Detection of Mouse Mammary Tumor Virus RNA in BALB/c Tumor Cell Lines of Nonviral Etiologies

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Abstract
A complementary DNA (cDNA) probe to mouse mammary tumor virus (MMTV) RNA was synthesized using calf thymus DNA oligonucleotides as a random primer. This probe was then used to study the expression of MMTV RNA in cell lines from BALB/c [mouse] tumors induced in vivo either spontaneously or in response to viral, chemical or hormonal stimuli. The cDNA had a length of approximately 400-500 nucleotides and specifically hybridized to MMTV RNA and BALB/c lactating mammary gland RNA, but not to Moloney leukemia virus RNA. Calf thymus DNA-primed cDNA could protect 50% of iodinated MMTV RNA from S1 nuclease digestion at cDNA-RNA ratios of 1:1 and 90% of labeled viral RNA at ratios of 10:1. Thermal denaturation of MMTV RNA-cDNA hybrids yielded a Tm [melting temperature] of 88.5.degree. C, indicative of a well-base-paired duplex. Screening of mouse mammary tumor cells for MMTV sequences revealed that 3 out of 5 lines of BALB/c origin had undetectable levels of viral RNA (< 3 molecules/cell) by RNA excess hybridization. Two of the three virus-negative cell lines were derived from tumors induced by the chemical carcinogen 7,12-dimethylbenz(.alpha.)anthracene, but the 3rd tumor occurred spontaneously. Two lines from tumors induced by either viral (mammary tumor virus) or hormonal (17-.beta.-estradiol) stimulus contained between 3 and 9 molecules of MMTV RNA/cell by both RNA excess and cDNA excess hybridization. Clonal derivatives of these tumor lines had levels of viral RNA comparable to those of their parental lines. Therefore, the presence of detectable MMTV RNA sequences is not a necessary requirement for the maintenance of all murine mammary gland neoplasias.