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Tumorigenesis by mouse mammary-tumor virus (MMTV) involves proviral disruption and transcriptional activation of a number of cellular oncogenes, generically termed Int. The frequencies with which different Int genes are activated in different mouse strains can be quite variable, and previous surveys have suggested that insertions at Int-2/Fgf-3 occur primarily in strains that develop pregnancy-dependent mammary tumors. To address this issue, we have determined the relative contributions of 5 known Int genes (Wnt-1, Wnt-3, Fgf-3, Fgf-4 and Int-3) in mammary tumors from virgin BR6 and multiparous BR6, BALB/cfBR6 and RIII mice. Whereas Fgf-3 was implicated in 66%, 80% and 92% of the tumors from the respective parous animals, only 20% of the tumors from virgin mice expressed Fgf-3. This reduced involvement of Fgf-3 was compensated by proviral insertions in Fgf-4, Int-3 and Wnt-3, but the frequency of Wnt-1 activation was relatively constant. These data strengthen the link between Fgf-3 and a pregnancy-dependent phenotype and suggest that, in the strains analyzed, the frequency of Int-gene activation was influenced more by the hormonal status than by the genetic background.

Low levels of anti-viral activity, mainly interferon alpha/beta (IFN-alpha/beta), are regularly found in lymphoid tissues of BALB/c mice infected with the C3H strain of mammary tumor virus. At the time of tumor development, significant amounts of anti-viral activity were detected in homogenates of spleen and mammary tumors, but not in blood and normal mammary glands. This activity is pH2-resistant and neutralized by antibody to IFN/alpha-beta. The pathogenetic role of IFN in mammary carcinogenesis was investigated in 2 ways: (a) by treating virus-infected newborn mice with antibody to IFN-alpha/beta, and (b) by giving either the latter antibody or IFN-alpha/beta to virus-free animals transplanted with pre-neoplastic lesions. Mice were treated only for 2 months, starting either 1 week after birth or immediately after tumor transplant. In case (a), treatment with antibody to IFN-alpha/beta shortened the incubation period of mammary carcinomas and decreased the mean survival time. In case (b), anti-IFN antibody did not significantly affect the development of mammary tumors. However, exogenous IFN-alpha/beta markedly reduced both tumor incidence and mortality rate. These results indicate that endogenous IFN-alpha/beta plays a crucial role in the in vivo restriction of the early infectious phase of spontaneous carcinogenesis and that relatively high doses of IFN-alpha/beta may inhibit the progression of pre-neoplastic lesions.

Mammary cancer in mice is characterized by progression through defined stages of preneoplasia, with the most common preneoplastic stage being the hyperplastic alveolar nodule (HAN). We determined the relative levels of RNA expression of various cellular proto-oncogenes and endogenous mouse mammary tumor virus genes in outgrowths and tumors of three sublines of the transplantable D1 HAN preneoplastic outgrowth line. The three sublines differed in relative tumor-producing capabilities. Subline D1B produced a high incidence of tumors with short latency periods, whereas sublines D1C and D1D produced low incidences of tumors with long latency periods. No consistent alteration in proto-oncogene expression correlated with relative tumorigenicity, although tumors frequently contained higher levels of one or more proto-oncogene transcripts as compared with preneoplastic tissue. Slightly elevated (2- to 6-fold) levels of different oncogene transcripts were detected in 13 of 17 tumors as compared with outgrowth tissue, including abl (2 tumors), fps (5 tumors), Ha-ras (6 tumors), and Ki-ras (8 tumors). One tumor contained 45 times more Ki-ras-specific RNA than outgrowth tissue because of a comparable amplification of Ki-ras DNA sequences. Elevated levels of Ha-ras occurred more frequently in tumors of a high-incidence subline than in a less-aggressive subline (5/10 vs 1/7), but this difference was not statistically significant. However, consistent changes in MMTV expression accompanied progression from preneoplastic tissues to mammary tumors. All 17 tumors displayed reduced levels of the MMTV-specific long terminal repeat (LTR) transcript (1.6 kb) as compared with HAN tissue; tumors with moderate levels of LTR transcript expressed the 3.8-kb envelope message as well, one not detected in HANs. Expression of the LTR transcript is apparently influenced by factors in addition to the methylation status of endogenous mouse mammary tumor virus genes, which was similar in outgrowths and tumors. As the survey of representative proto-oncogenes failed to identify a uniform change between HAN and tumors, it is likely that other genes are involved in tumor progression in the mammary gland.

A colony of BALB/c mice consisting of two sublines with a high incidence of mammary tumors was examined for the presence of a mammary tumor virus (MuMTV). The mammary tumor incidences in the two sublines were 18% and 35% at average tumor age 19-20 months. Over a period of 8 years, their milk at third to tenth lactations were monitored for the presence of MuMTV antigen,and the milk and tumors were examined for the presence of B particles. Neither antigen nor B particles were found. Milk and tumor extracts from the higher mammary tumor lines were also assayed for MuMTV bioactivity by intraperitoneal inoculation of weanling C57BL, BALB/c, and RIIIf females. No response was obtained, except possible in RIIIf. Both the MuMTV antigen incidence and the tumor incidence in inoculated RIIIf mice were somewhat elevated over controls. The question remains unanswered as to whether there is an active MuMTV in our colony of tumor-bearing BALB/c mice and, if there is, whether it is associated with B particles.

BALB/cfC3H female mice which develop mammary tumor virus (MTV)-induced mammary tumors possess not only serun antibodies directed against virus-associated antigens which are expressed on mammary tumor cells but also antibodies directed against antigenic specificities which are unique to each tumor. The possible range of unique specificities is considerable; a survey of serum samples from 18 tumor-bearing females revealed only two which reacted with a tumor-unique specificity of another isologous MTV-induced mammary tumor. A retrospective study revealed that in multiparous females, the serum activity against tumor-unique antigenicity could be detected 1 to 4 months before the tumor was grossly visible. On the other hand, in virgin females, the serum activity was detectable only when the tumor was visible. On the basis of this difference in time of origin of reactivity, we speculate that the mechanism of tumor formation in parous females may involve a longer preneoplastic or latent neoplastic period than in virgin females; in the latter the change from normal to overtly neoplastic appears to be more abrupt.

Urethane administrated in the drinking water of breeding female mice of five inbred strains (DDf, KF, C3Hf, ddY and C57BL) leads to a higher incidence of mammary tumors at an extremely early age in four of these strains (DDf, KF, C3Hf and ddY). In the case of the DDf strain the tumors are adenoacanthomas, but in the case of the other strains adenocarcinomas are also observed. The effect of urethane in the drinking water on expression of endogenous retroviral structural proteins of the B and C type (MTV and MuLV) was studied in the milk by an immunodiffusion test and in organ extracts by radioimmunoassays (for MTVp27, MTVgp52 and MuLVp30). Organs tested include salivary glands, thymus and spleen from female and male animals. In addition, prostate and seminal vesicles were tested in male animals and uterus and mammary glands in females. Mammary tumors induced by urethane were also treated. Urethane does not induce or enhance endogenous retroviral antigen expression, either in these organs or in the mammary gland, one of the target organs for tumorigenesis by this agent.

Selenium in its organic and inorganic forms has been shown to inhibit the development of chemically induced, spontaneous and transplanted tumors. The present investigation was performed to study the effect of selenium (4 micrograms per ml of drinking water) on tumorigenesis of adenovirus-type-9-induced breast fibroadenomas and on 1,2-dimethylhydrazine-induced bowel carcinogenesis in WF rats. It was found that identical treatment with Se under identical conditions and with no obvious toxic effects on the rats (1) resulted in inhibition of DMH-induced large-bowel carcinogenesis; (2) facilitated induction of small-bowel cancer by the same carcinogen in the same animals, and (3) greatly facilitated induction of breast fibroadenoma by adenovirus type 9 in the same strain of rats. The effect of Se treatment on DMH-induced large-bowel carcinogenesis confirms previous findings and proves that the opposite effect on fibroadenoma development is not due to differences in e.g. effective dose, animal strains or condition of the animals. It is not yet clear through which mechanisms Se exerts these effects.

Transplantable mouse tumors used in many areas of cancer research generally produce several host-range classes of infectious type-C virus. The release of virus from such tumors should be considered in the design and evaluation of experiments in which they are used.

A study was undertaken to determine whether activation of expression of silent endogenous mouse mammary tumor virus (MMTV) proviruses may occur during tumor induction by a chemical carcinogen. A series of transplantable mammary tumors induced in BALB/c mice by treatment with dimethylbenz(alpha)anthracene (DMBA), pituitary isograft, or both was examined. The results obtained suggest that chemical carcinogens may induce mammary tumors through more than one pathway. Two of 9 tumor lines produced virus-specific products at levels above those observed during the course of normal mammary gland development. One tumor contained high levels of MMTV-specific envelope [3.8 kilobase (kb)] and genomic length (8.9 kb) RNAs. This tumor expressed core- and envelope-related proteins detectable by immunoblotting (including p28, gp52, and gp36), displayed an acquired provirus with a restriction map different from those of described exogenous MMTV strains, and contained abundant virus particles. The other tumor that expressed high levels of MMTV gene products contained envelope-specific (3.8 kb) and long-terminal-repeat-specific (1.6 kb) messages but no full-length RNA. It exhibited an aberrant 39 kDa, envelope-related protein, but no virus particles. Methylation data implicated the usually silent endogenous Mtv-8 provirus as the source of the abnormal envelope protein. None of the tumors expressed RNA from the putative mammary oncogenes, int-1 or int-2. We propose that chemical carcinogens may activate different cellular genes by mutation and that, in a subset of DMBA-induced mammary tumors, the target genes include endogenous MMTV proviruses that are normally not expressed. The effect on provirus expression varies from tumor to tumor, but is stable over passage of a given tumor. MMTV may be of etiological importance in the genesis of those DMBA-induced tumors which contain high levels of MMTV-specific products, but its action in the BALB/c system is not mediated through enhanced expression of the int-1 or int-2 preferred integration regions.