RESUMO
We have developed an experimental model of mammary carcinogenesis in which the administration of medroxyprogesterone acetate (MPA) to female BALB/c mice induces progestin-dependent ductal metastatic mammary tumors with high levels of estrogen receptor (ER) and progesterone receptor (PR). Through selective transplants in untreated mice, we have obtained progestin-independent variants, still expressing high levels of ER and PR. Primary cultures of the MPA-induced carcinomas C4-HD and C7-HI were set up, and after 3-4 months, several different cell lines were obtained. Four of these, MC4-L1, MC4-L2, MC4-L3, and MC4-L5 were established from C4-HD and a fifth, MC7-L1, from C7-HI. All cells were of epithelial origin, as demonstrated by electron microscopy and by immunocytochemical identification of cytokeratin and cadherin. In vitro MC4-L1, MC4-L3, and MC4-L5 showed a typical epithelial morphology; when transplanted in vivo, they originated metastatic carcinomas with different degrees of differentiation. MC4-L2 and MC7-L1 deviated from the standard epithelial picture; they disclosed a spindle-shaped morphology in vitro and in vivo gave rise to a biphasic spindle cell/tubular carcinoma and an anaplastic carcinoma, respectively; both lines gave rise to metastases. This differential morphology correlated with a higher degree of aggressiveness, as compared with MC4-L1, MC4-L3, and MC4-L5. ERs and PRs were detected by binding, immunocytochemistry, and Western blot. In vitro, MC4-L2 and MC7-L1 were stimulated by MPA (nM to microM) and 17beta-estradiol (nM and 10 nM); no significant stimulation was observed in MC4-L1, MC4-L3, and MC4-L5 under the same experimental conditions. In vivo, MPA significantly stimulated tumor growth in all epithelioid lines but not in MC4-L2 and MC7-L1. A progestin-dependent growth pattern was confirmed for MC4-L1, MC4-L3, and MC4-L5 in successive transplants, whereas MC4-L2 and MC7-L1 behaved as progestin independent. This is the first description of mouse mammary carcinoma cell lines expressing ER and PR. The different in vitro hormone responses as compared with in vivo and the differential effects of 17beta-estradiol in the parental tumors and in cell lines render these lines useful tools for the in vitro and in vivo study of hormone regulation of tumor growth and metastases.
Assuntos
Carcinoma Ductal de Mama/patologia , Neoplasias Mamárias Experimentais/patologia , Neoplasias Hormônio-Dependentes/patologia , Células Tumorais Cultivadas , Animais , Carcinoma Ductal de Mama/metabolismo , Diferenciação Celular/fisiologia , Divisão Celular/efeitos dos fármacos , Divisão Celular/fisiologia , Modelos Animais de Doenças , Estradiol/farmacologia , Feminino , Imuno-Histoquímica , Neoplasias Mamárias Experimentais/metabolismo , Acetato de Medroxiprogesterona/farmacologia , Camundongos , Camundongos Endogâmicos BALB C , Microscopia Eletrônica , Transplante de Neoplasias , Neoplasias Hormônio-Dependentes/metabolismo , Receptor ErbB-2/metabolismo , Receptores de Estrogênio/metabolismo , Receptores de Progesterona/metabolismoRESUMO
The copper hazard was evaluated by means of a 7-day toxicity test with Bufo arenarum embryos. The LC50 and LC10 values from 24 to 168 h of exposure were approx. 0.085 and 0.05 mg Cu2+/1, respectively, while the LC90 resulted in 0.155 mg Cu2+/1 but in this case from 96 h onwards the LC90 diminished up to approx. 0.105 mg Cu2+/1. These data plotted as Toxicity Profiles (TOP) provide a better understanding of concentration and time-dependent thresholds. For instance, exposure threshold occurs within the first 24 h of treatment while for concentration thresholds LC10 and LC90 seem to be more meaningful than LC50 because the S.D. of this last value is overlapping those of LC10 and LC90 for most of the exposure period evaluated. Toxicity data corresponds to a pH of 6.8 which is normal for the maintaining media. Combined treatments of copper and zinc point out a beneficial effect of zinc proportional to the zinc concentration in the maintaining media, e.g. 100% of protection was achieved with 30 mg Zn2+/1 for a copper concentration exerting 90% of mortality. The presence of Cu2+ did not enhance Zn2+ toxicity. The results are discussed in terms of water quality criteria for wildlife and human health protection purposes.
Assuntos
Cobre/administração & dosagem , Cobre/toxicidade , Embrião não Mamífero/efeitos dos fármacos , Poluentes Químicos da Água/administração & dosagem , Poluentes Químicos da Água/toxicidade , Zinco/administração & dosagem , Animais , Bufo arenarum/embriologia , Relação Dose-Resposta a Droga , Interações Medicamentosas , Ecossistema , Saúde Ambiental , Feminino , Água Doce , Humanos , Dose Letal Mediana , Masculino , Concentração Máxima PermitidaRESUMO
Two thirds of breast cancers express estrogen receptors (ER). ER alpha (ERα) mediates breast cancer cell proliferation, and expression of ERα is the standard choice to indicate adjuvant endocrine therapy. ERbeta (ERß) inhibits growth in vitro; its effects in vivo have been incompletely investigated and its role in breast cancer and potential as alternative target in endocrine therapy needs further study. In this work, mammary epithelial (EpH4 and HC11) and breast cancer (MC4-L2) cells with endogenous ERα and ERß expression and T47-D human breast cancer cells with recombinant ERß (T47-DERß) were used to explore effects exerted in vitro and in vivo by the ERß agonists 2,3-bis (4-hydroxy-phenyl)-propionitrile (DPN) and 7-bromo-2-(4-hydroxyphenyl)-1,3-benzoxazol-5-ol (WAY). In vivo, ERß agonists induced mammary gland hyperplasia and MC4-L2 tumour growth to a similar extent as the ERα agonist 4,4',4''-(4-propyl-(1H)-pyrazole-1,3,5-triyl) trisphenol (PPT) or 17ß-estradiol (E2) and correlated with higher number of mitotic and lower number of apoptotic features. In vitro, in MC4-L2, EpH4 or HC11 cells incubated under basal conditions, ERß agonists induced apoptosis measured as upregulation of p53 and apoptosis-inducible factor protein levels and increased caspase 3 activity, whereas PPT and E2 stimulated proliferation. However, when extracellular signal-regulated kinase 1 and 2 (ERK ½) were activated by co-incubation with basement membrane extract or epidermal growth factor, induction of apoptosis by ERß agonists was repressed and DPN induced proliferation in a similar way as E2 or PPT. In a context of active ERK ½, phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K)/RAC-alpha serine/threonine-protein kinase (AKT) signalling was necessary to allow proliferation stimulated by ER agonists. Inhibition of MEK ½ with UO126 completely restored ERß growth-inhibitory effects, whereas inhibition of PI3K by LY294002 inhibited ERß-induced proliferation. These results show that the cellular context modulates ERß growth-inhibitory effects and should be taken into consideration upon assessment of ERß as target for endocrine treatment.