MOLECULAR BASIS OF PATHOGENESIS OF MURINE LEUKEMIA VIRUS

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Abstract

We have been in a unique position to study various cellular genes involved in the development of mouse B cell lymphomas due to the availability of tumors and tumor-derived cell lines from NFS.V+ mice and other strains of mice expressing ecotropic virus at high levels. NFS mice do not normally express ecotropic virus due to the lack of the viral genome in their DNA. However, high virus expression was achieved in NFS mice infected with ecotropic virus or congenic mice from AKR or C58 strains with ecotropic genes. Insertional mutagenesis by proviral integration proved to be quite useful for identifying involved cellular genes. Using Southern analysis to compare each tumor DNA with its respective tail DNA, we determined if there were new ecotropic proviral integrations for each of these tumors. In order to enumerate all new integrations or to rule out concealed integrations, multiple restriction endonuclease digestions were sometimes necessary. About 90% of the total tumor DNAs had new somatic integration of ecotropic MuLV. We observed that not all of the novel integrations in the tumors appeared in the cell lines derived from them; on the other hand cell lines sometimes harbored novel integrations not obvious in the primary tumor. To begin with we chose two tumors and their corresponding cell lines to molecularly clone both 3' and 5' cellular sequences adjacent to the viral genome using the ecotropic virus-specific probe sequence.In one instance, the primary tumor had one novel viral integration whereas the cell line had a unique integration site, but one which was different from the tumor. We molecularly cloned and characterized both novel sites and the flanking cellular DNA was sequenced. In the second case there were six novel integration sites in the primary tumor and eight in the cell line of which four were in common with the primary tumor. We molecularly cloned 5 sites from the cell line. Sequencing of 10 host viral junction fragments and repeated blast searches revealed that these are hitherto unidentified sequences. Unique sequence probes were derived from the flanking cellular DNA in one of these clones. Screening of over 650 tumor DNAs revealed one other rearrangement involving the same cellular sequence. This DNA sequence maps in the middle of mouse chromosome 5. In order to identify the cellular gene(s) which may be involved, we are analyzing BAC clones containing about 130 kb of cellular sequences using this probe. Using the IPCR technique, 3 other common integration sites were molecularly identified from NFS.V+ and AKXD lymphomas. The sites were cloned and unique sequence probes were developed. Screening of DNAs from NFS. V+ tumors to determine their commonality in these tumors is in progress. We will continue to develop unique sequence probes from the molecularly cloned integration sites to determine their frequency of involvement as well as to determine which genes are involved.