A study of adenosine 3':5'-cyclic monophosphate, sodium butyrate and cortisol as inducers of HeLa alkaline phosphatase

The role of adenosine 3′:5′-cyclic monophosphate in the cortisol-mediated induction of HeLa 65 alkaline phosphatase was investigated. Although growth of these cells with 0.5–1.0 mmN₆,O²′-dibutyryl adenosine 3′:5′-cyclic monophosphate induces a 5- to 8-fold increase in cellular phosphatase activity after 72 hr, neither cAMP nor theophylline induce at concentrations up to 1 mm. Sodium butyrate induces the enzyme as well as dibutyryl cAMP. Moreover, induction kinetics show sodium butyrate to be a more efficient inducer than dibutyryl cAMP, inducing activity as quickly as cortisol. This suggests that the butyric acid cleaved from dibutyryl cAMP by HeLa cells is the mediator of induction when the cyclic nucleotide derivative is used.     

Effects of short chain fatty acid,sodium butyrate,on osteoblastic cells and osteoclastic cells

• 1.1. Sodium butyrate increased alkaline phosphatase (ALP) activity of cloned osteoblastic cell line MC3T3-El by the stimulation of de novo enzyme synthesis.
• 2.2. Sodium butyrate did not affect mature osteoblastic cells but affected preosteoblastic cells.
• 3.3. Sodium butyrate decreased tartrate resistant acid phosphatase (TRACP)-positive multinucleated cells (MNC) formation from bone marrow cells. This related to the cytotoxicity of sodium butyrate on bone marrow cells.

     

Mitotic responses of the anterior pericardial wall of Biomphalaria glabrata (Mollusca) subjected to challenge

Using light microscope autoradiography and electron microscopy we studied the effect of juvenile hormone III (JHIII) and β-ecdysone insect molting hormone (β-ecd) on the replication of Tipula iridescent virus (TIV) in suspension cultured cells of Estigmene acrea. JHIII at a concentration of 87.5 μg/ml completely inhibited viral DNA synthesis, but upon removal of JHIII, [³H]thymidine was incorporated into the cytoplasm as detected by autoradiography and virions in developmental stages from the same cell samples were-readily seen by electron microscopy. β-ecd at a concentration of 17.5 μg/ml, unlike JHIII, permitted viral DNA synthesis in the presence of the hormone although at a reduced level when compared to TIV-infected cells. But the presence of β-ecd seemed to prevent capsid formation, although islands similar in fine structure to those of viroplastic centers were seen by electron microscopy. Once β-ecd was removed from the medium, TIV-inoculated cells appeared to synthesize new virions in a normal pattern. Both hormones inhibited host cell DNA synthesis in noninfected cells.     

The activation of KSHV lytic cycle blocks autophagy in PEL cells

This study confirms that autophagy is activated concomitantly with KSHV lytic cycle induction, and that autophagy inhibition by BECN1 knockdown reduces viral lytic gene expression. In addition, we extend previous observations and show that autophagy is blocked at late steps, during viral replication. This is indicated by the lack of colocalization of autophagosomes and lysosomes and by the LC3-II level that does not increase in the presence of bafilomycin A₁ in primary effusion lymphoma (PEL) cells induced to enter the lytic cycle, either by TPA/sodium butyrate (BC3 and BCBL1) or by doxycycline (TRExBCBL1-Rta). The autophagic block correlates with the downregulation of RAB7, whose silencing with specific siRNA results in an autophagic block in the same cells. Finally, by electron microscopy analysis, we observed viral particles inside autophagic vesicles in the cytoplasm of PEL cells undergoing viral replication, suggesting that they may be involved in viral transport.     

Virus DNA replication and protein synthesis in Amsacta moorei entomopoxvirus-infected Estigmene acrea cells

Amsacta moorei entomopoxvirus DNA synthesis was detected in Estigmene acrea cells by [³H]thymidine incorporation 12 hr after virus inoculation. Hybridization of ³²P-labeled Amsacta entomopoxvirus DNA to the DNA from virus-infected cells indicated that viral-specific DNA synthesis was initiated between 6 and 12 hr after virus inoculation. A rapid increase in the rate of virus DNA synthesis was detected from 12 to 24 hr after virus inoculation. Amsacta entomopoxvirus protein biosynthesis in E. acrea cells was studied by [su35S]methionine incorporation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Extracellular virus and virus-containing occlusion bodies were first detected in virus-infected cell cultures 18 hr after virus inoculation. Thirty-seven virus structural proteins, ranging in molecular weight from 13,000 to 208,000 were detected in both occluded and nonoccluded forms of the virus. The biosynthesis of virus structural proteins increased rapidly from 18 to 34 hr after infection. A major viral-induced protein corresponding in molecular weight to viral occlusion body protein (110,000) was detected approximately 24 hr after virus inoculation.     

Cellular functions are required for the synthesis and integration of avian sarcoma virus-specific DNA.

We have used two experimental strategies to test the role of cellular functions in the synthesis and integration of virus-specific DNA in cells infected by avian sarcoma virus.First, quail embryo fibroblasts, placed in stationary phase (G₀) by prolonged serum starvation, did not support the efficient synthesis of viral DNA during the first 24–48 hr after infection. Synthesis of viral DNA was impaired according to at least two parameters: the amount of DNA was diminished, particularly the amount of the plus-strand DNA (identical in polarity to the viral genome); and the length of both minus and plus strands was reduced in the stationary cells. In parallel cultures fed with fresh serum, over two thirds of the cells were able to reenter the cell cycle within 24 hr, and viral DNA of normal size was synthesized.Second, density labeling of viral and cellular DNA with BUdR was used to determine whether cellular DNA synthesis was required for integration of viral DNA. In both quail embryo fibroblasts released from G₀ by serum replacement and randomly growing duck embryo fibroblasts, viral DNA was integrated only into cellular DNA replicated during the infection.Our results indicate that serum-starved cells lack a factor (or factors) required for the efficient and complete synthesis of ASV-specific DNA. We have not been able to establish whether such factor(s) are present in growing cells only during S phase. Integration of viral DNA appears to require cellular DNA synthesis; this may be due to a requirement for a factor (or factors) present in adequate concentration only during S phase or to a requirement for the structural changes in cellular DNA that accompany replication.     

Studying the enucleation process, DNA breakdown and telomerase activity of the K562 cell lines during erythroid differentiation in vitro

During erythropoiesis, some organelles such as mitochondria and nucleus are lost by autophagy and enucleation processes in the presence of macrophages in vivo. In vitro production of erythrocytes has raised many questions about the mechanism of enucleation. The aim of this work was to study the DNA breakdown, enucleation, hemoglobin synthesis and telomerase activity of K562 cells during erythroid differentiation. For these purposes, K562 cells were induced to differentiate by erythropoietin + rhGM-CSF, DMSO, and sodium butyrate separately up to 14 d. In different time intervals, hemoglobin synthesis was evaluated by benzidine staining and RT-PCR for γ-globin gene expression. DNA breakdown was analyzed by 4′,6-diamidino-2-phenylindole (DAPI) staining, DNA ladder electrophoresis and comet assay. The telomerase activity was evaluated by TRAP assay. Our result indicated that, sodium butyrate and DMSO inhibited K562 cell growth about 50–60% in comparison to untreated control cells. The percentage of benzidine-positive cells was about 45% in the presence of sodium butyrate after 10 d. Densitometric analysis of RT-PCR and calculated data indicated a 1.5-fold increase in relative γ-globin gene expression at 96 h, in the presence of 1 mM sodium butyrate in comparison with untreated cells. DAPI staining did not reveal any evidence of internal lysis of the nucleus during erythroid differentiation at first wk, but this was obvious in the second wk. DNA laddering pattern was not observed in differentiated cells during 14 d. In comet assay, the percentage of DNA in tail, tail length, and tail moment were significantly different between untreated and treated cells (p?<?0.05). Telomerase activity was inhibited up to 90.3% during erythroid differentiation of these cells.     

Restricted infectivity of ecotropic type C retroviruses in mouse teratocarcinoma cells: Studies on viral DNA intermediates

Replication of Gross strain N-tropic type C retrovirus was markedly restricted in a pluripotential undifferentiated embryonal cell line (PCC₄) of murine teratocarcinoma, whereas the same virus could cause productive infection in a myoblast-derived differentiated line (PCD₁) of the same tumor origin. To investigate the restriction mechanism, we compared the initial viral DNA formation in these two cell lines. Analyses by means of a modified Hirt extraction procedure and a modified Southern gel transfer method indicated that PCC₄ and PCD₁ cells supported the synthesis of viral DNA intermediates after inoculation of the Gross virus. In both cells, a linear DNA duplex (form III viral DNA) appeared at 4 hr, reached a maximal level at 8–9 hr, and declined rapidly thereafter, while two closed-circular supercoiled DNA duplexes (form I viral DNA) showed their appearance, increase and decline in the 8–24 hr period. During the period from 34 to 78 hr after virus inoculation, another burst of viral DNA synthesis occurred in PCD₁ cells, presumably due to secondary virus infection, while at this period both form III and form I viral DNAs became undetectable in PCC₄ cells. The Hirt supernatant DNAs prepared from PCD₁ and PCC₄ cells 10 hr after virus inoculation were equally infectious for NIH3T3 cells in a DNA transfection assay. Both PCD₁ and PCC₄ cells were very poor recipients for DNA transfection, although one positive result with PCD₁ cells might suggest a difference between the two cell types in this aspect. These results indicate that restriction of type C retrovirus in undifferentated embryonl carcinoma cells occurs at a step subsequent to formation and maturation of viral DNA intermediates.     

The enhancement of phase 2 enzyme activities by sodium butyrate in normal intestinal epithelial cells is associated with Nrf2 and p53

Dietary fiber fermentation by the colonic bacterial flora produces short-chain fatty acids, acetate, propionate and butyrate. Among them, butyrate is considered to be the major energy substrate for colonocytes and, at least in rats, seems to protect against colonic carcinogenesis. In this study, we examined the effect and the mechanisms of short-chain fatty acids on the activity of phase 2 enzymes. Sodium butyrate increased phase 2 enzyme activities in normal rat small intestine epithelial cells, Glutathione S-transferase and NAD(P)H:quinone oxidoreductase (NQO) in a dose-dependent manner_; however, other short-chain fatty acids did not increase them. The mechanism of the induction of phase 2 enzymes with sodium butyrate sodium butyrate, but not other short-chain fatty acids was related to the increase of NF-E2-related factor 2 (Nrf2) nuclear translocation and the decrease in the levels of nuclear fraction p53. Sodium butyrate also caused enhancement of Nrf2 mRNA levels and suppression of p53 mRNA levels. Sodium butyrate enhances the activities of phase 2 enzymes via an increase in the Nrf2 protein levels in the nucleus and a decrease in the mRNA and protein levels of p53.     

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.     

Induction of alkaline phosphatase and the glycoprotein hormone α subunit in HeLa cells by inhibitors of DNA polymerase

Levels of the glycoprotein hormone α subunit and alkaline phosphatase activity were increased in cultures of HeLa S₃ cells exposed to aphidicolin (0.2–10 μg/ml) or phosphonoformic acid (0.1–3 mm), inhibitors of DNA polymerase α. Induction was dependent on both the concentration and duration of exposure to the inhibitors and was prevented by cycloheximide and actinomycin D. Limited characterization of the induced α subunit and alkaline phosphatase activity suggest that they are similar to the uninduced proteins expressed by this cell line. Induction of both proteins by aphidicolin and phosphonoformic acid was enhanced by the simultaneous addition of 3 mm sodium butyrate but was depressed by 1 mm hydroxy urea. In contrast, both butyrate and hydroxy urea cause induction of these proteins when added alone to HeLa cultures. It is unlikely that a direct relationship exists between protein induction and the inhibition of DNA synthesis produced by aphidicolin and phosphonoformic acid since the concentrations required to produce half-maximal induction are 5 to 10 times greater than those needed to inhibit replication by 50%.     

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.     

Effects of cycloheximide on virus RNA replication in an inducible line of polyoma-transformed rat cells.

In this article, we describe two distinct effects of cycloheximide (CH), a potent inhibitior of protein synthesis, on the replication of polyoma virus (PV) DNA in an inducible line of PV-transformed rat cells (LPT cells). Exposure of LPT cells to CH causes up to an 8 fold increase in the cellular concentration of PV DNA determined by molecular hybridization. The same treatment inhibits cell division and chromosomal DNA replication. However, the amount of chromosomal DNA per cell is not affected by the drug. In LPT cells treated with mitomycin C (MMC), PV DNA replication is enhanced after 7 hr. During the period extending from 7 hr to 24 hr, the concentration of virus DNA increases at least 100 fold. CH added to the cells 0-7 hr after treatment with MMC inhibits the replication of PV DNA by 90-100%. The inhibition is less effective in cells exposed to CH from 7 hr and on. The inhibitory effect is reversible: virus DNA synthesis is resumed after removal of CH from the growth medium. Thus CH acts as an inducer of virus DNA synthesis in cells whose resident viral genome is repressed, but inhibits the autonomous replication of the activated genome following induction with MMC.     

Lack of effect of butyrate on S phase DNA synthesis despite presence of histone hyperacetylation

The effects of sodium butyrate on [³H]thymidine incorporation and cell growth characteristics in randomly growing and synchronized HeLa S₃ cells have been examined in an attempt to determine what effects, if any, butyrate has on S phase cells. Whereas 5 mM sodium butyrate rapidly inhibits [⁵H]thymidine incorporation in a randomly growing cell populations, it has no effect on incorporation during the S phase in cells synchronized by double thymidine block techniques. This lack of effect does not result from an impaired ability of the S phase cells to take up butyrate, since butyrate administration during this period leads to histone hyperacetylation that is identical with that seen with butyrate treatment of randomly growing cells. Furthermore, the ability to induce such hyperacetylation with butyrate during an apparently normal progression through S phase indicates that histone hyperacetylation probably has no effect on the overall process of DNA replication. Temporal patterns of [³H]thymidine incorporation and cell growth following release from a 24-h exposure to butyrate confirm blockage of cell growth in the G1 phase of the cell cycle. Thus, the inhibition by butyrate of [³H]thymidine incorporation in randomly growing HeLa S₃ cell populations can be accounted for solely on the basis of a G1 phase block, with no inhibitory effects on cells already engaged in DNA synthesis or cells beyond the G1 phase block at the time of butyrate administration.     

Cancer cell sensitization to fas-mediated apoptosis by sodium butyrate

Cancer cells often resist Fas-mediated apoptosis even when the Fas receptor is expressed at the cell surface. We show here that human and rat colon cancer cells undergo massive apoptosis when they are exposed to soluble Fas ligand in the presence of sodium butyrate, an agent that induces by itself only a low rate of apoptosis. Sodium butyrate potentiates Fas-dependent apoptosis in seven out of eight colon cancer cell lines. Sodium butyrate does not increase Fas receptor cell surface expression and does not modify cell levels of Bcl-2, Bcl-xL, Bcl-xS and Bax. Sodium butyrate also induces tumor cell sensitization to the apoptotic effect of the combination of TNF-alpha and IFN-gamma, but it does not modify the level of the FADD/Mort1 adaptator molecule, at the connection between Fas- and TNF-dependent apoptosis pathways. Because the clinical toxicity of butyrate is low, its ability to enhance Fas-signal delivery in cancer cells could be of therapeutic interest.     

Selective inhibition by sodium butyrate of glucocorticoid-induced tyrosine aminotransferase synthesis in hepatoma tissue-cultured cells

Sodium butyrate in a 5 mM concentration prevents the induction of tyrosine aminotransferase in hepatoma culture cells, without affecting the basal level of the enzyme. This effect is reversible immediately after the removal of butyrate, or after a lag, if butyrate was present for more than 2 h. Neither the amount of cellular RNA nor the rate of total RNA synthesis were affected by sodium butyrate. Furthermore, butyrate does not inhibit protein synthesis: [35S]methionine incorporation into proteins, measured in a reticulocyte lysate system, shows no significant difference between the translation capacity of the RNAs from butyrate-treated cells and from dexamethasone-induced or uninduced cells. Nevertheless, when tyrosine aminotransferase was isolated from the translation products by its specific antiserum and analyzed by gel electrophoresis, we observed that the amount of the enzyme synthetized in the presence of RNAs from dexamethasone/butyrate-treated cells was strongly diminished relative to that synthesized in the presence of RNA from dexamethasone-induced cells. These experiments indicate that the treatment of the cells with butyrate decreases the activity of the specific messenger RNA for tyrosine aminotransferase to a level close to the basal level.     

Sodium butyrate selectively inhibits host cell glycoprotein synthesis in human fibroblasts infected with cytomegalovirus

Host cell as well as viral DNA synthesis in human fibroblasts infected with human cytomegalovirus was found to be largely resistant even to high concentrations sodium butyrate. Likewise, production of viral progeny was reduced by 1–2 orders of magnitude but not abolished. On the other hand, the drug allowed (modified) glycosylation only of viral polypeptides whereas t h a t of host proteins was suppressed. Immunofluorescence studies on living cells suggested that butyrate may interfere with processing and intracellular transport of virus-specific surface membrane antigens.     

Induction of cellular transglutaminase biosynthesis by sodium butyrate

Cellular transglutaminase activity was induced in simian virus-transformed human embryonic lung fibroblasts (WI-38 VA13A) by sodium butyrate. The level of enzyme activity approached a maximum by 6 days; 9–11-fold higher in the presence of sodium butyrate (1 mM) than in its absence. The observed increases in cellular transglutaminase activity could be entirely accounted for by equivalent increases in the levels of enzyme protein measured by inhibition enzyme-linked immunosorbent assay. Sodium butyrate also increased the rate of enzyme synthesis, but had no effect on the rate of cellular transglutaminase degradation. The increase in the rate of enzyme synthesis was matched by an increased level of translatable transglutaminase mRNA as measured in a cell-free translation system. Our results suggest that sodium butyrate regulates cellular transglutaminase at the pretranslational level.     

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.