Purpose. To compare systemic intravenous and local intratracheal delivery of doxorubicin (DOX), antisense oligonucleotides (ASO) and small interfering RNA (siRNA). Methods. "Neutral" and cationic liposomes were used to deliver DOX, ASO, and siRNA. Liposomes were characterized by dynamic light scattering, zeta-potential, and atomic force microscopy. Cellular internalization of DOX, ASO and siRNA was studied by confocal microscopy on human lung carcinoma cells. In vivo experiments were carried out on nude mice with an orthotopic model of human lung cancer. Results. Liposomes provided for an efficient intracellular delivery of DOX, ASO, and siRNA in vitro. Intratracheal delivery of both types of liposomes in vivo led to higher peak concentrations and much longer retention of liposomes, DOX, ASO and siRNA in the lungs when compared with systemic administration. It was found that local intratracheal treatment of lung cancer with liposomal DOX was more efficient when compared with free and liposomal DOX delivered intravenously. Conclusions. The present study outlined the clear advantages of local intratracheal delivery of liposomal drugs for the treatment of lung cancer when compared with systemic administration of the same drug.

Friedreich's ataxia is caused by the massive expansion of GAA⅐TTC repeats in intron 1 of the frataxin (X25) gene. Our prior investigations showed that long GAA⅐TTC repeats formed very stable triplex structures which caused two repeat tracts to adhere to each other (sticky DNA). This process was dependent on negative supercoiling and the presence of divalent metal ions. Herein, we have investigated the formation of sticky DNA from plasmid monomers and dimers; sticky DNA is formed only when two tracts of sufficiently long (GAA⅐TTC) n (n ‫؍‬ 59 -270) are present in a single plasmid DNA and are in the direct repeat orientation. If the inserts are in the indirect (inverted) repeat orientation, no sticky DNA was observed. Furthermore, kinetic studies support the intramolecular nature of sticky DNA formation. Electron microscopy investigations also provide strong data for sticky DNA as a single long triplex. Hence, these results give new insights into our understanding of the capacity of sticky DNA to inhibit transcription and thereby reduce the level of frataxin protein as related to the etiology of Friedreich's ataxia.Friedreich's ataxia (FRDA) 1 is the most common hereditary ataxia. The gene for this autosomal recessive neurodegenerative disease has been mapped to chromosome 9q13-q21.1 by linkage studies (1, 2). The clinical features and molecular biology FRDA have been reviewed (3-10). The FRDA gene, X25, contains seven exons and encodes a 210-amino acid protein called frataxin (8). The vast majority of FRDA patients have an expanded GAA⅐TTC repeat in the first intron of the frataxin gene. Normal alleles have 6 -34 repeats of GAA⅐TTC repeats, but FRDA-associated alleles have 66 -1,700 or more GAA⅐TTC repeats (3,8,10). FRDA is the only triplet repeat disease that has a recessive inheritance pattern and is caused by the expansion of a GAA⅐TTC repeat (9).The age of onset and the severity of the disease are correlated with the length of the GAA⅐TTC repeats (8,11,12). Patients carrying expanded GAA⅐TTC repeats in both alleles have reduced levels of frataxin, and an inverse correlation exists between the size of the GAA⅐TTC repeat and amount of the frataxin protein (13,14). This reduction in the amount of the frataxin protein is caused by a diminution in the amount of the frataxin mRNA (8,15,16). The amount of the X25 mRNA is inversely related to the length of the GAA⅐TTC repeat both in vitro (17) and in vivo (18). Virtually all workers in the field have proposed that the long GAA⅐TTC repeats form triplexes (three stranded DNA structures) that are involved in the transcriptional inhibition (16 -22). In fact, recent investigations (22) directly demonstrated that sticky DNA (a complex triplex formed at GAA⅐TTC repeats; described below) inhibits transcription in vitro, and in vivo investigations (18, 23) are consistent with these results.Substantial prior investigations (19, 24 -29) showed that shorter GAA⅐TTC inserts (16 -58 repeats) adopt triplex structures. DNA triplexes are formed at polypurine⅐polypyrimidine (R⅐Y) t...

Abstract:We discovered that the abundant human nuclear protein poly(ADP-ribose) polymerase (hPARP-1) binds to intramolecular DNA quadruplexes in vitro with high affinity and with a stoichiometry of two proteins for one quadruplex. Using an enzymatic assay, we have shown that hPARP-1 gets catalytically activated upon binding to G-quadruplexes localized at the c-kit promoter and human telomere regions. This is the first example of a truly functional quadruplex-protein interaction which has possible implications in understanding hPARP-1 mediated mechanisms of transcription regulation and telomere end protection. 2It has been known for several decades that DNA sequences containing a high density of contiguous guanines grouped as clusters are able to adopt four-stranded secondary structures named guanine (G)-quadruplexes or tetraplexes. 1Converging in silico and in vitro data have recently revealed high prevalence of such G rich DNA sequences throughout the human genome. 2-4The identification of prokaryotic and eukaryotic proteins that interact with these motifs also reinforces the hypothesis that quadruplexes do form in vivo and that their formation is biologically relevant. and more recently regulation of gene transcription, [10][11][12] there is noticeable absence of examples for DNA quadruplex induced modulation of nuclear protein(s) function. When exploring protein-quadruplex interactions, attention has been almost exclusively focused on the effects of the natural DNA binding protein on the quadruplex structure and proteins have been classified according to their ability to stabilise, destabilise or promote the formation of DNA quadruplexes. 6-7Unnatural Cys 2 His 2 zinc finger proteins have also been engineered that bind to quadruplex DNA with high affinity [13][14] and block the biochemical functions of DNA polymerase and telomerase via quadruplex stabilization. , the exact role quadruplexes may play remains unknown.Herein, we discovered that the abundant human nuclear protein poly(ADP-ribose) polymerase (hPARP-1) binds to intramolecular DNA quadruplexes in vitro with high affinity and gets catalytically activated upon binding to G-quadruplexes. This is the first example of a truly functional quadruplex-protein interaction which has possible implications in understanding hPARP-1 mediated mechanisms of transcription regulation and telomere end protection.Our first goal was to demonstrate the interaction between hPARP-1 and an intramolecular promoter DNA quadruplex. Two G-rich sequences have been identified within the nuclease hypersensitive region of the human proto-oncogene c-kit that encodes for a tyrosine kinase receptor. Both sequences are 31 nucleotides apart from each other and were shown to form Gquadruplex structures in vitro. [21][22] As a model system we have chosen to study in detail the interaction between hPARP-1 and the quadruplex from the c-kit promoter which proved the most stable (c-kit-1). 21However we also investigated the interaction with other intramolecular quadruplexes, including the seco...

The polypurine⅐polypyrimidine sequence requirements for the formation of sticky DNA were evaluated in Escherichia coli plasmid systems to determine the potential occurrence of this conformation throughout biological systems. A mirror repeat, dinucleotide tract of (GA⅐TC) 37 , which is ubiquitous in eukaryotes, formed sticky DNA, but shorter sequences of 10 or 20 repeats were inert. (GGA⅐TCC) n inserts (where n ‫؍‬ 126, 159, and 222 bp) also formed sticky DNA. As shown previously, the control sequence (GAA⅐TTC) 150 (450 bp) readily adopted the X-shaped sticky structure; however, this structure has never been found for the nonpathogenic (GAAGGA⅐TCCTTC) 65 of the same approximate length (390 bp). A sequence that is replete with polypurine⅐polypyrimidine tracts that can form triplexes and slipped structures but lacks long repeating motifs (the 2.5-kbp intron 21 sequence from the polycystic kidney disease gene 1) was also inert. Interestingly, tracts of (GAA⅐TTC) n (where n ‫؍‬ 176 or 80) readily formed sticky DNA with (GAAGGA⅐TCCTTC) 65 cloned into the same plasmid when the pair of inserts was in the direct, but not in the indirect (inverted), orientation. The stabilities of the triple base (Watson-Crick and Hoogsteen) interactions in the DNA/DNA associated triplex region of the sticky conformations account for these observations. Our results have significant chemical and biological implications for the structure and function of this unusual DNA conformation in Friedreich's ataxia.The salient clinical and molecular biological features of Friedreich's ataxia (FRDA) 1 as well as the properties of DNA threestranded structures (triplexes) and sticky DNA were reviewed in the companion paper (1). Because this novel DNA structure was discovered (2) in the long GAA⅐TTC mutation in intron 1 of the frataxin gene, which is responsible for most cases of FRDA (3-7), we wished to determine the sequence requirements for its stabilization. Sticky DNA is a polypurine⅐polypurine⅐ polypyrimidine (R⅐R⅐Y) triplex; hence, the features must include a mirror repeat sequence. However, the role of the distribution of purines and pyrimidines on the complementary strands has not been explored. Also, because a (GAA-GGA⅐TCCTTC) 65 tract is present in the same intron, the significance of the DNA sequence on triplex and sticky DNA formation may have further relevance. At present, the function of this unusual repeating hexanucleotide tract is unclear, especially because it does not track through family pedigrees with the disease or inhibit transcription (2, 8 -10).The effect of GGA⅐TCC-interrupted triplets in long GAA⅐TTC repeat tracts was investigated (10) to determine some of the sequence requirements for sticky DNA and to evaluate further the veracity of its long GAA⅐GAA⅐TTC triplex structure. Studies were conducted on a family of seven periodically substituted inserts (all ϳ130 repeats in length) which contain 0, 4, 7, 8, 11, 20, or 50% substitution of GAA⅐TTC with GGA⅐TCC triplets. A relatively small amount of substitution (less than 11%) ...