Inhibition of DNA polymerase alpha expression and cell growth, a possible triple helix mechanism.
The antiproliferative effects mediated by a 14-mer homopyrimidine oligonucleotide (5' CTTTCT-CTTTTCTC3'), designed to form DNA triplex with a purine region of the DNA polymerase alpha promoter, were evaluated on the human breast cancer cell line MDA-MB 231. In order to stabilize the triple complex under physiologic conditions, replacement of cytosines by methylcytosines in the oligomer sequence was carried out. Band-shift analyses demonstrated a complete triplex formation between the radiolabeled target duplex DNA and the methylcytosine-modified oligomer at the concentration of 0.1 microM under physiologic pH and temperature. A single exposure of MDA-MB 231 cells to 0.5 microM methylcytosine-modified oligonucleotide was able to markedly reduce the cell number and the percentage of cells in DNA synthesis up to 58% and 66%, respectively, compared with controls. Furthermore, a 48% reduction in the amount of the DNA polymerase alpha mRNA was reported after treatment with the oligomer. In conclusion, data from the present study demonstrate that an oligonucleotide to DNA polymerase alpha promoter, designed to form a triple helix with target double-stranded DNA, inhibits the expression of the reporter gene at the biologic and molecular levels, suggesting a possible triplex-mediated mechanism of action.
Alama
A
A
Dipartimento di Farmacologia Sperimentale, Istituto Nazionale Ricerca Cancro, Genova, Italy.
Barbieri
F
F
Cagnoli
M
M
Mazzei
M
M
Grandi
T
T
Nicolin
A
A
D001483:Base Sequence; D001943:Breast Neoplasms / Q000473:pathology; D002455:Cell Division / Q000187:drug effects; D004257:DNA Polymerase II / Q000096:biosynthesis / Q000187:drug effects* / Q000235:genetics; D005260:Female; D006801:Humans; D008969:Molecular Sequence Data; D009841:Oligonucleotides / Q000235:genetics / Q000494:pharmacology*; D011485:Protein Binding; D017510:Protein Folding; D014407:Tumor Cells, Cultured
D009841:Oligonucleotides; D004257:DNA Polymerase II