Quantitative analysis of histone H1 degrees protein synthesis in HTC cells.

H1 degrees, a member of histone H1 family associated with cell growth arrest and differentiation, is barely expressed in most mammalian cells in culture. Depending on the cell type, serum deprivation or drugs, such as sodium butyrate, significantly increase H1 degrees mRNA level and H1 degrees protein accumulates. However, probably because of a lack of a simple quantitative procedure, little is known about the relationship between H1 degrees mRNA content and its effective translation rate. Using a rat hepatoma cell line and sodium butyrate as a model system, we attempted to evaluate this in different cellular conditions by measuring H1 degrees synthesis with a rapid quantitative procedure we described previously. We found that although the amount of H1 degrees mRNA rapidly increased and then stabilized under sodium butyrate treatment, its transcription was delayed and H1 degrees protein was synthesized in a progressive wave. Butyrate removal from cell culture confirmed that mRNA level and protein synthesis were independently regulated, and provided evidence that sodium butyrate would not directly target the translation apparatus. In contrast, during the S phase of the cell cycle, H1 degrees gene transcription and protein synthesis were concomitantly activated. Taken together these data provide evidence that H1 degrees accumulation results from an increase of its synthesis and that, depending on conditions, a cell exhibits a H1 degrees translation efficiency which may or may not reflect the mRNA level.

A protein phosphatase is involved in the inhibition of histone deacetylation by sodium butyrate.

Treatment of cells with sodium butyrate is known to increase histone acetylation by inhibiting deacetylases. Here we have observed, in cultured hepatoma cells, that the potent serine-threonine phosphatase inhibitors, okadaic acid or calyculin A, inhibited phosphatase activity and concomitantly decreased the histone acetylation classically maintained by sodium butyrate. These results suggest that a protein phosphatase may mediate the sodium butyrate effect on deacetylases. Since we have previously found that such a protein would also mediate the sodium butyrate effect on gene expression, we propose that a phosphatase activity constitutes an early and essential step in the sodium butyrate-triggered signalling pathway.

The effects of sodium butyrate on transcription are mediated through activation of a protein phosphatase.

In this study we have investigated the molecular mechanism by which sodium butyrate modulates gene expression when added to cultured cells. As a model system we used hepatoma tissue culture cells in which sodium butyrate treatment increases histone H1(0) mRNA level and decreases c-myc mRNA level. Because we observed that stimulation of histone H1(0) gene expression could take place in the absence of protein neosynthesis, we hypothesized that sodium butyrate induced a post-translational modification of a factor involved in the transcription process. Using different types of well known kinase and phosphatase inhibitors, we studied the implication of kinase or phosphatase activity in this pathway. Interestingly, cell treatment with potent serine-threonine-phosphatase inhibitors, calyculin A or okadaic acid, prevented the regulation of both histone H1(0) and c-myc gene expressions by sodium butyrate. On the other hand, the tyrosine phosphatase inhibitor, vanadate, or the protein kinase C inhibitor, staurosporine, did not significantly modify sodium butyrate effects. Using protein phosphatase 1 and 2A for in vitro assays, we found a 45% increase of phosphatase activity after cell treatment by sodium butyrate, possibly due to a protein phosphatase 1-type protein phosphatase. These data strongly suggest that signaling pathway(s) triggered by sodium butyrate to modulate gene expression involve(s) a serine-threonine-phosphatase activity.

Characterization, purification and cDNA cloning of a rat perchloric-acid-soluble 23-kDa protein present only in liver and kidney.

A novel protein was extracted with 5% perchloric acid from rat liver and kidney. It is absent from other rat organs. Its apparent molecular mass is 23 kDa as determined by HPLC gel filtration. A single band, corresponding to 10 kDa, was observed after SDS/PAGE, suggesting that the protein consists of two subunits with similar molecular masses. This protein can neither be phosphorylated by ATP, nor acetylated. The sequence of the cDNA encoding this protein was determined. Southern-blot analysis showed that the corresponding gene spanned at least 10 kb and contained at least five introns. Zoo-blot analysis at medium stringency strongly suggests that the gene has been conserved during evolution. The amino-acid sequence of this protein with a highly conserved region is similar to that of a heat-shock protein.

[Molecular and cellular action of butyrate]. / Action moléculaire et cellulaire du butyrate.

Butyrate has a dramatic effect on transformed cells in culture. This effect disappears as soon as butyrate is removed from the medium. The other short chain fatty acids are much less effective. Butyrate produces an arrest of cell proliferation at the early G1 phase of the cell cycle. The effect is very general and may be used for cell growth synchronization. This compound increases the expression of the c-fos oncogene and inhibits the expression of c-myc in all phases of the cell cycle. Butyrate modulates the expression of several genes. In general it induces the expression of markers of cell differentiation. Many studies have been devoted to hemoglobin synthesis which is induced in erythroleukemia cells. In general it induces the synthesis of embryonic and of fetal hemoglobin, and delays and even suppresses the switch to adult hemoglobin, which could be useful for the treatment of sickle cell anemia and beta thalassemia. This effect of butyrate seems to require specific DNA regulatory sequences. Butyrate induces the synthesis of alkaline phosphatase, placental and intestinal isozymes, especially in cells where these syntheses are ectopic. It has the same effect on peptidic hormone syntheses and also on receptors of thyroid hormone and insulin. It stimulates their synthesis in cells which are poor in receptor and inhibits the synthesis in cells which have high amounts of these receptors. The use of antibiotics and of the run on method strongly suggest that butyrate acts at the transcriptional level. Butyrate inhibits the induction of proteins, including enzymes, by steroid hormones as has been shown for the induction of tyrosine aminotransferase by glucocorticoids, of ovalbumin and transferrin by estradiol in chick oviduct. Butyrate strongly alters cell morphology, usually it produces an enlargement of the cells with formation of protrusions. In HTC cells alteration of nucleoli and of the nuclear shape are observed. All these alterations are reversible and the cells recover the normal morphology upon removal of butyrate. These alterations result at least partly from modifications of the cytoskeleton: induction of vimentin and cytokeratin, formation of microfilaments, of microtubules and of actin fibers. The external matrix is also modified, as are the cell surface glycoproteins, and gangliosides. Most of these alterations are consistent with the loss of transformation characteristics of the cell. The mechanism of action of butyrate has been studied by many authors. It has been well established that butyrate induces an hyperacetylation of histones by inhibiting histone deacetylases, which is consistent with its stimulatory effect on gene expression.4+ and would require transacting proteins. The use of butyrate in therapeutics would require the synthesis of new molecules including butyrate but more active and metabolized at a slower rate. Several such molecules have been synthesized: monobutyrate 3 (or 6) monoacetate glucose, pivalyloxymethyl-butyrate. The use of such molecules in human therapeutics has been suggested, especially in hematology (sickle cell anemia, beta thalassemia) and in cancerology.

Sodium butyrate inhibits c-myc and stimulates c-fos expression in all the steps of the cell-cycle in hepatoma tissue cultured cells.

Sodium butyrate decreases the c-myc mRNA and increases the c-fos transcript level in HTC cells. This effect is independent of the cell-cycle phase. Actinomycin D suppresses the effect on c-fos. Cycloheximide increases both mRNA levels. Sodium butyrate suppresses the effect on c-myc and potentializes the effect on c-fos mRNAs. This suggests that sodium butyrate acts at the transcriptional level and that its effect does not result from the arrest of the cells at the G1 phase.

Increased level of the mitochondrial ND5 transcript in chemically induced rat hepatomas.

We have constructed a cDNA library from a hepatoma cell line (HTC cells) and isolated the clones corresponding to mRNAs present at a much higher level in hepatomas than in normal hepatocytes. The characterization of one of these clones is described in this paper. This clone is homologous to part of the mitochondrial ND5 gene (a subunit of NADH-ubiquinone oxidoreductase). The level of this mRNA was found increased in HTC cells and in hepatocytes from diethylnitrosamine-treated rats long before the development of tumors and strongly increased in carcinoma nodules as compared to hepatocytes from nontreated rats. Southern blot analysis showed a mitochondrial DNA heterogeneity in hepatomas with an alteration of the structure of part of the molecules.

RNAs containing mitochondrial ND6 and COI sequences present an abnormal structure in chemically induced rat hepatomas.

We have constructed a cDNA library prepared from an hepatoma cell line (HTC cells) and isolated a clone, pHT 13, which corresponds to mRNAs present at a much higher level in rat hepatomas than in normal hepatocytes. The sequence of the pHT 13 insert has been previously published (Nucleic Acids Res. 1988, 16,10935). This clone contains mitochondrial DNA sequences with an abnormal organization, since it includes part of the NADH dehydrogenase subunit 6 (ND6) and of the cytochrome oxidase subunit I (COI) genes separated by 230 bases instead of 9 kb in mitochondrial genome from normal hepatocytes. In this work we show (1) that RNAs homologous to this clone are present in hepatoma cells but not in normal hepatocytes, (2) that a 3 kb fragment of tumor mitochondrial DNA contains both the ND6 and the COI sequences. The abnormal structure of the DNA is confirmed by Southern blot analysis which shows that distinct types of mitochondrial DNAs are present in hepatoma cells.

DNA sequences homologous to mitochondrial genes in nuclei from normal rat tissues and from rat hepatoma cells.

Using specific probes we show that sequences homologous to NADH dehydrogenase Subunit 6, and Cytochrome oxidase Subunits I, II, and III mitochondrial genes are present in nuclear DNA from various tissues. These mitochondrial-like sequences are also present in rat hepatoma nuclear DNA but with an abnormal organization and a higher copy number than in normal hepatocytes.

Isolation and characterization of complementary DNA clones for genes overexpressed in chemically induced rat hepatomas.

In order to characterize the genes overexpressed in an hepatoma cell line, the HTC cells, and in diethylnitrosamine induced solid hepatomas, we constructed a complementary DNA library from HTC cells and performed differential screening with probes from HTC cells, from malignant nodules obtained 70 weeks after the carcinogen treatment, and from hepatocytes from normal rat liver. Eight clones corresponding to messenger RNAs (mRNAs) much more expressed in hepatomas than in hepatocytes from normal liver were isolated. Three, clones pHT 71, pHT 13, and pHT 26, were further analyzed by the study of their corresponding transcripts in hepatocytes from regenerating liver and in the hepatocytes from the nontumorous parts of the liver. Clone pHT 71 corresponds to a single 2.3-kilobase mRNA which is present in high levels in carcinoma nodules in hepatoma cell lines, in the nontumorous parts of the liver, and in hepatocytes isolated from regenerating liver 30 h after partial hepatectomy. Clone pHT 13 hybridizes with three distinct transcripts 3.8, 2.6, and 1.6 kilobases long. High levels of the 3.8- and 1.6-kilobase mRNAs are present in carcinoma nodules, in hepatoma cell lines, and in the nontumorous parts of the liver. However, the levels of these RNAs are similar in hepatocytes from regenerating liver and in hepatocytes obtained from normal rat liver. Clone pHT 26 corresponds to a 0.6-kilobase mRNA which exists at a high level only in cancer nodules and in hepatoma cell lines. We were unable to observe any cross-hybridization between these clones and the oncogenes which have been found to be expressed in hepatomas (c-fos, c-Ha-ras, c-Ki-ras, N-ras, and c-myc). The mRNAs corresponding to the three clones have not been detected in various tissues from normal adult rats. Our study shows that a high level of these mRNAs might be associated with rat liver carcinogenesis.

Changes in poly (A) + RNA translational pattern during chemically induced hepatocarcinogenesis.

Hepatocarcinoma was induced by administration of diethylnitrosamine to rats. The rats were sacrificed 70 weeks after the administration and the carcinoma nodules were separated from the perinodular parenchymental cells after perfusion of liver with collagenase. The in vitro translational pattern of mRNAs from hepatocellular carcinomas, from perinodular hepatocytes and from regenerating liver after partial hepatectomy were compared by one- and two-dimensional electrophoreses to the pattern obtained with RNA from normal hepatocytes. An increased synthesis of several peptides was observed with RNAs from carcinoma and from regenerating liver and to a lesser extent with RNA from perinodular hepatocytes, which suggests that the increase in synthesis is at least partly related to cell proliferation. A decreased synthesis of several other peptides was observed with RNA from carcinoma nodules and to a lesser extent with RNA from perinodular hepatocytes, but not with RNA from regenerating liver, which suggests that this decrease in synthesis is related to some transformation specific process. These changes are observed as soon as 22 weeks after carcinogen administration. These observations also suggest that at least part of the perinodular hepatocytes have some characteristics of the transformed cells.

Expression of c-fos oncogene during hepatocarcinogenesis, liver regeneration and in synchronized HTC cells.

We have studied the expression of c-fos gene in rat hepatoma induced by DENA. An increase of c-fos mRNA concentration was observed after 8 days, but the maximal 5- to 6-fold increase was observed after 70 weeks. This increase was found in perinodular hepatocytes as well as in cancer nodules. c-fos expression was also enhanced during liver regeneration at a period corresponding to cell proliferation. In HTC cells the arrest of the cell cycle at early G1 phase by addition of sodium butyrate was accompanied by a strong increase of c-fos gene expression. However the c-fos mRNA rapidly decreased after removal of sodium butyrate during the progression of the cells in the cell cycle and increased transiently when the cells entered again in G1 phase.

The 25 kDa protein kinase inhibitor present in liver cell is absent in fast growing HTC cells and is induced in sodium butyrate treated cells.

We have recently characterized a cAMP independent protein kinase inhibitor in rat liver. This inhibitor is absent or inactive in fast growing HTC cells and is induced according to exponential kinetics by sodium butyrate, a compound which arrests cell growth at the G1 phase of the cell cycle. It is suggested that the inhibitor could be involved in cell growth regulation.

Effect of sodium butyrate on the complexity and the translation activity of hepatoma tissue-cultured cell RNAs.

We have studied the effect of sodium butyrate on RNA populations and on the in vitro translation pattern of RNA from Hepatoma Tissue-Cultured (HTC) cells. Since sodium butyrate inhibits cell growth we have used stationary cells as control. Molecular hybridization study of cDNA with polyadenylated RNAs shows that sodium butyrate induces the formation of additional 15% new RNA sequences, essentially in the class of abundant sequences and of sequences of intermediary abundance. This drug also decreases 5-10 times the frequency of the sequences of intermediary abundance. One- and two-dimensional polyacrylamide gel electrophoreses of the translational products of RNAs show that sodium butyrate induces or strongly increases the synthesis of 13 polypeptides and decreases the synthesis of 6 polypeptides, which is consistent with the effect of sodium butyrate on RNA populations and could result from the effect of this drug at both transcriptional and early post-transcriptional steps. Electrophoretic analysis of the in vitro translation products of RNAs from cells submitted to sodium butyrate and transferred to a normal medium for various lengths of time shows that most of the polypeptides return to a normal pattern after a certain length of time which varies according to the polypeptide.

Correlated increase of the expression of the c-ras genes in chemically induced hepatocarcinomas.

The expression of the c-Ha-ras, the c-Ki-ras and the N-ras genes was measured by the dot blot technique in rat liver tumors induced by a short diethylnitrosamine (DENA) treatment and in the surrounding liver cells. A 2 to 25 times higher level of transcript was found as well in the surrounding cells, as in the tumor cells, as compared to the level in hepatocytes. In addition the increase of expression was parallel for the three ras genes. We conclude that this enhanced expression can be attributed to an epigenetic mechanism and it can, in certain cases, be dissociated from cell proliferation.

The small chromatin fragments released by micrococcal nuclease from hepatoma tissue cultured cell nuclei are strongly enriched in coding DNA sequences and are related to an actively transcribed single-stranded DNA fraction.

It was shown with the use of specific probes that mild micrococcal nuclease digestion released from chromatin actively-transcribed genes as small nucleosome oligomers. In the present work we demonstrate that most if not all of the active genes are accessible to the nuclease. It was found that the short released fragments are greatly enriched in transcribed DNA sequences, the most enriched being the dimers of nucleosomes since 35% of their DNA could be hybridized to cytoplasmic RNA. The results of cDNA-DNA hybridizations indicate that the monomers and dimers of nucleosomes contain most of the DNA sequences which encode poly(A+) RNAs, however larger released fragments include some transcribed sequences, while the nuclease resistant chromatin is considerably impoverished in coding sites. These evidences are the finding that about 25% of the DNA from the dimers of nucleosomes are exclusively located in this class of fragments, tend to prove that the active chromatin regions are attacked in a non-random way by micrococcal nuclease. We have previously isolated, without using exogenous nuclease, an actively transcribed genomic fraction amounting to 1.5-2% of the total nuclear DNA, formed of single-stranded DNA. In the present study we show that all or nearly all the single-stranded DNA sequences could be reassociated with the DNA fragments present in the released monomers and dimers of nucleosomes. Our observations confirmed our previous finding that the greatest part of single-stranded DNA selectively originates from the coding strand of genomic DNA.

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.

Effect of sodium butyrate on the hepatoma cell cycle: possible use for cell synchronization.

Exposure of HTC cells to sodium butyrate caused inhibition of growth. The site of growth inhibition was studied by time-lapse cinematography and [3H]thymidine incorporation studies. Evidence is presented that sodium butyrate affected the cell cycle at a specific point immediately after mitosis. Inasmuch as it does not modify the interphase duration after its removal, butyrate may be used for HTC synchronization.