Incidence Of Spontaneous Mammary Tumors Research Articles

R-loop functions in Brca1-associated mammary tumorigenesis

Deleterious accumulation of R-loops, a DNA-RNA hybrid structure, contributes to genome instability. They are associated with BRCA1 mutation-related breast cancer, an estrogen receptor α negative (ERα-) tumor type originating from luminal progenitor cells. However, a presumed causality of R-loops in tumorigenesis has not been established in vivo. Here, we overexpress mouse Rnaseh1 (Rh1-OE) in vivo to remove accumulated R-loops in Brca1-deficient mouse mammary epithelium (BKO). R-loop removal exacerbates DNA replication stress in proliferating BKO mammary epithelial cells, with little effect on homology-directed repair of double-strand breaks following ionizing radiation. Compared to their BKO counterparts, BKO-Rh1-OE mammary glands contain fewer luminal progenitor cells but more mature luminal cells. Despite a similar incidence of spontaneous mammary tumors in BKO and BKO-Rh1-OE mice, a significant percentage of BKO-Rh1-OE tumors express ERα and progesterone receptor. Our results suggest that rather than directly elevating the overall tumor incidence, R-loops influence the mammary tumor subtype by shaping the cell of origin for Brca1 tumors.

Impairment of the non-homologous end joining and homologous recombination pathways of DNA double strand break repair: Impact on spontaneous and radiation-induced mammary and intestinal tumour risk in Apcmin/+ mice

Female Apcmin/+ mice carrying the BALB/c variant of Prkdc or heterozygous knockout for Xrcc2, were sham- or 2Gy X-irradiated as adults to compare the effect of mild impairments of double–strand break (DSB) repair pathways, non-homologous end joining (NHEJ) and homologous recombination (HR) respectively on spontaneous and radiation–induced mammary and intestinal tumorigenesis. Mice with impaired NHEJ showed no difference in incidence of spontaneous mammary tumours, compared with matched controls, (2.46 fold, P=0.121) and significantly less following irradiation (radiation–induced excess; 0.35 fold, P=0.008). In contrast mice with impaired HR presented with significantly less spontaneous mammary tumours than matched controls (0.33 fold, P=0.027) and significantly more following irradiation (radiation-induced excess; 3.3 fold, P=0.016). Spontaneous and radiation-induced intestinal adenoma multiplicity in the same groups were significantly greater than matched controls for mice with impaired NHEJ (sham; 1.29 fold, P<0.001, radiation–induced excess; 2.55 fold, P<0.001) and mice with impaired HR showed no significant differences (sham; 0.92 fold, P=0.166, radiation-induced excess; 1.16, P=0.274). Genetic insertion events were common in spontaneous tumours from NHEJ impaired mice compared with matched controls. γH2AX foci analysis suggests a significantly faster rate of DSB repair (MANOVA P<0.001) in intestinal than mammary tissue; apoptosis was also higher in irradiated intestine.To conclude, results suggest that pathway of choice for repair of spontaneous and radiation-induced DSBs is influenced by tissue type. NHEJ appears to play a greater role in DSB repair in intestinal tissue since impairment by functional change of Prkdc significantly increases the rate of mis-repair in intestinal but not mammary tissue. HR appears to play a greater role in DSB repair in adult mammary tissue since impaired HR results in significant changes in mammary but not in the intestinal tumorigenesis. This indicates that early DNA damage response and repair is important for cancer susceptibility and plays a role in determining tissue specificity of cancer risk.

Abstract LB-190: Growth hormone signaling in mammary stem and progenitor cells

Abstract The role of growth hormone (GH) in development of the normal mammary gland has been convincingly demonstrated in studies that show dramatically impaired mammary development in spontaneous dwarf rats and growth hormone receptor knock-out (GHR-KO) mice. There is also evidence that GH signaling may have a role in breast cancer. Animals treated with GH develop hyperproliferative lesions in the mammary gland and have increased incidence of spontaneous mammary tumors. Conversely, animals deficient in GH signaling are resistant to carcinogen-induced cancers, including mammary carcinoma. In humans, there is an increased incidence of cancer in patients with acromegaly (syndrome caused by excess GH production by pituitary gland) and a decreased incidence in patients with Laron syndrome (GH deficiency). In this study, we show that GH may increase the risk of breast cancer by stimulating stem and progenitor cells to enter the cell cycle. Using immunohistochemistry and immunostaining coupled with flow cytometry analysis, we found that GHR is expressed in stem and progenitor cells in the normal breast epithelium. We have previously shown that cells with ALDH activity from the human mammary epithelium have functional properties of stem/progenitor cells. Two thirds of these ALDH positive cells express GHR. GHR positive mammary epithelial cells are capable to initiate mammosphere formation in suspension culture and can generate both luminal and myoepithelial cell lineages in vitro. GH treatment increased mammosphere formation in a dose dependent manner. Although the total number of progeny was increased in these conditions, cell dynamics analysis using PKH26 dye dilution showed that the ratios between stem cells, early and late progenitor cells were not changed. GH treatment reduced the number of cells in quiescent state (G0) and decreased p21 protein levels, a cell-cycle regulator previously associated with stem cells quiescence. Taken together, these results indicated that GH treatment did not change symmetry of self-renewal cell divisions but increased the number of cycling mammary stem/progenitor cells. We found that GH is present in normal human mammary epithelial cells both in vitro and in vivo. In vitro, GH is secreted by mammary epithelial cells upon progesterone stimulation. We propose a model in which GH, estrogen and progesterone establish a paracrine signaling loop that stimulates GHR positive/steroid receptor negative stem and progenitor cells to enter the cell cycle. We propose that increased levels or prolonged exposure steroid hormones and/or GH would lead eventually to an expanded undifferentiated population at higher risk for transformation through oncogenic hits. Consistent with this concept, analysis of 180 cases of clinical precursor lesions (DCIS) showed an expanded GHR+ cell population in 92% of cases, with expression in 50-100% of the cell population in more than half of cases. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-190. doi:1538-7445.AM2012-LB-190

Intraductal pegylated liposomal doxirubicin may achieve long term protection in HER2/NEU transgenic mice by restricting mammary gland outgrowth

This work is based on the hypothesis that intraductal injection can treat existing lesions and prevent future breast cancers originating in the epithelial cells lining the breast ducts. We have previously demonstrated our ability to access the entire mammary gland through the teat in mouse and rat mammary tumor models. Further, we showed that several other common chemotherapeutic drugs such as 5 fluorouracil, carboplatin, and methotrexate were effective, but not to the same extent as PLD. Thus, intraductal injection of PLD has potential in the prevention and neo-adjuvant therapy of breast cancer. Recently, we observed that in addition to rendering the mammary glands tumor free for more than 3 months after all of the control animals had developed multiple tumors, mammary glands of PLD treated mice were stunted in their growth. Instead of the florid growth with multiple branches and side branches studded with terminal end buds, akin to a spring tree seen in normal glands the PLD treated mammary gland presented the aspect of an autumn tree. In the transgenic mice, the epithelial cells in the entire mammary gland express the MMTV-Her2/neu transgene and at even higher levels during pregnancy. This raised the concern that stimulation of proliferation of these cells, for example, by pregnancy, may result in a higher incidence of spontaneous mammary tumors in the PLD treated mice. What then is the response of this treated mammary gland to a new pregnancy? To test this concept, we induced pregnancy in PLD treated mice. Her2/neu mice treated with intraductal PLD, like their untreated controls, had normal deliveries, with pups of normal weight and number in the litter. However, unlike control mice, their pups survived for less than one week. Long term follow-up showed that pregnancy did not increase the incidence of tumors in PLD treated mice. Their mammary glands showed a very poor proliferative response to the pregnancy hormones and remained stunted in their growth. It is likely that PLD not only affected the eradication of preneoplasias but also resulted in a depletion of normal mammary gland stem cells This concept is being tested by determining the difference in stem cell content between untreated and PLD treated glands by transplantation of serial dilutions of epithelial cells from sham and PLD treated glands into cleared mouse fat pads, examination of markers of stem cells, such as ALDHA, etc. If proven to be the case, these findings raise the possibility that long term protection may be achieved by intraductal injection of PLD to women at high risk of developing breast cancer.

Clonal origin of spontaneous multiple mammary tumors in mice with cellular mosaicism.

The clonal origin of spontaneous multiple mammary tumors in mice was examined. For this purpose, hybrid female mice (Pgk-1b/Pgk-1a), F1[SHN(Pgk-1b) X C3H/He (Pgk-1a)], with X-chromosome inactivation mosaicism with regard to the phosphoglycerate kinase (PGK)-1 isozyme together with a high incidence of spontaneous mammary tumors were constructed. Thirty-seven of 45 mammary tumors (82%) in mosaic mice had a single phenotype of PGK, indicating monoclonal origin. The multiple mammary tumors formed in these mice varied in PGK type, indicating independent cellular origins.

Access to Exercise

The incidence of spontaneous tumors in Sprague-Dawley rats has ranged from 45–71% with female rats having double the percentage compared with males. Mammary tumors account for most of the tumors. Tumors may induce cancer and other life-shortening diseases, making the study of tumor development and prevention a thriving area of research. It is unknown how the gene-environment interaction influences spontaneous tumor development. Animals that reside solely in a standard cage are extremely sedentary and this trait may enhance tumerigenesis. On the other hand, animals with access to physical activity may inhibit tumor development. PURPOSE: This study compared tumorigenesis-related gene expression and the number, size, and age-associated incidence of spontaneous mammary tumors in female Sprague-Dawley rats with different access to physical activity: 1) none outside a standard cage, (SED) 2) twice-weekly physical activity in a large box (PA), and 3) regular running wheel exercise (EX). METHODS: 108 female rats were divided into three treatment groups and investigated and compared for tumor growth and development. Affymetrix microarray and RMA expression analysis identified genes with significant >2 fold changes in gene expression. Real-time quantitative reverse transcriptase polymerase chain reaction (qPCR) was used for microarray verification. RESULTS: Palpable tumors began to develop in all groups at around 59 weeks. SED animals had tumors that were greater in number and size compared with PA and EX. At 98 weeks, 14 EX rats had 36 tumors, 22 PA rats had 52 tumors, and 23 SED rats had 66 tumors. At 98 weeks, the SED group had the lowest percentage of tumor free survival compared with PA and EX. Transforming growth factor alpha (TGF?) and Rab40c (a member of the RAS oncogene family) were 2 fold greater in expression in EX than in SED (p <0.05). CONCLUSION: Animals that resided solely in cages with no access to physical activity outside a cage, demonstrated greater number and size of spontaneous mammary tumors while access to physical activity and exercise for almost two years reduced spontaneous tumorigenesis in female rats. Surprisingly, TGFα and Rab40c, both of which are associated with tumorigenesis and carcinogenesis, were up-regulated in the exercised animals.

Melatonin and mammary cancer: a short review.

Melatonin is an indolic hormone produced mainly by the pineal gland. The former hypothesis of its possible role in mammary cancer development was based on the evidence that melatonin down-regulates some of the pituitary and gonadal hormones that control mammary gland development and which are also responsible for the growth of hormone-dependent mammary tumors. Furthermore, melatonin could act directly on tumoral cells, as a naturally occurring antiestrogen, thereby influencing their proliferative rate. The first reports revealed a low plasmatic melatonin concentration in women with estrogen receptor (ER)-positive breast tumors. However, later studies on the possible role of melatonin on human breast cancer have been scarce and mostly of an epidemiological type. These studies described a low incidence of breast tumors in blind women as well as an inverse relationship between breast cancer incidence and the degree of visual impairment. Since light inhibits melatonin secretion, the relative increase in the melatonin circulating levels in women with a decreased light input could be interpreted as proof of the protective role of melatonin on mammary carcinogenesis. From in vivo studies on animal models of chemically induced mammary tumorigenesis, the general conclusion is that experimental manipulations activating the pineal gland or the administration of melatonin lengthens the latency and reduces the incidence and growth rate of mammary tumors, while pinealectomy usually has the opposite effects. Melatonin also reduces the incidence of spontaneous mammary tumors in different kinds of transgenic mice (c-neu and N-ras) and mice from strains with a high tumoral incidence. In vitro experiments, carried out with the ER-positive MCF-7 human breast cancer cells, demonstrated that melatonin, at a physiological concentration (1 nM) and in the presence of serum or estradiol: (a) inhibits, in a reversible way, cell proliferation, (b) increases the expression of p53 and p21WAF1 proteins and modulates the length of the cell cycle, and (c) reduces the metastasic capacity of these cells and counteracts the stimulatory effect of estradiol on cell invasiveness; this effect is mediated, at least in part, by a melatonin-induced increase in the expression of the cell surface adhesion proteins E-cadherin and beta(1)-integrin. The direct oncostatic effects of melatonin depends on its interaction with the tumor cell estrogen-responsive pathway. In this sense it has been demonstrated that melatonin down-regulates the expression of ERalpha and inhibits the binding of the estradiol-ER complex to the estrogen response element (ERE) in the DNA. The characteristics of melatonin's oncostatic actions, comprising different aspects of tumor biology as well as the physiological doses at which the effect is accomplished, give special value to these findings and encourage clinical studies on the possible therapeutic value of melatonin on breast cancer.

Final report on the safety assessment of Hydrogenated Cottonseed Oil, Cottonseed (Gossypium) Oil, Cottonseed Acid, Cottonseed Glyceride, and Hydrogenated Cottonseed Glyceride.

Hydrogenated Cottonseed Oil, Cottonseed (Gossypium) Oil, Cottonseed Acid, Cottonseed Glyceride, and Hydrogenated Cottonseed Glyceride are cosmetic ingredients derived from Cottonseed Oil and used as skin-conditioning agents and surfactants. Nonoils known to be toxic that may be found in cottonseed oils include gossypol, aflatoxin, and cyclopropenoid fatty acids (CPFA). Toxic heavy metal and/or polychlorinated biphenyl (PCB) or other pesticide contamination is also possible. Cottonseed Oil was nontoxic in acute oral toxicity studies in rats. In a short-term study, rabbits that had been fed 2% Cottonseed Oil for 7 weeks had significantly lower blood chemistry parameters (compared to wheat bran controls) and significantly more stored hepatic vitamin A (compared to rabbits fed other fats). Cottonseed Oil controls used as vehicles in two parenteral studies produced negative results. Hydrogenated Cottonseed Oil tested in formulation did not produce dermal or ocular irritation in rabbits. An oral-dose reproductive study tested up to 30% Cottonseed Oil (with 1% CPFAs) and reported no adverse effects on sexual maturity and reproductive performance of the F0 generation; changes were noted in the F1 generation but reproductive capacity was not altered. Parenteral-dose reproductive studies reported no adverse effects. Cottonseed Oil was not mutagenic. Cottonseed Oil did not induce aberrant crypt foci when given orally to mice, but in other studies, it increased the incidence of spontaneous mammary tumors in rats and mice. Mice fed 20% Hydrogenated Cottonseed Oil during induction and promotion of photocarcinogenesis had significantly lower tumor incidence compared to mice fed 20% sunflower oil. Hydrogenated Cottonseed Oil in formulation (up to approximately 21%) was neither an irritant nor sensitizer in clinical studies. Limited clinical data indicated that Cottonseed Oil does not contain allergic protein. Based on the available data, it was concluded that these ingredients may be used safely in cosmetic formulations if established limits on gossypol, heavy metals, and pesticide concentrations are not exceeded.

Final Report on the Safety Assessment of Hydrogenated Cottonseed Oil, Cottonseed (Gossypium) Oil, Cottonseed Acid, Cottonseed Glyceride, and Hydrogenated Cottonseed Glyceride

Hydrogenated Cottonseed Oil, Cottonseed (Gossypium) Oil, Cottonseed Acid, Cottonseed Glyceride, and Hydrogenated Cottonseed Glyceride are cosmetic ingredients derived from Cottonseed Oil and used as skin-conditioning agents and surfactants. Non-oils known to be toxic that may be found in cottonseed oils include gossypol, aflatoxin, and cyclopropenoid fatty acids (CPFA). Toxic heavy metal and/or polychlorinated biphenyl (PCB) or other pesticide contamination is also possible. Cottonseed Oil was nontoxic in acute oral toxicity studies in rats. In a short-term study, rabbits that had been fed 2% Cottonseed Oil for 7 weeks had significantly lower blood chemistry parameters (compared to wheat bran controls) and significantly more stored hepatic vitamin A (compared to rabbits fed other fats). Cottonseed Oil controls used as vehicles in two parenteral studies produced negative results. Hydrogenated Cottonseed Oil tested in formulation did not produce dermal or ocular irritation in rabbits. An oral-dose reproductive study tested up to 30% Cottonseed Oil (with 1% CPFAs) and reported no adverse effects on sexual maturity and reproductive performance of the F0 generation; changes were noted in the F1 generation but reproductive capacity was not altered. Parenteral-dose reproductive studies reported no adverse effects. Cottonseed Oil was not mutagenic. Cottonseed Oil did not induce aberrant crypt foci when given orally to mice, but in other studies, it increased the incidence of spontaneous mammary tumors in rats and mice. Mice fed 20% Hydrogenated Cottonseed Oil during induction and promotion of photocarcinogenesis had significantly lower tumor incidence compared to mice fed 20 % sunflower oil. Hydrogenated Cottonseed Oil in formulation (up to 21 %) was neither an irritant nor sensitizer in clinical studies. Limited clinical data indicated that Cottonseed Oil does not contain allergic protein. Based on the available data, it was concluded that these ingredients may be used safely in cosmetic formulations if established limits on gossypol, heavy metals, and pesticide concentrations are not exceeded.

Aryl hydrocarbon hydroxylase activity in spontaneous mammary tumors in rat.

The development of spontaneous mammary tumors was observed for about 2 years in a group of 25 female Sprague-Dawley rats aged over 1 year at the beginning of the study. All younger females in our animal facility were similarly monitored. In old females, the incidence of spontaneous mammary tumors was 64%. The parity of rats did not protect them from tumorigenesis, but the proportion of malignant tumors was higher in virgin (57%) than in parous (13%) rats. Activities of cytochrome P450IA1-dependent enzyme (aryl hydrocarbon hydroxylase, AHH) and NADPH-cytochrome P450 reductase (NCR) were determined in microsome fractions isolated from livers, lungs, uteri and tumors of rats. AHH and NCR activities in tumors and uteri were low compared to those in livers or lungs. In tumors, the activity distributions were wide, even in different tumors of the same animal the AHH activities varied as widely as between different animals. The activities in benign and malignant tumors were not statistically significantly different. No correlation with liver, lung or uterine activities was found either. With ageing of the rat, the AHH activities in tumors, liver and lungs decreased. The behavior of AHH in spontaneous mammary tumors in rats seems to be similar to that found in chemically induced tumors and seems to show individual regulation, possibly altered by tumorigenesis in each individual tumor.

SUPPRESSION OF TUMORIGENESIS AND TUMOR-GROWTH OF MOUSE MAMMARY-GLANDS BY A TRADITIONAL CHINESE HERBAL MEDICINE, SHO-SAIKO-TO

Hyperplastic alveolar nodule (HAN) is a representative preneoplastic state in mammary glands of mice. We investigated the effect of Sho-saiko-to (SST; a Japanese modified traditional Chinese herbal medicine containing seven medicinal plants) on the formation and growth of HAN and mammary tumour growth in SHN virgin mice which showed a high incidence of spontaneous mammary tumours. Chronic oral administration of SST reduced the number and the area of HAN and mammary thymidylate synthetase (TS) activity with slight decrease of serum prolactin level. In the established mammary tumours of mice, 20-day administration of SST reduced the activity of TS and thymidine kinase (TK) with slight reduction of serum prolactin level, though the significant change of mammary-tumour growth was not observed. These results indicate that SST may prevent the mammary tumourigenesis by suppression of the de novo pathway for pyrimidine nucleotide synthesis and the mammary tumour growth by suppression of the salvage pathway as well as the de novo pathway in mice.

Effect of ascorbic acid on incidence of spontaneous mammary tumors and UV-light-induced skin tumors in mice

Two large-scale studies of the effect of different amounts of L-ascorbic acid in the food on tumor-free survival have been conducted. One involved the incidence of spontaneous mammary tumors in RIII mice, with seven ascorbic acid and three control groups, 50 mice per group. With increasing ascorbic acid in the diet, there was a highly significant delay before appearance of the first tumor. Median age at first tumor was 82.5 wk in ad libitum controls, 124.9 wk in the highest-dose ascorbate group. The proportion of mice with tumors was also reduced. The other study involved dermal neoplasms in mice irradiated with ultraviolet light. A pronounced effect of vitamin C in decreasing the incidence and delaying the onset of malignant lesions was observed with high statistical significance. By 20 wk approximately five times as many mice had developed serious lesions in the zero-ascorbate as in the high-ascorbate group.

Melatonin, a suppressor of spontaneous murine mammary tumors

The effect of prolonged, oral melatonin treatment on spontaneous mammary tumor development in female C3H/Jax mice was studied. Melatonin was administered at a dose of 25 micrograms/mouse/day from 21 to 44 days of age and 50 micrograms/mouse/day from day 45 onwards. By the age of 12 months, 62.5% of control animals developed tumors as opposed to 23.1% in the melatonin treated group (P less than 0.02). In both control and treated mice, the thoracic pairs of mammary glands were obviously more susceptible to spontaneous mammary tumor development, as at least 50% of the total tumors developed in this region. Reduction in submaxillary and pituitary gland weights of treated animals was observed at necropsy (P less than 0.001). Decreased serum 17-beta-estradiol (E2) levels in melatonin treated mice (P less than 0.05) and a marked reduction in [3H]thymidine incorporation into DNA of melatonin-treated mammary glands (P less than 0.02) positively correlated with the sparse mammary gland development seen in these mice. These observations indicate that at the given dose level, melatonin modulates the degree of development of the mammary epithelium, subsequently reducing spontaneous mammary tumor incidence in these high risk mice.

Actions of an endogenous antitumorigenic agent on mammary tumor development and modeling analysis of its capacity for interacting with DNA

Antineoplaston A10 (3-phenylacetylamino-2,6-piperidinedione) is an agent derived from human urine which is remarkable for its antineoplastic activity and lack of toxicity. This study deals with the discovery of a hormonal component to the action of A10 on rodent tumor formation and approaches to the delineation of its mechanism of action. Oral administration of A10 dramatically delays onset of spontaneous mammary tumor incidence in female and orchidectomized male C3H + mice. The action in the male qualitatively mimics that of androgens. In the rat, A10 ingestion is very effective in preventing carcinogen-induced mammary tumors, but does not cause regression of pre-established tumors. It selectively blocks the occurrence of the estrogen-sensitive subpopulation of tumors, acting in a fashion completely analogous to that of tamoxifen; in contrast to tamoxifen, however, A10 has no measurable affinity for the estrogen receptor. Modeling studies demonstrate a stereoselective capacity for interaction between A10 and specific deoxyribonucleotide base pair sequences. The potential affinity is lower than that for classical intercalating antitumour agents, and the stereospecificity differs from that which we have previously established for estrogens. We offer an interpretation of the data in terms of direct effect of A10 at the genomic level to alter the cellular responsiveness to steroid hormones.

Age related differences in immunocompetence and incidence of mammary adenocarcinoma in murine mammary tumor virus-infected C3H/Bi mice

In breeder C3H/Bi female mice, infected neonatally by murine mammary tumor virus (MTV), the incidence of spontaneous mammary tumors is greater than 95% between 5 and 9 months of age. In young (2–3 months) female the probability for developing a tumor in the next month is negligible, higher than 80% in mice of middle age (5–6 months) but lower than 4% in aged (10–12 months) females. The age-related changes of some immune functions of spleen cells from these tumor free female mice have been evaluated. While the proliferative capacity of cells to Phytohemagglutinin (PHA) increases, the T cell-dependent antibody response against sheep red blood cells (SRBC) and the antibody-dependent cellular cytotoxicity (ADCC) are significantly decreased in 5–6-month-old mice as compared to the young (2–3 months) female mice. The antibody response against SRBC and the mitogenic response to PHA decline markedly in 10–12-month-old mice but the ADCC increases in this group of mice. In addition, assays with monoclonal anti-Lyt-1 and anti-Lyt-2 antibodies indicate that percentage of Lyt 1 − 2 + cells (suppressor and cytotoxic T cells) is lower in 10–12-month-old female as compared to 5–6-month-old animals. These results show that the immune alterations observed in 10–12-month-old C3H/Bi mice are not closely associated with an increase in incidence of spontaneous tumors and suggest that a high non-T killer cell activity could protect some of these older C3H/Bi female mice against mammary tumor development.

Effects of X-irradiation, ovariohysterectomy and estradiol-17β on incidence, benign/malignant ratio and multiplicity of rat mammary neoplasms—A preliminary report

An overview is given of the effects of X-irradiation, ovariohysterectomy and estradiol-17β administration on mammary tumorigenesis in females of 3 rat strains, viz. the WAG/Rij, BN/BiRij and SD. The 3 rat strains differed significantly in their spontaneous mammary tumor incidence. Female SD rats had the highest incidence (47%) and female BN/BiRij rats the lowest (17%). Female WAG/Rij rats had an intermediate incidence of 29%. The benign/malignant ratio in female WAG/Rij, BN/BiRij and SD rats was 1.0, 2.0 and 7.3, respectively. The average number of mammary gland neoplasms per untreated tumor-bearing female was 1.2 in the WAG/Rij, 1.0 in the BN/BiRij and 1.6 in the SD, whereas the respective maximum numbers were 2, 1 and 5. Ovariohysterectomy almost entirely prevented mammary tumor formation in all 3 rat strains, whereas estrogen treatment enhanced it. In addition, estrogen treatment resulted in an increased number of mammary tumors per tumor-bearing female and changed the benign/malignant ratio into the direction of malignant. X-irradiation increased the mammary tumor incidence in all 3 rat strains, especially of the benign tumors. Estrogen potentiated the effect of irradiation. An effect of irradiation on mammary tumorigenesis was not observed in ovariohysterectomized females of all 3 rat strains.

Effect of dietary ascorbic acid on the incidence of spontaneous mammary tumors in RIII mice.

A study of the effect of different amounts of L-ascorbic acid (vitamin C), between 0.076% and 8.3%, contained in the food has been carried out with ten groups of RIII mice (seven ascorbic acid and three control groups), with 50 mice in each group. With an increase in the amount of ascorbic acid there is a highly significant decrease in the first-order rate constant for appearance of the first spontaneous mammary tumor after the lag time to detection by palpation. There is also an increase in the lag time. The mean body weight and mean food intake were not significantly different for the seven ascorbic acid groups. Striking differences were observed between the 0.076% ascorbic acid and the control groups (which synthesize the vitamin): smaller food intake, decreased lag time, and increased rate constant of appearance of the first mammary tumor. This comparison cannot be made experimentally for guinea pigs and primates because the control groups would develop scurvy.

Interaction of the heterozygous nude gene with the asplenia trait in mammary tumorigenesis.

The BALB/c mouse strain has been shown to contain endogenous mouse mammary tumor virus (MMTV) proviral sequences. However, no exogenous MMTV particles have been detected in their tissues. Female BALB/c mice from our colonies exhibit a very low incidence of spontaneous mammary tumors (SMT); less than 1% at up to 20 mo of age. Immunodeficient BALB/c mice heterozygous for the nude gene (nu/+, +/+), for the dominant hemimelia gene associated with asplenia (+/+, Dh/+), or for both traits (nu/+, Dh/+) have been examined for SMT incidence and the presence of MMTV proviruses. Based on restriction digestion with Eco RI, Bam HI, and Pst I, the immunodeficient mice have an MMTV provirus copy number and organization identical to the BALB/cCrgl strain. This MMTV DNA pattern is distinct from the MMTV proviruses in C3H/He, C57BL/6J and CBA/CaJ mice, which were parental strains of the immunodeficient mutants. Normal female BALB/c or BALB/c heterozygous for the asplenic trait do not develop significant numbers of SMT at up to 19 mo of age. In contrast, an incidence of 23.8% and 57.7% SMT was observed in BALB/c nu/+ heterozygotes, and in BALB/c nu/+, Dh/+ heterozygotes, respectively. These results indicate that agenesis of the spleen, concomitant with the presence of the heterozygous nude gene, contribute to a high incidence of SMT in the low-SMT BALB/c mouse strain.

Selenium interactions with carcinogens

Although selenium was once considered to be a toxic, undesirable and carcinogenic element, it is now recognized as an essential element with anticarcinogenic properties. Epidemiological studies in the United States have shown an inverse relationship between selenium intake and certain forms of cancer in humans, but other factors must be considered since cancer is not higher in people living in selenium-deficient areas of the world (Finland, New Zealand, and Keshan disease area, China). Under most dietary conditions, selenium has been shown to reduce the spontaneous mammary tumor incidence in an inbred strain of mice. In general, selenium will counteract to various degrees, the chemical carcinogens used to produce lesions of the skin [coaltar, 3-methylcholanthrene, α-pyrene, and 7,12 dimethylbenz(a) anthracene (DMBA)], liver (3-methyl-4-dimethyl-aminoazobenzene, aflatoxin B 1, and 2-acetylaminofluorene), mammary gland (DMBA and N-methyl-N-nitrosurea), and intestinal tract [1,2-dimethylhydrazine,bis(2-oxopropyl) nitrosamine, and azoxymethane]. Dietary factors, such as fat, will modify the protective effects of selenium. High dietary unsaturated fats, for example, markedly increase the mammary tumors in rats treated with DMBA, and selenium will reduce the tumor incidence but not to the level of rats fed a low fat diet. Other factors known to affect the anticarcinogenic effects of selenium include synthetic antioxidants, vitamin E, vitamin A and ascorbic acid. The mechanisms of selenium counteraction of carcinogens remain unknown.

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.