Dissociation of telomerase activity and telomere length maintenance in primitive human hematopoietic cells

Abstract

Primitive human hematopoietic cells have low endogenous telomerase activity, yet telomeres are not maintained. In contrast, ectopic telomerase expression in fibroblasts and other cells leads to telomere length maintenance or elongation. It is unclear whether this disparity can be attributed to telomerase level or stems from fundamentally different telomere biology. Here, we show that telomerase overexpression does not prevent proliferation-associated telomere shortening in human hematopoietic cells, pointing to the existence of cell type-specific differences in telomere dynamics. Furthermore, we observed eventual stabilization of telomere length without detectable changes in telomerase activity during establishment of two leukemic cell lines from normal cord blood cells, indicating that additional cooperating events are required for telomere maintenance in immortalized human hematopoietic cells.

Fig. 1.

Absence of telomere shortening during short-term culture of normal human hematopoietic cells. (A) Telomerase activity of GFP- and hTERT-transduced Lin^- CB cells assessed by the telomeric repeat amplification protocol. Cumulative days in culture are indicated. CHAPS is a negative buffer control, and TSR8 is a positive control. Telomerase activity of HeLa cells is shown for comparison. IC, internal control for Taq amplification of telomere extension products. (B) Proliferation (Upper) and mean telomere length (Lower) of GFP-transduced (▵) and hTERT-transduced (○) Lin^- CB cells during culture.

Fig. 2.

TLS-ERG-expressing Lin^- CB cells have low telomerase activity and exhibit telomere shortening. (A) Proliferation of Neo-transduced (▵) and TLS-ERG-transduced (○) cells from a representative experiment. (B) Telomerase activity of Neo- and TLS-ERG-transduced cells. Controls are as described in Fig. 1 A. Cumulative PD are indicated below each lane. (C) Mean telomere length of TLS-ERG-transduced cells determined by flow-FISH. All data are from the same experiment.

Fig. 3.

hTERT overexpression does not prevent telomere shortening in TLS-ERG-expressing CB cells during extended culture. (A) Telomerase activity of cells transduced with Neo, TLS-ERG, GFP, or hTERT as indicated. Cumulative days in culture and PD are indicated above and below each lane, respectively. Controls are as described in Fig. 1 A. (B) Mean telomere length of cells transduced with Neo (▴), TLS-ERG (•), TLS-ERG plus GFP (▵), and TLS-ERG plus hTERT (○), as determined by flow-FISH. All data are from the same experiment.

Fig. 4.

Eventual stabilization of telomere length in hTERT-transduced hematopoietic cells without a concomitant change in telomerase activity. (A) (Upper) Flow cytometric analysis showing expression profiles of CD14, CD15, CD33, CD34, and CD38, and May-Grünwald-Giemsa-stained cytospin preparation of cells expressing TLS-ERG after 89 and 85 days of culture, respectively. (Scale bar: 20 μm.) (Lower) The same analysis for cells expressing TLS-ERG plus hTERT (T2a cells) after 357 and 382 days of culture, respectively. (B) Mean telomere length of cells transduced with Neo (▴), TLS-ERG (•), TLS-ERG plus GFP (▵), and TLS-ERG plus hTERT (T2a cells) (○), as determined by flow-FISH. (C) Telomerase activity of T2a cells after cumulative days in culture indicated. Protein extract from 50-5,000 cells was used for each sample as shown. PD are indicated below. Controls are as described in Fig. 1A.

Fig. 5.

Absence of telomere length maintenance in TLS-ERG-expressing cells with elevated levels of endogenous telomerase activity. (A) Telomerase activity of TEX cells after cumulative days in culture indicated. Protein extract from 50-5,000 cells was used where indicated. PD are shown below. Controls are as described in Fig. 1 A. (B) Mean telomere length of TEX cells as determined by flow-FISH (○), compared with relative telomerase activity during culture (▵) normalized to level after 26 days (4 PD). TFU, telomere fluorescence units.