Genomes of inbred mouse strains like C57Bl6(B6), C57Bl10(B10) and their sublines harbor only one copy of the N-tropic ecotropic murine leukemia viral genome. These genomes occationally yield infectious virus, but in old age. In order for the spontaneously activated virus to spread, p30 region of the gag gene require change either by mutation or by recombination to produce B-tropic virus which can infect host cells. Spontaneous lymphoma in these strains are rare. B6 strain is widely used to develop and study genetically altered mice. Such genetically altered mice have a much higher incidence of lymphoma. Administration of soluble cytochrome C in the presence of interleukin-6 with miniosmotic pump causes T cell expansion in T-cell receptor(TCR) transgenic Rag2-/- B10.A mice. Between 3 and 12 months after immunization, a high proportion of the treated mice develop T cell lymphomas. A series of 15 such lymphomas have been analyzed. In order to determine some common mechanisms of T cell transformation and to identify genes governing distinctive behavior, whole genome expression analysis using oligonucleotide microarrays have been applied. Expression of endogenous retrovirus has been detected. One or more novel integration of retrovirus with an altered gag occured in 50% of the tumor studied. In order to identify the cellular gene(s) where novel integration occured, host viral junction fragment has been cloned by an inverse PCR technique. These studies are in progress. We are also studying aging B6 and B6;129 mice to understand the tumor incidences, type of tumors and the contribution of retrovirus for the activation and inactivation of cellular genes that may play a part in the development of such tumors. B6 mice inoculated with helper free BM5 def-neo virus developed primary mediastinal (thymic) diffuse large B-cell lymphomas. We are analysing DNAs derived from such tumors to determine gene(s) involved in the development of these lymphomas. We are also studying murine leukemia virus induced B-cell lymphomas. 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 bacteriophage lambda cloning system and 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. 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. Recently a new retroviral insertion site that is the most frequent genetic alteration in AKXD B-cell leukemias has been described. This insertion site is designated as Lvis1 and is responsible for 2% of AKXD leukemias. Utilizing an unique sequence probe from Lvis1, we analyzed DNAs from over four hundred B-cell lymphomas of various classes from NFS.V+ mice. Lymphomas classified as MZL and CBCC yield 35% alteration at the Lvis1 locus. It has been suggested that upregulation of HEX and mEg5 may be a contributory factor for such B-cell leukemia develoment. Using NFS.V+ congenic derived tumors, we are trying to corroborate and expand on these findings.
| Hori, M; Xiang, S; Qi, C F et al. (2001) Non-Hodgkin lymphomas of mice. Blood Cells Mol Dis 27:217-22 |
| Morse 3rd, H C; Qi, C F; Chattopadhyay, S K et al. (2001) Combined histologic and molecular features reveal previously unappreciated subsets of lymphoma in AKXD recombinant inbred mice. Leuk Res 25:719-33 |
