Does a Sentinel or a Subset of Short Telomeres Determine Replicative Senescence?

The proliferative life span of human cells is limited by telomere shortening, but the specific telomeres responsible for determining the onset of senescence have not been adequately determined. We here identify the shortest telomeres by the frequency of signal-free ends after in situ hybridization with telomeric probes and demonstrate that probes adjacent to the shortest ends colocalize with γH2AX-positive DNA damage foci in senescent cells. Normal BJ cells growth arrest at senescence before developing significant karyotypic abnormalities. We also identify all of the telomeres involved in end-associations in BJ fibroblasts whose cell-cycle arrest at the time of replicative senescence has been blocked and demonstrate that the 10% of the telomeres with the shortest ends are involved in >90% of all end-associations. The failure to find telomeric end-associations in near-senescent normal BJ metaphases, the presence of signal-free ends in 90% of near-senescent metaphases, and the colocalization of short telomeres with DNA damage foci in senescent interphase cells suggests that end-associations rather than damage signals from short telomeres per se may be the proximate cause of growth arrest. These results demonstrate that a specific group of chromosomes with the shortest telomeres rather than either all or only one or two sentinel telomeres is responsible for the induction of replicative senescence

New therapeutic approach for brain tumors: Intranasal delivery of telomerase inhibitor GRN163

The blood-brain barrier is a substantial obstacle for delivering anticancer agents to brain tumors, and new strategies for bypassing it are greatly needed for brain-tumor therapy. Intranasal delivery provides a practical, noninvasive method for delivering therapeutic agents to the brain and could provide an alternative to intravenous injection and convection-enhanced delivery. We treated rats bearing intracerebral human tumor xeno-grafts intranasally with GRN163, an oligonucleotide N3′→P5′thio-phosphoramidate telomerase inhibitor. 3′-Fuorescein isothiocyanate (FITC)–labeled GRN163 was administered intranasally every 2 min as 6 μl drops into alternating sides of the nasal cavity over 22 min. FITC-labeled GRN163 was present in tumor cells at all time points studied, and accumulation of GRN163 peaked at 4 h after delivery. Moreover, GRN163 delivered intranasally, daily for 12 days, significantly prolonged the median survival from 35 days in the control group to 75.5 days in the GRN163-treated group. Thus, intranasal delivery of GRN163 readily bypassed the blood-brain barrier, exhibited favorable tumor uptake, and inhibited tumor growth, leading to a prolonged lifespan for treated rats compared to controls. This delivery approach appears to kill tumor cells selectively, and no toxic effects were noted in normal brain tissue. These data support further development of intranasal delivery of tumor-specific therapeutic agents for brain tumor patients

Antiadhesive Effects of Grn163L - An Oligonucleotide N3 '-> P5 ' Thio-Phosphoramidate Targeting Telomerase

We determined previously that a novel human telomerase RNA (hTR) antagonist, GRN163L, inhibited the tumorigenic potential of A549-luciferase (A549-luc) lung cancer cells in vitro and in vivo. Further studies revealed that A549-luc cells were also morphologically altered by GRN163L. A549-luc cells treated before cell attachment with a single dose of GRN163L only weakly attached to the substrate and remained rounded, whereas control mismatch-treated cells exhibited typical epitheloid appearance and adhesion properties. These morphologic changes were independent of hilt expression and telomerase inhibition and were unrelated to telomere length. This effect is dependent on the molecular properties of the lipid moiety, the phosphorothioate backbone, and the presence of triplet-G sequences within the GRN1163L structure. Altered adhesion was manifested by a 50% reduction in rapid cellular attachment and a 3-fold decrease in total cell spreading surface area. Administration of a single dose of GRN163L (15 mg/kg) at the time of cell inoculation, using an in vivo model of lung cancer metastasis, resulted in significant reductions in tumor burden at days 13, 20, and 27 of tumor progression. Thus, the potent antimetastatic effects of GRN163L may be related, in part, to the antiadhesive effects of this novel cancer therapeutic conferred via specific structural determinants and that these effects are independent of telomerase inhibition or telomere shortening.WoSScopu