To develop a model system in which we could induce GCRs, we used the rare restriction enzyme I-SceI, whose 18 bp recognition sequence is not normally present in the human or mouse genome, to produce a single DNA DSB within a mammalian cell, based on the hypothesis that improper repair of these breaks could lead to GCRs. This enzyme has been used in a series of elegant studies to produce specific, non-random GCRs mediated by homologous recombination in mammalian cells. We generated a construct that expressed the Herpes simplex virus type I thymidine kinase (TK) gene under the control of the constitutive EF1a promoter, with the recognition sequence for the I-SceI restriction enzyme placed between the EF1a promoter and the TK gene. This pEF1aTK vector was introduced into the U937 cell line, and stable transfectants were selected with G418. Transfectants with a single copy of the pEF1aTK vector were identified by Southern blot, and those that expressed TK under control of the EF1alpha promoter were identified based on sensitivity to ganciclovir (GCV). One clone (named F5) was chosen for further study. To estimate the efficiency of I-SceI cleavage, we transfected F5 cells with a vector that conferred hygromycin resistance (HygroR) and expressed I-SceI. We found that half of the 18 hygroR clones showed altered DNA sequences flanking the I-SceI cleavage site, indicating that the DNA had been cleaved and subsequently repaired in these clones. We next carried out a series of experiments that utilized the negative selection provided by the expression of TK. Cells were transfected with an I-SceI expression vector and selected with GCV (to select for cells that had lost TK expression) and G418 (to select against clones that had deleted the entire pEF1aTK vector). Using a combination of Southern blots, forward PCR, and inverse PCR, we characterized over 100 independent clones recovered from 4 experiments. Most of the clones had small deletions and showed evidence consistent with non-homologous end joining (NHEJ), such as microhomology or local sequence inversions. Nine clones had insertions of sequence that matched distant regions of the genome; all of these sequences were transcribed regions, and one contained a LINE sequence. A single clone fused alpha satellite sequences that could not be uniquely mapped on the human genome to the TK region; viable cells from this clone were unavailable to confirm a potential GCR via fluorescence in situ hybridization (FISH). We considered the possibility that the U937 cell line, which has a fairly normal karyotype [47 XX, t(10;11)] may not be an ideal choice for these types of experiments, and that a structurally unstable cell line might be more likely to produce GCR. The NCI 60 cell line panel has been assayed for numerical and structural instability; OVCAR8 showed the highest level of ongoing structural instability. We generated OVCAR-8 subclones that harbored a single copy of the pEF1aTK vector, and transfected these clones with I-SceI expression vectors. However, similar to the results with the U937 clones, all 31 GCV-resistant (GCVR) clones had small interstitial deletions and features of repair via NHEJ. An additional reason that we did not recover any clear GCR from the above experiments may be that the frequency of spontaneous DNA DSBs was too low. Therefore, we increased the number of DNA DSB in the OVCAR8 subclone by treating the cells with agents known to cause DNA DSB (bleomycin or etoposide), 48 hours after transfection of the I-SceI expression vector. We recovered fewer GCVR clones, possibly due to toxicity of bleomycin or etoposide. We characterized 15 bleomycin clones and 7 etoposide clones; all were interstitial deletions, many with features of NHEJ. We also considered the possibility that a potential disadvantage of the above approach is that it utilizes a negative selection system, and will generate clones that delete small (i.e. greater than 100bp) portions of the EF1a promoter or TK cDNA, leading to lack of TK expression and GCVR. Therefore, we developed a complementary vector that allowed positive selection. This vector contains a hygromycin phosphotransferase gene (HygroR; confers resistance to hygromycin) preceded by an I-SceI recognition sequence, but no promoter region. This promoter-less cassette also contains a G418R cassette to allow for selection of cells that have integrated the vector. We transfected this vector into OVCAR 8 cells, and identified clones that integrated a single copy. As anticipated, since the HygroR gene lacks a promoter, these cells are hygromycin sensitive. These cells were then transfected with an I-SceI expression vector, and selected with hygromycin, in the hopes of recovering rare clones that had undergone a GCR, and juxtaposed a promoter from a distant genomic region, thus allowing expression of the HygroR gene, leading to hygromycin resistance. No HygroR clones were recovered from control experiments with pBluescript transfection, and we recovered few clones following transfection with the I-SceI expression vector. Thus far, all 12 of the clones analyzed have been vector capture events, in which a portion of SV40 regulatory sequences derived from the I-SceI expression vector have become juxtaposed to the hygroR gene. Because I-SceI cleaves DNA as a homodimer, it is possible that weak interactions between monomeric subunits prevent the cleaved DNA strands from separating. Therefore, we attempted to produce GCR using an additional method for producing DNA DSB. Illegitimate V(D)J recombination has long been recognized as a potential source of DNA DSB at chromosomal translocation breakpoints. The introduction of RAG1, RAG2, and E2A was sufficient to promote V(D)J recombination in non-lymphoid cells. We modified the pEF1aTK vector by inserting a heptamer/spacer/nonamer V(D)J recombination signal sequence (RSS) from the human IGK locus adjacent to the I-SceI site. The modified vector (named pEF1aTK15) was transfected into U937 cells, and we selected stable transfectants that had integrated a single copy of the vector and expressed TK. Two independent clones, named P8 and P16 were studied. Because the cell lines had both an I-SceI site as well as a RSS, we were able to assay differences in the GCVR clones produced by either I-SceI cleavage or RAG-mediated cleavage. We recovered 18, 3, and 0 GCVR clones that had disrupted the EF1a-TK cassette following transfection of the P8 clones with I-SceI, RAG1/RAG2/E2A, or pBluescript, respectively. Similar to prior results, the 18 clones produced by I-SceI cleavage all had small interstitial deletions between the EF1a promoter and the TK gene, many with features characteristic of NHEJ. In contrast, the clones produced by transfection of RAG1/RAG2/E2A generated large deletions, of 80-135 kb, all with signs of NHEJ, including microhomology of 2-6 bp. Results from the P16 cell line were similar; clones generated with I-SceI showed interstitial deletions of 34-3611, whereas clones generated by RAG1/RAG2/E2A contained deletions of 266-262,683 bp. We looked for cryptic heptamer/nonamer sequences that could have signaled a DNA DSB near the deletion junction, and in several cases identified 5/7 or 6/7 matches to a consensus heptamer sequence within 100 nucleotides of the junc [summary truncated at 7800 characters]
| Beachy, Sarah H; Aplan, Peter D (2010) Mouse models of myelodysplastic syndromes. Hematol Oncol Clin North Am 24:361-75 |
| Harper, David P; Aplan, Peter D (2008) Chromosomal rearrangements leading to MLL gene fusions: clinical and biological aspects. Cancer Res 68:10024-7 |
| Cheng, Yue; Zhang, Zhenhua; Slape, Christopher et al. (2007) Cre-loxP-mediated recombination between the SIL and SCL genes leads to a block in T-cell development at the CD4- CD8- to CD4+ CD8+ transition. Neoplasia 9:315-21 |
| Aplan, Peter D (2006) Chromosomal translocations involving the MLL gene: molecular mechanisms. DNA Repair (Amst) 5:1265-72 |
| Aplan, Peter D (2006) Causes of oncogenic chromosomal translocation. Trends Genet 22:46-55 |
| Lin, Ying-Wei; Perkins, Jonathan J; Zhang, Zhenhua et al. (2004) Distinct mechanisms lead to HPRT gene mutations in leukemic cells. Genes Chromosomes Cancer 39:311-23 |
| Lin, Ying-Wei; Aplan, Peter D (2004) Leukemic transformation. Cancer Biol Ther 3:13-20 |
