native O-GlcNAc glycoproteins on the tumor cell surface. Peptide immunization of tumor-bearing mice had a moderate effect on tumor regression. Inclusion of interleukin 12 in the immunization regimen stimulated complete elimination of tumor cells in all of the mice tested, whereas interleukin 12 administration alone afforded no tumor growth inhibition. Adoptive transfer of immune T cells into tumor-bearing nude mice indicates a role for CD8+ T cells in tumor regression. This work postulates that peptide mimetics of glycosylated tumor rejection antigens might be further developed for immune therapy of cancer.
p53 Pathway in Renal Cell Carcinoma Is Repressed by a Dominant Mechanism
Authors: Gurova Katerina V. , Hill Jason E. , Razorenova Olga V. , Chumakov Peter M. , Gudkov Andrei V.
Published: September 2005

Renal cell carcinoma (RCC) rarely acquires mutations in p53 tumor suppressor gene, suggesting that p53 signaling in this tumor type might be repressed by some other mechanism. In fact, all four RCC-derived cell lines we tested maintained wild-type p53 but were not capable of transactivating p53-responsive reporters and endogenous p53-responsive genes. p53 protein in RCC showed normal response to genotoxic stress, including accumulation, nuclear translocation, and activation of specific DNA binding. Functional and expression analysis of Mdm2, MdmX, and Arf showed lack of involvement of these p53 regulators in the observed defect of p53 function in RCC. However, activation of p53-mediated transactivation could be achieved by extremely high levels of p53 attained by lentivirus vector-driven transduction, suggesting the involvement of a dominant inhibitor in repression of p53-dependent transactivation in RCC. Consistently, p53 inactivation prevailed in the hybrids of RCC cells with the cells possessing fully functional p53. Remarkably, cells of normal kidney epithelium also caused partial p53 repression in cell fusion experiments, suggesting that RCC-specific p53 repression may be based on an unknown dominant mechanism also acting in normal kidney tissue.
Role of Immature Myeloid Gr-1+ Cells in the Development of Antitumor Immunity
Authors: Li Qingsheng , Pan Ping-Ying , Gu Peidi , Xu Dongping , Chen Shu-Hsia
Published: September 2005

One of the mechanisms by which tumor cells evade the immune system is the lack of proper antigen-presenting cells. Improvement in host immunity against tumor cells can be achieved by promoting the differentiation of dendritic cells (DCs) from immature myeloid cells (Gr-1+Ly-6C+F4/80+) that accumulate in the bone marrow and lymphoid organs of mice with large tumor burdens. The enriched immature myeloid cells inhibit T-cell proliferation and tumor-specific T-cell response, which can be reversed by the differentiation of immature myeloid cells or depletion of F4/80+ cells. Sorted Gr-1+/F4/80+ immature myeloid cells differentiated into CD11c+ cells that express CD80 and I-A/I-E (MHC class II) in the presence of recombinant murine granulocyte macrophage colony-stimulating factor (GM-CSF). Furthermore, intratumoral gene delivery of GM-CSF not only promoted the differentiation of carboxyfluoroscein succinimidyl ester-labeled immature myeloid cells into CD11c+ cells with the characteristics of mature DCs (CD80+, I-A/I-E+) but also enhanced innate natural killer and adaptive cytolytic T-cell activities in mice treated with interleukin (IL)-12 and anti-4–1BB combination therapy. More importantly, intratumoral delivery of GM-CSF and IL-12 genes in combination with 4–1BB costimulation greatly improved the long-term survival rate of mice bearing large tumors and eradicated the untreated existing hepatic tumor. The results suggest that inducing the maturation of immature myeloid cells, thus preventing their inhibitory activity and enhancing their antigen-presenting capability, by GM-CSF gene therapy is a critically important step in the development of effective antitumor responses in hosts with advanced tumors.
Use of Replication-Conditional Adenovirus as a Helper System to Enhance Delivery of P450 Prodrug-Activation Genes for Cancer Therapy
Authors: Jounaidi Youssef , Waxman David J.
Published: September 2005

Cytochrome P450 (CYP) gene transfer sensitizes tumor xenografts to anticancer prodrugs such as cyclophosphamide (CPA) without a detectable increase in host toxicity. Optimal prodrug activation is achieved when a suitable P450 gene (e.g., human CYP2B6) is delivered in combination with NADPH-cytochrome P450 reductase (P450R), which encodes the flavoenzyme P450 reductase. We sought to improve this gene therapy by coordinated delivery and expression of P450 and P450R on a single bicistronic vector using an internal ribosomal entry site (IRES) sequence. Retrovirus encoding a CYP2B6-IRES-P450R expression cassette was shown to induce strong P450-dependent CPA cytotoxicity in a population of infected 9L gliosarcoma cells. Adeno-P450, a replication-defective, E1/E3 region-deleted adenovirus engineered to express CYP2B6-IRES-P450R, induced intracellular CPA 4-hydroxylation, and CPA cytotoxicity, in a broad range of human cancer cell lines. However, limited Adeno-P450 gene transfer and CPA chemosensitization was seen with certain human tumor cells, notably PC-3 prostate and HT-29 colon cancer cells. Remarkable improvements could be obtained by coinfecting the tumor cells with Adeno-P450 in combination with Onyx-017, an E1b-55k gene-deleted adenovirus that selectively replicates in p53 pathway-deficient cells. Substantial increases in gene expression were observed during the early stages of viral infection, reflecting an apparent coamplification of the Adeno-P450 genome, followed by enhanced viral spread at later stages, as demonstrated in cultured tumor cells, and in A549 and PC-3 solid tumor xenografts grown in scid mice. This combination of the replication-defective Adeno-P450 with a replication-conditional and tumor cell-targeted helper adenovirus dramatically improved the low gene transfer observed with some human tumor cell lines and correspondingly increased tumor cell-catalyzed CPA 4-hydroxylation, CPA cytotoxicity, and in vivo antitumor activity in a PC-3 tumor xenograft model. The use of tumor-selective, replicating adenovirus to promote the spread of replication-defective gene therapy vectors, such as Adeno-P450, substantially increases the therapeutic potential of adenoviral delivery systems, and should lead to increased activity and enhanced tumor selectivity of cytochrome P450 and other gene-directed enzyme prodrug therapies.