Telomerase activity: a biomarker of cell proliferation, not malignant transformation

Abstract

Telomerase activity is readily detected in most cancer biopsies, but not in premalignant lesions or in normal tissue samples with a few exceptions that include germ cells and hemopoietic stem cells. Telomerase activity may, therefore, be a useful biomarker for diagnosis of malignancies and a target for inactivation in chemotherapy or gene therapy. These observations have led to the hypothesis that activation of telomerase may be an important step in tumorigenesis. To test this hypothesis, we studied telomerase activity in isogeneic samples of uncultured and cultured specimens of normal human uroepithelial cells (HUCs) and in uncultured and cultured biopsies of superficial and myoinvasive transitional cell carcinoma (TCC) of the bladder. Our results demonstrated that four of four TCC biopsies, representing both superficial and myoinvasive TCCs, were positive for telomerase activity, but all samples of uncultured HUC were telomerase negative. However, when the same normal HUC samples were established as proliferating cultures in vitro, telomerase activity was readily detected but usually at lower levels than in TCCs. Consistent with the above observation of the telomerase activity in HUCs, telomeres did not shorten during the HUC in vitro lifespan. Demonstration of telomerase in proliferating human epithelial cells in vitro was not restricted to HUCs, because it was also present in prostate and mammary cell cultures. Notably, telomerase activity was relatively low or undetectable in nonproliferating HUC cultures. These data do not support a model in which telomerase is inactive in normal cells and activated during tumorigenic transformation. Rather, these data support a model in which the detection of telomerase in TCC biopsies, but not uncultured HUC samples, reflects differences in proliferation between tumor and normal cells in vivo.

Figure 1

Telomerase activity is not detected in ureteral tissues. Results of the TRAP assay to detect telomerase activity in ureter, ureteral mucosa, and ureteral submucosa from two tissue samples (Ureters 1 and 2) are shown. Controls included a sample of every extract treated with RNase (−/+ RNase), the reaction mixture with no cell extract added (Mix), and samples containing extracts from both a telomerase-negative (TCC 96–2) and a telomerase-positive (TCC 94–10) TCC line. A 10-bp DNA ladder is included in lane M for size reference.

Figure 2

Telomerase activity is detected in proliferating HUCs. Results of the TRAP assay to test telomerase activity in two independent HUC cell cultures (HUCs 1 and 2) established from explant cultures of Ureters 1 and 2, respectively, as shown in Fig. 1, are shown. Telomerase activity was analyzed in both proliferating (Prolif) and senescent (Sensc) cultures. The control lanes are as described in Fig. 1.

Figure 3

Association of telomerase activity with HUC proliferation. The TRAP assay results shown compare telomerase activity in isogeneic samples of uroepithelial cells. Samples included uncultured mucosa (muc), proliferating (Prolif) HUC, senescent (Sensc) HUC, and confluent (Conf) HUC cultures. The mucosa and all cultures were derived from ureter 1. The control lanes are as described in Fig. 1.

Figure 4

Telomerase activity is detected in proliferating epithelial cells. Results of the TRAP assay comparing proliferating cultures of ureter (HUC), bladder (BEC), and mammary (MEC) epithelial cells (A) and prostate (PEC) epithelial cells (B). The control lanes are as described in the legend to Fig. 1.

Figure 5

Telomerase activity in uncultured and cultured TCCs. Representative results of the TRAP assay comparing uncultured biopsies (Biop) of TCCs and proliferating TCC cultures (Cult) from three TCCs (TCCs 1, 2, and 3) are shown. TCCs 1 and 3 are superficial grade I, and TCC 2 is myoinvasive grade III. The controls are as described in Fig. 1.

Figure 6

Lack of telomere shortening in HUC in vitro and in vivo. Results of Southern blot analysis to determine whether telomere length changes after serial passage of HUC in vitro by using cultures (HUCs 3 and 4) established from two different ureters. The donor of ureter 3 was 53 years old, and the donor of ureter 4 was 23.

Figure 7

Lack of correlation between telomerase activity and telomere length in two TCC lines. A Southern blot analysis is shown demonstrating shorter telomeres (3–5 kb) in a TCC line that shows telomerase activity (P) compared with a TCC line that is negative (N) for telomerase activity (5–10 kb) (Figs. 1, 2, 3, 4, 5 above). Telomere length of early-passage (P1) and senescent (P4) cultures of HUC 5 are shown for comparison. The donor of ureter 5 was 17 years old.