Gudmundur Thordarson

Short-term exposure to pregnancy levels of estrogen prevents mammary carcinogenesis

It is well established that pregnancy early in life reduces the risk of breast cancer in women and that this effect is universal. This phenomenon of parity protection against mammary cancer is also observed in rodents. Earlier studies have demonstrated that short-term administration of estradiol (E) in combination with progesterone mimics the protective effect of parity in rats. In this study, the lowest effective E dosage for preventing mammary cancer was determined. Rats were injected with N-methyl-N-nitrosourea at 7 weeks of age; 2 weeks later, the rats were subjected to sustained treatment with 20 μg, 100 μg, 200 μg, or 30 mg of E in silastic capsules for 3 weeks. Treatments with 100 μg, 200 μg, and 30 mg of E resulted in serum levels of E equivalent to those of pregnancy and were highly effective in preventing mammary cancer. E treatment (20 μg) did not result in pregnancy levels of E and was not effective in reducing the mammary cancer incidence. In another set of experiments, we determined the effect of different durations of E with or without progesterone treatments on mammary carcinogenesis. These experiments indicate that a period as short as one-third the period of gestation is sufficient to induce protection against mammary carcinogenesis. The pioneering aspect of our study in contrast to long-term estrogen exposure, which is thought to increase the risk of breast cancer, is that short-term sustained treatments with pregnancy levels of E can induce protection against frank mammary cancer.

Mammary Phenotypic Expression Induced in Epidermal Cells by Embryonic Mammary Mesenchyme

The goal of this research was to establish methods for inducing mammary epithelial differentiation from nonmammary epithelium. For this purpose, mid-ventral or dorsal epidermis (skin epithelium; SKE) from 13-day rat or mouse embryos was associated with 13-day embryonic mouse mammary mesenchyme (mammary gland mesenchyme; MGM) (mouse MGM+rat or mouse SKE). The resultant MGM+SKE recombinants as well as controls (homotypic mouse mammary recombinants, homotypic mouse skin recombinants and mouse mammary mesenchyme by itself) were grafted under the renal capsule of syngeneic or athymic female nude mouse hosts. Most female hosts were induced to undergo lactogenesis by grafting an adult pituitary which elicited a state of hyperprolactinemia. Tissue recombinants of mouse MGM+rat or mouse SKE grown for 1 month in vivo formed a hair-bearing keratinized skin from which mammary ductal structures extended into the mesenchyme. The ducts were composed of columnar luminal epithelial cells as well as basal, actin-positive myoepithelial cells. When grown in pituitary-grafted hosts, the ductal epithelial cells expressed casein and alpha-lactalbumin as judged by immunocytochemistry. The expression of caseins in MGM+SKE recombinants was confirmed by Western blot. The epithelial cells in mouse MGM+rat SKE recombinants expressing milk proteins were shown to be rat cells while the surrounding connective tissue was composed of mouse cells based upon staining with Hoechst dye 33258. Using mammary-specific markers, these studies confirmed the earlier morphological studies of Propper and unequivocally demonstrated for the first time that embryonic mammary mesenchyme can induce morphological and functional mammary differentiation from nonmammary epithelium.

Insulin-like growth factor (IGF)-I obliterates the pregnancy-associated protection against mammary carcinogenesis in rats: evidence that IGF-I enhances cancer progression through estrogen receptor-α activation via the mitogen-activated protein kinase pathway

IntroductionPregnancy protects against breast cancer development in humans and rats. Parous rats have persistently reduced circulating levels of growth hormone, which may affect the activity of the growth hormone/insulin-like growth factor (IGF)-I axis. We investigated the effects of IGF-I on parity-associated protection against mammary cancer.MethodsThree groups of rats were evaluated in the present study: IGF-I-treated parous rats; parous rats that did not receive IGF-I treatment; and age-matched virgin animals, which also did not receive IGF-I treatment. Approximately 60 days after N-methyl-N-nitrosourea injection, IGF-I treatment was discontinued and all of the animal groups were implanted with a silastic capsule containing 17β-estradiol and progesterone. The 17β-estradiol plus progesterone treatment continued for 135 days, after which the animals were killed.ResultsIGF-I treatment of parous rats increased mammary tumor incidence to 83%, as compared with 16% in parous rats treated with 17β-estradiol plus progesterone only. Tumor incidence and average number of tumors per animal did not differ between IGF-I-treated parous rats and age-matched virgin rats. At the time of N-methyl-N-nitrosourea exposure, DNA content was lowest but the α-lactalbumin concentration highest in the mammary glands of untreated parous rats in comparison with age-matched virgin and IGF-I-treated parous rats. The protein levels of estrogen receptor-α in the mammary gland was significantly higher in the age-matched virgin animals than in untreated parous and IGF-I-treated parous rats. Phosphorylation (activation) of the extracellular signal-regulated kinase-1/2 (ERK1/2) and expression of the progesterone receptor were both increased in IGF-I-treated parous rats, as compared with those in untreated parous and age-matched virgin rats. Expressions of cyclin D1 and transforming growth factor-β3 in the mammary gland were lower in the age-matched virgin rats than in the untreated parous and IGF-I-treated parous rats.ConclusionWe argue that tumor initiation (transformation and fixation of mutations) may be similar in parous and age-matched virgin animals, suggesting that the main differences in tumor formation lie in differences in tumor progression caused by the altered hormonal environment associated with parity. Furthermore, we provide evidence supporting the notion that tumor growth promotion seen in IGF-I-treated parous rats is caused by activation of estrogen receptor-α via the Raf/Ras/mitogen-activated protein kinase cascade.

Prevention of mammary carcinogenesis by short-term estrogen and progestin treatments

IntroductionWomen who have undergone a full-term pregnancy before the age of 20 have one-half the risk of developing breast cancer compared with women who have never gone through a full-term pregnancy. This protective effect is observed universally among women of all ethnic groups. Parity in rats and mice also protects them against chemically induced mammary carcinogenesis.MethodsSeven-week-old virgin Lewis rats were given N-methyl-N-nitrosourea. Two weeks later the rats were treated with natural or synthetic estrogens and progestins for 7–21 days by subcutaneous implantation of silastic capsules.ResultsIn our current experiment, we demonstrate that short-term sustained exposure to natural or synthetic estrogens along with progestins is effective in preventing mammary carcinogenesis in rats. Treatment with 30 mg estriol plus 30 mg progesterone for 3 weeks significantly reduced the incidence of mammary cancer. Short-term exposure to ethynyl estradiol plus megesterol acetate or norethindrone was effective in decreasing the incidence of mammary cancers. Tamoxifen plus progesterone treatment for 3 weeks was able to confer only a transient protection from mammary carcinogenesis, while 2-methoxy estradiol plus progesterone was effective in conferring protection against mammary cancers.ConclusionsThe data obtained in the present study demonstrate that, in nulliparous rats, long-term protection against mammary carcinogenesis can be achieved by short-term treatments with natural or synthetic estrogen and progesterone combinations.

Tamoxifen inhibition of estrogen receptor-α-negative mouse mammary tumorigenesis

Tamoxifen reduces the relative risk of breast cancer developing from specific premalignant lesions. Many breast cancers that arise after tamoxifen treatment are estrogen receptor-α (ER-α)–negative, although premalignant lesions such as atypical ductal hyperplasia are highly ER-α–positive. The p53 null mouse mammary epithelial transplant model is characterized by ER-α–positive premalignant lesions that give rise to both ER-α–positive and ER-α–negative tumors. Given this progression from ER-α–positive to ER-α–negative lesions, we tested the ability of tamoxifen to block or delay mammary tumorigenesis in several versions of this model. In groups 1 and 2, p53 null normal mammary epithelial transplants were maintained in virgin mice. In groups 3 to 5, the p53 null and mammary transplants were maintained in mice continuously exposed to high levels of progesterone. In groups 6 and 7, transplants of the premalignant outgrowth line PN8a were maintained in virgin mice. Tamoxifen blocked estrogen signaling in these mice as evidenced by decreases in progesterone-induced lateral branching and epithelial proliferation in the mammary epithelium. Tamoxifen did not alter the elevated levels of progesterone in the blood while significantly reducing the circulating level of prolactin. Tamoxifen reduced tumor incidence in p53 null normal mammary epithelial transplants maintained in virgin mice from 55% to 5% and in progesterone-stimulated mice from 81% to 21%. The majority of the resultant tumors were ER-α–negative. Tamoxifen also significantly delayed tumorigenesis in the ER-α–positive high premalignant line PN8a from 100% to 75%. These results show that tamoxifen delays the emergence of ER-α–negative tumors if given early in premalignant progression.

Refractoriness to mammary carcinogenesis in the parous mouse is reversible by hormonal stimulation induced by pituitary isografts

We have previously reported that mouse mammary epithelial cells transformed in vitro yield tumors which vary qualitatively and quantitatively as a function of the mitogenic environment in which the cells are propagated at the time of carcinogen treatment. One milieu supportive of transformation in vitro was medium supplemented with progesterone and prolactin as the mitogens. We have performed parallel studies in which virgin mice were isografted with pituitaries resulting in elevated serum titers of progesterone and prolactin. After carcinogen treatment, these mice developed mammary tumors which included those identical genotypically and phenotypically to tumors induced in vitro in cells grown in progesterone and prolactin during carcinogen exposure. Our current working hypothesis is that the mitogenic environment around the time of carcinogen administration can modulate the incidence and phenotype of the resultant tumors. To further test this hypothesis, we have evaluated the susceptibility of hormonally-stimulated parous mice to chemically induced mammary carcinogenesis since parity is known to significantly reduce the susceptibility of the mouse mammary gland to carcinogenesis. Virgin or multiparous BALB/c mice were isografted with two pituitaries. Five weeks after surgery, the mice were injected with N-methyl-N-nitrosourea (MNU; 50 micrograms/g i.v.). Mammary carcinomas arose in 85% (11/13) with a median latency of 22.8 weeks and 1.9 tumors per virgin mouse and 80% (24/30) with a median latency of 22.1 weeks at a frequency of 1.9 tumors per parous mouse. Only 14% (2/14) of the non-isografted, age-matched parous controls developed tumors when injected with MNU. Fourteen parous mice receiving only pituitary isografts (no MNU), did not develop mammary carcinomas within the 7-month period of the study. These results demonstrate that parous BALB/c mice are refractory to MNU-induced mammary carcinogenesis and that this refractoriness is not permanent, but can be overcome by hormonal stimulation mediated by pituitary isografts.

Development of a placental cell culture system for studying the control of mouse placental lactogen II secretion

Fetal placental tissue from 11 days pregnant mice was dissociated in collagenase and DNase solution and then separated on a 40 per cent Percoll gradient. Trophoblast cells banded at a density of 1.05 g/ml. When cultured on rat tail collagen, these cells formed colonies of mono- and binucleate cells varying in size from 40 to 70 microns. At the time of plating, about 13 per cent of the trophoblast cells secreted mouse placental lactogen II (mPL-II) as determined by reverse haemolytic plaque assay. The ratio of mPL-II-producing cells increased significantly in culture and reached 63 per cent after 48 h. The secretion of mPL-II increased continuously during six days of culture, whereas the total protein release was highest after the first day, declined the second day and then remained relatively constant for the last four days of culture. The DNA content of the cells did not change significantly during the six-day period. When the trophoblast cells were incubated with insulin (1 ng/ml to 5 micrograms/ml), a modest but significant reduction in mPL-II secretion was observed. No change in the mPL-II secretion was seen when epidermal growth factor was administered to the culture in concentrations from 1 ng/ml to 10 micrograms/ml. It is concluded that this in vitro culture system is suitable for studying both mPL-II secretion and the differentiation of mPL-II-producing cells.

Estrogen Can Prevent Breast Cancer by Mimicking the Protective Effect of Pregnancy

Our data, based on the MNU-exposed parous studies and STET/STEPT rats, indicate that these three phenotypes are alike in terms of their susceptibility to MC. All are: susceptible to initiation and development of latent cancers; have a drastically reduced incidence of overt mammary cancers; have a decreased promotional environment with persistently reduced circulatory levels of GH and PRL, as well as a decreased expression of ERα and PRs in the MEC. Finally, all protocols can develop a high incidence of overt mammary cancer with increased promotion with E2 ± P.

Microarray Analysis of Estrogen-induced Protection Against Breast Cancer

Pregnancy early in life reduces the risk of breast cancer (BC) in women and this effect is universal. This phenomenon is also observed in rodents. We have shown that a treatment with high pregnancy levels of 17β-estradiol (E2) for 7 to 21 days is effective in conferring protection against mammary carcinogenesis. We determined the difference in gene expression between hormone-protected rats and unprotected rats. Nine weeks old female Lewis rats were treated with 10 microgram (unprotected), or 200 microgram (protected) of E2 in silastic capsules for 3 weeks. Control rats received silastic capsules with no hormone. The rats were terminated 8 weeks after the removal hormone treatment. Mammary RNA was used for microarray analysis. Using Agilent Rat cDNA Microarrays, with 14,815 unique clones, we have analyzed genes from Rattus norvegicus and Rattus rattus which are annotated as “mRNA” or “gene EST” in GenBank. The genes involved in growth promotion like interleukin 18, kit oncogene, thyrotropin stimulating hormone receptor, cyclin dependent kinases etc. were down regulated in the protected group compared to the unprotected groups. In contrast, genes involved in growth inhibition like early growth response 1, insulin-like growth factor binding proteins and genes involve in apoptosis and DNA repair like T-cell death activated gene, CD47, histone acetyltransferase were up regulated in the protected animals compared to the unprotected. These findings define a pattern of gene expression that could serve to determine the efficacy of protective hormone treatments and help identify potential biomarkers for prevention of mammary cancers.

Mouse Placental Lactogens: Characterization and Regulation of Expression in Trophoblast Cells

The fetus develops in an environment where respiration, alimentation, and excretory functions are provided by the placenta. In addition, the placenta is the site of synthesis and release of a wide array of steroid and protein hormones that are important for the maintenance of pregnancy and the well-being of the developing fetus. The hormones produced by the placenta share structural and functional overlap with hormones produced by the hypothalamus, pituitary, ovaries, and adrenals. Thus, the placenta has developed the capacity to compress the endocrine function of several endocrine organs into one functional endocrine unit. The evidence shows considerable differences among species relative to the presence or absence of a given placental hormone. For example, the human placenta produceshuman chorionic gonadotropin (hCG), a protein hormone that shares structural and functional activity with pituitaryluteinizing hormone (LH). On the other hand, the mouse pla¬centa appears to be devoid of the ability to produce a CG that is similar to mouse LH.