Dissociation among in vitro telomerase activity, telomere maintenance, and cellular immortalization

Abstract

The immortalization of human cells is a critical step during tumorigenesis. In vitro, normal human somatic cells must overcome two proliferative blockades, senescence and crisis, to become immortal. Transformation with viral oncogenes extends the life span of human cells beyond senescence. Such transformed cells eventually succumb to crisis, a period of widespread cellular death that has been proposed to be the result of telomeric shortening. We now show that ectopic expression of the telomerase catalytic subunit (human telomerase reverse transcriptase or hTERT) and subsequent activation of telomerase can allow postsenescent cells to proliferate beyond crisis, the last known proliferative blockade to cellular immortality. Moreover, we demonstrate that alteration of the carboxyl terminus of human telomerase reverse transcriptase does not affect telomerase enzymatic activity but impedes the ability of this enzyme to maintain telomeres. Telomerase-positive cells expressing this mutant enzyme fail to undergo immortalization, further tightening the connection between telomere maintenance and immortalization.

Figure 1

hTERT expression confers telomerase activity in transformed human cells. (A) T-Ag-transformed HEK cells were infected with control or hTERT-expressing retroviruses. Total RNA (40 μg) was isolated from the resulting polyclonal populations and clonal isolates and assayed for hTERT expression by an RNase protection assay using antisense probes specific for the 3′ untranslated portion region of hTERT (endogenous) or the terminal 275 bp of the hTERT cDNA (hTERT). The latter probe protects identical length RNA fragments when hybridized with both endogenous and ectopic hTERT mRNA. Yeast tRNA and a human β-actin probe demonstrate the specificity of the probe and the presence of equal amounts of RNA, respectively. (B) Cytosolic cellular extracts (0.2 or 0.02 μg) prepared from the parental line postcrisis, control, or hTERT-infected populations, or hTERT-infected clonal isolates were assayed for telomerase activity. As a negative control, 2 μg of all extracts tested was heat treated (HT) to inactivate telomerase before telomere repeat amplification protocol assay.

Figure 2

hTERT expression permits telomere maintenance and allows T-Ag-transformed HEK cells to overcome crisis. (A) Genomic DNA was isolated from the parental line and the control and hTERT-infected populations and hybridized with a telomeric probe to visualize the TRF. The relative migration of molecular weight markers are on the left. (B) Culture growth (pd) versus time (days) are shown for polyclonal populations of T-Ag-transformed HEK cells infected with the control retrovirus (○) or the hTERT-expressing retrovirus (•). (C) Culture growth (pd) versus time (days) are shown for clonal isolates infected with the control retrovirus (open symbols: ○, □, ⋄, ▵, ▿) or the hTERT-expressing retrovirus (closed symbols: •, ■, ♦, ▴, ▾).

Figure 3

hTERT-HA expression restores telomerase activity in postsenescent human cells. (A) Control or hTERT-HA expression vectors were stably introduced into a T-Ag-transformed HEK cell clone, yielding multiple clonal populations. Total RNA (40 μg) was isolated and assayed for hTERT-HA expression by an RNase protection assay using an antisense hTERT probe that recognizes and distinguishes endogenous hTERT and retroviral hTERT-HA transcripts. Results from a representative control and a high hTERT-HA-expressing clone are shown. Yeast tRNA and a human β-actin probe demonstrate the specificity of the probe and the presence of equal amounts of RNA, respectively. (B) Control or hTERT-HA expression vectors were stably introduced into p21^+/+ and p21^−/− fibroblasts, yielding multiple clonal populations. Representative clones from each infection are shown. Total cellular extracts were immunoblotted with an anti-HA antibody to visualize hTERT-HA. (C) Cytosolic cellular extracts prepared from the same control (0.2 μg) and hTERT-HA-transfected HEK clones (2, 0.2, or 0.02 μg) or from the parental line postcrisis were assayed for telomerase activity. As a negative control, 2 μg of all the extracts tested was heat treated (HT) to inactivate telomerase before telomere repeat amplification protocol assay. (D) Four micrograms of cytosolic cellular extracts prepared from p21^+/+ or p21^−/− fibroblast clones was assayed for telomerase activity. Telomerase-positive HeLa cell cytosolic extracts (4, 2, and 1 μg) are included as a positive control.

Figure 4

Expression of hTERT-HA does not result in telomere maintenance. (A) The TRF of a representative control, low, and high hTERT-HA-expressing clones at late passage are shown compared with the parental line at the time of transfection. The relative migration of molecular weight markers is indicated on the right. (B) Genomic DNA was isolated from hTERT-HA-expressing fibroblast clones one passage before terminal growth arrest and hybridized with a telomeric probe to visualize the TRF. Cultures of uninfected, normal (p21^+/+) cells in senescence and p21^−/− cells in crisis are included to show the telomere length at terminal growth arrest. (C) Individual telomeres (yellow) of a metaphase spread of chromosomes (blue) of a high hTERT-HA-expressing clone before crisis were identified by fluorescent in situ hybridization with a telomeric probe. Arrow denotes a chromosome end that fails to hybridize to the probe.

Figure 5

hTERT-HA expression does not immortalize transformed cells. (A) Culture growth (pd) versus time (days) is plotted for individual clonal populations of T-Ag-transformed HEK cells transfected with the empty vector clone 1 (○), 2 (□), 3 (▵), and 4 (◊) or (B) with the hTERT-HA expression vector, yielding clones with either low telomerase activity (<50% of the activity detected in immortal T-Ag-transformed HEK cells): clone 1 (○), 2 (□), 3 (▵), and 4 (◊) or high levels or telomerase activity (>90% of the telomerase activity detected in immortal T-Ag-transformed HEK cells): clone 5 (•) and 6 (■). (C) The level of both the transfected hTERT-HA and endogenous hTERT mRNAs was measured in a postcrisis, high hTERT-HA-expressing clone by an RNase protection assay for hTERT. This antisense probe identifies both endogenous and ectopically expressed hTERT-HA as shown in total RNA isolated from the telomerase positive human cell line 293 and a murine NIH 3T3 cell line stably expressing hTERT-HA. Yeast tRNA and a human β-actin probe demonstrate the specificity of the probe and the presence of equal amounts of RNA, respectively. (D and E) Culture growth (pd) versus time (days) are shown for normal (p21^+/+) fibroblasts (D) or postsenescent (p21^−/−) fibroblasts (E) that either do (■) or do not (○, ◊) express the hTERT-HA protein. The pd values of p21^−/− cells were redefined after the second gene targeting event (26) and thus do not correspond to those of p21^+/+ cells.