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Department of Pathology, University of Wales College of Medicine, Heath Park, Cardiff CF14 4XN, United Kingdom
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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20 pd and was not bypassed by hTERT expression. Abrogation of p53 function, by expression of human papillomavirus type 16 E6, led to an extension of life span, implying that replicative senescence in NHAs was p53-dependent but telomere-independent. human papillomavirus type16 E6 expression promoted additional growth of up to 12 pd, until a second telomere-independent PLB (termed MINT) was imposed associated with elevated p16INK4A levels. A proportion of cells escaped from MINT lost p16INK4A expression and achieved approximately an additional 25 pd until a crisis-like third PLB (M2) was reached. Expression of hTERT in post-MINT cells allowed these cells to become immortal and bypass this third PLB. The in vitro PLBs appear, in order of occurrence, dependent upon p53, p16INK4A, and telomere erosion, a situation that mirrors an equivalent order of mutational events during tumor progression in vivo. This study describes a model that provides a plausible explanation for the selective pressures driving mutational events in this tumor type and provides direct evidence of a p53-dependent, telomere-independent PLB. |
INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Two PLBs, replicative senescence (M1; Refs. 2
, 3
) and crisis (M2; 4
), have been well-characterized in vitro in many cell types, including HDFs. Under optimal culture conditions fibroblasts undergo 40–70 pd (depending on donor age) before entering a stable proliferative arrest state (M1) in which they remain viable for many months or years (2)
. HDFs can bypass M1 by expression of a variety of DNA tumor virus proteins, including SV40 T antigen (5
, 6)
, and a combination of both HPV-16 E6 and E7 genes, both of which target a common set of cell cycle regulatory tumor suppressor gene products, notably p53 and pRB (7
, 8)
. Fibroblasts that bypass M1 in this way are capable of at least an additional 30 pd, after which additional expansion is limited by the second PLB termed crisis or M2, which is characterized by widespread cell death. Escape from M2 depends on stabilization of telomere length by reactivation of telomerase or by an alternative mechanism (9
, 10)
. Expression of the catalytic subunit of telomerase (hTERT) alone is sufficient to bypass M1 or M2 and immortalize fibroblasts (11)
.
Additional investigation has revealed that intermediate arrest states (termed MINT) can be imposed through selective abrogation of either p53 or pRB alone (12) . The intermediate states of growth arrest induced by loss of p53 (MINTE6) or pRB (MINTE7) function are very different. MINTE6 is reminiscent of M1 senescence and is potentially mediated by p16INK4A expression, whereas the expansion of cell cultures at MINTE7 is limited by increasing cell death and a phenotype similar to M2 (12) . Similar MINT states have been observed in Li-Fraumeni syndrome fibroblasts (13) , where spontaneous loss of the remaining copy of wild-type p53 also results in growth arrest at a point intermediate between M1 and M2.
The initial aim of this study was to investigate factors affecting life span in astrocytes. It was designed in such a way as to recapitulate the genetic events during the clonal evolution of tumors in vivo, using serial analysis of both clonal and polyclonal populations, with extensive characterization of cells at all stages of their life span. Our results led to a model of multiple PLBs that is strikingly consistent with those suggested by the pattern of genetic changes associated with disease progression in vivo. This study also uncovered a novel p53-dependent, telomere-independent PLB.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Retroviral Gene Transfer.
Genes were transduced into NHAs using amphotropic retroviral vectors. The catalytic subunit of telomerase, hTERT cloned into pBABE puro, was used as previously described (14
, 15)
and the empty vector used as a control. A retrovirus expressing HPV-16 E6 protein (HPV-16 E6 neo; Ref. 7
) conferring resistance to G418 was used, which allowed dual drug selection in later experiments. A retrovirus based on pBABE neo was used as a vector control. For infection, cells were plated in 60-mm dishes at a density of 105 cells/dish and 2 days later exposed to retrovirus-containing medium from near-confluent producer cells, containing Polybrene at a concentration of 8 µg/ml. Two days after infection, cells were serially diluted and replated at densities appropriate to produce polyclonal and monoclonal populations. For drug selection, G418 was used at a concentration of 400 µg/ml and puromycin at 2.5 µg/ml. To create dual infected cells, NHAs were infected sequentially with pBABE neo at pd 7, a G418-resistant pooled population expanded, and then infected at pd 13 with pBABE puro, at which point individual drug-resistant clones were isolated.
Detection of SA ß-Gal Activity.
Two days after plating on coverslips endogenous SA ß-gal activity (16)
was assessed histochemically using 5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside as the substrate.
Assessment of DNA Synthesis by BrdUrd Incorporation.
Cells were plated onto coverslips and re-fed with Dulbecco’s modified Eagle medium 2 days before the assay. Cells were labeled by incubation in 50 µM BrdUrd for 1 h, after which nuclear incorporation was detected by immunoperoxidase immunocytochemistry as described previously (12)
. The proportion of labeled nuclei (LI) was determined from a count of 500 cells taken from three coverslips and the SE of the proportion calculated.
Immunocytochemistry.
For p21WAF1 detection, NHAs were plated onto coverslips 2 days before analysis, fixed in 4% paraformaldehyde (10 min) and then pretreated with 50 mM glycine (10 min), 0.2% Triton X-100 (10 min), and 0.3% H2O2 (3 min). Nonspecific binding was blocked with 2% horse serum (30 min). Anti-p21WAF1 antibodies (clone 6B6; Cambridge Bioscience, Cambridge, United Kingdom; Ref. 17
) were applied followed by a mouse-specific avidin-biotin-peroxidase system (Novocastra). Sites of antibody binding were visualized by the deposition of brown polymer after incubation in diaminobenzidine-hydrogen peroxide solution.
Telomerase and Telomere Length Assays.
TRAP assays and TRF length measurements were performed as described previously (18)
. For TRAP assays, protein extracts were prepared from trypsinized NHAs and diluted to 5000 cell equivalents/reaction, where an oligonucleotide substrate was extended by telomerase, amplified by PCR, and products separated on 10% polyacrylamide gels. Products were visualized by Sybr Gold staining and detected by fluorimaging (Storm; Amersham Biosciences). For TRF analyses, 1 µg of genomic DNA extracted using a Nucleon kit (Amersham) was digested with HinfI and RsaI, separated on 0.5% agarose gels, which were denatured with 0.5 M NaOH and neutralized with 0.5 M Tris (pH 8.0) containing 1.5 M NaCl. The gels were dried and probed with 5'-CCCTAACCCTAACCCTAA-3' end-labeled with 
32P-ATP. TRFs were detected by phosphorimaging.
Immunoblotting.
Cells were lysed for 5 min at 4°C in a buffer containing 1% NP40 in 150 mM NaCl, 50 mM Tris (pH 8.0), 5 mM EDTA supplemented with 1 mM phenylmethylsulphonyl fluoride, 0.01 mg/ml aprotinin, and 0.01 mg/ml leupeptin. Protein samples (20 µg each) were separated on 12% SDS PAGE gels and transferred onto polyvinylidene difluoride (Millipore). Primary antibodies raised against p53 (DO-1 (19)
, p27KIP1 (K250; Transduction Laboratories), p21WAF1 (clone 6B6; Cambridge Bioscience; Ref. 17
), and p16INK4A (DCS 50; Calbiochem; Ref. 20
) were applied. Antibody binding was detected by goat antimouse peroxidase conjugate and visualized by the enhanced chemiluminescence detection system (Amersham Biosciences). The filter was subsequently stained with India ink and loaded protein quantified by using a Bio-Rad imaging densitometer with Molecular Analyst software. All immunoblots were performed at least three times.
Analysis of Methylation Status of p16INK4A Promoter.
Genomic DNA was modified using a CpGenome DNA modification kit followed by detection using a p16INK4A CpG WIZ Amplification kit (both Intergen). Methylation-specific PCR primers (21)
were used to distinguish methylated bisulphite-modified DNA from unmethylated modified DNA at a 5' CpG island in the p16INK4A promoter region. The following changes were made to the manufacturer’s protocol. DNA (2 µg) was modified with bisulphite and resuspended in 20 µl of a 10 mM Tris buffer, pH8, containing 1 mM EDTA buffer. DNA (200 ng) was added to PCR reactions and incubated at 95°C for 5 min (hot start) before addition of 1 unit of Taq DNA polymerase (Promega). PCR products were separated on 1.5% agarose gels.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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55 days (Fig. 1)
. Morphologically young NHAs (pd 8) were thin and elongated (Fig. 2A)
. Approximately 50 ± 2.6% of cells were immunopositive for p21WAF1 staining (Fig. 2B)
and showed a BrdUrd LI of 31 ± 2.3% (Fig. 2C)
after a 1 h pulse, whereas 46 ± 2.2% of cells showed SA ß-gal (Fig. 2D)
.
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was accompanied by high p21WAF1 (85 ± 1.6%) immunostaining (Fig. 2F)
, a low BrdUrd LI (1 ± 0.5%; Fig. 2G
), an elevated SA ß-gal index (90 ± 1.3%; Fig. 2H
).
Replicative Senescence in NHAs Is Telomere Independent.
Telomere length was analyzed in astrocytes at pd 8, 16, and 20 using the TRF assay (18)
. The assay detects all telomeric sequences that are displayed as smears, indicative of differences in telomere length between both individual chromosomes and cells within the population. The TRF assay indicated that there appeared to be a modest amount of telomere shortening during the replicative life span of the culture (Fig. 3A)
. In the M1 sample (20 pd), a proportion of the smear comprises lower molecular weight species. However, the overall signal is of a higher molecular weight with a wider distribution of telomeres compared with senescent HDFs where TRFs as low as 2 kb are detected (22)
.
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These data showed that forced expression of hTERT in NHAs conferred telomerase activity on normally negative NHAs. Twenty-two individual NHA.hTERT subclones and 26 NHA.puro subclones were isolated and their proliferative life spans determined (Fig. 3C)
. NHA.hTERT subclones underwent an additional 5–12 pd after infection similar to NHA.puro that accumulated an additional 5–11 pd, with no significant difference between the two sample sets (P = 0.8). Therefore, reactivation of telomerase in NHAs failed to confer an extension of life span. Furthermore, at the end of their life span, both cell populations were morphologically similar (Fig. 3D)
and had the appearance of cells in M1. Therefore, unlike in fibroblasts, hTERT expression failed to extend the replicative life span of NHAs.
Replicative Senescence in NHAs Is p53 Dependent.
Although hTERT expression in HDFs allows these cells to become immortal, ablating the function of either or both of the tumor suppressor proteins pRB and p53 can also confer significant extension of cellular life span (12)
. M1 in astrocytes was associated with high levels of p21WAF1 expression, suggesting that senescence was p53 dependent (Fig. 2F)
. To test this hypothesis, HPV-16 E6, which targets p53 for degradation, was introduced into NHAs at pd 7 using a neoR amphotropic retrovirus or the empty vector as a control. Infected cells were then seeded at a range of densities in G418 to allow selection and analysis of pooled populations and individual colonies. NHAs infected with HPV-16 E6 (designated NHA.E6) initially divided rapidly, showed similar morphology to young cells, and they continued to divide past the point at which NHA.neo cells entered M1. When analyzed at a point after the control NHAs senesced, the E6 cycling populations show morphology (Fig. 4A)
, similar to young astrocytes (Fig. 2A)
and as expected low levels of p21WAF1 expression were seen (8 ± 1.6%) consistent with E6 abrogation of p53 function (Fig. 4B)
. The cells had a BrdUrd LI of 31 ± 2.7% (Fig. 4C)
and a SA ß-gal index of 40 ± 2.8% (Fig. 4D)
.
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.
In another experiment NHAs were infected 1–2 pd before senescence (19 pd). As predicted, these reached MINT after a similar life span extension (Fig. 4)
. Populations of pooled colonies reached MINT after an extra 12 pd (data not shown). Therefore, overall E6 confers a limited but statistically significant life span extension of 7–12 pd (P < 0.001). Astrocytes at MINT were morphologically very similar to cells at M1 and were enlarged and flattened with a characteristic granular cytoplasm (Fig. 4E)
and p21WAF1 levels remained very low (1 ± 0.7%; Fig. 4F
), indicating continued abrogation of p53 by HPV-16 E6. The cells had a low BrdUrd LI (3 ± 1.2%, Fig. 4G
) and a very high SA ß-gal index (90 ± 2.1%; Fig. 4H
).
Expression of hTERT in Cells Expressing HPV-16 E6 Fails to Prevent MINT.
M1 senescence in NHAs was associated with, but not dependent upon, telomere erosion (Fig. 3A)
. Unless telomerase is reactivated, the additional growth past M1 induced by E6 expression would be expected to be accompanied by ongoing telomere erosion, raising the possibility that MINT is telomere dependent. NHA.E6 cultures at pd 13 were thus infected with pBABE.hTERT using the puromycin resistance gene as a second selectable marker to yield cells termed NHAE6.hTERT. Control NHA.E6 cultures were also infected with a retrovirus carrying the puromycin resistance gene alone, yielding cells termed NHA.E6.puro. Drug-resistant populations were expanded on a clonal basis. Six independent clones were analyzed and TRAP assays confirmed the NHA.E6.hTERT cells were telomerase positive (data not shown). NHA.E6 cells were telomerase negative (data not shown). All NHA.E6.hTERT clones entered a MINT-like state after a similar number of pd as NHA.E6, indicating that growth arrest was independent of telomerase status (data not shown).
Frequent Escape from MINT Associated with Loss of p16INK4A Expression.
Unlike M1, from which we never observed spontaneous escape (Fig. 5, A–D)
, the E6 induced MINT state frequently failed to act as a permanent barrier to proliferation (Fig. 5, G and H)
. In several examples, both clonal and polyclonal cultures began to reenter the cell cycle after residing at MINT for a period ranging between 15 and 40 days (Fig. 5, G and H)
. These cells remain telomerase negative (data not shown). TRF analysis of cells that had escaped from MINT indicated that this new burst of proliferation was associated with additional telomere shortening (Fig. 5I)
. The cells continued to divide until they reached an M2 or crisis-like state, presumably because of critical telomere erosion (Fig. 5, G, H, and J)
. No additional stable or transient life span barriers were observed during progression to M2.
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. The expression of p21WAF1 mirrored that seen immunocytochemically with high levels at M1 (Fig. 2F)
and loss of expression when E6 was present (Figs. 4, B and F
, and 6B
). Analysis of p27KIP1 in NHA revealed that there was a 3-fold decrease in NHA.E6 cells at MINT compared with those at M1 (Fig. 6B)
. There was no detectable change in p27KIP1 levels in cells that escaped from MINT.
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. Escape from MINT was initially associated with reduced p16INK4A expression, as shown by NHA.E6 post-MINT cycling cells at 35 pd, where p16INK4A levels are 6-fold reduced compared with MINT (Fig. 6B)
. This underestimates the degree of p16INK4A down-regulation measured on a per-nuclei basis (the presumed site of action) because of the reduced total cell volume of cycling post MINT cells. The residual p16INK4A expression in these initial post-MINT samples is mostly likely explained by the presence of p16INK4A-positive MINT cells still present in these mixed cultures. With additional growth of post-MINT cells, these residual MINT cells would be expected to be diluted out, and indeed, p16INK4A levels rapidly decrease to undetectable levels with ongoing proliferation (data not shown and Fig. 7B
). Thus, the most likely conclusion is that escape from MINT is associated with loss of expression of p16INK4A. In astrocytes, p16INK4A is the only cell cycle regulatory protein analyzed where levels change significantly when the cells escape from MINT.
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. All NHAE6.hTERT clones had long telomeres by the time they had progressed past MINT (the earliest time at which samples could be assessed; an example of one clone is shown in Fig. 5I
). This telomere extension is particularly striking when comparing an NHA.E6.hTERT clone at 60 pd to a NHA.E6 clone at crisis after the same number of divisions (Fig. 5I)
. The growth of two NHA.E6.hTERT clones is shown in Fig. 7A
. To date, these have accumulated >170 pd and continue to divide and thus can be considered immortal. Analysis by immunoblotting of these clones at pd 88 and 65 showed undetectable levels of p16INK4A expression (Fig. 7B)
, implying that escape from MINT in cells expressing telomerase was also dependent on loss of this cyclin-dependent kinase inhibitor. The potential mechanism of p16INK4A down-regulation was investigated. The methylation status of the p16INK4A promoter was determined using bisulphite modification followed by PCR (21)
. Methylated p16INK4A was present in cells that had escaped MINT (Fig. 7C
, Lane 3) and subsequently become immortal after hTERT expression (Fig. 7C
, Lane 4). Thus, a combination of a loss of p16INK4A expression, abrogation of p53, and maintenance of telomere length appears necessary for cellular immortality in NHA. |
DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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3 pd; Ref. 23
) or relatively high (e.g. fibroblasts, >70 pd; Ref. 2
). Human astrocytes have been reported to have a proliferative life span somewhere in between these two extremes (24)
. In our hands, NHAs achieved 20 pd before entering replicative senescence. This state was reminiscent of M1 senescence in fibroblasts, being associated with high p21WAF1 and SA ß-gal-labeling indices. NHAs are telomerase negative, but although some telomere erosion is seen in the cell divisions before senescence in NHAs, the final arrest state does not appear to depend on telomere erosion. Previously, we have shown that forced expression of telomerase (using a retroviral vector encoding hTERT) is sufficient to immortalize HDFs (14 , 15 , 25) . In contrast, similar experiments in NHAs did not lead to any detectable extension of cellular life span, despite successful delivery of telomerase. We thus conclude that this first senescence-like PLB does not require telomere erosion.
Interestingly, some researchers have reported immortalization of NHAs after forced expression of telomerase. However, whereas in this study, we followed several independent hTERT-expressing clones, these previous studies focused on bulk pooled colonies (24 , 26) . For this reason, it cannot be discounted that in these other studies additional spontaneous changes occurred within one or more cells within this population, conferring a growth advantage, which then expanded to become predominant in the culture. This possibility, although not commented on previously, is made all of the more plausible by our data on subsequent PLBs in astrocytes. Our own data on clonal cultures, which clearly demonstrate the failure of hTERT to confer life span extension in any of the 22 NHA clones isolated, strongly implies that M1 senescence is telomere independent and that telomerase expression alone is not sufficient to immortalize primary astrocytes.
NHAs are not the first cell type to show telomere-independent senescence. Although telomere maintenance is ultimately necessary for cellular immortality, several other cell types do not show life span extension after forced expression of telomerase. Instead, before telomere erosion becomes a barrier to cell division, they enter a telomere-independent growth arrest state. Examples include thyroid follicular epithelial cells (25)
, pancreatic islet ß cells (27)
, keratinocytes (28)
, mammary epithelium (29)
, and uroepithelial cells (30)
. Interestingly, where tested, many of these cell types show no alteration in their cellular life span after experimental abrogation of p53 function (31
, 32)
. Unexpectedly, however, our own data (using HPV-16 E6 oncoprotein) reveals that NHA cellular life span is extended, by 12 pd, after abrogation of p53 function. This provides a highly unusual example of a human cell that shows p53-dependent but telomere-independent senescence. Although a p53-dependent telomere-independent PLB has been described in keratinocytes (33)
, it is important to note that in this example the barrier is not the initial (M1) senescence but rather a downstream PLB (with M1 involving instead p16INKA/pRB). Overexpression of wild-type cyclin-dependent kinase 4 in HDFs allows these cells to escape M1 through hyperphosphorylation of pRB (34)
. Our preliminary data (not shown) indicates that NHAs expressing wt CDK4 remain in M1, implying that M1 is a pRB-independent event. Therefore, NHAs are novel for providing a human example of a cell type that naturally undergoes p53-dependent telomere-independent senescence.
A Second Telomere-independent PLB in NHAs Involving p16INK4A.
Abrogation of p53 function confers life span extension but is insufficient to immortalize NHAs. Instead, E6-expressing NHAs eventually reach a second PLB we term MINT. A similar intermediate PLB has been demonstrated in E6-expressing HDFs (12)
, and in this example, MINT is characterized by a 3-fold increase in p16INK4A expression compared with its level in M1. Although a modest increase in p16INK4A is seen in MINT versus M1 astrocytes, the most compelling evidence for a role of p16INK4A in MINT comes from the analysis of rare spontaneous clones that escape MINT. To date, every independent escapee from MINT in NHAs grown in astrocyte medium has been associated with complete loss of detectable p16INK4A expression. Thus, although it remains unclear if up-regulation of p16INK4A is causal in the MINT growth arrest, it seems likely that loss of p16INK4A expression allows cells to reenter proliferation. Analysis of the promoter region of p16INK4A suggests that down-regulation of protein levels are caused by extensive methylation, a phenomenon frequently observed in tumors, including gliomas (35)
.
Another difference is seen between NHAs and other cell types when considering p27KIP1. Our previous studies have shown that primary thyrocytes senesce with high levels of p27KIP1. Expression of an activated form of Ha-RAS allows thyrocytes to bypass senescence and is associated with a reduction of p27KIP1 expression. In contrast, no changes in p27KIP1 expression are seen in NHA.E6 cells that emerge from MINT.
The nature of the cell division counter that triggers MINT in NHAs remains unknown. Although in some fibroblast strains MINT can be bypassed by forced expression of telomerase (36) this does not appear to be the case in NHAs, because MINT still occurs in clones expressing both hTERT and E6. This second PLB thus appears to be telomere-independent, and can be bypassed by spontaneous loss of p16INK4A expression.
A Third PLB in NHAs That Is Telomere Driven.
A third PLB is eventually reached by NHAs that have escaped MINT. Such cells continued to proliferate with no additional intermediate arrest states until they reached a crisis-like state some 30 pd later. Overall, the combined effect of E6 expression and p16INK4A loss can add 40 pd to the life span of NHAs. In parallel experiments using NHA.E6 cells that also express hTERT, escape from MINT was again seen. However, in this case, no subsequent entry into crisis was evident, but rather the cells continued to proliferate indefinitely, and have now undergone sufficient additional divisions (>170 pd) that we consider them immortal. Thus, this third crisis-like PLB appears to be telomere driven and, as such, would mirror the M2 crisis seen in fibroblasts when both the p53 and pRB pathways have been abrogated.
A Representative in Vitro Model of Multistep Human Glioma Development.
Although highly malignant tumors derived from astrocytes can appear to arise de novo, there is commonly a well-defined progression from the potentially curable benign grade I lesion through increasing degrees of malignancy, defined on pathological criteria as grade II (low-grade), grade III (anaplastic), and ultimately grade IV (glioblastoma multiforme).
This is associated with the stepwise accumulation of genetic abnormalities, a common sequence (1) , being, in grade I–IV, respectively: (a) growth factor and/or receptor overexpression (presumably the initiating event); (b) p53 mutation; (c) disruption of the Rb pathway (frequently through loss of p16 expression); and (d) telomerase activation.
As indicated in Fig. 8
, this is provocatively consistent with the sequence of events in our in vitro model, wherein exposure to high levels of growth factor (particularly platelet-derived growth factor and epidermal growth factor) in culture medium initially switches on proliferation, analogous to the endogenous stimulation provided by growth factor (receptor) overexpression in vivo, after which there is a series of PLBs overcome successively by experimental abrogation of p53, spontaneous loss of p16 through promoter methylation, and finally telomerase (hTERT) expression.
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If so, this raises the interesting possibility that at least some of the classic clinicopathological features of malignant progression in this tumor type, increasing growth rate and invasiveness, might simply be explained as secondary results of the overall relaxation of growth control consequent on loss of these inhibitory pathways.
In summary, we propose a tumor model consistent with the molecular genetics of glioma development and a new addition to the existing array of PLBs in human cells, namely a p53 dependent, telomere-independent induced state of growth arrest. The nature of the signal that activates p53 to impose this barrier is unclear.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 This work was supported by a Cancer Research UK studentship (to R. J. E.) and program grant (to D.W-T., D. K.). 
3 To whom requests for reprints should be addressed, at Department of Pathology, University of Wales College of Medicine, Heath Park, Cardiff CF14 4XN, United Kingdom. Phone: 44-29-20-744849; Fax: 44-29-20-745121; E-mail: Jonescj{at}cf.ac.uk
4 The abbreviations used are: NHA, normal human astrocyte; PLB, proliferative life span barrier; HDF, human diploid fibroblast; pd, population doubling; HPV, human papillomavirus; SA ß-gal, senescence-associated ß-galactosidase; BrdUrd, bromodeoxyuridine; LI, labeling index; TRAP, telomeric repeat amplification protocol; TRF, terminal restriction fragment.
Received 2/28/03. Revised 5/23/03. Accepted 6/ 4/03.
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) were routinely cultured over a period of 75 days. During that time they reached replicative senescence (M1) after 55 days.
HindIII digest; Lanes 1–3, NHA DNA extracted from cells at 8, 16, and 20 (senescent) pd, respectively. B, TRAP assays of NHA infected with hTERT, the catalytic subunit of telomerase, or a vector control. Cell extracts (5000 cell equivalents/reaction) were assayed for telomerase activity using the TRAP assay. C, control telomerase-positive 293 cell extract. When extracts are pretreated at 85°C for 10 min, telomerase activity is abolished (negative control). All samples successfully amplified a control substrate (internal telomerase amplification standard; *). Sample 1, NHA.hTERT-pooled colonies. Sample 2, NHA.puro pooled colonies. C, Bar chart showing the total life span of NHA.hTERT (
) in vitro. Cells were infected at pd 13. D, phase contrast photomicrographs of NHA.puro (i) and NHA.hTERT at pd 20 (ii).
) and two individual subclones infected with HPV-16 E6 and hTERT (
, 
), which continue to proliferate and can be considered immortal. B, immunoblot of NHA expressing HPV-16 E6 at MINT and two representative NHAE6.hTERT immortal subclones at pd 88 and 65 probed with anti-p16INK4A (DCS 50) antibodies (i). The level of protein loading was determined by India ink staining (ii). C, down-regulation of p16INK4A expression is associated with promoter methylation. i, unmethylated primer set was used to amplify bisulphite modified unmethylated p16INK4A DNA. ii, methylated primer set was used to amplify methylated bisulphite-modified p16INK4A DNA. Lane 1, (i) methylated and (ii) unmethylated control DNA; Lane 2, NHA pd 6; Lane 3, NHA.E6 clone pd 43, and Lane 4, NHAE6.hTERT clone pd 145.