Introduction Diverse microarray and sequencing technologies have been widely used to characterise the molecular changes in malignant epithelial cells in breast cancers. Such gene expression studies to identify markers and targets in tumour cells are, however, compromised by the cellular heterogeneity of solid breast tumours and by the lack of appropriate counterparts representing normal breast epithelial cells.

Two experiments were conducted to assess the endogenous fraction of purine derivative (PD) excretion, urinary recovery, and intestinal digestibility of purines in Nellore heifers. For both experiments, 8 Nellore heifers fitted with ruminal and abomasal cannulas were allocated to two 4 × 4 Latin squares. The diets were based on corn silage and concentrate (60 and 40% DM basis, respectively); feces and urine samples were obtained by total collection, and abomasal DM flow was estimated using indigestible NDF as an internal marker. In Exp. I, 4 of the 8 heifers (BW 258 ± 20 kg) were also fitted with ileal cannula. The planned treatments were 4 different DMI: 1.2, 1.6, 2.0, and 2.4% of BW (DM basis). The endogenous losses and purine recovery as urinary PD were estimated using linear regression between daily urinary PD excretion (Y) and daily abomasal flow of purine bases (X), expressed in millimoles per kilogram of BW(0.75). In Exp. II, the same 8 Nellore heifers (BW of 296 ± 15 kg) were fed at 1.37% BW (DM basis). The treatments were the infusion of purines (RNA from torula yeast, type VI, Sigma) into the abomasum in increasing amounts (0, 33, 66, and 100 mmol/d). All statistical analyses were performed using the PROC MIXED procedure in SAS. In Exp. I, the DMI range was 1.16 to 1.84% of BW and did not affect (P > 0.05) the apparent RNA digestibility in the small intestine, which had a mean of 75.6%, and a true digestibility of 93.0%. The mean ratio of the N-RNA to the total-N in the ruminal bacteria was 0.137. The daily urinary PD excretion (Y, mmol/kg of BW(0.75)) was a function of RNA flow in the abomasum (X, mmol/kg of BW(0.75)): Y = 0.860X + 0.460, where 0.860 and 0.460 were the PD recovery of purines and the endogenous fraction (in mmol/kg of BW(0.75)), respectively. In Exp. II, the daily urinary PD excretion was a function of RNA flow in the abomasum: Y = 0.741X + 0.301, where 0.741 and 0.301 were the recovery of PD in urine of infused purines and the endogenous losses (in mmol/kg of BW(0.75)), respectively. In conclusion, our data suggest that in Nellore heifers the respective values of endogenous PD excretion (mmol/kg of BW(0.75)), urinary recovery of the purines absorbed in the abomasum, and true digestibility of RNA in the small intestine were 0.30, 0.80, and 0.93.

IntroductionBreast cancer currently accounts for more than one-quarter of all female cancers and, despite the great progress in treatment observed in the past few years, the need for identification of new gene targets that can be used for diagnosis, prognosis and therapy is evident. A previous study identified the transcription factor NR4A1 as a gene upregulated in primary breast cancer compared with normal tissue by microarray analysis and sequencing technologies. The purpose of the study was to identify the role of NR4A1 in normal mammary epithelial and breast cancer cell biology.MethodsNR4A1 expression in breast tumours was assessed by semiquantitative and real-time PCR using RNA from normal and tumour samples or breast cancer cell lines. Immunohistochemistry on tissue microarrays was performed to check NR4A1 protein expression in breast tumours. MCF-10A and 226L normal mammary epithelial cells as well as the tumour lines PMC42, ZR-75-1 and MDA-MB-231 were transduced with full-length NR4A1, and the ability of NR4A1-overexpressing cells to migrate was tested using scratch wound or transwell migration assays. Proliferation was measured using the MTT and BrdU assays, while apoptosis was determined by the Annexin V assay. The ability of the cells to adhere to extracellular matrix was tested by adhesion assays and integrin cell surface expression was measured by flow cytometry. Activation of the FAK as well as ERK1/2 and PI3K pathways was checked by western blotting.ResultsBreast tissue microarray analysis showed NR4A1 expression in primary tumours, which was reduced in higher grade and metastatic tumours. Ectopic expression of NR4A1 in MCF-10A, 226L, PMC42 and ZR-75-1 cells led to reduced ability of the cells to migrate, while no differences were observed in their proliferation and apoptotic index. NR4A1 expression altered the ability of the MCF-10A cells to adhere to the extracellular matrix and affected cell surface expression of integrins.ConclusionsNR4A1 acts as an antimigratory factor in two normal mammary epithelial and two breast cancer cell lines tested. It is therefore possible that NR4A1 acts as an antimigratory factor in breast tumours, and further studies should be conducted to understand the mechanisms involved.