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J. Biol. Chem., Vol. 279, Issue 42, 43634-43645, October 15, 2004
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From the Children's Cancer Institute Australia for Medical Research, Randwick, New South Wales 2031, Australia
Received for publication, March 3, 2004 , and in revised form, August 9, 2004.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Compelling evidence implicates the gradual shortening of telomeres as an upstream event in the initiation of CDK inhibitor activity and induction of senescence. Telomeres are specialized chromosomal-end structures that prevent abnormal chromosomal fusions and rearrangements (10). In normal human cells telomeres shorten with each cell division. When telomeres become critically short, chromosomal instability ensues and p53-dependent DNA damage signals initiate the senescence program. Senescence may also be triggered by telomere-independent stresses, such as DNA damage caused by irradiation and cytotoxic drugs or chemically induced oxidative stress (11). In addition, premature senescence may be induced by expression of oncogenic ras and raf, which signal via the mitogen-activated kinase cascade to induce expression of p16INK4a and p53 (12–14).
In contrast to telomere attrition observed in normal somatic cells, 
80 –90% of cancer cells and immortal cell lines have telomeres stabilized or elongated by the enzyme telomerase (15). Telomerase is a ribonuclear protein complex that has a reverse transcriptase (hTERT) as a catalytic domain (16). Ectopic expression of hTERT was shown to be sufficient to reconstitute telomerase enzyme activity, elongate telomeres, and extend the lifespan of normal human fibroblasts and retinal epithelial cells (17–20). In addition, it was reported that expression of hTERT immortalized specific strains of fibroblasts, epithelial, and endothelial cells, without the requirement for molecular alterations in p53/p21cip1 and pRB/p16INK4a pathways, and without phenotypic changes associated with carcinogenesis (21–23). However, other investigators have found reconstitution of telomerase enzyme activity to be insufficient for immortalization keratinocytes, mammary, and adenoid epithelial cells, as well as bone marrow, brain, and mammary-derived endothelial cells (24–29). In the case of mammary epithelial cells and keratinocytes, inactivation of p16INK4a expression was necessary for hTERT-transduced cells to proliferate beyond senescence (24). The apparent inability of hTERT to immortalize specific cell types was proposed to be caused by inadequate culture conditions, which induced p16INK4a expression (30). However, more recent investigations found no difference in the replicative capacity of hTERT-transduced keratinocytes and bone marrow endothelial cells cultured under alternative culture conditions (29, 31). Overall, these results suggest that a telomere-independent barrier may operate to prevent immortalization of some strains of human epithelial and endothelial cells.
There is also a growing body of evidence indicating that expression of catalytically active telomerase is insufficient for immortalization of some strains of human fibroblasts. Our previous study, as well as independent investigations, demonstrated that several different stains of fibroblasts expressing hTERT ceased proliferating after a significantly extended lifespan (32–35). In our study of hTERT-transduced MRC5 lung fibroblasts (MRC5hTERT), growth arrest was not permanent, as subsets of mass cultured cells eventually resumed proliferation, and immortal cell lines were established. Similarly, Noble et al. (35) have recently shown that hTERT-transduced human foreskin fibroblasts undergo a period of reduced growth during immortalization. The growth lags observed in hTERT-transduced fibroblast cultures are consistent with the requirement for co-operating molecular events for immortalization. During the period of delayed growth, MRC5hTERT cells exhibited features that were characteristic of cells driven beyond senescence and into crisis by viral oncogenes, which inactivate the p53 and pRB tumor suppressor pathways (6, 9, 36, 37). Substantial evidence indicates that crisis in viral oncogene transformed cells is a consequence of critical telomere shortening (38–40). However, the growth barrier observed in hTERT-transduced, telomerase positive cultures provides evidence for a telomere-independent crisis. While crisis in oncogene transformed cells has been extensively investigated, hTERT-driven crisis remains largely uncharacterized.
In the present study, 67 MRC5hTERT clones were established to enable investigation of hTERT-driven crisis. Our results demonstrate that all MRC5hTERT clones that had an extended lifespan were subject to a telomere-independent crisis, while only 28% of lifespan extended clones escaped crisis and were immortalized. Immortalization was associated with inactivation of p16INK4a, which was not reversed by expression of oncogenic ras. However, the regulation of pRB phosphorylation and function of the p53/p21cip1 pathway was normal in immortal cells. Our results indicate that inactivation of p16INK4a may be necessary to overcome telomere-independent crisis during immortalization of MRC5 fibroblasts.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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-Minimal Essential Medium (MEM) with 2 mM L-glutamine, 100 units /ml penicillin and 100 µg/ml streptomycin (Invitrogen Life Technologies) plus 10% fetal bovine serum (FBS) (Trace Scientific, VIC, Australia) at 37 °C/5% CO2. MRC5hTERT clones were established by plating the MRC5hTERT-1 mass cultured cells at 66 population doublings (PDs) at a concentration of 20 cells/ml in a 96-well plate and identifying wells containing single cells under an inverted microscope. For colony formation in soft agarose, cells were plated at 10,000/ml in 0.33% agarose (Seaplaque, Cambrex, Rockland, ME)/20% FBS/MEM over a preformed layer of 0.5% agarose/20% FBS/MEM and incubated at 37 °C/5% CO2. Colonies of greater than 50 cells each were scored under an inverted microscope on day 14.
Telomerase Assay—Telomerase expression was quantified using the PCR-based telomeric repeat amplification protocol (TRAP) as described previously (15). Protein was extracted by lysing cells in CHAPS lysis buffer (10 mM Tris, pH 7.5, 1 mM MgCl2, 1 mM EGTA, 0.1 mM benzamidine, 5 mM 
-mercaptoethanol, 0.5% CHAPS, 10% glycerol) on ice for 30 min. Supernatant was collected following centrifugation at 12,000 x g for 30 min at 4 °C. Aliquots of 2 µg of protein were assayed in 25-µl reactions, which included TS primer, NT primer, and 0.01 amol of internal PCR control (TSNT). Each TRAP assay included a negative control (CHAPS buffer), 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 with 0.01% (v/v) SYBR® Green 1 nucleic acid gel stain (Molecular Probes, St. Louis, MI). Images were captured by laser scanning using a Typhoon 9410 Variable Mode Imager (Molecular Dynamics, Sunnyvale, CA) and analyzed using ImageQuant (Molecular Dynamics). Telomerase activity in each sample, or total product generated (TPG) was calculated as a fraction of TPG in the neuroblastoma control: TPG = 100 x [(T – B)/TSNT]/[(NB – B)/TSNTNB], where abbreviations are as follows; T is sample signal, B is background of negative control, TSNT is the internal control signal, NB is the neuroblastoma signal, and TSNTNB is the signal of the internal control band of the neuroblastoma control.
Telomeric Restriction Fragment (TRF) Assay—Genomic DNA was extracted by standard phenol/chloroform procedure and precipitated with 0.3 M sodium acetate and 2 volumes of ethanol. For TRF assays, 10 µg of DNA was digested with Hinf and RsaI restriction enzymes (New England Biolabs, Beverly, MA) for 16 h at 37 °C, followed by electrophoresis through a 0.7% agarose gel. Gels were depurinated, denatured, neutralized, and transferred to Hybond-N+ nylon membrane (Amersham Biosciences). Transfer was by capillary action using 20x SSC for 16 h, before fixing the transferred DNA to the membrane by UV cross-linking (120 mJ) (Stratagene, La Jolla, CA). Chemiluminescence detection of telomeric restriction fragments was performed using the Telo-TAGGG Telomere Length Assay (Roche Applied Science). In brief, the membrane was blocked 30 min at 42 °C and hybridized for 3 h at 42 °C with a digoxigenin (DIG)-labeled telomere probe. Further washes were performed according to the manufacturer's instruction before incubating with an anti-DIG alkaline phosphatase secondary antibody for 30 min at room temperature. CDP-star substrate (Roche Applied Science) was added for detection of telomeres, and the membrane was exposed to x-ray film (Fuji Film, Japan). Images were scanned on a flat bed scanner and TRF signals quantified using MacBas (Fuji). Mean TRF lengths were determined according to the following formula [
(Si/Li)]/[Si], where abbreviations are as follows; Si is TRF signal at a given location after background subtraction, Li is the corresponding length at position i (41).
Western Blot Analysis—Protein lysates were collected from cell pellets by incubation on ice in 10 mM Tris, pH 8.0, 150 mM NaCl, 1 mM EDTA, 1% Nonidet P-40, 1% protease inhibitor (Sigma) for 30 min. Protein concentrations were determined with the BCA assay (Pierce). For detection of p16INK4a, p21cip1, pRB, and total actin expression, aliquots of 25 µg of protein were separated by SDS-PAGE through a 15% polyacrylamide gel and transferred to polyvinylidine difluoride (PVDF) membrane (Millipore, Bedford, MA). For detection of pRB phosphorylation status, 50 µg of protein was separated on 7%, polyacrylamide gel and transferred to PVDF membrane. Membranes were stained with 0.1% (w/v) Ponceau S (Sigma) in 5% aqueous acetic acid to confirm even transfer of proteins, then destained in demineralized water. Membranes were blocked in 5% skim milk and hybridized to the following primary antibodies: mouse anti-human p21cip1 monoclonal antibody (BD PharMingen, San Diego, CA) 1/500, rabbit anti-human p16INK4a polyclonal antibody (BIOSOURCE, Nivelles, Belgium) 1/1000, rabbit anti-human actin 1/50000, mouse anti human RB antibody (BD PharMingen) 1/500. Membranes were washed in 20 mM Tris, pH 7.5, 150 mM NaCl, 0.05% (v/v) Tween-20, and hybridized to the corresponding horseradish peroxidase-conjugated secondary antibodies; anti-rabbit (Amersham Biosciences) 1:10000 or anti-mouse (Amersham Biosciences) 1:5000, for 1 h at room temperature. Chemiluminescence detection was performed using Supersignal (Pierce) according to the manufacturer's instructions and images were acquired on x-ray film or Versdoc Imaging System (Bio-Rad).
Retroviral Transduction—Stable retroviral producers, which generate high titers of the replication defective retroviral vectors L9IGFP and L9NrIGFP, were previously established and generously provided by A/Prof. Geoff Symonds and Dr. Alla Dolnikov (Children's Cancer Institute Australia for Medical Research) (42). The L9NrIGFP vector encodes mutant N-ras (N-ras13, G-C transversion at position 763) and a yellow fluorescence protein reporter gene GFPtpz (referred to as YFP in this report) that are linked by an IRES. Expression of N-ras protein was previously shown to correlate with YFP fluorescence (42). L9IGFP, which encodes YFP alone, was used as a control. For simplicity, the L9NrIGFP and L9IGFP vectors are referred to as Nras-YFP and YFP vectors, respectively, in the present report. Retroviral vectors, encoding wild-type p16INK4a (p16wt), p16INK4a with an inactivating missense mutation (p16M53T) and the control vector, pBABEpuro, were generously provided by Dr. Gordon Peters (Imperial Cancer Research Fund Laboratories, London, UK) (43). Expression and function of p16wt and p16M53T were previously demonstrated in retrovirally transduced fibroblasts (43). In the present study, plasmid retroviral vectors were transfected into Phoenix A packaging cells (obtained from American Type Culture Collection with permission from Garry Nolan of Stanford University) using a standard calcium phosphate transfection procedure. MRC5 cells, and MRC5hTERT cells were transduced by overnight incubation in viral supernatant supplemented with 8 µg/ml polybrene (Sigma) for three consecutive days. Cells transduced with Nras-YFP and YFP were enumerated by flow cytometry using a FACS Caliber flow cytometer (BD PharMingen). Cells transduced with p16wt, p16M53T, and pBABEpuro were selected by growth in 0.8 µg/ml puromycin (Sigma) in 10% MEM.
-Galactosidase Staining—Cells were assayed for senescence-associated -galactosidase activity (SA--gal) at pH 6.0 as previously described (44). In brief, cells were first rinsed with phosphate-buffered saline, then fixed in 2% formaldehyde, 0.2% glutaraldehyde, and stained with 1 mg/ml 5-bromo-4-chloro-3-indolyl -D-galactopyranoside (X-gal) (Sigma) for 12–16 h at 37°C. Cells expressing -galactosidase activity (stained blue) were scored under an inverted microscope. At least three fields of 50–200 cells were scored for each sample assayed. Values are given are means with S.D.
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RESULTS |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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-gal activity (a hallmark of senescence), or underwent cell death and displayed morphologic features of apoptosis (Table I and Fig. 1c). 11 out of 39 lifespan-extended clones (28%) escaped crisis, continued to proliferate beyond 220 PD (which represented more than a 3-fold increase in lifespan) and were considered immortal (Fig. 1d). Two additional immortal clones (clones D1 and D2) were established by limiting dilution of the MRC5hTERT-1 mass culture at 396 PDs. These clones were established to ensure that our analyses included clones that were most favored by the immortalization process.
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92 PD) verses post-crisis and immortal clones (
141 PD) (Student's t test, p = 0.9).
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Modulation of p16INK4a and p21cip1 in MRC5hTERT Clones and Mass Culture—Western blot analysis was performed to investigate the expression of p16INK4a and p21cip1 during immortalization of MRC5hTERT cells (Fig. 3). As expected, both p21cip1 and p16INK4a were markedly elevated in senescent nontransduced MRC5 cells (PD 63) compared with earlier passage MRC5 cells (PD 41) (Fig. 3a, lanes 7 and 8). p16INK4a accumulated in MRC5hTERT mass cultured cells to reach very high levels at 97 PD, which was prior to crisis, and then returned to low levels by 124 PDs. In the Western blot shown in Fig. 3a, p16INK4a was undetectable in post-crisis cells at 154 and 260 PD (Fig. 3a, lanes 5 and 6). However, further analyses, using an antibody from an alternate company (BIOSOURCE), revealed low levels of p16INK4a expression in post-crisis cells at 147, 159, and 181 PDs (data not shown). Irrespective of the antibody used, p16INK4a was undetectable at eight different time points between 203–420 PDs. Thus, although down-regulation of p16INK4a was detectable immediately after crisis, complete silencing of p16INK4a in MRC5hTERT mass culture did not correspond with escape from crisis, but appeared to occur between 181 and 203 PDs. Time course analyses of p21cip1 expression in MRC5hTERT mass cultured cells showed that p21cip1 was induced by 63 PD and reached levels comparable to senescent cells by 124 PD, when the culture exhibited dramatic cell death and slowed growth (Fig. 3a, lane 4). Expression of p21cip1 was reduced to low levels in post-crisis cells, but was not completely extinguished.
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Expression and Phosphorylation of pRB during Immortalization of MRC5hTERT Cells—The phosphorylation status of pRB was also investigated (Fig. 4a). Our results confirmed that pRB was in a hypophosphorylated state in senescent, non-transduced MRC5s (PD 63) (lane 7) and up-regulated and partially phosphorylated in earlier passage MRC5s (PD57) (lane 6). In the MRC5hTERT-1 mass culture, pRB was down-regulated and in its active, hypophosphorylated form during crisis (lanes 2 and 3), then up-regulated and phosphorylated in post-crisis cells (lanes 4 and 5). These changes were consistent with the up-regulation of p16INK4a and p21cip1 at crisis and down-regulation of these CDK inhibitors in post-crisis cells.
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MRC5hTERT Response to Genotoxic Stress—Dysfunction of G1/S checkpoint control may render cells less sensitive to genotoxic stress, such as ionizing irradiation. Hence, expression of p21cip1 and its transcriptional activator, p53, were investigated following exposure of MRC5hTERT cells to ionizing irradiation. In preliminary experiments, it was determined that p53 accumulated 3 h after exposure to 10 Gy irradiation, while p21cip1 was maximally induced 24 h after irradiation in non-transduced MRC5 cells (data not shown). We next tested the response of pre- and post-crisis MRC5hTERT-1 mass-cultured cells, as well clones 2, 12, 49, and D1 to irradiation (Fig. 5a and additional data not shown). In all samples, including the immortal clone D1, p53 accumulated 3-h post-irradiation, and p21cip1 was induced 24 h after irradiation. These data indicate that the p53/p21cip1 response to genotoxic stress was functionally normal in post-crisis MRC5hTERT cells, and that inactivation of this checkpoint was not necessary for immortalization. Phosphorylation and expression of pRB in response to irradiation was also investigated. Western blot analysis demonstrated that pRB was phosphorylated prior to irradiation, then became hypophosphorylated 18 h after irradiation. This phosphorylation pattern was observed in non-transduced MRC5 cells, as well as pre- and post-crisis MRC5hTERT cells, indicating that phosphorylation of pRB in response to genotoxic stress was unperturbed during immortalization (Fig. 5b).
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gal (Fig. 6d). In contrast to the earlier passage cells, late passage MRC5hTERT (414) cells transduced with N-ras-YFP (MRC5hTERT(414)N-ras) did not undergo arrest and did not exhibit a statistically significant increase in SA--gal activity.
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4-fold) than MRC5hTERT(414)YFP cells (p = 0.0017) (Fig. 6e). MRC5hTERT(414)Nras colonies were also larger (up to 500 µM in diameter) than MRC5hTERT(414)YFP colonies. Neither MRC5hTERT(414)YFP nor MRC5hTERT(414)-Nras cells formed tumors when 5 x 106 cell were subcutaneously implanted in nude mice. The mice were monitored for tumor formation for 5 months. In contrast, tumors formed within 3 weeks in control mice that were inoculated with 5 x 106 HT1080 fibrosarcoma cells. Thus, the combination of telomerase activity, inactivation of p16INK4a, and expression of oncogenic N-ras in MRC5 cells was not sufficient for tumor formation.
MRC5hTERT Response to p16INK4a Expression—To determine whether loss of p16INK4a was instrumental in the immortalization process, immortal and pre-crisis MRC5hTERT cells were transduced with retroviral vectors encoding p16wt and p16M53T (43). Transduction with the p16wt vector was sufficient to halt proliferation of immortal MRC5hTERT-1 ((MRC5hTERT (400)) cells. Indeed, the p16wt vector was as effective in immortal MRC5hTERT-1 cells as pre-crisis cells (MRC5hTERT (75)) cells (Fig. 7a) and normal MRC5s (additional data not shown). Growth-arrested cells adopted a senescent morphology and exhibited SA--gal activity (Fig. 7b). The Met to Thr mutation in the p16M53T vector completely abrogated the growth suppressive activity of p16INK4a, as previously reported (43). These results demonstrate that reconstitution of p16INK4a restores immortal MRC5hTERT cells to a phenotype that is indistinguishable from pre-crisis cells and support the possibility that loss of p16INK4a was a critical event in the immortalization process.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Analysis of telomerase activity in MRC5hTERT clones indicates that crisis was unlikely due to the level of telomerase enzyme activity. A suboptimal level of telomerase was detected in MRC5hTERT clone 1, which underwent a senescent-like arrest at crisis. However, this clone was an exception, as it expressed a lower level of telomerase than all other clones tested and was the only clone that did not have extended telomeres. In contrast to clone 1, another 2 clones that underwent delayed senescence at crisis expressed higher levels of telomerase and had hyperextended telomeres (greater than 20 Kbp). A recent report has shown that very high levels of exogenously expressed telomerase may induce a senescent-like arrest in hTERT-transduced fibroblasts (48). However, high levels of telomerase did not appear to be the cause of senescent-like arrest or crisis in MRC5hTERTs cultures, since there was no selection for lower levels of telomerase in post-crisis clones. Indeed the highest levels of telomerase were detected in some of the post-crisis samples. The apparent discrepancy between our report and the study by Gorbunova et al. (48) may be explained by differences in methodology or the different fibroblast strains used in the two studies. It is also possible that the level of telomerase activity expressed in MRC5hTERT cells was not as high as the levels of telomerase expressed by hTERT-transduced fibroblasts in the study by Gorbunova et al. (48). Finally, we cannot exclude the possibility that MRC5hTERT clones expressing very high levels of telomerase were among the 28 clones that senesced prior to 80 PDs (did not have an extended lifespan) and therefore were not included in this analysis. TRF analysis revealed very long telomeres in 9/10 clones. These hyperextended telomeres did not appear to be detrimental to cell proliferation, or to be the cause of crisis, since there was no selection for shorter telomere lengths in post-crisis and immortal clones. Overall, our results indicate no consistent relationship between telomerase activity or telomere length and the onset of crisis.
The unexpected proliferative arrest of 70% of the 26 MRC5hTERT clones in our previous study precluded rigorous study of molecular events responsible for growth arrest (45). In the present study we show that growth arrest and cell death at crisis was likely due to the up-regulation of p16INK4a and p21cip1, with consequent hypophosphorylation of pRB. It was previously demonstrated that both p16INK4a and p21cip1 are up-regulated during replicative senescence, as well as in oncogene-induced premature senescence (2, 3, 12, 13, 49). Up-regulation of p21cip1 has also been associated with growth arrest induced by oxidative stress (50), while p16INK4a may be induced by suboptimal culture conditions, such as growth in chemically defined media with low (0.25%) serum (30). Suboptimal culture conditions were unlikely to be the cause of crisis in MRC5hTERT cultures, since 10% serum was included in the culture media and the onset of crisis in MRC5hTERT cells and senescence in MRC5 cells was very similar in 2 different media formulations. In addition, non-transduced MRC5s underwent replicative senescence at 65 PDL when telomeres were around 5.0 Kbp, which is consistent with expectations for fibroblasts grown under optimal culture conditions.
Following crisis, p16INK4a and p21cip1 were down regulated, while pRB became phosphorylated. Our result showing repression of p16INK4a in post-crisis MRC5hTERT cells is consistent with a recent report that demonstrated down-regulation of p16INK4a in post-crisis human foreskin fibroblasts that were immortalized with hTERT (35). Our results showing that restoration of p16INK4a in post-crisis MRC5hTERT cells induced growth arrest and features of senescence, provides further evidence that loss of p16INK4a was a critical event in the immortalization process. The functional significance of the down-regulation of p21cip1 in MRC5hTERT cells is less clear, since p21cip1 was repressed in clones that escaped crisis, as well as in clones that permanently arrested in a senescent-like state. Furthermore, p21cip1 was readily induced by irradiation in very late passage cells. In the study by Noble et al. (35) a defective p53/p21cip1 response to DNA damage was found in 1 of 3 hTERT-immortalized clones. In contrast, we found no defects in the p53/p21cip1 DNA damage response in the post-crisis MRC5hTERT mass culture and four post-crisis clones, including one clone that was passaged for more than 400 PDs. This result may reflect inherent differences in MRC5 lung fibroblasts compared with the foreskin fibroblasts employed in the study by Noble et al. (35).
It is noteworthy that the pattern of p16INK4a and p21cip1 expression observed in MRC5hTERT clones that underwent senescent-like arrest at crisis was analogous to normal fibroblasts during replicative senescence, where p21cip1 is transiently induced and high levels of p16INK4a are sustained (51). One proposed explanation for these observations is that transient high levels of p21cip1 act to initiate senescence, while high levels of p16INK4a sustain the senescent phenotype (51). In contrast to p21cip1, inactivation of p16INK4a corresponded well with immortalization. Expression of p16INK4a was reduced in clones that escaped crisis, but not clones that arrested in a senescent-like state. However, the timing of p16INK4a inactivation did not precisely correspond with escape from crisis, as a low level of p16INK4a was detected in early post-crisis cells. This may be a reflection of the long half-life of p16INK4 protein, unsynchronized repression of p16INK4a in subsets of cells and/or retention of p16INK4a in subpopulations of cells that did not escape crisis and were carried over in the culture. These possibilities are consistent with the gradual outgrowth of p16INK4a-negative cells in the mass culture.
It was previously shown that expression of hTERT does not overcome ras-induced premature senescence in human fibroblasts (52). The present study demonstrates that immortal p16INK4a-negative MRC5hTERT cells were refractory to ras-induced de-repression of p16INK4a and senescent-like growth arrest. Our results are in agreement with recent studies that showed fibroblasts derived from individuals who were genetically deficient at the INK4a locus were resistant to ras-induced growth arrest (43, 53). Similar to immortal MRC5hTERT cells, the cells from the INK4a-deficient individuals retained p53/p21cip1 function. These results strongly implicate p16INK4a as the critical mediator of ras-induced growth arrest. However, a contrasting report that examined the effects of p16INK4a inactivation by overexpressing a mutant cdk4-cyclin D fusion protein concluded that inactivation of p21cip1 in addition to abrogation of p16INK4a was necessary for overcoming ras induced growth arrest (54). There are several possible explanations for this apparent discrepancy, such as different levels of ras expression, different methodologies and vectors, and/or spontaneous molecular changes or inherent differences in the various cell strains employed.
Transduction with oncogenic ras facilitated anchorage independent growth of p16INK4a-negative MRC5hTERT cells. However, the cloning efficiency was very low and the colonies were very small compared with other transformed cell lines that we have tested (29). Brookes et al. (53), reported more robust colony formation when INK4a-deficient fibroblasts were cotransfected with hTERT and ras, while Wei et al. (54) indicated that abolition of both p16INK4a and p21cip1 were necessary for anchorage-independent growth of hTERT-transduced fibroblasts. Considerable variations in the materials and methods used for this assay are likely to account for differences obtained in these independent investigations. Despite the variations among these reports on the requirements for anchorage independent growth, there is a long-standing consensus that several molecular alterations are required for acquisition of a tumorigenic phenotype (1). Our results, showing that N-ras transduced p16INK4a-negative MRC5hTERT cells were nontumorigenic in immunocompromised mice are consistent with this notion. It is likely that inactivation of the P53/p21 pathway and additional molecular events would be required for transformation of MRC5 cells to a tumorigenic phenotype.
Down-regulation of p16INK4a in late passage MRC5hTERT cells coincided with increased expression of pRB. The increase in pRB expression was not simply a reflection of proliferative status. Previous investigations have shown that high p16INK4a may transcriptionally repress pRB in transformed cell lines and tumor-derived cells (55). Our results extend these findings by showing that pRB was elevated in p16INK4a-negative cells when compared with the corresponding p16INK4a-positive cells. These results are consistent with a model in which pRB and p16INK4a expression is reciprocally regulated via a feedback loop (55, 56).
Despite inactivation of p16INK4a and increased expression of pRB, pRB was returned to a hypophosphorylated state following irradiation of immortal MRC5hTERT cells. This was likely due to the activity of p21cip1, which remained functional throughout the immortalization process. These results show that hypophosphorylation of pRB in response to stress is not an accurate indicator of p16INK4a function. Moreover, our results illustrate the apparent redundancy of p16INK4a in the DNA damage-mediated regulation of pRB phosphorylation. The observation of a normal pRB phosphorylation pattern in p16INK4a-negative MRC5hTERT-1 cells is also important in relation to the prevailing notion that p16INK4a regulates cell lifespan by modulating phosphorylation of pRB. Our observations are consistent with a recently proposed model of the molecular control of senescence, whereby pRB was placed in a linear pathway downstream from P53 and p21cip1, with p16INK4a forming an additional branch at the level of pRB (54). This model implies that inactivation of p16INK4a would not prevent pRB-mediated growth arrest in p53/p21cip1 competent cells. Nevertheless, a critical role for p16INK4a inactivation in immortalization of primary human fibroblasts is strongly implicated by our results and others, which have demonstrated selection for p16INK4a inactivation during immortalization of human fibroblasts (33, 35, 57, 58). Specifically, our investigations indicate that inactivation of p16INK4a may be necessary to overcome telomere-independent crisis, which appears to be an integral event during hTERT-mediated immortalization of MRC5 fibroblasts.
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FOOTNOTES |
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To whom correspondence should be addressed: Children's Cancer Institute Australia for Medical Research, P. O. Box 81 High St., Randwick, NSW 2031, Australia. Tel.: 61-2-9382-0048; Fax: 61-2-9382-1850; E-mail: k.mackenzie{at}unsw.edu.au.
1 The abbreviations used are: CDK, cyclin-dependent kinase; FBS, fetal bovine serum; MEM, minimal essential medium; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; PDs, population doublings; X-gal, 5-bromo-4-chloro-3-indolyl--D-galactopyranoside; SA--gal, senescence-associated -galactosidase activity; FACS, fluorescent-activated cell sorting; TRAP, telomeric repeat amplification protocol; GFP, green fluorescent protein; YFP, yellow fluorescent protein; TERT, telomerase reverse transcriptase; RB, retinoblastoma; TRAP, telomeric repeat amplification protocol; TRF, telomeric restriction fragment; S.D., standard deviation.
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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