PNAEmu can significantly reduce Burkitt's lymphoma tumor burden in a SCID mice model: cells dissemination similar to the human disease.

In human Burkitt's Lymphoma (BL) BRG cells, a t(8;14) translocation, placing c-myc near the Emu enhancer of the H chain locus, causes tumor expansion. Earlier, we showed that a peptide nucleic acid complementary to the Emu sequence (PNAEmu), specifically inhibited the expression of translocated c-myc and impaired the growth of BRG cells-induced subcutaneous tumors in mice suffering from severe combined immunodeficiency (SCID). In this study, the therapeutic potential of PNAEmu was evaluated in a systemic mouse model. BRG-BL cells transfected with the luciferase gene were inoculated intravenously into SCID mice resulting in a preferential expansion, similar to the one of human adult patients, in the abdominal cavity, central nervous system and bone marrow. The mice were chronically injected intraperitoneally either with PNAEmu or with control PNA. The treatment was stopped when the control animals developed severe neurological symptoms. As detected both by inspection at necropsy and imaging, overall tumor growth in PNAEmu-treated mice decreased by >80%. Histological and immunohistochemical studies showed, only in PNAEmu-treated mice, a substantially reduced BL cell growth at the major sites of invasion and vast areas of necrosis in the lymphomatous tissues, with concomitant c-myc expression downregulation. Altogether, the data support the therapeutic potential of PNAEmu in human adult BL.

Lack of mutagenicity and clastogenicity of PNAEmu-NLS targeted to a regulatory sequence of the translocated c-myc oncogene in Burkitt's lymphoma.

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.

Inhibition of Burkitt's lymphoma cells growth in SCID mice by a PNA specific for a regulatory sequence of the translocated c-myc.

In Burkitt's lymphoma (BL) cells due to a t(8;14) chromosomal translocation c-myc is often placed in proximity to the Emu enhancer of the Ig locus and upregulated. We demonstrated that in BL cells a peptide nucleic acid (PNA), complementary to intronic Emu sequences (PNAEmuwt), specifically blocks the expression of the c-myc oncogene under the Emu enhancer control and inhibits BL cell growth in culture. Here, we investigated whether PNAEmuwt was also able to block tumor growth in SCID mice inoculated with human BL cell lines. After subcutaneous inoculum in mice BL cells reproducibly form tumors. Both pre-treatment of BL cells with PNAEmuwt before inoculum and chronic intravenous administration of PNAEmuwt to mice already inoculated with BL cells selectively caused increased latency of tumor appearance and decreased final tumor size. Tumors from PNAEmuwt-treated animals showed substantial areas of cell necrosis and of c-myc downregulation. Inhibition of tumor growth was specific and was not observed with PNAEmumut carrying sequence mutations and in BL cell lines where the translocated c-myc is not under the control of the Emu enhancer. These data confirm the potential therapeutic value of PNA targeted to regulatory non-coding regions.

Dihydrotestosterone as a selective cellular/nuclear localization vector for anti-gene peptide nucleic acid in prostatic carcinoma cells.

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.

Contrasting effects of PNA invasion of the chimeric DMMYC gene on transcription of its myc and PVT domains.

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.

Selective hypermethylation of transcribed nucleosomal DNA by sodium butyrate.

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.

Recovery of transcriptionally active chromatin restriction fragments by binding to organomercurial-agarose magnetic beads. A rapid and sensitive method for monitoring changes in higher order chromatin structure during gene activation and repression.

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.

Modulation of colonic epithelial cell proliferation, histone acetylation, and luminal short chain fatty acids by variation of dietary fiber (wheat bran) in rats.

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).

Effects of N-methyl-N-nitrosourea on transcriptionally active and inactive nucleosomes: macromolecular damage and DNA repair.

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.

Factors affecting nucleosome structure in transcriptionally active chromatin. Histone acetylation, nascent RNA and inhibitors of RNA synthesis.

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)

Target tissue DNA damage in inbred mouse strains with different susceptibility to the colon carcinogen 1,2-dimethylhydrazine.

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.

Specific targets of alkylating agents in nuclear proteins of cultured hepatocytes.

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.