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Peptide nucleic acids (PNAs) are synthetic homolog of nucleic acids in which the phosphate-sugar polynucleotide backbone is replaced by a flexible polyamide. They bind complementary polynucleotide sequences with higher affinity and specificity than their natural counterparts. PNAs linked to the appropriate nuclear localization signal (NLS) peptide have been used to selectively down-regulate the expression of several genes in viable cells. For example in Burkitt's lymphoma (BL) cells the c-myc oncogene is translocated in proximity to the Emu enhancer of the Ig gene locus and upregulated. PNAs complementary to the second exon of c-myc or to the Emu enhancer sequence (PNAEmu-NLS), selectively and specifically block the expression of the c-myc oncogene and inhibit cell growth in vitro and in vivo. PNAEmu-NLS administration to mice did not exhibit toxic effects even at the highest concentration allowed by the experimental conditions. Because of the accumulating data confirming PNAEmu-NLS potential therapeutic value, PNAEmu-NLS was evaluated for the inability to induce mutations in tester strains of Salmonella typhimurium, Escherichia coli, and at the hprt locus in Chinese hamster ovary cells (CHO). Moreover, the induction of chromosomal aberrations in CHO cells and of micronuclei in human lymphocytes were investigated. We may conclude that PNAEmu-NLS neither induces mutations nor has clastogenic effects as detectable by treatment under the standard test conditions.

Peptide nucleic acids (PNAs) may be a potent tool for gene function studies in medically important parasitic organisms, especially those that have not before been accessible to molecular genetic knockout approaches. One such organism is Entamoeba histolytica, the causative agent of amebiasis, which infects about 500 million people and is the cause of clinical disease in over 40 million each year, mainly in the tropical and subtropical world. We used PNA antisense oligomers to inhibit expression of an episomally expressed gene (neomycin phosphorotransferase, NPT) and a chromosomal gene (EhErd2, a homolog of Erd2, a marker of the Golgi system in eukaryotic cells) in axenically cultured trophozoites of E. histolytica. Measurement of NPT enzyme activity and EhErd2 protein levels, as well as measurement of cellular proliferation, revealed specific decreases in expression of the target genes, and concomitant inhibition of cell growth, in trophozoites treated with micromolar concentrations of unmodified antisense PNA oligomers.

Peptide nucleic acids (PNAs) are synthetic structural analogues of DNA and RNA that, if allowed to enter the cell, bind to the complementary polynucleotide sequence and inhibit DNA transcription and mRNA translation. Although PNAs have a very limited ability in penetrating nuclei of living cells, there are indications that covalent linkage of the PNA to appropriate vectors, e.g., a nuclear localization signal, permits access to the genome. Here we test the ability of dihydrotestosterone (T) covalently linked to PNA to act as a vector for targeting c-myc DNA to prostatic cancer cell nuclei. LNCaP cells, which express the androgen receptor gene, and DU145 cells, in which the androgen receptor gene is silent, offer a model to test this biologically active hormone as a cell-specific vector. T vector was covalently linked to the NH2-terminal position of a PNA complementary to a unique sequence of c-myc oncogene (PNAmyc-T). To localize PNAmyc-T and vector-free PNA within the cells, a rhodamine (R) group was attached at the COOH-terminal position (PNAmyc-R, PNAmyc-TR); cellular uptake was monitored by confocal fluorescence microscopy. PNAmyc-R was detected only in the cytoplasm of both prostatic cell lines, whereas PNAmyc-TR was localized in nuclei as well as in cytoplasm of LNCaP cells. In contrast, PNAmyc-TR uptake in DU145 cells was minimal and exclusively cytoplasmic. In LNCaP cells, MYC protein remained unchanged by exposure to vector-free PNAmyc, whereas a significant and persistent decrease was induced by PNAmyc-T. In DU145 cells, MYC expression was unaltered by PNAmyc with or without the T vector. Our data show that the T vector facilitates cell-selective nuclear localization of PNA and its consequent inhibition of c-myc expression. These findings suggest a strategy for targeting of cell-specific anti-gene therapy in prostatic carcinoma.

Peptide nucleic acids (PNA) are synthetic homologs of nucleic acids in which the phosphate-sugar polynucleotide backbone is replaced by a flexible polyamide. In this study, a PNA construct was employed as an anti-gene agent in intact cells in culture. The cell lines studied were derived from Burkitt's lymphomas (BL) that presented a translocated and hyperexpressed c-myc oncogene. A 17-mer anti-myc PNA, complementary to a unique sequence located at the beginning of the second exon of the oncogene, and was covalently linked at its N terminus to a nuclear localization signal (NLS) (PNA-myc(wt)-NLS). When BL cells were exposed to PNA-myc(wt)-NLS, the anti-gene construct was localized predominantly in the cell nuclei and a rapid consequent downregulation of c-myc expression occurred. Under these conditions, both completion of a productive cell cycle and apoptosis were inhibited.

A peptide nucleic acid (PNA) complementary to a unique DNA sequence in the second exon of the human myc proto-oncogene was tested for its effects on transcription in colonic adenocarcinoma cells in which myc had been amplified and rearranged. A prominent rearrangement in this human cell line (COLO320-DM) involves the insertion of exon 1 of the PVT gene, which is normally located 57 kb downstream, into the first myc intron. We compared the effects of PNA invasion of the resulting chimeric gene (DMMYC) on sense and antisense transcription of its myc and PVT domains. Run-on transcription experiments showed that PNA binding to the unique myc sequence was highly specific and strongly inhibited sense transcription of four unique myc sequences downstream of the PNA.DNA hybridization site, the extent of inhibition at each sequence depending on the duration of exposure to PNA, and the distance between the downstream myc sequence and the PNA block. The same PNA also inhibited antisense transcription of unique myc sequences upstream of the binding site, confirming that transit of the RNA polymerase II complexes was impaired in both directions. The inhibitory effect of PNA on upstream antisense transcription extended beyond the recombination site into the contiguous PVT domain of the chimeric DMMYC gene. In contrast, the same PNA did not inhibit PVT transcription in a cell line (Raji lymphoma) in which PVT rearrangement did not involve the myc locus.

Previous studies have shown that treatment of cultured fibroblasts with millimolar concentrations of sodium butyrate results in increased methylation of cytosine residues in DNA. In this study, active nucleosomes were fractionated from the inactive ones by organomercurial agarose column chromatography. DNA in each fraction was hydrolyzed to its constituent bases and subjected to HPLC analysis in order to determine the 5-methylcytosine content. In control cells, the active nucleosomal DNA was hypomethylated (0.97 +/- 0.27% 5-methyleytosine) when compared with the inactive DNA fraction (1.61 +/- 0.15%). This result was not unexpected since DNA hypermethylation is generally associated with gene inactivation. Treatment of cells with sodium butyrate, however, resulted in increased methylation of the active nucleosomal DNA such that it was comparable to that of the inactive fraction of control cells (1.73 +/- 0.02% 5-methylcytosine). A much smaller increase in 5-methylcytosine content was detected in the inactive DNA fraction of sodium butyrate-treated cells (from 1.61 to 1.89%). Removal of the sodium butyrate followed by a chase in butyrate-free medium for up to 120 h failed to reverse the butyrate-induced hypermethylation. Reversal was achieved only after continuous culture in butyrate-free medium for 10 days.

The unfolding of nucleosomes along transcriptionally active DNA sequences uncovers previously shielded cysteinyl-thiol groups of histone H3 molecules located at the center of the nucleosome core. This change in conformation and SH reactivity of nucleosomes along transcribed DNA sequences makes it possible to separate active from inactive nucleosomes by mercury affinity chromatography. The binding of thiol-reactive nucleosomes to an organomercurial-agarose column has been shown previously to reflect, with accuracy, both the timing and extent of transcription of the associated DNA sequences (Chen, T. A., and Allfrey, V. G. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 5252-5256). Here, we extend this experimental approach to the analysis of higher order chromatin structures. Large chromatin fragments released by treating isolated nuclei with restriction endonucleases are fractionated on mercurated agarose magnetic beads that capture nucleosomes with accessible histone H3 thiols, but do not react with the hidden H3 thiols of the compactly beaded nucleosomes of inactive genes. The SH-reactive domains of c-myc and other genes are rapidly separated from the non-SH-reactive restriction fragments by the magnetic bead technique. The new method also overcomes a major limitation of mercurated agarose column chromatography, which is not suitable for studies of higher order chromatin structure because large chromatin fragments occlude the mercury column; occlusion is not a problem in magnetic separations using suspended mercurated agarose beads. Here, we describe the synthesis of mercurated agarose magnetic beads with high capacity for SH groups and test their application to the recovery of chromatin restriction fragments of c-myc and the growth arrest gene gas1.

The effect of increasing amounts of wheat bran (0, 5, 10, 20%) in AIN-76 semisynthetic diet on colonic luminal short chain fatty acids, epithelial cell histone acetylation, and cytokinetics, was studied for 2 weeks in groups of 10 male Sprague-Dawley rats. Luminal contents were removed from the colon at sacrifice, quick frozen, and analyzed for short chain fatty acids by gas-liquid chromatography. Histone acetylation was assessed in cells isolated from the same animals. Cell proliferation was measured after a short pulse in vivo with [3H]thymidine. Colonic luminal butyrate levels were lower in the 0 and 20% fiber groups, and higher in the 5 and 10% fiber groups. In contrast, cell proliferation, as determined by labeling index, was higher in the 0 and 20% fiber groups, and lower in the 5 and 10% fiber groups. This resulted in a significant inverse correlation between luminal butyrate levels and colonic cell proliferation. In addition, there was a positive linear correlation between luminal butyric acid levels and colon epithelial cell histone acetylation. From these data it was concluded that colonic butyrate levels can be modulated by the addition of wheat bran to the diet and that butyrate can modulate DNA synthesis (calculated as labeling index) in the proliferative compartments of colonic crypts. The localization of dividing cells was unchanged and no induction of terminal differentiation was detectable (contrary to what has been observed for transformed cells in culture).

We previously reported a separation, on an organomercurial column, of transcriptionally inactive nucleosomes (class 1) from those containing active gene sequences (classes 2 and 3). In this paper, we analyzed nucleosomal damage caused by exposure of HeLa S3 cells in suspension culture to the directly alkylating carcinogen N-methyl-N-nitrosourea (MNU). The extent and site of methylation induced by the compound in nucleosomal DNA and RNA were determined by cell incubation in the presence of [3H]MNU. The highest amount of damage was detected in DNA of class 3 nucleosomes, while RNA alkylation was comparable in all nucleosomal classes. Cellular capacity for repair of MNU-induced DNA strand breaks (estimated after a short pulse with [3H]thymidine) was found to be higher in active nucleosomal fractions (classes 2 and 3) than in the inactive fraction (class 1). Our data support the postulate that chromatin primary structure plays a role in modulating carcinogen damage to chromosomal macromolecules and in DNA strand breakage and repair mechanisms. Some of these initial steps are believed to be critical in the process of carcinogenesis.

The nucleosomes of transcriptionally active genes can be separated from those of inactive genes by affinity chromatography on organomercury-agarose (Hg-agarose) columns. The basis for this separation is the difference in accessibility of the sulfhydryl groups of histone H3 and certain non-histone proteins in active and inactive chromatin. A new procedure distinguishing between different modes of binding of transcriptionally active nucleosomes to the Hg-agarose column has been applied to study several factors which might influence the binding reaction. Nucleosomes that bind to the column because of salt-labile associations with SH-reactive non-histone proteins, such as the high-mobility-group proteins, HMG-1 and HMG-2, were released by adding 0.5 M NaCl to the eluting buffer. The remaining nucleosomes, in which reactive histone H3 thiol groups can bind covalently to the organomercury, were then displaced from the column by 10 mM dithiothreitol. Both Hg-agarose-bound fractions contain the transcriptionally active DNA sequences of the cell, but inactive nucleosomes, such as those containing alpha-globin DNA, pass through the column. The histones of both Hg-agarose-bound fractions have significantly higher levels of acetylation than do histones of the unbound fraction, but the content of tri- and tetra-acetylated H3 and H4 is significantly higher in the nucleosomes with reactive H3 thiols. The rate of turnover of histone N-acetyl groups is also far greater in the Hg-agarose-bound nucleosomes than in the unbound nucleosomes. Although the overall levels of histone acetylation can be increased significantly by incubating HeLa cells in the presence of the deacetylase inhibitor, 5 mM sodium butyrate, this treatment has little if any effect on the total number of nucleosomes retained on the Hg-agarose column. However, the ability of Hg-agarose chromatography to detect localized changes in chromatin structure is evidenced by an 11-fold increase in the Hg-agarose binding of nucleosomes containing the DNA of the butyrate-inducible alkaline phosphatase gene, compared to the Hg-agarose-bound nucleosomes of control cells. Although nascent RNA chains are present in the Hg-agarose-bound nucleosomes released by dithiothreitol, binding of the SH-reactive nucleosomes to the Hg-agarose column is not dependent on the presence of proteins associated with nascent RNA chains, since binding does not decrease following removal of the nascent transcripts by ribonuclease treatment.(ABSTRACT TRUNCATED AT 400 WORDS)

The methylating carcinogen 1,2-dimethylhydrazine (DMH) CAS 540.73.8 is highly organ-specific and, under certain experimental conditions, produces a high incidence of adenocarcinoma in the colon of rodents. We have tried to assess the possibility that part of the organ-specifity in the carcinogenic effect of DMH could be attributed to its metabolism by specific microsomal enzymes. In particular, we compared the in vitro effects of DMH in the presence of either colon or liver microsomes from animals that had been treated with microsomal enzyme inducers. V79 Chinese hamster cells were used as the target to evaluate the damage to the genetic material, as judged by (1) formation of adducts of DNA bases and (2) amino acid modifications in nuclear proteins using [Me-14C]DMH and appropriate analytical detection systems. Our results tend to support the above postulated hypothesis.

We have established a specific correlation between the carcinogenic potency of a series of alkylating agents, with a mechanism of reaction ranging between Ingold's SN1-SN2 (ENU greater than MNU = MNNG greater than EMS greater than DMS = MMS) (Vogel et al., 1979; Bartsch et al., 1983) and specific target sites in the amino acids of nuclear proteins of cultured hepatocytes. More potent carcinogens, that react predominantly with an Ingold's SN1 mechanism, mainly alkylate the amino group of lysine and the guanido group of arginine. Weaker carcinogens, reacting with a mechanism closely resembling an Ingold's SN2, mainly alkylate the sulfhydryl group of the cysteine and the 3 position of the imidazolic ring of histidine. A compound with an intermediate type of reactivity alkylates, to a comparable extent, all 4 of the above-described positions. Although stable DNA damage brought about by alkylating carcinogens is considered to be the most likely cause of neoplastic transformation, epigenetic modifications may also play an important role in the process, especially because of their extreme stability. We have verified the existence of a linear correlation between the Swain-Scott substrate constant (S) of each compound and the amount of alkylation produced at the specific target sites. This type of correlation could be the basis of a 'short-term' genotoxicity assay in a battery of complementary tests.

The alkylating carcinogen 1,2-dimethylhydrazine (DMH) induces colonic tumors with high incidence. The sites of in vivo modification of target macromolecules were studied using [methyl-3H]DMH. Carcinogen binding to subcellular fractions of colonic epithelial cells was analyzed at time intervals ranging from 10 min to 36 hr, with particular emphasis on the alkylation of nuclear constituents. DMH modifies not only nucleic acids but also histones and other DNA-binding proteins in the target cells. Separation of the 3H-methylated amino acids showed aberrant methylation patterns after exposure to [3H]DMH, as compared with methylations observed with [methyl-3H]methionine as a natural methyl group donor. DMH-modified histone H1 contained methylated lysin, arginine, and histidine residues not normally found, and other histones had abnormal methylarginine contents. High-mobility-group proteins and other nuclear proteins contained methyllysine residues not normally detected. Proteins known to be associated with template-active and more accessible DNA sequences, such as the high-mobility group proteins and the multiacetylated forms of histones H3 and H4, were preferentially damaged after exposure to [3H]DmH. The result suggests that carcinogen-induced chromosomal damage is not random and may selectively affect proteins in the actively transcribing or replicating genes in the target cells. That damage affects the amino acids most likely to be involved in DNA binding.

Unfolding of the nucleosomes in transcriptionally active chromatin uncovers the sulfhydryl groups of histone H3 and permits the selective recovery of the unfolded nucleosomes by mercury-affinity chromatography. This new technique has been used to compare the nucleosomal proteins and their postsynthetic modifications in the unfolded and the compactly beaded nucleosomes of HeLa cells in logarithmic growth, and at different stages of the growth cycle. The Hg-bound nucleosomes are shown to be deficient in replicating DNA sequences, but to remain associated with fragments of nascent RNA chains (or RNP particles) during gradient centrifugations. Both nucleosome fractions contain a full complement of "core" histones but differ with respect to postsynthetic modifications. The Hg-bound nucleosomes contain high levels of the tri- and tetra-acetylated forms of histones H3 and H4. The unbound nucleosomes are deficient in acetylated histones but enriched in phosphorylated H2A. In synchronized HeLa cells, histone H2A and H4 gene sequences occur in the Hg-bound nucleosomes during the S-phase when their transcription takes place, but not in the G2-phase when the genes are repressed.

Mice from strains with different susceptibility to the colon-specific carcinogen 1,2-dimethylhydrazine (DMH) were tested for DNA damage in liver, kidney and colon after administration of the compound at a dosage that has been reported to induce a high incidence of adenocarcinoma in the colon of rodents. DNA breaks were evaluated from their elution rate constant according to the alkaline elution technique. We found that 4 h after administration of the carcinogen there was a substantial and comparable DNA damage in liver and kidney of all strains examined. Conversely, colon DNA damage was hardly above control levels in the carcinogen-resistant strains. The highest DNA damage was detected in the most susceptible strain and was slightly lower in the two other susceptible strains. We propose that the extent of DNA breakage in a target organ could be one of the factors determining organ-specific and strain-specific susceptibility to DMH.

We have compared liver, kidney and colon DNA damage, as single strand breaks, in mice with different strain-dependent susceptibility to the colon-specific carcinogen 1,2-dimethylhydrazine (DMH). The mouse strains studied were: AKR/J, DBA2 totally resistant; CD1, C57BL/6N moderately susceptible; SWR/J very susceptible to DMH-induced carcinogenesis. DNA breaks were estimated from the elution rate constant (K) according to the alkaline elution technique. At 4 h after carcinogen administration a substantial and comparable DNA damage was found in liver and kidney in all the strains examined. The DNA fragmentation index, however, reached a maximum value at 2 h after treatment in the liver of the most susceptible strain (SWR/J). About 50% of the liver DNA damage detected in all five strains 4 h after DMH administration persisted at 24 h after treatment and was totally repaired at 72 h. Kidney DNA damage decreased in 48 h toward the range of control values. In colon epithelial cells (the carcinogen target tissue) 2 and 4 h after DMH administration the amount of DNA single strand breaks was correlatable with the strain sensitivity to the carcinogen. In the time interval studied (2-72 h after DMH administration) the decrease of colon DNA damage was linear in the resistant strains. In contrast, in the more susceptible strain (SWR/J), the amount of DNA breaks remained high up to 24 h after treatment and returned to background level at 72 h.

Duck erythrocyte chromatin has been treated with DNAase 1 under conditions that are known to digest selectively the structural genes coding for globin mRNAs. This limited digestion releases specific sets of nonhistone chromosomal proteins that are not preferentially released during limited digestion with micrococcal nuclease, which does not selectively attack the globin sequences. Analysis of nucleosome monomer and multimer peaks separated on sucrose gradients after limited digestion with micrococcal nuclease shows that the proteins which are released by DNAase 1 digestion remain associated with the chromatin subunits and can be removed by extraction in 0.5 M NaCl. These proteins are tentatively identified as members of the high mobility group (HMG) proteins (originally described by Goodwin, Sanders and Johns, 1973) in terms of their extractability, electrophoretic characteristics and amino acid composition.

In HeLa cells which have been exposed to 5 mM sodium butyrate for 21 h, the level of histone acetylation is greatly increased as compared to control cells (Riggs, M.G., Whittaker, R.G., Neumann, J.R., and Ingram, V.R. (1977) Nature 268, 462-464). Our experiments indicate that the increase in the relative amounts of multiacetylated forms of histones H4 and H3 following butyrate treatment is the result of an inhibition of histone deacetylase activity.

We have tried to establish a correlation between the carcinogenic potency of four methylating compounds and their specific target sites in chromatin. We have therefore compared the nuclear metabolism of two relatively weak carcinogens radioactively labelled: dimethyl sulphate (DMS) and methyl methanesulphonate (MMS), and two potent carcinogens: N-methyl-N-nitrosourea (MNU) and N-methyl-N'-nitro-N-nitrosoguanosine (MNNG) in cultured primary hepatocytes and the V79 Chinese hamster cell line. Cysteine (when present), and to a lesser extent histidine, were methylated by MMS and DMS not only in the total acid-soluble nuclear protein (H) but also in purified histones H1 and H3. These compounds had the same effect not only on total non-histone nuclear protein (NH) but also on purified HMG1 and HMG2 (nuclear non-histone proteins with high electrophoretic mobility). Traces of methylarginine and methylated lysine could be detected in all samples. MNU and MNNG predominantly methylated lysine and arginine residues, the former being found mostly in acid soluble, the latter in non-histone nuclear protein. Methylated cysteine and histidine were present in trace amounts. Our preliminary data suggest specific amino acid methylation at the nuclear protein level for carcinogens with different potencies, similar to what has been found for DNA bases.

Nuclei were isolated from colonic epithelial tumors induced in rats by the administration of 1,2-dimethylhydrazine. The nuclei were fractionated according to buoyant density by centrifugation in discontinuous sucrose density gradients. Nuclei differing in density differ in size, nonhistone protein-to-DNA ratio, and DNA synthetic activity. Their distribution in a density gradient also reflects their histological localization in the layers of the intestinal mucosa, as judged by the nuclear capacity for [3H]thymidine incorporation into DNA in vivo. Different nuclear classes isolated from the tumors contain characteristic complements of nuclear nonhistone proteins. Particularly striking accumulations of two protein classes with molecular weights of ca. 44,000 and 62,000 occur during carcinogenesis. These proteins are not uniformly distributed throughout all nuclear classes derived from the tumors. They are not at all prominent in normal colonic epithelial nuclei or in epithelial cells surroundign the tumors, or in the liver nuclei of animals treated with 1,2-dimethylhydrazine. Procedures for the differential extraction of these protein classes are described. Similar nuclear proteins have been detected in human colonic tumors and in a human cell line (HT-29) derived from an adenocarcinoma of the colon. The selective accumulation of such proteins in colonic tumor nuclei may have diagnostic value.

We have tried to establish a correlation between the carcinogenic potency of two alkylating compounds and specific target sites in chromatin. We have therefore compared the nuclear metabolism of radioactively-labelled methylmethanesulfonate (MMS), a relatively weak carcinogen and N-methylnitrosourea (MNU), a highly potent carcinogen in cultured primary hepatocytes which have, high microsomal drug-metabolizing activity and in V79 Chinese hamster cells which have low microsomal enzymatic activity. The modification of specific amino acid residues in acid-soluble nuclear proteins (H) and non-histone nuclear proteins (NH) was studied after exposing the cells to various doses of alkylating agents overnight. We found that at all doses, mainly the cysteine (Cys), but also to a lower extent the histidine (His) residues are methylated in both H and NH protein fractions by MMS. At high doses of MMS, traces of methylarginine and methylated lysines could be detected. MNU predominantly methylates lysine and arginine residues, the former being found mostly in H, the latter in NH. Although both hepatocytes and V79 cells metabolized radioactively-labelled carcinogen, a higher percentage of counts were incorporated by the hepatocytes; 'unusually' methylated amino acids were detectable in the hepatocyte proteins with relatively low doses of the alkylating agents but not in V79 cells. In the presence of exogenous microsomes, during exposure of V79 cells to the alkylating agents, the amount of amino acid methylation is qualitatively and quantitatively similar to that found in hepatocytes. Our data suggest a specific mechanism of protein methylation, at the level of target amino acids, for carcinogens with different potencies similar to what has been found for DNA bases. A component of the microsomal fraction (S9) may be able to enhance this effect.

In addition to its known effect in suppressing the deacetylation of the nucleosomal core histones, sodium butyrate in the concentration range 0.5 to 15 mM causes a selective inhibition of [32P]phosphate incorporation into histones H1 and H2A of cultured HeLa S3 cells. No commensurate general inhibition of phosphorylation is seen in the non-histone nuclear proteins of butyrate-treated cells, but phosphorylation patterns are altered and 32P-uptake may be stimulated, as well as inhibited, depending upon the protein fraction analyzed. The degree of inhibition of histone phosphorylation in intact cells increases progressively as the butyrate concentration is raised from 0.5 to 15 mM. The effect is time-dependent and fully reversible. Butyrate has no effect on the kinetics of phosphate release from previously phosphorylated histones of cultured cells, nor does it significantly alter the rate of dephosphorylation of 32P-labeled histone H1 by endogenous phosphatases in vitro. Despite the suppression of [32P]phosphate incorporation into histones H1 and H2A of butyrate-treated cells, Na-butyrate does not inhibit the in vitro activities of either type I or type II cyclic AMP-dependent protein kinases, or the cAMP-independent H1 kinase associated with cell cycle progression. This suggests that the butyrate effect on histone phosphorylation in vivo is indirect and may involve an alteration in substrate accessibility or a modulation of systems affecting kinase activities. The poly(ADP)-ribosylation of HeLa histones is not inhibited by 5 mM Na-butyrate. Cells exposed to butyrate show an impaired methylation of lysine and arginine residues in their histones and nuclear hnRNP particles, respectively.

The properties of two classes of nonhistone nuclear proteins (NHNP) whose appearance has been correlated with the process of carcinogenesis in the colonic epithelium of rats given 1,2-dimethylhydrazine (DMH) have been investigated. NHNP isolated from adenocarcinomas of the colon were tested for their binding to homologous DNA. The tumor-specific protein class TNP1 (molecular weight ca. 44,000) shows high affinity for DNA while the second class of tumor-specific proteins (TNP2; molecular weight ca. 62,000) does not bind to DNA. When tumor chromatin is subjected to a limited digestion with DNAse I under conditions that are known to preferentially digest active genes, the TNP1 proteins are selectively released, suggesting that this protein fraction is associated with the actively transcribing portions of the genome. The complexity of both tumor-specific protein classes has also been analyzed by two-dimensional gel electrophoresis. This method reveals a high degree of complexity both in TNP1 and TNP2. Protein classes similar to TNP1 and TNP2 that are also found in human colonic adenocarcinomas are not detectable in polyps from familial polyposis-affected patients at times when no sign of malignancy has yet appeared.

The complement of nuclear non-histone proteins in epithelial cells of the colon is progressively altered during the course of carcinogenesis induced by 1, 2-dimethylhydrazine, until finally the nuclear proteins of tumor cells are easily distinguishable from those of the surrounding normal tissue. These changes in nuclear protein compostion reflect earlier differences in the rates of synthesis of individual protein species. Radioisotopic double-labeling experiments show that the synthesis of nuclear proteins of molecular weights 44,000 and 62,000 is selectively accelerated within 4 weeks after administration of the carcinogen, long before any morphological indications of malignancy appear.

Sodium cyanate, which in its tautomeric acidic form, isocyanic acid, acts as a protein carbamylating reagent, has been previously shown to inhibit selectively both DNA and protein synthesis in a variety of solid tumors. We have now compared its effects on protein synthesis in normal colonic epithelium and in colon tumors induced by the administration of 1,2-dimethylhydrazine to rats. The incorporation of 3H-amino acids into cytoplasmic and nuclear protein fractions was suppressed to a much greater extent in the tumor tissue than in colonic epithelial tissue surrounding the tumors of cyanate-treated rats. Despite its effect on tumor protein synthesis in whole animals, cyanate had little or no effect on cultured cells (HT-29) derived from a human adenocarcinoma of the colon, nor on other malignant cell lines such as HeLa S3 cells, chick fibroblasts transformed by the Rous sarcoma virus, mouse Ehrlich ascites tumor cells, or rat Novikoff hepatoma cells. However, the administration of cyanate i.p. does suppress amino acid incorporation by Novikoff hepatoma cells in the peritoneal cavity of rats. The implication that the mechanism of cyanate inhibition of protein synthesis in tumors may require its in vivo metabolism or utilization to produce a postsynthetic modification of circulatory factors is discussed.

Sodium cyanate, a selective inhibitor of protein synthesis in animal tumors in situ, has no comparable effect when added to tumor cells in culture. However, a rapid inhibition of protein synthesis in cultured tumor cells can be achieved by reaction of cyanate with the drug-metabolizing system of liver microsomes. Procedures are described for the induction, preparation, and testing of an active cytochrome P-450 fraction which converts cyanate to a short-lived dialyzable metabolite that selectively inhibits amino acid incorporation in tumor cells without a corresponding effect on protein synthesis in normal cells. The suppression of tumor protein synthesis is not due to a general toxicity reaction because thymidine incorporation into the DNA of tumor cells is not inhibited by the cyanate metabolite. When HeLa cells are exposed to sodium butyrate, a substance reported to suppress the malignant phenotype in several tumor cell lines, they lose their sensitivity to the cyanate metabolite. Chick fibroblasts which are normally insensitive to the cyanate metabolite become cyanate sensitive after transformation by the Schmidt-Ruppin strain of the Rous sarcoma virus.