CANCER GENOMICS & PROTEOMICS
Volume 5, Number
5, September-October 2008
CONTENTS
PAGE
Secondary Structure at a Hot Spot for DNA Methylation in DNA from Human Breast Cancers. J. CLARK, S.S. SMITH (Duarte, CA, USA)
241
Comparative Clinical and Transcriptomal Profiles of Breast Cancer Between French and South Mediterranean PatientsShow Minor but Significative Biological Differences. N. CHALABI, D.J. BERNARD-GALLON,Y.-J. BIGNON, THE BREAST MED CONSORTIUM: F. KWIATKOWSKI, M. AGIER, V. VIDAL, V. LAPLACE-CHABAUD, V. SYLVAIN-VIDAL, V. BERTHOLET, F. DE LONGUEVILLE, M. LACROIX, G. LECLERCQ, J. REMACLE, C. SIBILLE, N. ZAMMATEO, N. BEN JAAFAR, A. SEFIANI, K. OULDIM, A. MEGARBANE, N. JALKH, W. MAHFOUDH, W. TROUDI, A. BEN AMMAR-EL GAIED, L. CHOUCHANE (Clermont- Ferrand; Saint-Beauzire, France; Namur, Belgium; Rabat, Maroc; Bayrout, Liban; Tunis, Tunisie)
253
Tissue Microarrays of Human Tumor Xenografts: Characterization of Proteins Involved in Migration and Angiogenesis for Applications in the Development of Targeted Anticancer Agents. V. SMITH, G.J. WIRTH, H.- H. FIEBIG, A.M. BURGER (Freiburg, Germany; Geneva, Switzerland; Detroit, MI, USA)
263
Improved Effector Function of Leukemia-specific T-lymphocyte Clones Trained with AML-derived Dendritic Cells. F.R. SCHUSTER, R. BUHMANN, S. REUTHER, B. HUBNER, C. GRABRUCKER, A. LIEPERT, R. REIBKE, P. LICHTNER, T. YANG, T. KROELL, H.-J. KOLB, A. BORKHARDT, H. SCHMETZER (Dusseldorf; Munich, Germany)
275
Genomic Changes of the 55 kDa Subunit of DNA Polymerase ε in Human Breast Cancer. Q. ZHOU, R. EFFATI, K. TALVINEN, H. POSPIECH, J.E. SYVAOJA, Y. COLLAN (Turku; Oulu; Joensuu, Finland)
287
CANCER GENOMICS & PROTEOMICS 5: 241-252 (2008)
Secondary Structure at a Hot Spot for DNA Methylation in DNA from Human Breast Cancers
JARROD CLARK, STEVEN S. SMITH
City of Hope, 1500 E. Duarte Rd., Duarte, CA, U.S.A.
Abstract: The VNTR at c-Ha-ras resides in a hotspot for DNA methylation on chromosome 11 in human tumors, where it is flanked by two MspI restriction sites. We have investigated the nature of the MspI site polymorphism at the c-Ha-ras VNTR observed in variety of tumors including breast cancer.We find that the MspI site 5' to the VNTR is present in a Non-B DNA structure with single-strand character that renders it accessible to bisulfite modification under native conditions, while the MspI site 3' to the VNTR appears to reside in a normal B-form structure that is inaccessible to bisulfite. The non-B DNA structure accounts for the observed polymorphism since MspI cannot cleave single-stranded DNA and control experiments show that the MspI sites were neither mutated nor abnormally methylated. Southern blotting showed that structural polymorphism was present in tumor DNA and tumor adjacent normal tissue DNA but absent from lymphocyte DNA from the same patients. We conclude that the non-B DNA structural polymorphism detected in human tumors near the c-Ha-ras VNTR is a self-perpetuating epigenetic mark that manifests itself spontaneously during breast carcinogenesis in a methylation hot spot.
Comparative Clinical and Transcriptomal Profiles of Breast Cancer Between French and South Mediterranean Patients Show Minor but Significative Biological Differences
N. CHALABI1,2, D.J. BERNARD-GALLON1,2, Y.-J. BIGNON1,2,3 and THE BREAST MED CONSORTIUM: F. KWIATKOWSKI1, M. AGIER1,6, V. VIDAL1,6, V. LAPLACE-CHABAUD1,6, V. SYLVAIN-VIDAL1,6, V. BERTHOLET7, F. DE LONGUEVILLE7, M. LACROIX7, G. LECLERCQ7, J. REMACLE7, C. SIBILLE8, N. ZAMMATEO8, N. BEN JAAFAR9, A. SEFIANI9, K. OULDIM9, A. MÉGARBANÉ10, N. JALKH10, W. MAHFOUDH11, W. TROUDI11, A. BEN AMMAR-EL GAÏED11, L. CHOUCHANE12
1Département d'Oncogénétique, Centre Jean Perrin, 63011 ClermontFerrand Cedex 01; 2Centre de Recherche en Nutrition Humaine (CRNH), 63009 ClermontFerrand Cedex 01; 3Université d'Auvergne, 63001 Clermont Ferrand 1; 4Diagnogene, Division d'Imaxio, Biopôle ClermontLimagne, 63360 SaintBeauzire, France; 5Université de Namur, URBC, 5000 Namur; 6Eppendorf Array Technologies, 5000 Namur, Belgium; 7Département de Génétique et de Biologie Moléculaire, Institut National d'Hygiene, Rabat, Maroc; 8Unité de Génetique Médicale, Université SaintJoseph, Bayrout, Liban; 9Laboratoire d'Immunologie, Institut Pasteur de Tunis; 10Laboratoire d'ImmunoOncologie Moléculaire, Faculté de Médecine de Monastir, Université de Monastir, Tunis, Tunisie.
Abstract: Background: In Western countries, breast cancer incidence and mortality are higher than in Mediterranean countries. These differences have been ascribed to environmental factors but also to late-stage diagnostic and biological specific characteristics. Patients and Methods: Between September 2002 and September 2005, we collected clinical data by phone counselling 180 French and Mediterranean breast cancer patients and performed microarray experiments. Results: Characteristics of breast cancer in patients from Lebanon, Tunisia and Morocco were more aggressive (more SBR grade III and positive node invasion) and patients were 10 years younger at diagnosis. Sixteen differentially expressed genes such as MMP9, VEGF, PHB1, BRCA1, TFAP2C, GJA1 and TFF1 were also found. Additionally, an up-regulation of cytokeratins KRT8 and KRT18 may indicate a luminal B subtype in "South" (Lebanon, Tunisia and Morocco) tumors while "North" (France) tumors may more frequently be luminal A type. Conclusion: This study allowed the identification of specific clinical and transcriptomic parameters in patients from South Mediterranean countries.
Tissue Microarrays of Human Tumor Xenografts: Characterization of Proteins Involved in Migration and Angiogenesis for Applications in the Development of Targeted Anticancer Agents
VICTORIA SMITH1, GREGORY J. WIRTH2, HEINZ-HERBERT FIEBIG1,3, ANGELIKA M. BURGER
1Institute for Experimental Oncology, Oncotest GmbH, Freiburg, Germany; 2Clinic of Urology, University of Geneva, Switzerland; 3Tumor Biology Center at the University of Freiburg, Clinic for Medical Oncology, Freiburg, Germany; 4Barbara Ann Karmanos Cancer Institute and Department of Pharmacology, Wayne State University School of Medicine, Detroit, MI, U.S.A..
Abstract: As new target-directed anticancer agents emerge, preclinical efficacy studies need to integrate target-driven model systems. This approach to drug development requires rapid and reliable characterization of the new targets in established tumor models, such as xenografts and cell lines. Here, we have applied tissue microarray technology to patient-derived, re-growable human tumor xenografts. We have profiled the expression of five proteins involved in cell migration and/or angiogenesis: vascular endothelial growth factor (VEGF), matrix metalloproteinase 1 (MMP1), protease activated receptor (PAR1), cathepsin B, and â1 integrin in a panel of over 150 tumors and compared their expression levels to available patient outcome data. For each protein, several target overexpressing xenografts were identified. They represent a subset of tumor models prone to respond to specific inhibitors and are available for future preclinical efficacy trials. In a "proof of concept" experiment, we have employed tissue microarrays to select in vivo models for therapy and for the analysis of molecular changes occurring after treatment with the anti-VEGF antibody HuMV833 and gemcitabine. Whereas the less angiogenic pancreatic cancer PAXF736 model proved to be resistant, the highly vascularized PAXF546 xenograft responded to therapy. Parallel analysis of arrayed biopsies from the different treatment groups revealed a down-regulation of Ki-67 and VEGF, an altered tissue morphology, and a decreased vessel density. Our results demonstrate the multiple advantages of xenograft tissue microarrays for preclinical drug development.
Improved Effector Function of Leukemia-specific T-lymphocyte Clones Trained with AML-derived Dendritic Cells
IEDHELM R. SCHUSTER1* RAYMUND BUHMANN2,3*, SUSANNE REUTHER1, BERND HUBNER1, CHRISTINE GRABRUCKER3, ANJA LIEPERT3, ROLAND REIBKE3, PETER LICHTNER2, TING YANG2, TANJA KROELL3, HANS-JOCHEM KOLB3, ARNDT BORKHARDT1, HELGA SCHMETZER2,3
1Department of Pediatric Oncology, Hematology and Immunology, Heinrich Heine University Medical Center, Dusseldorf; 2HelmholtzZentrum Munchen, German Research Center for Environmental Health; 3Department for Hematopoietic Transplantation, University of Munich, Germany
Abstract: Recently it was shown that myeloid leukemic cells can be induced to differentiate into leukemia-derived dendritic cells (DCleu), regaining the stimulatory capacity of professional DCs while presenting the leukemic antigen repertoire. But so far, the induced antileukemic T-cell responses have varied in specificity and efficacy, or have even mediated opposite effects. In an attempt to further characterize the DC/DCleu induced T-cell response pattern, immunoscope spectratyping, a novel and powerful tool to detect T-cell receptor (TCR) rearrangements was used in combination with functional flow cytometry and non-radioactive fluorolysis assays. Human leucocyte antigen (HLA) matched donor T-cells were repeatedly stimulated, either with leukemic blasts (French-American-British, FAB M4eo) or the corresponding blast-derived DCs. Functional comparison revealed no significant difference in their T-cell stimulatory capacity, while the DC/DCleu fraction favored T-cells with a higher lytic activity, comprising a higher proportion of T-memory CD45R0+ cells. Stimulation with blasts and DC/DCleu induced a similar TCR restriction pattern, while stimulation with DC/DCleu favored the CD4 T-cell subset and seemed to cause a higher grade of restriction. In conclusion, a combined strategy using spectratyping with functional tests might not only provide useful information about the specificity and efficacy of the induced T-cell response, but also pave the way to gain effective T-cell clones for therapeutic use.
Genomic Changes of the 55 kDa Subunit of DNA Polymerase ε in Human Breast Cancer
QI ZHOU1, REZA EFFATI1, KATI TALVINEN1, HELMUT POSPIECH2, JUHANI E. SYVAOJA2,3, YRJO COLLAN1
1Department of Pathology, University of Turku, FIN-20520 Turku; 2Biocenter Oulu and Department of Biochemistry, P.0.Box 3000, University of Oulu, FIN-90014 Oulu; 3Department of Biology, P.O, Box 111, University of Joensuu, FIN-80101 Joensuu, Finland
Abstract: Background: DNA polymerases (Pols) represent potential candidates for cancer genes because of their central functions in DNA metabolism. Defects of some DNA Pols have shown cancer associations, but data on DNA polymerase (Pol) ε is limited. Materials and Methods: Twenty-four human breast cancer DNA samples and four control DNA samples were examined for possible mutation in the entire coding region of the 55 kDa small subunit of the human DNA Pol ε gene using polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) analysis of the DNA and sequence analysis. In addition, 20 control DNAs were studied with PCR-SSCP for the end of intron 18 and exon 19 region. Results: An AATT deletion was found at one location in intron 18 in 2 out of the 24 breast cancer cases (8%), but in none of the control cases. In addition, a single base transition was found in the cancer DNAs in intron 14, but the same changes were also found in the control DNAs, suggesting polymorphism. Conclusion: Specific changes might occur in the 55 kDa small subunit DNA sequence of DNA Pol ε in breast cancer. The deletion at the region of intron-exon junction may not affect the protein code, but could potentially influence splicing efficiency and expression levels, possibly impairing the function of Pol ε DNA.
