Immunization of mice against murine mammary tumor virus infection and mammary tumor development.

Formalin-inactivated whole murine mammary tumor virus (MuMTV), VuMTV membranes, the acid-soluble component of MuMTV, and purified MuMTV glycoprotein with a molecular weight of 55,000 (gp55; also designated as gp52) were used as vaccines in an attempt to identify the MuMTV antigen(s) that can protect mice from exogenous MuMTV infection and subsequent tumor development. Formalin-inactivated whole MuMTV, MuMTV membranes, and purified MuMTV gp55 were effective immunogens, whereas the acid-soluble component of MuMTV (which consists mainly of MuMTV gp55) failed to protect mice from challenge with live virus. These results suggest that (a) MuMTV gp55 is the major immunizing antigen and (b) its native conformation must be maintained for it to be an effective vaccine.     

Effect of a mouse mammary tumor virus-derived protein vaccine on primary tumor development in mice.

The vaccines used in this study were derived from purified murine mammary tumor virus (MuMTV) preparations. Approximately 60% of the protein fractions consisted of the major viral membrane glycoprotein gp52. Inoculation sc of 10 microgram MuMTV-S-derived vaccine significantly delayed the appearance of primary mammary tumors in GR and BALB/cfC3H mice (strains with high incidences of mammary cancer); in BALB/c and C3Hf mice, which have a moderate tumor incidence at an advanced age, this treatment resulted in a slight and substantial acceleration, respectively, of primary tumor development. The induced cellular immune reactivity for vaccination, as measured with the in vivo Winn test and the in vitro leukocyte adherence inhibition assay, was strongest in the GR strain as compared to the BALB/c strain. The titer of antibodies to tumor cells, as estimated by membrane immunofluorescence, was also higher in the GR strain. In BALB/cfC3H mice, the influence of different vaccination schemes with an MuMTV-O-derived protein vaccine on primary tumor development was studied. Before sc injection, the vaccine was precipitated on alum. A dose of 10 microgram vaccine resulted in a 61% decrease in tumor incidence. Two or five additional booster injections with 1 microgram protein vaccine had no beneficial effect, although the amount of antibody measured was increased after boosting.     

Naturally occurring humoral immunity to murine mammary tumor virus (MuMTV) and MuMTV GP52 in mice with low mammary tumor incidence.

Antibodies to murine mammary tumor virus (MuMTV) were found in sera from male and female mice by means of a radiolabelled intact MuMTV precipitation assay. These antibodies were demonstrated both in strains of mice that have a high incidence of mammary tumors and transmit the highly oncogenic MuMTV via the milk (C3H) and in mice that have been foster-nursed to remove the highly oncogenic milk-borne MuMTV (C3Hf) and subsequently have a decreased mammary tumor incidence. Antibodies to MuMTV were readily demonstrated in C3H mice at 6 weeks of age, whereas only marginal antibody activity was detected in C3Hf mice of less than 15 weeks of age. Antibody levels increased with age in both strains, but in C3Hf mice the immune response was also accelerated by pregnancy. Several feral and BALB/c NIV mouse sera with high (¹²⁵I)MMTV precipitating titers also precipitated the major MuMTV envelope glycoprotein (gp52). Maximum precipitation of the (¹²⁵I)gp52 was >50% with a 20% endpoint binding titer of 1:40, whereas the same sera precipitated >80% of (¹²⁵I)MMTV with a titer of >1:2560. These naturally occurring antibodies were specific for gp52 since only MuMTV and purified MuMTV gp52 competed for binding of radiolabelled antigens by limiting dilutions of mouse sera. MuMTV gp52 was found in both C3H and C3Hf mice, predominantly in organs which had secretory functions, such as submaxillary glands and mammary tissue of females and submaxillary, coagulating, and vesicular glands and vas deferens of males. Extracts of other tissues were negative for MuMTV gp52. Neither gp52 nor naturally occurring antibodies for MuMTV were found in BALB/c or C57BL/6 mice.     

Gene expression screening for specific genes associated with mouse mammary tumor development

The preneoplastic hyperplastic alveolar nodule is a frequent and well-characterized precursor to mammary tumors in the mouse. Although the biological characteristics of the preneoplastic state have been understood for many years, the molecular alterations associated with preneoplasia and tumorigenicity are unknown. We applied the technique of differential display of mRNA to two closely matched cell populations of mammary preneoplasias that differed only in their tumorigenic potential. Two mRNAs were isolated that were overexpressed only in tumorigenic preneoplasias and in tumors and not in normal pregnant mammary gland or in nontumorigenic preneoplasias. Partial nucleotide sequencing indicated that one of the mRNAs had not yet been described, whereas the second mRNA was highly homologous to a relatively uncharacterized gene termed pT-2. These results illustrate the utility of the differential display method for isolating and identifying uniquely expressed genes from tissues maintained in the microenvironment where tumors arise naturally.     

Murine mammary tumor virus seroepidemiology in BALB/cfC3H mice: correlation with tumor development

Serum reactivity to murine mammary tumor virus (MuMTV) was inversely related to mammary tumor risk in 8-to 22-week-old BALB/cfC3H breeding females. Mice at low tumor risk exhibited high-titered serum reactivity to MuMTV (50% end point, greater than or equal to 1:40 by radioimmunoassay) approximately 3-6 months earlier than did the mice at high tumor risk. Maternal MuMTV antibody levels were correlated with the serum anti-MuMTV reactivity of their neonatal offspring (2 wk of age). Serologic antiviral reactivity in infected mice did not change during periods of pregnancy and lactation. All infected animals had detectable serum MuMTV reactivity by 33 weeks of age. The virus-neutralizing capabilities of some of these sera were tested, Sera from some of the young, low-tumor-risk animals that had MuMTV-precipitating antibodies also had virus-neutralizing activity. Conversely, none of the sera from the high-tumor-risk animals had detectable MuMTV-neutralizing antibodies.     

Bcl-2 expression delays mammary tumor development in dimethylbenz(a)anthracene-treated transgenic mice

Bcl-2 is known to have dual antiproliferative and antiapoptotic roles. Overexpression of Bcl-2 in the mammary gland using a whey acidic protein (WAP) promoter-driven Bcl-2 transgene inhibits apoptosis in the mammary gland during pregnancy, lactation, and involution, and also counteracts apoptosis induced by overexpression of a mutant p53 transgene (WAP-p53 172 R-L). WAP-Bcl-2 mice and nontransgenic controls were treated with the carcinogen dimethylbenz(a)anthracene (DMBA). Surprisingly, the nontransgenic mice developed mammary tumors with decreased latency. Tumors arising in WAP-Bcl-2 mice displayed substantially reduced levels of proliferation relative to those seen in nontransgenic mice (P < 0.015), perhaps resulting in the observed increase in tumor latency following carcinogen treatment. This WAP-Bcl-2 mouse tumor model reflects the situation seen in some human breast cancers overexpressing Bcl-2, where expression of Bcl-2 has been shown to correlate with a lower proliferative index in tumors.     

Primary tumor immunity in nude mice

Tumor implants grew better in radiated or in newborn nu/nu mice than in adult nu/nu controls when, and only when, the tumors were demonstrably immunogenic in normal mice. This result suggests primary immunity. No evidence of immunological memory was found by immunization-challenge type experiments in the nude mice.     

Importance of epidermal growth factor in implantation and growth of mouse mammary tumor in female nude mice

In female nude mice, epidermal growth factor (EGF) was present at a mean concentration of 42.8 +/- 16.9 (SD) ng/mg wet tissue in the submandibular gland and 0.28 +/- 0.13 ng/ml in the plasma. Sialoadenectomy (removal of the submandibular glands) decreased circulating EGF to undetectable levels (less than 0.1 ng/ml). The possible role of EGF in transplantation and growth of mouse mammary tumors in nude mice was examined by sialoadenectomy, anti-EGF treatment, and EGF replacement therapy. The success rate of transplantation of a spontaneous mouse mammary tumor into nude mice was 55% in normal females and 17% in sialoadenectomized animals. Anti-EGF treatment of sialoadenectomized mice completely abolished the implantation of the tumor. Daily administration of EGF at a dose of 5 micrograms per mouse to both normal and sialoadenectomized animals enhanced the success rate of tumor implantation to more than 80%. Sialoadenectomy and/or anti-EGF treatment of tumor-bearing nude mice reduced the growth of implanted mammary tumors. These results suggest that EGF is important for implantation and growth of spontaneous mouse mammary tumor in female nude mice.     

Iron enhances tumor growth. Observation on spontaneous mammary tumors in mice.

Iron is essential for the growth of all cells, including tumor cells. The authors previously reported that a variety of transplantable tumors grew faster and larger in mice that were on an iron-rich diet compared with those on an iron-deficient diet. In this study the authors examined the relationship between iron in the diet and development of tumors in mice that are known to develop spontaneous tumors--C3H/HeN-MTV+(C3H-MTV+) mice that were congenitally infected with mammary tumor virus. These mice have a greater than 96% chance of developing mammary tumors between the ages of 7.2 and 9.2 months. Fifteen C3H-MTV+ weanlings were given a low-iron diet (5 mg iron/kg diet), and 15 were given diets with normal amounts of iron (180 mg Fe/kg diet). Thirteen of the 15 mice from the low-iron group and all 15 mice from the normal-iron group developed tumors. The average tumor growth rate in the normal-iron group was 112%/wk, compared with 62%/wk for the low-iron group. The difference in tumor growth rate between the two groups was significant (P = 0.02 by Student's t test). In this study, low iron intake did not prevent tumor development, but the results confirm the authors' previous report that iron nutrition of the host affects tumor growth; tumors grow better in an iron-rich environment. High levels of iron in the diet may enhance tumor growth, and this should be considered when treating patients with cancer.     

Prolactin gene-disruption arrests mammary gland development and retards T-antigen-induced tumor growth

Prolactin (PRL), interacting with other hormones from the pituitary, gonad, and placenta, activates specific signals that drive the appropriately timed morphological and functional development of the mammary gland. A mouse model of isolated PRL deficiency (PRL-/-) was created by gene disruption in an effort to further understand the molecular basis of mammary gland development and breast cancer. Whereas primary ductal growth was normal in PRL-/- mice, ductal arborization was minimal (branches/mm2=1.5+/-0.5), and lobular budding was absent. Replacement therapy with PRL injections stimulated a modest degree of lobular budding and ductal arborization (3.75+/-0.9). Pituitary transplants to the kidney capsule of PRL-/- mice restored lobular budding and ductal arborization, to the full extent of that seen in control animals (20. 3+/-5.5). Pregnancy, established by mating progesterone-treated PRL-/- females with PRL-/- males, led to complete morphological development of the mammary gland, appropriate to the gestational stage. PRL treatment stimulated tyrosine phosphorylation and DNA binding activity of Stat5a, but not Stat1 in PRL-/- or PRL+/- females, and Stat5a, but not Stat1, was elevated by estradiol within 24 h. PRL-deficient mice were crossed with mice expressing a dominant oncogene (polyoma middle-T antigen driven by the MMTV promoter, PyVT mice). Palpable (1 mm3) tumors were detected an average of 9 days earlier in hormonally normal females (PRL+/-:PyVT) compared with littermates that were PRL-deficient (PRL-/-:PyVT). The growth rate of PyVT-induced tumors was 30% faster in PRL+/-, than in PRL-/- females.     

Continuous intravenous fucose treatment of rat mammary tumor

Thirteen male, adult, 400-gm Lewis-Wistar rats received implants of a transplantable mammary tumor, which had arisen spontaneously in rats being treated with N-N Dimethyl B-Aziridino propionamide. Three rats (controls) received no treatment, five were given dextrose (8.7 mg/kg/day) and five given L-fucose (7.6 mg/kg/day) for four days. The sugars were adminstered by continuous intravenous infusion through a catheter placed in the superior vena cava which was maintained in position with a swivel harness apparatus allowing complete freedom of motion during therapy. There was a 20% reduction in tumor size (mean diameter) during fucose infusion, while the controls increased by 5%. The fucose did produce a diuresis but no toxicity was encountered. At sacrifice, on the 23rd day of tumor growth, bone marrow, liver, kidney, and spleen were normal histologically. Tumor fucose was approximately doubled in those receiving fucose, as compared to the control and dextrose treated animals.