2-5A antisense directed against telomerase RNA produces apoptosis in ovarian cancer cells.
OBJECTIVE
RNase L is converted to an active form upon binding short 2',5'-oligoadenylates (2-5A). To direct RNase L to an RNA target, 2-5A is attached to an antisense oligonucleotide (2-5A antisense). This chimera can be directed against telomerase-an RNA-protein complex that elongates telomeric DNA and is involved in cellular immortalization. Our objective is to investigate the effect of 2-5A antisense by targeting telomerase RNA (hTR) in the ovarian cancer cell line, HEY-1B.


METHODS
Baseline RNase L levels and telomerase activities were measured in both HEY-1B and normal ovarian epithelial cells (NOE). Cells were treated daily with chimeric oligonuclotides (ODN) directed against four different hTR sites, or control ODNs including nonchimeric antisense, 2-5A fused to a mismatched sequence, or inactive 2-5A fused to antisense. At 48 h, apoptosis was evaluated using the TUNEL assay. After six daily ODN administrations, telomerase activity was redetermined, and at 7 days viability counts were obtained.


RESULTS
Both cell lines expressed similar levels of RNase L. Hey-1B displayed telomerase activity while NOE did not. After 7 days of transfection, 2-5A antisense ODNs caused profound cell death in the HEY-1B cells, but not in the NOE cells. This effect was seen regardless of hTR target site, and ODN controls showed no significant decrease in cell viability in either cell line. HEY1B cells treated with 2-5A antisense against hTR showed a decrease in telomerase activity and a profound induction of programmed cell death.


CONCLUSIONS
The results suggest that 2-5A antisense directed against telomerase RNA results in apoptotic cell death in ovarian cancer cells, but not normal ovarian epithelial cells. The 2-5A antisense strategy may hold a considerable advantage over the conventional antisense approach in targeting cancer-causing genes.
Kushner
D M
DM
Department of Gynecology & Obstetrics, The Cleveland Clinic Foundation, Cleveland, Ohio, 44195, USA.
Paranjape
J M
JM
Bandyopadhyay
B
B
Cramer
H
H
Leaman
D W
DW
Kennedy
A W
AW
Silverman
R H
RH
Cowell
J K
JK
D000227:Adenine Nucleotides / Q000378:metabolism / Q000627:therapeutic use*; D002470:Cell Survival / Q000187:drug effects; D004722:Endoribonucleases / Q000378:metabolism / Q000627:therapeutic use*; D005260:Female; D006801:Humans; D009363:Neoplasm Proteins / Q000032:analysis / Q000037:antagonists & inhibitors*; D016376:Oligonucleotides, Antisense / Q000378:metabolism / Q000627:therapeutic use*; D009843:Oligoribonucleotides / Q000378:metabolism / Q000627:therapeutic use*; D010051:Ovarian Neoplasms / Q000201:enzymology / Q000628:therapy*; D012334:RNA, Neoplasm / Q000037:antagonists & inhibitors; D011993:Recombinant Fusion Proteins / Q000627:therapeutic use; D019098:Telomerase / Q000032:analysis / Q000037:antagonists & inhibitors*; D014407:Tumor Cells, Cultured
D000227:Adenine Nucleotides; D009363:Neoplasm Proteins; D016376:Oligonucleotides, Antisense; D009843:Oligoribonucleotides; D012334:RNA, Neoplasm; D011993:Recombinant Fusion Proteins; C023505:2',5'-oligoadenylate; D019098:Telomerase; D004722:Endoribonucleases; C031956:2-5A-dependent ribonuclease