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.

An amplification of tandem CAG trinucleotide sequences in DNA due to errors in DNA replication is involved in at least four hereditary neurodegenerative diseases. The CAG triplet repeats when translated into protein give rise to tracts of glutamine residues, which are a prominent feature of many transcription factors, including the TATA-binding protein of transcription factor TFIID. We have used a biotin-labeled, complementary peptide nucleic acid (PNA) to invade the CAG repeats in intact chromatin and then employed a method for the selective isolation of transcriptionally active chromatin restriction fragments containing the PNA-DNA hybrids. The PNA-containing chromatin fragments were captured on streptavidin-agarose magnetic beads and shown to contain all the CAGPNA hybrids of the active chromatin fraction. DNA hybridization experiments using a DNA probe specific for unique sequences downstream of the CAG-tandem repeats confirmed that the PNA-DNA hybrids contained the transcribed gene for the TATA-binding protein.In contrast, no hybridization signal was detected with a DNA probe specific for the c-myc protooncogene, which is amplified and transcriptionally active in COLO 320DM cells but lacks CAG tandem repeats.Peptide nucleic acids (PNAs) are structural homologues of DNA and RNA in which the entire phosphate-sugar backbone has been replaced by a flexible peptide backbone consisting of 2-aminoethyl glycine units linked to the purine and pyrimidine bases (1-3). The absence of phosphate groups in the PNA molecule facilitates its invasion of negatively charged DNA duplexes containing complementary base sequences (4-6). When PNA hybridizes to a targeted DNA sequence, one strand of DNA may be rapidly displaced to form a D-loop (1, 5, 6), while the PNA binds to its complementary DNA sequence by WatsonCrick base pairing (7). PNAs show greater discrimination and form more stable associations with DNA than the corresponding DNADNA duplexes (7). PNA invasion of DNA strands to form stable PNADNA hybrids has profound implications for both positive and negative control of gene activity. For example, it has been shown that DNA loops displaced as a consequence of PNA binding act as artificial transcription promoters (8), but PNA binding to the transcribed strand of simian virus 40 DNA blocks transcript elongation beyond the site of PNA DNA duplex formation (9).Here, we explore the potential of a biotinylated PNA probe to invade DNA triplet repeat sequences in the chromatin of transcriptionally active genes. By using a PNA specific for multiple CAG repeats, together with a method for the separation of transcriptionally active chromatin restriction fragments from inactive chromatin restriction fragments (10), we show that the PNA probe can hybridize effectively to CAG triplet repeats in intact chromatin and that transcriptionally active chromatin fragments containing the PNA-DNA hybrids can be captured quantitatively on streptavidin-coated magnetic beads.An amplification of CAG triplet repeats occurs in at least four i...

CD10 constitutes a favourable prognostic marker for childhood acute lymphoblastic leukaemia. Since correlations between CD10, cell cycle and apoptotic abilities were demonstrated in various cell types, we investigated whether differences existed in the cycling/apoptotic abilities of CD10-positive and CD10-negative B acute lymphoblastic leukaemia cells. Twenty-eight cases of childhood acute lymphoblastic leukaemia (mean age of 6.8 years) were subdivided into two groups according to high (17 cases, 93.2+4.5%, MRFI 211+82 CD10-positive cells) or low (11 cases, 11.5+6.2%, MRFI 10+7 CD10-negative cells) expression of CD10. CD10-positive acute lymphoblastic leukaemia cells were cycling cells with elevated c-myc levels and propensity to apoptosis, whereas CD10-negative acute lymphoblastic leukaemia cells had lower cycling capacities and c-myc levels, and were resistant to apoptosis in vitro. A close correlation between all these properties was demonstrated by the observations that the few CD10-positive cells found in the CD10-negative acute lymphoblastic leukaemia group displayed elevated c-myc and cycling capacities and were apoptosis prone. Moreover, exposure of CD10-positive acute lymphoblastic leukaemia B cells to a peptide nucleic acid anti-gene specific for the second exon of c-myc caused inhibition of c-myc expression and reduced cell cycling and apoptotic abilities as well as decreased CD10 expression.

The DNA sequence of the genes for the androgen receptor (AR) and TATA-binding protein (TBP), like many other genes encoding transcription factors, contains a series of tandem CAG repeats. Here we explore the capacity of complementary peptide nucleic acids (PNAs) to invade the CAG triplets of the AR and TBP genes in human prostatic cancer cells and show that the PNAs readily entered the nuclei of lysolecithin-permeabilized cells and effectively inhibited sense transcription of unique AR and TBP DNA sequences downstream of the site of PNA.DNA hybridization, but not upstream of that site. These PNAs had little or no effect on transcription of the c-myc gene, which lacks a CAG triplet domain. Conversely, a PNA complementary to a unique sequence of the c-myc gene did not inhibit transcription of the AR or TBP genes but did inhibit c-myc transcription. Comparisons of PNA effects on sense and antisense transcription of the AR, TBP, and c-myc genes confirm that progression of the RNA polymerase complex beyond the site of PNA.DNA hybridization is impaired in both directions. Suppression of the AR gene results in refolding of a transcriptionally active nucleosome containing a unique 17-mer AR DNA sequence.