Activation of a MMTV/mdr3 Fusion Transcript from a Cryptic Viral Promoter is Stimulated by Mdr-derived Sequences Located in Intron I

In P388/VCR-10 cells, resistance to cytotoxic drugs is caused by the overexpression of the mdr3 gene, in absence of gene amplification. The gene is transcriptionally activated following integration of a full-length mouse mammary tumor virus (MMTV) within intron I, upstream of the coding region of the gene. This integration results in the production of MMTV/mdr3 fusion transcripts that originate from the antisense 5'LTR of the provirus. The mechanism of mdr3 activation in these cells remains unclear since it cannot be accounted for either by activation from the normal MMTV promoter or by activation of the mdr3 promoter by MMTV enhancer sequences. Subcloning and sequence analysis of the genomic region encompassing the 5' LTR of the provirus with adjacent mdr3 sequences up to exon 2 showed that the LTR had not undergone small rearrangements or deletions. Transfections of fusion plasmids containing this genomic fragment and the reporter gene luciferase showed the presence of transcriptionally active sequences in that region. Deletions of 5' and 3' sequences from this fragment have shown that the antisense LTR itself has little contribution to the activation of the luciferase gene, whereas the mdr3 derived sequences that include part of intron I and the beginning of exon 2 strongly activated luciferase expression when inserted in either orientations upstream of the reporter gene. These results suggest the presence of an activator element within intron I of mdr3 capable of activating transcription from a cryptic start site present in the antisense MMTV LTR. Derepression of this activator sequence within intron I by a mechanism involving integration of a transposable element may be a prerequisite to transcriptional activation of the gene which is silent in the parental P388 cells. Further support for a derepression mechanism of activation in P388 cells is provided by the identification of independent genomic rearrangements in the 5' region of mdr3 in additional MDR P388 derivatives analyzed in this study.