Telomere restoration and extension of proliferative lifespan in dyskeratosis congenita fibroblasts

Abstract

Dyskeratosis congenita (DC), an inherited bone marrow failure syndrome, is caused by defects in telomerase. Somatic cells from DC patients have shortened telomeres and clinical symptoms are most pronounced in organs with a high cell turnover, including those involved in hematopoiesis and skin function. We previously identified an autosomal dominant (AD) form of DC that is caused by mutations in the telomerase RNA component (TER). In this study, we evaluated whether retroviral expression of TER and/or telomerase reverse transcriptase (TERT), the catalytic component of telomerase, could extend telomere length and rescue AD DC cells from a phenotype characteristic of early senescence. Exogenous TER expression, without TERT, could not activate telomerase in AD DC skin fibroblasts. Transduction of TERT alone, however, provided AD DC cells with sufficient telomerase activity to extend average telomere length and proliferative capacity. Interestingly, we found that expression of TER and TERT together resulted in extension of lifespan and higher levels of telomerase and longer telomeres than expression of TERT alone in both AD DC and normal cells. Our results provide evidence that AD DC cells can be rescued from defects in telomere maintenance and proliferation, and that coexpression of TERT and TER together provides a more efficient means to elongate telomeres than expression of TERT alone. Similar strategies may be useful for ameliorating the detrimental effects of telomere shortening in AD DC and other diseases associated with telomerase or telomere defects.

Fig. 1

Premature senescence and short telomeres in AD DC (DC-HSF-1) as compared to normal (N-HSF-1) fibroblasts. (A) DC-HSF-1 cells proliferated for approximately half the lifespan of normal cells. (B) Quantification of telomere signal in normal and AD DC fibroblasts at early passage and senescence. Telomere signal was ascertained by real-time PCR methodology as described in the Experimental procedures. The T : S ratio represents the ratio of telomere signal over that of a single gene copy control (all relative to the T : S ratio of N-HSF-1 at early passage). All error bars represent standard error of the mean from three replicate assays.

Fig. 2

Reconstitution of telomerase in TER negative cells. (A) The TER/eGFP FIV lentiviral construct. The TER gene along with a U3 small nucleolar (sno) RNA polymerase II promoter was inserted into the replication defective eGFP FIV construct. (B) The TER/eGFP FIV and eGFP vector alone were pseudotyped with VSV-G and were transduced into TERT expressing, TER negative VA13 cells. Transduction resulted in high telomerase activity in these cells, indicating functionality of the introduced TER gene. (C) Northern blot showing the accumulation of mature TER of approximately 450 bases in cells transduced with the TER/eGFP FIV as compared to eGFP FIV alone. RPLP0 is an internal control for RNA loading.

Fig. 3

Reconstitution of TER, telomerase, and telomere length in AD DC fibroblasts. Cells were transduced with vector, TER alone, TERT alone, or TER and TERT together. AD DC cells are in black and normal cells in white. (A) TER levels in transduced cells as measured by QRT-PCR. Values are relative to vector transduced normal cells (N-Vector). (B) Telomerase activity as measured by a quantitative PCA TRAP assay [in cell equivalents as compared to the activity of a standard E6/E7 immortalized human skin keratinocytes (HSK) cell line]. (C) Relative telomere length in transduced cells. Quantification of telomere signal was ascertained by real-time PCR methodology. The T : S ratio represents the ratio of telomere signal over that of a single gene copy control (all relative to the T : S ratio of N-Vector). All error bars represent standard error of the mean from three replicate assays.

Fig. 4

(A) Telomere length over long-term passaging in TERT and TERT/TER transduced AD DC (DC) and normal (N) fibroblast clones. E, early passage (P5, ~15 pd postcloning); L, later passage (P25, ~75 pd postcloning). Three clones of each cell type were analyzed. Telomere length was assessed as in Fig. 3 with a quantitative PCR assay. Relative T : S ratio is the ratio of telomere over single gene signal made relative to early passage normal (N) fibroblasts. All values represent the average of three replicate assays. Error bars are standard error of the mean. (B) Telomerase activity in early and late passages of TERT and TERT/TER transduced DC cells. ‘E’ and ‘L’ designations are similar to those described in 4A. Error bars represent standard error of the mean for three replicate assays.

Fig. 5

Telomerase reconstitution ‘rejuvenates’ AD DC fibroblasts. Normal and DC fibroblasts were transduced with vector, TER, TERT, or TERT/TER as described in the Experimental procedures. Photographs were taken within three passages after selection (×100 magnification).

Fig. 6

Telomere length as assessed by Q-FISH. (A) Representative metaphases of vector only AD DC fibroblasts (Vector-DC-HSF-1), TERT expressing AD DC fibroblast clone at early passage (TERT-DC-HSF-1 clone A), and TERT-TER expressing AD DC fibroblast clone at early passage (TERT-TER-DC-HSF-1 clone C). Q-FISH was performed using a PNA telomere-specific probe (see Experimental procedures). (B) Histogram representation of telomere signals from > 400 telomeres per cell type of transduced early passage AD DC fibroblasts including Vector-DC-HSF-1, TERT-DC-HSF-1 clone A, and TERT-TER-DC-HSF-1 clone C (top three panels) and transduced early passage normal (N-HSF-1) fibroblasts Vector-N-HSF-1, TERT-N-HSF-1 clone A, and TERT-TER-N-HSF-1 clone C (bottom three panels). The Y-axis on each graph represents frequency of events while the X-axis represents telomere length (as ascertained by calibration with controls). Average telomere lengths are shown beneath each graph. (C) Extensive elongation and a normal distribution of telomere length in later passage TERT-TER DC cells.

Fig. 6

Telomere length as assessed by Q-FISH. (A) Representative metaphases of vector only AD DC fibroblasts (Vector-DC-HSF-1), TERT expressing AD DC fibroblast clone at early passage (TERT-DC-HSF-1 clone A), and TERT-TER expressing AD DC fibroblast clone at early passage (TERT-TER-DC-HSF-1 clone C). Q-FISH was performed using a PNA telomere-specific probe (see Experimental procedures). (B) Histogram representation of telomere signals from > 400 telomeres per cell type of transduced early passage AD DC fibroblasts including Vector-DC-HSF-1, TERT-DC-HSF-1 clone A, and TERT-TER-DC-HSF-1 clone C (top three panels) and transduced early passage normal (N-HSF-1) fibroblasts Vector-N-HSF-1, TERT-N-HSF-1 clone A, and TERT-TER-N-HSF-1 clone C (bottom three panels). The Y-axis on each graph represents frequency of events while the X-axis represents telomere length (as ascertained by calibration with controls). Average telomere lengths are shown beneath each graph. (C) Extensive elongation and a normal distribution of telomere length in later passage TERT-TER DC cells.

Fig. 7

Effects of telomerase on telomere length at individual chromosome arms. Graphs represent telomere length on the ‘p’ and ‘q’ arms from individual chromosomes of early passage cells expressing eGFP only (A), TER (B), or TERT (C). The distribution of telomere length at individual chromosome arms in 7–20 metaphase cells is expressed in box plots. In each box plot, the stars represent the first and 99th percentile of the telomere length values, the lines represent the 10th and 90th percentile, and the boxes represent the 25th and 75th percentile. Median values are given by the small box and the 50th percentile of the telomere length distribution by the horizontal bar in the box.