Reconstitution of active telomerase in primary human foreskin fibroblasts: effects on proliferative characteristics and response to ionizing radiation.
OBJECTIVE
Telomere shortening has been proposed to trigger senescence, and since most primary cells do not express active telomerase, reactivation of telomerase activity was proposed as a safe and non-transforming way of immortalizing cells. However, to study radiation responses, it is as yet unclear whether cells immortalized by telomerase reactivation behave in a similar manner as their parental primary cells.


METHODS
Primary human foreskin fibroblasts were transfected with the human catalytic subunit of telomerase, the reverse transcriptase (hTERT), and their growth characteristics and response to DNA damage were characterized.


RESULTS
The sole expression of the human hTERT was sufficient to immortalize the human foreskin fibroblasts. With time in culture, the immortalized cells almost doubled their average telomeric length and the clonal population contained almost no post-mitotic fibroblasts anymore. Up to 300 population doublings, no alterations compared with the parental primary cells were seen in terms of clonogenic radiosensitivity, DNA double-strand break repair, radiation-induced increases in p53 and p21(WAF-1,CIP-1) expression, and the G1/S and G2/M cell cycle checkpoints. Moreover, mitogen-induced mitotic arrest of fibroblasts was still possible in the hTERT-immortalized clones.


CONCLUSIONS
Immortalizing fibroblasts by reconstitution of active telomerase seems a good, reliable manner to generate a large source of cells with a radiation damage response similar to the primary cells.
Kampinga
H H
HH
Department of Radiation and Stress Cell Biology, DCB, Faculty of Medical Sciences, University of Groningen, Groningen 9713 AV, The Netherlands. h.h.kampinga@med.rug.nl
Van Waarde-Verhagen
M A W  H
MA
Van Assen-Bolt
A J
AJ
Nieuwenhuis
B
B
Rodemann
H P
HP
Prowse
K R
KR
Linskens
M H K
MH
D000222:Adaptation, Physiological / Q000528:radiation effects; D017209:Apoptosis / Q000528:radiation effects; D002455:Cell Division / Q000528:radiation effects; D002460:Cell Line; D002470:Cell Survival / Q000528:radiation effects; D004268:DNA-Binding Proteins; D004307:Dose-Response Relationship, Radiation; D004789:Enzyme Activation / Q000528:radiation effects; D005347:Fibroblasts / Q000166:cytology / Q000201:enzymology* / Q000528:radiation effects*; D006801:Humans; D008297:Male; D010413:Penis / Q000166:cytology / Q000201:enzymology / Q000528:radiation effects; D011839:Radiation, Ionizing; D011994:Recombinant Proteins / Q000378:metabolism; D012867:Skin / Q000166:cytology / Q000201:enzymology / Q000528:radiation effects; D019098:Telomerase / Q000235:genetics / Q000378:metabolism*; D014162:Transfection
D004268:DNA-Binding Proteins; D011994:Recombinant Proteins; D019098:Telomerase