CCAAT/Enhancer binding protein delta (c/EBPdelta) regulation and expression in human mammary epithelial cells: I. "Loss of function" alterations in the c/EBPdelta growth inhibitory pathway in breast cancer cell lines

"Loss of function" alterations in growth inhibitory signal transduction pathways are common in cancer cells. In this study, we show that growth arrest (GA) treatments--serum and growth factor withdrawal and growth inhibitory IL-6 family cytokines (Interleukin-6 and Oncostatin M (OSM))--increase STAT3 phosphorylation (pSTAT3), increase CCAAT enhancer binding protein delta (C/EBPdelta) gene expression and induce GA of primary, finite-lifespan human mammary epithelial cells (HMECs), and immortalized breast cell lines (MCF-10A and MCF-12A). In contrast, serum and growth factor withdrawal from human breast cancer cell lines (MCF-7, SK-BR-3, T-47D, and MDA-MB-231) for up to 48 h induced a relatively modest increase in pSTAT3 levels and C/EBPdelta gene expression and resulted in varying levels of GA. In most breast cancer cell lines, IL-6 family cytokine treatment increased pSTAT3 levels and C/EBPdelta gene expression, however, growth inhibition was cell line dependent. In addition to "loss of function" alterations in growth inhibitory pathways, breast cancer cell lines also exhibit "gain of function" alterations in growth signaling pathways. The Akt growth/ survival pathway is constitutively activated in T-47D and MCF-7 breast cancer cells. The Akt inhibitor LY 294,002 significantly enhanced T-47D growth inhibition by serum and growth factor withdrawal or IL-6 family cytokine treatment. Finally, we show that activation of the pSTAT3/C/EBPdelta growth control pathway is independent of estrogen receptor status. These results demonstrate that "loss of function" alterations in the pSTAT3/C/EBPdelta growth inhibitory signal transduction pathway are relatively common in human breast cancer cell lines. Defective activation of the pSTAT3/ C/EBPdelta growth inhibitory signal transduction pathway, in conjunction with constitutive activation of the Akt growth stimulatory pathway, may play a synergistic role in the etiology or progression of breast cancer.     

tPA/gGH双基因转染山羊乳腺上皮细胞表达分析

人组织纤溶酶原激活剂(tissue-type plasminogen activator,tPA)是一种被广泛应用于临床的溶栓药物。双基因共整合入生物体内能够产生协同作用,从而提高目的基因的表达水平。但是目前,利用gGH基因与tPA基因共整合以期提高tPA表达水平的相关研究较少。为筛选获得tPA高表达的tPA/gGH双基因整合的单克隆转基因山羊乳腺上皮细胞株,本研究以β-casein基因作为调控序列,构建乳腺特异性表达载体PCL25/gGH,并通过电转染将tPA和gGH双基因共转染山羊乳腺上皮细胞;通过G418筛选获得抗性细胞株,经PCR检测获得转基因单克隆细胞株;利用催乳素诱导tPA表达,收集48 h后细胞诱导液进行ELISA()和Western blotting检测并分析其tPA表达水平。结果表明,共获得142株抗性单克隆细胞,其中有53株tPA单基因整合细胞株,34株tPA/gGH双基因整合细胞株,双基因整合率达23.9%(34/142)。共检测出29株细胞能够表达tPA,其中单基因表达细胞为12株,表达率为22.6%(12/53);双基因表达细胞为17株,表达率为50.0%(17/34);且单基因细胞表达tPA含量为7.5~52.0μg/mL,而双基因细胞表达tPA含量为40~360μg/mL,明显高于单基因表达水平。本研究通过电转染的方式成功获得了tPA/gGH双基因整合的单克隆山羊乳腺上皮细胞株,并证明双基因整合的细胞株表达tPA水平明显提高,为后期制备高表达转基因山羊奠定了基础。     

Expression of CCAAT/enhancer binding protein C/EBPalpha, beta and delta in rat adipose stromal-vascular cells in vitro.

Stromal-vascular (S-V) cells from rat inguinal fat depots were isolated and cultured in medium containing fetal bovine serum (FBS) and differentiated in defined medium until lipid accumulation was apparent. C/EBPalpha, beta and delta levels were evaluated for different growth conditions and at different times using Western blots. Immediately after isolation C/EBPalpha, beta and delta could not be detected in S-V cells. After seeding for 24 h in Dulbecco's modified Eagle's medium (DMEM) with FBS, C/EBPalpha, beta and delta could all be detected. Cells at day 1 of culture in insulin, transferrin, triiodothyronine and selenium (ITTS) had increased levels of C/EBPalpha and continued steady high levels to day 6 of culture. Cultures grown in DMEM alone, with no ITTS, showed C/EBPalpha levels similar to ITTS cultures at day 1 and day 3; however, levels diminished after day 3. DMEM cultures also showed lipid accumulation at day 6; however, the number of cells and the amount of lipid cell were reduced from levels observed in ITTS cultures. C/EBPbeta was expressed uniformly throughout the culture period in either DMEM or ITTS cultures while C/EBPdelta expression was higher with DMEM treatment than with ITTS. Treatment of 2 day DMEM cultures with FBS increased levels of C/EBPbeta and delta but significantly reduced levels of C/EBPalpha. Immunocytochemical analysis of S-V cells at day 1 of culture showed a similar percentage of cells stained in DMEM cultures and ITTS cultures. However, by day 6 of culture the percentage of cells staining positively for C/EBPalpha in DMEM had been reduced by one half while in ITTS the percent positive cells remained about the same. Our results indicate that ITTS is not necessary for the induction of C/EBPalpha and accumulation of lipid in S-V cells. However, ITTS is responsible for maintaining C/EBPalpha and enhanced lipid accumulation. Because C/EBPalpha, beta and delta expression occurs very early in cell culture and C/EBPalpha and delta expression continues to increase in DMEM without any apparent inducing agents, our results suggest that these factors may be expressed by the same cells in vivo before being placed in culture. Thus, a large fraction of S-V cells may be further along in the differentiation program than 3T3 cells are when they begin differentiation.     

8-Hydroxyquinoline inhibits iNOS expression and nitric oxide production by down-regulating LPS-induced activity of NF-kappaB and C/EBPbeta in Raw 264.7 cells

In activated macrophage, large amounts of nitric oxide (NO) are generated by inducible nitric oxide synthase (iNOS), resulting in acute or chronic inflammatory disorders. In Raw 264.7 cells stimulated with lipopolysaccharide (LPS) to mimic inflammation, 8-hydroxyquinoline (8HQ) inhibited the LPS-induced expression of both iNOS protein and mRNA in a parallel dose-dependent manner. 8HQ did not enhance the degradation of iNOS mRNA. To investigate the mechanism by which 8HQ inhibits iNOS gene expression, we examined the activation of MAP kinases in Raw 264.7 cells. We did not observe any significant change in the phosphorylation of MAPKs between LPS alone and LPS plus 8HQ-treated cells. Moreover, 8HQ significantly inhibited the DNA-binding activity of nuclear factor-kappaB (NF-kappaB) and CCAAT/enhancer-binding protein beta (C/EBPbeta), but not activator protein-1 and cAMP response element-binding protein. Taken together, these results suggest that 8HQ acts to inhibit inflammation through inhibition of NO production and iNOS expression through blockade of C/EBPbeta DNA-binding activity and NF-kappaB activation.