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PubMed1986

Induction of functional beta-adrenergic receptors in rat aortic smooth muscle cells by sodium butyrate.

Rat aortic smooth muscle cells in culture (A-10; ATCC CRL 1476) exhibited low levels of beta-adrenergic receptors as determined by specific binding of [125I]cyanopindolol ([125I]CYP) and marginal stimulation of adenylate cyclase in plasma membranes by (-)isoproterenol. When these cells were exposed to 5 mM sodium butyrate, the number of beta-adrenergic receptors and the beta-agonist-stimulated adenylate cyclase activity increased markedly. However, basal, GTP, Gpp(NH)p, and fluoride-stimulated activities did not change. The induction of beta-adrenergic receptors and beta-agonist stimulated adenylate cyclase activity was time- and dose-dependent, and was relatively specific for sodium butyrate. Propionate and valerate were less effective than butyrate, while isobutyrate, succinate, and malonate were ineffective. The induction involved RNA and protein synthesis because induction was prevented by treatment with cycloheximide, puromycin, and actinomycin D. Butyrate did not cause a general increase in cell surface receptors, because the number of vasopressin receptors did not change. The sustained presence of butyrate appeared to be necessary for the maintenance of the induced beta-receptors. When butyrate was removed, receptor number and beta-agonist-stimulated adenylate cyclase activity were decreased by 90% over 24 hr. We conclude that the poor response of rat aortic smooth muscle cell plasma membranes to beta-adrenergic agonists is due to the presence of a low number of beta-adrenergic receptors. Butyrate markedly increased the number of beta-receptors which resulted in a proportional increase in beta-agonist-stimulated adenylate cyclase activity. The increase in receptor number was dependent on RNA and protein synthesis. Butyrate treatment did not affect the activity of the cyclase unit and the efficiency of coupling between the receptors and the guanine nucleotide regulatory protein, Ns.

Adenylyl Cyclases↗
PubMed1987

Effects of plasma aldosterone on butyrate absorption and metabolism in the rabbit proximal colon.

Butyrate absorption in the proximal colon of the anaesthetized rabbit was evaluated by measuring the variations in the concentration of butyrate in colonic loops and in arterial and venous plasmas; metabolic conversions were studied using (3,4-14C) butyrate. Interrelations between butyrate absorption and metabolism and the excretory cycle of the rabbit were examined, as well as the effects of exogenous aldosterone, the hormone generally implicated in the diurnal rhythm of the fecal excretion. The colonic tissue metabolized butyrate via 2 main pathways. They were of differing intensity according to the 2 phases of the excretory cycle. When the plasma level of aldosterone was high (during hard faeces production), the butyrate was mainly oxidized to CO2, yielding energy for metabolic processes. When the plasma level of aldosterone was lower (during soft production), butyrate was also oxidized to CO2 but it was a better source of free amino acids. Exogenous aldosterone (30 micrograms/kg) enhanced absorption and oxidative metabolism of the butyrate, which occurred normally when hard faeces were elaborated.

Aldosterone↗
PubMed1985

Stimulation of deoxyribonucleic acid excision repair in human fibroblasts pretreated with sodium butyrate.

The effect of pretreatment with sodium butyrate on DNA excision repair was studied in intact and permeable confluent (i.e., growth-inhibited) diploid human fibroblasts. Exposure to 20 mM sodium butyrate for 48 h increased subsequent ultraviolet (UV)-induced [methyl-3H]thymidine incorporation by intact AG1518 fibroblasts by 1.8-fold and by intact IMR-90 fibroblasts by 1.2-1.3-fold. UV-induced incorporation of deoxy[5-3H]cytidine, deoxy[6-3H]cytidine, and deoxy[6-3H]uridine, however, showed lesser degrees of either stimulation or inhibition in butyrate-pretreated cells. This result suggested that measurements of butyrate's effect on DNA repair synthesis in intact cells are confounded by simultaneous changes in nucleotide metabolism. The effect of butyrate on excision repair was also studied in permeable human fibroblasts in which excision repair is dependent on exogenous nucleotides. Butyrate pretreatment stimulated UV-induced repair synthesis by 1.3-1.7-fold in permeable AG1518 cells and by 1.5-2-fold in permeable IMR-90 cells. This stimulation of repair synthesis was not due to changes in repair patch size or composition or in the efficiency of DNA damage production but rather resulted from a butyrate-induced increase in the rate of damage-specific incision of DNA. The increased rate of incision in butyrate-pretreated cells could be due either to increased levels of enzymes mediating steps in excision repair at or before incision or to alterations in chromatin structure making damage sites in DNA more accessible to repair enzymes.

Adenosine Triphosphate↗
PubMed1992

The effect of sodium butyrate on the growth characteristics of human cervix tumour cells.

Sodium butyrate has been shown to affect cell proliferation, and, at concentrations above approximately 0.5 mM, to cause cell death in some tumour cell lines. When combined with cytotoxic drugs increase in chemosensitivity has been observed. We are presently carrying out a study of the combined effects of sodium butyrate and cytotoxic drugs on cultured cervix tumour cells. To provide a baseline for this study we have carried out a systematic investigation of the effects of sodium butyrate alone on the growth characteristics of cervix tumour cells cultured as multicell spheroids. This has shown that concentrations of n-butyrate of 0.005 mM to 0.50 mM decrease cell proliferation without inducing cell death, the effect increasing with increasing concentration. Butyrate concentrations greater than 0.50 mM cause cell death after a period of 5 to 15 days exposure, dependent on concentration. Concentrations of 0.010 mM and above cause fragmentation of, and increased cell shedding from, multicell spheroids, suggesting an effect on the cell surface. Concentrations of butyrate greater than 0.10 mM cause a considerable increase in the synthesis of cytokeratin, as shown by reaction with cytokeratin antibody. Correlated with this is a marked increase in cell size, concentrations of butyrate of 2.0 or 3.0 mM leading to an approximate doubling of cell diameter, followed by cell disintegration. The effects of butyrate less than 0.25 mM are readily reversible. At concentrations greater than 0.25 mM the effects are reversible up to a limit of about 7 to 20 days depending on concentration, even when cytokeratin synthesis has been induced.

Butyrates↗
PubMed1995

Stimulation of tissue-type plasminogen activator gene expression by sodium butyrate and trichostatin A in human endothelial cells involves histone acetylation.

We have previously shown that the pleiotropic agent sodium butyrate strongly stimulates tissue-type plasminogen activator (t-PA) expression in human umbilical vein endothelial cells (HUVEC). Here we provide the following evidence that the butyrate-induced t-PA expression in HUVEC involves histone H4 acetylation. (1) t-PA induction by butyrate occurs at the transcriptional level and does not require new protein synthesis, indicating a direct effect. (2) t-PA induction by butyrate can be fully mimicked by a specific, structurally unrelated, histone deacetylase inhibitor, trichostatin A. (3) At optimally stimulatory conditions, a combination of butyrate and trichostatin A does not enhance t-PA production more than each of the compounds alone, indicating that both compounds act through a common regulatory mechanism. (4) Induction of t-PA transcription by butyrate and trichostatin A was found to be preceded by histone H4 acetylation; at suboptimal inducing concentrations of butyrate and trichostatin A, the degree of acetylation of histone H4 caused by each agent was similarly reduced. These results are consistent with a role for histone H4 acetylation in t-PA induction by butyrate in HUVEC.

Acetylation↗
PubMed1995

n-Butyrate mediation of ganglioside expression of human and murine cancer cells demonstrates relative cell specificity.

1. n-Butyrate, a short chain fatty acid produced by colonic fermentation, induces differentiation in human neoplastic cell lines, and reduces expression in vitro of a sialyltransferase that glycosylates N-linked glycoproteins in hepatoblastoma cells. Gangliosides are amphipathic, sialylated glycosphingolipids that undergo profound changes in many transformed cells and may protect neoplastic cells from host immune surveillance. Colonic mucosal cells are exposed to luminal short-chain fatty acid concentrations of up to 80 mmol/l, and there is some evidence that short-chain fatty acids may alter ganglioside expression in colon cancer cells. 2. Because of the importance of gangliosides in cancer pathogenesis, we investigated the effects of n-butyrate on ganglioside expression of colonic (human and murine) and non-colonic cancer cells. 3. Three separate colon cancer cell lines (LS174T, T84 and MCA-38), when butyrate treated, demonstrated striking amplification of specific individual gangliosides. However, the total lipid-bound sialic acid content of gangliosides of butyrate-treated LS174T cells diminished. In contrast to earlier reports, n-butyrate did not mediate expression of all gangliosides and specifically did not mediate expression of GM3. This effect persisted even after removal of butyrate. 4. In contrast, exposure of extracolonic cells to butyrate, including cervical cancer (HeLa) and laryngeal cancer (HEp-2) cell lines in this study and hepatoblastoma cells (Hep G2) in our previous work, caused no detectable changes in ganglioside expression. 5. In conclusion, our results indicate a relative tissue specificity of butyrate-mediated alterations in ganglioside expression that is not universal but is limited to specific gangliosides.

Animals↗
PubMed1996

Sodium butyrate is a potent modulator of smooth muscle cell proliferation and gene expression.

Sodium butyrate is a small, naturally occurring molecule with demonstrated activity on cell growth and differentiation. However, its effect on smooth muscle cells had not been examined. We have found that sodium butyrate and its more stable in vivo analogue tributyrin are potent DNA synthesis and cell proliferation inhibitors. The inhibitory activity of sodium butyrate was not mediated by an elevation of endogenous cAMP levels, a known pathway involved in SMC growth-arrest and maintenance of the contractile phenotype. Consistent with the concept that its activity is mediated by a cAMP-independent pathway, butyrate was able to augment the maximum DNA synthesis inhibitory effect of various agents that elevated intracellular cAMP levels. Additionally, butyrate present for just the initial 8 h or present as late as 16 h after serum addition was able to inhibit DNA synthesis. By contrast, the cAMP analogue, 8Br-cAMP had to be present throughout the entire G0 to S phase of the cell cycle to effectively inhibit DNA synthesis. These results indicated that sodium butyrate inhibited SMC growth through a cAMP-independent mechanism. We also found that sodium butyrate was unable to abrogate the expression of the serum-inducible genes c-fos, c-myc, Ki-Ras and PS4, but was able to directly stimulate the expression of PS4 and thrombospondin. These results indicate that a number of important early G1 events initiated by serum growth factors are unaltered by sodium butyrate and that this compound is able to directly stimulate the expression of certain genes normally associated with SMC proliferation.

Adenosine Monophosphate↗
PubMed1998

Slow-release pellets of sodium butyrate increase apoptosis in the colon of rats treated with azoxymethane, without affecting aberrant crypt foci and colonic proliferation.

We investigated whether sodium butyrate, administered orally as gastroresistant slow-release pellets to rats, could affect markers of colon carcinogenesis. F344 male rats were fed a high-fat diet (230 g/kg corn oil, wt/wt) and treated with two injections (1 wk apart) of azoxymethane (15 mg/kg sc) or saline. Rats were then divided into two groups: one received the diet with 1.5% (wt/wt) sodium butyrate for 10 weeks to provide 150 mg butyrate/day, and one group received no butyrate. At the end of this period, rats were sacrificed, and colonic proliferative activity, number of aberrant crypt foci (ACF), and apoptosis were assessed in the colon. The proliferative activity and ACF induction were not affected by butyrate pellet administration. On the contrary, in rats treated with butyrate, apoptotic index increased from 0.12 +/- 0.12 to 0.81 +/- 0.10 (means +/- SE, p < 0.05). The short-chain fatty acid concentration was significantly increased in the feces of rats treated with butyrate. In conclusion, the increase in the mucosal apoptotic index suggests that gastroresistant butyrate pellets have a beneficial effect against colon carcinogenesis. However, because butyrate pellets did not modify proliferation or ACF induction, this conclusion should be confirmed in long-term carcinogenesis experiments.

Animals↗
PubMed1997

Bcl-2 deregulation leads to inhibition of sodium butyrate-induced apoptosis in human colorectal carcinoma cells.

Epidemiological studies have linked dietary fiber to the prevention of human colorectal cancer and suggest that short chain fatty acids such as butyric acid, which is produced by fermentation of dietary fiber in the large intestine, may be an important mediator of the protective effects of fiber. We investigated the role of Bcl-2 deregulation on the sensitivity of colorectal carcinoma cells to undergo butyrate-induced apoptosis. Here we report an inverse relationship between the levels of Bcl-2 and the sensitivity of colorectal carcinoma cell lines to undergo apoptosis in response to butyrate. Overexpression of Bcl-2 in colorectal carcinoma DiFi cells resulted in suppression of butyrate-induced apoptosis and enhanced cell survival in response to butyrate. Butyrate-induced apoptosis was accompanied by inhibition of expression of a 30 kDa protein (p30, immunorecognized by anti-Bcl-2 mAb) and this cellular effect of butyrate was inhibited by Bcl-2 overexpression. These findings suggest that deregulation of Bcl-2 in human colorectal carcinoma cells confers resistance to induction of apoptosis by butyrate, a dietary micronutrient.

Apoptosis↗
PubMed1997

Butyrate alters activity of specific cAMP-receptor proteins in a transgenic mouse colonic cell line.

There is great interest in utilizing butyrate as a chemotherapeutic agent. To elucidate its mechanism of action, the effect of butyrate on cAMP receptor protein kinase (PKA) activity in young adult mouse colon (YAMC) cells isolated from transgenic mice bearing a temperature sensitive mutation of the SV40 large T antigen gene was investigated. Conditionally immortalized cultures were plated at the permissive temperature (33 degrees C) or growth arrested by incubation at the nonpermissive temperature (39 degrees C). In addition, cells were incubated at 33 degrees C with or without 1 mmol/L butyrate for 24 h. Butyrate treatment reduced cell proliferation by 28% and enhanced apoptosis by 350% compared with cultures not exposed to butyrate. The PKA type I/II isozyme activity ratio was lower (P < 0.05) in cells incubated with butyrate. The relative level of PKA I isozyme was higher in proliferating cells at 33 degrees C (63% of total PKA), while the relative level of PKA II was higher in nonproliferating cells undergoing apoptosis at 39 degrees C (59% of total PKA). Neither incubation conditions (33 vs. 39 degrees C) nor butyrate treatment altered total PKA activity. When YAMC cells were incubated with 8-CI-cAMP, an activator of PKA II, growth was markedly inhibited in cells at both temperatures. Consistent with in vitro data, increased PKA I isozyme levels were associated with dysregulated growth in vivo. Specifically, the relative level of PKA I isozyme was three- to fivefold higher in rat colonic tumors compared with normal nontransformed colonic mucosa. These data indicate that the biological effects of butyrate on colonocyte proliferation and apoptosis are associated with changes in PKA isozyme-dependent signal transduction, and the YAMC cell line is a relevant model to examine the molecular mechanisms by which dietary-derived factors affect relative cancer risk.

Animals↗
PubMed1993

Ileal and colonic epithelial metabolism in quiescent ulcerative colitis: increased glutamine metabolism in distal colon but no defect in butyrate metabolism.

Previous studies have shown that butyrate is an important energy source for the distal colon, and that its metabolism may be defective in ulcerative colitis (UC). A similar metabolic defect in the ileum might account for the occurrence of 'pouchitis' in UC patients after colectomy. A method has been developed that allows the measurement of metabolism in ileocolonoscopic biopsy specimens, and this has been used to assess butyrate and glutamine metabolism in quiescent UC and controls. Preliminary experiments showed optimal metabolism of butyrate at 1 mmol/l. In controls glutamine metabolism was greater in the ascending (mean (SD)) (4.9 (3.2) nmol/h/micrograms protein) than in the descending colon (1.4 0.7)) (p < 0.05, Mann-Whitney U test), but butyrate metabolism was similar in the two regions (ascending 62.6 (44.2), descending 51.5 (32.0)). Consequently ratios of butyrate/glutamine metabolism were higher in the descending colon (20.6 (14.3)) than in the ascending colon (14.3 (9.6)) (p < 0.05). In UC, rates of butyrate metabolism were similar in the ascending (92.5 (58.3) nmol/h/micrograms protein) and descending (93.3 (115)) colon, and these were not significantly different from controls. In UC, glutamine metabolism was similar in the ascending (6.2 (7.7) nmol/h/micrograms protein) and descending colon (7.8 (7.9)); the metabolism in the descending colon was significantly greater than in controls (p < 0.01). Butyrate (135 (56) nmol/h/microgram protein) and glutamine (24.1 (16.2)) metabolism in the ileum in UC, were not significantly different from control values (butyrate 111 (57), glutamine 15.5 (15.6)). These results confirm that there is regional variation of nutrient utilisation throughout the colon, but they do not support the hypothesis that UC is caused by a deficiency of butyrate metabolism.

Adolescent↗
PubMed1989

Sodium butyrate induces pyroglutamyl peptidase I and decreases thyrotropin-releasing hormone receptors in GH3 cells.

The effect of sodium butyrate treatment on TRH-degrading enzymes and TRH receptors in GH3 cells was investigated. The specific activity of pyroglutamyl peptidase I (EC 3.4.19.3) was increased by exposure to sodium butyrate in a time- and concentration-dependent manner, whereas the specific activity of prolyl endopeptidase (EC 3.4.21.26) was unchanged. The maximal effect occurred at a concentration of 1 mM sodium butyrate and 16 h after exposure. The increase was reversible upon removal of sodium butyrate from the cell culture. Cycloheximide totally blocked the stimulation, indicating that the increase was due to new protein synthesis. Sodium butyrate had no effect on pyroglutamyl peptidase I activity in the AtT-20 cell line. [methyl-3H]TRH binding to intact GH3 cells was reduced to 70% of the control value when cells were exposed to 1 mM sodium butyrate for 8 h. A maximal decrease in binding to 40% of the control value occurred after 16 h of exposure. The Kd of [methyl-3H]TRH binding was not changed. Sodium butyrate altered GH3 cell morphology, but the morphological changes occurred after alterations of pyroglutamyl peptidase I activity and [methyl-3H]TRH-binding sites. Other agents known to alter GH3 cell morphology had no effect on pyroglutamyl peptidase I activity. These results indicate that sodium butyrate can in some respects mimic the action of T3 on GH3 cells. Moreover, they provide further evidence that the activity of pyroglutamyl peptidase I, but not prolyl endopeptidase, is subject to regulation in the GH3 cell.

Aminopeptidases↗
PubMed1992

Effects of butyrate on cell cycle progression and polyploidization of various types of mammalian cells.

We studied the effect of butyrate on cell cycle progression and polyploidization in three fibroblast (rat 3Y1, human IMR-90, and human embryo lung HEL) and two epithelial (human embryo kidney HEK and monkey kidney BSC-1) cells. In these cells, except for 3Y1, G1 arrest with butyrate was incomplete, and the production of tetraploid cells was detectable in the presence of butyrate. G2 arrest with butyrate was also incomplete in HEL and BSC-1 cells, and the number of HEL cells increased in the presence of butyrate. On the contrary, most BSC-1 cells that divided in the presence of butyrate were unstable and the number of attached cells decreased. These results indicate that the effect of butyrate on cell cycle progression varies with the cell type and that polyploidization can be induced by a single treatment with butyrate.

Animals↗
PubMed1996

Effects of sodium butyrate on glucose transporter and glucose-phosphorylating enzyme gene expression in RINm5F insulinoma cells.

RINm5F insulinoma cells show a defective physiological insulin secretory response to glucose stimulation. The short chain carbonic acid sodium butyrate induced a growth arrest during a 72-h tissue culture period. In contrast to control RINm5F cells, 2 mM glucose increased insulin secretion by more than 70% in these sodium butyrate-treated cells (1 mM) without any further increase of the secretory rate between 2 and 20 mM glucose. This effect of sodium butyrate on insulin secretion was assessed in comparison with its effect on gene expression of the GLUT1 and GLUT2 glucose transporter, hexokinase type I and type II, glucokinase and insulin. Sodium butyrate at a 1 mM concentration decreased GLUT1 gene expression by nearly 50%, but did not induce gene expression of the low-affinity GLUT2 glucose transporter above the detection limit. Furthermore, sodium butyrate increased glucokinase gene expression by more than 50% and hexokinase type II gene expression by more than 100%, while insulin gene expression was increased only by 24%. Hexokinase type II enzyme activity was increased by more than 100% without a concomitant significant change of the glucokinase enzyme activity. Sodium butyrate (2 mM) caused effects comparable with those of 1 mM sodium butyrate. Thus the improved insulin secretory responsiveness of RINm5F insulinoma cells after sodium butyrate treatment at low non-physiological millimolar glucose concentrations can be interpreted as a result of an increased hexokinase-mediated metabolic flux rate through the glycolytic chain.

Blotting, Northern↗
PubMed1992

n-butyrate reduces the expression of beta-galactoside alpha 2,6-sialyltransferase in Hep G2 cells.

n-Butyrate, a short chain fatty acid that is produced by colonic bacterial fermentation, is detectable in portal blood and induces differentiation in various human neoplastic cell lines. Earlier reports indicated approximately 20-fold induction in vitro by n-butyrate of the sialyltransferase that catalyzes terminal glycosylation of GM3 ganglioside in HeLa and colon cancer cells. We previously isolated a 1.3-kilobase cDNA for a human beta-galactoside alpha 2,6-sialyltransferase, for which N-linked glycoproteins are the acceptors. We report here that treatment of Hep G2 cells with 5 mM n-butyrate for 24 h reduced beta-galactoside alpha 2,6-sialyltransferase mRNA levels by approximately 90%. Reductions in mRNA level were followed by approximately 75 and approximately 90% reductions, respectively, in specific beta-galactoside alpha 2,6-sialyltransferase enzyme activity after treatment for 24 and 36 h with 5 mM n-butyrate. However, in contrast with earlier reports of enhanced ganglioside synthesis in response to n-butyrate treatment, incubation of Hep G2 cells with n-butyrate did not alter the ganglioside pattern as assessed by thin layer chromatography of lipids extracted from treated cells. Nuclear run-on reactions indicated that the rate of transcription of beta-galactoside, alpha 2,6-sialyltransferase was not altered by treatment with 5 mM n-butyrate for 24 h, but the effects of this treatment on cytoplasmic levels of beta-galactoside alpha 2,6-sialyltransferase mRNA were largely negated by co-treatment with actinomycin D or cycloheximide. Therefore, our results show that n-butyrate reduces expression of mature beta-galactoside alpha 2,6-sialyltransferase mRNA by post-transcriptional mechanisms.

Antigens, CD↗
PubMed1992

Effect of sodium butyrate on estrogen receptor and epidermal growth factor receptor gene expression in human breast cancer cell lines.

Estrogen receptor (ER) binding has been shown to decrease in breast cancer cell lines exposed to sodium butyrate; however, the underlying mechanisms are unknown. In MCF-7 breast cancer cells, butyrate caused a rapid time- and concentration-dependent decrease in ER mRNA levels, apparent by 3 h at 3 mM butyrate. ER gene transcription rate was decreased and cycloheximide co-treatment did not relieve this inhibitory effect, suggesting that the butyrate effect was not dependent on ongoing protein synthesis. In both MCF-7 and T-47D cells the decrease in ER mRNA was mirrored by an increase in the level of epidermal growth factor receptor (EGF-R) mRNA. A marked inverse relationship exists between ER and EGF-R in human breast cancer biopsies and cell lines, and the reciprocal modulation of these genes by butyrate suggests that the expression of ER and EGF-R may be co-regulated. This relationship was further investigated in lines expressing only one or the other receptor. In the ER-positive EGF-R-negative line, MDA-MB-134-VI, butyrate exposure decreased ER mRNA levels, implying that the regulation of ER mRNA by butyrate is independent of EGF-R expression. However, butyrate decreased EGF-R mRNA in two ER-negative lines, MDA-MB-231 and HBL-100. As this effect differed from that in ER-positive lines, the regulation of EGF-R may depend on the expression of ER. The possibility that ER and EGF-R gene expression are closely linked has implications in the understanding of progression of human breast cancers to a hormone-independent phenotype and for the use of ER and EGF-R levels as independent prognostic indicators.

Breast Neoplasms↗
PubMed1986

DNA hypermethylation in sodium butyrate-treated WI-38 fibroblasts.

Sodium butyrate is very often used to alter gene expression in cultured cells. In this study, we examined the effects of this compound on various cellular events in WI-38 human embryonic lung fibroblasts in culture. During a 16-20-h treatment at sodium butyrate concentrations of between 5 and 20 mM, no adverse effects on cell morphology were observed. However, cell division and DNA synthesis were reversibly inhibited, the latter by 85, 80, and 70% at sodium butyrate concentrations of 5, 10, and 20 mM, respectively. Although overall protein synthetic activity was not significantly affected, RNA synthesis decreased to 76% of the control values at a sodium butyrate concentration of 5 mM. Butyrate treatment also caused hypermethylation of DNA cytosines as determined by differential digestion by MspI/HpaII restriction endonucleases and by high performance liquid chromatography analysis of the DNA. The 5-methylcytosine content of the DNA in untreated WI-38 fibroblasts was 2.94 +/- 0.46% of total cytosine residues, while in cultures treated with 5, 10, and 20 mM sodium butyrate, these values were 5.76 +/- 0.28, 5.91 +/- 0.37, and 6.8 +/- 0.44%, respectively. An interesting feature is that this hypermethylation occurred in DNA which was synthesized in the presence of sodium butyrate (newly synthesized) as well as in DNA which had been synthesized before butyrate administration (pre-existing DNA). The hypermethylated state was conserved only in the former situation, since the methylcytosines were rapidly lost in the subsequent generation in the latter case. It would therefore appear that methylcytosines are maintained after cell replication only if they are generated on newly synthesized DNA.

Butyrates↗
PubMed1987

Depletion of sodium butyrate from the culture medium of Friend erythroleukemia cells undergoing differentiation.

Friend erythroleukemia cells can be induced to undergo erythroid differentiation by a variety of unrelated compounds. The fact that sodium butyrate causes reversible alterations in growth, morphology, and biochemistry in many cell systems prompted us to reexamine its pattern of induction of differentiation and to compare it to that of dimethyl sulfoxide (DMSO) and hexamethylbisacetamide (HMBA). By the fourth day of induction, a peak in hemoglobin accumulation was reached in the cultures treated with each of these potent inducers. Differences, however, were noted in cultures in which there had been no change of medium for 7 days. Whereas DMSO or HMBA induced cultures reached a stationary stage of growth and maintained a high percentage of benzidine positive cells, butyrate treated cultures resumed active growth and showed a marked decrease in the percentage of benzidine positive cells. However, the actual number of terminally differentiated cells remained relatively constant. The addition of fresh butyrate to 4-day treated cultures prevented the decrease in the percentage of benzidine positive cells. Measurement of [14C]butyrate uptake into the cells showed a decrease in the incorporation of the inducer with time coincident with the decrease in the percentage of benzidine positive cells and of the butyrate in the medium. Incorporation of [3H]thymidine into cells undergoing differentiation for 4 days indicated that butyrate treated cells, but not cells treated with DMSO or HMBA were capable of active DNA synthesis and growth after removal of the inducers. These data suggest that butyrate, a natural fatty acid, is metabolized by the cells and with time its concentration is reduced to a level below that required to stimulate differentiation. Additional evidence to support this notion are the results obtained with conditioned medium (CM) from induced cultures. CM-DMSO and CM-HMBA retained the capacity to induce differentiation whereas CM-butyrate lost its potency with time.

Animals↗