bluuebunny/crossref_metadata_2025 huggingface dataset:
Tumor Suppressor VDUP1 Increases p27kip1 Stability by Inhibiting JAB1
Authors: Jeon Jun-Ho , Lee Kee-Nyung , Hwang Chae Young , Kwon Ki-Sun , You Kwan-Hee , Choi Inpyo
Published: September 2005

Vitamin D3 up-regulated protein 1 (VDUP1) is a stress-response gene that is up-regulated by 1,25(OH)2D3 in many cells. It has been reported that VDUP1 expression is reduced in many tumor cells and the enforced expression of VDUP1 inhibits cell proliferation by arresting cell cycle progression. Here, we found that VDUP1−/− fibroblast cells proliferated more rapidly compared with wild-type cells with reduced expression of p27kip1, a cyclin-dependent kinase inhibitor. JAB1 is known to interact with p27kip1 and to decrease the stability of p27kip1. VDUP1 interacted with JAB1 and restored JAB1-induced suppression of p27kip1 stability. In this process, VDUP1 blocked the JAB1-mediated translocation of p27kip1 from the nucleus to the cytoplasm. In addition, VDUP1 inhibited JAB1-mediated activator protein-1 activation and cell proliferation. Taken together, these results indicate that VDUP1 is a novel factor of p27kip1 stability via regulating JAB1.
Kaposi's Sarcoma–Associated Herpesvirus Viral IFN Regulatory Factor 1 Inhibits Transforming Growth Factor-β Signaling
Authors: Seo Taegun , Park Junsoo , Choe Joonho
Published: September 2005

Kaposi's sarcoma–associated herpesvirus, also called human herpesvirus 8, has been implicated in the pathogenesis of Kaposi's sarcoma, body cavity–based primary effusion lymphoma, and some forms of multicentric Castleman's disease. The Kaposi's sarcoma–associated herpesvirus open reading frame K9 encodes viral IFN regulatory factor 1 (vIRF1), which functions as a repressor of IFN-mediated signal transduction. vIRF1 expression in NIH 3T3 cells leads to transformation and consequently induces malignant fibrosarcoma in nude mice, suggesting that vIRF1 is a strong oncoprotein. Here, we show that vIRF1 inhibited transforming growth factor-β (TGF-β) signaling via its targeting of Smad proteins. vIRF1 suppressed TGF-β-mediated transcription and growth arrest. vIRF1 directly interacted with both Smad3 and Smad4, resulting in inhibition of their transactivation activity. Studies using vIRF1 deletion mutants showed that the central region of vIRF1 was required for vIRF1 association with Smad3 and Smad4 and that this region was also important for inhibition of TGF-β signaling. In addition, we found that vIRF1 interfered with Smad3-Smad4 complex formation and inhibited Smad3/Smad4 complexes from binding to DNA. These results indicate that vIRF1 inhibits TGF-β signaling via interaction with Smads. In addition, the data indicate the TGF-β pathway is an important target for viral oncoproteins.
Mutant V599EB-Raf Regulates Growth and Vascular Development of Malignant Melanoma Tumors
Authors: Sharma Arati , Trivedi Nishit R. , Zimmerman Melissa A. , Tuveson David A. , Smith Charles D. , Robertson Gavin P.
Published: September 2005

Activating mutations of the B-RAF gene are observed in >60% of human melanomas. Approximately 90% of these mutations occur in the activation segment of the kinase domain as a single-base substitution that converts a valine to glutamic acid at codon 599 (V599E) in exon 15. This mutation causes activation of the kinase as well as downstream effectors of the mitogen-activated protein kinase–signaling cascade, leading to melanoma tumor development by an as yet unknown mechanism. In this study, we have identified the role of V599EB-Raf in melanoma tumor development by characterizing the mechanism by which this mutant protein promotes melanoma tumorigenesis. Small interfering RNA targeted against B-Raf or a Raf kinase inhibitor (BAY 43-9006) was used to reduce expression and/or activity of V599EB-Raf in melanoma tumors. This inhibition led to reduced activity of the mitogen-activated protein kinase–signaling cascade and inhibited tumor development in animals. Targeted reduction of mutant V599EB-Raf expression (activity) in melanoma cells before tumor formation inhibited tumorigenesis by reducing the growth potential of melanoma cells. In contrast, reduction of mutant V599EB-Raf activity in preexisting tumors prevented further vascular development mediated through decreased vascular endothelial growth factor secretion, subsequently increasing apoptosis in tumors. These effects in combination with reduced proliferative capacity halted growth, but did not shrink the size of preexisting melanoma tumors. Thus, these studies identify the mechanistic underpinnings by which mutant V599EB-RAF promotes melanoma development and show the effectiveness of targeting this protein to inhibit melanoma tumor growth.
Knockdown of STAT3 Expression by RNA Interference Inhibits the Induction of Breast Tumors in Immunocompetent Mice
Authors: Ling Xiaoyang , Arlinghaus Ralph B.
Published: September 2005

Constitutively activated STAT3 is involved in the formation of multiple types of tumors including breast cancer. We examined the effects of Stat3 protein knockdown by RNA interference using a dicistronic lentivirus small hairpin (shRNA) delivery system on the growth of mammary tumors in BALB/c mice induced by the 4T1 cell line. A single exposure of 4T1 cells to shRNA/STAT3 lentivirus transduced 75% of the cells with green fluorescent protein (GFP) within 96 hours. In cells selected for GFP expression, neither Stat3 protein nor phosphotyrosine Stat3 was detected. Tumor formation induced by injecting 4T1 cells into the mammary fat pad was blocked by expression of the shRNA for STAT3 whereas all mice injected with 4T1 cells expressing only GFP efficiently formed tumors. c-Myc expression was reduced 75% in cells expressing greatly reduced levels of Stat3 compared with the GFP control. Of interest, the level of activated Src, which is known to activate Stat3, was virtually eliminated but the level of the Src protein itself remained the same. Importantly, expression of Twist protein, a metastatic regulator, was eliminated in STAT3 knockdown cells. Invasion activity of STAT3 knockdown cells was strongly inhibited. However, the proliferation rate of cells in Stat3 knockdown cells was similar to that of the GFP control; the cell cycle was also not affected. We conclude from these studies that activated Stat3 protein plays a critical role in the induction of breast tumors induced by 4T1 cells by enhancing the expression of several important genes including c-Myc and the metastatic regulator Twist. These studies suggest that stable expression of small interfering RNA for STAT3 has potential as a therapeutic strategy for breast cancer.
An Expression Signature Classifies Chemotherapy-Resistant Pediatric Osteosarcoma
Authors: Mintz Michelle B. , Sowers Rebecca , Brown Kevin M. , Hilmer Sara C. , Mazza BethAnne , Huvos Andrew G. , Meyers Paul A. , LaFleur Bonnie , McDonough Wendy S. , Henry Michael M. , Ramsey Keri E. , Antonescu Cristina R. , Chen Wen , Healey John H. , Daluski Aaron , Berens Michael E. , MacDonald Tobey J. , Gorlick Richard , Stephan Dietrich A.
Published: September 2005

Osteosarcoma is the most common malignant bone tumor in children. Osteosarcoma patients who respond poorly to chemotherapy are at a higher risk of relapse and adverse outcome. Therefore, it was the aim of this study to identify prognostic factors at the time of diagnosis to characterize the genes predictive of poor survival outcome and to identify potential novel therapeutic targets. Expression profiling of 30 osteosarcoma diagnostic biopsy samples, 15 with inferior necrosis following induction chemotherapy (Huvos I/II) and 15 with superior necrosis following induction chemotherapy (Huvos III/IV), was conducted using Affymetrix U95Av2 oligonucleotide microarrays. One hundred and four genes were found to be statistically significant and highly differentially expressed between Huvos I/II and III/IV patients. Statistically significant genes were validated on a small independent cohort comprised of osteosarcoma xenograft tumor samples. Markers of Huvos I/II response predominantly were gene products involved in extracellular matrix (ECM) microenvironment remodeling and osteoclast differentiation. A striking finding was the significant decrease in osteoprotegerin, an osteoclastogenesis inhibitory factor. Additional genes involved in osteoclastogenesis and bone resorption, which were statistically different, include annexin 2, SMAD, PLA2G2A, and TGFβ1. ECM remodeling genes include desmoplakin, SPARCL1, biglycan, and PECAM. Gene expression of select genes involved in tumor progression, ECM remodeling, and osteoclastogenesis were validated via quantitative reverse transcription-PCR in an independent cohort. We propose that osteosarcoma tumor–driven changes in the bone microenvironment contribute to the chemotherapy-resistant phenotype and offer testable hypotheses to potentially enhance therapeutic response.
Role of Glycogen Synthase Kinase 3β in Rapamycin-Mediated Cell Cycle Regulation and Chemosensitivity
Authors: Dong JinJiang , Peng Junying , Zhang Haixia , Mondesire Wallace H. , Jian Weiguo , Mills Gordon B. , Hung Mien-Chie , Meric-Bernstam Funda
Published: September 2005

The mammalian target of rapamycin is a serine-threonine kinase that regulates cell cycle progression. Rapamycin and its analogues inhibit the mammalian target of rapamycin and are being actively investigated in clinical trials as novel targeted anticancer agents. Although cyclin D1 is down-regulated by rapamycin, the role of this down-regulation in rapamycin-mediated growth inhibition and the mechanism of cyclin D1 down-regulation are not well understood. Here, we show that overexpression of cyclin D1 partially overcomes rapamycin-induced cell cycle arrest and inhibition of anchorage-dependent growth in breast cancer cells. Rapamycin not only decreases endogenous cyclin D1 levels but also decreases the expression of transfected cyclin D1, suggesting that this is at least in part caused by accelerated proteolysis. Indeed, rapamycin decreases the half-life of cyclin D1 protein, and the rapamycin-induced decrease in cyclin D1 levels is partially abrogated by proteasome inhibitor N-acetyl-leucyl-leucyl-norleucinal. Rapamycin treatment leads to an increase in the kinase activity of glycogen synthase kinase 3β (GSK3β), a known regulator of cyclin D1 proteolysis. Rapamycin-induced down-regulation of cyclin D1 is inhibited by the GSK3β inhibitors lithium chloride, SB216763, and SB415286. Rapamycin-induced G1 arrest is abrogated by nonspecific GSK3β inhibitor lithium chloride but not by selective inhibitor SB216763, suggesting that GSK3β is not essential for rapamycin-mediated G1 arrest. However, rapamycin inhibits cell growth significantly more in GSK3β wild-type cells than in GSK3β-null cells, suggesting that GSK3β enhances rapamycin-mediated growth inhibition. In addition, rapamycin enhances paclitaxel-induced apoptosis through the mitochondrial death pathway; this is inhibited by selective GSK3β inhibitors SB216763 and SB415286. Furthermore, rapamycin significantly enhances paclitaxel-induced cytotoxicity in GSK3β wild-type but not in GSK3β-null cells, suggesting a critical role for GSK3β in rapamycin-mediated paclitaxel-sensitization. Taken together, these results show that GSK3β plays an important role in rapamycin-mediated cell cycle regulation and chemosensitivity and thus significantly potentiates the antitumor effects of rapamycin.
A Noninvasive Approach for Assessing Tumor Hypoxia in Xenografts: Developing a Urinary Marker for Hypoxia
Authors: Nelson Daniel W. , Cao Hongbin , Zhu Yonghua , Sunar-Reeder Bulbin , Choi Clara Y.H. , Faix James D. , Brown J. Martin , Koong Albert C. , Giaccia Amato J. , Le Quynh-Thu
Published: September 2005

Tumor hypoxia modifies the efficacy of conventional anticancer therapy and promotes malignant tumor progression. Human chorionic gonadotropin (hCG) is a glycoprotein secreted during pregnancy that has been used to monitor tumor burden in xenografts engineered to express this marker. We adapted this approach to use urinary β-hCG as a secreted reporter protein for tumor hypoxia. We used a hypoxia-inducible promoter containing five tandem repeats of the hypoxia-response element (HRE) ligated upstream of the β-hCG gene. This construct was stably integrated into two different cancer cell lines, FaDu, a human head and neck squamous cell carcinoma, and RKO, a human colorectal cancer cell line. In vitro studies showed that tumor cells stably transfected with this plasmid construct secrete β-hCG in response to hypoxia or hypoxia-inducible factor 1α (HIF-1α) stabilizing agents. The hypoxia responsiveness of this construct can be blocked by treatment with agents that affect the HIF-1α pathways, including topotecan, 1-benzyl-3-(5′-hydroxymethyl-2′-furyl)indazole (YC-1), and flavopiridol. Immunofluorescent analysis of tumor sections and quantitative assessment with flow cytometry indicate colocalization between β-hCG and 2-(2-nitro-1H-imidazol-1-yl)-N-(2,2,3,3,3-pentafluoropropyl)acetamide (EF5) and β-hCG and pimonidazole, two extrinsic markers for tumor hypoxia. Secretion of β-hCG from xenografts that contain these stable constructs is directly responsive to changes in tumor oxygenation, including exposure of the animals to 10% O2 and tumor bed irradiation. Similarly, urinary β-hCG levels decline after treatment with flavopiridol, an inhibitor of HIF-1 transactivation. This effect was observed only in tumor cells expressing a HRE-regulated reporter gene and not in tumor cells expressing a cytomegalovirus-regulated reporter gene. The 5HRE β-hCG reporter system described here enables serial, noninvasive monitoring of tumor hypoxia in a mouse model by measuring a urinary reporter protein.
Inhibition of c-Jun-N-terminal-Kinase Sensitizes Tumor Cells to CD95-Induced Apoptosis and Induces G2/M Cell Cycle Arrest
Authors: Kuntzen Christian , Sonuc Nilüfer , De Toni Enrico N. , Opelz Christine , Mucha Simon R. , Gerbes Alexander L. , Eichhorst Sören T.
Published: September 2005

Loss of susceptibility to apoptosis signals is a crucial step in carcinogenesis. Therefore, sensitization of tumor cells to apoptosis is a promising therapeutic strategy. c-Jun-N-terminal-kinases (JNK) have been implicated in stress-induced apoptosis, but may also contribute to survival signaling. Here we show that CD95-induced apoptosis is augmented by the JNK inhibitor SP600125 and small interfering RNA directed against JNK1/2. SP600125 potently inhibited methyl methane sulfonate–induced phosphorylation of c-Jun, but had minimal effect on apoptosis alone. In contrast, it strongly enhanced CD95-mediated apoptosis in six of eight tumor cell lines and led to a G2/M phase arrest in all cell lines. SP600125 enhanced cleavage of caspase 3 and caspase 8, the most upstream caspase in the CD95 pathway. JNK inhibition up-regulates p53 and its target genes p21Cip1/Waf1 and CD95. However, although HCT116 p53−/− cells and p21+/+ cells were less sensitive to CD95 stimulation than their p53+/+ and p21−/− counterparts, p53 and p21 were not involved in the JNK-mediated effect. JunD, which was described to be protective in tumor necrosis factor–induced apoptosis, was not regulated by JNK inhibition on the protein level. When transcription was blocked by actinomycin D, JNK inhibition still enhanced apoptosis to a comparable extent. We conclude that JNK inhibition has antitumor activity by inducing growth arrest and enhancing CD95-mediated apoptosis by a transcription-independent mechanism.
Antiangiogenic Concentrations of Paclitaxel Induce an Increase in Microtubule Dynamics in Endothelial Cells but Not in Cancer Cells
Authors: Pasquier Eddy , Honore Stéphane , Pourroy Bertrand , Jordan Mary Ann , Lehmann Maxime , Briand Claudette , Braguer Diane
Published: September 2005

Microtubule-targeted drugs such as paclitaxel exhibit potent antiangiogenic activity at very low concentrations, but the mechanism underlying such an effect remains unknown. To understand the involvement of microtubules in angiogenesis, we analyzed the dynamic instability behavior of microtubules in living endothelial cells [human microvascular endothelial cells (HMEC-1) and human umbilical vascular endothelial cells (HUVEC)] following 4 hours of paclitaxel treatment. Unexpectedly, antiangiogenic concentrations of paclitaxel (0.1-5 nmol/L) strongly increased microtubule overall dynamicity in both HMEC-1 (86-193%) and HUVEC (54-83%). This increase was associated with increased microtubule growth and shortening rates and extents and decreased mean duration of pauses. The enhancement of microtubule dynamics by paclitaxel seemed to be specific to antiangiogenic concentrations and to endothelial cells. Indeed, cytotoxic concentration (100 nmol/L) of paclitaxel suppressed microtubule dynamics by 40% and 54% in HMEC-1 and HUVECs, respectively, as observed for all tested concentrations in A549 tumor cells. After 4 hours of drug incubation, antiangiogenic concentrations of paclitaxel that inhibited endothelial cell proliferation without apoptosis (1-5 nmol/L) induced a slight decrease in anaphase/metaphase ratio, which was more pronounced and associated with increased mitotic index after 24 hours of incubation. Interestingly, the in vitro antiangiogenic effect also occurred at 0.1 nmol/L paclitaxel, a concentration that did not alter mitotic progression and endothelial cell proliferation but was sufficient to increase interphase microtubule dynamics. Altogether, our results show that paclitaxel mediates antiangiogenesis by an increase in microtubule dynamics in living endothelial cells and suggest that the impairment of interphase microtubule functions is responsible for the inhibition of angiogenesis.
Functional Expression and Mutations of c-Met and Its Therapeutic Inhibition with SU11274 and Small Interfering RNA in Non–Small Cell Lung Cancer
Authors: Ma Patrick C. , Jagadeeswaran Ramasamy , Jagadeesh Simha , Tretiakova Maria S. , Nallasura Vidya , Fox Edward A. , Hansen Mark , Schaefer Erik , Naoki Katsuhiko , Lader Alan , Richards William , Sugarbaker David , Husain Aliya N. , Christensen James G. , Salgia Ravi
Published: September 2005

Non–small cell lung cancer (NSCLC) is a difficult disease to treat. The c-Met receptor is an attractive potential target for novel therapeutic inhibition in human cancers. We provide strong evidence that c-Met is overexpressed, activated, and sometimes mutated in NSCLC cell lines and tumor tissues. Expression of c-Met was found in all (100%) of the NSCLC tumor tissues examined (n = 23) and most (89%) of the cell lines (n = 9). Sixty-one percent of tumor tissues strongly expressed total c-Met, especially adenocarcinoma (67%). Specific expression of phospho-Met (p-Met) [Y1003] and [Y1230/1234/1235] was seen by immunohistochemistry. p-Met expression was preferentially observed at the NSCLC tumor invasive fronts. c-Met alterations were identified within the semaphorin domain (E168D, L299F, S323G, and N375S) and the juxtamembrane domain (R988C, R988C + T1010I, S1058P, and alternative splice product skipping entire juxtamembrane domain) of a NSCLC cell line and adenocarcinoma tissues. We validated c-Met as potential therapeutic target using small interfering RNA down-regulation of the receptor expression by 50% to 60% in NSCLC cells. This led to inhibition of p-Met and phospho-AKT and up to 57.1 ± 7.2% cell viability inhibition at 72 hours. The selective small molecule inhibitor of c-Met SU11274 inhibited cell viability in c-Met-expressing NSCLC cells. SU11274 also abrogated hepatocyte growth factor–induced phosphorylation of c-Met and its downstream signaling. Here, we provide first direct evidence by small interfering RNA targeting and small molecule inhibitor that c-Met is important in NSCLC biology and biochemistry. These results indicate that c-Met inhibition will be an important therapeutic strategy against NSCLC to improve its clinical outcome.
RASSF1A Suppresses the c-Jun-NH2-Kinase Pathway and Inhibits Cell Cycle Progression
Authors: Whang Young Mi , Kim Yeul Hong , Kim Jun Suk , Yoo Young Do
Published: September 2005

Some oncogenes, such as activated Ras, cause the malignant transformation of lung cells. c-Jun-NH2-kinase (JNK) activation is essential for the oncogenic function of these cells. In this study, we show that RASSF1A inhibits the growth of lung cancer cells by blocking the JNK pathway. The exogenous expression of RASSF1A suppressed JNK phosphorylation, and cells stably transfected with RASSF1A showed reduced JNK and c-Jun phosphorylation and Cyclin D1 down-regulation. An in vitro kinase assay showed that the exogenous expression of RASSF1A inhibited JNK activity and that JNK activity suppression due to ectopically expressed RASSF1A was revived by RASSF1A siRNA treatment. Based on our data, we suggest that RASSF1A exerts a tumor-suppressing effect by blocking oncogene-mediated JNK activation in lung cells.
Histone Deacetylase Inhibitors Decrease DNA Methyltransferase-3B Messenger RNA Stability and Down-regulate De novo DNA Methyltransferase Activity in Human Endometrial Cells
Authors: Xiong Yuning , Dowdy Sean C. , Podratz Karl C. , Jin Fan , Attewell John R. , Eberhardt Norman L. , Jiang Shi-Wen
Published: September 2005

It is well known that the histone deacetylase (HDAC) inhibitor trichostatin A (TSA) acts synergistically with the DNA methyltransferase (DNMT) inhibitor 5-aza-2′-deoxycytidine (ADC) to reactivate DNA methylation-silenced genes. Moreover, in several studies, TSA was capable of inducing DNA demethylation even in the absence of ADC. Here we describe a mechanism by which HDAC inhibitors affect DNA methylation through their regulation on DNMT3B, a methyltransferase responsible for de novo DNA methylation. Using quantitative real-time PCR and Western blot analysis, we show that TSA down-regulates DNMT3B mRNA and protein expression in human endometrial cancer cells. This decrease in DNMT3B mRNA results in a significant reduction in de novo methylation activities. Further experiments indicated that TSA decreases DNMT3B mRNA stability and reduces its half-life from ∼4 to 2.5 hours. We established that protein synthesis is required for posttranscriptional regulation, suggesting the involvement of an RNase and/or key mRNA stabilization factor(s) controlling the DNMT3B mRNA stability. Therefore, TSA may not only modify histone acetylation, but also potentially alter DNA methylation. Since the HDAC inhibitors are frequently used in epigenetic studies and are considered to be promising anticancer drugs, these new findings will have implications in both laboratory and clinical settings.
Targeted Measles Virus Vector Displaying Echistatin Infects Endothelial Cells via αvβ3 and Leads to Tumor Regression
Authors: Hallak Louay K. , Merchan Jaime R. , Storgard Chris M. , Loftus Joseph C. , Russell Stephen J.
Published: September 2005

Targeting tumor-associated vascular endothelium by replication-competent viral vectors is a promising strategy for cancer gene therapy. Here we describe the development of a viral vector based on the Edmonston vaccine strain of measles virus targeted to integrin αvβ3, which is expressed abundantly on activated but not quiescent vascular endothelium. We displayed a disintegrin, M28L echistatin that binds with a high affinity to integrin αvβ3 on the COOH terminus of the viral attachment (H) protein and rescued the replication-competent recombinant virus by reverse genetics. The new targeted virus was named measles virus echistatin vector (MV-ERV). Its native binding to CD46 was purposefully retained to allow virus infection of tumor cells expressing this receptor. MV-ERV correctly displayed echistatin on the outer surface of its envelope and produced interesting ring formation phenomena due to cell detachment upon infection of susceptible Vero cells in vitro. MV-ERV grew to 106 plaque-forming units/mL, slightly lower than the parental Edmonston strain of measles virus (MV-Edm), but it selectively infected Chinese hamster ovary cells expressing integrin αvβ3. It also selectively infected both bovine and human endothelial cells on matrigels and unlike MV-Edm, MV-ERV infected newly formed blood vessels in chorioallantoic membrane assays. In animal models, MV-ERV but not the control MV-Edm caused the regression of s.c. xenografts of resistant multiple myeloma tumors (MM1) in severe combined immunodeficient mice. The tumors were either completely eradicated or their growth was significantly retarded. The specificity, potency, and feasibility of MV-ERV infection clearly show the potential use of MV-ERV in gene therapy for targeting tumor-associated vasculature for the treatment of solid tumors.
A Novel Telomere Structure in a Human Alternative Lengthening of Telomeres Cell Line
Authors: Marciniak Robert A. , Cavazos David , Montellano Richard , Chen Qijun , Guarente Leonard , Johnson F. Brad
Published: September 2005

Cancer cells require mechanisms to maintain telomeres. Most use telomerase, but 5% to 20% of tumors use alternative lengthening of telomeres (ALT), a telomerase-independent mechanism that seems to depend on recombination. ALT is characterized by amplification of telomere TTAGGG repeats to lengths beyond 50 kb, by elevated rates of telomere recombination, and by nuclear structures called ALT-associated promyelocytic leukemia bodies. In Saccharomyces cerevisiae, survivors of telomerase inactivation also use recombination to maintain telomeres. There are two types of survivors, which differ in telomere structure. The first possesses telomere repeats and the Y′ subtelomeric element amplified together as a tandem array at chromosome termini (type I), and the other possesses amplification of telomeric repeats alone (type II), similar to previously described human ALT cells. Here, we describe the first human ALT cell line having “tandem array” telomeres with a structure similar to that of type I yeast survivors. The chromosome termini consist of a repeat unit containing ∼2.5 kb of SV40 DNA and a variable amount of TTAGGG sequence repeated in tandem an average of 10 to 20 times. Similar to previously described ALT cells, they show evidence of telomere recombination, but unlike standard ALT cells, they lack ALT-associated promyelocytic leukemia bodies and their telomeres are transcribed. These findings have implications for the pathogenesis and diagnosis of cancer.
Epigenetic Reactivation of Tumor Suppressor Genes by a Novel Small-Molecule Inhibitor of Human DNA Methyltransferases
Authors: Brueckner Bodo , Garcia Boy Regine , Siedlecki Pawel , Musch Tanja , Kliem H. Christian , Zielenkiewicz Piotr , Suhai Sandor , Wiessler Manfred , Lyko Frank
Published: September 2005

DNA methylation regulates gene expression in normal and malignant cells. The possibility to reactivate epigenetically silenced genes has generated considerable interest in the development of DNA methyltransferase inhibitors. Here, we provide a detailed characterization of RG108, a novel small molecule that effectively blocked DNA methyltransferases in vitro and did not cause covalent enzyme trapping in human cell lines. Incubation of cells with low micromolar concentrations of the compound resulted in significant demethylation of genomic DNA without any detectable toxicity. Intriguingly, RG108 caused demethylation and reactivation of tumor suppressor genes, but it did not affect the methylation of centromeric satellite sequences. These results establish RG108 as a DNA methyltransferase inhibitor with fundamentally novel characteristics that will be particularly useful for the experimental modulation of epigenetic gene regulation.
Mimicry of a Cellular Low Energy Status Blocks Tumor Cell Anabolism and Suppresses the Malignant Phenotype
Authors: Swinnen Johannes V. , Beckers Annelies , Brusselmans Koen , Organe Sophie , Segers Joanna , Timmermans Leen , Vanderhoydonc Frank , Deboel Ludo , Derua Rita , Waelkens Etienne , De Schrijver Ellen , Van de Sande Tine , Noël Agnès , Foufelle Fabienne , Verhoeven Guido
Published: September 2005

Aggressive cancer cells typically show a high rate of energy-consuming anabolic processes driving the synthesis of lipids, proteins, and DNA. Here, we took advantage of the ability of the cell-permeable nucleoside 5-aminoimidazole-4-carboxamide (AICA) riboside to increase the intracellular levels of AICA ribotide, an AMP analogue, mimicking a low energy status of the cell. Treatment of cancer cells with AICA riboside impeded lipogenesis, decreased protein translation, and blocked DNA synthesis. Cells treated with AICA riboside stopped proliferating and lost their invasive properties and their ability to form colonies. When administered in vivo, AICA riboside attenuated the growth of MDA-MB-231 tumors in nude mice. These findings point toward a central tie between energy, anabolism, and cancer and suggest that the cellular energy sensing machinery in cancer cells is an exploitable target for cancer prevention and/or therapy.
Survivin as a Radioresistance Factor, and Prognostic and Therapeutic Target for Radiotherapy in Rectal Cancer
Authors: Rödel Franz , Hoffmann Jörg , Distel Luitpold , Herrmann Martin , Noisternig Timmo , Papadopoulos Thomas , Sauer Rolf , Rödel Claus
Published: September 2005

Apoptosis levels have been shown to predict tumor response to preoperative radiochemotherapy in rectal cancer. Recently, the prominent role of survivin, a structurally unique member of the inhibitor of apoptosis protein family, has been shown in colorectal cancer tumorigenesis and prognosis. In this study, we investigated whether survivin plays a direct role in mediating radiation resistance. We used short interfering RNA molecules to decrease survivin in radioresistant SW480 and intermediately radioresistant HCT-15 colorectal cancer cells. This resulted in a significant decrease of survivin mRNA and protein expression with a maximum at 24 to 48 hours after transfection. If irradiated during this sensitive period, an increased percentage of apoptotic cells and an increased caspase 3/7 activity in parallel with a decreased cell viability and a reduced clonogenic survival was shown. These effects were more pronounced in the radioresistant SW480 cell line with a radiation-induced cytotoxicity enhancement factor at 10% and 50% survival of 1.8 to 2.2 for SW480 and 1.5 to 1.7 for HCT-15, respectively. Furthermore, transfection with survivin short interfering RNA increased levels of G2-M arrest and levels of DNA double-strand breaks in irradiated cells. These observations indicate that cell cycle and DNA repair mechanisms may be associated with apoptosis induction in tumor cells that are otherwise resistant to killing by radiation. In a translational study of 59 patients with rectal cancer treated with a combination of radiotherapy and chemotherapy, increased survivin expression was inversely related to the levels of apoptosis, and was also associated with a significantly higher risk of a local tumor recurrence.
Multiphoton Microscopy of Endogenous Fluorescence Differentiates Normal, Precancerous, and Cancerous Squamous Epithelial Tissues
Authors: Skala Melissa C. , Squirrell Jayne M. , Vrotsos Kristin M. , Eickhoff Jens C. , Gendron-Fitzpatrick Annette , Eliceiri Kevin W. , Ramanujam Nirmala
Published: September 2005

This study characterizes the morphologic features and the endogenous fluorescence in the stratified squamous epithelia of the 7,12-dimethylbenz(a)anthracene-treated hamster cheek pouch model of carcinogenesis using multiphoton laser scanning microscopy (MPLSM). MPLSM allows high-resolution, three-dimensional image data to be collected deeper within thick tissue samples with reduced phototoxicity compared with single-photon imaging. Three-dimensional image stacks of normal (n = 13), precancerous (dysplasia, n = 12; carcinoma in situ, n = 9) and cancerous tissue [nonpapillary squamous cell carcinoma (SCC), n = 10, and papillary SCC, n = 7] sites in the hamster cheek pouch were collected in viable, unsectioned tissue biopsies at a two-photon excitation wavelength of 780 nm. Five features were quantified from the MPLSM images. These included nuclear density versus depth, keratin layer thickness, epithelial thickness, and the fluorescence per voxel in the keratin and epithelial layers. Statistically significant differences in all five features were found between normal and both precancerous and cancerous tissues. The only exception to this was a lack of statistically significant differences in the keratin fluorescence between normal tissues and papillary SCCs. Statistically significant differences were also observed in the epithelial thickness of dysplasia and carcinoma in situ, and in the keratin layer thickness of dysplasia and SCCs (both nonpapillary and papillary). This work clearly shows that three-dimensional images from MPLSM of endogenous tissue fluorescence can effectively distinguish between normal, precancerous, and cancerous epithelial tissues. This study provides the groundwork for further exploration into the application of multiphoton fluorescence endoscopy in a clinical setting.
Tbx2 Is Overexpressed and Plays an Important Role in Maintaining Proliferation and Suppression of Senescence in Melanomas
Authors: Vance Keith W. , Carreira Suzanne , Brosch Gerald , Goding Colin R.
Published: September 2005

The INK4a and ARF genes found at the CDKN2A locus are key effectors of cellular senescence that is believed to act as a powerful anticancer mechanism. Accordingly, mutations in these genes are present in a wide variety of spontaneous human cancers and CDKN2A germ line mutations are found in familial melanoma. The TBX2 gene encoding a key developmental transcription factor is amplified in pancreatic cancer cell lines and preferentially amplified and overexpressed in BRCA1 and BRCA2 mutated breast tumors. Overexpression of Tbx2 and the related factor Tbx3, which is also overexpressed in breast cancer and melanomas, can suppress senescence in defined experimental systems through repression of ARF expression. However, it is not known how Tbx2 mediates its repressive effect nor whether endogenous Tbx2 or Tbx3 perform a similar antisenescence function in transformed cells. This is a particularly important question because the loss of CDKN2A in many human cancers would, in principle, bypass the requirement for Tbx2/3-mediated repression of ARF in suppressing senescence. We show here that Tbx2 is overexpressed in melanoma cell lines and that Tbx2 targets histone deacetylase 1 to the p21Cip1 (CDKN1A) initiator. Strikingly, expression of an inducible dominant-negative Tbx2 (dnTbx2) leads to displacement of histone deacetylase 1, up-regulation of p21Cip1 expression, and the induction of replicative senescence in CDKN2A-null B16 melanoma cells. In human melanoma cells, expression of dnTbx2 leads to severely reduced growth and induction of senescence-associated heterochromatin foci. The results suggest that the activity of endogenous Tbx2 is critically required to maintain proliferation and suppress senescence in melanomas.
A Novel Transgenic Mouse Model Reveals Humanlike Regulation of an 8-kbp Human TERT Gene Promoter Fragment in Normal and Tumor Tissues
Authors: Ritz Julia M. , Kühle Olaf , Riethdorf Sabine , Sipos Bence , Deppert Wolfgang , Englert Christoph , Günes Cagatay
Published: September 2005

Telomerase activity is repressed in most human somatic tissues during differentiation processes but strongly up-regulated in most human tumors. Regulation of human telomerase activity primarily occurs at the level of transcriptional initiation of the TERT gene, which encodes the catalytic subunit of telomerase. We have generated a novel transgenic mouse model to study the regulation of the human TERT gene promoter in an in vivo system. For this purpose, we have cloned an 8.0-kbp human TERT promoter fragment in front of the bacterial lacZ reporter gene (hTERTp-lacZ), which encodes the β-galactosidase enzyme. Expression of the reporter gene was monitored by reverse transcription–PCR analysis, 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside staining of whole mount preparations, and histologic sections. We find that the activity of the human TERT promoter in most normal mouse tissues recapitulates the expression of the hTERT gene in normal human tissues and is under tighter control when compared with the endogenous mouse TERT gene expression. In testis, where highest lacZ expression was observed, the expression of the reporter gene was restricted to the spermatogonial stem cells and the spermatocytes. Intriguingly, we find increased levels of lacZ expression in mammary tumors of hTERTp-lacZ × p53+/− bitransgenic mouse mammary tumor model. Thus, this transgenic mouse model provides a suitable in vivo system to analyze the expression of the human TERT gene under physiologic conditions and during tumorigenesis.
DNA Ligase III as a Candidate Component of Backup Pathways of Nonhomologous End Joining
Authors: Wang Huichen , Rosidi Bustanur , Perrault Ronel , Wang Minli , Zhang Lihua , Windhofer Frank , Iliakis George
Published: September 2005

Biochemical and genetic studies support the view that the majority of DNA double-strand breaks induced in the genome of higher eukaryotes by ionizing radiation are removed by two pathways of nonhomologous end joining (NHEJ) termed D-NHEJ and B-NHEJ. Whereas D-NHEJ depends on the activities of the DNA-dependent protein kinase and DNA ligase IV/XRCC4, components of B-NHEJ have not been identified. Using extract fractionation, we show that the majority of DNA end joining activity in extracts of HeLa cells derives from DNA ligase III. DNA ligase III fractionates through two columns with the maximum in DNA end joining activity and its depletion from the extract causes loss of activity that can be recovered by the addition of purified enzyme. The same fractionation protocols provide evidence for an additional factor strongly enhancing DNA end joining and shifting the product spectrum from circles to multimers. An in vivo plasmid assay shows that DNA ligase IV–deficient mouse embryo fibroblasts retain significant DNA end joining activity that can be reduced by up to 80% by knocking down DNA ligase III using RNA interference. These in vivo and in vitro observations identify DNA ligase III as a candidate component for B-NHEJ and point to additional factors contributing to NHEJ efficiency.
Hypersialylation of β1 Integrins, Observed in Colon Adenocarcinoma, May Contribute to Cancer Progression by Up-regulating Cell Motility
Authors: Seales Eric C. , Jurado Gustavo A. , Brunson Brian A. , Wakefield John K. , Frost Andra R. , Bellis Susan L.
Published: September 2005

Colon adenocarcinomas are known to express elevated levels of α2-6 sialylation and increased activity of ST6Gal-I, the Golgi glycosyltransferase that creates α2-6 linkages. Elevated ST6Gal-I positively correlates with metastasis and poor survival, and therefore ST6Gal-I–mediated hypersialylation likely plays a role in colorectal tumor invasion. Previously we found that oncogenic ras (present in roughly 50% of colon adenocarcinomas) up-regulates ST6Gal-I and, in turn, increases sialylation of β1 integrin adhesion receptors in colon epithelial cells. However, we wanted to know if this pattern held true in vivo and, if so, how β1 hypersialylation might contribute to colon tumor progression. In the present study, we find that β1 integrins from colon adenocarcinomas consistently carry higher levels of α2-6 sialic acid. To explore the effects of increased α2-6 sialylation on β1-integrin function, we stably expressed ST6Gal-I in a colon epithelial cell line lacking endogenous ST6Gal-I. ST6Gal-I expressors (with α2-6 sialylated β1 integrins) exhibited up-regulated attachment to collagen I and laminin and increased haptotactic migration toward collagen I, relative to parental cells (with completely unsialylated β1 integrins). Blockade of ST6Gal-I expression with short interfering RNA reversed collagen binding back to the level of ST6Gal-I nonexpressors, confirming that α2-6 sialylation regulates β1 integrin function. Finally, we show that β1 integrins from ST6Gal-I expressors have increased association with talin, a marker for integrin activation. Collectively, these findings suggest that β1 hypersialylation may augment colon tumor progression by altering cell preference for certain extracellular matrix milieus, as well as by stimulating cell migration.
Versatile Prostate Cancer Treatment with Inducible Caspase and Interleukin-12
Authors: Nikitina Ekaterina Yu. , Desai Smruti A. , Zhao Xiuqin , Song Weitao , Luo Annie Z. , Gangula Rama D. , Slawin Kevin M. , Spencer David M.
Published: September 2005

To establish optimized conditions for immunity against prostate cancer, we compared the efficacy of multiple approaches in autochthonous and s.c. transgenic adenocarcinoma of the mouse prostate (TRAMP)-based models. Mice immunized with interleukin (IL)-12–containing apoptotic, but not necrotic TRAMP-C2 cell–based, vaccines were resistant to TRAMP-C2 tumor challenge and re-challenge, independently of the route of vaccination (s.c. or i.p.). Administration of γ-irradiated TRAMP-C2 cells preinfected with adenovirus containing both B7-1 and IL-12 genes, unlike adenovirus containing B7-1 alone, considerably protected C57BL/6 mice from TRAMP-C2 tumor growth and extended the life span of TRAMP mice. Vaccines that included dendritic cells, instead of IL-12, were equally efficient. Whereas injections of ligand-inducible caspase-1– and IL-12–containing adenoviruses cured small s.c. TRAMP-C2 tumors, nanopump-regulated delivery of viruses led to elimination of much larger tumors. The antitumor immune responses involved CD4+-, CD8+-, and natural killer cells and were strengthened by increasing the number of vaccinations. Intraprostatic administration of inducible caspase-1– and IL-12–containing adenoviruses resulted in local cell death and improved survival of adenocarcinoma-bearing TRAMP mice. Thus, tumor cell apoptosis induced by caspase in situ and accompanied by IL-12 is efficient against prostate cancer in a preclinical model.
8-Chloro-Cyclic AMP–Induced Growth Inhibition and Apoptosis Is Mediated by p38 Mitogen-Activated Protein Kinase Activation in HL60 Cells
Authors: Ahn Young-Ho , Jung Joong Mok , Hong Seung Hwan
Published: September 2005

8-Chloro-cyclic AMP (8-Cl-cAMP), which is known to induce growth inhibition, apoptosis, and differentiation in various cancer cell lines, has been studied as a putative anticancer drug. However, the mechanism of anticancer activities of 8-Cl-cAMP has not been fully understood. Previously, we reported that the 8-Cl-cAMP-induced growth inhibition is mediated by protein kinase C (PKC) activation. In this study, we found that p38 mitogen-activated protein kinase (MAPK) also plays important roles during the 8-Cl-cAMP-induced growth inhibition and apoptosis. SB203580 (a p38-specific inhibitor) recovered the 8-Cl-cAMP-induced growth inhibition and apoptosis, whereas other MAPK inhibitors, such as PD98059 (an extracellular signal-regulated kinase–specific inhibitor) and SP600125 (a c-Jun NH2-terminal kinase–specific inhibitor), had no effect. The phosphorylation (activation) of p38 MAPK was increased in a time-dependent manner after 8-Cl-cAMP treatment. Furthermore, SB203580 was able to block PKC activation induced by 8-Cl-cAMP. However, PKC inhibitor (GF109203x) could not attenuate p38 activation, indicating that p38 MAPK activation is upstream of PKC activation during the 8-Cl-cAMP-induced growth inhibition. 8-Chloro-adenosine, a metabolite of 8-Cl-cAMP, also activated p38 MAPK and this activation was blocked by adenosine kinase inhibitor. These results suggest that 8-Cl-cAMP exerts its anticancer activity through p38 MAPK activation and the metabolite(s) of 8-Cl-cAMP mediates this process.
Lower Induction of p53 and Decreased Apoptosis in NQO1-Null Mice Lead to Increased Sensitivity to Chemical-Induced Skin Carcinogenesis
Authors: Iskander Karim , Gaikwad Amos , Paquet Marilene , Long Delwin J. , Brayton Cory , Barrios Roberto , Jaiswal Anil K.
Published: September 2005

NAD(P)H:quinone oxidoreductase 1 (NQO1) is a cytosolic protein that catalyzes metabolic detoxification of quinones and protects cells against redox cycling and oxidative stress. NQO1-null mice deficient in NQO1 protein showed increased sensitivity to 7,12-dimethylbenz(a)anthracene– and benzo(a)pyrene-induced skin carcinogenesis. In the present studies, we show that benzo(a)pyrene metabolite benzo(a)pyrene-trans-7,8-dihydrodiol-9,10-epoxide and not benzo(a)pyrene quinones contributed to increased benzo(a) pyrene-induced skin tumors in NQO1-null mice. An analysis of untreated skin revealed an altered intracellular redox state due to accumulation of NADH and reduced levels of NAD/NADH in NQO1-null mice as compared with wild-type mice. Treatment with benzo(a)pyrene failed to significantly increase p53 and apoptosis in the skin of NQO1-null mice when compared with wild-type mice. These results led to the conclusion that altered intracellular redox state along with lack of induction of p53 and decreased apoptosis plays a significant role in increased sensitivity of NQO1-null mice to benzo(a)pyrene-induced skin cancer.
Potent Selection of Antigen Loss Variants of B16 Melanoma following Inflammatory Killing of MelanocytesIn vivo
Authors: Sanchez-Perez Luis , Kottke Timothy , Diaz Rosa Maria , Ahmed Atique , Thompson Jill , Chong Heung , Melcher Alan , Holmen Sheri , Daniels Gregory , Vile Richard G.
Published: September 2005

We have reported that i.d. injection of plasmids encoding hsp70 and a suicide gene transcriptionally targeted to melanocytes generates specific proinflammatory killing of melanocytes. The resulting CD8+ T cell response eradicates systemically established B16 tumors. Here, we studied the consequences of that CD8+ T cell response on the phenotype of preexisting tumor. In suboptimal protocols, the T cell response selected B16 variants, which grow extremely aggressively, are amelanotic and have lost expression of the tyrosinase and tyrosinase-related protein 2 (TRP-2) antigens. However, expression of other melanoma-associated antigens, such as gp100, was not affected. Antigen loss could be reversed by long-term growth in culture away from immune-selective pressures or within 96 hours by treatment with the demethylating agent 5-azacytidine (5-Aza). When transplanted back into syngeneic animals, variants were very poorly controlled by further vaccination. However, a combination of vaccination with 5-Aza to reactivate antigen expression in tumors in situ generated highly significant improvements in therapy over treatment with vaccine or 5-Aza alone. These data show that inflammatory killing of normal cells activates a potent T cell response targeted against a specific subset of self-antigens but can also lead to the immunoselection of tumor variants. Moreover, our data indicate that emergence of antigen loss variants may often be due to reversible epigenetic mechanisms within the tumor cells. Therefore, combination therapy using vaccination and systemic treatment with 5-Aza or other demethylating agents may have significant therapeutic benefits for antitumor immunotherapy.