DNA-binding studies of AV-153, an antimutagenic and DNA repair-stimulating derivative of 1,4-dihydropiridine.

UNLABELLED: The ability to intercalate between DNA strands determines the cytotoxic activity of numerous anticancer drugs. Strikingly, intercalating activity was also reported for some compounds considered to be antimutagenic. The aim of this study was to determine the mode of interaction of DNA with the antimutagenic and DNA repair-stimulating dihydropyridine (DHP) AV-153. DNA and AV-153 interactions were studied by means of UV/VIS spectroscopy, fluorimetry and infrared spectroscopy. Compound AV-153 is a 1,4 dihydropyridine with ethoxycarbonyl groups in positions 3 and 5. Computer modeling of AV-153 and DNA interactions suggested an ability of the compound to dock between DNA strands at a single strand break site in the vicinity of two pyrimidines, which was confirmed in the present study. AV-153 evidently interacted with DNA, as addition of DNA to AV-153 solutions resulted in pronounced hyperchromic and bathochromic effects on the spectra. Base modification in a plasmid by peroxynitrite only minimally changed binding affinity of the compound; however, induction of single-strand breaks using Fenton's reaction greatly increased binding affinity. The affinity did not change when the ionic strength of the solution was changed from 5 to 150 mM NaCl, although it increased somewhat at 300 mM. Neither was it influenced by temperature changes from 25 to 40°C, however, it decreased when the pH of the solution was changed from 7.4 to 4.7. AV-153 competed with EBr for intercalation sites in DNA: 116 mM of the compound caused a two-fold decrease in fluorescence intensity. FT-IR spectral data analyses indicated formation of complexes between DNA and AV-153. The second derivative spectra analyses indicated interaction of AV-153 with guanine, cytosine and thymine bases, but no interaction with adenine was detected. CONCLUSIONS: The antimutagenic substance AV-153 appears to intercalate between the DNA strands at the site of a DNA nick in the vicinity of two pyrimidines.

Structure and analysis of the 5' flanking region of the human interleukin-2 gene.

To investigate the structural and functional organization of the human interleukin-2 locus, the nucleotide sequence of 9339 bp of the 5' flanking region of this gene has been determined. Computer search analysis reveals five stretches with a high degree of homology between the human and mouse 5' flanking sequence, including a very distant 5' region. In this region additional binding sites for potential transcription factors were found that are identical to known regulatory sequences. The possible roles of these putative regulatory elements in the interleukin-2 gene regulation remain to be proven. The 5' end of the sequence contains almost full-length LINE element. LINE consensus open reading frames ORF1 and ORF2 in the reported sequence are interrupted by insertions, deletions or in-frame nonsense mutations. Comparative analysis of the ratio of codon changes that result in amino acid replacement to those that are silent revealed a high silent mutation frequency throughout the consensus ORF1 3' end, suggesting that this region is probably under selection for protein function.

[Cloning and nucleotide sequence of the 5'-flanking area of the human interleukin-2 gene]. / Klonirovanie i nukleotidnaia posledovatel'nost' 5'-flankiruiushchei oblasti gena interleikin-2 cheloveka.

We have cloned human interleukin-2 gene and sequenced its 1'-flanking region (-1940 to -936). The region contains promoter-like structures having a high degree of homology with the real promoter.