[Telomerization as a method of obtaining immortal human cells preserving normal properties].
Most human somatic cells have no telomerase activity. This leads to terminal underreplication of chromosomes and, hence, proliferative ageing of cells. We studied the consequences of introduction of the gene of the catalytic component of human telomerase hTERT in the normal fibroblasts of adult human skin. The expression of this gene led to the appearance of telomerase activity in the fibroblasts, elongation of telomeres (to the size characteristic of the embryonic cells), and immortalization. The cells retained their normal karyotype. The activity of ribosomal genes remained unchanged: the degree of their methylation, abundance, and transcriptional activity (two clones were studied). The cells did not undergo significant changes after transition over the Hayflick's limit, retained the constant rate of proliferation (one of the clones was followed to the level of 200 duplications of the population), and resembled, in appearance, young diploid human fibroblasts. The initial cells and cells transfected by an empty vector could pass through no more than 68 duplications, their proliferation slowed down and they acquired the morphology characteristic for the ageing cells. The telomerized cells retained the normal capacity of entering the proliferative rest as a result of serum starvation. Telomerization did not eliminate the contact inhibition of proliferation but led to an increased saturating density of cells, which reached the levels characteristic for the early embryonic cells. The long-term suppression of the telomerase function by azidothymidine led to a shortening of telomeres and significantly slowed down cell proliferation. The cells that did not divided for a long time were enlarged, preserved their viability, and resembled, in appearance, the ageing cells. In the test on heterokaryons (index of telomerase activity on the chromosomes inside the cell), the telomerized cells behaved as other immortal cells. All these data suggest that the telomerized cells preserved the normal mechanisms of regulation of cell proliferation.
Egorov
E E
EE
Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Vavilova 32, Moscow, 119991 Russia. yegorov@eimb.ru
Terekhov
S M
SM
Vishniakova
Kh S
KhS
Karachentsev
D N
DN
Kazimirchuk
E V
EV
Tsvetkova
T G
TG
Veĭko
N N
NN
Smirnova
T D
TD
Makarenkov
A S
AS
El'darov
M A
MA
Meshcheriakova
Iu A
IuA
Liapunova
N A
NA
Zelenin
A V
AV
D018929:Cell Culture Techniques / Q000379:methods; D002455:Cell Division / Q000187:drug effects / Q000235:genetics; D002461:Cell Line, Transformed / Q000166:cytology* / Q000502:physiology*; D016922:Cellular Senescence / Q000502:physiology; D016895:Culture Media, Serum-Free / Q000494:pharmacology; D004268:DNA-Binding Proteins; D005347:Fibroblasts / Q000166:cytology / Q000187:drug effects / Q000502:physiology; D020459:Genes, rRNA; D006801:Humans; D007621:Karyotyping; D019098:Telomerase / Q000235:genetics / Q000378:metabolism*; D016615:Telomere / Q000502:physiology*; D014162:Transfection
D016895:Culture Media, Serum-Free; D004268:DNA-Binding Proteins; D019098:Telomerase