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ONCOLOGY RESEARCH
AN INTERNATIONAL JOURNAL
INCORPORATING ANTI-CANCER DRUG DESIGN

ABSTRACTS
VOLUME 15, NUMBER 3

Oncology Research, Volume 15, pp. 113-128
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Copyright © 2005 Cognizant Comm. Corp.
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Activation of Cyclin D1 by Estradiol and Spermine in MCF-7 Breast Cancer Cells: A Mechanism Involving the p38 MAP Kinase and Phosphorylation of ATF-2

Joan S. Lewis,1* Veena Vijayanathan,1 T. J. Thomas,1,3 Richard G. Pestell,5 Chris Albanese,5 Michael A. Gallo,2,3,4 and Thresia Thomas2,3,4

1Department of Medicine, University of Medicine and Dentistry of New Jersey-Robert Wood Johnson Medical School, New Brunswick, New Jersey, NJ 08903, USA
2Environmental and Occupational Medicine, University of Medicine and Dentistry of New Jersey-Robert Wood Johnson Medical School, New Brunswick, New Jersey, NJ 08903, USA
3The Cancer Institute of New Jersey, University of Medicine and Dentistry of New Jersey-Robert Wood Johnson Medical School, New Brunswick, New Jersey, NJ 08903, USA
4Environmental and Occupational Health Sciences Institute, University of Medicine and Dentistry of New Jersey-Robert Wood Johnson Medical School, New Brunswick, NJ 08903, USA
5Department of Oncology, Lombardi Cancer Center, Georgetown University, Washington, DC 20057, USA

Estradiol (E2) and the naturally occurring polyamines (putrescine, spermidine, and spermine) play important roles in breast cancer cell growth and differentiation. We examined the effects of E2 and spermine on the phosphorylation and DNA binding of activating transcription factor-2 (ATF-2) in MCF-7 breast cancer cells. ATF-2 is a transcription factor involved in estrogenic regulation of cyclin D1 gene, and thereby cell cycle progression. DNA affinity immunoblot assays showed a six- to eightfold increase in the binding of ATF-2 to a 74-mer ATF/CRE oligonucleotide (ODN1) from cyclin D1 promoter in the presence of 4 nM E2 and 0.5 mM spermine, compared to untreated control. Individual treatments with E2 or spermine caused a twofold or lower increase in ATF-2 binding to ODN1. Immunoblotting with phospho-ATF-2 antibody showed that increased DNA binding of ATF-2 was associated with its phosphorylation. A p38 MAP kinase inhibitor, PD169316, inhibited ATF-2 phosphorylation. In contrast, the MEK-ERK1/2 inhibitor, PD98059, or the JNK inhibitor, SP600125, had no significant effect on DNA binding of ATF-2. Cyclin D1 promoter (-1745CD1) activity increased by approximately 12-fold (above control) in the presence of E2 and spermine, compared to a sixfold increase in the presence of E2 alone and a twofold increase with spermine. Cells transfected with a dominant negative mutant of ATF-2 showed decreased transactivation of cyclin D1 promoter in response to E2 and spermine. These results indicate that spermine can enhance E2-induced cell signaling and cyclin D1 transcription by activation of the p38 MAP kinase and phosphorylation of ATF-2, contributing to breast cancer cell proliferation.

Key words:ATF-2; Cyclin D1; Estradiol; Polyamines; MCF-7 cells

Address correspondence to Thresia Thomas, 125 Paterson Street, Clinical Academic Building, Room 7092, New Brunswick, NJ 08903, USA. Tel: (732) 235-8458; Fax: (732) 235-8473; E-mail: thomasth@umdnj.edu

*Present address: Fox Chase Cancer Center, 333 Cottman Drive, Philadelphia, PA 19111.




Oncology Research, Volume 15, pp. 129-138
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Copyright © 2005 Cognizant Comm. Corp.
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In Vitro Antitumor Structure-Activity Relationships of threo/trans/threo/trans/erythro bis-Tetrahydrofuranic Acetogenins: Correlations With Their Inhibition of Mitochondrial Complex I

José R. Tormo,1 Nuria DePedro,1 Inmaculada Royo,1 Isabel Barrachina,2 M. Carmen Zafra-Polo,2 Cristina Cuadrillero,1 Pilar Hernández,1 Diego Cortes,1,2 and Fernando Peláez1

1CIBE-Merck Research Laboratories, MERCK, SHARP & DOHME de ESPAÑA S.A., C/Josefa Valcárcel, 38, Madrid 28027, Spain
2Departament de Farmacologia Facultat de Farmàcia, Universitat de València, Avgd. Vicent Andrés Estellés s/n, Burjassot 46100, València, Spain

Annonaceous acetogenins are known to be cytotoxic against tumor cell lines by virtue of their inhibition of mitochondrial complex I. We decided to conclude part of our recent revisions of the different structure-activity relationships (SARs) found within these compounds with a detailed description of the cytotoxic activity, and correlations with the inhibition of the target enzyme, of the broadest subclass of this family of natural products, the bis-tetrahydrofuranic acetogenins (bis-THF ACGs) of threo/trans/threo/trans/erythro relative configuration. Five naturally occurring ACGs and more than 10 semisynthetic analogs were tested against the MCF-7 (breast), A-549 (lung), HepG2 (liver), HT-29 (colon), MES-SA (ovary), and a multidrug-resistant (MDR-MES-SA/Dx5) cell lines using the MTT cytotoxicity assay to determine if the mitochondrial complex I inhibition correlated with the in vitro antitumor potency of the most common ACGs. Results indicated that a previously observed trend for other subclasses of ACGs between the ED50 of the cytotoxicity assay and the polarity of compounds was not present in this set and that there were several specific interactions that enhanced the antitumor activity. For example, some of the guanacone derivatives prepared were two orders of magnitude more potent than the parent compound for specific cell lines.

Key words: Annonaceous acetogenins; Mitochondrial complex I; Structure-activity relationships; Cytotoxic activity

Address correspondence to José R. Tormo, CIBE-Merck Research Laboratories, MERCK, SHARP & DOHME de ESPAÑA S.A., C/Josefa Valcárcel, 38, Madrid 28027, Spain. Tel: 34.91.321.04.92; E-mail: jose_tormo@merck.com




Oncology Research, Volume 15, pp. 139-150
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Mutant IkBa Suppresses Hypoxia-Induced VEGF Expression Through Downregulation of HIF-1a and COX-2 in Human Glioma Cells

Yoshihira Kimba,1 Tatsuya Abe,1 Jian Liang Wu,1 Ryo Inoue,1 Minoru Fukiki,1 Kimitoshi Kohno,2 Hidenori Kobayashi,2

1Department of Neurosurgery, Oita University School of Medicine, 1-1, Idaigaoka, Hasama-machi, Oita, 879-5593 Japan
2Department of Molecular Biology, University of Occupational and Environmental Health, School of Medicine, Fukuoka 807-8555, Japan

Our previous study demonstrated that mutant IkBa (IkBaM) could inhibit glioma angiogenesis and tumorigenesis through the downregulation of vascular endothelial growth factor (VEGF) and IL-8. However, the pathways involved in VEGF expression are not well understood. Growing evidence indicates that hypoxia-inducible factor-1a (HIF-1a) and cyclooxygenases-2 (COX-2) play important roles in this progression. In this study, we first examined the expressions of hypoxia-induced genes in human glioma cells transfected with IkBaM (IN500DM) or control plasmid (IN500D) in vitro. We found that hypoxic stress induced the expressions of HIF-1a, COX-2, and VEGF, and that IkBaM completely suppressed these expressions in vitro. Next, we injected these glioma cells into nude mice. After 3 weeks, the mice were moved to a hypoxic chamber (10% oxygen) for 3, 12, 24, 48, 96, or 144 h. The expressions of HIF-1a, COX-2, and VEGF in vivo were then analyzed by Northern blot and immunohistochemistry. IkBaM suppressed the expression of hypoxia-induced HIF-1a gene in vivo, but hypoxic stress induced the expression of COX-2 after 72 h. VEGF induction followed after 96 h of hypoxia in IN500DM cells. These findings suggest that VEGF expression appears to be regulated through dual interdependent mechanisms involving HIF-1 and COX-2 genes, and IkBaM could inhibit VEGF expression through these two pathways. Thus, IkBaM is identified as a pivotal factor in angiogenesis and is a potential target for neoplasm therapy.

Key words: Hypoxia; HIF-1H; VEGF; NF-kB; COX-2; Angiogenesis

Address correspondence to Tatsuya Abe, Department of Neurosurgery, Oita University School of Medicine, Idaigaoka 1-1, Hasama, Oita, 879-5593, Japan. Tel: +81-97-586-5862; Fax: +81-97-586-5869; E-mail: abet@med.oita-u.ac.jp




Oncology Research, Volume 15, pp. 151-160
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Influence of p53 Status on the HSV-Tk/GCV-Induced Bystander Effect in a Panel of Human Ovarian Carcinoma Cell Lines

Ingrid J. van Dillen,3 Nanno H. Mulder,1 Wim J. Sluiter,2 Coby Meijer,1 Steven de Jong,1 Jadranka Loncarek,4 Marc Mesnil,5 Erik F. J. de Vries,3 Willem Vaalburg,3 and Geke A. P. Hospers,1

1Department of Medical Oncology, Groningen University Hospital, PO Box 30.001, 9700 RB Groningen, The Netherlands
2Department of Pathology, Groningen University Hospital, PO Box 30.001, 9700 RB Groningen, The Netherlands
3PET Center, Groningen University Hospital, PO Box 30.001, 9700 RB Groningen, The Netherlands
4Department of Biochemistry and Molecular Biology, Faculty of Pharmacy and Biochemistry, University of Zagreb, Ante Kovacica 1, 10 000 Zagreb, Croatia
5Equipe Communications Jonctionnelles, Laboratoire des Biomembranes et Signalisation Cellulaire, CNRS-UMR 6558, Universite de Poitiers, 40, Avenue du Recteur Pineau, 86022 Poitiers, cedex 5, France

The p53 protein is mutated in 50% of all human tumors and plays a key role in apoptosis, cell cycle, and the expression of several genes. We investigated if p53 mutations influence the herpes simplex virus thymidine kinase (HSV-tk)/ganciclovir (GCV)-induced bystander effect (BE). Additionally, we studied some of the underlying mechanisms. GCV sensitivity and BE were studied in a human ovarian carcinoma cell line transfected with an empty vector (A2780/cmv) or a vector containing a p53 hotspot mutation at codon 175 (A2780/m175) or 248 (A2780/m248). In addition, expression levels of two nucleoside analogue transporters, multidrug resistance-related protein 4 and 5 (MRP4/MRP5), were determined. Finally, differences in gap junctional intercellular communication (GJIC) were studied by determining connexin 43 (Cx43) expression and by modulating GJIC with 18-α-glycyrrhetinic acid. Our results showed that compared to A2780/cmv, GCV sensitivity was significantly decreased in A2780/m175 and A2780/m248. Additionally, a significant BE (relative increase in cell kill) was found in A2780/cmv and A2780/m248. In contrast, an increased survival was observed in A2780/m175. No MRP4 or MRP5 expression was detected. However, all A2780 cell lines expressed Cx43. Modulating the GJIC significantly decreased the BE in A2780/cmv and A2780/m248. In conclusion, HSV-tk/GCV-induced BE is influenced by p53 mutations. Differences in GJIC could be one of the underlying mechanisms.

Key words: Herpes simplex virus thymidine kinase; Ganciclovir; Bystander effect; p53; Gap junctional intercellular communication; Multidrug resistance

Address correspondence to Geke A. P. Hospers, Department of Medical Oncology, Groningen University Hospital, PO Box 30.001, 9700 RB Groningen, The Netherlands. Tel: +31-50-3612821; Fax: +31-50-3614862; E-mail: g.a.p.hospers@int.azg.nl




Oncology Research, Volume 15, pp. 161-168
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Copyright © 2005 Cognizant Comm. Corp.
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Caspases-Dependent Cleavage of Mitotic Checkpoint Proteins in Response to Microtubule Inhibitor

Kwan-Hyuck Baek,1* Hyun-Jin Shin,1* Sook-Jung Jeong,1 Joong-Won Park,1 Frank McKeon,2 Chang-Woo Lee,1 and Chang-Min Kim1

1Research Institute, National Cancer Center, Goyang, Gyeonggi, 411-764, Korea
2Department of Cell Biology, Harvard Medical School, Boston, MA 02115, USA

The mitotic checkpoint ensures the fidelity of chromosomal segregation by delaying the onset of anaphase until all chromosomes are aligned on the metaphase plate. After sustained mitotic arrest, however, cells eventually exit mitosis without the mitotic checkpoint being silenced. These cells then undergo apoptosis, an event that is important for prevention of the chromosomal instability observed in human cancers. An interesting question is to establish the biochemical link between the mitotic checkpoint and the subsequent apoptotic cell death. Here, we found that following prolonged spindle damage, the mitotic checkpoint kinases such as Bub1 and BubR1 were cleaved through a mechanism sensitive to caspases inhibitor. Interestingly, the expression of these mutants resistant to caspases-dependent cleavage led to increased apoptosis after sustained mitotic arrest, and a correspondingly more efficient elimination of the polyploid population than that seen in cells expressing wild-type proteins. These findings provide the novel biochemical properties of mitotic checkpoint proteins through its cleavage by caspases-dependent manner.

Key words: Mitotic checkpoint proteins; Apoptotic cell death; Caspase-mediated cleavage; Polyploidy

Address correspondence to Chang-Woo Lee, Research Institute, National Cancer Center, Goyang, Gyeonggi, 411-764, Korea. Tel: 82-31-920-2385; Fax: 82-31-920-1949; E-mail: cwlee@ncc.re.kr

*These two authors contributed equally to this work.




Oncology Research, Volume 15, pp. 169-
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Copyright © 2005 Cognizant Comm. Corp.
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Tumor Transition Zone: A Putative New Morphological and Functional Hallmark of Tumor Aggressiveness

Oscar D. Bustuoabad,1* Raúl A. Ruggiero,1* Pedro di Gianni,1 M. Gabriela Lombardi,1 Carolina Belli,1 Gabriela V. Camerano,1 Graciela Dran,1 Carolina Schere-Levy,1 Héctor Costa,1 Martín A. Isturiz,1 Marina Narvaitz,1 Nico van Rooijen,3 Victoria de los A. Bustuoabad,1 and Meiss, Roberto P.2

1División Medicina Experimental, Instituto de Investigaciones Hematológicas, Academia Nacional de Medicina, Pacheco de Melo 3081, 1425 Buenos Aires, Argentina
2Departamento de Patología, Centro de Estudios Oncológicos, Academia Nacional de Medicina, Pacheco de Melo 3081, 1425 Buenos Aires, Argentina
3Department of Cell Biology and Immunology, Faculty of Medicine, Vrije Universiteit, Amsterdam, The Netherlands

A small primary or secondary tumor load can occasionally induce more deleterious effects than a histologically identical larger one. In the four murine models studied herein this enhanced tumor aggressiveness could not be attributed to NRAS mutations or other hereditary changes, differential vascularization of live tumor tissues, or necrosis content. Instead, the main tumor feature associated with a more aggressive behavior was the presence of a high number of vessels, sometimes filled with inflammatory cells, inside a tumor area, which we have identified and designated as the transition zone between the live and the necrotic zones. Our experiments suggest that during tumor growth, different cachectic factors are produced within the transition and necrotic zones by dying tumor cells and by tumor infiltrating macrophages only reaching the general circulation through the vessels present in the transition zone. Therefore, a small tumor displaying high vascularization of its transition area could be harmful to its host, while, in contrast, a large tumor could behave as a relatively benign one if its transition zone exhibited little or no vascularization, and in consequence its cachectic factors remained "trapped." Similar histological images to those observed in mice were seen in a significant percentage of human cancer biopsies, raising the possibility that such images might have a prognostic value.

Key words: Tumor transition zone; Vascularization; Inflammation; Tumor aggressiveness

Address correspondence to Roberto P. Meiss, Departamento de Patología, Centro de Estudios Oncológicos, Academia Nacional de Medicina, Pacheco de Melo 3081, 5to. piso, 1425, Buenos Aires, Argentina. Tel/Fax: 54-11-4805-8176; E-mail: patologia@oncologia.anm.edu.ar

*These two authors contributed equally to this work.