Telomere maintenance in laser capture microdissection-purified Barrett's adenocarcinoma cells and effect of telomerase inhibition in vivo.
OBJECTIVE
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.


METHODS
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 beta-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.
Shammas
Masood A
MA
Department of Surgery, Wayne State University and Karmanos Cancer Institute, Detroit, Michigan 48201, USA.
Qazi
Aamer
A
Batchu
Ramesh B
RB
Bertheau
Robert C
RC
Wong
Jason Y Y
JY
Rao
Manjula Y
MY
Prasad
Madhu
M
Chanda
Diptiman
D
Ponnazhagan
Selvarangan
S
Anderson
Kenneth C
KC
Steffes
Christopher P
CP
Munshi
Nikhil C
NC
De Vivo
Immaculata
I
Beer
David G
DG
Gryaznov
Sergei
S
Weaver
Donald W
DW
Goyal
Raj K
RK
D000230:Adenocarcinoma / Q000378:metabolism / Q000648:ultrastructure*; D000818:Animals; D017209:Apoptosis; D001471:Barrett Esophagus / Q000378:metabolism / Q000648:ultrastructure*; D045744:Cell Line, Tumor; D016922:Cellular Senescence; D004847:Epithelial Cells / Q000648:ultrastructure; D005260:Female; D006801:Humans; D007834:Lasers; D051379:Mice; D016513:Mice, SCID; D042282:Microdissection; D009368:Neoplasm Transplantation; D019098:Telomerase / Q000037:antagonists & inhibitors* / Q000378:metabolism; D016615:Telomere / Q000648:ultrastructure*
D019098:Telomerase