Covalent binding of 17β-estradiol and retinoic acid to proteins in the human breast cancer cell line MCF-7

Summary Both retinoic acid and 17β-estradiol formed covalent bonds with proteins of the human breast cancer cell line MCF-7. Two-dimensional gel patterns of the labeled proteins were unique for each ligand. There were four major retinoylated proteins in MCF-7 consisting of two doublets with molecular masses of 37 kDa and 20 kDa. These proteins were designated 37a, 37b, 37c, and 20d. The extent of retinoylation was very low in a 55 kDa protein that we previously identified in the human myeloid leukemia cell line HL60 [Takahashi, N. and Breitman, T. R. (1989) J. Biol. Chem. 264, 5159–5163]. These results indicated that the protein substrates for retinoylation may vary among cell-types. About 10 proteins were labeled from 17β-estradiol. Two of these proteins had mobilities that were identitied to the retinoylated proteins 37a and 20c. These results indicate that in MCF-7 cells there are two proteins that can be retinoylated and labeled from estradiol. The demonstration that some ligands of the steroid/thyroid receptor family are covalently linked to cellular proteins suggests new mechanisms for the many effects of these agents on cells. This study is the first report showing that estradiol or one of its metabolic products covalently binds to proteins in the human breast cancer cell line MCF-7. Two of the proteins labeled from radioactive estradiol comigrate with proteins labeled from radioactive retinoic acid. These results suggest new mechanisms of action for the steroid and thyroid hormones. EDITOR’S STATEMENT This study is the first report showing that estradiol or one of its metabolic products covalently binds to proteins in the human breast cancer cellline MCF-7. Two of the proteins labeled from radioactive estradiol comigrate with proteins labeled from radioactive retinoic acid. These results suggest new mechanisms of action for the steroid and thyroid hormones.     

Elimination of cells with abnormal nuclei in human epidermoid carcinoma cell line A431 by α-lipoic acid

Skin is usually exposed to adverse environmental conditions that may cause pathological cell proliferation and malignant transformation. Antioxidants are able to affect these processes and eliminate transformed cells. The purpose of this work was to investigate the effect of α-lipoic acid (ALA) on human epidermoid carcinoma cell line A431. It was found that 100, 200, 300, 500 μM ALA added for 24, 48, 72 h inhibited cell proliferation and stimulated apoptosis. Most dying cells have abnormal nuclei (micronuclei, giant nuclei, nuclei with buds). Electron microscopy showed that cells with normal nuclear phenotypes after treatment with 200 μM ALA for 48 h had ultrastructural organizations typical for control cells. Thus, α-ALA not only triggers the apoptosis of carcinoma cells, but it may also activate the mechanism for eliminating cells with abnormal numbers of chromosomes.     

Studies of α-protein in human cell cultures

Summary α-Protein growth fraction (AGF) eliminates the 60- to 90-day adaptive phase required to establish actively growing cultures of HeLa (Gey), human heart (Girardi), KB (Eagle) and other established cell lines in serum-free chemically defined medium A₃. AGF is effective at less than 0.4 μg per ml. By using the procedures described in the text, it is possiblee to culture HeLa cells is very simple media such as Eagle's basal medium. The properties of AGF are such that it may be adsorbed on glass or plastic flasks. Glass flasks treated with AGF retain full activity after washing with acetone, and treatment with ethyl ether and chemically defined medium. Adsorbed AGF is destroyed by trypsin. AGF can detoxify protamines, polylysines or histones. It will reverse the aggregation response induced by adding complexes composed of carboxymethylcellulose (CMC) and basic proteins. The results support the contention that highly adsorptive AGF functions at the cell surface and is capable of modifying the response of the cell to its environment.     

Metabolic flux rearrangement in the amino acid metabolism reduces ammonia stress in the α1-antitrypsin producing human AGE1.HN cell line

This study focused on metabolic changes in the neuronal human cell line AGE1.HN upon increased ammonia stress. Batch cultivations of α(1)-antitrypsin (A1AT) producing AGE1.HN cells were carried out in media with initial ammonia concentrations ranging from 0mM to 5mM. Growth, A1AT production, metabolite dynamics and finally metabolic fluxes calculated by metabolite balancing were compared. Growth and A1AT production decreased with increasing ammonia concentration. The maximum A1AT concentration decreased from 0.63g/l to 0.51g/l. Central energy metabolism remained relatively unaffected exhibiting only slightly increased glycolytic flux at high initial ammonia concentration in the medium. However, the amino acid metabolism was significantly changed. Fluxes through transaminases involved in amino acid degradation were reduced concurrently with a reduced uptake of amino acids. On the other hand fluxes through transaminases working in the direction of amino acid synthesis, i.e., alanine and phosphoserine, were increased leading to increased storage of excess nitrogen in extracellular alanine and serine. Glutamate dehydrogenase flux was reversed increasingly fixing free ammonia with increasing ammonia concentration. Urea production additionally observed was associated with arginine uptake by the cells and did not increase at high ammonia stress. It was therefore not used as nitrogen sink to remove excess ammonia. The results indicate that the AGE1.HN cell line can adapt to ammonia concentrations usually present during the cultivation process to a large extent by changing metabolism but with slightly reduced A1AT production and growth.     

Enhancement of glycosylation of cellular glycoconjugates in the squamous carcinoma cell line MDA886Ln by β-all-trans retinoic acid

Retinoids have been shown to inhibit the growth and modulate the glycosylation of head and neck squamous cell carcinoma (HNSCC) cells including the MDA886Ln cells. To examine the effects of -all-trans retinoic acid (RA) on glycoconjugates in HNSCC MDA886Ln cells, the cells were grown in the absence or presence of 1 µM RA and then labeled with tritiated monosaccharides, extracted and analysed by polyacrylamide gel electrophoresis and fluorography. RA increased markedly the incorporation of [³H]-glucosamine, [³H]-galactose, and [³H]-mannose into numerous cellular glycoconjugates, however, the incorporation of [³H]-fucose and [³H]-leucine was almost unaffected by RA. RA increased the incorporation of glucosamine and galactose but not mannose into high molecular weight (HMW) glycoconjugates of about 220 and 500–600 kDa. To analyse the steady state level of glycoconjugates by lectin blotting, extracts of unlabeled cells were separated by gel electrophoresis and the gels were probed with¹²⁵I-labeled wheat germ agglutinin (WGA) andMaackia amurensis (MA) agglutinin. Both lectins were found to bind to numerous glycoconjugates including the HMW glycoconjugates, whereas¹²⁵I-peanut agglutinin bound only to the HMW glycoconjugates. RA treatment increased the binding of all three lectins to the HMW glycoconjugates. These findings demonstrate that RA enhanced the incorporation of specific monosaccharides into a variety of glycoconjugates and in particular into HMW mucin-like glycoconjugates. This effect of RA may be the result of induction of a more normal differentiation state of the HNSCC cells.     

Phytanic acid α-oxidation in human cultured skin fibroblasts

We studied the oxidation of [1-¹⁴C]phytanic acid, 3-methyl substituted fatty acid, to pristanic acid and ¹⁴CO₂ in human skin fibroblasts. The specific activity for α-oxidation of phytanic acid in peroxisomes was 29- and 124-fold higher than mitochondria and endoplasmic reticulum. This finding demonstrates for the first time the presence of fatty acid α-oxidation enzyme system in peroxisomes.     

Altered distribution of the nuclear receptor rarβ accompanies proliferation and differentiation changes caused by retinoic acid in Caco-2 cells

Summary All epithelial cells require retinoic acid for growth, maintenance, and differentiation. Although the epithelial cells that line the gastrointestinal tract are exposed to extreme retinoid concentration fluctuations in luminal fluid, whether proliferation and differentiation in these cells are significantly affected is not known. We have investigated this question using Caco-2 cells as a model because, although they are derived from a colon adenocarcinoma, they differentiate spontaneously in a manner similar to enterocytes in the small intestine. We found that retinoic acid caused maximum inhibition of cell growth and ornithine decarboxylase activity during the proliferative period. Retinoic acid increased brush border enzyme activities only in differentiating cells but stimulated transglutaminase activity in cells at all stages. In untreated proliferating cells, we found an early peak of transglutaminase activity that has not been reported before. Retinoic acid in intestinal cells acts through its nuclear receptor, RARβ. The nuclear distribution of this receptor has not been demonstrated. In this study, we show that RARβ responds to increasing concentrations of retinoic acid with a shift to the nuclear membrane in undifferentiated cells and progressive aggregation, diffusion, and loss in differentiated cells. We conclude that retinoic acid can inhibit proliferation and stimulate differentiation in Caco-2 cells depending on concentration and cell stage, and that these effects are accompanied by changes in distribution, as well as by the loss of RARβ.     

Effects of decorin on the expression of α-smooth muscle actin in a human myofibroblast cell line

Myofibroblasts are metabolically and morphologically distinctive fibroblasts expressing α-smooth muscle actin (α-SMA), and their activation plays a key role in development of the fibrotic response. In an activated state, myofibroblasts cease to proliferate and start to synthesize large amounts of extracellular component proteins. The expression of α-SMA correlates with the activation of myofibroblasts. Decorin, a member of the small leucine-rich proteoglycan gene family, has been implicated in the negative control of cell proliferation primarily by upregulating the expression of p21, a potent inhibitor of cyclin-dependent kinase. In order to examine the effect of decorin on myofibroblast cell growth, we rendered a human lung myofibroblast cell line, MRC-5, quiescent by either cell–cell contact or serum starvation, and examined the relationship between decorin and α-SMA expression in these cells. The expression of decorin in cells made quiescent by serum starvation was lower than that in cells made quiescent by cell–cell contact. In contrast, the expression of α-SMA in cells made quiescent by cell–cell contact was lower than that in cells made quiescent by serum starvation. Furthermore, forced expression of decorin was accompanied by a suppression of α-SMA expression, whereas knocking down of decorin expression by RNA interference increased the expression of α-SMA.     

Decalpenic acid induces early osteoblastic markers in pluripotent mesenchymal cells via activation of retinoic acid receptor γ

Decalpenic acid is a natural small molecule previously isolated from the fermentation broth of fungi that induces early osteoblastic markers in pluripotent mesenchymal cells. Treatment of mouse pluripotent mesenchymal C3H10T1/2 cells with decalpenic acid gave rise to a morphological change similar to that induced by the treatment with retinoic acid, i.e. the cells adopted a more elongated spindle shape. Using a retinoic acid response element reporter and receptor activity assays, we show that decalpenic acid is a new retinoid with selectivity towards retinoic acid receptors γ and α. The induction of early osteoblastic markers by decalpenic acid was significantly inhibited by treatment with the retinoid antagonist, LE540, or with small interfering RNA-mediated knockdown of retinoic acid receptor γ. These results demonstrated that decalpenic acid induces early osteoblastic markers in pluripotent mesenchymal cells through activation of retinoic acid receptor γ.     

Binding receptors for α-l-fucosidase in human B-lymphoid cell lines

An established mechanism for directing newly made acid hydrolases to lysosomes involves acquisition of mannose 6-phosphate residues by the carbohydrate portion of acid hydrolases followed by binding to specific membrane-bound transport receptors and delivery to lysosomes. Two distinct phosphomannosyl receptors (CI-MPR and CD-MPR) have been identified. Alternative mechanisms for trafficking acid hydrolases exist. This report examines means for the possible receptor-mediated intracellular transport of -l-fucosidase in lymphoid cells. The binding of -l-fucosidase to intact cells and to total cell membrane preparations, in conjunction with immunoassays of solubilized membrane preparations, revealed the presence of CI-MPR and CD-MPR on human lymphoid and fibroblast cell lines. The mean level of CD-MPR in nine lymphoid cell lines was 7.2-fold greater than CI-MPR. The mean level of CI-MPR in two fibroblast lines was 3.8-fold greater than CD-MPR. The mean content of CI-MPR was 19.5-fold greater in the fibroblasts than in the lymphoid cells. The CD-MPR content of fibroblasts and lymphoid cells was nearly equivalent. Among these cell lines were a fibroblast and a lymphoid line from the same individual. These results indicate that human B-lymphoid cells are deficient in CI-MPR and suggest that modulation of expression of CI-MPR and CD-MPR in lymphoid cells differs from that in fibroblasts, including cell lines with identical genomes. No specific receptor capable of binding -l-fucosidase independent of mannose 6-phosphate was demonstrable, despite published results that support the existence of a mannose 6-phosphate independent trafficking mechanism in lymphoid cells for this enzyme.     

Induction of apoptosis in human T leukemic cell line CEM-6T by tripter-ine;comparison with arsenic oxide and hydroxyl camptothecin

In China, the herbal plant Lei gong teng (tryipterygium wolfordii Hook F) is widely used in rheumatic diseases, indicating that some compounds in this plant might have effects on immune cells. We have reported that one of the compound from this plant, tripterine could induce apoptosis in the human mast leukemic cell line HMC-1 (Chin J of Hematol 20:146,1999). To further explore the potential value of triperterine in the treatment of leukemia, we cultured human T leukemia cell line CEM-6T in vitro with tripterine, or arsenic oxide, or hydroxyl camptothecin at various concentration ranging from 0.0625 μM to 128 μM DNA electrophoresis and annexin V labeling were used to detect apoptosis. The protein levels of Bcl-2,     

Isothiocyanate–drug interactions in the human adenocarcinoma cell line Caco-2

Loss or mutation of the PTEN (phosphatase and tensin homologue deleted on chromosome 10) gene is associated with resistance to epidermal growth factor receptor (EGFR) inhibitors. However, the mechanism underlying remains elusive. In this study, we aimed to explore whether sensitivity to the EGFR tyrosine kinase inhibitor (TKI) is affected by PTEN status in endometrial cancer cells. PTEN siRNA and the PTEN gene were transfected into HEC-1A and Ishikawa endometrial cancer cells using lentiviral vectors. Cells were treated under various concentrations of RG14620 and rapamycin, which are EGFR and mammalian target of rapamycin (mTOR) inhibitors, respectively. The IC(50) of RG16420 was determined by using the MTT method. Cell apoptosis and the cell cycle were studied, and activation of EGFR, AKT, and p70S6 were detected by Western blot analysis. Loss of PTEN promoted cell proliferation and led to significant increases in the levels of EGFR, phospho-EGFR, AKT, phospho-AKT, and phospho-mTOR proteins. Ishikawa and HEC-1A(PTENkd) cells that displayed loss and inactivation of PTEN function were resistant to RG14620. HEC-1A and Ishikawa(PTEN) cells with intact PTEN were sensitive to RG14620. The combination of two inhibitors was more effective than both monotherapies, particularly in carcinoma cells with PTEN dysfunction. Decreased phospho-EGFR protein expression was observed in all cell lines that were sensitive to RG14620. Decreased phospho-AKT and phospho-p70S6 protein expression was observed in PTEN-intact cells that were sensitive to RG14620. PTEN loss results in resistance to EGFR TKI, which was reversed by PTEN reintroduction or mTOR inhibitor treatment. The combined treatment of EGFR TKI and the mTOR inhibitor provided a synergistic effect by promoting cell death in PTEN-deficient and PTEN-intact endometrial cancer cells, particularly in PTEN-deficient carcinoma cells with up-regulated EGFR activation.     

Prion infection promotes extensive accumulation of α-synuclein in aged human α-synuclein transgenic mice

In neurodegenerative disorders of the aging population, misfolded proteins, such as PrP^Sc, α-synuclein, amyloid β protein and tau, can interact resulting in enhanced aggregation, cross seeding and accelerated disease progression. Previous reports have shown that in Creutzfeldt-Jakob disease and scrapie, α-synuclein accumulates near PrP^Sc deposits. However, it is unclear if pre-existing human α-synuclein aggregates modified prion disease pathogenesis, or if PrP^Sc exacerbates the α-synuclein pathology. Here, we inoculated infectious prions into aged α-synuclein transgenic (tg) and non-transgenic littermate control mice by the intracerebral route. Remarkably, inoculation of RML and mNS prions into α-synuclein tg mice resulted in more extensive and abundant intraneuronal and synaptic α-synuclein accumulation. In addition, infectious prions led to the formation of perineuronal α-synuclein deposits with a neuritic plaque-like appearance. Prion pathology was unmodified by the presence of α-synuclein. However, with the mNS prion strain there was a modest but significant acceleration in the time to terminal prion disease in mice having α-synuclein aggregates as compared with non-tg mice. Taken together, these studies support the notion that PrP^Sc directly or indirectly promotes α-synuclein pathology.     

Apoptosis of human leukemic HL—60 cells induced to differentiate by treatment with RA or DMSO

In present study,we studied the effect of all-trans retinoic acid(ATRA)and dimethylsulfoxide(DMSO)on the induction of apoptosis in HL-60 cell line.Based on morphological changes by Hochest 33342 staining and identification of internuclesomal NDA celeavage by gel electrophoresis,we observed aberrant nuclear chromatin condensation and ladder-like pattern of DNA degradation. Using Flow Cytometric method.We found sub-G1 peak in RA-treated HL-60 cells starting 5 to 6d after the initiation of the treatment However,Such an obvious apoptotic peak was not identified in DMSO-differentiated cells.Combining the research accomplished before.our study approves further that apoptosis could be a common mode of death of terminally differentiated HL-60 cells.     

Effects of ascorbic acid and ��-carotene on HepG2 human hepatocellular carcinoma cell line

Recent studies have demonstrated that vegetable rich diets have protective effects on the occurrence and prognosis of various cancers. In addition to dietary intakes, ascorbic acid and β-carotene are also taken as supplements. The aim of this study was to assess effects of ascorbic acid, β-carotene and their combinations on human hepatocellular carcinoma cell line HepG2. Ascorbic acid and β-carotene were applied to cells as plasma peak concentrations (70 and 8 μM, respectively) and their half concentrations (35 and 4 μM, respectively) for 24 and 48 h. Genotoxic and cytotoxic effects of ascorbic acid and β-carotene were evaluated by alkali single cell gel electrophoresis (SCGE), acridine orange/ethidium bromide staining patterns of cells (apoptosis and necrosis) and lipid peroxidation (thiobarbituric acid reactive substances, TBARS). Results of the SCGE demonstrated that both ascorbic acid and β-carotene caused DNA damage on HepG2 which were also concordant to increased apoptosis and necrosis of cells. Increased TBARS values also demonstrated increased lipid peroxidation in these cells. Results of the present study demonstrates that when dietary intakes of ascorbic acid and β-carotene and their relevant achievable plasma level concentrations were considered, both ascorbic acid and β-carotene induce genotoxic and cytotoxic damage on HepG2 together with increased oxidative damage in contrast to their protective effect on healthy cells. This may be correlated to oxidative status and balance of ROS in hepatocellular carcinoma cells.     

Expression of α1-adrenergic receptor subtypes in heart cell culture

Three ₁AR subtypes have been cloned so far and are designated as _1a, _1b, and _1d. Organspecific distribution pattern and subtype-specific effects are known but not fully understood. To address a cell-type specific expression pattern in the heart we investigated expression pattern of ₁AR subtypes on RNA and proteinlevel in heart tissue, cultured cardiomyocytes and nonmyocytes of the rat. Each ₁ARsubtype mRNA was present in neonatal and adult rat heart culture but the relative distribution pattern was significantly different. While the _1aAR subtype is preferentially expressed in adult cardiomyocytes, the _1bAR subtype was preferentially expressed in the nonmyocyte cell fraction. The RTPCR results were confirmed by Westernblotting (_1b) and immunocytochemical studies. Incubation with an ₁agonist (phenylephrine) for 72 h led to a significant reduction of the _1bAR in neonatal heart cell culture on both mRNA and protein level. In contrast, incubation with an ₁antagonist (prazosin) induced a 1.6 fold upregulation of the _1aAR mRNA without significant effects on radioligand binding and functional assay. The results indicate a distribution pattern of the ₁AR subtype which is specific for cell type and ontogeny of the rat heart and may be regulated by adrenergic agents.