Temporary ex vivo inhibition of the expression of the human oncogene HER2 (NEU) by a triple helix-forming oligonucleotide.
A 28-base phosphodiester triple helix-forming oligonucleotide, mostly G and A containing, targeted to a polypurine tract interrupted by a purine-pyrimidine inversion, situated upstream from the TATA box of the promoter of the human HER2 gene, was conceived by computer modeling. The "energetically best choice" was oligo 28(C), which formed the triple helix in vitro, as proved by gel retardation and Fourier transform infrared spectroscopy. When administered as a complex with lipofectin, fluorescence confocal microscopy and electrophoresis confirmed the delivery and persistence of this unprotected oligonucleotide inside MCF7 (breast cancer) cells. At a concentration of 2 microM, the oligonucleotide reduced within 6 h the HER2 mRNA level to 42% (Northern blot) but did not interfere with the transcription of a housekeeping gene, glyceraldehyde-3-phosphate dehydrogenase. During the first day of administration at 0.22 microM, it lowered to 59% the HER2 protein in treated, as compared to nontreated, cells (ELISA). The effect was sequence specific when compared to that of five different negative controls, and it was target selective when compared to the expression of a related, nontargeted protein, the epidermal growth factor receptor. By day 2, the inhibitory effect was overcome by replenishment reactions.
Porumb
H
H
Laboratoire de Spectroscopie Bio-moléculaire, URA CNRS 1430, Université Paris-Nord, Bobigny, France.
Gousset
H
H
Letellier
R
R
Salle
V
V
Briane
D
D
Vassy
J
J
Amor-Gueret
M
M
Israël
L
L
Taillandier
E
E
D001483:Base Sequence; D015152:Blotting, Northern; D001943:Breast Neoplasms / Q000188:drug therapy / Q000235:genetics / Q000378:metabolism; D004586:Electrophoresis; D004797:Enzyme-Linked Immunosorbent Assay; D015870:Gene Expression / Q000187:drug effects*; D006801:Humans; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D016376:Oligonucleotides, Antisense / Q000737:chemistry / Q000494:pharmacology*; D010714:Phosphatidylethanolamines / Q000494:pharmacology; D011687:Purines / Q000494:pharmacology; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000096:biosynthesis / Q000235:genetics*; D012680:Sensitivity and Specificity; D017550:Spectroscopy, Fourier Transform Infrared
D016376:Oligonucleotides, Antisense; D010714:Phosphatidylethanolamines; D011687:Purines; D018719:Erb-b2 Receptor Tyrosine Kinases; C020888:1,2-dielaidoylphosphatidylethanolamine