Effect of simulated American,Bulgarian, and Japanese human diets and of selenium supplementation on the incidence of virally induced mammary tumors in female mice

In attempts to simulate the effects of diet on human breast cancer development groups of female C3H mice infected with mammary tumor virus (MMTV-) were maintained on diets formulated to resemble the typical American, Bulgarian, and Japanese human diets. The incidence of mammary tumors was the highest (84%) in group of mice receiving the simulated meat- and fat-rich American diet, which was also low in selenium (Se content: 0.15 ppm). The appearance of mammary tumors was delayed in the mice maintained on the simulated Bulgarian diet, and the final tumor incidence (27%) paralleled the correspondingly lower Bulgarian breast cancer incidence. The simulated Bulgarian diet contained more Se (0.25 ppm), and was lower in fat, meat, and sugar and higher in complex carbohydrates (cereals) than the simulated American diet. In the mice fed the simulated Japanese diet, the appearance of mammary tumors was also delayed, and the tumor incidence was diminished to 47%. In this diet, fish meal was a major source of Se, which is known to have low bioavailability. Additional supplementation of the Japanese-type diet with bioavailable Se (1 ppm) lowered the tumor incidence to 10%. Based on these studies, recommendations are made for breast cancer risk reduction by dietary means.     

Calorie consumption level influences development of C3H/Ou breast adenocarcinoma with indifference to calorie source

To analyze simultaneously the influence attributable to calorie consumption level and percentage of dietary fat on the spontaneous development of mammary adenocarcinoma, virgin female C3H/Ou mice were separated into five dietary groups. Four groups of mice were fed purified diets either ad libitum (16-18 kcal/mouse/day) or restricted 40% in calorie consumption (10-11 kcal/mouse/day), and diets contained either 4.5%, 7.5%, 67%, or 68% calories from fat. Mice that consumed isocaloric diets developed breast malignancy at a comparable pace. Consuming a diet in which fats were present only at levels sufficient to satisfy the threshold requirement of essential fatty acids, 4.5-7.5% of the total calories, or alternatively where dietary fat represented greater than 67% of the total calories consumed, did not significantly alter the tendency for breast tumor development. The pace and frequency with which tumors occurred reflected the host's level of calorie consumption. Mice consuming a high caloric diet, low or high in fat, tended to have a shortened latency to breast tumor formation, an increased incidence of breast tumors, elevated serum prolactin levels, elevated levels of antibodies to mouse mammary tumor virus, and elevated circulating immune complex levels.     

Selenium and cancer: effects of selenium and of the diet on the genesis of spontaneous mammary tumors in virgin inbred female C3H/St mice

Inbred female C3H/St mice exhibit the normal incidence of spontaneous mammary adenocarcinoma of 80--100% if they are maintained on a standard commercial laboratory diet containing 0.15 ppm of selenium with meat and dried skimmed milk as major sources of protein. The tumor incidence drops to 42% if animals of the same strain are kept on a diet containing 0.45 ppm of selenium, with fishmeal as the main source of protein. The tumor incidence declines further to 25, 19 and 10% if the animals in addition receive 0.1, 0.5, and 1.0 ppm of selenium in the drinking water. Selenium supplementation at these levels has no noticable adverse effects on weight-grains and survival of the mice. Selenium supplmented groups of animals also remained tumor-free for longer periods than the unsupplemented controls. The results of this study indicate that a diet rich in seafoods and cereals provides more selenium and may in turn lower the probability of cancer development. Reference is made to the average human diet in the U.S.A., which only contains 0.07--0.15 ppm of selenium due to the comparatively low consumption of cereals and seafoods. An equivalent mouse diet would not have any cancer-protecting effect in the C3H/St mice of our study. Australian workers have reported significantly lower tumor incidence in a different strain of C3H mice if it was kept in Australia rather than in the U.S.A. We have found that the Australian feed contained three times more selenium than that employed in the U.S.A. and propose that this difference in selenium content was primarily responsible for these previous observations.     

A novel mechanism of resistance to mouse mammary tumor virus infection

Exogenous mouse mammary tumor virus (MMTV) is carried from the gut of suckling pups to the mammary glands by lymphocytes and induces mammary gland tumors. MMTV-induced tumor incidence in inbred mice of different strains ranges from 0 to as high as 100%. For example, mice of the C3H/HeN strain are highly susceptible, whereas mice of the I/LnJ strain are highly resistant. Of the different factors that together determine the susceptibility of mice to development of MMTV-induced mammary tumors, genetic elements play a major role, although very few genes that determine a susceptibility-resistance phenotype have been identified so far. Our data indicate that MMTV fails to infect mammary glands in I/LnJ mice foster nursed on viremic C3H/HeN females, even though the I/LnJ mammary tissue is not refractory to MMTV infection. Lymphocytes from fostered I/LnJ mice contained integrated MMTV proviruses and shed virus but failed to establish infection in the mammary glands of susceptible syngeneic (I x C3H.JK)F(1) females. Based on the susceptible-resistant phenotype distribution in N(2) females, both MMTV mammary gland infection and mammary gland tumor development in I/LnJ mice are controlled by a single locus.     

Mouse mammary tumor virus proviral sequences congenital to C3H/Sm mice are differentially hypomethylated in chemically induced, virus-induced, and spontaneous mammary tumors

C3H/Sm mice have lost the exogenous milk-borne mouse mammary tumor virus (MMTV) characteristic of the C3H strain and have a very low (1.5%) incidence of spontaneous mammary tumors, yet they are highly susceptible to mammary carcinogenesis by either chemical carcinogens or infection with the milk-borne virus. We have analyzed the MMTV proviral DNA content of normal tissues and of spontaneous, virus-induced, and chemically induced mammary tumors by restriction endonuclease digestion and Southern blot analysis. Although the results clearly showed additional MMTV sequences in the virus-induced tumor which are not present in normal liver DNA, none of the spontaneous or chemically induced tumors could be shown to contain either newly acquired exogenous or amplified endogenous MMTV sequences. Interestingly, mammary tumors arising in C3H/Sm mice treated simultaneously with infectious MMTV (C3H) and dimethylbenz[a]anthracene (DMBA) possessed new exogenous MMTV DNA even though no quantitative change in tumor production was observed when these mice were compared with C3H/Sm mice treated with DMBA alone (Smith et al., Int. J. Cancer 26:373-379, 1980). Our data indicate that the endogenous MMTV proviral units are extensively methylated in normal tissues, such as livers and normal nonlactating mammary glands. In the absence of MMTV (C3H), we found that in the rare, spontaneously occurring C3H/Sm mammary tumors, certain endogenous MMTV sequences were specifically hypomethylated. Hypomethylation of endogenous MMTV sequences was also noted in the chemically induced mammary tumors, even though radioimmune competition assays for MMTV gp52 and p28 are negative (Smith et al., Int. J. Cancer 27:81-86, 1981). Our results support the conclusion that amplification of endogenous MMTV sequences is not intrinsic to C3H/Sm mouse mammary tumors arising spontaneously or after induction by chemicals. On the other hand, integration of exogenous MMTV DNA into the genome was a constant feature of mammary tumors developing in MMTV (C3H)-infected C3H/Sm mice, even when DMBA was used as the carcinogen. Hypomethylation of some endogenous MMTV sequences is characteristic of C3H/Sm mammary tumors, whether spontaneous or induced by chemicals, which suggests that these sequences are located in actively transcribing regions of the tumor cell genome.     

Molecular cloning, characterization, and genetic mapping of an endogenous murine mammary tumor virus proviral unit I of C3H/He mice.

We have cloned and characterized a novel endogenous murine mammary tumor virus proviral unit of the C3H/He strain of mice. The cloned proviral unit is 16 kilobase pairs (kbp) in size and is composed of a 5.6-kbp 5' EcoRI segment of an endogenous provirus with 10.4-kbp flanking cellular sequences. A comparison of the restriction map of the cloned proviral DNA with that of an endogenous provirus of the GR strain of mice has revealed minor differences in restriction sites on the two proviruses. The restriction enzyme SstI, which does not cleave the 5' EcoRI fragment of GR DNA, cleaves the C3H/He proviral sequences once; MspI has an additional site in the C3H/He proviral sequences. By using a subcloned fragment containing unique cellular sequences as a hybridization probe, we (i) mapped the C3H/He proviral unit to chromosome 14 by using mouse-hamster somatic cell hybrids, and (ii) demonstrated that this proviral unit is also present in the genome of DBA/2 mice. From these results we conclude that the C3H/He strain of mice acquired this proviral unit from DBA stock by genetic transmission. Our data also indicate that the murine mammary tumor virus sequences present in the gag-specific proviral unit of C3H/He mice extend at least 2.45 kbp downstream of the EcoRI site in the genomic DNA. Since the structural organization and chromosomal location of this proviral unit are distinct from those of previously reported proviral units represented by similar-sized (16.7-kbp) EcoRI fragments, we tentatively propose to designate this proviral unit Mtv-7a.     

Effects of dietary selenium and of lead on the genesis of spontaneous mammary tumors in mice

Selenium added to the diet significantly lowers the incidence of spontaneous mammary adenocarcinoma in female inbred C3H/St mice infected with the Bittner Milk Factor. Lead, 5 ppm, added to the drinking water in the form of the acetate, diminishes the uptake of selenium and reduces its anticarcinogenic effects, causing mammary tumors to appear with the same high incidence as in Se-unsupplemented controls. At higher lead concentrations in the drinking water (25 ppm), the overall tumor incidence is lowered, but tumor growth is significantly accelerated and the survival of tumor-bearing mice is shortened. Under the conditions of administration chosen, lead acts as a selenium antagonist and lowers the concentrations of selenium in liver, kidney, and spleen. The deposition of selenium, copper, and arsenic in bone is increased as compared to lead-unexposed controls.     

The effects of physiological adaptations to calorie restriction on global cell proliferation rates

Calorie restriction (CR) reduces the rate of cell proliferation in mitotic tissues. It has been suggested that this reduction in cell proliferation may mediate CR-induced increases in longevity. However, the mechanisms that lead to CR-induced reductions in cell proliferation rates remain unclear. To evaluate the CR-induced physiological adaptations that may mediate reductions in cell proliferation rates, we altered housing temperature and access to voluntary running wheels to determine the effects of food intake, energy expenditure, percent body fat, and body weight on proliferation rates of keratinocytes, liver cells, mammary epithelial cells, and splenic T-cells in C57BL/6 mice. We found that ～20% CR led to a reduction in cell proliferation rates in all cell types. However, lower cell proliferation rates were not observed with reductions in 1) food intake and energy expenditure in female mice housed at 27°C, 2) percent body fat in female mice provided running wheels, or 3) body weight in male mice provided running wheels compared with ad libitum-fed controls. In contrast, reductions in insulin-like growth factor I were associated with decreased cell proliferation rates. Taken together, these data suggest that CR-induced reductions in food intake, energy expenditure, percent body fat, and body weight do not account for the reductions in global cell proliferation rates observed in CR. In addition, these data are consistent with the hypothesis that reduced cell proliferation rates could be useful as a biomarker of interventions that increase longevity.     

In vitro pregnenolone metabolism by mouse adrenal gland: II-Biosynthesis of androgens

Adrenal gland homogenates from four different strains of mice were incubated with (4-14 C)-pregnenolone and a NADPH generating system. The most important androgen synthesized was dehydroepiandrosterone; testosterone and progesterone were synthesized to a lesser extent and the production of androstenedione was very low. The highest synthetic activities were found in the high mammary tumor strain of mice (C3H x RIII) Fl; they were increased by ovariectomy, particularly when performed at two months of age. In the other strains, they were lower, specially in the low mammary tumor strain C 57 BL. However, the 3 beta-hydroxysteroid dehydrogenase / delta 5, 4 isomerase activity was not modified by ovariectomy in the high mammary tumor strain whereas it was increased in the low mammary tumor strains. These results indicate that the androgen synthesis in mouse adrenal depends on factors such as age, sex, endocrine status (ovariectomy) but also on susceptibility to mammary tumor development.     

Establishment and characterization of a new murine mammary tumor cell line,balb/c-MC

Summary A new murine mammary tumor cell line (BALB/c-MC) was established from a spontaneous mammary tumor in a 17-mo.-old female mouse of the low mammary cancer strain BALB/cHe. The cell line was derived from a papillary adenocarcinoma. In monolayer culture the line exhibits a pavementlike arrangement of cells and forms “domes” or “hemicysts” as the cells become confluent. The cell line rapidly forms tumors when transplanted into young syngeneic BALB/cHe mice. The subcutaneous injection of 10⁶ cells resulted in the development of mammary tumors (typical papillary adenocarcinomas) in 33 of 37 (87% recipients within 2 to 3 mo. after injection. These mammary tumors also metastasize to lung [14 of 33 (42%) of recipients] during this time. The number of chromosomes in this cell line is hyperdiploid (average of 43, range 39 to 44).     

Tumour incidence, growth, reproduction and longevity in female C3H mice fed polyunsaturated ruminant-derived foodstuffs

Earlier studies with the random-bred Quackenbush mouse strain showed that human-type diets based on linoleic acid-enriched foodstuffs derived from ruminants fed protected polyunsaturated oils have no detrimental effects on growth, reproduction or longevity. Tumour incidence and time of onset of tumour development have now been studied in the inbred, tumour-prone mouse strain C3H, in addition to growth, reproduction and longevity. Mice were fed a polyunsaturated human diet, a conventional human diet, or mouse cubes. The results with C3H mice tended to confirm those with Quackenbush strain mice-growth rates and reproductive productivities were very similar in the two groups eating human diets. Mice on the conventional human diet tended to survive better to about 60 weeks of age than mice on the polyunsaturated diet or the cube diet, after which the mortality rates of the mice on the three diets were similar. The degree of unsaturation of the dietary fat had no significant effect on the incidence of tumours. The tumour incidence was about 40% which, taken in conjunction with the average age of onset (about 80 weeks), suggested that the NIV virus rather than the MMTV virus was responsible. It would appear that the high-fat human diets had no effect on the incidence of mammary tumours caused by this virus.     

Adipose tissue and the effects of fat and calories on breast tumorigenesis in rats

A high fat diet fed ad libitum will promote breast tumorigenesis in rats while caloric restriction of the same high fat diet counteracts this promotional effect. The present study examined the effects of dietary fat and calorie intake on adipose tissue weight and fatty acid composition and on tumor incidence and development. The sites of adipose tissue chosen were the mammary fat pad, representing adipose tissue in the immediate location of the studied tumor, and the abdominal fat depot which in humans has been associated with an increased risk of breast cancer. High (20% corn oil) and low (5% corn oil) fat test diets were offered ad libitum and at 40% restriction levels. In agreement with prior studies, caloric restriction of both high and low fat diets led to marked decreases in tumor incidence (63 to 68% versus 21%), tumor burden (1.84 to 2.05 versus 0.37 to 0.43 tumors/rat), and tumor weight (7.1 to 11.9 versus 1.4 to 2.2 g) at the time of sacrifice (133 days post-DMBA). While final body weights were reduced in proportion to the level of caloric restriction (290 to 291 g versus 184 to 201 g), abdominal fat (8.8 to 9.2 versus 0.9 to 1.6 g), and mammary fat weights (3.1 to 4.1 versus 0.7 to 2.0 g) were reduced markedly in association with the decrease in tumorigenesis. While both tumor and mammary fat were enriched with linoleate reflecting the fatty acid composition of dietary fat, the ratio of arachidonic acid to linoleic acid was higher in tumor tissue than in surrounding normal mammary tissue in both the phospholipid (0.78 versus 0.18) and neutral lipid fractions (0.22 versus 0.03). These observations are consistent with the concept that increases in fat tissue mass in abdominal and mammary fat depots may mediate some of the promotional effects of high fat and high calorie diets. Restriction of dietary fat and calories to reduce body fat and strategies to modify the composition of stored lipids in fat depots may offer nutritional approaches to breast cancer prevention and treatment.     

Influence of caloric restriction and exercise on tumorigenesis in rats

Underfeeding or caloric restriction have been shown to inhibit the growth of spontaneous, transplanted, or chemically induced tumors in rats and mice. At 40% caloric restriction, growth of 7,12-dimethylbenz(a)anthracene-induced mammary and 1,2-dimethylhydrazine-induced colonic tumors is inhibited significantly even when the restricted diet contains twice as much fat as the control diet. Some inhibitory effects become evident even at 10% caloric restriction. In studies involving high fat diets, we find that rats receiving 20% fat ad libitum exhibit significantly higher 7,12-dimethylbenz(a)anthracene-induced mammary tumor incidence, multiplicity, and weight than rats ingesting the same amount of fat daily, but in a diet containing 25% fewer calories. In a study of intermittent ad libitum and restrictive feedings, chemically induced tumorigenicity varies inversely with feed efficiency. Exercise has also been shown to inhibit tumor growth. Sedentary rats fed ad libitum have a 108% higher incidence of 1,2-dimethylhydrazine-induced colon tumors than rats fed ad libitum but subjected to vigorous treadmill exercise. Caloric flux (either reduced intake or increased outflow) appears to reduce tumorigenicity in rodents.     

Reduction of enhanced mammary carcinogenesis in LA/N-cp (corpulent) rats by energy restriction

Restriction of energy intake significantly reduces mammary tumorigenesis in normal rats exposed to carcinogens. Genetically obese LA/N-cp (corpulent) female rats were given 7,12-dimethylbenz[a]anthracene and fed purified diets ad libitum or restricted to 60% of the ad libitum caloric intake. Phenotypically lean littermates were also fed ad libitum. Obese animals developed large mammary tumors more rapidly than genetically normal rats so that 100% of the animals had tumors in less than 16 weeks. Only 21% of the lean animals developed tumors; the energy restricted obese animals had a tumor incidence of 27%. Although obese rats fed the restricted diet weighed significantly less than those fed ad libitum, percent body fat was not reduced, indicating that lean tissue was affected more. Obese animals were markedly hyperinsulinemic (1003 +/- 193 microunits/ml) and energy restriction reduced this to 328 +/- 41; the lean animals had insulin levels of 12 +/- 2. Tumor-bearing rats had higher insulin levels than rats without tumors. These data suggest that body fatness is not directly associated with risk of carcinogenesis. Lean body mass, adipose tissue mass, and their interaction with insulin in its capacity as a growth factor rather than body fatness per se may be determinants of tumor promotion.     

Exogenous mouse mammary tumor virus proviral DNA isolated from a kidney adenocarcinoma cell line contains alterations in the U3 region of the long terminal repeat.

Mouse mammary tumor virus (MMTV) is a B-type retrovirus which induces predominantly mammary carcinomas after a relatively long latency period. To date, very little is known about the reasons for the strict tissue specificity of MMTV. The BALB/cf/Cd strain of mice, which was infected with milk-borne MMTV (C3H), shows a high incidence of kidney adenocarcinomas, and our data suggest that MMTV might be involved in the formation of these tumors. Newly integrated exogenous MMTV proviruses were found in the genome of transplanted tumor cells as well as in the DNA of a cell line derived from one tumor, but not in normal cells of BALB/cf/Cd mice. The MMTV DNA in these tumor cells was transcribed and viral RNA synthesis was strongly stimulated by glucocorticoid hormones. Viral structural polypeptides, comparable in size and antigenicity to MMTV polypeptides of infected mammary tumor cells were synthesized and processed normally in the cell line and were organized correctly into intracytoplasmic particles. Heteroduplex analysis of the molecularly cloned MMTV proviral DNAs of kidney and mammary tumor origin revealed a high degree of homology in the gag, pol, and env genes. A striking difference, however, was observed in the U3 region of the two LTRs that might relate to the different tissue specificity of the two viruses.     

Influence of fatty acid‐free diet on mammary tumor development and growth rate in HER‐2/neu transgenic mice

Numerous investigations have found a relationship between higher risk of cancer and increased intake of fats, while results of clinical studies of fat reduction and breast cancer recurrence have been mixed. A diet completely free of fats cannot be easily administered to humans, but experimental studies in mice can be done to determine whether this extreme condition influences tumor development. Here, we examined the effects of a FA‐free diet on mammary tumor development and growth rate in female FVB‐neu proto‐oncogene transgenic mice that develop spontaneous multifocal mammary tumors after a long latency period. Mice were fed a fatty acid‐free diet beginning at 112, 35, and 30 days of age. In all these experiments, tumor appearance was delayed, tumor incidence was reduced and the mean number of palpable mammary tumors per mouse was lower, as compared to standard diet‐fed mice. By contrast, tumor growth rate was unaffected in mice fed the fatty acid‐free diet. Plasma of mice fed the fatty acid‐free diet revealed significantly higher contents of oleic, palmitoleic and 20:3ω9 acids and lower contents of linoleic and palmitic acids. In conclusion, these findings indicate that a FA‐free diet reduces tumor incidence and latency but not tumor growth rate, suggesting that a reduction in dietary FAs in humans may have a protective effect on tumorigenesis but not on tumors once they appear. J. Cell. Physiol. 228: 242–249, 2013. © 2012 Wiley Periodicals, Inc.     

Effects of Enriched Environment on COX-2, Leptin and Eicosanoids in a Mouse Model of Breast Cancer

Cyclooxygenase-2 (COX-2) and adipokines have been implicated in breast cancer. This study investigated a possible link between COX-2 and adipokines in the development of mammary tumors. A model of environmental enrichment (EE), known to reduce tumor growth was used for a syngeneic murine model of mammary carcinoma. 3-week-old, female C57BL/6 mice were housed in standard environment (SE) or EE cages for 9 weeks and transplanted orthotopically with syngeneic EO771 adenocarcinoma cells into the right inguinal mammary fat pad. EE housing influenced mammary gland development with a decrease in COX-2 expressing cells and enhanced side-branching and advanced development of alveolar structures of the mammary gland. Tumor volume and weight were decreased in EE housed mice and were associated with a reduction in COX-2 and Ki67 levels, and an increase in caspase-3 levels. In tumors of SE mice, high COX-2 expression correlated with enhanced leptin detection. Non-tumor-bearing EE mice showed a significant increase in adiponectin levels but no change in those of leptin, F₂-isoprostanes, PGF_2α, IL-6, TNF-α, PAI-1, and MCP-1 levels. Both tumor-bearing groups (SE and EE housing) had increased resistin, IL-6, TNF-α, PAI-1 and MCP-1 levels irrespective of the different housing environment demonstrating higher inflammatory response due to the presence of the tumor. This study demonstrates that EE housing influenced normal mammary gland development and inhibited mammary tumor growth resulting in a marked decrease in intratumoral COX-2 activity and an increase in the plasma ratio of adiponectin/leptin levels.     

Effect of bromocriptine on the ovarian cycle of two inbred strains of mice

Mice of the C3H/Sy (high incidence of spontaneous mammary cancer) and AKR/Sy (low incidence of spontaneous mammary cancer) inbred strains, which have different hormonal profiles, were injected daily with bromocriptine for 1 month. The treatment increased the duration of the ovarian cycle of the AKR/Sy mice, whereas that of the C3H/Sy mice was not affected. It is suggested that the effect of bromocriptine on the ovarian cycle depends on the concentrations of plasma progesterone reached in each strain of mouse.     

Effects of amphetamine on the development of MTV-induced mammary tumors in female mice.

Female C3H/He mice carrying the mammary tumor virus (MTV) were monitored for mammary tumor incidence and latent periods while submitted to a daily subcutaneous injection with amphetamine (0,4 mg/kg/day). Results show that amphetamine caused an increase in incidence and a decrease in latency of tumors compared with placebo. There was also appreciated a correlation with the lethality of mice.