Dose-responsive induction of mammary gland carcinomas by the intraperitoneal injection of 1-methyl-1-nitrosourea

Dose-response relationships for the induction of mammary tumors by a single i.p. injection of 1-methyl-1-nitrosourea (MNU) were studied. Groups of 30 female Sprague-Dawley rats were given i.p. injections of 50, 37.5, 25, 12.5, or 0 mg MNU/kg body weight at 50 days of age. Animals were palpated for tumor detection twice weekly throughout a 28-week observation period. Administration of MNU i.p. caused no acute toxicity. Both benign and malignant mammary tumors were induced by MNU, but malignant tumors appeared earlier and at a faster rate than benign tumors. The incidence and numbers of mammary carcinomas increased whereas median cancer-free time decreased with increasing dose of MNU. Approximately twice as many mammary cancers were observed in the cervical-thoracic as in the abdominal-inguinal mammary gland chains irrespective of carcinogen dose, while the frequency of tumor occurrence in the left versus right chains was similar. Tumor latency decreased with increasing dose of MNU, but the quartiles for time to detection of all tumors within each carcinogen dose group were similar irrespective of anatomical region in which the tumors occurred. The mammary tumor response attained via i.p. injection was similar but the coefficient of variation for tumor multiplicity within a carcinogen dose group was lower in comparison to that observed when MNU was administered i.v. or s.c. Among these techniques for carcinogen injection, the i.p. route is the most rapid method and offers an added advantage of ease of administration with quantitative, reproducible delivery of the desired amount of carcinogen and a decrease in variability of tumor response among animals within a treatment group. This method is well suited for the technically less experienced investigator and for those in need for a rapid method of injecting MNU to large numbers of animals.     

Effect of carcinogen dose and age at administration on induction of mammary carcinogenesis by 1-methyl-1-nitrosourea.

The objective of the work reported in this paper was to determine if the tumorigenic response to 1-methyl-1-nitrosourea (MNU) in the mammary gland varied with age of administration and was dose dependent when the carcinogen was injected prior to 50 days of age. Using a recently developed method for mammary tumor induction, MNU was injected i.p. at doses ranging from 25 to 75 mg/kg at 35 days of age or 50 mg/kg at 28, 35 or 42 days of age. Treatment with MNU resulted in induction of both benign and malignant mammary tumors. The incidence of mammary gland adenocarcinomas was 100% at and above the 50 mg/kg dose of MNU, irrespective of the age at which carcinogen was administered. The number of cancers increased in proportion to carcinogen dose, whereas cancer latency decreased as the MNU dose increased. In rats injected with 50 mg MNU/kg body weight at 28, 35 or 42 days of age, differences among groups in cancer incidence, number or latency were not statistically significant. Metastases of mammary neoplasms to lung, liver and spleen were observed in rats injected with MNU at 35 or 42 days of age. These data indicate (i) the dose responsiveness of MNU-induced mammary carcinogenesis in rats initiated prior to 50 days of age; (ii) the lack of effect of age at initiation if prior to 50 days on final tumor outcome; and (iii) that the age at which MNU is injected may affect the metastatic potential of the mammary carcinomas that are induced.     

Estradiol and progesterone can prevent rat mammary cancer when administered concomitantly with carcinogen but do not modify surviving tumor histology,estrogen receptor alpha status or Ha-ras mutation frequency

An early full-term pregnancy is protective against mammary cancer in both humans and rodents. Treating rats with two hormones of pregnancy, estradiol and progesterone, for 5 weeks renders the rat mammary glands refractory to carcinogenesis. Our objectives was to determine if a shortened regimen (3 weeks) would be as effective as the 5-week regimen and to determine if the mammary gland was vulnerable to carcinogenic insult during the hormone treatments. We also examined cancers that survived the chemopreventive regimen to see if those tumors were particularly aggressive compared to control tumors (i.e., less differentiated, estrogen receptor alpha (ER alpha)-negative or harbored mutations in Ha-ras). In the first experiment, Lewis rats were injected with N-methyl-N-nitrosourea (MNU, 50 mg/kg) at 50 days of age. At 60 days of age, the rats were either mated and allowed to nurse their young for 3 weeks, treated with hormone vehicle for 5 weeks, or 17 beta-estradiol (E, 20 micrograms) and progesterone (P, 4 mg) 5 times per week for 3 or 5 weeks. All the rats exposed to MNU but not estradiol and progesterone developed multiple mammary cancers. Pregnancy reduced multiplicity to 0.40 cancers/rat. Treatments of estradiol and progesterone for 3 or 5 weeks reduced cancer multiplicity and increased latency to a similar degree as pregnancy. Mammary cancers from each group displayed a similar spectra of histologic class, estrogen receptor alpha (ER alpha) content and Ha-ras mutation status. In the second experiment, 50-day-old rats were treated for five weeks with either estradiol and progesterone or vehicle as above beginning at 60 days of age and treated with MNU at 50, 64, 78 or 92 days of age. In each case, estradiol and progesterone treatments resulted in significantly reduced mammary tumor frequency. These results demonstrate that a three-week regimen of estradiol and progesterone can protect the mammary gland from chemically-induced carcinogenesis even when carcinogen exposure occurs concomitant with estradiol and progesterone stimulation.     

Mammary tumor induction by N-methyl-N-nitrosourea in genetically resistant Copenhagen rats.

The Copenhagen rat is completely resistant to mammary cancer induction by N-methyl-N-nitrosourea (MNU) when the carcinogen is administered during sexual development, a period when other strains of rats are normally susceptible to mammary gland carcinogenesis. Here we administered 30 mg/kg MNU i.p. to two groups of neonatal (2-3-day-old) Copenhagen rats. One group (group B, 18 animals) received no further treatment, while the other group (group C, 17 animals) received a second dose of 30 mg/kg MNU via the tail vein at 50 days of age. About 30% of the rats in group B and about 70% of those in group C developed mammary carcinomas before they were 1 year of age. About one-half of the tumors in both groups were cribriform adenocarcinomas and one-half were adenosquamous carcinomas. The latter tumor type has not been observed previously in susceptible rat strains. The ability to induce these mammary tumors in the Copenhagen rat suggests that the putative mammary carcinoma suppressor gene is functionally inactive in neonatal animals or is inactivated when these animals are treated with MNU.     

Centrosome amplification and overexpression of aurora A are early events in rat mammary carcinogenesis

The cells of many solid tumors have been found to contain supernumerary centrosomes, a condition known as centrosome amplification. Centrosome amplification, accompanied by the overexpression of an associated kinase, Aurora A (AurA), has been implicated in mechanisms leading to mitotic spindle aberrations, aneuploidy, and genomic instability. Using a well-established rat mammary model favorable for experimental carcinogenesis, we analyzed centrosome amplification as a cellular marker for early stages of transformation and its regulation by the kinase ratAurA. Parity or treatment with estrogen and progesterone conferred resistance to tumorigenesis, as well as to overexpression of ratAurA and to centrosome amplification. ratAurA, cloned from a rat mammary gland cDNA library, is a bona fide Ser/Thr kinase, and sequence comparison demonstrated high homology to members of the entire AurA kinase family. Using immunocytochemical localization with confocal microscopy, we found ratAurA to be localized at the centrosome in normal and neoplastic tissues of the rat mammary gland. Normal ductal epithelium and stromal cells displayed an expected complement of one to two centrosomes/cell, whereas comparable cells in methylnitrosourea-treated animals displayed significantly elevated centrosome numbers. In tumors, 46% of cells showed more than two centrosomes/cell, and ratAurA expression levels coincided with higher centrosome numbers. Both centrosome numbers and ratAurA expression were permanently elevated. Centrosome amplification was found to occur at a very early, premalignant stage prior to detectable lesions after treatment with methylnitrosourea, a condition that was not detected in mammary glands of rats made refractory to the carcinogen via pregnancy or estrogen and progesterone treatment. Our results indicate that hormones influence kinase expression, and progesterone had the major effect on ratAurA expression levels. Cumulatively, these results suggest that ratAurA overexpression and centrosome amplification were linked to tumor development and progression and may serve as early markers in tumorigenesis.     

Inhibition of mammary gland tumors by short-term treatment of estradiol-3-benzoate associated with down-regulation of estrogen receptor ERalpha and ERbeta

Epidemiological evidence indicates that estrogens are one of the risk factors of breast cancer. However, there have been reports that pre-pubertal estrogen exposure is related to reduced breast cancer risk. These discrepancies made us investigate the time-point and duration of estrogen exposure. Our studies focus on the effect of estradiol-3-benzoate (EB) on the mammary gland that was exposed to carcinogens. Ninety-six female Sprague-Dawley rats were randomly divided into 6 groups. Animals at 7 weeks of age were injected with 7,12-dimethylbenz[a]anthracene (DMBA) in groups 1, 2 and 3 or N-methyl-N-nitrosourea (MNU) in groups 4, 5 and 6. One week later, the animals were subjected to sustained treatment with 0 micro g (groups 1 and 4), 30 micro g (groups 2 and 5) or 300 micro g (groups 3 and 6) of EB containing pellets for 4 weeks. All animals were sacrificed at 5 weeks or 21 weeks after carcinogen treatment, for the examination of mammary gland differentiation or mammary gland tumors, respectively. At 21 weeks after carcinogen treatment, the incidence of mammary tumors in group 2 was significantly decreased (P<0.05). EB treatment decreased the multiplicity of DMBA- or MNU-induced mammary gland tumors. At 5 weeks after carcinogen treatment, there were increased branchings of the mammary gland, and there was also a decrease of ERalpha and ERbeta in EB treatment groups. Taken together with these results, we conclude that EB has an inhibitory effect on mammary carcinogenesis, and it suggests that this inhibition may be associated with the differentiation of mammary gland and modulation of ERalpha and ERbeta.     

Influence of caffeine on development of benign and carcinomatous mammary gland tumors in female rats treated with the carcinogens 7,12-dimethylbenz(a)anthracene and N-methyl-N-nitrosourea

The effect of chronic caffeine consumption (500 mg/liter of drinking water) on the initiation and promotion stages of 7,12-dimethylbenz(a)anthracene (DMBA) (a low dose, 0.5 mg/100 g body weight, i.v.) and N-methyl-N-nitrosourea (MNU) (a standard dose, 2.5 mg/100 g body weight, i.v.) induced mammary gland tumorigenesis in female Sprague-Dawley rats was determined. In the initiation studies, caffeine was administered for 30 days prior to and for 3-4 days after carcinogen treatment (carcinogens administered at 55-57 days of age); in the promotion studies, caffeine was administered beginning 3-4 days after carcinogen treatment and until experiment termination (DMBA study and MNU study, 48 and 26 weeks after carcinogen treatment, respectively). In the DMBA study, there were 62-73 rats/group, in the MNU study, 40 rats/group. Eighty-nine % of the mammary tumors induced by DMBA were benign (adenomas, fibroadenomas, often with cystic secretory activity), 11% were carcinomas (intraductal and invasive); virtually all of the MNU-induced mammary tumors were carcinomas (approximately 99%). Caffeine consumption during the initiation stage in the DMBA-treated rats resulted in a significant decrease in the mean number of mammary carcinomas per rat (50% reduction, P less than 0.01) and mean number of benign mammary tumors per rat (28% reduction, P less than 0.05); caffeine consumption during the promotion stage significantly decreased the mean number of benign mammary tumors per rat (57% reduction, P less than 0.001) while not significantly influencing mammary carcinoma number. In contrast, caffeine consumption during either the initiation or promotion stages of MNU-treated rats did not significantly influence this tumorigenic process. The influence of caffeine on urinary and fecal excretion of tritiated DMBA and on rat mammary gland development at the time of carcinogen treatment also was determined. Slightly reduced levels of tritium in 24-h urinary samples were observed in caffeine-treated animals (P = 0.06). No significant effect of caffeine on 24- to 96-h fecal or 48- to 96-h urinary excretion of the isotope was observed. No apparent effect of caffeine on rat mammary gland development (number of ducts, degree of lobuloalveolar development) was observed. That caffeine significantly suppresses the initiation stage of DMBA-induced rat mammary gland tumorigenesis, while not influencing this stage when MNU is used as a carcinogen, suggests that caffeine acts via an alteration in carcinogen (DMBA) activation. The lack of a pronounced effect of caffeine on tritiated DMBA excretion, however, does cast some doubt on this mechanism.(ABSTRACT TRUNCATED AT 400 WORDS)     

Distribution of epidermal growth-factor receptors in normal and neoplastic mammary tissues

Epidermal growth factor (EGF) is considered to be mitogenic for proliferation of mammary glands in animals. The action of EGF is mediated by specific EGF receptors (EFG-R). In the present study, we investigated distribution of EGF receptors during various physiological stages of mammary glands, N-methyl-N-nitrosourea (MNU)-induced mammary tumors in rats and human breast cancer samples. EGF receptor concentrations were determined by Scatchard analyses in the membrane fraction of the tissues. Results showed increased EGF receptor levels in the structurally differentiated mammary tissues from pregnant rats; whereas lower concentrations were observed in the functionally differentiated glands from lactating rats. EGF receptors were absent in the majority of the tumors induced by MNU. The loss of EGF receptor was not observed during the first 20 days post carcinogen treatment, but appeared to be correlated with the onset of the tumor. Consistent with the literature, the majority of the steroid receptor positive human breast cancer samples were EGF receptor negative, whereas steroid receptor negative samples contained EGF receptors. These results suggest that the loss of EGF receptors in ovarian hormone dependent mammary tumors does not occur gradually during carcinogenesis but appears to be a characteristic of hormone dependent mammary tumor cells.     

Induction of invasive carcinomas of the seminal vesicles and coagulating glands of F344 rats by administration of N-methylnitrosourea or N-nitrosobis(2-oxopropyl)amine and followed by testosterone propionate with or without high-fat diet

The potential modifying effects of testosterone propionate (TP) and high-caloric high-fat diet (20% corn oil, HF) on rat accessory sex gland carcinogenesis were investigated. Male F344 rats were treated five times at 4-week intervals with N-methylnitrosourea (MNU) i.v. or N-nitrosobis(2-oxopropyl)amine (BOP) s.c., each injection following 2 weeks pretreatment with dietary ethinyl estradiol. After completion of this carcinogen administration stage, animal groups received subcutaneous implantation of Silastic tubes filled with 40 mg TP with or without HF for 40 weeks. Carcinomas of the seminal vesicles and/or coagulating glands were induced in 5, 39 and 56% of rats given MNU alone, MNU and TP, and MNU and HF plus TP respectively. No equivalent tumors were found in rats given MNU and HF. In the BOP-treated groups, 11% of animals receiving TP but no HF diet demonstrated seminal vesicle carcinomas and 6% of rats receiving TP plus HF diet had coagulating gland carcinoma. Thus while TP exerted a strong enhancing effect on tumor growth in the seminal vesicles and coagulating glands, high caloric HF did not manifest any significant influence.     

Suppression by pregnancy of chemically induced preneoplastic cells of the rat mammary gland

Since a previous study suggested that pregnancy either eliminated preneoplastic cells or increased their latency period in rat mammary glands, additional experiments were performed to determine the fate of these cells. Following administration of the carcinogen dimethylbenzanthracene, few mammary cancers appeared after rats completed pregnancy and lactation. Because these results are similar to those previously obtained with N-methyl-N-nitrosourea (MNU), the effect of pregnancy appears to be independent of the carcinogen used to induce cancer. For rats dosed with MNU, relatively few cancers developed either during an extended observation following pregnancy and lactation or following administration of prolactin, a growth stimulator of mammary cancers. In the latter experiment, the average number of mammary cancers per rat in the prolactin-treated virgin rats was 7.1, while prolactin-treated parous rats had only 0.8 cancers per rat; i.e., 89% fewer cancers. Thus, pregnancy appears to suppress many of the preneoplastic cells induced by carcinogens in the mammary gland of rats.     

The histogenesis of mammary tumours induced in the rat by chemical carcinogens

126 Wistar female rats were given carcinogenic compounds, 2-acetylaminofluorene (2-AAF), 20-methylcholanthrene (MC) and 7,12-dimethylbenz(a)anthracene (DMBA), for induction of breast tumors to determine the site of origin and biological behavior of mammary carcinoma in the rat. 18 virgin rats served as control material of a corresponding age to the tumor-bearing rats. A dissecting microscope was used to identify early neoplastic lesions in the breast glands for histological study. 17 weeks after carcinogen treatment, breast gland tumors, especially adenocarcinomas became evident by palpation. Mammary carcinoma was found to be multifocal in origin, arising almost exclusively in ducts, ductules, and end-buds without the intervention of any recognizable lesion preceding the appearance of tumor cells. Fibroadenomas and adenomas occurred in rats with generally well-developed breast glands, while mammary carcinomas occurred in younger rats with less developed breast glands. For successful tumor induction, the important factors to consider are the architectural state of the mammary gland, the action of hormonal factors, genetically-determined susceptibility, and the carcinogenic agent and its dosage. Mast cells did not play a significant role in tumor formation, nor were significant myoepithelial cell contributions found in the early carcinomas and fibroadenomas.     

Effect of selective ablation of proliferating mammary epithelial cells on MNU induced rat mammary tumorigenesis

Proliferating cells within the terminal end buds of the virgin female rat mammary gland are the most susceptible to chemical carcinogen induced tumorigenesis. We hypothesized that selective ablation of proliferating cells in the mammary gland would reduce mammary tumor incidence upon carcinogen challenge. Selective ablation of proliferating cells was achieved by intraductal injections of Adv-RSV-tk and gancyclovir administration. Despite efficient viral transduction of the thymidine kinase protein and the apparent elimination of >90% of the prolif-erating cells, the rats exhibited a higher incidence of MNU induced mammary tumors arising with shorter latency as compared to control animals. Several possible explanations of the puzzling relationship between elimination of cycling cells and increased tumor incidence are discussed and alternative strategies for the prevention of breast cancer are proposed.     

Progression of rat mammary development with age and its relationship to carcinogenesis by a chemical carcinogen

The progression of mammary gland development in Sprague-Dawley rats between 5 and 440 days of age was studied. Tumorigenesis of the mammary gland after administration of 7,12-dimethylbenz(a)anthracene (DMBA) was also investigated during different prepubertal and postpubertal stages. The mammary gland starts as a single primary duct on day 5 and with age branches and grows into a complete gland. The growth of the gland was observed to start from the nipple area and progress into the empty fat pad. This growth was in the form of club-shaped end-buds which, after the onset of puberty, became gradually transformed into alveolar buds. [³H]-thy-midine labelling index (LI.) was high (19%) in prepubertal glands, but it eventually declined to 8% at 60-65 days. The postpubertal gland at the age of 150 days was determined to be practically mitotically static, but at 440 days, there was another increase in LI. There was a direct relationship between mammary tumorigenesis by DMBA and LI. in the mammary gland at the time of treatment with the carcinogen. Up to 55 days of age, tumorigenesis was 90-100%, and LI. was also high during this period. The carcinogen was practically ineffective at 150 days, when the mammary gland was non-proliferative. Though the total tumor induction was high in the prepubertal rats, the majority of the tumors (up to 84%) were benign fibro-adenomas, while almost all the tumors in postpubertal rats were adenocarcinomas. Thus, a relationship was observed between the presence of ovarian hormones and the induction of malignant tumors. It was concluded that cell proliferation index at the time of carcinogen treatment is important for tumor induction, but whether or not the tumor is malignant is determined by the presence or absence of ovarian hormones.     

Post-initiation treatment of Indole-3-carbinol did not suppress N-methyl-N-nitrosourea induced mammary carcinogenesis in rats.

The consumption of cruciferous vegetables (the Family of Cruciferae) such as cabbage, broccoli and Brussels sprouts has been shown to have cancer chemopreventive effects in humans and experimental animals. Indole-3-carbinol (I3C), one component of cruciferous vegetables, has been shown to exert cancer chemopreventive influence in liver, colon, and mammary tissue when given before or concurrent with exposure to a carcinogen. However in some reports, there has been evidence that consumption of I3C after carcinogen treatment might be associated with tumor promotion in some tissues. There have been no reports, to our knowledge, of post-initiation effects of I3C in the N-methyl-N-nitrosourea (MNU)-induced mammary tumor model in rats. Our studies were performed to examine this question. Ninety-six, 4-week-old female Sprague-Dawley rats were randomly divided into five groups. The animals of groups 1, 2 and 3 received an intraperitoneal injection of MNU at the age of 50 days. The animals of groups 4 and 5 were injected with saline only at the same time. Animals of groups 1 and 2 were given diet containing 100 ppm and 300 ppm I3C from week 1 until week 25 after MNU treatment. The animals of group 4 were given basal diet containing 300 ppm I3C without MNU treatment. All animals were killed at week 25. The incidences of mammary tumors in the groups 1, 2 and 3 were 95.8% (23/24), 83.3% (20/24) and 82.4% (28/34), respectively. The average number of tumors in the tumor bearing rats of the MNU and I3C 300 ppm group (group 2; 3.85+/-0.63) was higher than that in the MNU alone group (group 3; 2.46+/-0.31). These results represented that exposure to I3C after carcinogen treatment did not suppress development of mammary tumors.     

Susceptibility of lean and obese Zucker rats to tumorigenesis induced by N-methyl-N-nitrosourea

To address the possible involvement of hyperinsulinemia in breast cancer development, we have examined the susceptibility of lean and obese Zucker rats to N-methyl-N-nitrosourea (MNU)-induced mammary cancer. Fifty-day-old female lean or obese Zucker rats received intraperitoneal (i.p.) injections of 37.5 or 20 mg/kg MNU, respectively. We showed in separate experiments that these doses produce similar levels of DNA methylation in the mammary epithelial cells of the lean and obese animals. Over the course of 29 weeks following MNU treatment, half of the lean rats developed carcinomas of the mammary gland, demonstrating that they are of intermediate susceptibility to mammary tumorigenesis. During this period, the obese rats developed hyperinsulinemia and insulin resistance as expected. Although palpable tumors developed at a similar rate in the lean and obese rats, only 10% of the obese animals developed mammary carcinomas. The obese rats, however, developed a high incidence (63.3%) of epidermal cysts that occurred mainly in the region of the mammary glands. A 13.3% incidence of colon carcinomas was also found in the obese rats. These results suggest that the development of hyperinsulinemia does not render the obese Zucker rats more susceptible to mammary gland carcinogenesis. Our observation of colon carcinomas in obese, but not lean rats, however, is consistent with evidence that hyperinsulinemia promotes colon cancer in rodents and humans.     

Mammary carcinogenesis induced byN-methyl-N-nitrosourea (MNU) and medroxyprogesterone acetate (MPA) inBALB/c mice

Summary MPA induces mammary tumors in virgin BALB/c mice with an average latency of 52 weeks. In order to determine whether the simultaneous administration of a chemical carcinogen, N-methyl-N-nitrosourea (MNU), shortened the latency of MPA-induced tumors, a total of 60 virgin female BALB/c mice were treated with either MNU+MPA or MNU or MPA. The experiment lasted 7 months. The incidence and latency of mammary tumors were significantly different between the 3 groups: 15/19 (79%) in MNU+MPA-treated mice with a latency of 154±19 days; 3/20 (15%) in MNU-treated mice with a latency of 179±7 days; 0/20 (tumors only start appearing after 10 months) in MPA-treated mice. Histologically, MNU+MPA-induced tumors were similar to the few tumors observed in MNU-treated mice: most of them were type B adenocarcinomas with a high degree of necrosis and calcification. Only one of the MNU+MPA-induced tumors expressed high levels of ER and PR and proved to be MPA-responsive in further passages. All the other tumors showed low or non-detectable levels of ER and PR together with an independent pattern of tumor growth. In MNU-treated mice the only tumor that was transplanted proved to be hormone independent and had low levels of PR and ER. In both MNU and MNU+MPA treated mice lung adenocarcinomas were detected. Cystic uterine glandular hyperplasias were observed in all animals. It can be concluded that MPA and MNU potentiate their carcinogenic effect in mammary gland.Abbreviations MPA Medroxyprogesterone Acetate - MNU N-methyl-N-nitrosourea - ER Estrogen Receptors - PR Progesterone Receptors     

Parous mammary glands exhibit distinct alterations in gene expression and proliferation responsiveness to carcinogenic stimuli in Lewis rats

Early full-term pregnancy affords lifetime protection against the development of breast cancer. Parity-induced protection can be reproduced in a carcinogen-induced rat mammary carcinoma model, but the molecular mechanisms of this protection against carcinogenic stimuli in rat mammary glands have not been fully characterized. To gain a better understanding of these molecular mechanisms, we used an oligonucleotide microarray to examine gene expression in parous and age-matched virgin (AMV) mammary glands of Lewis rats before and after carcinogen (N-methyl-N-nitrosourea; MNU) treatment. Parous mammary glands before MNU treatment showed up-regulation of multiple differentiation-related genes, such as whey acidic protein (Wap), casein beta (Csn2), casein gamma (Csng), lipopolysaccharide binding protein (Lbp), secreted phosphoprotein 1 (Spp1) and glycosylation-dependent cell adhesion molecule 1 (Glycam1). Also, parous mammary glands before MNU treatment exhibited down-regulation of growth-related genes such as regenerating islet-derived 3 alpha (Reg3a), mesothelin (Msln), insulin-like growth factor 2 (Igf2) and insulin-like growth factor binding protein 4 (Igfbp4). After MNU treatment, AMV mammary glands exhibited up-regulation of growth-related genes, such as Msln, cell division cycle 2 homolog A (Cdc2a), Igf2, Igfbp4, stathmin 1 (Stmn1) and homeobox, msh-like 1 (Msx1), whereas expression of these genes remained low in parous mammary glands. AMV mammary glands also exhibited marked up-regulation of Cdc2a and Stmn1 in response to MNU. After MNU treatment, the PCNA labeling index increased significantly in AMV mammary epithelial cells (13.7+/-1.1%), but remained low in parous mammary glands (3.6+/-0.4%). The response of AMV mammary glands to carcinogenic stimuli includes up-regulation of growth-related genes and increased cell proliferation. The lack of a similar response in parous mammary glands may explain parity-induced protection against mammary tumor development.     

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 μg/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.