Extra mouse mammary tumor proviruses in DBA/2 mouse lymphomas acquire a selective advantage in lymphocytes by alteration in the U3 region of the long terminal repeat
Journal of Virology
We determined the nucleotide sequences of the long terminal repeats (LTRs) from mouse mammary tumor virus (MMTV) proviruses acquired in two DBA/2 mouse lymphoma cell lines, MLA and DL-8. Proviruses from MLA contained a 352-base-pair deletion from nucleotides 669 to 1020 in the U3 region of the LTR, whereas the LTR alteration of the DL-8 provirus involved both a similar 360-base-pair deletion and generation of a tandem repeat region consisting of sequences of flanking deletions. To assess the
... s. To assess the function of the rearranged LTRs, we constructed plasmids in which normal and rearranged LTRs drove the reporter chloramphenicol acetyltransferase gene and transfected them into T-cell lines (Jurkat, Molt-3, and DL-8) and the mammary tumor cell line T47D. Both rearranged LTRs were transcriptionally active, but normal LTRs were not active in either the presence or absence of glucocorticoids in all T-cell lines. In T47D cells, however, the MLA provirus LTR showed the same glucocorticoidor progestin-dependent transcriptional activity as did normal LTRs. The DL-8 provirus LTR acquired a novel enhancer(s) by rearrangement and thus had a high basal transcriptional activity in T47D cells. The results of chloramphenicol acetyltransferase assays using plasmids with various chimeric MMTV LTRs revealed that the rearranged LTRs had lost their negative regulatory element and contained an enhancer element that was highly homologous to the enhancer A element of polyomavirus (from nucleotides 525 to 558). GR but not C3H mouse MMTV contained this enhancer. These results elucidate some of the molecular mechanisms involved in the selection of mutant MMTVs with rearranged LTRs in lymphoma cells. Mouse mammary tumor virus (MMTV) is known to be associated with the mammary gland epithelium and induce mammary adenocarcinoma through the activation of cellular int genes by insertion of proviruses (9, 35). However, there is also extensive literature documenting the presence of MMTV information in other types of tumors, including lymphoid leukemia (1, 11, 29, 34, 39, 47, 49, 54) , kidney adenocarcinoma (14, 51), and pituitary tumors (38, 45). Many investigators have reported the amplification of extra MMTV proviruses in T-cell leukemia in GR (30-32), DBA/2 (23), BALB/c (22), and C57BL/6 (11, 12, 20) mice, and most of these extra proviruses have site-specific rearrangements in their long terminal repeats (LTRs). Furthermore, Ball et al. have recently shown that such a mutated MMTV (DMBA-LV) induces T-cell lymphomas (1-3) . The LTR rearrangements reported so far consist of deletions of a similar size at similar sites in the U3 region of the LTR, and in some cases of a substitution resulting in unique tandem repeats. In addition, the U3 region of the LTR plays a crucial role in determining the tissue tropism and tumorigenicity of C-type retroviruses (6, 8, 25) . Hsu et al. (20) and Theunissen et al. (44) have shown that LTR rearrangements can cause a shift in the cell-type-specific expression pattern of MMTV. In this study, we examined the structure and function of MMTV LTRs isolated from two DBA/2 mouse lymphoma cell lines, MLA and DL-8, in comparison with those from wild-type MMTV from C3H and GR mice. The results show that these extra MMTV proviruses acquired a selective advantage in T cells by generation of a new enhancer sequence in addition to removal of reported negative regulatory elements (20, 33). We also discuss the possibility that * Corresponding author. these mutant MMTVs are the causative agents of murine leukemia. MATERIALS AND METHODS Cell lines. Two cell lines, MLA (47, 49) and DL-8, were established from spontaneous lymphomas of DBA/2 mice in our laboratory. Initially, MLA was classified as a T-cell line (49), but a later investigation of cell surface markers showed that it was actually a B-cell line (43). DL-8 was classified as a T-cell line. The other cell lines used included Jurkat and Molt-3 (human T-cell lines) and T47D (a human mammary tumor cell line). All cell lines were grown in RPMI medium supplemented with 10% fetal calf serum. Molecular cloning of MMTV proviruses and DNA sequencing. Genomic libraries were constructed from MLA and DL-8 cell lines by using EMBL3 arms and 15to 22kilobase-pair (kb) cellular DNA fragments as described previously (28, 53) . The X bacteriophage clones containing MMTV sequences were screened by the plaque hybridization technique (4), using MMTV U3-specific DNA (the 1.0-kb PstI-SacI fragment of the C3H MMTV LTR) as a probe. Of 30 LTR-containing phages isolated from the MLA genomic library, two clones, A MLA1 and MLA2, were found to have the whole provirus structure and altered LTRs (see Results). From the DL-8 library, a phage clone containing a newly integrated provirus carrying altered LTRs was isolated and designated X DL-8. From A DL-8, a 1.4-kb BglII fragment containing the entire 3' LTR (1.1 kb) plus some cellular flanking DNA (0.3 kb) was also isolated and subcloned into the BamHI site of pUC119. This subclone was designated P8PaLTR.