Long telomeres bypass the requirement for telomere maintenance in human tumorigenesis

Abstract

Despite the importance of telomere maintenance in cancer cell survival via the elongation of telomeres by telomerase reverse transcriptase (TERT) or alternative lengthening of telomeres (ALT), it had not been tested directly whether telomere maintenance is dispensable for human tumorigenesis. We engineered human tumor cells containing loxP-flanked hTERT to enable extensive telomere elongation prior to complete hTERT excision. Despite unabated telomere erosion, hTERT-excised cells formed tumors in mice and proliferated in vitro for up to 1 year. Telomerase reactivation or ALT was not observed, and the eventual loss of telomeric signal coincided with loss of tumorigenic potential and cell viability. Crisis was averted via the reintroduction of active but not inactive hTERT. Thus, telomere maintenance is dispensable for human tumorigenesis when telomere reserves are long. Yet, despite telomere instability and the presence of oncogenic RAS, human tumors remain susceptible to crisis induced by critically short telomeres.

Figure 1. An hTERT-excisable tumorigenic cell line

(A) Western analysis of whole cell lysates (50 µg) from HA5, HT (HA5 + TERT) and HTR (HT + RAS) cells at indicated population doubling level (PDL). (B) RT-PCR analysis of hTERT, hph, and GAPDH at indicated PDL. (C) Analysis of telomerase activity of cell lysates (200, 100, 50 ng) at indicated PDL. LB, negative buffer control; CTL, HeLa cell lysate positive control; IC, internal control PCR product. (D) Replicative lifespan of HA5, HT and HTR cells. HT or HTR cells were immortal. (E) Anchorage-independent colony growth at indicated PDL (n=3). 293T cells were a positive control for colony formation. Statistical significance between HA5 (no colonies formed) and HT or HTR cell lines as indicated (n=3, *** p<0.001; ns, p>0.05, power^{(1-β err prob)}>0.99, α^actual=0.05, two-tailed). (F) TRF analysis of average telomere length at increasing PDL. 293T cells were included as a control. Weighted mean telomere lengths (kbp) are indicated below each lane. (G) Schematic of elements introduced into HA5 cells, at indicated PDL.

Figure 2. Excision of hTERT from tumor cells with short telomeres

(A) Western analysis of cell lysates (50 µg) in HTR^EP (Early Passage) cells transfected with Cre recombinase or empty vector control at indicated population doubling level (PDL). (B) RT-PCR analysis of hTERT, hph, and GAPDH at indicated PDL. (C) Replicative lifespan of indicated cell lines. HTR^{EP Vec} remained immortal. (D) Anchorage-independent colony formation at indicated PDL. 293T cells were included as a positive control, and HA5 as a negative control. HTR^{EP Cre-4} at PDL 42 (no colonies) differed significantly from HTR^{EP Cre-4} at PDL 8 (n=3, * p<0.05, power^{(1-β err prob)}=1.0, α^actual=0.05, two-tailed). (E) RT-PCR analysis of hTERT, Sh Ble (zeocin) and GAPDH in tissue extracted from renal capsule (RC) or subcutaneous (SC) injection sites, or normal adjacent kidney (NK). (F) TRF analysis of average telomere length at increasing PDL. Weighted mean telomere lengths (kbp) are indicated below each lane. (G) Schematic of elements introduced into HT cells, at indicated PDL. (H) Incidence of tumor formation of indicated cell lines in immunodeficient mice (see Experimental Procedures for details).

Figure 3. Excision of hTERT from tumorigenic cells with elongated telomeres

(A) Telomerase activity in cell lysates (200 ng) from HTR^Cre and HTR^Vec clonal cell lines at indicated PDL, controls as specified in Figure 2. (B) RT-PCR analysis of hTERT, hph, and GAPDH at indicated PDL. HA5 cells were included as a negative control. (C) Replicative lifespan of each clonal line, as indicated. HTR^Vec cells remained immortal. (D) Anchorage-independent colony growth at increasing PDL, including HA5 and HTR cells as controls (n=4 each), and 293T cells (n=3). Difference between the latest and earliest PDL within each line as indicated (**, p<0.01; ***, p<0.001, power^{(1-β err prob)}=1.0, α^actual=0.05, two-tailed). (E) TRF analysis of average telomere length at indicated PDL. Weighted mean telomere lengths (kbp) are indicated below each lane. (F) Analysis of telomere integrity. X-axis, individual lines and respective PDL; y-axis, average number of telomere signal-free ends (SFE) per metaphase (n=10). Brackets indicate a statistically significant difference (p<0.001, power^{(1-β err prob)}=1.0, α^actual=0.038–0.044). HTR^Vec at PDL 169 possessed no SFE. (G) Relative telomere length of the lines depicted in (F). X-axis, telomere fluorescence intensity in arbitrary units; y-axis, frequency of events. Early PDL (light grey), late PDL (dark grey). Graphs are scaled equivalently. (H) RT-PCR analysis of hTERT, Sh Ble (zeocin resistance) and GAPDH in normal adjacent kidney (NK) or renal capsule (RC). The water control (H₂O) is the same as in Figure 2E, lane 11.

Figure 4. Ability of hTERT to rescue crisis in hTERT-excised cells

(A) Wild-type (WT) or mutant (Q169A; D868A, D869A) hTERT or empty vector (Vec) were introduced into HTR^{EP Cre} cells and analyzed for telomerase activity (200, 100, 50 ng lysate). (B) Replicative lifespan of cell lines as indicated above. hTERT WT cells remained immortal. (C) Anchorage-independent growth of cell lines as indicated (n=4). Statistical significance compared with vector controls as indicated (***, p<0.001, power^{(1-β err prob)}=1.0, α^actual=0.05, two-tailed). Controls and axis labels as in Figure 2.