Endothelin-1 acts as an autocrine growth factor for normal human keratinocytes

Colorectal cancers are often composed of cell types representing various differentiated cell lineages, however little is known concerning the relationship of differentiation and drug resistance in these cancers. The present study was performed to develop and characterize a stable, differentiated clone of the human colon cancer cell line LS174T and to characterize the drug resistance of this cell line in relation to its undifferentiated parental cell line. LS174T cell line was treated with the differentiating agent sodium butyrate (0.5 mM) for 30 days, then recultured in standard medium. Foci of flat-appearing cells appeared and were isolated using cloning rings, and subcloned. One subclone was designated LS174T-D. The LS174T-D clone maintains a stable, differentiated phenotype in standard culture conditions in the absence of sodium butyrate. It is characterized by the formation of a polarized monolayer with dome formation and the presence of prominent apical microvilli and tight junctions. This cell line demonstrated reduced growth in soft agar and nude mice compared with the parental cell line. LS174T-D cells expressed immunoreactive intestinal mucin antigens and brush border enzymes dipeptidyl aminopeptidase (DAP)-IV and aminopeptidase. The activities of DAP-IV and aminopeptidase were increased 5.6-fold and 3.4-fold, respectively, in LS174T-D compared with parental cells. Proliferation assays demonstrated that, compared with the parental cell line, LS174T-D cells were more resistant to doxorubicin (93-fold), cisplatin (23-fold), 5-fluorouracil (12-fold), 5-fluorodeoxyuridine (31-fold), and methotrexate (12.5-fold). Intracellular uptake of (³H)-5-fluorodeoxyuridine did not differ significantly in the differentiated and undifferentiated cell lines. Levels of mdr-1 p-glycoprotein measured by Western blot and RNA Northern blot assays were also similarly low in both cell lines. However, total glutathione content and glutathione-S-transferase activities were increased in LS174T-D cells by sixfold and threefold, respectively, compared with parental cells. Depletion of glutathione by pretreatment with DL-buthionine sulfoximine reversed LS174T-D resistance to cisplatin. Long-term treatment with sodium butyrate induces or selects for colon cancer cells with features of enterocytic differentiation. This stably differentiated cell line is associated with glutathione-mediated multidrug resistance, and provides a model for further studies of differentiation in normal and cancerous colon. © 1994 Wiley-Liss, Inc.

1 This article is a US Government work and, as such, is in the public domain in the United States of America

     

Enhanced transglutaminase activity in transformed human lung fibroblast cells after exposure to sodium butyrate

The low level of transglutaminase activity in virus-transformed human embryonic lung fibroblasts (WI-38 VA13A) increased markedly when cells were exposed to sodium butyrate. The effect of sodium butyrate was time- and concentration-dependent and fully reversible. Transformed cells exposed for 5 days to 1 mM sodium butyrate had fewer cells, showed an 8–10-fold higher transglutaminase activity, and stained more abundantly for transglutaminase and pericellular fibronectin than control cells when examined by indirect immunofluorescence. Non-transformed cells (WI-38) showed only a 2–4-fold increase in transglutaminase activity when treated in a similar manner. Studies with metabolic inhibitors revealed the increase in activity was the result of synthesis of new protein. Kinetic studies showed the affinity of putrescine for the enzyme was essentially unchanged but the number of active sites increased 9-fold following exposure to sodium butyrate. Enhanced transglutaminase activity returned to control levels within 7 days after subculture and sodium butyrate removal. These findings suggest that sodium butyrate offers a potential model system to understand better the role of transglutaminase in cells in culture; particularly growth control in transformed cells.     

Effect of sodium butyrate on cell proliferation and cell cycle in porcine intestinal epithelial (IPEC-J2) cells

Conflicting results have been reported that butyrate in normal piglets leads either to an increase or to a decrease of jejunal villus length, implying a possible effect on the proliferation of enterocytes. No definitive study was found for the biological effects of butyrate in porcine jejunal epithelial cells. The present study used IPEC-J2 cells, a non-transformed jejunal epithelial line to evaluate the direct effects of sodium butyrate on cell proliferation, cell cycle regulation, and apoptosis. Low concentrations (0.5 and 1 mM) of butyrate had no effect on cell proliferation. However, at 5 and 10 mM, sodium butyrate significantly decreased cell viability, accompanied by reduced levels of p-mTOR and PCNA protein. Sodium butyrate, in a dose-dependent manner, induced cell cycle arrest in G0/G1 phase and reduced the numbers of cells in S phase. In addition, relative expression of p21, p27, and pro-apoptosis bak genes, and protein levels of p21Waf1/Cip1, p27Kip1, cyclinD3, CDK4, and Cleave-caspase3 were increased by higher concentrations of sodium butyrate (1, 5, 10 mM), and the levels of cyclinD1 and CDK6 were reduced by 5 and 10 mM butyrate. Butyrate increased the phosphorylated form of the signaling molecule p38 and phosphorylated JNK. In conclusion, the present in vitro study indicated that sodium butyrate inhibited the proliferation of IPEC-J2 cells by inducing cell cycle arrest in the G0/G1 phase of cell cycles and by increasing apoptosis at high concentrations.     

The isolation of HTC variant cells which can replicate in butyrate. Changes in histone acetylation and tyrosine aminotransferase induction

We have obtained a number of variant HTC cells which are capable of vigorous replication in the presence of 6 mM sodium butyrate. These cells show characteristic changes in histone acetylation. H2A/H2B are no longer modified and the turnover of histones H3/H4 acetate is about 4-fold greater than in control HTC cells at the same butyrate concentration. Histone deposition continues successfully even though histones H3/H4 become hyperacetylated upon association with the chromatin. Prompt deacetylation of new histones does not appear to be a prerequisite for successful deposition processes. Initial enzymatic studies indicate that not only do the butyrate-resistant cells show an increased deacetylase activity (on a per cell basis), but also the enzyme is less sensitive to sodium butyrate under in vitro assay conditions. In contrast to control HTC cells in 6 mM butyrate in which dexamethasone induction of tyrosine aminotransferase is inhibited, the butyrate-resistant variant cells are capable of tyrosine aminotransferase induction even in the presence of butyrate. The implications of these observations are discussed.     

Effect of sodium butyrate on S-100 protein levels and the cAMP response

Sodium butyrate (NaB), when added to cell cultures, produces a variety of morphological and biochemical changes. We examined its effects, in nM concentrations, on the expression of two glioma cell-associated proteins, glial fibrillary acidic protein (GFAP) and S-100 protein in human glioma-derived cell line (RF), and of S-100 protein in the C6 rat glioma cell line. GFAP levels decreased by about 50% in the RF cell line, and S-100 protein levels decreased protein levels decreased by about 40% after treatment with 1 mM NaB for 48 h. In the C6 rat glioma cell line, isoproterenol with theophylline was found to increase S-100 levels by two-fold over basal levels. NaB was found to inhibit the induction of S-100 protein but exhibited no effect on the basal levels of the protein. Other short chain fatty acids, including sodium propionate and sodium isobutyrate, exhibited partial inhibitory activity. NaB, at an EC50 of 1 mM, was also found to inhibit both the beta-adrenergic and the forskolin-mediated increase in cAMP levels in these cells. This suggests that NaB may inhibit cells from expressing S-100 protein by attenuating cAMP levels.     

Modulation of thyroid hormone nuclear receptors by short-chain fatty acids in glial C6 cells. Role of histone acetylation

We have studied the effect of butyrate and other short-chain fatty acids on thyroid hormone nuclear receptors in C6 cells, a rat glioma cell line. Exposure of C6 cells to butyrate leads to increased levels of L-triiodothyronine (T3) in the nuclear and extranuclear compartments. The rise in nuclear binding is not merely a reflection of the higher cellular hormone content, and Scatchard analysis of T3 binding to isolated nuclei reveals that butyrate increases receptor number without changing affinity. The effect on the receptor is quantitatively important: a 48-h incubation with 2 mM butyrate increases nuclear binding by 2-3-fold, and 5 mM butyrate by 3-5-fold. Other short-chain fatty acids were found to similarly influence both nuclear receptor and extranuclear T3 levels with the following potency: butyrate greater than valerate greater than propionate greater than acetate. On the contrary, ketone bodies were ineffective. Butyrate increases receptor levels by decreasing receptor degradation, since the apparent t1/2 of receptor disappearance increased by approximately 3-fold in cells incubated with 2 mM butyrate for 48 h. The regulation of receptor number might be secondary to an action of butyrate on regions of the chromatin to which the receptor associates. We then examined the effect of butyrate on histone acetylation. The fatty acid had little effect in increasing the level of multiacetylated forms of H3 and H4 histone when studied in acid-urea gels, but it markedly inhibited the turnover of [3H] acetate from the histone fraction. There was a striking similarity in the dose-response of butyrate for increasing receptor levels and inhibiting histone deacetylation. Furthermore, a very close correlation between receptor levels and [3H]acetate release was also found when different short-chain fatty acids were used. We thus conclude that the effect of butyrate on the receptor could be explained by a modification of the chromatin structure of C6 cells secondary to acetylation.     

Effect of sodium butyrate on the production of serotonin, histamine and glycosaminoglycans by cultured murine mastocytoma cells

Effect of sodium butyrate on the cellular serotonin, histamine and glycosaminoglycans (GAGs) contents of mastocytoma p-815 cells was examined. When mastocytoma p-815-4 cells were cultured for 4 days in the presence of butyrate at 2 mM, the optimal concentration for the induction of granulopoiesis in this cell line, the cellular serotonin, histamine and GAGs contents increased markedly: serotonin increased almost 7 times, histamine 140 times and total GAGs 10 times. Chondroitinase ABC-resistant GAGs, heparin and/or heparan sulfate, increased 21 times. These cellular products reached at 3 or 4 days culture their maximal levels which depended on the amount of butyrate added up to 2 mM. The effectiveness of butyrate varied among the cloned cell lines: serotonin and histamine contents did not increase in mastocytoma p-815-6 cells, in which butyrate failed to induce granulopoiesis.     

Sodium butyrate stimulates the synthesis of firefly luciferase in transfected CHO cells but levels of BiP chaperone are unaffected

A stably transfected CHO cell line (LUCLEAD) was used where the coding region of native Firefly luciferase was linked to the 3'-UTR of the bovine growth hormone, and the 5'-nucleotides coding for the albumin signal peptide were linked to the N-terminal end of the luciferase coding region. Incubation of cells with 1 or 2 mM sodium butyrate (SB) for 72 h had no effect on cell growth since cultures reached confluency at the same time as control cells. Although cell cultures incubated with SB at a concentration of 4 mM were only about 60% confluent the luciferase content was about 5-fold higher than that in control cells. Cells incubated with either 1 or 2 mM SB showed intermediate levels of luciferase content. The amount of the chaperone BiP in the cells was not affected by incubation with SB. The results indicate that SB can be used to effectively promote synthesis of recombinant luciferase.     

Associated effects of sodium butyrate on histone acetylation and estrogen receptor in the human breast cancer cell line MCF-7

Sodium butyrate at a concentration of 5mM causes significant hyperacetylation of the core histones in the human breast cancer cell line MCF-7. Histone hyperacetylation was achieved in rapidly-growing cells at 40% confluency after 24 hours in 5mM sodium butyrate. More nearly confluent cells did not reach as high a level of histone hyperacetylation. Upon assaying the estrogen receptors, both cytosolic and KCl-extractable nuclear, we found that butyrate treatment had lowered the estrogen receptor levels in both compartments. To our knowledge this is the first report of an effect of sodium butyrate on estrogen receptor levels.     

Erythroid Differentiation of K562 Cells Resistant to 2-(4"-Dimethylaminostyryl)quinoline-1-Oxide or 4-Nitroquinoline 1-Oxide Is Considerably Induced after Thymidine Treatment

We studied the effect of certain differentiation inducers—thymidine, sodium butyrate, and dimethyl sulfoxide—on the cells of parental line K562 and the derived sublines resistant to quinoline xenobiotics 2-(4-dimethylaminostyryl)quinoline 1-oxide or 4-nitroquinoline 1-oxide. The cells of the both derived sublines demonstrate cross-resistance to these xenobiotics, while the subline resistant to 2-(4"-dimethylaminostyryl)quinoline 1-oxide is also resistant to ethidium bromide and colchicine. Treatment of cells of the sublines with thymidine but neither sodium butyrate nor dimethyl sulfoxide for 2 or 4 days considerably increases intracellular content of hemoglobin as compared to the parental cells K562. The revealed effect is due to increased hemoglobin content in the cells rather than to the increased proportion of hemoglobin-containing cells, which results from decreased proliferation rate observed in all cases.     

Biochemical studies on cell fusion. I. Lipid composition of fusion- resistant cells

A series of stable cell mutants of mouse fibroblasts were previously isolated (Roos, D. S. and R. L. Davidson, 1980, Somatic Cell Genet., 6:381-390) that exhibit varying degrees of resistance to the fusion-inducing effect of polyethylene glycol (PEG), but are morphologically similar to the parental cells from which they were derived. Biochemical analysis of these mutant cell lines has revealed differences in whole cell lipid composition which are directly correlated with their susceptibility to fusion. Fusion-resistant cells contain elevated levels of neutral lipids, particularly triglycerides and an unusual ether-linked lipid, O-alkyl, diacylglycerol. This ether lipid is increased approximately 35-fold over parental cells in the most highly PEG-resistant cell line. Fusion-resistant cells also contain more highly saturated fatty acyl chains (ratio of saturated to polyunsaturated fatty acids [S/P ratio] approximately 4:1) than the parental line (S/P ratio approximately 1:1). Cells which are intermediate in their resistance to PEG have ether lipid and fatty acid composition which is intermediate between the parental cells and the most fusion-resistant mutants. In a related communication (Roos, D. S. and P. W. Choppin, 1985, J. Cell. Biol., 100:1591-1598) evidence is presented that alteration of lipid content can predictably control the fusion response of these cells.     

Production of analytical quantities of recombinant proteins in Chinese hamster ovary cells using sodium butyrate to elevate gene expression.

Sodium butyrate was used to enhance expression levels and thereby facilitate the generation of analytical quantities of nine different tissue plasminogen activator (tPA) analogues expressed under the control of the cytomegalovirus immediate early (CMV IE) promoter by the Chinese hamster ovary (CHO) mammalian expression system. Production involved growth in roller bottles, using serum free or low serum media formulations, together with repetitive, sodium butyrate inductions. Average inductions in the presence of sodium butyrate ranged from 2 to 9-fold relative to uninduced controls, using cell lines with no previous butyrate exposure. Retardation of growth rate by butyrate minimized the need to split cells during the production runs, extending longevity of roller bottles containing cells secreting at induced levels. SDS-PAGE analyses indicate a consistently high percentage of single-chain material. Measurements of specific activity and fibrinogen fragment enhancement for one of the analogues demonstrate that neither of these two critical parameters are affected by production in the presence of butyrate. Induction kinetic data and growth curves for the expression of sCD4 under control of the SV40 early promoter demonstrate that the benefits of butyrate can be realized with different promoters and heterologous genes, and are additive when used in conjunction with an amplified cell line constitutively expressing at elevated levels. This work demonstrates the practical application of sodium butyrate in the production of analytical quantities of protein from the CHO expression system, and suggests a role for sodium butyrate in commercial scale processes as well.     

Butyrate metabolism upstream and downstream acetyl-CoA synthesis and growth control of human colon carcinoma cells.

Butyrate is a short chain fatty acid (SCFA) produced by bacterial fermentation of dietary fibers in the colon lumen which severely affects the proliferation of colon cancer cells in in vitro experiments. Although butyrate is able to interfere with numerous cellular targets including cell cycle regulator expression, little is known about butyrate metabolism and its possible involvement in its effect upon colon carcinoma cell growth. In this study, we found that HT-29 Glc-/+ cells strongly accumulated and oxidized sodium butyrate without producing ketone bodies, nor modifying oxygen consumption nor mitochondrial ATP synthesis. HT-29 cells accumulated and oxidized sodium acetate at a higher level than butyrate. However, sodium butyrate, but not sodium acetate, reduced cell growth and increased the expression of the cell cycle effector cyclin D3 and the inhibitor of the G1/S cdk-cyclin complexes p21/WAF1/Cip1, demonstrating that butyrate metabolism downstream of acetyl-CoA synthesis is not required for the growth-restraining effect of this SCFA. Furthermore, HT-29 cells modestly incorporated the 14C-labelled carbon from sodium butyrate into cellular triacylglycerols and phospholipids. This incorporation was greatly increased when D-glucose was present in the incubation medium, corresponding to the capacity of hexose to circulate in the pentose phosphate pathway allowing NADPH synthesis required for lipogenesis. Interestingly, when HT-29 cells were cultured in the presence of sodium butyrate, their capacity to incorporate 14C-labelled sodium butyrate into triacylglycerols and phospholipids was increased more than twofold. In such experimental conditions, HT-29 cells when observed under an electronic microscope, were found to be characterized by an accumulation of lipid droplets in the cytosol. Our data strongly suggest that butyrate acts upon colon carcinoma cells upstream of acetyl-CoA synthesis. In contrast, the metabolism downstream of acetyl-CoA [i.e. oxidation in the tricarboxylic acid (TCA) cycle and lipid synthesis] likely acts as a regulator of butyrate intracellular concentration.     

Expression of rat liver glutathione-S-transferase GSTA5 in cell lines provides increased resistance to alkylating agents and toxic aldehydes

The glutathione-S-transferases (GST) are a major contributor to the eukaryotic cell's defences against chemical and oxidative stress. However, the role of individual GST isoenzymes in conferring resistance to xenobiotics has not been fully determined. We have examined the effect of the rat GSTA5 isoenzyme in the detoxication of alkylating agents and aldehydes by constructing a cell line in which it is stably expressed. The hamster fibroblast cell line V79 was transfected with a construct expressing GSTA5 from the CMV promoter. A stable clone (V79-GSTA5) was isolated after selecting for the neomycin phosphotransferase gene present on the introduced DNA. The cell line showed significantly increased levels of resistance towards the alkylating agents chorambucil and melphalan. Levels of resistance were 4-6-fold greater in V79-GSTA5 cells than in control cells. Increased levels of resistance were also observed towards the lipid peroxidation product acrolein (IC(50)=80 microM compared with 17 microM in control cells). The V79-GSTA5 cells also showed a 4-fold increase in resistance to trans, trans muconaldehyde (IC(50)=4 micro compared with l microM for control cells). GSTA5 did not protect against 4-hydroxynonenal, but it did provide greater levels of protection to hydrogen peroxide, with an IC(50) of 380 microM in V79-GSTA5 compared with 180 microM in control cells. In contrast, V79-GSTA5 cells were more sensitive to methyl glyoxal, suggesting that a methyl glyoxal-glutathione conjugate is more toxic that the parental compound. These data contribute towards the evaluation of the role of GSTA5 in the detoxication of these compounds.     

Induction of blood coagulation factor VIII by sodium butyrate in Balb/c 3T3 cells

Balb/c 3T3 cells have long been used in cell biological studies. When cells were treated with 2 mM sodium butyrate they became flattened and their nuclei became small relative to the cytoplasm. Electron micrographically, intracellular organellae developed significantly: the number of mitochondria increased and membranous structures, namely the rough-surfaced endoplasmic reticulum and Golgi apparatus, developed markedly. Blood coagulation factor VIII was induced in the presence of 2 mM sodium butyrate in a cell sonicate, but not in culture medium. The amount of this factor was almost 100-fold that in the nontreated cells 4 days after culture initiation, indicating that the cloned Balb/c 3T3 cells were of endothelial origin.     

Spectroscopic and kinetic evidence for a cyclic geminal diamine intermediate in the reaction of O-aminoserine with pyridoxal 5'-phosphate in alkali

HT-29, a cell line derived from a human colon carcinoma, exhibits very low alkaline phosphatase activity. The enzyme is thermolabile and is of the intestinal type. Hyperosmolality and/or sodium butyrate induce increased levels of activity. The increase is most pronounced with HT-29 cells growing in hyperosmolar medium containing sodium butyrate. Under these conditions specific activity rises over 1000-fold. The effect of hyperosmolality is blocked by cycloheximide and that of sodium butyrate by thymidine, cordycepin, and cycloheximide. By contrast to other human cancer cell lines, the enzyme of HT-29 is not influenced by cell density and glucocorticoid hormones. 5-Bromo-2′-deoxyuridine and inhibitors of DNA synthesis cause a slight increase in specific activity.     

Effect of apolipoprotein E-free high density lipoproteins on cholesterol metabolism in cultured pig hepatocytes

We studied cholesterol synthesis from [14C]acetate, cholesterol esterification from [14C]oleate, and cellular cholesterol and cholesteryl ester levels after incubating cells with apoE-free high density lipoproteins (HDL) or low density lipoproteins (LDL). LDL suppressed synthesis by up to 60%, stimulated esterification by up to 280%, and increased cell cholesteryl ester content about 4-fold. Esterification increased within 2 h, but synthesis was not suppressed until after 6 h. ApoE-free HDL suppressed esterification by about 50% within 2 h. Cholesterol synthesis was changed very little within 6 h, unless esterification was maximally suppressed; synthesis was then stimulated about 4-fold. HDL lowered cellular unesterified cholesterol by 13-20% within 2 h and promoted the removal of newly synthesized cholesterol and cholesteryl esters. These changes were transient; by 24 h, both esterification and cellular unesterified cholesterol returned to control levels, and cholesteryl esters increased 2-3-fold. HDL core lipid was taken up selectively from 125I-labeled [3H]cholesteryl ester- and ether-labeled HDL. LDL core lipid uptake was proportional to LDL apoprotein uptake. The findings suggest that 1) the cells respond initially to HDL or LDL with changes in esterification, and 2) HDL mediates both the removal of free cholesterol from the cell and the delivery of HDL cholesteryl esters to the cell.     

Histone deacetylation in nuclei isolated from hepatoma tissue culture cells. Inhibition by sodium butyrate.

Nuclei from hepatoma tissue culture (HTC) cells were isolated by standard methods and incubated in media commonly used for nuclease digestions (DNAase I and micrococcal nuclease) and for in vitro RNA synthesis. During the incubation, histones can be deacetylated from both control cells and cells treated with 6 mM sodium butyrate to enhance the levels of histone acetylation. Deacetylation of histone is much more apparent in nuclei isolated from sodium butyrate-treated cells. Inclusion of 6 mM sodium butyrate in the incubation medium effectively inhibits the endogenous deacetylase activity acting on histones H3 and H4, whereas sodium acetate at the same concentration has very little inhibitory effect.