Skip to main content

CHARACTERIZATION OF SOMATIC MUTATIONS DURING RADIATION INDUCED OSTEOSARCOMAGENESIS

Objective

THE PROJECT REPRESENTS A CONTINUATION OF A COLLABORATION UNDER THE COORDINATION OF THE EUROPEAN LATE EFFECTS PROJECT (EULEP) BETWEEN OUR LABORATORY AND THE RESEARCH GROUPS OF DR. V. ERFLE AT GESELLSCHAFT FUER STRAHLEN- UND UMWELTFORSCHUNG (GSF), NEUHERBERG, FRG, AND OF DR. J. MERREGAERT AT STUDIENZENTRUM VOOR KERNENERGIE (SCK/CEN), MOL, BELGIUM. OUR ROLE IN THESE STUDIES IS TO PERFORM MOLECULAR CHARACTERIZATION OF RETROVIRUSES AND CELLULAR GENES ISOLATED DURING RADIATION OSTEOSARCOMAGENESIS AND DERIVED FROM THE LABORATORIES OF GSF AND SCK/CEN.
INTERNAL ALPHA- AND BETA-RADIATION IN BONE TISSUE BY ADMINISTRATION OF OSTEOTROPIC RADIONUCLIDES LEADS TO THE DEVELOPMENT OF OSTEOSARCOMAS AT HIGH FREQUENCIES IN A NUMBER OF INBRED STRAINS OF MICE. THE DEVELOPMENT OF SUCH TUMOURS IS FREQUENTLY ASSOCIATED WITH THE EXPRESSION OF ENDOGENOUS TYPE C RETROVIRUS. VIRUS CAN BE ISOLATED BOTH FROM BONE TISSUE EARLY AFTER THE START OF IRRADIATION AND FROM TUMOURS. THESE UNCLONED VIRUS ISOLATES CAN INDUCE OSTEOMAS, LYMPHOMAS, AND OSTEOPETROSES BY INJECTION INTO NEWBORN MICE. THESE OBSERVATIONS RAISE THE POSSIBILITY THAT PROVIRAL GENES ARE INVOLVED IN THE DEVELOPMENT OF THE PRIMARY RADIATION INDUCED TUMOURS. THE MUTATED PROVIRAL GENES MAY INFLUENCE ANOTHER GROUP OF CELLULAR GENES INVOLVED IN CARCINOGENESIS, THE ONCOGENES. AN EXAMPLE OF THIS TYPE OF GENE IS THE FOS GENE, WHICH WAS FIRST ISOLATED FROM RETROVIRUSES EXPRESSED IN MURINE OSTEOSARCOMAS.
THIS PROJECT AIMS AT IDENTIFYING GENETIC CHANGES SPECIFICALLY ASSOCIATED WITH THE DEVELOPMENT OF BONE TUMOURS. THE ANALYSIS WILL FOCUS UPON MUTATION IN PROVIRAL GENES AND IN OTHER CELLULAR GENES ASSOCIATED WITH INTEGRATED PROVIRUSES.
One section of the research concerned the structure and function of murine leukemia viruses associated with bone tumours and with lymphoid tumours. The viruses characterised with respect to their pathogenic properties in laboratory mouse strains and their genomic structures have been analysed by partial or complete nucleotide sequence analysis. A hypervariable region has been identified in the part of U3 associated with transcriptional enhancement.
THE ANALYSES WILL INCLUDE THE FOLLOWING STEPS:
A. VIRUS ISOLATION
I. ENDOGENOUS VIRUSES WILL BE ISOLATED AS MOLECULAR CLONES FROM A LAMBDA PHAGE LIBRARY OF THE MOUSE GENOME.
II. VIRUSES EXPRESSED IN RADIATION INDUCED OSTEOSARCOMAS. VIRUSES WILL BE ISOLATED DIRECTLY FROM OSTEOSARCOMA CELLS GROWN IN CULTURE. INDIVIDUAL VIRUS CLONES WILL BE DERIVED BY INFECTION OF MURINE AND NON-MURINE EMBRYO FIBROBLASTS AT END-POINT DILUTION. FOR SELECTED ISOLATES MOLECULAR CLONING OF PROVIRAL DNA WILL BE PERFORMED.
B. VIRUS CHARACTERIZATION
1. BIOLOGICAL PROPERTIES
THE HOST RANGE AND GROWTH PROPERTIES OF THE VIRUSES ISOLATED WILL BE ANALYSED IN CELL CULTURE SYSTEMS. CHARACTERIZATION OF THE VIRUSES WITH RESPECT TO THEIR ABILITY TO INDUCE TUMORUS IN ANIMALS AND TO TRANSFORM CELLS IN CULTURE WILL TAKE PLACE IN THE LABORATORIES AT GSF AND SCK/CEN.
2. ANALYSIS OF GENOME STRUCTURE
RNA FROM UNCLONED AND CLONED VIRUS ISOLATES WILL BE SUBJECTED TO PARTIAL NUCLEOTIDE SEQUENCE USING RNASE T1 FINGER PRINTING TECHNIQUES.THIS ANALYSIS WILL GIVE AN INDICATION OF THE HETEROGENEITY OF THE VIRAL RNA AND OF THE CONTENT OF SEQUENCES RELATED TO THE GENOMIC RNA OF WELL-CHARACTERIZED ISOLATES.
COMPLETE NUCLEOTIDE SEQUENCE ANALYSIS OF SELECTED REGIONS WILL BE PERFORMED BY THE DIDEOXYNUCLEOTIDE CHAIN TERMINATION TECHNIQUE. FOR VIRUSES NOT AVAILABLE AS MOLECULAR CLONES, THE ANALYSIS WILL USE SYNTHETIC DNA OLIGONUCLEOTIDES COMPLEMENTARY TO THE RNA GENOME AS PRIMERS IN A CHAIN ELONGATION REACTION CATALYZED BY REVERSE TRANSCRIPTASE. FOR VIRUSES AVAILABLE AS MOLECULAR CLONES STANDARD NUCLEOTIDE SEQUENCE ANALYSIS USING SUBCLONING IN M13 VECTORS WILL BE PERFORMED.
C. ANALYSIS OF THE INTEGRATION AND EXPRESSION OF RETROVIRUSES IN BONE TUMOUR CELLS.
A PANEL OF RECOMBINANT DNA CLONES CARRYING SMALL INSERTS OF THE MURINE LEUKAEMIA VIRUS GENOME HAS BEEN DERIVED IN THIS LABORATORY. RADIOACTIVELY LABELLED PROBES FROM INDIVIDUAL CLONES WILL BE USED TO MONITOR THE STRUCTURE OF INTEGRATED PROVIRUSES AND THE PATTERN OF RNA EXPRESSION IN OSTEOSARCOMA CELLS GROWN IN CULTURE. THIS ANALYSIS WILL INVESTIGATE THE POSSIBILITIES OF TUMOUR SPECIFIC VIRAL INTEGRATION SITES AND OF THE OCCURRENCE OF TRANSCRIPTS OF A STRUCTURE UNIQUE TO THE TUMOUR CELLS. DETAILED ANALYSES OF CRITICAL GENETIC REGIONS WILL BE PERFORMED USING STANDARD NUCLEOTIDE MAPPING AND SEQUENCING TECHNIQUES.

Funding Scheme

CSC - Cost-sharing contracts

Coordinator

AARHUS UNIVERSITET
Address
Ringgade 1-3
8000 Arhus
Denmark