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Salmonella is a Gram-negative, facultative anaerobe that is a common cause of host intestinal infections. Salmonella grows under aerobic and anaerobic conditions, and it has been proven capable of inhibiting tumor growth. However, the molecular mechanism by which Salmonella inhibits tumor growth is still unclear. Angiogenesis plays an important role in the development and progression of tumors. We investigated the antitumor effect of Salmonella in a syngeneic murine tumor model. Hypoxia-inducible factor-1 (HIF-1)α plays a significant role in tumor angiogenesis. We examined the molecular mechanism by which Salmonella regulated vascular endothelial growth factor (VEGF), which is an important angiogenic factor. The expression of VEGF in tumor cells was decreased by treatment with Salmonella. The conditioned medium from Salmonella-treated cells inhibited the proliferation of endothelial cells. Salmonella inhibited the expression of HIF-1α as well as downregulated its upstream signal mediator protein kinase B (AKT). Salmonella significantly inhibited tumor growth in vivo, and immunohistochemical studies of the tumors revealed decreased intratumoral microvessel density. These results suggest that Salmonella therapy, which exerts anti-angiogenic activities, represents a promising strategy for the treatment of tumors., Competing Interests: The authors declare no conflict of interest.

Therapeutic attenuated strains of Salmonella Typhimurium target and eradicate tumors in mouse models. However, the mechanism of S. Typhimurium for tumor targeting is still poorly understood. We performed a high-throughput screening of single-gene deletion mutants of S. Typhimurium in an orthotopic, syngeneic murine mammary model of breast cancer. The mutants under selection in this system were classified into functional categories to identify bacterial processes involved in Salmonella accumulation within tumors. Niche-specific genes involved in preferential tumor colonization were identified and exemplars were confirmed by competitive infection assays. Our results show that the chemotaxis gene cheY and the motility genes motAB confer an advantage for colonization of Salmonella within orthotopic syngeneic breast tumors. In addition, eutC, a gene belonging to the ethanolamine metabolic pathway, also confers an advantage for Salmonella within tumors, perhaps by exploiting either ethanolamine or an alternative nutrient in the inflamed tumor environment., Competing Interests: The authors declare no conflicts of interest.

Background/aim: Cancer treatment with attenuated Salmonella enterica Typhimurium (S. Typhimurium) has gained momentum in recent years. However, the effectiveness of this treatment has not been explored in autochthonous models. We report the efficacy of S. Typhimurium in mice with autochthonous mammary tumors., Materials and Methods: S. Typhimurium attenuated by deletion of cyclic adenosine monophosphate signaling, SalpNG.1, was injected into female BALB-neuT tumor-bearing mice. Mice were monitored for efficacy and sacrificed for mechanistic studies., Results: In treated mice, seven-week post-treatment tumor burden was reduced by 85% and median survival was increased by 88%. Efficacy was correlated with increased tumor-infiltrating CD8 and natural killer cells. In addition, SalpNG.1 treatment caused a systemic increase of monocytic myeloid-derived suppressor cells that accumulated to high numbers within tumor tissue. Bacteria were not detected in tumor tissue, suggesting that the observed efficacy was due to a systemic rather than a tumor-specific effect of the bacteria., Conclusion: S. Typhimurium treatment reduces tumor burden and increases survival in an autochthonous breast cancer model., (Copyright© 2015 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.)

The intracellular metabolism and cytostatic activity of the anticancer drug gemcitabine (2',2'-difluoro-2'-deoxycytidine; dFdC) was severely compromised in Mycoplasma hyorhinis-infected tumor cell cultures. Pronounced deamination of dFdC to its less cytostatic metabolite 2',2'-difluoro-2'-deoxyuridine was observed, both in cell extracts and spent culture medium (i.e. tumor cell-free but mycoplasma-containing) of mycoplasma-infected tumor cells. This indicates that the decreased antiproliferative activity of dFdC in such cells is attributed to a mycoplasma cytidine deaminase causing rapid drug catabolism. Indeed, the cytostatic activity of gemcitabine could be restored by the co-administration of tetrahydrouridine (a potent cytidine deaminase inhibitor). Additionally, mycoplasma-derived pyrimidine nucleoside phosphorylase (PyNP) activity indirectly potentiated deamination of dFdC: the natural pyrimidine nucleosides uridine, 2'-deoxyuridine and thymidine inhibited mycoplasma-associated dFdC deamination but were efficiently catabolized (removed) by mycoplasma PyNP. The markedly lower anabolism and related cytostatic activity of dFdC in mycoplasma-infected tumor cells was therefore also (partially) restored by a specific TP/PyNP inhibitor (TPI), or by exogenous thymidine. Consequently, no effect on the cytostatic activity of dFdC was observed in tumor cell cultures infected with a PyNP-deficient Mycoplasma pneumoniae strain. Because it has been reported that some commensal mycoplasma species (including M. hyorhinis) preferentially colonize tumor tissue in cancer patients, our findings suggest that the presence of mycoplasmas in the tumor microenvironment could be a limiting factor for the anticancer efficiency of dFdC-based chemotherapy. Accordingly, a significantly decreased antitumor effect of dFdC was observed in mice bearing M. hyorhinis-infected murine mammary FM3A tumors compared with uninfected tumors.

Background: Myeloid-derived suppressor cells (MDSCs) are present in large numbers in blood of mice and humans with cancer, and they strongly inhibit T-cell and natural killer (NK) cell responses, at young and old age. We found that a highly attenuated bacterium Listeria monocytogenes (Listeria(at))-infected MDSC and altered the immune-suppressing function of MDSC., Methods: Young (3 months) and old (18 months) BALB/cByJ mice with metastatic breast cancer (4T1 model) were immunised with Listeria(at) semi-therapeutically (once before and twice after tumour development), and analysed for growth of metastases and primary tumour, in relation to MDSC-, CD8 T-cell and NK cell responses., Results: We found that Listeria(at)-infected MDSC, which delivered Listeria(at) predominantly to the microenvironment of metastases and primary tumours, where they spread from MDSC into tumour cells (infected tumour cells will ultimately become a target for Listeria-activated immune cells). Immunotherapy with Listeria(at) significantly reduced the population of MDSC in blood and primary tumours, and converted a remaining subpopulation of MDSC into an immune-stimulating phenotype producing IL-12, in correlation with significantly improved T-cell and NK cell responses to Listeria(at) at both ages. This was accompanied with a dramatic reduction in the number of metastases and tumour growth at young and old age., Conclusions: Although preclinical studies show that immunotherapy is less effective at old than at young age, our study demonstrates that Listeria(at)-based immunotherapy can be equally effective against metastatic breast cancer at both young and old age by targeting MDSC.

Breast cancer patients are susceptible to infections such as candidiasis. Due to the importance and the role of matrix metalloproteinases (MMP) in breast cancer progression and its correlation with tumor metastasis, we analyzed the serum level of MMPs -2, -3, -9 and tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) in breast cancer bearing mice in the presence of systemic Candida albicans infection. Female BALB/c mice were divided into 4 groups: group I had tumor + candidiasis; group II, tumor only; group III, candidiasis only and group IV as negative control. Tumor tissue was separated from stock breast cancer bearing mice and transplanted subcutaneously into the groups I and II mice. Two weeks after tumor transplantation, groups I and III were infected with Candida albicans by intravenous injection. One week after systemic infection, the sera of the experimental groups were prepared and analyzed with ELISA for MMP-2, -3, -9 and TIMP-1 levels. The results showed that the levels of MMP-3, MMP-9 and TIMP-1 were increased in groups I, II and III, as compared to the control group. However, the level of MMP-2 was decreased in mice infected with Candida albicans and in infected mice bearing tumor. These data suggest that candidiasis may have a positive effect on tumor progression and metastasis.

Many studies have demonstrated that intravenously administered bacteria can target and proliferate in solid tumors and then quickly be released from other organs. Here, we employed the tumor-targeting property of Escherichia coli Nissle 1917 to inhibit mouse B16 melanoma and 4T1 breast tumors through the expression of azurin protein. For this purpose, recombinant azurin-expressing E. coli Nissle 1917 was developed. The levels of in vitro and in vivo azurin secretion in the engineered bacterium were determined by immunochemistry. Our results demonstrated that B16 melanoma and orthotopic 4T1 breast tumor growth were remarkably restrained and pulmonary metastasis was prevented in immunocompetent mice. It is worth noting that this therapeutic effect partially resulted from the antitumor activity of neutrophils and lymphocytes due to inflammatory responses caused by bacterial infections. No toxicity was observed in the animal during the experiments. This study indicates that E. coli Nissle 1917 could be a potential carrier to deliver antitumor drugs effectively for cancer therapy.

Background: An effective cancer therapeutic must selectively target tumours with minimal systemic toxicity. Expression of a cytotoxic protein using Salmonella typhimurium would enable spatial and temporal control of delivery because these bacteria preferentially target tumours over normal tissue., Methods: We engineered non-pathogenic S. typhimurium to secrete murine TNF-related apoptosis-inducing ligand (TRAIL) under the control of the prokaryotic radiation-inducible RecA promoter. The response of the RecA promoter to radiation was measured using fluorometry and immunoblotting. TRAIL toxicity was determined using flow cytometry and by measuring caspase-3 activation. A syngeneic murine tumour model was used to determine bacterial accumulation and the response to expressed TRAIL., Results: After irradiation, engineered S. typhimurium secreted TRAIL, which caused caspase-3-mediated apoptosis and death in 4T1 mammary carcinoma cells in culture. Systemic injection of Salmonella and induction of TRAIL expression using 2 Gy gamma-irradiation caused a significant delay in mammary tumour growth and reduced the risk of death by 76% when compared with irradiated controls. Repeated dosing with TRAIL-bearing Salmonella in conjunction with radiation improved the 30-day survival from 0 to 100%., Conclusion: These results show the pre-clinical utility of S. typhimurium as a TRAIL expression vector that effectively reduces tumour growth and extends host survival.

The successful application of live bacteria in cancer therapy requires a more detailed understanding of bacterial interaction with the tumour microenvironment. Here, we analysed the effect of Escherichia coli K-12 colonization on the tumour microenvironment by immunohistochemistry and fluorescence microscopy in the murine 4T1 breast carcinoma model. We described the colonization of tumour-bearing mice, as well as the spatiotemporal distribution of E. coli K-12 in the 4T1 tumour tissue over a period of 14 days. The colonization resulted within 3 days in large avascular necrotic tissue, redistribution of hypoxic areas and an enhanced collagen IV deposition within the colonized tumour tissue, which changed the tumoral perfusion of systemically injected immunoglobulins. In addition, E. coli K-12 colonization led to the redistribution of tumour-associated macrophages, forming a granulation tissue around bacterial colonies, and also to an increase in TNFalpha and matrix metalloproteinase 9 expression. Colonization of 4T1 tumours by E. coli K-12 resulted in strong reduction of pulmonary metastatic events. These new insights will contribute to the general understanding of the tumour-microbe cross-talk and to the design of bacterial strains with enhanced anticancer efficiency.

Inflammation associated with bacterial infections is a risk factor for cancers in humans, yet its role in breast cancer remains poorly understood. We have previously shown that innate immune inflammatory response against intestinal bacteria is sufficient to induce colon cancer. Here we report that infecting Rag2-deficient C57BL/6 Apc(Min/+) mice with an intestinal bacterial pathogen, Helicobacter hepaticus, significantly promotes mammary carcinoma in females and enhances intestinal adenoma multiplicity by a tumor necrosis factor alpha (TNFalpha)-dependent mechanism. The mammary and intestinal tumor development as well as the increase in proinflammatory mediators is suppressed by adoptive transfer of interleukin 10-competent CD4+CD45RB(lo)CD25+ regulatory (T(R)) cells. Furthermore, prior exposure of donor mice to H. hepaticus significantly enhances antitumor potency of their T(R) cells. Interestingly, these microbially experienced T(R) cells suppress tumorigenesis more effectively in recipient mice irrespective of their tumor etiology. These data suggest that infections with enteric pathogens enhance T(R)-cell potency and protect against epithelial cancers later in life, potentially explaining paradoxical increases in cancer risk in developed countries having more stringent hygiene practices. The possibility that dysregulated gut microbial infections in humans may lead to cancer in anatomically distant organs, such as breast, highlights the need for novel immune-based strategies in cancer prevention and treatment.

Mouse mammary tumor virus is a replication-competent B-type murine retrovirus responsible for mammary gland tumorigenesis in some strains of laboratory mice. Mouse mammary tumor virus is transmitted horizontally through the milk (exogenous or milk-borne virus) to susceptible offspring or vertically through the germ line (endogenous provirus). Exogenously acquired and some endogenous mouse mammary tumor viruses are expressed at high levels in lactating mammary glands. We show here that there is packaging of the endogenous Mtv-1 virus, which is expressed at high levels in the lactating mammary glands of C3H/HeN mice, by the virions of exogenous C3H mouse mammary tumor virus [MMTV(C3H)]. The mammary tumors induced in C3H/HeN mice infected with exogenous MMTV (C3H) virus contained integrated copies of recombinant virus containing a region of the env gene from an endogenous virus. This finding indicates that there was copackaging of the Mtv-1 and MMTV(C3H) RNAs in the same virions. Moreover, because Mtv-1 encodes a superantigen protein with a V beta specificity different from that encoded by the exogenous virus, the packaging of Mtv-1 results in an infectious virus with a broader host range than MMTV(C3H).

Lymphocytic infiltrates of BALB/c C4 hyperplastic alveolar nodule (HAN) have elevated natural killer (NK) activity, which correlates positively with the progression of C4 HAN to tumor: C4 HAN produces an infectious mouse mammary tumor virus, MMTV(C4), which encodes a superantigen that activates and deletes T-cells with the V beta 2 segment in the T-cell receptor. In this report, NK activation by both MMTV(C4) and MMTV(C4) superantigen was tested. NK activity was measured in naive BALB/c mice, BALB/c mice depleted of V beta 2+ T-cell, or V beta 2-transgenic mice after they received injections of either purified MMTV(C4) or MMTV(C4)-infected splenocytes. Elevated NK activity was observed in BALB/c mice receiving MMTV(C4) or MMTV(C4)-infected splenocytes. Depletion of V beta 2+, but not V beta 8+, T-cells by specific anti-V beta hybridoma before injection of MMTV(C4)-infected cells reduced but did not eliminate NK activation. NK activation in V beta 2-transgenic mice occurred before massive CD4 T-cell deletion took place and was more pronounced than that in the nontransgenic littermates. These results indicate that MMTV activates NK cells through superantigen-dependent and -independent pathways and supports the role of MMTV(C4) in the augmented NK activity observed in C4 HAN infiltrates. The progression of C4 HAN to tumor represents a model system for the analysis of how tumorigenesis may be affected by lesion-associated viruses.

Mammary tumours developed in 110 (95.7%) of 115 DDD/1-Mtv-2/Mtv-2 (DDD/1-Mtv-2) and 24 (47.1%) of 51 DDD/1fDDD/1-Mtv-2 (DDD/1fMtv-2) force-bred female mice during a one-year period. The mean tumour age +/- SE was 220 +/- 7 and 269 +/- 7 days, respectively. These tumours were examined for responsiveness to pregnancies by comparing their growth after transplantation between virgin and breeding recipients. Of 73 tumours from DDD/1-Mtv-2 mice, 9 (12%) were completely pregnancy-dependent (CPD), 3 (4%) pregnancy-dependent (PD), 9 (12%) pregnancy-responsive (PR), and 52 (71%) pregnancy-independent (PI), and of 25 tumours from DDD/1fMtv-2 mice, one (4%) was CPD, one (4%) PR, and 23 (92%) PI. Although most tumours were heterogeneous in morphology and there was no clear relation between morphology and PD properties, most CPD tumours were type P and considered to be connected with mammary plaques. When 9 CPD tumours from DDD/1-Mtv-2 mice were serially transplanted in breeders, 6, 2 and one progressed to lose pregnancy dependence within 3, 8 and 18 generations, respectively. DDD/1-Mtv-2 mice will provide a model for studies on progression of mammary tumours from hormone-dependent to autonomous states.

Transgenic mice carrying the Wnt-1 protooncogene modified for expression in mammary epithelial cells exhibit hyperplastic mammary glands and stochastically develop mammary carcinomas, suggesting that additional events are necessary for tumorigenesis. To induce such events and to identify the genes involved, we have infected Wnt-1 transgenic mice with mouse mammary tumor virus (MMTV), intending to insertionally activate, and thereby molecularly tag, cooperating protooncogenes. Infection of breeding female Wnt-1 transgenics decreased the average age at which tumors appeared from approximately 4 months to approximately 2.5 months and increased the average number of primary tumors per mouse from 1-2 to > 5. A smaller effect was observed in virgin females, and infection of transgenic males showed no significant effect on tumor latency. More than half of the tumors from the infected breeding group contained one or more newly acquired MMTV proviruses in a pattern suggesting that most cells in tumors arose from a single infected cell. Analyses of provirus-containing tumors for induced or altered expression of int-2/Fgf-3, hst/Fgf-4, int-3, and Wnt-3 showed activation of int-2 in 39% of tumors, hst in 3%, and both int-2 and hst in 3%. DNA analyses with probes for protooncogenes and MMTV confirmed that the activations resulted from proviral insertions. There was no evidence for proviral insertions at the int-3, Wnt-3, or Wnt-1 loci. These findings provide further evidence that fibroblast growth factors Int-2 and Hst can cooperate with Wnt-1, another secreted factor, in mammary tumorigenesis, and they illustrate the capacity of this system to identify cooperating oncogenes.

DDD/1 mice free from exogenous mouse mammary tumor virus (MMTV) do not develop any neoplastic mammary lesions. In GR mice, the expression of Mtv-2, an endogenous proviral MMTV, leads to 100% incidence of mammary ductal hyperplasias and tumors. An Mtv-2 congenic line, DDD/1-Mtv-2/Mtv-2, was established by introducing Mtv-2 from GR into DDD/1 to elucidate its function. Development of mammary plaques (MPQ) characterized by ductal hyperplasias was investigated in 152 congenic females on day 17 to 19 of the first pregnancy. The incidence of MPQ was 48.0% and most MPQ-positive mice (75.3%) had only one MPQ. Generally, MPQ were small in size: the diameter was as small as < or = 3 mm in 77.6% of them. Of 84 MPQ implanted into intact fat pads, 43 (51.2%), 38 (45.2%) and 3 (3.6%) showed undetectable, pregnancy-dependent and autonomous growths; respectively when the hosts underwent pregnancy. Almost all MPQ produced normal-appearing ductal-alveolar outgrowths in mammary epithelium-divested or cleared fat pads of virgins. MPQ implanted into cleared fat pads were very similar to normal mammary glands in the responses to progesterone (P) and estradiol (E) alone or in combination except for association of ductal hyperplasias in 4 of 12 MPQ under E+P treatment. These findings revealed the preneoplastic nature of MPQ. Exogenous MMTV proviruses were demonstrated in all MPQ. The int-2 DNA rearrangement was found in 2 of 10 MPQ but in none of 9 mammary carcinomas and the int-1 DNA rearrangement in none of 10 MPQ but in 5 of 10 carcinomas. It is thus likely that the Mtv-2 gene participates in a very early stage of mammary tumorigenesis not directly but indirectly through insertion mutation of host genes, while the cellular oncogenes, int-2 and int-1, may contribute to preneoplastic transformation of mammary epithelium and progression from preneoplastic to more malignant states, respectively.

Oncogenic viruses demonstrating a strict tropism for the mammary gland provide special opportunities to study the susceptibility of this tissue to neoplasia. In rats, human adenovirus type 9 (Ad9) elicits mammary fibroadenomas that are similar to common breast tumors in women, as well as phyllodes-like tumors and mammary sarcomas. By constructing recombinant adenoviruses between Ad9 and Ad26 (a related nontumorigenic virus), it was shown that the Ad9 E4 region was absolutely required to produce these mammary tumors. This indicates that an adenovirus gene located outside the classic transforming region (E1) can significantly influence the in vivo oncogenicity of an adenovirus. Consistent with a direct role in mammary gland oncogenesis, the Ad9 E4 region also exhibited transforming properties in vitro. Therefore, the Ad9 E4 region is a viral oncogene specifically involved in mammary gland tumorigenesis.

We found that the number of peripheral blood polymorphonuclear leukocytes (PMN) dramatically increased in both sarcoma 180 (S-180) and MM-46 mammary carcinoma (MM-46) ascites tumor-bearing mice, and mice required a remarkable resistance to Candida albicans infection via intravenous route. When the resistance was determined by number of cells of C. albicans in the kidney, a significant decrease in the number of fungal cells was observed in the kidneys of infected ascites tumor-bearing mice. An increase of active oxygen levels of PMN from ascites tumor-bearing mice was observed, suggesting that this factor is important in developing of resistance in ascites tumor-bearing mice. Additionally, a culture supernatant of tumor cells co-cultivated with bone-marrow cells in vitro increased the number of granulocytes and macrophages differentiated from the bone-marrow cells.

The uniformly lethal development of mammary tumors in polyomavirus-infected adult female nude mice was prevented by adoptive cell transfer of polyomavirus-immune splenocytes or peritoneal cells. Transferred immune cells also lowered the growth rate of emerging tumors. The induction of other relatively less frequent tumors of the skin and bone was decreased as well. Using in situ hybridization of whole-body sections as well as hybridization of nucleic acids from the mammary glands, we show for the first time that transferred immune cells, but not normal cells, virtually eliminated virus signal in the whole mouse and in the mammary glands. Since infected and tumorous mammary glands produce very little infectious virus, it appears that a major mechanism mediating the prevention of polyomavirus oncogenesis involves the immunological elimination of nonproductively and persistently infected cells.

The distribution of mouse mammary tumor virus (MMTV) antigens was studied by the immunoperoxidase method in the II-TES and I-TES mouse strains as well as their progenitors, CS and DBA/2 strains. In the II-TES, I-TES and CS strains, and BALB/c mice foster-nursed with these strains, MMTV antigens were found not only in epithelial cells of the mammary glands but also in those of other tissues including the seminal vesicle, vas deferens, epididymis, prostate, parotid, submandibular, lacrimal, sebaceous, and urethral glands. In DBA/2 and BALB/cfDBA/2 mice, however, the MMTV antigens were found only in the mammary glands. Electron microscopic examination showed MMTV particles in these organs. When we examined the presence of Mtv-1 and 2 proviruses, which are known to be responsible for MMTV expression, in the genomes of the II-TES, I-TES, CS and DBA/2 strains by Southern blotting, Mtv-2 was not found in any of the mice and Mtv-1 was found in the II-TES and DBA/2 mice but not in the I-TES and CS mice. Instead, four new endogenous MMTV loci, which have never previously been reported in laboratory mouse strains, were detected in the genomes of the II-TES, I-TES and CS strains. One (designated Mtv-42) was common in the three strains and the other three (designated Mtv-43, 44 and 45) were common in the II-TEX and I-TES strains or the II-TES and CS strains. These results thus suggest that new endogenous MMTV loci may be responsible for MMTV expression in a variety of tissues of these three strains.

The frequency with which int-1 and int-2 are rearranged in mouse mammary tumors by mouse mammary tumor virus (MMTV)-induced insertional mutagenesis is a consequence of the host genetic background. In 75% of C3H mammary tumors, int-1 is rearranged by MMTV insertion, whereas only 30% of BALB/cfC3H tumors contain a virus-induced rearrangement of int-1. This difference is significant (P less than 0.005) and could not be accounted for by the potentially additive effect of the genetically transmitted Mtv-1-encoded virus in C3H mice. Similarly, MMTV-induced rearrangement of the int-2 gene in mammary tumors of the R111 mouse strain (59%) occurred at a significantly (P less than 0.025) higher frequency than in BALB/cfR111 (25%) mammary tumors. Moreover, in BALB/cfR111 mammary tumors, there is evidence that rearrangement of int-1 and int-2 does not occur independently (P less than 0.025). These results suggest that the long history of inbreeding for high tumor incidence of C3H and R111 mouse strains has selected for the fixation of host mutations which either complement the action of the particular int gene or affect the sensitivity of specific subpopulations of mammary epithelium to infection by particular strains of MMTV.

Transcripts related to the human carcinoembryonic antigen were found in mRNA isolated from both dimethylbenzanthracene-induced and mouse mammary tumor virus-induced mammary tumors. A cDNA library was prepared from a dimethylbenzanthracene-induced tumor, and a clone was isolated by hybridization with a human carcinoembryonic antigen cDNA probe. Its sequence, when compared to those of members of the human carcinoembryonic antigen gene family, was most homologous to the sequence of the human biliary glycoprotein (BGP) gene. Thus, this clone appears to encode a portion of the mouse biliary glycoprotein gene. Southern blot analysis of EcoRI-digested mouse cellular DNA with this probe detected four restriction fragments, all of which appear to be located on mouse chromosome 7, Northern blot analysis using the mouse probe demonstrated that related mRNA species were expressed in some normal adult mouse tissues.

During the culturing of a mouse mammary tumor cell line, MuMT73, maintained in vitro for more than a decade, we found morphological heterogeneity in its cells; some showed contact inhibition in their growth, some formed domes and some grew criss cross and piled up. In trying to clone the cell line to isolate cells showing contact inhibition or dome formation, we were able to establish six clonal cell lines. These six cell lines were categorically divided into three groups according to their phenotypical behavior, Groups A, B and C. Group A (clones 1, 5 and 7) cells had a property of contact inhibition. They induced no tumor when injected into the subcutaneous tissue of the back, nor even when injected into the mammary fat pads or under the kidney capsule of syngenic or nude mice, and therefore were thought to be non-malignant in nature. They were positively stained by anti-keratin antiserum and had mouse mammary tumor viruses (MMTVs). Group B (clone 6) cells grew in a crisscross pattern and piled up, and they induced tumors when injected into the subcutaneous tissue of the back of mice. Group C (clones 3 and 4) cells formed domes in their growth and induced some tumors in the mammary fat pads and under the kidney capsule of KSN nude mice. In Southern blots with MMTV-env probe, numerous exogenous MMTV proviruses were detected in these cell lines. The insertion patterns of these proviruses in cells of non-malignant clonal lines (Group A) resembled those of malignant lines (Group B), except one band (about 26 Kb), but were considerably different from those of intermediate lines (Group C). On the other hand, no difference was detected in Southern blots with int-1 or int-2 probes among the non-malignant, intermediate and malignant clonal cell lines.

Chronic energy-intake restriction inhibits mouse mammary tumor virus (MMTV)-induced mammary tumors in C3H/Ou mice by greater than 90%. We have shown that associated with suppression of mammary tumorigenesis there is a reduction or inhibition of circulating prolactin, MMTV particles expressed, and MMTV mRNA transcription in mammary glands (and in most organs tested). To understand the concerted action of prolactin, energy-consumption level, and MMTV on inducing mammary tumors, experiments were designed to control prolactin and energy levels in order to evaluate their effects on MMTV mRNA expression. Mice on restricted diets were grafted with adenohypophyses, and mice fed ad libitum were treated with the dopaminomimetic agent octahydrobenzo [g]quinoline. Adenohypophyseal grafting significantly increased prolactin in dietary (energy)-restricted mice, and this effect was associated with an increase in MMTV mRNA expression within the mammary gland; a linear correlation between prolactin levels and MMTV mRNA expression in the mammary gland was found. Conversely, elimination of the nocturnal peak of circulating prolactin by i.p. injection of dopaminomimetic octahydrobenzo [g]quinoline to mice fed ad libitum delayed (by 8 weeks) and reduced (even as long as 25 weeks) mammary gland MMTV mRNA expression. These findings associate prolactin influences with MMTV mRNA production in mice and help explain the link between chronic energy-intake restriction and reduced MMTV gene expression.

We have isolated a common insertion site, Wnt-3, for proviruses of the mouse mammary tumor virus (MMTV). Of mammary tumors induced by the GR variant of MMTV, 5% contains a provirus at Wnt-3, which is located on mouse chromosome 11. The gene is transcribed into a 3.8-kilobase (kb) mRNA in tumors with nearby proviral insertions but not in tumors with proviruses at other loci or in most adult tissues. Normal expression of Wnt-3 is detected in mouse embryos (with a peak around day 12 of gestation) and at low levels in adult brain. The transcriptional unit of the Wnt-3 gene spans approximately 55 kb, with a first intron of 36 kb. The deduced amino acid sequence of the Wnt-3 protein is 47% identical to the int-1/Wnt-1 gene product.

Mouse mammary tumor virus (MMTV)-induced mammary adenocarcinomas can develop from several different premalignant precursors common in GR mice. Insertion mutagenesis of the mammary protooncogenes int-1 and int-2 was studied in this multistep system by analyzing samples from various stages of neoplastic development for novel int-1 and int-2 restriction fragments generated by MMTV provirus integration. int-1 and int-2 insertion mutations were observed in both premalignant lesions and malignant tumors. Some of the tumors with insertion mutations were experimentally derived from insertion mutation-free premalignant precursors. Each class of neoplasm examined had a characteristic frequency of int-1 and int-2 insertion mutations; however, no correspondence was observed between neoplasm morphology and mutation of either gene. These results indicate that insertion mutation of the int-1 and int-2 loci by MMTV provirus can be involved in the earliest identifiable stages of neoplastic development as well as during progression of premalignant lesions to tumors. Insertion mutation of int-1 and int-2 is therefore not stage specific in this system.

Chronic energy intake restriction (CEIR) reduces mouse mammary tumor virus (MMTV)-induced mammary tumors in C3H/Ou mice. Fewer than 10% of C3H/Ou mice developed mammary tumors during 88 wk of study when subjected to CEIR regardless of calorie source (fat vs. carbohydrate). By contrast, 100% of mice fed ad libitum diets relatively high in fat or carbohydrate or a commercial diet developed tumors by 35-40 wk. MMTV proviral DNA transcription was shown to be activated in spleen, liver, lung, kidney, small intestine, and mammary gland of mice consuming these diets ad libitum. By contrast, these messages were suppressed by CEIR in all tissues analyzed except spleen. MMTV proviral messages in liver and mammary gland increased with age in full-fed mice and were suppressed by CEIR. These findings suggest that the nutritional regulation of MMTV proviral DNA expression is tissue-specific. In CEIR mice the suppressed MMTV proviral DNA transcripts in mammary gland and liver increased with time in association with the delayed onset of mammary tumors. Mammary tumorigenesis in C3H mice is associated with integration of MMTV proviral DNA, which appears to activate a putative mammary tumor protooncogene, int-1. CEIR apparently decreases the frequency of viral reintegration adjacent to the int-1 gene and thus inhibits expression of int-1 and probably an initiation step in mammary tumorigenesis. Expression of other putative protooncogenes, int-2 and ras, in liver tissue was also reduced by CEIR. These findings indicate that both initiation and promotion of mammary tumorigenesis are influenced by CEIR in C3H/Ou mice.

We examined the effects of mouse mammary tumor virus integration on the multiple RNA transcripts expressed from the int-2 proto-oncogene in virally induced breast tumors. Proviral insertion either upstream or downstream of the gene could simultaneously activate transcription from three dissimilar int-2 promoters. In some tumors, the activating provirus lies within the transcription unit and disrupts the structures of the various RNAs. Insertions in the 5' region of the gene had complex effects depending on the orientation and position of the provirus relative to the three promoters and intron-exon boundaries. RNase protection experiments identified transcripts initiated in the viral long terminal repeat, at normal and cryptic sites in the int-2 sequences, and from cryptic promoters in an inverted provirus. AT the 3' end, insertions occurred within the untranslated trailer and provided alternative termination signals that substituted for one or both of the normal the poly(A) addition sites. However, in no instance, of the 20 tumors analyzed in detail, did a provirus perturb the presumed open reading frame of the gene. These data strongly implicate the normal product of the int-2 gene, which is related to the fibroblast growth factor family, as a contributory factor in virally induced mammary tumors.

Lactating mammary glands and hormonally induced mammary tumors of BALB/c mice from three geographically separated breeding colonies were examined by molecular hybridization, using murine mammary tumor virus (MuMTV) cDNA representing the entire viral genome to determine the amount of MuMTV-related RNA expressed in these tissues. The RNA extracted from these tissues by the classical sodium dodecyl sulfate-pronase, phenol-chloroform procedure (method 1) contained barely detectable levels of MuMTV-related sequences. In contrast, both normal lactating mammary glands and hormonally induced mammary tumors of these mice were found to contain approximately one to two copies of the MuMTV genome per cell by using a new procedure in which the RNA was extracted with guanidine derivatives (method 2). No significant differences in the MuMTV-related RNA content of the BALB/c mammary tissues were observed regardless of their colony of origin. Our results suggest that expression of MuMTV RNA does not change in BALB/c mammary glands during transformation to a malignant state and that MuMTV expression does not play a role in tumorigenesis in these mice. In view of the increased recovery of MuMTV-related RNA from BALB/c mice with method 2, we compared the level of MuMTV RNA expression in lactating mammary glands and mammary tumors of other mouse strains, including C57BL/6 and RIII, using both extraction methods. Yields of MuMTV-related RNA from mammary tissues increased by as much as 35- to 40-fold, using method 2 as compared with method 1. Therefore method 2, involving guanidine derivatives, appears to be method of choice for MuMTV-related RNA extraction from the mammary tissues of certain strains of mice, particularly those expressing relatively low levels of MuMTV RNA.

The possible interaction of environmental factors with the endogenous mouse mammary tumor virus (MMTV) genome in the development of mammary tumors in the low-tumor-incidence BALB/c mouse strain was examined. Tumors were induced in virgin female animals by treatment with chemical carcinogen 7,12- dimethylbenz[alpha]anthracene or urethan, with or without prolonged hormonal stimulation, or by X-irradiation. Concomitant hormonal stimulation resulted in increased tumor incidences compared with those induced by chemical carcinogen treatment alone. The frequency of tumor induction by irradiation alone or in combination with urethan or prolactin stimulation was very low. MMTV expression in the mammary tumors was assayed by nucleic acid hybridization and by immunohistochemical staining. Depending upon the treatment group, 0 to 89% of the tumors contained detectable levels of MMTV RNA (>/=0.0005% of the total cellular RNA). Tumors which contained detectable viral transcripts exhibited only low levels of MMTV RNA, which did not appear to represent the accumulation of RNA sequences homologous to the entire MMTV genome; synthesis of MMTV structural proteins was detected in only one tumor. Viral RNA-positive tumors were generally associated with a longer latent period. MMTV RNA expression occurred in tumors classified histologically as adenoacanthomas, as well as in mammary adenocarcinomas, although the cell types in the adenoacanthomas expressing viral RNA were not identified. It does not appear that expression of the endogenous MMTV genome is required for maintenance of all mammary tumors in BALB/c mice, although partial genome expression undetectable by the methods employed cannot be ruled out. Linear regression analyses were performed. The mean time to tumor appearance and the percentage of tumors which were MMTV RNA positive were found to vary linearly as a function of the total dose of 7,12-dimethylbenz[alpha]anthracene administered. The percentage of tumors which were MMTV RNA positive was also shown to be linearly related to the mean time to tumor appearance. These relationships provide a basis for predictions in the BALB/c system related to these parameters.

An indirect immunoperoxidase method is described, which can readily detect viral antigens in paraffin sections of primary, transplanted, and metastatic mammary tumors of mice. In addition to having the obvious advantage of not being limited to fresh specimens, immunoperoxidase staining of paraffin sections proved to be superior in many respects when compared with immunofluorescence and frozen sections. Immunoperoxidase staining of paraffin sections is permanent and provides the kind of histological detail required for precise cytological identification and localization with light microscopy. All of 25 tumors and 4 metastatic lesions showed evidence of glycoprotein gp52 as well as other mouse mammary tumor viral antigens. The pattern and intensity of the stain were related to the degree of histologic differentiation of the tumor. Wide variations in expression of viral antigens by individual malignant cells were observed within the same tumor.

A DNA species with buoyant densities greater than mouse cellular DNA was found associated with intracytoplasmic A particles (CAP) isolated from mouse mammary tumor virus-infected mouse mammary tumors and mouse Leydig cell tumors which produce CAP but no complete mouse mammary tumor virus virions. This DNA species was absent in identically prepared tissue fractions from tumors which did not contain CAP. Treatment of CAP-associated DNA with pancreatic RNase A did not alter the buoyant density although a reduction in apparent molecular weight (broadening of the DNA band at equilibrium) was observed upon analytical equilibrium sedimentation in CsCl. The molecular weight of untreated CAP-associated DNA was estimated to range from 0.8 x 10(6) to 3.1 x 10(6). Base composition analysis showed CAP-DNA to possess an approximate guanine plus cytosine content of 38%. Ninety percent of CAP-associated DNA eluted as single-stranded molecules upon hydroxyapatite column chromatography, a characteristic that accounts in part for its higher buoyant density in neutral CsCl compared to native double-stranded mouse DNA. In two preparations, CAP-DNA had a sedimentation coefficient of 7 to 8S.

The int-1 locus appears to be involved in over 80% of C3H exogenous mouse mammary tumor virus (MMTV)-induced mouse mammary tumors, and the int-2 locus appears to be involved in approximately 10% of these tumors. Analysis of 46 C3Hf mammary tumors resulting from endogenous, rather than exogenous, MMTV infection revealed that only 41% expressed int-1 RNA, while 2% expressed int-2 RNA. Our results suggest that in addition to the int-1 and int-2 loci, other loci may be involved in endogenous-MMTV-induced mammary tumors of the C3Hf mouse.

The int-1 gene is often activated by proviral insertion in mouse mammary tumors. Direct evidence for the normal function of this gene and its role in tumorigenesis has therefore been lacking. To examine possible biological effects of int-1 activation in in vitro cell systems, we have constructed recombinant molecules of genomic int-1 DNA, transcriptionally activated by retroviral promoters. Transfection of these constructs into cuboidal RAC311C mammary cells leads to morphological transformation of the cells and rapid tumorigenicity.

Electrophoretic data from both sodium dodecyl sulfate-polyacrylamide gels (SDS-PAGE) and acid-urea gels reveal a protein in purified murine mammary tumor virus (MuMTV) which co-migrates with purified chick skeletal muscle actin. 125I-labeling of intact and disrupted virus preparations shows that the actin-like protein is not artifactually adsorbed to the outside of virions during isolation. Quantitative SDS-PAGE and examination of negatively stained preparations show that the actin cannot be accounted for by a contaminating population of virus-free vesicles. The ultrastructure of mammary epithelial cells and of Rous sarcoma virus-transformed chick embryo fibroblasts shows that virus extrusion is associated with filament-containing cellular processes. In particular, MuMTV is released from the ends of long microvilli which contain a bundle of 6-8-nm microfilaments and share other structural features with intestinal microvilli. We suggest that virus nucleoids require an interaction with host cell contractile proteins for their extrusion from the cell.

The strain BALB/cNIV/Crgl was developed by infecting BALB/c/Crgl mice with mouse mammary tumor virus from C3Hf mice. A BALB/c normal mammary duct was transplanted into the gland-free fat pad of a hormone-stimulated female C3Hf X BALB/c F1 mouse. A hyperplastic alveolar nodule was found in the BALB/c ductal outgrowth and was transplanted into another hybrid gland-free fat pad. The resultant hyperplastic alveolar outgrowth was finally transplanted to female BALB/c mice. The hyperplastic alveolar outgrowth contained an exogenous, infectious mouse mammary tumor virus named the nodule-inducing virus, which was thought to be derived from the endogenous low oncogenic mouse mammary tumor virus found in C3Hf mice. The hyperplastic alveolar outgrowth-bearing BALB/c mice were inbred for four generations, and one family was selected as the strain BALB/cNIV/Crgl. It was found that (a) the mouse mammary tumor virus found in the BALB/cNIV strain was milk transmitted, but not transmitted by infected males; (b) the BALB/cNIV breeding females had a low tumor incidence (40%) and a longer latent period (14 months) than did female BALB/cfC3H mice (92% at 8 months); (c) the BALB/cNIV nodule outgrowths had low tumor-producing capabilities (50%) and longer latent periods (13.4 months) than did nodule outgrowths derived from female BALB/cfC3H mice (100% at 7.7 months).

All inbred strains of mice transmit one or more copies of mouse mammary tumor virus (MMTV) DNA integrated as proviral sequences. This complicates efforts to define viral-induced mammary carcinogenesis. Here we report the use of surgical nonlethal splenectomy in tissue typing mice and the development of an MMTV-negative mouse strain. The MMTV-negative strain allows study of the involvement of non-MMTV genes in mammary carcinogenesis. In addition, it can be used as a sterile background into which MMTV variants can be introduced. Through the techniques described here, mice containing single MMTV loci or specific combinations can be specially chosen and rapidly developed. In this manner, the oncogenecity of particular MMTV variants may be assessed.

Mouse mammary tumor cells (Mm5Mt/c1) produced mouse mammary tumor virus (MMTV) (type B retrovirus) at early passages in culture. They apparently produced predominently type C retrovirus(es) at late passages in culture. The decline in MMTV production during passage was not accompanied by an apparent decrease in the synthesis or accumulation of intracellular MMTV-specific RNA. As judged by the concentration of RNA (mol/titer) x half-time (sec) of hybridization of cellular RNA with MMTV complementary DNA, the intracellular concentration MMTV-specific RNA did not change significantly during passage in culture. Since late-passage RNA, particularly polysomal poly(adenylic acid)-containing RNA, yielded lower apparent maximum hybridization of MMTV complementary DNA as compared with early-passage RNA, it was possible that the complexity or base sequence of MMTV-specific RNA in late-passage cultures was not identical with that in early-passage cultures. The data on thermal transitions of hybrids were in accord with this possibility. In contrast, the steady-state level of type C virus-specific RNA in late-passage cultures was orders of magnitude higher than that in early-passage cultures. The production of type C retrovirus(es) at later passages was apparently accompanied by increased transcription of type c retrovirus DNA.

There is now good evidence that the induction of mammary carcinomas by mouse mammary tumour virus (MMTV) involves provirus activation of specific cellular genes. Thus, a high percentage of virally induced tumours contain an acquired MMTV provirus in either of two defined integration regions, termed int-1 and int-2, and provirus insertion is accompanied by expression of specific RNA transcripts from these regions. We show here that in some recurring, pregnancy-dependent mammary tumours provirus integration within int-2 has already occurred at the earliest appearance of the tumour and may therefore represent an important step in the development of neoplasia. As judged by the distribution of the acquired MMTV proviruses, the tumours recurring at any one site represent the same clonal population of cells as the original tumour and remain clonal during cycles of proliferation and regression, including the transition to hormone-independent status. These data suggest that either the expression of the int-2 locus or the function of this putative oncogene must remain responsive to hormones and that some additional event must be responsible for the transition to autonomous growth.

Mammary tumors in the GR mouse strain are caused by the expression of an endogenous provirus of the mouse mammary tumor virus (MMTV). The tumors progress from a hormone-dependent growth phase to autonomous, hormone-independent growth. We studied proviral insertion of MMTV at the int-1 and int-2 mammary oncogenes and the transcription of these genes during progression of a series of transplanted mammary tumors. During the hormone-dependent phase, 6 of 15 transplanted tumors were positive for proviral insertion at int-1 or int-2 or both. These tumors were oligoclonal with respect to the fraction of tumor cells with novel int-1 and int-2 restriction fragments and, apparently, consisted of different tumor cells with proviruses integrated at different oncogenes, including genes that are not yet known. In 10 tumors we detected expression of the int genes, indicating that most tumors contain minor populations of cells with int-1 or int-2 activations. On transplantation the tumors remained oligoclonal during the hormone-dependent phase. The hormone-independent variants of the tumors emerged as clonal outgrowths of cells with MMTV proviruses that could be traced back in the hormone-dependent tumors, but not always those of cells that were positive for insertions near int-1 or int-2. The maintenance of oligoclonality during the hormone-dependent phase suggests a growth-controlling effect of different populations of cells on each other. The clonal, hormone-independent tumors that arise later seem to be the result of mutations that are unrelated to int activation.

The RAC cell lines are derived from a mammary tumor induced by the mouse mammary tumor virus (MMTV). Polygonal cells have retained many characteristics of epithelial cells and induce adenocarcinomas; cuboidal cells are poorly tumorigenic; and elongated cells produce highly malignant sarcoma-like tumors. MMTV proviral integrations, one of which has cis-activated the int-2 gene, demonstrated that the lines are clonal descendents from a single tumor cell. Polygonal cells have a high constitutive expression of MMTV and contain int-2 RNA. In contrast, the cuboidal and the elongated cells show no detectable expression of int-2, even in the presence of glucocorticoid hormone. Transcription of MMTV in these cells is also low but can be stimulated by dexamethasone, albeit to levels lower than in polygonal cells. Thus, expression of int-2 seems to be caused by an enhancing activity on the MMTV provirus which is not dependent on steroid hormone and is specific for mammary tumor cells with epithelial characteristics. Progression in this cell system does not require sustained expression of int-2.

The prototype virus of the type D retroviruses is the Mason-Pfizer monkey virus (MPMV). MPMV was originally isolated from a breast carcinoma of a female rhesus monkey (an Old World monkey). MPMV is of obvious importance in that it is the only retrovirus thus far isolated from a mammary tumor of a primate and has been shown to have transforming potential for primate cells in vitro. Subsequent to the isolation of MPMV viruses morphologically and immunologically indistinguishable from MPMV have been isolated from normal placenta and lactating mammary glands of other rhesus monkeys in captivity. Recently, viruses morphologically resembling MPMV have been isolated from a langur monkey (another Old World monkey) and from squirrel monkeys (a New World monkey). Based on nucleic acid hybridization studies, the latter 2 viruses represent endogenous viruses in their species of origin, whereas MPMV appears to be a horizontally related to the langur monkey isolate. Studies on the immunological relatedness of the type D retroviruses have demonstrated interspecies cross-reactivities between the major internal and external proteins of the viruses. Furthermore, these viruses also share cross-reactivity of their major external glycoproteins with those of the type C baboon endogenous virus. These interspecies reactivities can also be demonstrated in natural sera from both imported and laboratory-bred monkeys. The demonstration of these interspecies cross-reactivities shared by distantly related primate retroviruses provides a means for detecting determinants that are representative of all primate retroviruses presently known and yet to be isolated and may provide new assays for detection of a human retrovirus.

The milk fat-globule membrane and the mouse mammary-tumour virus isolated from the milk of infected Swiss mice have been investigated for their content in gangliosides. When compared on the lipid phosphorus basis, viral envelope is found to contain more than twice as much lipid-bound sialic acid as fat-globule membrane. The ganglioside patterns of these two structures appear rather similar, except for the occurrence in fat-globule membrane of a low ganglioside homolog, presumably GM2, not detected in viral envelope. A common and dominant trait is the presence in both structures, as the main ganglioside, of a component which has been so far characterized as a disialoganglioside, having the same neutral glycolipid moiety as GD1a, but with both sialic acid residues displayig to Clostridium perfringens and Vibrio cholerae neuraminidase, the susceptibility typical of terminal sialic acid residues.

In the mouse strain GR, the Mtv-2 gene controls the expression of large amounts of mammary tumor virus (MTV) antigens in the milk at first lactation. It also controls the early appearance of mammary tumors. We have investigated the number of MTV proviral sequences associated with this Mtv-2 gene by nucleic acid hybridization between MTV [(3)H]cDNA and DNA from GR, B10, and GR-Mtv-2(-) mice. B10 and GR-Mtv-2(-) mice lack Mtv-2 gene expression. The molecular hybridizations revealed that the DNA of GR mice contains 12 copies of MTV proviral sequences, whereas only 4 copies are present in the DNA of B10 and GR-Mtv-2(-) mice. We therefore conclude that the Mtv-2 gene in the GR mouse strain is associated with eight additional MTV proviral sequences. The four Mtv proviral sequences in the GR-Mtv-2(-) DNA might represent another Mtv gene in the GR mouse. Different amounts of MTV RNA are detected in mammary glands at first lactation of B10 and GR-Mtv-2(-) mice, even though both contain four copies of MTV proviral sequences. This indicates a difference between these two mouse strains either in the regulation of expression of these MTV proviral sequences or in the location of these sequences in the murine genome.

The effect of the monovalent carboxylic ionophore monensin on the biosynthesis, intracellular transport, and surface expression of the glycoproteins of Mason-Pfizer monkey virus was examined. Cells treated with monensin at concentrations of 10(-7) or 10(-6) M continued to synthesize virus particles, which from electron microscopic studies appeared to bud normally from the plasma membrane of the cells. However, the particles released had an altered buoyant density in sucrose gradients and were noninfectious. These noninfectious virions had a normal complement of non-glycosylated polypeptides but showed a significantly reduced amount of glycosylated proteins. The gp70 and gp20 polypeptides appeared to be completely absent, and a heterogeneous, higher-molecular-weight protein was observed on the virions instead. Studies on intracellular protein synthesis indicated that the precursor (Pr86env) to gp70 and gp20 is synthesized normally but is not cleaved to the mature proteins. Immunofluorescence studies showed, however, that the uncleaved molecule is expressed on the cell surface. In this system, therefore, Mason-Pfizer monkey virus glycoprotein migration appears to occur in the presence of monensin, whereas the cleavage and insertion of the glycoproteins into virions are inhibited.

In BALB/cfC3H and BALB/cfRIII virgin female mice, i.e., BALB/c mice carrying milk-transmitted C3H and those carrying RIII murine mammary tumor virus infection, respectively, mammary noduligenesis and tumorigenesis are remarkably different. Focal hyperplastic alveolar changes or hyperplastic alveolar nodules were removed from BALB/cfRIII or BALB/cfC3H breeding female donors and transplanted into the gland-free mammary fat pads of syngeneic BALB/c virgin female recipients with or without hormonal supplementation. The results obtained have shown significant differences in: (a) the morphological type of outgrowths; (b) the incidence of malignant transformations; and (c) the hormonal responsiveness of the tumors arisen after pituitary transplants.

Intracytoplasmic A particles (CAP), previously identified as probably cytoplasmic nucleocapsid precursor structures to mouse mammary tumour virus (MMTV), possess both DNA binding and DNA unwinding activities, CAP proteins bind to both single-stranded (ss)- and double-stranded (ds)DNA, with the ssDNA slightly preferred. This activity was linear over a 30-fold concentration of A particle protein and was not affected by NaCl concentrations as high as 0.6 M or pH changes over a wide range. DNA binding by CAP proteins was sensitive to heat or addition of sodium dodecyl sulphate (SDS) and was neutralized by pre-incubation of CAP with anti-MMTV p14, but not by anti-MMTV p27, p10 or anti-mouse casein. Incubation of CAP with dsDNA led to unwinding of the double helix as measured by its increased sensitivity to S1 nuclease digestion. This activity was also linear over a several-fold concentration of A particle protein and was heat labile. It was not inhibited by pre-incubation of CAP with either anti-MMTV p14 or with anti-MMTV, anti-MMTV p27 and anti-MMTV p10. DNA unwinding was inhibited by anti-A particle antiserum and to a lesser extent by anti-CAP p20-18.

The role of steroids in promoting cell proliferation is well established but the molecular mechanisms are not clear. The S115 mouse mammary tumour cell line provides a model system for molecular studies in vitro in that it exhibits in tissue culture both a positive proliferative response to androgens and a change from a transformed phenotype in the presence of androgen to a normal phenotype when androgen is removed. We have considered here the possible involvement of mouse mammary tumour virus (MMTV) in these processes. We have demonstrated the presence in S115 cells of MMTV-related sequences which are transcribed into RNA only in the long-term presence of androgen. Prolonged culture in the absence of androgen, which results in loss of proliferative response to androgen, is accompanied by loss of MMTV-related RNA and increased methylation of MMTV-related sequences.