2-5A antisense therapy directed against human telomerase RNA inhibits telomerase activity and induces apoptosis without telomere impairment in cervical cancer cells

Abstract

Human telomerase RNA ( hTR ), an important component of telomerase, is a possible target of telomerase -based cancer gene therapy. The present study was undertaken to assess the efficacy of antisense hTR therapy using newly developed 2 -5A ( 5 0 -phosphorylated 2 0 -5 0 -linked oligoadenylate ) -linked oligonucleotides against cervical cancer cells. ME180 and SiHa cells were treated with 2 -5A -linked antisense hTR designed to complement the region of hTR between residues 76 and 94. The hTR expression, telomerase activity, cell viability, and apoptosis were then examined. The 2 -5A anti -hTR effectively degraded hTR and inhibited telomerase activity. The 2 -5A mutant anti -hTR and the anti -hTR without 2 -5A were not capable of inhibiting telomerase activity. Inhibition of telomerase by 2 -5A anti -hTR rapidly decreased cell viability only in telomerase -positive cells within 3 -6 days after the treatment, when telomere length has not yet been shortened. This inhibition was associated with apoptosis, possibly through activation of caspase family members. These findings suggest that 2 -5A -linked antisense -hTR therapy has a potent telomeraseinhibitory effect associated with a cytocidal effect from caspase -induced apoptosis, and may therefore be a potential tool in telomerase -based gene therapy against cervical cancers.

Telomerase inhibitor GRN163L inhibits myeloma cell growth in vitro and in vivo

“…Although in some malignancies such as cervical cancer the inhibition of telomerase induces apoptotic cell death within a few days, without inducing significant telomere shortening, 30 cell death in most cancer cells occurs after a lag period of 3À5 weeks, probably required for reduction in telomere length below critical limit. 18,[24][25][26][27]29,31 Moreover, we observed a marked reduction in telomere length, leading to complete erosion of telomeres on a subset of chromosomes in myeloma cells following GRN163L treatment.…”

“…The Invariant Set Normalization method was used to normalize arrays at probe level to make them comparable, and the model-based method was used for probe selection and to compute expression values. 29,30 These expression levels were assigned standard errors based on replicates, which were subsequently used to compute 90% confidence intervals of fold changes in intergroup comparisons. The lower confidence bounds of 'fold change' were conservative estimates of the actual changes.…”

“…[29][30][31][32][33][34][35] Approximately 8 weeks following bone implantation, 5 Â 10 6 human IL-6-dependent INA6 cells were injected directly into human bone in SCID-hu host. Production of human paraprotein in mouse serum was monitored as an indicator of myeloma cell growth.…”

Efficient growth inhibition of HPV 16 E6-expressing cells by an adenovirus-expressing p53 homologue p73β

“…The universal cdk inhibitor p21 WAF1/CIP1 , expressed from an adenovirus, has been found to be very effective in inhibiting the growth of cancer cells expressing HPV E6 gene or human cervical cancerderived cancer cell lines (Prabhu et al, 1998;Tsao et al, 1999). Antisense approach against HPV E6/E7 oncogene or telomerase RNA has been found to be effective in inhibiting the growth of cervical cancer cells (Hamada et al, 1996;Yatabe et al, 2002). We have attempted p73-based approach for efficient inhibition of growth of cervical cancer cells.…”

Telomerase and its potential for therapeutic intervention

“…An antisense construct expressing 185 nucleotides of hTERC was able to shorten telomeres in HeLa cells over 23-26 population doublings via the reduction of telomerase activity and induced apoptosis (Feng et al, 1995). ODNs tagged with 2 0 -5 0 -oligoadenylate (2-5A) to bind and cleave hTERC caused apoptosis in glioma, prostate, cervical, bladder and ovarian cancer cells within 4-5 days (Kondo et al, 1998;Kushner et al, 2000;Koga et al, 2001;Yatabe et al, 2002). To enhance the binding properties to hTERC sequences and stability, 2 0 -O-methyl-RNA oligomers were developed (Herbert et al, 1999).…”