Inhibition of Erythroleukemia Cell Growth by Triplex-forming RNAs.

OBJECTIVE

We have previously reported that oligodeoxyribonucleotides, designed to bind in a triplex fashion to a specific p53 binding site homology, inhibit the proliferation of colon cancer cells in vitro and in vivo. The present study was designed to extend these observations and to determine whether ribonucleic acid (RNA) generated from a retroviral vector (RVV) and possessing a corresponding triplex forming site can, in a similar fashion, inhibit proliferation of p53-null K-562 leukemia cells. Viral vectors may offer advantages over oligonucleotides for tumor treatment. RVVs have the potential to be taken up more efficiently than oligonucleotides and to be expressed continuously and long-term, circumventing the need for repeated and frequent oligomer administration.

EXPERIMENTAL DESIGN

The p53-null human erythroleukemia cell line, K-562, was stably transfected with a tetracycline-repressible p53 expression construct (p53/pUHD10-3). p53 protein in these cells is expressed in the absence of tetracycline but down-regulated upon tetracycline treatment. Triplex-forming oligonucleotides [Hoog 1 (experimental) and Hoog 3 (control)] were cloned into RVVs in order to generate triplex-forming fusion mRNAs. Naive K-562 cells and p53/pUHD10-3-transfected K-562 cells (with and without tetracycline treatment) were infected with viruses that express the triplex-forming RNAs. Cell growth was measured by BrdU incorporation into DNA.

RESULTS

RVVs encoding Hoog 1, in both orientations, inhibit the growth of naive K-562 cells and p53-transfected, tet-repressed K-562 cells. p53 expression in K-562 cells decreases growth to the same extent as Hoog 1 RVV treatment. However, Hoog 1-RVV does not further inhibit growth of p53-expressing K-562 cells. Treatment with an RVV encoding the control, Hoog 3, has no growth inhibitory effect.

CONCLUSION

Triple helix-forming RNAs directed to a p53 consensus sequence homology reduce leukemia cell proliferation, suggesting a novel method of treatment.