Telomere Maintenance in Laser Capture Microdissection–Purified Barrett's Adenocarcinoma Cells and Effect of Telomerase Inhibition In vivo

Abstract

Purpose: The aims of this study were to investigate telomere function in normal and Barrett's esophageal adenocarcinoma (BEAC) cells purified by laser capture microdissection and to evaluate the effect of telomerase inhibition in cancer cells in vitro and in vivo. Experimental Design: Epithelial cells were purified from surgically resected esophagi. Telomerase activity was measured by modified telomeric repeat amplification protocol and telomere length was determined by real-time PCR assay. To evaluate the effect of telomerase inhibition, adenocarcinoma cell lines were continuously treated with a specific telomerase inhibitor (GRN163L) and live cell number was determined weekly. Apoptosis was evaluated by Annexin labeling and senescence by h-galactosidase staining. For in vivo studies, severe combined immunodeficient mice were s.c. inoculated with adenocarcinoma cells and following appearance of palpable tumors, injected i.p. with saline or GRN163L. Results: Telomerase activity was significantly elevated whereas telomeres were shorter in BEAC cells relative to normal esophageal epithelial cells. The treatment of adenocarcinoma cells with telomerase inhibitor, GRN163L, led to loss of telomerase activity, reduction in telomere length, and growth arrest through induction of both the senescence and apoptosis. GRN163L-induced cell death could also be expedited by addition of the chemotherapeutic agents doxorubicin and ritonavir. Finally, the treatment with GRN163L led to a significant reduction in tumor volume in a subcutaneous tumor model. Conclusions: We show that telomerase activity is significantly elevated whereas telomeres are shorter in BEAC and suppression of telomerase inhibits proliferation of adenocarcinoma cells both in vitro and in vivo.

Shortened telomere length in white matter oligodendrocytes in major depression: potential role of oxidative stress

“…Genomic DNA was isolated from captured cells using a silica-gel membrane based QIAamp DNA Micro Kit (Qiagen, USA) as described previously (Shammas et al, 2008) with modification. To prevent generation of abasic sites on the DNA and to minimize oxidative damage, 50 μM phenyl-tert-butyl nitron (SIGMA, USA) was supplemented (O'Callaghan et al, 2008).…”

Evidence for Enhanced Telomerase Activity in Barrett's Esophagus with Dysplasia and Adenocarcinoma

“…More recently, Barclay et al (2005) evaluated telomerase activity along with hTERT and splice variants in BE and adenocarcinoma and suggested a significant increase in telomerase activity occurring in patients with BE and adenocarcinoma. However, neither hTERT mRNA levels nor hTERT mRNA splicing patterns correlate with telomerase enzyme activity, as it depends on posttranscriptional and posttranslational modification of hTERT, which includes phosphorylation of hTERT protein, assembly into telomerase holoenzyme, and its association with other proteins such as hsp90 and p23 etc (Shammas et al, 2008).…”

“…Activation of telomerase results in maintenance of telomere length, immortalization of cells, and the eventual development of a malignant clone (Harley et al, 1996;Blackburn 2010). Telomerase activity is low or undetectable in normal somatic tissues in which telomeres are not extended and, therefore, undergo progressive shortening with cell division (Shammas et al, 2008). Telomerase activity is seen in 85-95% of biopsies of various tumor types but is absent in normal somatic cells and in normal tissue adjacent to cancers (Kim et al, 1994).…”

Emerging therapeutic targets in esophageal adenocarcinoma

“…However, due to the disproportional ratios of telomerase present in normal versus tumor cells, telomerase-targeted therapy can be an effective anticancer therapy while sparing normal tissue [59, 61]. Recently, inhibition of telomerase, and therefore shortening of telomeres, has been considered as a mechanism to target tumor growth in Barrett's induced EAC [62, 63]. …”