Mass Cultured Human Fibroblasts Overexpressing hTERT Encounter a Growth Crisis Following an Extended Period of Proliferation

Experimental Cell Research 259, 336 –350 (2000) doi:10.1006/excr.2000.4982, available online at http://www.idealibrary.com on

Mass Cultured Human Fibroblasts Overexpressing hTERT Encounter a Growth Crisis Following an Extended Period of Proliferation Karen L. MacKenzie,* ,1 Sonia Franco,* Chad May,† Michel Sadelain,† and Malcolm A. S. Moore* *James Ewing Laboratory of Developmental Hematopoiesis and †Department of Human Genetics, Sloan-Kettering Cancer Institute, New York, New York 10021

During the process of immortalization, at least two mortality checkpoints, M1 and M2, must be bypassed. Cells that have bypassed M1 (senescence) have an extended life span, but are not necessarily immortal. Recent studies have shown that ectopic expression of the catalytic subunit of telomerase (hTERT) enables normal human cells to bypass senescence (M1) and oncogene transformed cells to avert crisis (M2) and become immortal. However, it is unclear whether hTERT expression is sufficient for normal human fibroblasts to overcome both M1 and M2 and become immortal. We have investigated the role of telomerase in immortalization by maintaining mass cultures of hTERT-transduced primary human fetal lung fibroblasts (MRC-5 cells) for very long periods of time (more than 2 years). In the present studies, up to 70% of MRC-5 cells were transduced with retroviral vectors that express hTERT. hTERT-transduced cells exhibited high levels of telomerase activity, elongation of telomeres, and proliferation beyond senescence. However, after proliferating for more than 36 population doublings (PDLs) beyond senescence, the overall growth rate of hTERT-expressing cells declined. During theses periods of reduced growth, hTERT-transduced MRC-5 cells exhibited features typical of cells in crisis, including an increased rate of cell death and polyploidy. In some instances, very late passage cells acquired a senescence-like phenotype characterized by arrest in the G1 phase of the cell cycle and greatly reduced DNA synthesis. At the onset of crisis, hTERTtransduced cells expressed high levels of telomerase and had very long telomeres, ranging up to 30 kb. Not all cells succumbed to crisis and, consequently, some cultures have proliferated beyond 240 PDLs, while another culture appears to be permanently arrested at 160 PDLs. Late passage MRC-5 cells, including postcrisis cells, displayed no signs of malignant transformation. Our results are consistent with the model in which telomerase and telomere elongation greatly extends cellular life span without inducing malignant 1 To whom correspondence and reprint requests should be addressed at Stem Cell Biology Program, Children’s Cancer Institute Australia for Medical Research, High Street (PO Box 81), Randwick, NSW 2031 Australia. Fax: 61(2) 9382-1850. E-mail: [email protected].

0014-4827/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.

changes. However, these investigations also indicate that hTERT-expressing cells may undergo crisis following an extended life span and that immortality is not the universal outcome of hTERT expression in normal diploid fibroblasts. © 2000 Academic Press

INTRODUCTION

Normal human cells undergo a finite number of population doublings (PDLs) in vitro before they enter an irreversible postmitotic state referred to as senescence [1]. The telomere hypothesis of replicative senescence proposes that the shortening of telomeres with each cell division is central to the machinery of the mitotic clock that determines when cells senesce [2]. Telomeres are specialized structures at chromosomal ends that are composed of TTAGGG DNA repeats [3]. Telomeric DNA is folded into loops through association with telomere binding proteins [4 –7]. Telomeres cap chromosomal ends and thereby function to prevent abnormal chromosomal fusions and rearrangements. However, each time a cell divides, the most distal part of the chromosome is incompletely duplicated and the telomere becomes shorter [8]. Critically short telomeres are dysfunctional and enable formation of aberrant chromosomal structures which activate p53-mediated DNA damage signals resulting in growth arrest or senescence [9]. Senescence is one safeguard against excessive proliferation, which is the hallmark of cancer cells. The transformation events that lead to unchecked proliferation have been modeled in vitro as a multistep process in which at least two mortality checkpoints must be overcome [10]. The first checkpoint of this process, referred to as mortality stage 1 (M1), appears to be regulated by the CDK inhibitors p21 CIP1/WAF1 and p16 INK4a which function in pathways of the tumor suppresser genes p53 and RB, respectively [11]. Inactivation of these tumor suppressor pathways, either as a consequence of genetic mutation or through interactions with viral oncogenes, enables cells to bypass M1 and proliferate for an additional 20 – 40 PDL beyond

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senescence. However, cells which bypass senescence in this way are still subject to telomeric shortening [12, 13]. Furthermore, these cells continue to age, accumulate chromosomal abnormalities, and eventually succumb to the second mortality checkpoint (M2) known as crisis. Crisis is generally described as a dynamic process in which continued proliferation is counteracted by increased cell death [10, 14]. Eventually the culture declines as the rate of cell death overrides proliferation. This end point at crisis is in clear contrast to senescence, during which cells do not proliferate or undergo active cell death; senescent cells arrest in G1, remain intact and viable, and are metabolically active [15]. As a very rare, spontaneous event, a clone may escape crisis and proliferate indefinitely. The rate that human fibroblasts expressing the SV40 large T antigen (SV40 LT) escape crisis was calculated to be 1 in 3 ⫻ 10 7 [14]. The majority of cells that escape crisis express the enzyme telomerase [12], which functions to maintain telomeric structures by catalyzing addition of TTAGGG repeats [16, 17]. A small subset of immortal cells are telomerase negative; however, these cells have very long telomeres as a result of an alternative telomere lengthening mechanism (ALT) that may involve recombination [18]. Similarly, most tumor cells either express telomerase and have stable telomeres or have long telomeres that are maintained by ALT [19, 20]. In contrast to immortal cell lines and tumor cells, normal human somatic cells express negligible amounts of telomerase. These observations suggest that telomerase and/or telomere maintenance is an important aspect of the immortal phenotype. Telomerase is a ribonuclear protein complex that has a reverse transcriptase (hTERT) as a catalytic domain. The recent cloning of both hTERT and the RNA component (hTR) of human telomerase [21, 22] has provided an opportunity to directly investigate the role of telomerase and telomere maintenance in replicative life span. While hTR is abundantly expressed in both normal and tumor cells, hTERT expression was shown to correlate with telomerase activity, suggesting that hTERT is the limiting component of the enzyme. Indeed, overexpression of hTERT was shown to be sufficient to reconstitute telomerase activity in normal human diploid cells [23, 24]. Reconstitution of telomerase activity, through ectopic expression of hTERT, enables normal human fibroblasts and retinal epithelial, mesothelial, and endothelial cells to bypass senescence [25–29]. However, hTERT expression is not sufficient to extend the life span of mammary epithelial cells or keratinocytes [29, 30]. For the latter cell types, loss of p16 INK4a, in addition to hTERT expression, appears to be necessary for life span extension. Overexpression of hTERT was also shown to enable SV40 LT-transformed human cells to overcome crisis and become

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immortal [31–33]. These observations suggest that telomerase and/or telomere maintenance are crucial to both the M1 and M2 checkpoints. However, even though telomerase activity extends the life span of certain cell types, it does not necessarily follow that telomerase expression is sufficient for immortalization. In the present study, we have monitored hTERTtransduced fibroblasts for extended periods of time (more than 2 years) in order to address the question of immortality. Using high-titer retroviral vectors expressing hTERT, we established six cultures where up to 150,000 cells/culture were transduced. All 6-hTERTtransduced cultures bypassed senescence and proliferated for at least an additional 36 PDL, confirming that telomerase activity and/or telomere elongation extends the life span of normal human fibroblasts. However, each of these cultures eventually entered a cell death phase which resembled crisis. Cells undergoing crisis continued to express telomerase and had long telomeres. Some subpopulations of cells continued to proliferate beyond periods of crisis, reaching more than 250 PDL beyond senescence. These results demonstrate that telomerase activity and/or telomere elongation may not be sufficient for immortalization of human fibroblasts. MATERIALS AND METHODS Cell culture. MRC-5 human fetal lung fibroblasts were purchased from American Type Culture Collection (Manassas, VA) and cultured in minimal essential medium (MEM) (Gibco BRL Life Technologies, Grand Island, NY) with 2 mM glutamine and Earl’s balanced salt solution plus 10% fetal bovine serum (FBS) (Hyclone, Logan, UT). Retroviral producer cells and HeLa cells were maintained in Dulbecco’s modified Eagle’s medium with 10% FBS. All cells were grown at 37°C/5% CO 2. For colony formation in agarose, MRC-5 cells were plated at 50,000/ml in 0.33% agarose/20% FBS/ MEM over a preformed layer of 0.5% agarose/20% FBS/MEM and incubated at 37°C/5% CO 2. HeLa cells were cultured in agarose under identical conditions except they were plated at 500 cells/ml. Colonies were scored on day 14. Retroviral vectors and viral producer cells. The control retroviral vector, MGFP, was constructed by subcloning the coding sequence for the enhanced green fluorescence protein (eGFP) into the PmlI site of MFG [34]. For construction of the bicistronic vectors MB2TIG and MTIG, an EcoRI fragment containing the coding sequence for human telomerase reverse transcriptase cDNA was excised from the plasmid pGRN145. The ends of hTERT cDNA were enzymatically filled using Klenow enzyme and inserted into the PmlI site of either MFG(B2) or MFG to construct MB2TIG and MTIG, respectively. The MB2TIG and MTIG vectors differ by a point mutation (B2) in the 5⬘-untranslated region. This B2 mutation may enable more efficient expression in stem cells. No difference in expression was noted in MRC-5 cells transduced with MTIG verses MB2TIG (MRC5/TIG verses MRC5/B2TIG cells, respectively). Retroviral plasmids were transfected into 293GPG cells [35] using a standard calcium phosphate transfection procedure. Transfected 293GPG cells produce retrovirus packaged into a vesicular stomatitis virus– glycoprotein (VSV-G)-derived envelope. To establish stable producer cell lines, PG13 cells [36] were infected with viral supernatant from 293GPG transfectants. MRC-5 cells were transduced by overnight incubation in viral supernatant supplemented with 8 ␮g/ml polybrene (Sigma,

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St. Louis, MO). Transduced cells were enumerated by flow cytometric analysis for GFP expression using a Becton-Dickinson FACScan. Protein extraction and telomerase expression. Telomerase expression was quantified using the PCR-based telomeric repeat amplification protocol (TRAP) as described previously [19, 37]. Briefly, protein was extracted by lysing cells in Chaps buffer on ice for 30 min. Supernatant was collected following centrifugation at 12,000g for 30 min at 4°C. Protein concentration was determined by the Bradford assay. Aliquots of 2 ␮g of protein were assayed in 25-␮l reactions which included an [␥- 32P]ATP end-labeled TS primer and 0.01 amol of an internal PCR control (TSNT). The TSNT PCR product is 36 bp and runs 14 bp below the smallest size-fractionated TRAP-derived species. Each TRAP assay also included a negative control (H 2O), a positive control (protein extract from the SK-N-SH neuroblastoma cell line), and the R8 quantification oligonucleotide. PCR products were fractionated through a 12.5% polyacrylamide gel and visualized by phosphorimaging. The following formula was used to calculate the amount of telomerase activity or total product generated (TPG) for each sample as a fraction of TPG in the neuroblastoma control: TPG ⫽ 100 ⫻ [(T ⫺ B)/CT]/[(NB ⫺ B)/CNB], where T is the radioactive signal in test extract, B is background activity in the negative control, CT is activity of the TSNT band in the test sample, NB is the signal from neuroblastoma cell line, and CNB is the activity of the TSNT band in the neuroblastoma control. Telomeric restriction fragment (TRF) assay and Southern blot analysis. Genomic DNA was extracted by standard phenol/chloroform procedure and precipitated with 0.3M sodium acetate and 2 vol of ethanol. TRF assays were performed as previously described [38]. In brief, 5–10 ␮g of DNA was digested with MspI and RsaI (Boehringer Mannheim, Indianapolis, IN) for 16 h at 37°C, followed by electrophoresis through a 0.5% agarose gel. Gels were then depurinated, denatured neutralized, and transferred to Nytran nylon membranes (Schleider and Schuell, Keene, NH) using Southern blot technique. Membranes were prehybridized in 6⫻ SSPE/0.5% SDS/5⫻ Denhardt’s/20␮g/ml tRNA for 3 h at 55°C and then hybridized in 6⫻ SSPE/1% SDS/5⫻ Denhardt’s with a 5⬘ [␥- 32P]ATP end-labeled telomeric oligonucleotide probe (TTAGGG) 4 for 16 h at 55°C. Membranes were washed three times in 6⫻ SSPE/0.1% SDS at room temperature and once at 55°C for 60 s. Finally, the membranes were rinsed in 5⫻ SSPE and then exposed to phosphorimaging plates overnight. Mean and peak TRFs were analyzed as previously reported [38]. Southern blot analysis for retroviral integration sites was carried out by digesting 10␮g of genomic DNA with BglII (Boehringer Mannheim), fractionating through a 0.8% agarose gel and transferring to Gene Screen Plus nylon membranes (NEN Research Products, Boston, MA) using a standard alkaline transfer procedure. Prehybridized was performed for 0.5 to 2 h at room temperature in hybridization solution containing 0.6 M NaCl, 0.18 M Na 2HPO 4, 0.6mM EDTA, 1% N-lauryl sarcosine, and 10% dextran sulfate. Membranes were then hybridized in this solution with [␣- 32P]dCTP-labeled hTERT probe which was an EcoRI fragment of pGRN145. Hybridization was performed at 65°C for 12–24 h. Hybridized membranes were washed twice in 2⫻ SSPE (300 mM NaCl, 18 mM NaH 2PO 4, 2 mM EDTA, pH 7.4) for 5 min at room temperature, once in 2⫻ SSPE/0.5% SDS for 15 min at 65°C, and then once in 0.1⫻ SSPE at 65°C before exposure to imaging plates. ␤-Galactosidase staining. Cells were assayed for ␤-galactosidase activity at pH 6.0 as previously described [39]. In brief, cells were first rinsed with PBS and then fixed in 2% formalydehyde/0.2% glutaraldehyde and stained with 1 mg/ml X-gal (Boehringer Mannheim) in 40 mM citric acid/sodium phosphate, pH 6.0/5 mM potassium ferrocyanide/5 mM potassium ferricyanide/150 mM NaCl/2 mM MgCl. The reaction proceeded for 12–16 h at 37°C. Cell cycle analysis and BrdU incorporation. One million cells that were in exponential growth were pulsed with 20 ␮M BrdU for 2.5 h. The cells were then harvested, fixed in 70% ethanol, and stained with 10 ␮g/ml propidium iodide and FITC-labeled anti-BrdU

antibodies (Becton-Dickinson, San Jose, CA) as described previously (40). Cells were analyzed by two-color fluorescence on a BectonDickinson FACScan.

RESULTS

Retroviral Transduction and Reconstitution of Telomerase Activity in MRC-5 Cells Three retroviral vectors were constructed to study the role of hTERT in the immortalization process. The vectors MB2TIG and MTIG are bicistronic vectors that encode hTERT and eGFP. MGFP is a control vector that expresses only eGFP. MRC-5 cells are primary human fetal lung fibroblasts which express the RNA component of telomerase but not hTERT and therefore do not have telomerase enzyme activity [23]. In three independent experiments, MRC-5 cells were transduced with retroviral vectors packaged in either VSV-G or GALV envelopes. In each experiment, mockinfected (MRC5/Mock 1-3) and MGFP-infected cells (MRC5/GFP 1-3) served as controls. Three days after infection, transduction efficiency was assessed by FACS analysis (Fig. 1a). High levels of eGFP expression were detected in MRC5/GFP cells and lower levels of eGFP were detected in MRC5/TIG and MRC5/B2TIG cultures. The lower intensity of eGFP expression in MRC5/TIG and MRC5/B2TIG cultures was due to the 3⬘ position of the eGFP-coding sequence relative to the internal ribosomal entry site and hTERT cDNA. The percentage of eGFP-positive cells indicated that up to 70 and 95% of MRC-5 cells were transduced with MTIG and MGFP, respectively. High levels of telomerase activity were detected in all cultures that were transduced with either MTIG (MRC5/TIG 1-2) or MB2TIG (MRC5/B2TIG 1-4) (Fig. 1b). In contrast, no significant telomerase activity was detected in mock-infected and MGFP-infected cultures. A summary of FACS and TRAP results from the three experiments is provided in Table 1. Proliferation and Cell Death in MRC5/TIG and MRC5/B2TIG Cultures Control and hTERT-transduced cells were maintained in culture and PDLs were enumerated. Growth curves showing proliferation of transduced and control MRC-5 cells in three independent experiments are shown in Fig. 2. All mock-infected and MRC5/GFP cells ceased to proliferate by 62.5 ⫾ 3.5 PDL. At this stage of the cultures, MRC5/Mock and MRC5/GFP cells remained attached to the culture flask and exhibited morphologic features that are typical of senescent fibroblasts. Senescent MRC5/Mock and MRC5/GFP cells became enlarged, flat, and nonrefractile Furthermore, late passage MRC5/Mock and MRC5/GFP cells stained positive for senescence-associated ␤-galactosidase (SA

hTERT-TRANSDUCED FIBROBLAST IMMORTALIZATION BY TELOMERASE

FIG. 1. eGFP expression and telomerase activity in cells transduced with retroviral vectors. (a) FACS analysis for eGFP expression in MRC-5 cells 3 days after retroviral infection in Experiment 1. M1 defines eGFP-positive cells. The percentage of eGFP-positive cells is indicated within each graph. (b) TRAP assay for telomerase activity in MRC-5 cells 3 days after retroviral infection (Experiment 1). Positive controls are R8 and NB. Negative controls are water and nontransduced MRC-5 cells (MRC5/Mock-1). Telomerase activity was normalized to an internal control and quantified as a percentage of telomerase activity in NB.

␤-gal) [39], a biochemical marker of senescence. In contrast to MRC5/Mock and MRC5/GFP cells, all six MRC5/TIG and MRC5/B2TIG cultures retained normal morphology and readily bypassed senescence. Thus, overexpression of hTERT and induction of telomerase activity extended the life span of MRC-5 cells. At various times after senescence, hTERT-transduced cultures underwent a growth crisis that was principally characterized by an overall reduction in cellular expansion (Fig. 2). In some instances, reduced growth appeared to be due to an increased rate of cell death, where foci of dying cells were observed among patches of proliferating cells. Cells in this death phase were contracted, rounded, and loosely attached to the culture flask (Fig. 3). This morphology was clearly dis-

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tinct from senescent cells. We also observed periods when cellular growth was reduced without apparent cell death. In these instances, the cells exhibited morphologic characteristics that were more typical of senescent cells. Cells in growth crisis stained positive for SA-␤-gal, regardless of morphology (Figs. 3f–3h). To varying degrees, periods of cell death or reduced growth were observed in all hTERT cultures. Although the growth curve of MRC5/TIG-2 does not clearly demonstrate a period of reduced growth, small foci of dying cells were observed in this culture from PDL 210 onward (Fig. 3g). The two crisis phenotypes (cell death verses senescence-like) were not mutually exclusive. In some instances we observed both dying cells and senescent-like cells simultaneously within a culture, while in the MRC5/B2TIG-1 culture two different phenotypes were observed during different stages. In the MRC5/B2TIG-1 culture, a period of massive cell death was followed by a static phase where remaining cells were attached to the flask, but did not proliferate. In some cultures, subsets of cells continued to proliferate beyond periods of crisis, such that over 300 PDLs were attained. However, in the MRC5/B2TIG-1 culture no net expansion was observed beyond crisis. At the present time, all remaining cultures show sporadic signs of focal cell death or slowed growth. These results suggest that hTERT expression may be insufficient for immortalization. Slowed growth and cell death in postsenescent MRC5/TIG and MRC5/B2TIG cultures did not appear to be due to adverse culture conditions since early passage control MRC-5 cells that were maintained in the same media remained healthy. In addition, reduced growth and cell death was not apparent in all postsenescent hTERT cultures at any one time. Nevertheless, to rule out the possibility that cell death was a culture artifact, stocks of postsenescent hTERT cells from earlier passages (10 – 40 PDL precrisis) were thawed from liquid nitrogen. The thawed cells exhibited similar growth kinetics and entered the cell death phase within 1– 6 PDL of the earlier passage cells (Table 2). Furthermore, in parallel with the original cultures, thawed MRC5/TIG-1 cells resumed proliferation after an extended crisis period while thawed MRC5/B2TIG-1 cells could not be propagated beyond crisis (data not shown). These results indicate that cell death in hTERT cultures was a programmed event and not due to adverse culture conditions. Cell Cycle Analysis To further characterize the altered growth kinetics in late passage hTERT cultures, DNA cell cycle analyses and BrdU incorporation studies were performed (Figs. 4a– 4c). For these studies, early passage and senescent nontransduced MRC-5 cells were used as

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TABLE 1

Exp

Cell strain

FACS a % eGFP⫹

1

MRC5/Mock-1 MRC5/GFP-1 MRC5/TIG-1 MRC5/B2TIG-1 MRC5/Mock-2 MRC5/GFP-2 MRC5/TIG-2 MRC5/B2TIG-2 MRC5/Mock-3 MRC5/GFP-3 MRC5/B2TIG-3 MRC5/B2TIG-4

0.0 92.1 70.6 63.7 0.0 99.2 7.3 32.0 0.0 91.3 6.3 1.6

2

3

Telomerase a % NB control

Senescence (PDL)

Crisis (PDL)

0 0 73 47 2 ND 16 45 4 3 53 20

67 67 — — 64 54 — — 62 61 — —

— — 108 124 — — 210 98

155 139

Crisis 2 b (PDL) thawed

114 123

ND 94

156 ND

Note. ND, not determined. a FACS and TRAP were performed 3 days postinfection. b Onset of crisis in cells that were thawed from earlier passage (10 – 40 PDLs precrisis).

controls. Analysis of early passage MRC-5 cells demonstrated that about 30% were in S phase and incorporated BrdU during a 2.5-h pulse. In contrast, late passage MRC-5 cells accumulated in G1 and less than 10% of these cells synthesized DNA and incorporated BrdU. Precrisis hTERT cells (e.g., MRC5/B2TIG-3 at PDL 105 and MRC5/TIG-2 at 201 PDLs) produced similar DNA profiles to early passage MRC-5 cells, where 36% of the cells were detected in S phase and 24% of the cells incorporated BrdU. Cells analyzed during periods of growth crisis exhibited two distinct cycling profiles. The cell cycle profiles of MRC5/TIG-2 and MRC5/B2TIG-2 assayed during a cell death phase, where foci of cells were contracting and detaching from the flask, were similar to early passage control MRC-5 cells, with greater than 30% of the cells in S phase and incorporating BrdU. MRC5/B2TIG-1 and MRC5/ B2TIG-4 were also analyzed during a period of reduced growth and exhibited cell cycle kinetics that were very similar to senescent cells. These cells accumulated in G1, with fewer cells in S phase and less incorporation of BrdU. Cells with an enlarged and flattened morphology (senescence-like morphology) were noted in the MRC5/B2TIG-1 and MRC5/B2TIG-4 at the time the assays were performed. DNA analysis also revealed hypotetraploid subpopulations within cultures that where engaged in, or had emerged from, a cell death phase. During a period when a substantial proportion of MRC5/B2TIG-2 cells (approximately 30% by visual inspection) were undergoing cell death, 35.9% of this culture was hypotetraploid. These hypotetraploid cells had a DNA index (DI) of 1.76 and coefficient of variation (CV) of 3.1 relative to the rest of the culture. A hypotetraploid subpopulation, with a DI of 1.86 (CV ⫽ 4.3) was also detected in MRC5/TIG-1 cultures postcrisis (PDL 167).

FIG. 2. Proliferation of transduced and control MRC-5 cells. Growth curves for transduced and mock-infected MRC-5 cells from three independent experiments. Cells were harvested, counted, and split every 7 days.

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FIG. 3. Cell morphology and SA-␤-galactosidase staining. Photomicrographs of MRC-5 cells taken with phase contrast to illustrate morphology: (a) nontransduced cells at PDL 51.3, (b) MRC5/B2TIG-2 cells with normal morphology at PDL 169.5, (c) MRC5/B2TIG-1 cells in crisis at PDL 135 PDL, and (d) nontransduced cells senescing at PDL 68. SA-␤-galactosidase staining; (e) control MRC-5 cells at PDL 43; (f) control MRC-5 cells at PDL 68; (g) MRC5/TIG-2 cells in crisis at PDL 226; and (h) MRC5/B2TIG-2 cells in crisis at PDL 192. Blue cells indicate SA-␤-galactosidase activity (e– h). All magnifications are 100⫻.

TABLE 2

At this time point, 53.9% of the MRC5/TIG-1 culture was hypotetraploid. However, the proportion of hypotetraploid cells in the MRC5/TIG-1 culture was reduced to less than 10% by PDL 210, indicating overgrowth of the abnormal cells with normal diploid cells. Hypotetraploid populations appeared to be proliferating, since a significant percentage of these cells were in S phase and incorporated BrdU (Figs. 4a and 4c). Chicken erythroid nuclei were spiked into DNA

Telomeres at the onset of crisis Cells

PDLs

Peak TRF (kb)

TRF a range (kb)

MRC5/TIG-1 MRC5/B2TIG-1 MRC5/TIG-2

111 125 92

20.0 19.4 22.7

13.9330.0 13.3330.0 16.6330.0

a

The range of TRF measurements extended beyond 30 kb for each sample.

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FIG. 4. Cell cycle analysis. (a) Examples of cell cycle profiles for control and hTERT-transduced MRC-5 cells; (b) summary of cell cycle analysis for control and hTERT-transduced MRC-5 cells; and (c) FACS analysis showing BrdU incorporation and DNA staining with propidium iodide. Cells were pulsed with BrdU for 2.5 h before labeling with FITC-conjugated BrdU antibody and counterstaining with propidium iodide.

staining solutions to standardize DNA content measurements. In this way we determined that, with the exception of the MRC5/TIG-1 and MRC5/B2TIG-2 hypotetraploid subsets, all MRC5/TIG and MRC5/B2TIG cultures had a normal diploid DNA content. When compared to early passage, nontransduced MRC-5 cells, the DI of MRC5/TIG and MRC5/B2TIG cultures ranged from 0.98 to 1.02 (data not shown). In these analyses CVs were always less than 5. Telomerase and Telomeres Telomerase activity in hTERT-transduced cells was measured at numerous time points using the semiquantitative TRAP assay. Following retroviral infection of MRC-5 cells, telomerase activity steadily increased to reach maximum levels at about the time control cultures senesced. At this time, telomerase activity in MRC5/B2TIG-1 and MRC5/TIG-1 cultures was equal to, or greater than telomerase activity in the NB tumor cell line (Fig. 5a). A similar pattern of telomerase expression was seen for MRC5/B2TIG-2 and MRC5/TIG-2, although telomerase levels were generally lower for these cells owing to the lower transduc-

tion efficiency (data not shown). The apparent increase in telomerase activity during the first 30 – 40 PDL after transduction probably reflects selection for hTERT-expressing, transduced cells over nontransduced, telomerase-negative cells. It is also significant that telomerase activity remained high during crisis. Telomere measurements were made by TRF analysis of genomic DNA isolated from mock-infected and transduced cells (Figs. 5b and 5c). TRF measurements showed that telomeres of MRC5/Mock-1 and MRC5/ GFP-1 cells shortened at similar rates: 72 and 75 bp/ PD, respectively. By extrapolating these data, we estimated that the telomeres of MRC-5 cells at harvest (PDL 0) were about 10 kb and shortened to about 5 kb at senescence (PDL 62.5). Telomeres of MRC-5 cells were rapidly elongated following transduction with either MB2TIG or MTIG. At the time control cultures underwent senescence, the peak telomere length measurements for four of the hTERT cultures was around 15 kb. The average rate of telomere elongation in hTERT cultures up to this time was 129 ⫾ 31 bp/PDL. During crisis, peak TRF measurements for MRC5/ TIG-1, MRC5/B2TIG-1, and MRC5/B2TIG-2 were

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FIG. 4—Continued

19 –23 kb, with a range of 13 to greater than 30 kb. Upon further culture, the telomere dynamics in hTERT cultures became more variable. While the telomeres of MRC5/TIG-2 cells extended to beyond 30 kb at 143 PDL, telomeres in MRC5/TIG-1 cultures reached a maximum length of 21 kb at 120 PDL and then shortened to 15 kb over the next 15 PDLs. Fluctuations in telomere lengths while the cells were in crisis may

reflect outgrowth or loss of clones with different length telomeres. A detailed analysis of clonal variations in telomere length for MRC5/TIG-1 and MRC5/B2TIG-1 cultures will be reported elsewhere [41]. Clonal Analysis by Retroviral Integration Sites Southern blot analysis of retroviral integration sites was performed to study clonal evolution within MRC5/

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large numbers of transduced clones, we expected the Southern hybridization to produce smears at early time points. Consistent with this notion, a smear was produced by hybridization of MRC5/B2TIG-1 DNA isolated at PDL 67. However, unique fragments, indicating dominant clones, were detected as early as 67 PDLs in MRC5/TIG-1 cultures and 74 PDLs in MRC5/ B2TIG-1 cultures. By PDL 74, the dominant clone in the MRC5/TIG-1 culture was overgrown by another clone which dominated the culture for at least an additional 50 PDLs. Indeed the latter clone was still present in the MRC5/TIG-1 culture during crisis. The MRC5/B2TIG-1 clone detected at 74 PDLs dominated the MRC5/B2TIG-1 culture for at least 30 PDLs, but was overgrown by another clone while the culture was in crisis (PDL 117–132). A number of weakly hybridizing fragments, indicating minor clones, were also de-

FIG. 5. Telomerase and telomeres. (a) Cells were harvested at different time points and assayed for telomerase activity using the TRAP assay; (b) an example of TRF analysis of genomic DNA isolated from MRC5/Mock-1, MRC5/TIG-1, and MRC5/B2TIG-1 cells harvested at different PDLs. White bars indicate peak telomere lengths in each sample. (c) Graphic representation of peak telomere lengths quantified by TRF measurements in Experiments 1 and 2.

TIG-1 and MRC5/B2TIG-1 cultures (Fig. 6a). For these investigations, genomic DNA was digested with BglII and hybridized to an hTERT probe. Southern blots were washed at high stringency. With this technique, each hybridizing fragment represents a single proviral integrant. Genomic DNA isolated from nontransduced MRC-5 cells was used as a negative control and producer cells were used as a positive control (additional data not shown). A compilation of our Southern analyses is shown graphically in Fig. 6b. By multiplying the transduction efficiency by the starting number of cells, we estimate that there were 146,000 transduced cells in the MRC5/TIG-1 culture and 94,000 cells transduced in the MRC5/B2TIG-1 culture immediately after infection. Considering these

FIG. 6. Southern analysis of transduced clones. (a) An example of a Southern blot of genomic DNA from MRC5/B2TIG-1 and MRC5/ TIG-1 cells at different stages of culture. Genomic DNA was digested with BglII and hybridized to an hTERT probe. Hybridizing fragments represent single retroviral integration sites and indicate individual clones. Strongly hybridizing restriction fragments indicate dominant clones, while weakly hybridizing fragments represent minor clones. (b) Compilation of Southern blot data acquired for MRC5/ TIG-1 and MRC5/B2TIG-1 cultures. Each point represents an individual clone. These results demonstrate clonal fluctuations within the mass cultures over time.

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tected in MRC5/TIG-1 and MRC5/B2TIG-1 cultures at various time points. However, detailed analysis of these clones is restricted by the limited sensitivity of this technique. Nevertheless, results from these Southern analyses indicate that clonal selection occurred during the culture of postsenescent cells. These data are corroborated by survival analysis of individual clones derived from these cultures [41]. Transformation Assays In MRC5/B2TIG-1 and MRC5/TIG-1 cultures, periods of crisis were followed by bursts steady of growth. This appeared to be due to overgrowth of dying or growth-arrested cells with proliferating cells. Although the postcrisis cells appeared to be morphologically unchanged, we performed a series of assays to determine whether the cells dominating late passage cultures had characteristics of malignant cells. First, we found no significant difference in the proliferative rate of postcrisis MRC5/TIG-1 and MRC5/B2TIG-1 cells compared to earlier passage cells and presenescent control cells (Fig. 7a). To compare growth factor requirements, we next measured proliferation in decreasing concentrations of serum. The results in Fig. 7b show that growth of postcrisis MRC5/TIG cells was serum dependent and extinguished in a dose-dependent way which paralleled the growth retardation measured for control cells and earlier passage hTERT cells. We also confirmed that the growth of cells in late passage hTERT cultures was contact inhibited, such that these cells grew to a similar cell density as early passage MRC-5 control cells (Fig. 7c). Late passage, nontransduced MRC-5 cells grew to a lower cell density, as expected for cells approaching senescence. Clonogenic assays were performed in soft agar to determine whether growth of hTERT-transduced cells was anchorage independent. For these assays HeLa cells were used as a positive control. HeLa cells formed large, compact colonies at a frequency of 11 ⫾ 7%, whereas hTERT-expressing MRC-5 cells formed very small colonies at a frequency of less than 0.1%. In four independent tests there was no significant difference between the number of small colonies formed by three different MRC5/TIG cultures, including postcrisis MRC5/TIG-1 cells and control MRC-5 cells (data not shown). Finally, we confirmed that late passage MRC-5 cells arrested in response to ␥-irradiation. Proliferation of early and late passage hTERT-expressing MRC-5 cells was partially inhibited at 350 rads and completely inhibited by 950 rads of ␥-irradiation (data not shown). This latter result indicates that the p53 cell cycle checkpoint was functional in postcrisis MRC-5 cells. Indeed, clonal deletion at late passage is consistent with normal function of checkpoint controls. Taken together, the results from these assays demonstrate no malignant changes in

FIG. 7. Transformation assays. (a) To determine growth rates, cells were harvested each week and counted. Data shown are average values plus SEM calculated from at least six individual time points. (b) Serum requirements were determined by seeding cells at 1 ⫻ 10 4 per well and then changing the medium to include different concentrations of serum once the cells had attached to the plate. Cells were counted 10 days later. The graph shows average values ⫾ SEM derived from three independent experiments. (c) To investigate growth arrest in response to cell– cell contact, cells were seeded in triplicate wells at 1 ⫻ 10 4 per well and grown in 10% serum. After 14 days, the cells in each well were harvested and individually counted. Each cell strain was evaluated in several independent experiments as indicated. Average values and SEM calculated from independent experiments are shown. Student’s t test was performed to compare the cell density of each cell strain with early passage MRC-5 cells.

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hTERT-transduced cells which dominated the cultures after 100 –200 PDLs. DISCUSSION

Previous studies have shown that constitutive expression of hTERT reconstitutes telomerase activity in vivo and extends the life span of primary human fibroblasts [25–27]. In those studies, vectors carrying drug resistance genes were utilized to select transduced clones in media containing antibiotics. Several telomerase-positive clones were reported to proliferate for at least 20 – 40 PDLs beyond senescence. Two later investigations reported growth of hTERT-transduced fibroblast clones for 30 –100 PDLs beyond senescence [42, 43]. However, together the latter studies by Jiang et al. [42] and Morales et al. [43] refer to only five clones and detailed growth kinetics were not provided in either report. In the absence of convincing data which demonstrate that immortality is the universal outcome for fibroblasts with an extended life span, it is not possible to conclude from previous studies that overexpression of hTERT is sufficient for immortalization. In the present investigations, we have employed high-titer retroviral vectors to induce expression of hTERT in human lung fibroblasts. No drug selection was applied and transduced fibroblasts were passaged as mass cultures. In the present report, we provide the growth kinetics of these mass cultures over an extensive period of time. Our most advanced cultures have been monitored for more than 280 PDLs, over approximately 2 years (or 220 PDLs over 700 days postsenescence). The most important consequence of this longer term follow up is the observation of cell death in later stages of the cultures. Although our study confirms that hTERT extends the life span of human fibroblasts, it also demonstrates for the first time that proliferation of hTERT-expressing fibroblasts is unstable after long periods of culture. In the present study, growth crisis was observed in all six postsenescent hTERT cultures. The time that crisis occurred varied between the different cultures; the earliest crisis was observed in MRC5/B2TIG-2 at 36 PDLs postsenescence and the latest crisis was first noted at about 145 PDLs postsenescence in the MRC5/TIG-2 culture. Although crisis was not apparent from the growth curve of MRC5/TIG-2, cell death of a small subset of cells was observed in this culture from 210 PDLs onward. Cell cycle analysis performed during growth crisis revealed two distinct types of growth impairment; one characterized by cell death concomitant with continued DNA synthesis and cell turnover and the other resembling senescence, with a large proportion of cells accumulating in G1 and very few cells synthesizing DNA. Although there are numerous reports that document the char-

acteristics of senescent cells [11, 15] fewer publications adequately describe cells in crisis. Moreover, past investigations that describe growth crisis refer to cells that express oncogenes [10, 14]. In this context, crisis is generally referred to as a period where cellular proliferation is balanced by cell death. Cell death at crisis is thought to occur as a consequence of conflicting signals, where oncogene-initiated signals for proliferation conflict with growth arrest mechanisms associated with cellular aging. However, a crisis phenotype was also described that is not associated with an increase in the rate of cell death but is characterized by features of senescent cells, including senescence-like morphology, G1 arrest, and SA␤-gal reactivity [44]. In addition, a recent report describes two distinct phenotypes for postsenescent cells in crisis; one phenotype resembles senescence while the other is associated with continued cell cycling, chromosomal aberrations, and a high frequency of cell death [45]. Thus, there appears to be two distinct phenotypes for cells in crisis, both of which were described in the present investigation. We noted hypotetraploid subpopulations in two cultures which were either in a period of crisis or had recently emerged from crisis. Hypotetraploid cells were in cycle, but appeared to be unstable since the proportion of hypotetraploid cells in one culture was substantially reduced at a later time point. Chromosomal instability, including polyploidy, is one well-documented characteristic of cells in crisis [12, 13, 44, 46 – 49]. Polyploid cells may evolve as a consequence of an attenuated mitotic spindle (G2) checkpoint, which normally prevents cells which have not completed mitosis to progress through G1 and into S phase [50]. Our observation that hypotetraploid cells were in S phase and incorporating BrdU is consistent with a loss of the mitotic spindle checkpoint control. Attenuation of the G2 checkpoint may have been a consequence of in vitro aging or telomere dysfunction. Replicative senescence and crisis are both proposed to be a consequence of telomeric shortening [2, 8, 12, 13, 51, 52]. In senescing cells and cells in crisis, very short telomeres signal growth arrest in response to apparent damaged DNA [9]. Telomeres in MRC5/TIG-1 and MRC5/B2TIG-1 cells elongated at a fairly consistent rate following retroviral transduction and during growth beyond senescence. At the onset of crisis telomeres were very long, ranging from 13 to over 30 kb, with modal TRF measurements over 19 kb. Telomeric shortening was therefore not responsible for crisis in hTERT cultures. These results are corroborated by our analysis of TRF lengths for individual clones [41]. Indeed, considering that at crisis telomeres were significantly longer than the estimated length of MRC-5 telomeres when the cells were first explanted, it seems plausible that the extreme length of the telomeres was

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responsible for growth inhibition. A recent investigation demonstrated that normal telomere function is not entirely dependent upon telomere length. Abnormal telomere structures are also incompatible with normal growth and can induce cell death in p53-competent cells [53]. One possible explanation for the cell death observed in our cultures is that the extra long telomeres in transduced MRC-5 cells did not form normal telomeric end structures and thereby initiated cell death. Aside from telomere dynamics, other molecular changes which occur during aging may have exerted growth restrictions on MRC5/TIG and MRC5/B2TIG cells. Of the vast array of epigenetic, genetic, and biochemical changes that have been documented in senescent cells, many of these are likely to be consequences, rather than causal factors, in the aging process. However, substantial evidence causally implicates reactive oxygen metabolites, which accumulate during in vitro passaging and cause irreversible oxidative damage, in cellular senescence and aging [54]. Also, extrachromosomal ribosomal DNA circles (ERCs), which accumulate in the nucleolus with each cell division, may be involved in the aging process. ERCs were proposed to inhibit cell proliferation by sequestering DNA replication factors [55]. Genetic mutations that may be randomly acquired during the extended life span could also initiate apoptotic or growth arrest signals through the normal G1 checkpoint controls. Our Southern analyses of retroviral integration sites revealed distinct restriction fragments, indicating dominant clones within the hTERT cultures. A number of less prominent clones were also detected in these analyses. These results suggest evolution toward oligoclonality soon after senescence. However, due to the limited sensitivity of this technique, we believe that these results underestimate the number of transduced clones that bypassed senescence. In support of this possibility, we have identified additional integration sites (that were not detected in the mass cultures) in several clones that were isolated by limiting dilution (data not shown). Dominant clones may be a consequence of transduction of faster growing cells or selection for cells that are more prone to life span extension. Clonal fluctuations observed during the propagation of these cultures support the possibility of selection. Evolution of clones within the hTERT mass cultures is also consistent with our survival analysis of individually isolated hTERT-transduced clones where only about one-third of the clones that bypassed senescence continued to proliferate beyond 130 PDLs [41]. The possibility that subsets of fibroblasts are more prone to hTERT-mediated life span extension and immortalization warrants further investigation. Putative factors that may predispose cells to bypass senescence upon induction of hTERT could also be related to the

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observed cell type specificity of hTERT. hTERT expression was shown to be sufficient to extend the life span of certain cell types but not others. It is striking that telomerase activity and telomere maintenance have differential effects in epithelial cells of different origins [25, 29, 30]. We cannot exclude the possibility that hTERT also has differential effects in various fibroblast strains as well as in individual cells within a strain. A molecular basis for susceptibility to hTERTmediated life span extension does not appear to be linked to transformation pathways, since in our investigations and in previous studies, postsenescent cells expressing hTERT exhibited no evidence of malignant transformation [42, 43]. An abundant body of evidence indicates that several molecular alterations must occur for normal human cells to bypass both senescence and crisis and acquire an immortal phenotype [11, 30, 56]. These necessary changes include, but may not be restricted to, repression of c-fos, inactivation of p53 and RB (or p21 CIP1/WAF1 and p16 INK4a), and activation of either telomerase activity or ALT [57–59]. In the present study, only one of these changes, the activation of telomerase activity, was evoked. It was therefore not necessarily surprising that immortalization was not the universal outcome for cells that expressed telomerase and bypassed senescence. Investigations which demonstrated that over expression of hTERT enables SV40 LT-transformed cells to avert crisis [31–33] do not conflict with our results since in the present investigation, hTERT expression was induced in cells that were normal and not oncogenically transformed. At no time did MRC5/TIG or MRC5/B2TIG cells exhibit a transformed phenotype. Furthermore, growth of MRC5/TIG and MRC5/ B2TIG cells was inhibited by ␥-irradiation, as expected for cells with normal G1 checkpoint controls. The transforming properties of SV40 LT are exerted (at least partially) through inactivation of tumor suppressor gene functions [58, 60]. In addition to life span extension, SV40 LT also induces genomic instability, phenotypic changes, and altered growth requirements [61, 62]. As a rare event, cells expressing SV40 LT antigen may escape crisis and become immortal [10, 14]. Activation of telomerase is clearly involved in the spontaneous immortalization of SV40 LT antigentransformed cells; however, it is likely that molecular alterations exerted by SV40 LT are also necessary for this process. In conclusion, our results indicate that elongation of telomeres permits life span extension, but is not necessarily sufficient for immortalization of human fibroblasts. Regardless of the long telomeres in MRC5/TIG and MRC5/B2TIG cells, many cells succumbed to cell death or growth arrest following a substantial extension of life span beyond senescence. Other cells appeared to have averted crisis and have proliferated for

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more than 280 PDLs. The events that signal growth arrest or cell death in the postsenescent cells still remain to be determined; however, two possibilities have been considered. These are (i) that the telomeres of very late passage cells were too long to be organized into normal telomeric end structures or (ii) that other genetic or metabolic changes associated with cellular aging activated checkpoint controls and caused growth crisis. Conversely, specific molecular and/or metabolic states may render subsets of cells more susceptible to telomerase-induced life span extension. It is plausible that replicative capacity is determined by the interplay of the cell cycle machinery with multiple factors which may include both telomeres and metabolites. We thank Ms. Dianna Ngok, Mr. Jason Anselmo, and Ms. Cuiwan Tan for technical assistance; Mr. Tom Delhory for assistance and advice with cell cycle analysis; and Geron Corporation for hTERT cDNA. This work was supported by NCI Grant CA59350, NHLBI Grant HL 61401, and the Gar Reichman Fund of Cancer Research Institute.

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