Effect of sialoadenectomy on medroxyprogesterone-acetate-induced mammary carcinogenesis in BALB/c mice. Correlation between histology and epidermal-growth-factor receptor content

To evaluate the possible involvement of the salivary glands in the modulation of medroxyprogesterone (MPA)-induced mammary tumorigenesis, 48 sialoadenectomized virgin BALB/c female mice and 47 controls were treated with 40mg MPA depot s.c. every 3 months for 1 year. Mammary tumors developed in 11 sialoadenectomized and in 34 control mice with similar latencies. In both groups, 75% of the tumors were ductal and progestin-dependent (PD) while the remainder were lobular and progestin-independent (PI). Epidermal growth factor (EGF) levels were measured in salivary glands (SG-EGF) and serum (S-EGF) in both groups. MPA induced a significant increase in SG-EGF and in S-EGF that became evident only after 1 month of MPA treatment. No increase in S-EGF was detected in MPA-treated sialoadenectomized mice, indicating that salivary glands are the major source of S-EGF. The presence of EGF receptors (EGF-R) was investigated in ductal PD and PI tumor lines and compared with 8 PI tumor lines of lobular origin. A significant difference in EGF-R content was found between lobular and ductal tumors. No increase in EGF-R was noted when ductal tumors became autonomous. EGF-R did not correlate with tumor growth rate and there was an inverse correlation between EGF-R and steroid receptors. When the effect of sialoadenectomy on tumor growth was tested in vivo in syngeneic transplants of 2 ductal PD, I ductal PI and 2 lobular PI mammary adenocarcinomas, it was not found to be significant when compared with the controls. It may be concluded that SG-EGF plays an important role in the induction of mammary adenocarcinomas by MPA, while it has no significant effect on the growth of established tumors.     

Promoter effect of medroxyprogesterone acetate (MPA) in N-methyl-N- nitrosourea (MNU) induced mammary tumors in BALB/c mice

The promoter effect of medroxyprogesterone acetate (MPA) on mammary carcinogenesis in female BALB/c mice was investigated using methylnitrosourea (MNU) as initiator. Nine out of 43 animals developed mammary carcinomas in the group treated with MNU (50 mg/kg) and MPA (administration of 40 mg every 3 months) starting 1 week after MNU administration. No tumors appeared in controls receiving only MNU or MPA during the time course of the experiment (9 months). The tumors were lobular adenocarcinomas showing different degrees of squamous differentiation with low or undetectable estrogen and progesterone receptors, and expressing epidermal growth factor receptors. These results support the hypothesis that MPA promotes the growth of MNU induced lesions.      

Differential expression of and responsiveness to transforming growth factor-beta (TGF-beta) isoforms in hormone-dependent and independent lines of mouse mammary tumors.

Transforming growth factor-beta2 (TGF-beta2) and -beta3 mRNA expressions were studied in ductal hormone-dependent (HD) and -independent (HI) in vivo lines of the medroxyprogesterone acetate (MPA)-induced mammary tumor model in Balb/c mice. MPA treatment of HD tumors induced a significant decrease in TGF-beta2 and -beta3 mRNA levels. Progression to an HI phenotype of ductal tumors was associated with reduced TGF-beta2 and -beta3 expressions, as compared with their HD counterparts. Exogenously added TGF-beta1, -beta2, and -beta3 (1 ng/ml) inhibited the proliferation of primary cultures of epithelial cells from ductal HD and HI tumors. In addition, TGF-beta expression and effects were studied in the other type of MPA-induced mammary tumors, which are of lobular origin and lack steroid hormone receptors and evidence an HI behavior. These lobular HI lines showed TGF-beta2 levels similar to those found in HD lines growing in MPA-treated mice. In contrast, TGF-beta3 mRNA levels were 12- to 20-fold higher than in HD tumors. Primary cultures of lobular HI epithelial cells required either TGF-beta concentrations of 10 ng/ml to show an inhibitory response, or were completely resistant to TGF-beta inhibition. Studies of the molecular mechanisms involved in reduction or loss of TGF-beta responsiveness in lobular HI tumors showed that cell surface type II TGF-beta receptor levels were lower in these tumors than those present in HD tumors. Our results support the hypothesis that TGF-beta could play a role as an autocrine growth inhibitor in HD and HI ductal tumors. Autonomous growth of lobular HI tumors could be favored by undetectable or low TGF-beta1 and -beta2 expressions and by reduced or lost sensitivity of epithelial cells to TGF-beta's antiproliferative effects. However, the extremely high levels of TGF-beta3 expression in lobular HI tumors, in spite of reduced sensitivity to TGF-beta3 inhibitory growth effect in tumor epithelial cells, suggest a net positive role for TGF-beta3 in these tumors.     

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     

Identification of women with an increased risk of developing radiation-induced breast cancer: a case only study

Introduction

Medroxyprogesterone acetate (MPA) induces estrogen receptor (ER)-positive and progesterone receptor (PR)-positive ductal invasive mammary carcinomas in BALB/c mice. We sought to reproduce this MPA cancer model in C57BL/6 mice because of their widespread use in genetic engineering. Within this experimental setting, we studied the carcinogenic effects of MPA, the morphologic changes in mammary glands that are induced by MPA and progesterone, and the levels of ER and PR expression in MPA-treated and progesterone-treated mammary glands. Finally, we evaluated whether the differences found between BALB/c and C57BL/6 mouse strains were due to intrinsic differences in epithelial cells.

Methods

The carcinogenic effect of MPA was evaluated in C57BL/6 mice using protocols proven to be carcinogenic in BALB/c mice. In addition, BALB/c and C57BL/6 females were treated with progesterone or MPA for 1 or 2 months, and mammary glands were excised for histologic studies and for immunohistochemical and Western blot evaluation of ER and PR. Hormone levels were determined by radioimmunoassay. Isolated mammary epithelial cells were transplanted into cleared fat pads of 21-day-old female Swiss nu/nu mice or control congenic animals.

Results

MPA failed to induce mammary carcinomas or significant morphologic changes in the mammary glands of C57BL/6 mice. The expression of ER-α and PR isoform A in virgin mice was surprisingly much higher in BALB/c than in C57BL/6 mammary glands, and both receptors were downregulated in progestin-treated BALB/c mice (P < 0.05). PR isoform B levels were low in virgin control mice and increased after progestin treatment in both strains. ER-β expression followed a similar trend. No differences in hormone levels were found between strains. Surprisingly, the transplantation of the epithelial mammary gland cells of both strains into the cleared fat pads of Swiss (nu/nu) mice abolished the mammary gland morphologic differences and the ER and PR differences between strains.

Conclusion

C57BL/6 mammary glands are resistant to MPA-induced carcinogenesis and to hormone action. MPA and progesterone have different effects on mammary glands. Low ER-α and PR-A levels in untreated mammary glands may be associated with a low-risk breast cancer profile. Although we cannot at this time rule out the participation of other, untested factors, our findings implicate the stroma as playing a crucial role in the strain-specific differential hormone receptor expression and hormone responsiveness.     

Induction of mammary adenocarcinomas by medroxyprogesterone acetate in BALB/c female mice

In a previous paper we reported that medroxyprogesterone acetate (MPA) decreased the incidence of foreign body tumorigenesis in BALB/c mice but that mammary adenocarcinomas appeared in some of the females. The experiment was repeated in 245 virgin females as follows: (1) 40 mice treated with 40 mg of MPA depot s.c. every 2 months during a whole year; (2) 117 mice bearing a foreign body (FB) and treated with MPA; (3) 46 mice bearing a FB; (4) 42 non-treated mice. Mammary adenocarcinomas developed in 16/40 in group 1 and 30/117 in group 2; no mammary tumors appeared in either control groups. The tumors were infiltrating adenocarcinomas often affecting more than one mammary gland; metastases were occasionally observed. Animals killed after 1 year of MPA treatment presented deciduomas. MPA also decreased the incidence of FB-induced sarcomas, confirming previous results.     

Relationship between menopausal hormone therapy and risk of ductal, lobular, and ductal-lobular breast carcinomas.

Combined estrogen and progestin hormone therapy (CHT) increases breast cancer risk, but this risk varies by breast cancer type. Several studies indicate that CHT is more strongly related to lobular carcinoma risk than to ductal carcinoma risk, but these studies have been limited in their assessments of recency and duration of use, and none included a centralized pathology review. We conducted a population-based case-control study consisting of 324 lobular, 196 ductal-lobular, and 524 ductal cases diagnosed from 2000 to 2004 and 469 controls ages 55 to 74 years old. Tissue specimens were centrally reviewed for 83% of cases. Associations between hormone use and breast cancer risk were evaluated using polytomous logistic regression. Current CHT users had 2.7-fold [95% confidence interval (95% CI), 1.7-4.2] and 3.3-fold (95% CI, 2.0-5.7) elevated risks of lobular and ductal-lobular carcinomas, respectively, regardless of tumor stage, size, or nodal status. Elevations in risk were observed only among users of CHT for > or =3 years. Among ductal-lobular cases, CHT increased risk of tumors that were > or =50% lobular (odds ratio, 4.8; 95% CI, 2.1-11.1) but not tumors that were <50% lobular (odds ratio, 1.9; 95% CI, 0.9-4.1). Current CHT users for > or =3 years have a substantially increased risk of lobular carcinomas. Although lobular carcinomas are less common than ductal carcinomas ( approximately 16% versus 70% of all invasive breast cancers in the United States), this duration is shorter than the 5 years of use widely cited to be needed to confer an increased risk of breast cancer overall. Further studies focusing on the etiology of lobular carcinomas are needed.     

Mouse mammary tumors induced by medroxyprogesterone acetate: immunohistochemistry and hormonal receptors

Mammary adenocarcinomas induced by medroxyprogesterone acetate (MPA) in female BALB/c mice were investigated as to their morphology and immunohistochemistry and their content of steroid, prolactin (PRL), and epidermal growth factor (EGF) receptors. Histologically, these tumors were mainly of ductal origin, since hyperplastic alveolar nodules were observed only in 3 cases. No viral particles were encountered in electron microscopic studies. Estrogen and/or progesterone, PRL, and EGF receptors were detected in MPA-induced tumors, as well as in the occasional spontaneous mammary tumors of multiparous females. EGF was detected, by a radioimmunoassay, in the cystic fluid of 12 mammary adenocarcinomas. MPA treatment was found to induce uterine secretory changes, glandular cystic hyperplasia, and eventually deciduomas that stained strongly for desmin and to a lesser degree for vimentin, suggesting a muscular differentiation. Consequently, MPA-induced adenocarcinomas can be considered as ductal tumors that possess estrogen and/or progesterone, PRL, and EGF receptors. Whether MPA induces tumor growth directly via progesterone receptors remains to be investigated.     

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

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

Progesterone receptor expression in medroxyprogesterone acetate-induced murine mammary carcinomas and response to endocrine treatment

Using medroxyprogesterone acetate (MPA) as a carcinogen, we were able to induce in BALB/c female mice, several progestin-dependent mammary ductal carcinomas that regress completely with estrogen or antiprogestins and are maintained by serial transplantations in syngeneic mice. Progestin-independent variants were subsequently generated or appeared spontaneously. Based on their response to estrogen or antiprogestins, we subdivided them into responsive progestin-independent (R-PI) variants which regress completely and unresponsive progestin-independent (UR-PI) carcinomas which are resistant to both families of compounds. In this study we have investigated progesterone receptor (PR) expression in six responsive progestin-dependent, six R-PI, and three UR-PI tumors. Progestin-dependent and R-PI tumors disclosed a higher expression of the PR_A isoform as compared with PR_B, as well as an additional band of 78 kDa that was not detected in uterine tissue; all were down-regulated by progestins. UR-PI tumors expressed lower levels of all bands in western blots, but were highly reactive by immunohistochemistry. PR RNA expression was detected in both, UR-PI and R-PI tumors. PR binding was comparable in progestin-dependent and R-PI tumors. In the three UR-PI tumors, only 29/61 (48%) of the samples evaluated showed low binding levels, the rest were negative. This report is the first to describe in an experimental model of breast cancer the expression of PR isoforms and their distribution. Our results suggest the expression of functionally altered isoforms in a subgroup of mammary carcinomas, which may explain their lack of hormone response.     

Hormone dependence of a mouse mammary tumor line inducedin vivo by medroxyprogesterone acetate

Summary The administration of MPA to virgin female BALB/c mice led to the development of mammary adenocarcinomas, which in furtherin vivo transplants gave rise to both MPA-dependent and MPA-independent lines. In this paper we chose one of the MPA-dependent lines with high contents of estrogen (ER) and progesterone (PR) receptors, and were able to demonstrate that a) the growth of these tumors could be manipulated by the administration or the withdrawal of the hormonal supply;b) PR were down-regulated in MPA-treated mice; c) progesterone had the same stimulatory effect as MPA on tumor growth; d) tumors did not grow in estrogen-treated mice; e) tumor growth was much lower in males than in females; f) the presence of the ovaries had a positive influence on tumor growth, even in the presence of MPA; g) the withdrawal of progestin pellets in ovariectomized mice usually led to complete remissions followed by regrowth of the tumors after several weeks; and h) the regrowing tumors maintained their steroid receptor pattern and (in 3 out of 4 cases) their hormone-dependent behavior in further passages.Abbreviations DMBA 7,12 dimethylbenz(a)anthracene - EB estradiol benzoate - ER estrogen receptors - MPA medroxyprogesterone acetate - P progesterone - PR progesterone receptors     

Mammary adenocarcinomas induced by medroxyprogesterone acetate: hormone dependence and EGF receptors of BALB/c in vivo sublines

Mammary adenocarcinomas were induced by medroxy-progesterone acetate (MPA) in female BALB/c mice. From 5 primary tumors, 9 different sublines were established by s.c. transplantation into syngeneic female mice; these developed after a long latent period (4-12 months). Each subline was transplanted both into 4 mice treated with 40mg of MPA depot (s.c. contralaterally to the tumor inoculum) and into 4 non-treated mice. Of the 9 sublines, 6 proved to be hormone-dependent (MPA-D) and 3 hormone-independent or autonomous (MPA-I). However, even the autonomous lines, when treated with MPA, showed a slight increase in growth. All MPA-D lines had a high content of ER (20-254 fmoles/mg of protein), PR (63-710), PRL-R (44-74) and low or non-detectable EGF-R. Of the 3 MPA-I sublines that were studied, 2 showed a high content of ER (16-125), PR (27-708), PRL-R (19-70) and EGF-R (29-65) while the other one had a low content of ER (0-36), PR (0-13), no EGF-R and moderate PRL-R (15-52). Spontaneous mammary tumors of BALB/c and C3H origin, which also showed an MPA-I pattern of tumor growth, had high levels of EGF-R. We postulate that MPA has a direct effect on mammary tumor cells in MPA-D lines and that the expression of EGF-R is correlated with an autonomous pattern of growth.     

Apigenin prevents development of medroxyprogesterone acetate-accelerated 7,12-dimethylbenz(a)anthracene-induced mammary tumors in Sprague-Dawley rats

The use of progestins as a component of hormone replacement therapy has been linked to an increase in breast cancer risk in postmenopausal women. We have previously shown that medroxyprogesterone acetate (MPA), a commonly administered synthetic progestin, increases production of the potent angiogenic factor vascular endothelial growth factor (VEGF) by tumor cells, leading to the development of new blood vessels and tumor growth. We sought to identify nontoxic chemicals that would inhibit progestin-induced tumorigenesis. We used a recently developed progestin-dependent mammary cancer model in which tumors are induced in Sprague-Dawley rats by 7,12-dimethylbenz(a)anthracene (DMBA) treatment. The flavonoid apigenin, which we previously found to inhibit progestin-dependent VEGF synthesis in human breast cancer cells in vitro, significantly delayed the development of, and decreased the incidence and multiplicity of, MPA-accelerated DMBA-induced mammary tumors in this animal model. Whereas apigenin decreased the occurrence of such tumors, it did not block MPA-induced intraductal and lobular epithelial cell hyperplasia in the mammary tissue. Apigenin blocked MPA-dependent increases in VEGF, and suppressed VEGF receptor-2 (VEGFR-2) but not VEGFR-1 in regions of hyperplasia. No differences were observed in estrogen or progesterone receptor (ER/PR) levels, or the number of estrogen receptor-positive cells, within the mammary gland of MPA-treated animals administered apigenin, MPA-treated animals, and placebo treated animals. However, the number of progesterone receptor-positive cells was reduced in animals treated with MPA or MPA and apigenin compared with those treated with placebo. These findings suggest that apigenin has important chemopreventive properties for those breast cancers that develop in response to progestins.     

Characterization of Mammary Plaques in DDD Mice Congenic for Mtv-2 Gene, DDD/1-Mtv-2/Mtv-2

DDD/1 mice free from exogenous mouse mammary tumor virus (MMTV) do not develop any neoplastic mammary lesions. In GR mice, the expression of Mtv-2 , an endogenous proviral MMTV, leads to 100% incidence of mammary ductal hyperplasias and tumors. An Mtv-2 congenic line, DDD/1- Mtv-2 / Mtv-2 , was established by introducing Mtv-2 from GR into DDD/1 to elucidate its function. Development of mammary plaques (MPQ) characterized by ductal hyperplasias was investigated in 152 congenic females on day 17 to 19 of the first pregnancy. The incidence of MPQ was 48.0% and most MPQ-positive mice (75.3%) had only one MPQ. Generally, MPQ were small in size: the diameter was as small as ≦3 mm in 77.6% of them. Of 84 MPQ implanted into intact fat pads, 43 (51.2%), 38 (45.2%) and 3 (3.6%) showed undetectable, pregnancy-dependent and autonomous growths; respectively when the hosts underwent pregnancy. Almost all MPQ produced normal-appearing ductal-alveolar outgrowths in mammary epithelium-divested or cleared fat pads of virgins. MPQ implanted into cleared fat pads were very similar to normal mammary glands in the responses to progesterone (P) and estradiol (E) alone or in combination except for association of ductal hyperplasias in 4 of 12 MPQ under E+P treatment. These findings revealed the preneoplastic nature of MPQ. Exogenous MMTV proviruses were demonstrated in all MPQ. The int-2 DNA rearrangement was found in 2 of 10 MPQ but in none of 9 mammary carcinomas and the int-1 DNA rearrangement in none of 10 MPQ but in 5 of 10 carcinomas. It is thus likely that the Mtv-2 gene participates in a very early stage of mammary tumorigenesis not directly but indirectly through insertion mutation of host genes, while the cellular oncogenes, int-2 and int-1 , may contribute to preneoplastic transformation of mammary epithelium and progression from preneoplastic to more malignant states, respectively.     

COX-2 is expressed in human pulmonary, colonic, and mammary tumors

BACKGROUND: The cyclooxygenase (COX) enzyme catalyzes the formation of prostaglandins, which can affect cell proliferation and alter the response of the immune system to malignant cells. The inducible form of COX, COX-2, has been shown to be important in carcinogenesis. METHODS: The authors studied COX-1 and -2 expression in 20 tumors of the lung, colon, and breast (60 total) by using commercially available monoclonal and polyclonal antibodies on formalin fixed, paraffin embedded tissue. Our evaluation also included seven carcinoma-associated colonic adenomas and 10 mammary ductal carcinomas in situ (DCIS). Quantitation of immunoreactivity was accomplished using an immunohistochemical scoring system that approximates the use of image analysis-based systems. RESULTS: Ninety percent of lung tumors (squamous cell carcinomas and adenocarcinomas), 71% of colon adenocarcinomas and 56% of breast tumors (DCIS and infiltrating ductal and lobular carcinomas) expressed COX-2 at a moderate to strong level, which was significantly different from the negligible expression in distant nonneoplastic epithelium (controls; P < 0.0001). Poorly differentiated histologic features were correlated with low COX-2 expression overall, especially in colon carcinomas. Among breast carcinomas, DCIS was more likely to express COX-2 than invasive carcinomas. Adenomatous colonic epithelium showed moderate COX-2 expression, as did adjacent nonneoplastic epithelium. COX-1 immunoreactivity was essentially weak to moderate in all tissues evaluated. CONCLUSIONS: COX-2 expression is upregulated in well and moderately differentiated carcinomas of the lung, colon, and breast whereas COX-1 appears to be constitutively expressed at low levels. A possible COX-2 paracrine effect is suggested by moderate immunoreactivity in adjacent nonneoplastic epithelium.     

Fluorescentin situ hybridization assessment of chromosome 8 copy number in breast cancer

Summary Conventional cytogenetics of breast and other solid tumors has been hampered by a number of factors. An analysis of breast tumor tissues was therefore undertaken using fluorescentin situ hybridization (FISH). A total of 34 specimens were analyzed using a chromosome 8-specific -satellite probe. Various approaches were tested and compared. Among 30 informative samples, 11 infiltrating ductal carcinomas, not otherwise specified (NOS), 5 ductal carcinomasin situ, 5 lobular carcinomas, 3 papillary carcinomas, and 6 benign lesions were studied. Of the 11 cases of infiltrating ductal carcinomas (NOS) analyzed, four cases showed 3 signals, one case showed 4 signals, and the rest showed 2 signals. Of the 5 cases of ductal carcinomain situ samples, 1 showed 3 signals and the other 4 cases showed 2 signals. All cases of lobular carcinomas, papillary carcinomas, and benign lesions showed 2 signals. We inferred from these data that 36% of the infiltrating ductal carcinomas (NOS) were trisomic and 9% were tetrasomic, whereas 20% of the ductal carcinomasin situ were trisomic. All samples from lobular carcinomas, papillary carcinomas, and the benign lesions were disomic. From our preliminary data, it can further be concluded that a subset of breast cancer is characterized by chromosome 8 trisomy. These data are consistent with an ever-increasing database on the association of chromosomal 8 trisomy with other cancers such as leukemia, lymphoma, prostate cancer, ovarian carcinoma, salivary gland tumor, malignant melanoma, desmoid tumors, and recently gestational trophoblastic disease. It is also noted that the ability to analyze formalin-fixed, paraffin-embedded archival material will enable a more comprehensive cytogenetic study of breast cancer than is currently available.     

Effect of medroxyprogesterone acetate (MPA) and serum factors on cell proliferation in primary cultures of an MPA-induced mammary adenocarcinoma

Summary The effect of progesterone (Pg), medroxyprogesterone acetate (MPA), estradiol (E₂), dihydrotestosterone (DHT) and dexamethasone (DEXA) was studied on thein vitro growth rate of a progestin-dependent (PD), estrogen-sensitive mammary tumor line originated in an MPA-treated BALB/c mouse (C4-HD), and on its estrogen-resistant variant (C4-HDR). The specificity of hormone action was further investigated using the anti-hormones RU-486 and hydroxyflutamide (FLU). Cell growth was evaluated in epithelial and fibroblastenriched cultures using³H-thymidine and/or autoradiography and immunocytochemistry. The results indicate that cell growth is directly stimulated by MPA and Pg at concentrations ranging from 10^–11 to 10^–7 M. RU486 prevented MPA-induced stimulation in concentrations 10 to 100 fold lower than those of MPA. When used alone, it inhibited cell proliferation only in concentrations higher than 10^–11 M. At nM concentrations, neither DEXA nor DHT stimulated³H-thyrnidine uptake except DEXA at 100 nM. MPA-induced stimulation was not reverted by micromolar concentrations of FLU. As for E₂ (10^–7–10^–9 M) it prevented MPA stimulation only in cultures of estrogen-sensitive tumors. Progesterone receptors (PR) (475 ± 115 fmoles/10⁵ cells, n = 5) and estrogen receptors (ER) (ND-115 fmoles/10⁵ cells, n = 5) were detected only in epithelial-enriched cultures. Serum from 7 day-MPA-treated mice induced a significant increase of³H-thymidine uptake; an increase was also obtained with serum from untreated ovariectomized animals to which 1 nM-100 nM concentrations of MPA had been added. The stimulatory effect of the exogenous MPA was much lower than that of the serum obtained from MPA-treated animals.It is concluded that MPA stimulates cell growth of primary cultures of MPA-induced PD tumors via PR. The results provide support for a direct effect of MPA which may be mediated or potentiated by serum factors.     

Influence of caffeine consumption on carcinomatous and normal mammary gland development in mice

The influence of caffeine consumption on the development of 7,12-dimethylbenz(a)anthracene-induced mammary carcinomas in BD2F1 female mice and spontaneous mammary carcinomas in nulliparous C3H mice was examined. Caffeine (250 and 500 mg/liter of drinking water) was administered to BD2F1 mice commencing 1 week after a series of 6 weekly 7,12-dimethylbenz(a)anthracene intubations, until experiment termination. Caffeine was administered to C3H mice (via drinking water) commencing at 8 weeks of age to experiment termination. In BD2F1 mice receiving 250 and 500 mg of caffeine, mammary carcinoma multiplicity (number of mammary carcinomas/mouse) was increased by 20 and 40%, respectively. In C3H mice receiving 250 and 500 mg caffeine, mammary carcinoma multiplicity was increased by 13 and 117%, respectively. In both BD2F1 and C3H mice, the higher dose level of caffeine resulted in a significant (P less than 0.05) increase in mammary carcinoma multiplicity. Caffeine consumption did not significantly effect the percentage of mice bearing mammary carcinomas or the mean latency period of mammary tumor appearance. In a second series of studies, the influence of caffeine consumption on mammary gland development in female BALB/c mice was assessed in vivo and in vitro (organ culture). In mice consuming caffeine (500 mg/liter of drinking water), mammary gland development was significantly (P less than 0.05) increased compared to control mice; this difference in mammae development was more conspicuous in mice treated with mammotropic hormones. In the organ culture studies, mammary glands derived from caffeine (500 mg/liter of drinking water) consuming BALB/c mice were more responsive in vitro to a mammotropic hormonal developmental growth stimulus than were mammae derived from control mice (P less than 0.05). These results provide evidence that caffeine consumption can enhance mammary tumorigenesis in C3H and carcinogen-treated BD2F1 female mice and, in addition, enhance developmental growth of the normal female mouse (BALB/c) mammary gland.     

Interactions between progestins and heregulin (HRG) signaling pathways: HRG acts as mediator of progestins proliferative effects in mouse mammary adenocarcinomas

The present study addressed links between progestin and heregulin (HRG) signaling pathways in mammary tumors. An experimental model of hormonal carcinogenesis, in which the synthetic progestin medroxyprogesterone acetate (MPA) induced mammary adenocarcinomas in female Balb/c mice, was used. MPA induced an in vivo up-regulation of HRG mRNA expression in progestin-dependent (HD) tumor lines. Mammary tumor progression to a progestin-independent (HI) phenotype was accompanied by a high constitutive expression of HRG. The HRG message arose from the tumor epithelial cells. Primary cultures of malignant epithelial cells from a HD tumor line were used to investigate HRG involvement on cell proliferation. HRG induced a potent proliferative effect on these cells and potentiated MPA mitogenic effects. Blocking endogenous HRG synthesis by antisense oligodeoxynucleotides (ASODNs) to HRG mRNA inhibited MPA-induced cell growth, indicating that HRG acts as a mediator of MPA-induced growth. High levels of ErbB-2 and ErbB-3 expression and low ErbB-4 levels were found in HD cells. Treatment of these cells with either MPA or HRG resulted in tyrosine phosphorylation of both ErbB-2 and ErbB-3. Furthermore, both HRG and MPA proliferative effects were abolished when cells were treated with ASODNs to ErbB-2 mRNA, providing evidence for a critical role of ErbB-2 in HRG-induced growth. Finally, blocking type I insulin-like growth factor receptor (IGF-IR) expression with ASODN resulted in the complete inhibition of HRG proliferative effect, demonstrating that a functional IGF-IR is required for HRG mitogenic activity. These results provide the first evidence of interactions between progestins and HRB/ErbB signal transduction pathways in mammary cancer and the first demonstration that IGF-IR is required for HRG proliferative effects.