Reconstituting Telomerase Activity Using the Telomerase Catalytic Subunit Prevents the Telomere Shorting and Replicative Senescence in Human Osteoblasts

Abstract

The rate of bone formation is largely determined by the number of osteoblasts, which in turn is determined by the rate of replication of progenitors and the life span of mature cells, reflecting the timing of death by apoptosis. However, the exact age-dependent changes of the cellular activity, replicative potential, and life span of osteoblasts have not been investigated to date. Here, we present evidence that the cellular activity, telomere lengths, and replicative life span of osteoblastic cells obtained from juxta-articular bone marrow gradually decrease with the advance of donor age. Recently, telomerase reverse transcriptase (hTERT) has been identified as a human telomerase catalytic subunit. We transfected the gene encoding hTERT into telomerase-negative human osteoblastic cells from donors and osteoblastic cell strain NHOst 54881 cells and showed that expression of hTERT induces telomerase activity in these osteoblastic cells. In contrast to telomerase-negative control cells, which exhibited telomere shortening and senescence after 10 -15 population doublings, telomerase-expressing osteoblastic cells had elongated telomere lengths and showed continued alkaline phosphatase activity and procollagen I C-terminal propeptide (

The role of chondrocyte senescence in osteoarthritis

“…Cells were allowed to reach confluence before harvesting for genomic DNA extraction. The average telomere length in primary chondrocytes was determined by terminal restriction fragment Southern blotting analysis as previously described (Yudoh et al, 2001). Briefly, chondrocyte cell lysates were digested with 500 µL of DNA extraction buffer [100 mM NaCl, 40 mM Tris-HCl pH 8, 20 mM EDTA, 0.5% sodium dodecyl sulphate (SDS)] containing 0.1 mg mL −1 Proteinase K. Genomic DNA was then extracted using phenol-chloroform.…”

Telomerase is not an oncogene

“…Transformed cells may also become immortal through the rare activation alternative (ALT) mechanisms of telomere maintenance. Spontaneous transition from hTERT transduced immortal normal cells to growth-deregulated transformed or tumorous cells has yet to be observed, but can be forced by expression of viral oncoproteins (broken arrows, bottom) (Migliaccio et al, 2000) Adrenocortical cells bovine, transformed adrenal gland yes 8 Osteoblasts, adult bone yes (Yudoh et al, 2001) Islet cells (beta enriched) adult primary pancreas no 9 (Halvorsen et al, 2000) (Matsunaga et al, 1999), and certain immune cells in AIDS (E ros et al, 1996) has been considered. The growing evidence that telomere-dependent replicative senescence may facilitate tumor initiation directly or indirectly, argues that telomerase activation may actually have a protective e ect on tumor initiation or progression in elderly patients.…”

Relevance and safety of telomerase for human tissue engineering

“…hTERT lifespan extension is also being investigated for the treatment of Duchenne muscular dystrophy by using genetically modified muscle satellite cells (6,7). Other cell types that have been lifespan-extended by telomerase in anticipation of tissue engineering applications include retinal pigment epithelia (8)(9)(10)(11)(12), dermal fibroblasts (13,14), vascular endothelium (15)(16)(17), bone marrow stromal cells (18)(19)(20)(21), osteoblasts (22,23), mesenchymal stem cells (24), and hepatic stellate cells (25,26). Using retroviral delivery of hTERT to human saphenous vein SMC, it is possible to culture autologous blood vessels for patients as old as 74 years (2).…”