The catalytic and the RNA subunits of human telomerase are required to immortalize equid primary fibroblasts

Abstract

Many human primary somatic cells can be immortalized by inducing telomerase activity through the exogenous expression of the human telomerase catalytic subunit (hTERT). This approach has been extended to the immortalization of cell lines from several mammals. Here, we show that hTERT expression is not sufficient to immortalize primary fibroblasts from three equid species, namely donkey, Burchelli's zebra and Grevy's zebra. In vitro analysis of a reconstituted telomerase composed by hTERT and an equid RNA component of telomerase (TERC) revealed a low activity of this enzyme compared to human telomerase, suggesting a low compatibility of equid and human telomerase subunits. This conclusion was also strengthened by comparison of human and equid TERC sequences, which revealed nucleotide differences in key regions for TERC and TERT interaction. We then succeeded in immortalizing equid fibroblasts by expressing hTERT and hTERC concomitantly. Expression of both human telomerase subunits led to telomerase activity and telomere elongation, indicating that human telomerase is compatible with the other equid telomerase subunits and proteins involved in telomere metabolism. The immortalization procedure described herein could be extended to primary cells from other mammals. The availability of immortal cells from endangered species could be particularly useful for obtaining new information on the organization and function of their genomes, which is relevant for their preservation.

Fig. 1

Morphology of donkey (a), Grevy’s zebra (b), and Burchelli’s zebra (c) primary fibroblasts at confluence. Senescent primary fibroblasts from donkey (d), Grevy’s zebra (e), and Burchelli’s zebra (f): cells have an increased size, are round shaped, lose side-by-side organization and vacuolize. hTERT transfected fibroblasts from donkey (g), Grevy’s zebra (h), and Burchelli’s zebra (i) showed a limited proliferative capacity, followed by a growth arrest and acquired a morphology similar to senescent primary fibroblasts. Immortalized fibroblasts from donkey (j) and Burchelli’s zebra (l) are characterized by a normal ordered organization similar to primary cells, while transfected cells from Grevy’s zebra (k) show a rounded morphology

Fig. 2

a PCR amplification of hTERT and PRKCI cDNA in primary fibroblasts from donkey, Burchelli’s and Grevy’s zebra and cells from the same species transfected with hTERT (+hTERT). b Top: Northern blot analysis of hTERC expression in primary fibroblasts from donkey (lane 1), Burchelli’s (lane 3), and Grevy’s zebra (lane 5), and in immortalized cells from the same species (lanes 2, 4, and 6). hTERC was detected by hybridization with a ³²P-labelled fragment of horse TERC. Bottom: Ethidium bromide staining of the agarose gel; the band corresponds to the 18S ribosomal RNA

Fig. 3

Schematic representation of the immortalization procedures for donkey (a), Grevy’s (b), and Burchelli’s zebra (c) fibroblasts. For each species, the lifespan in population doublings (PDs) of the primary fibroblasts, hTERT and hTERT/hTERC transfected cells is shown. In transfected cells, PDs were re-numbered after transfection

Fig. 4

TRAP analysis of telomerase activity in primary fibroblasts (P) from donkey (a), Burchelli’s (b), and Grevy’s zebra (c), and in cells from the same species transfected with hTERT only (TERT) or with both hTERT and hTERC (TERT + TERC). C+: positive control prepared using a protein extract from HeLa cells; C−: negative control, a heat inactivated protein extract was used in the assay; IC: internal amplification control; PE: amount of protein extract used for the assay; PD: number of population doublings. Telomerase activity was detected in cells transfected with both hTERT and hTERC, but not in primary fibroblasts or in cells transfected with hTERT only

Fig. 5

TRF length analysis in primary fibroblasts (P) and cells from donkey, Burchelli’s and Grevy’s zebra transfected with both hTERT and hTERC (TERT + TERC). RsaI-HinfI digested genomic DNA samples were separated by PFGE and TRFs were visualized by hybridization with a radioactively labeled telomeric probe. TRFs were anayzed at different number of population doublings (PD) for each cell line. In transfected cells, telomeres are clearly elongated compared to primary fibroblasts

Fig. 6

Telomeric fluorescent signal (red) variation in metaphase spreads from Burchelli’s zebra primary fibroblasts (a) and immortalized cells at PDs 13 (b) and PDs 68 (c) showing the elongation of telomeres with the increase of PDs. Chromosomes (blue) were stained with Hoechst 33258

Fig. 7

Chromosome number distribution in donkey (a), Grevy’s, (b) and Burchelli’s zebra (c) immortalized fibroblasts at different passages. Blue bars represent chromosome number distributions in primary fibroblasts. For each cell type, 100 metaphases were analyzed. The normal diploid number (2n) of the three species is indicated. A marked tendency to tetraploidy and subtetraploidy is observed in donkey and Grevy’s zebra immortalized cells

Fig. 8

In vitro reconstitution of telomerase with human TERT and horse TERC in the rabbit reticulocyte lysate (RRL) system. Telomerase activity was assayed using telomerase reconstituted without telomerase RNA (lane 2) or with horse (lane 3) or human (lane 4) TERC. Lanes 1 and 5 contain a size marker, corresponding to the primer radiolabeled at the 3′ end with [α-³²P]-dGTP. The number of nucleotides added by telomerase is indicated on the right. The total telomerase activity of the chimeric telomerase reconstituted with horse TERC and hTERT is 10 % relative to human telomerase and extension aborts after the first telomeric repeat addition (+4 product)

Fig. 9

Alignment of human (H. sapiens), horse (E. caballus), Burchelli’s zebra (E. burchelli), Grevy’s zebra (E. grevyi), donkey (E. asinus), and mouse (M. musculus) telomerase RNA sequences. Nucleotides conserved among the six species are in upper-case and mismatches are in red lower-case; nucleotides shared between the mouse and the equine sequences, but divergent from the human ones, are underlined; nucleotides on gray background correspond to the telomeric template; nucleotides relevant for telomerase activity are boxed (Chen and Greider 2003); conserved regions (CR1-8) as well as the two hypervariable regions (Chen et al. 2000) are shown