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The Herpes virus hominis growth is studied in Vero cells pretreated for 48 hrs with sodium butyrate 24 hour later. Total virus yield is at least ten times less in treated than in infected control cells. Butyrate has no direct effect on Herpes virus survival. However plaque numbers in pretreated cells are reduced by about 60 per 100.
The activity of the serine protease plasminogen activator (PA), which correlates with tumorigenicity and metastatic capacity, was examined using the 125I-labeled fibrin plate assay in cell extracts from four human glioma lines as a function of growth in vitro. Cell-associated inhibitory activity to plasmin and urokinase-type PA was also measured concurrently. The relative PA activities differed markedly among the lines, whereas inhibitory activities did not. Two lines, SNB-19 and SNB-75, exhibited maximal PA activities (1-6 m Plough units/micrograms protein) as cultures approached confluence, whereas two other lines, SNB-56 and SNB-78, expressed low PA activity at all times (less than 0.2 m Plough units/micrograms protein). The PA of SNB-19 cell extracts was predominantly urokinase-type PA. In addition to having the highest PA levels, SNB-19 and SNB-75 were the most clonogenic in soft agar and tumorigenic in nude mice. In contrast, SNB-56 and SNB-78 were poorly clonogenic in soft agar and were not tumorigenic in nude mice. Measured directly, inhibitory activities to plasmin, urokinase-type PA, and tissue-type PA were detected in SNB-19 (high PA) and SNB-56 (low PA) cell extracts. However, there were no qualitative or quantitative differences in inhibitor effects between SNB-19 and SNB-56 suggesting that the differences in PA activity between these lines resulted from changes in PA activity and were not due to differential plasminogen activator inhibitor effects. The ability of the differentiating agent sodium butyrate (NaB) to modulate total PA activity was also examined. Peak SNB-19 cell PA activity was decreased in a concentration (Ki, 0.75 mM) and time-dependent manner by the addition of nontoxic amounts of NaB. The dose-dependent decrease in PA activity induced by NaB was most likely due to an effect on PA itself, since the action of inhibitor on urokinase was unchanged in response to NaB. These results suggest that net cellular PA activity in glioma cells is a balance between relative PA activity and inhibitor(s) effects and that this balance can be modulated by sodium butyrate.
Mechanisms of leukemic cell clonal dominance may include aberrations of transmembrane signaling. In particular, neoplastic transformation has been associated with reduced capacity for hormone-stimulated adenylate cyclase activity. In the present study, prostaglandin E, a hormonal activator of adenylate cyclase that has antiproliferative activity in myeloid cells, and cholera toxin, an adenylate cyclase agonist that functions at a postreceptor site by activating the adenylate cyclase stimulatory GTP-binding protein (Gs), were studied for antiproliferative activity in two murine myeloid cell lines. FDC-P1, an interleukin 3 (IL 3)-dependent myeloid cell line and a tumorigenic IL 3-independent subline, FI, were resistant to these antiproliferative agents. The in vitro ability of the "differentiation" agent, sodium butyrate, to reverse their resistance to adenylate cyclase agonists was studied. The antiproliferative action of butyrate involved augmentation of transmembrane adenylate cyclase activity. Increased adenylate cyclase catalyst activity was the primary alteration of this transmembrane signaling group leading to the functional inhibitory effects on leukemia cells, although alterations in regulatory G-proteins appear to play a secondary role.
Two subpopulations of human colon tumor cells (clones A and D) which differ in their intrinsic sensitivity to X irradiation were grown for several passages in tissue culture medium containing the differentiation-inducing agent sodium butyrate (NaB, 2 mM). Values of the single-hit, multitarget survival curve parameters for non-NaB-treated clone A cells were n = 17.1, D0(Gy) = 0.81, and DQ(Gy) = 2.31; corresponding parameters for NaB-treated cells were 1.04, 1.16, and 0.05. For non-NaB-treated clone D cells, the survival parameters were n = 4.27, D0 = 1.05, and DQ = 1.52; corresponding parameters for NaB-treated cells were 1.19, 1.15, and 0.20. The large reduction in the DQ parameters of both clone A and D cells after NaB treatment indicates that sodium butyrate-induced cell maturation is accompanied by increase in radiation cell kill, particularly in the low-dose region of the survival curve.
The thyroid hormone receptor is a nuclear-associated protein which appears to mediate the actions of 3,5,3'-triiodo-L-thyronine (L-T3) and 3,5,3',5'-tetraiodo-L-thyronine (L-T4) in mammalian cells. In a previous study we reported that N-2-diazo-3,3,3-trifluoropropionyl-3,5,3'-triiodo-L-thyronine (L-T3-PAL) serves as an effective photoaffinity label probe of the receptor in GH1 cells, a growth hormone producting rat pituitary cell line. Irradiation of cells at 254 nm covalently cross-links L-[125I]T3-PAL to two molecular weight (Mr) nuclear receptor forms, an abundant 47,000 Mr component and a less abundant 57,000 Mr species (Pascual, A., Casanova, J., and Samuels, H. H. (1982) J. Biol. Chem. 257, 9640-9647). In this study we have explored a number of possible interrelationships of the different Mr receptor forms. Denaturing gel electrophoresis and autoradiography indicates that the 57,000 Mr form is a doublet species which differ in Mr by 1,000 to 2,000. The various receptor forms are not an artifact of the L-[125I]T3-PAL probe, and identical forms can be labeled at 310 nm using underivatized L-[125I]T4 with a 15-fold lower coupling efficiency. The 57,000 and 47,000 Mr receptor forms are not generated by indiscriminate proteolysis, UV peptide cleavage, or zero length protein-protein cross-linking by irradiation at 254 nm. Micrococcal nuclease excises both the 57,000 and 47,000 Mr forms, and receptor is not identified in the residual nuclear matrix fraction. Receptor is also not detected in the cytoplasmic fraction. By coupling dense amino acid labeling and photoaffinity labeling of receptor we determined a half-life of 2.4 h for the 57,000 Mr species and 5.6 h for the 47,000 Mr form while both species have similar relative synthetic rates. n-Butyrate has been previously shown to decrease receptor levels in GH1 cells. We demonstrate that n-butyrate decreases receptor levels primarily by shortening the half-life of the 47,000 Mr form.
Sodium n-butyrate has been used as a tool to study the presumptive effects of the in vivo hyperacetylation of histones from HeLa cells on the transcriptional activity of their isolated nuclei. No gross differences were found in either initiation or RNA synthetic capacity among the nuclei from control or butyrate treated cells. The electrophoretic pattern of the RNA synthesized in nuclear suspensions was markedly affected by prior exposure of the cells to the fatty acid.
Studies were carried out with HeLa S-3 cells to determine whether exposure of the cells to "differentiating agents" would modify the thermosensitivity of cells under the cellular conditions where there is no significant perturbation of the cell cycle distribution. Sodium butyrate, dibutyryl cyclic adenosine 3':5'-monophosphate (dbcAMP), and retinoic acid all afforded pronounced protective effects on the thermosensitivity of HeLa cells. The kinetics of expression of thermal resistance induced by the agents varied with different agents. Sodium butyrate (1 mM) was only capable of inducing the thermal resistance during the time of heating (42 degrees), while a minimum 8-hr exposure to dbcAMP (1 mM) and 48-hr exposure to retinoic acid (10 microM) prior to heating at 42 degrees were required to demonstrate the thermal resistance. The presence of dbcAMP and retinoic acid was not required during the heating. The thermal resistance acquired by the exposure of cells to dbcAMP and retinoic acid was reversible following the removal of the drugs with a time course varying with the duration of preexposure time. This difference in the kinetics of thermal resistance suggests that these agents may have a different mode of action for the induction of thermal resistance in HeLa cells.
14CO2 production from [l-14C]oleate, [l-14C]butyrate and [U-14C]proline by isolated rat hepatocytes was studied. In hepatocytes from fed rats, fatty acid and proline oxidation are stimulated in parallel by adrenaline, noradrenaline, vasopressin and angiotensin II. In contrast in hepatocytes from 24 h-starved rats these hormones stimulate proline oxidation whereas oleate and butyrate oxidation is hormone-insensitive. This suggests that 14CO2 production from [U-14C]proline and [l-14C]oleate is subject to independent endocrine control. In support of this in hepatocytes from fed rats, glucagon and dibutyryl cyclic AMP stimulate 14CO2 production from proline but inhibit 14CO2 production from [l-14C]oleate. The pathway of hepatic proline oxidation is discussed and it is suggested that 2-oxoglutarate dehydrogenase is one site of endocrine control of proline oxidation.
The in vitro interaction of butyric acid, crotonic acid and n-butylamine with rat liver glutathione S-transferase (GST) was studied, using glutathione (GSH) and 1-chloro-2,4-dinitrobenzene (CDNB) as substrates. Both acids inhibited the GST activity in crude extracts in a dose dependent manner; the amine did not. The GST isoenzymes were inhibited at different degrees. Kinetic studies never revealed competitive inhibition kinetics, with GSH nor with CDNB as the variable substrate. Titration of remaining GSH in appropriate incubation mixtures revealed no GST catalyzed conjugation with GSH. It is concluded that butyric and crotonic acid interact with GST by direct binding to these proteins via their carboxyl group. This binding could have a protective function against these compounds.
For determination of the extrachromosomal homologous DNA recombination efficiency, somatic cells of various lines have been transformed with plasmid DNAs which contain copies of neo-gene with non-overlapping deletions. Reconstruction of the neo-gene functional activity, which imparts a geneticin-resistant phenotype to cells, indicates that recombination has occurred. If dP1 and dR copies of the neo-gene are used, a single (reciprocal) exchange is necessary for reconstruction of the neo-gene by homologous DNA recombination, but a double exchange (gene conversion) is needed in the case of dP1 and dS copies. It is shown that in human cells of line HeLa and in mouse cells of line LMtk-, in contrast to the Chinese hamster cells of line A238, the frequency of double exchanges is comparable to that of the single DNA exchanges which is an evidence of participation in DNA recombination of gene conversion in addition to a single exchange mechanism. The treatment of cells with sodium butyrate and luminol exerts different influences on the rate of the single DNA exchanges and on that of gene conversion (double exchanges) in cells of lines LMtk- and HeLa, respectively. Essential distinctions in correlation of the single DNA exchange frequency and the gene conversion frequency in cells of the studied lines, and the possibility to distinguish between these mechanisms of recombination, under the treatment by sodium butyrate and luminol, may suggest the existence of two mechanisms of homologous DNA recombination in cultured animal cells, which function independently of one another, to a considerable extent.
In the present study we followed the changes in the intracranial pressure and EEG activity of rabbits caused by the butyric short-chain fatty acid infused as an 0.2 M solution at the rate of 12 ml/h. Changes in the rabbit's normal EEG of paroxysmal nature were initially observed, followed by a rise of intracranial pressure as a result of the edema that developed in the brain. Pretreatment of the animals with an inhibitor of the prostaglandin (PG) synthesis (indomethacin, i.v. 10 mg/kg b.w.) prevented the appearance of paroxysms. The intracranial pressure increased in the presence of indomethacin, but there was a tendency towards reduction of the maximal value reached in the control animals. We conclude that the PG system participates to a varying degree in the genesis of the butyrate-induced changes in the bioelectric cerebral activity and intracranial pressure.
The mode of cell death induced in the Y-79 human retinoblastoma cell line by sodium butyrate (SB), a short-chain fatty acid with potent inhibitory effects on the growth of many transformed cell lines, was investigated by fluorescence and transmission electron microscopy, agarose gel electrophoresis, and metabolic studies. While SB (< 1 mM) resulted in marked morphological differentiation, higher concentrations (1-4 mM) induced predominantly apoptotic involution in Y-79 in a concentration-dependent fashion after a latent period of 24 h. Dying cells displayed the characteristic morphology of apoptosis accompanied by DNA laddering with agarose gel electrophoresis. Extensive cell necrosis was apparent with 0.5 M SB. Induction of apoptosis and DNA laddering by SB was reduced by putative inhibitors of RNA and protein synthesis, but not putative endonuclease inhibitors. These results are important for understanding the mode of action of sodium butyrate as a potential cancer chemotherapeutic agent.
The effect of butyrate-induced differentiation on lipid composition of HT-29 cells were studied. It was observed that cell differentiation was associated with increased activity of alkaline phosphatase and changes in the lipid composition. Differentiated cells showed increased level of triacylglycerol, cholesteryl esters and decreased free cholesterol and phospholipids. Changes were also observed in individual phospholipid composition. It appears that butyrate induced differentiation is associated with increased esterification of neutral lipids.
Enterocyte differentiation occurs at the crypt-villus junction through the transcriptional activation of cell-specific genes, including the brush-border enzyme intestinal alkaline phosphatase (IAP) and the microvillar structural protein, villin. Based upon previous in vivo studies demonstrating that IAP and villin are differentially affected by alterations in epithelial growth state, we examined the regulation of these two genes in an in vitro model of enterocyte differentiation. HT-29 cells were maintained in DMEM + 10% FCS and treated with sodium butyrate to induce enterocyte differentiation. Cell count and [3H]thymidine measurements confirm that sodium butyrate causes a marked decrease in cellular growth state, consistent with the differentiation process. Northern blot analyses revealed time- and dose-dependent increases (P < 0.001) in steady-state IAP and villin mRNA levels. The increases in both IAP and villin expression were dependent upon the presence of one or more newly synthesized proteins. Post-confluence and serum starvation were used to cause cell cycle withdrawal prior to the differentiation process. As predicted from our previous in vivo data, villin expression was unaffected by changes in cellular growth state, whereas IAP expression was dramatically diminished under hypoproliferative conditions. We conclude that early withdrawal from the cell cycle alters the course of enterocyte differentiation, specifically decreasing IAP expression.
AML1 may play a role in growth and differentiation of cells along erythroid and/or megakaryocytic lineages, because a significant level of the AML1 gene is expressed in these cells. We overexpressed AML1a (without the transcription-activating domain) and AML1b (with the domain) proteins in K562 leukemia cells, which can be induced to differentiate into hemoglobin-producing cells and megakaryocytes. The AML1a-transfected K562 cells had a reduced capacity to differentiate in the presence of sodium n-butyrate but not in the presence of other inducers, such as hemin, 1-beta-D-arabinofuranosylcytosine, and herbimycin A. The AML1 antisense oligodeoxynucleotide but not the sense oligomer recovered its differentiation-inducing capacity in the presence of butyrate. On the other hand, AML1b conferred a similar differentiation-inducing capacity upon K562 cells transfected with vector alone. AML1a expression was associated with enhanced sensitivity to megakaryocytic differentiation induced by phorbol ester. These results provide evidence that AML1 proteins play a role in erythroid and megakaryocytic differentiation.
BACKGROUND: Intestinal mucosal turnover is a process of proliferation, differentiation, and apoptosis; the mechanisms remain largely undefined. The purpose of our study was to (1) assess the relationship between apoptosis and enterocyte differentiation and (2) determine whether the cell-cycle inhibitors, p21Waf1/Cip1 and p27Kip1, or the apoptosis inhibitors, Bcl-2 and Bcl-XL, may be involved. METHODS: Gut-derived Caco-2 cells were treated with sodium butyrate. Apoptosis was assessed by Hoechst stain, DNA laddering, and annexin V assay; differentiation was determined by alkaline phosphatase and sucrase activity. RNA and protein were analyzed for expression of p21Waf1/Cip1, p27Kip1, and members of the Bcl-2 family. RESULTS: Treatment of Caco-2 cells with sodium butyrate resulted in the concomitant induction of both differentiation (increased alkaline phosphatase and sucrase activity) and apoptosis. Increased levels of p21Waf1/Cip1 and p27Kip1 mRNA and protein were detected at 24 hours, occurring before apoptosis or differentiation; decreased mRNA levels of Bcl-2 and Bcl-XL were noted at 24 hours. CONCLUSIONS: Differentiation and apoptosis occurred simultaneously in Caco-2 cells, suggesting that apoptosis may be linked to enterocyte differentiation. The induction of p21Waf1/Cip1 and p27Kip1 and the down-regulation of Bcl-2 and Bcl-XL further suggest a link between the cell-cycle mechanisms regulating enterocyte differentiation and apoptosis.
BACKGROUND: Biologic agents that reverse early changes in the aerodigestive tract mucosa have potential treatment applications for patients with field cancerization of the upper aerodigestive tract. Sodium butyrate (BA) is a normal dietary constituent that induces differentiation and inhibits growth in several malignant cell types in vitro, but its effect on head and neck squamous cell carcinoma (HNSCC) has not been evaluated. METHODS: Using five HNSCC cell lines, the effects of BA on cell proliferation and apoptosis were examined by colorimetric and fluorescence-labeling methods, and the expression of differentiation markers and apoptosis-related proteins were analyzed using Western and Northern blotting, flow cytometry, and cell cycle analysis. RESULTS: BA-induced growth inhibition and apoptosis in HNSCC cells at millimolar concentrations. Apoptosis induction did not depend on the p53 status of the cell lines or on expression of members of the Bcl-2/Bax family. CONCLUSIONS: These results demonstrate that butyrate has activity against HNSCC in vitro and may have clinical applications for management of HNSCC patients.
Embryonic stem cells serve as a promising technology to obtain specific cell types for a number of biomedical applications. Because traditional techniques, such as embryoid body formation result in a wide array of differentiated cells such as hepatic, neuronal, and cardiac lineages, strategies have been utilized which favor cell-specific differentiation to generate more uniformity. In the present study, we have investigated the use of sodium butyrate in a monolayer culture configuration to mediate hepatocyte differentiation of murine embryonic stem cells. Several functional assays used to characterize hepatocyte function (viz. urea secretion, intracellular albumin content, cytokeratin 18, and glycogen staining) were used to analyze the differentiating cell population, suggesting the presence of an enriched population of hepatocyte-like cells. Since mature hepatocytes mediate energy metabolism predominantly through oxidative means as opposed to hepatocyte precursors, which are primarily glycolytic, we have performed a kinetic analysis of glycolytic and functional capacity to characterize the differentiated cells. In conjunction with mitochondrial mass and activity measurements, we show that Na-butyrate-mediated differentiated cells mediate energy metabolism predominantly through glycolysis. This metabolic and mitochondrial characterization can assist in evaluating stem cell differentiation and may prove useful in identifying key regulatory molecules in mediating further differentiation.