The DIM series of preneoplasms, developed from the BALB/c mammary cell line COMMA-D, and DIM-derived tumors were examined for evidence of mouse mammary tumor virus (MMTV) involvement in the tumorigenic process. DIM tissues were shown to be free of exogenous MMTV inasmuch as Southern blot analysis of DNAs extracted from DIM preneoplasms and tumors showed the lack of a PstI-generated 4.2-kilobase MMTV-gag-pol band. To examine the possibility of endogenous MMTV action via enhancer insertion, DNAs

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... rom DIM preneoplasms and tumors were restricted with EcoRI, BamHI, or SstI and subjected to Southern blot analysis using an MMTV-long terminal repeat probe. The normal endogenous proviral content was detected in all DIM tissues assayed; no new proviruses were found. To examine the possibility of endogenous MMTV gene product oncogenesis, MMTV transcripts were quantified by slot blot analysis and MMTV envelope proteins were assayed by immunohistochemical staining of DIM tissue sections. While all DIM tissues expressed MMTV-long terminal repeat transcripts, the level of expression did not correlate with tumorigenicity. Most frequently, MMTV-env-containing sequences were more abundant than the 1.6-kilobase long terminal repeat transcript, the latter being the most promising candidate for a MMTV gene product mediating mammary tumorigenesis. However, preneoplasms and tumors of only the DIM 4 line contained levels of MMTV Mr 52,000 or 36,000 glycoproteins detectable by immunoperoxidase staining. Electron microscopy did not reveal any virus particles in DIM 4 tissues. These data do not substantiate MMTV as a causative agent in the formation of the DIM preneoplasms or tumors. Thus while MMTV function may confuse the interpretation of causative events in the formation of other preneoplasms and neoplasms, the DIM preneoplasms represent a model system for the study of MMTV-independent mechanisms.