Cristina Rutigliano

The DNA-binding drugs mithramycin and chromomycin are powerful inducers of erythroid differentiation of human K562 cells

The human leukaemic K562 cell line can be induced in vitro to undergo erythroid differentiation by a variety of chemical compounds, including haemin, butyric acid, 5‐azacytidine and cytosine arabinoside. Differentiation of K562 cells is associated with an increased expression of embryo‐fetal globin genes, such as the ζ, ε and γ globin genes. Therefore the K562 cell line has been proposed as a useful in vitro model system to determine the therapeutic potential of new differentiating compounds as well as to study the molecular mechanism(s) regulating changes in the expression of embryonic and fetal human globin genes. Inducers of erythroid differentiation which stimulate γ‐globin synthesis could be considered for possible use in the experimental therapy of those haematological diseases associated with a failure in the expression of adult β‐globin genes. In this paper we demonstrated that the G + C selective DNA‐binding drugs chromomycin and mithramycin were powerful inducers of erythroid differentiation of K562 cells. Erythroid differentiation was associated with an increase in the accumulation of (a) Hb Gower 1 and Hb Portland and (b) γ‐globin mRNA.

Interaction of the Human NF-κB p52 Transcription Factor with DNA-PNA Hybrids Mimicking the NF-κB Binding Sites of the Human Immunodeficiency Virus Type 1 Promoter

We determined whether peptide nucleic acids (PNAs) are able to interact with NF-κB p52 transcription factor. The binding of NF-κB p52 to DNA-DNA, DNA-PNA, PNA-DNA, and PNA-PNA hybrid molecules carrying the NF-κB binding sites of human immunodeficiency type 1 long terminal repeat was studied by (i) biospecific interaction analysis (BIA) using surface plasmon resonance technology, (ii) electrophoretic mobility shift, (iii) DNase I footprinting, and (iv) UV cross-linking assays. Our results demonstrate that NF-κB p52 does not efficiently bind to PNA-PNA hybrids. However, a DNA-PNA hybrid molecule was found to be recognized by NF-κB p52, although the molecular complexes generated exhibited low stability. From the theoretical point of view, our results suggest that binding of NF-κB p52 protein to target DNA motifs is mainly due to contacts with bases; interactions with the DNA backbone are, however, important for stabilization of the protein-DNA complex. From the practical point of view, our results suggest that DNA-PNA hybrid can be recognized by NF-κB p52 protein, although with an efficiency lower than DNA-DNA NF-κB target molecules; therefore, our results should encourage studies on modified PNAs in order to develop potential agents for the decoy approach in gene therapy.

Biosensor technology and surface plasmon resonance for real-time detection of HIV-1 genomic sequences amplified by polymerase chain reaction.

BACKGROUND The recent development of biosensor technologies for biospecific interaction analysis enables the monitoring of a variety of molecular reactions in real time by surface plasmon resonance (SPR). If the ligand is a biotinylated single stranded DNA, this technology could monitor DNA-DNA hybridization. This approach could be of great interest in virology, since the hybridization step is oftenly required to confirm specificity of molecular diagnosis. OBJECTIVES To determine whether real-time molecular diagnosis of human immunodeficiency virus type I (HIV-1) could be performed using biosensors and SPR technology. STUDY DESIGN Specific hybridization of a biotinylated HIV-1 oligonucleotide probe immobilized on a sensor chip to single stranded DNA obtained by asymmetric polymerase-chain reaction (PCR) was determined using the BIAcore biosensor. RESULTS Direct injection of asymmetric PCR to a sensor chip carrying an internal HIV-1 oligonucleotide probe allows detection of hybridization by SPR using biosensor technology. This enabled us to apply a real-time, one-step, non-radioactive protocol to demonstrate the specificity of amplification of HIV-1 genomic sequences by PCR. CONCLUSION The procedure described in this study for HIV-1 detection is simple, fast (PCR and SPR analyses take 30 min), reproducible and could be proposed as an integral part of automated diagnostic systems based on the use of laboratory workstations and biosensors for DNA isolation, preparation of PCR reactions and analysis of PCR products.

Pyrazolo-triazoles as light activable dna cleaving agents

In view of the continuous interest in new DNA cleaving compounds, both for the development of new therapeutic agents and for the possible use as reagents in nucleic acids research, a few pyrazolo[3,4-d][1,2,3]triazole derivatives have been obtained and investigated for their antiproliferative activity and capability to cleave DNA, after light-activation. A possible in situ activation, i.e. in neoplastic tissues, of less cytotoxic derivatives, may lead to potential antitumor compounds endowed with high therapeutic indexes.

Targeting of the Sp1 binding sites of HIV-1 long terminal repeat with chromomycin: Disruption of nuclear factor · DNA complexes and inhibition of in vitro transcription

Sequence selectivity of DNA-binding drugs has recently been reported in a number of studies employing footprinting and gel retardation approaches. In this paper, we studied the biochemical effects of the sequence-selective binding of chromomycin to the long terminal repeat of the human immunodeficiency type I virus. Deoxyribonuclease I (E.C.3.1.21.1) footprinting, arrested polymerase chain reaction, gel retardation and in vitro transcription experiments have demonstrated that chromomycin preferentially interacts with the binding sites of the promoter-specific transcription factor Sp1. Accordingly, interactions between nuclear proteins and Sp1 binding sites are inhibited by chromomycin, and this effect leads to a sharp inhibition of in vitro transcription.

[2,1-c] [1,4]Benzodiazepine (PBD)-Distamycin Hybrid Inhibits DNA Binding to Transcription Factor Sp1

Abstract We designed and synthesized the hybrid 6, prepared combining the minor groove binders distamycin A and pyrrolo [2,1-c][1,4] benzodiazepine (PBD) 4, related to the natural occurring anthramycin (2) and DC-81 (3). In this paper, the effects of the compound 6 on molecular interactions between DNA and transcription factor Sp1 were studied. The results obtained demonstrate that PBD-distamycin hybrid is a powerful inhibitor of Sp1/DNA interactions.

In vitro and in vivo binding of a CC-1065 analogue to human gene sequences: a polymerase-chain reaction study.

In this paper we analyse the in vitro sequence selectivity of the CC-1065 analogue 2-[[5-[(1H-indol-2-yl]carbonyl)-1H-indol-2-yl] carbonyl]-7-methyl-1,2,8,8a-tetrahydrocyclopropa [c]-pyrrolo-[3,2-e]-indol-4-one (U-71184) employing the polymerase-chain reaction (PCR). In addition, we determined whether alteration of PCR by U-71184 is detected when DNA is isolated from cells cultured in the presence of this drug. As molecular model systems we employed the human estrogen receptor gene, the Ha-ras oncogene and the chromosome X-linked, (CGG)-rich fragile X mental retardation-1 gene. The first conclusion that can be drawn from the experiments reported in our paper is that U-71184 inhibits PCR in a sequence-dependent manner. A second conclusion of our experiments is that PCR performed on DNA from U-71184-treated cells is inhibited when the primers amplifying the estrogen receptor gene region are used. This approach might bring important information on both in vivo uptake of the drug by target cells and binding to DNA.

Selective binding to human genomic sequences of two synthetic analogues structurally related to U-71184 and adozelesin

In this paper, we analyse the in vitro sequence‐selectivity of two synthetic analogues of U‐71184 and adozelesin by polymerase chain reaction (PCR) performed on human genomic DNA. In addition, Dnase footprinting and nucleotide sequence analysis on arrested‐PCR products were performed to confirm sequence‐selective binding. Finally, the antitumor effects were studied in vitro on human leukemic L1210 cells. The binding activity of the two newly synthesized compounds to human gene sequences was compared with the CC‐1065 analogue U‐71184, the A+T sequence‐selective drug distamycin and the G+C sequence‐selective drugs mithramycin and chromomycin. As molecular model systems for in vitro DNA‐binding studies we used the human estrogen receptor gene and the Ha‐ras oncogene. In some experiments the PCR approach was performed using as target DNA a portion of the long terminal repeat (LTR) of the human immunodeficiency type 1 virus (HIV‐1). These genomic regions contain sequences that are different with respect to A+T/G+C ratios, being the upstream sequence of the human estrogen receptor gene A+T rich, while the Ha‐ras and HIV‐1 LTR sequences contain G+C–rich regions. The first conclusion that can be drawn from the experiments reported in our paper is that the two newly synthetized analogues of U‐71184 and adozelesin inhibit PCR‐mediated amplification of genomic regions in a sequence‐dependent manner. A second conclusion of our experiments is that these compounds are active inhibitors of tumor cell growth in vitro. Drug Dev. Res. 46:96–106, 1999.