Pharmacological and genetic analysis of chaperone proteins Hsp90 and p23 in telomerase and telomere biology

Telomeres are functional DNA-protein complexes located at the ends of linear chromosomes. They sequester chromosome ends from DNA repair pathways and prevent the loss of genomic material therefore maintaining the integrity of chromosomes. Telomeric DNA in humans is composed of many tandem repeats of the sequence TTAGGG that are gradually lost during replication due to the end replication problem. This regulates entrance into senescence and therefore determines cellular lifespan.;Telomerase is minimally composed of an RNA template (hTR) for synthesis of telomeric repeats, and a catalytic subunit, hTERT. Telomerase compensates for the loss of telomeric DNA by catalyzing the addition of telomeric repeats onto chromosome ends. While it is not expressed in normal somatic cells, telomerase is expressed in more than 85% of malignant cancers, making it an important target for cancer therapy.;We have previously shown that chaperones Hsp90 and p23, functionally associated with telomerase and are required for assembly of the active enzyme. Interestingly, Hsp90 and p23 remain associated with telomerase after folding, suggesting an additional role of chaperones in telomerase regulation. To explore this finding we have targeted Hsp90 and p23 using either a low concentration of hsp90 inhibitor radicicol or stable integration of siRNAs in metastatic prostate cancer cells. Depleting functional hsp90 or p23 has minimal effects on growth and viability in the short-term. However, tumorigenic cells cultured in low dose radicicol show a transient inhibition of telomerase followed by a period of adaptation and upregulation of the enzyme. Interestingly, continuous culturing of these cells and cells expressing long-term stable siRNA directed at hsp90 and p23 undergo dramatic telomere shortening in the presence of detectable telomerase activity. Suggesting chaperones may play additional roles in telomerase and telomere biology.;Interestingly, we observe that both radicicol treated cells and cells expressing siRNAs have high levels of nitric oxide synthase (NOS)-dependent free radical production and that removal of radicicol (an subsequently free radicals) leads to telomere elongation and prevents radicicol induced delayed apoptosis suggesting that reduced functional hsp90 or p23 disrupts free radical homeostasis and may contribute to telomere damage in these cells.