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Short Communication |
From the Department of Pathology,* Georgetown UniversityMedical Center, Washington, District of Columbia; and MillenniumPharmaceuticals, Incorporated,Cambridge, Massachusetts
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Abstract |
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 Top Abstract Materials and MethodsResultsDiscussionReferences |
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HPV-induced immortalization is closely linked to the increased activity of cellular telomerase, a ribonucleoprotein enzyme that synthesizes telomeric DNA.10-12 Indeed, overexpression of the hTERT component of this enzyme complex can induce immortalization of many human cell types,13,14 apparently by preventing the progressive loss of telomere length during DNA replication. The telomerase activity of HPV-immortalized cells seems to result at least in part from the expression of the HPV E6 protein, because the HPV E6 protein can induce telomerase in keratinocytes that lack detectable activity.15 More recent studies demonstrate that the induction of telomerase activity correlates directly with the E6-enhanced transcription of the hTERT promoter.16,17 E6-mediated activation of telomerase has also been shown to be independent of p53 degradation,15 eliminating the possibility that this transcription factor is a determinant of hTERT induction.
Although E6 can induce telomerase activity, it is clear that there are distinct cellular mechanisms operative during cell immortalization and transformation because E6-negative cervical carcinomas (as well as the vast majority of non-HPV-related tumors) also overexpress telomerase.18,19 To determine whether events in addition to E6 expression contribute to hTERT induction during HPV-mediated cellular immortalization, we performed the following experiments. Primary cervical cells were transduced with retroviruses containing the HPV-16 E6/E7 genes and then passaged in serum-free medium until cell immortalization had been achieved. At selected passages, the cervical cells were analyzed by quantitative techniques to correlate the expression of the E6 and E7 oncogenes with the level of hTERT mRNA and telomerase activity. Our results confirm that E6 does induce the rapid appearance of hTERT mRNA and telomerase in early-passage cervical cells, but that there is a subsequent progressive and profound increase in hTERT and telomerase expression during cell passaging that is independent of E6 protein levels.
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Materials and Methods |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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Primary human ectocervical keratinocytes were derived from fresh cervical tissue obtained after hysterectomy for benign uterine diseases. Standard trypsinization procedures were used to isolate the keratinocytes, which were cultured in serum-free keratinocyte medium supplemented with 50 µg/ml of bovine pituitary extract and 26 ng/ml of recombinant epidermal growth factor (Life Technologies, Inc., Grand Island, NY).20 Primary cultures were infected at passage 4 with high-titer LXSN retroviruses expressing either the HPV-6 or HPV-16 E6/E7 genes, with the HPV-16 E6 gene containing an AU1 epitope at the 3'-terminus to facilitate immunodetection.16 Control LXSN retroviruses expressed only the neomycin resistance gene. After infection, cells were selected with 50 µg/ml of G418 for 5 days and were subcultured at least once before extraction of RNA. All subsequent passages were performed at a split ratio of 1:4.
Immunoprecipitation and Western Blotting
Cell lysate (700 µg) was reacted with AU1 monoclonal antibody (BabCO) to immunoprecipitate the epitope-tagged HPV-16 E6 protein, or with ED17 monoclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA) to immunoprecipitate the HPV-16 E7 protein. The immunoprecipitated proteins were separated in 1.5-mm polyacrylamide Tris-glycine minigels (Invitrogen, Carlsbad, CA) and transferred to Immobilon-P membranes (Millipore, Bedford, MA). For E6 immunoblot analysis, the membrane was labeled in washing buffer (0.5% Triton X-100, 140 mmol/L NaCl, 10 mmol/L Na3PO4) in the presence of 2% bovine serum albumin with 0.4 µg/ml of HPV-16 E6 goat polyclonal antibody (N-17, Santa Cruz Biotechnology) for 90 minutes. For E7 analysis, the membrane was reacted with HPV-16 E7 mouse monoclonal antibody (ED17, Santa Cruz Biotechnology) in phosphate-buffered saline (PBS) containing 2% bovine serum albumin. After reaction with either alkaline phosphatase-conjugated mouse anti-goat IgG (GT34) antibody (Sigma Chemical Co., St. Louis, MO) or goat anti-mouse IgG antibody (Tropix, Bedford, MA) for 90 minutes, the E6 and E7 proteins were visualized with a chemiluminescent substrate (Tropix).
Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR)
Total RNA was extracted from ectocervical keratinocytes grown in 150-mm tissue culture dishes to 80% confluence using Trizol reagent according to instructions of the manufacturer (Life Technologies, Inc.). HeLa cells were used as a positive control. First-strand cDNA was synthesized as described previously21 with some modifications using 5 µg of RNA and 0.5 µg of oligo-dT by following the instructions of Superscript First-Strand Synthesis System for RT-PCR (Life Technologies, Inc.). Ten percent of the RNase H-treated cDNA product was subjected to PCR reaction for hTERT determination in a total volume of 50 µl containing 10 µmol/L sense primer (5'-CGGAAGAGTCTGGAGCAA-3') and 10 µmol/L anti-sense primer (5'-GGATGAAGCGGAGTCTGGA-3') as described in the manufacturer’s protocol. Initial denaturation for 3 minutes at 94°C was followed by 30 cycles of PCR amplification (94°C for 45 seconds, 57°C for 60 seconds, 72°C for 60 seconds). The same cDNA-samples were applied to PCR amplification for 36B4, a ribosomal phosphoprotein selected as a riboprobe for endogenous RNA control.22 To demonstrate purity of extracted RNA, PCR was performed without adding reverse transcriptase to samples. PCR products were separated by electrophoresis in 10% polyacrylamide gels.
Quantitative RT-PCR
DNase-treated total RNA (1 µg) was reverse-transcribed using
random hexamers and oligo-dT from the Superscript First-Strand
Synthesis System for RT-PCR (Life Technologies, Inc.). A control
reaction with no reverse transcriptase was performed to check for
contamination by genomic DNA. Multiplex PCR reactions contained
first-strand cDNA, recommended amounts of 18S rRNA Predeveloped Taqman
Assay Reagent and Taqman Universal PCR Master Mix (Applied Biosystems,
Foster City, CA), 900 nmol/L of sense primer
(5'-AAAGCATTGGAATCAGACAGCA-3') 900 nmol/L of anti-sense
primer(5'-TTCACAATCGGCCGCAG-3'), and 250 nmol/L
FAM-labeled probe (5'-CCTGCTGACGTCCAGACTCCGCTT-3'). PCR
reactions were performed in duplicates on the ABI Prism 7700 sequence
detector (50°C for 2 minutes, 95°C for 10 minutes, 40 cycles of
95°C for 15 seconds, followed by 60°C for 1 minute). Data were
analyzed with Sequence Detector v1.6.3. software (Applied Biosystems).
18S RNA was used as a housekeeping gene to normalize amounts of RNA.
For each reaction, expression was calculated as
2-
Ct, where Ct is the
difference between the Ct for the gene of
interest and the Ct for 18S RNA. The average
expression for the duplicates is reported with the SD.
Telomerase Activity Assay
Human cervical keratinocytes were grown in 100-mm tissue-culture dishes to 80% confluence, harvested by trypsinization, washed in cold PBS, and transferred to a microfuge tube. Cell pellets were lysed for 30 minutes on ice in 400 µl of telomeric repeat amplification protocol (TRAP) buffer (0.5% Chaps, 10 mmol/L Tris, pH 7.5, 1 mmol/L MgCl2, 1 mmol/L EGTA, 5 mmol/L ß-ME, 10% glycerol, 0.1 mmol/L 4-(2-amino-ethyl)benzene-sulfonyl fluoride hydrochloride (AEBSF). Lysates were centrifuged at 14,000 x g for 5 minutes at 4°C, the supernatant was transferred to a new tube, and protein concentration was determined (Bio-Rad, Richmond, CA). A TRAP assay18 was performed on 5 µg, 1.5 µg, 0.15 µg, and 0.015 µg of protein lysates as described, with some modifications. Lysates were incubated for 30 minutes at room temperature in a 50-µl reaction volume containing 1x PCR buffer (20 mmol/L Tris, pH 8.4, 50 mmol/L KCl), 100 ng telomerase substrate primer (5'-AATCCGTCGAGCAGAGTT-3'), 50 µmol/L each deoxynucleoside triphosphate (dATP, dTTP, dGTP, dCTP), 1.5 mmol/L MgCl2, and 0.5 µg T4 gene protein (Boehringer Mannheim, Indianapolis, IN). After initial denaturation for 4 minutes at 94°C 100 ng of downstream primer (5'-CCCTTACCCTTACCCTTACCCTAA-3') and 2.5 U of Taq DNA polymerase were added, followed by 31 cycles of PCR amplification (94°C for 30 seconds, 50°C for 30 seconds, 72°C for 45 seconds). HeLa cells (HPV-18-positive cervical cancer cell line) served as a positive control and IMR90 cells (normal embryonic lung fibroblast cell strain) as a negative control for telomerase activity, respectively. Twenty percent of the PCR products were separated on 10% nondenaturing polyacrylamide gels and visualized using the Gelcode color silver-staining kit (Pierce, Rockford, IL).
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Results |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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Primary cultures of human ectocervical keratinocytes were established from fresh cervical tissue as in Materials and Methods. After the fourth passage, cells were infected with three different LXSN retroviruses: an empty vector LXSN virus (control), LXSN containing low-risk HPV-6 E6/E7, and LXSN containing high-risk HPV-16 E6/E7. Expression of the HPV-6 and HPV-16 E6/E7 oncogenes by these retroviruses was confirmed by quantitative PCR and Northern blot analysis (data not shown). As expected, the LXSN control cells exhibited a very short life span, an average of eight population doublings. After transduction with low-risk HPV-6 E6/E7, cell proliferation was slightly extended to an average of 10 population doublings. In contrast, cells transduced with the high-risk HPV-16 E6/E7 genes proliferated indefinitely and have been maintained in vitro for more than 200 population doublings without evidence of a detectable crisis period. Two additional, independent experiments performed with this same protocol have given similar results.
High-Risk E6/E7 Oncoprotein Levels Remain Stable during in Vitro Cell Passaging
Generating immortalized cervical cells via repeated cell passaging
(as above) could potentially result in the selective outgrowth of a
subset of E6/E7-transduced cells. It was possible, therefore, that
late-passage cultures might consist of cells with a selective growth
advantage because of higher levels of viral oncoprotein expression. To
evaluate this possibility, we developed an
immunoprecipitation/immunoblotting technique that allowed us to measure
the amount of epitope-tagged E6 as well as E7 oncoprotein in cervical
cells and to determine whether there were indeed variations in
oncoprotein levels that were dependent on passage number (Figure 1)
. Our results were somewhat unexpected.
The levels of the E6 and E7 proteins showed virtually no change from
early passage (passage 4) through immortalization (passage 30).
Titration of cell lysates used in the immunoprecipitation demonstrated
that the detected levels of E6 and E7 proteins were within the linear
range for the immunoprecipitation/Western technique (data not shown).
Theseresults are consistent with the hypothesis that the transduced,
selected cells were uniformly expressing E6/E7 and that these levels of
expression did not change at later times. In addition, our data suggest
that there is no apparent growth advantage for cells containing higher
levels of E6/E7 than detected at early times.
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If the induction of hTERT mRNA (catalytic subunit of telomerase)
and telomerase activity required only E6 expression, then it would be
anticipated that they, like E6, would also remain constant throughout
cell passaging. To examine this possibility, we monitored hTERT mRNA
expression by reverse transcriptase-PCR amplification as well as
quantitative PCR amplification. RNA was isolated from control,
low-risk, and high-risk E6/E7-transduced cervical cells,
reverse-transcribed to generate cDNAs, and subjected to PCR (30 cycles)
with the hTERT primers indicated in Materials and Methods (Figure 2A)
. The expression of hTERT increased
progressively with cell passage, with the highest levels being detected
at passage 30. Analysis of the samples with a 36B4-specific riboprobe
confirmed the uniformity of the cDNA reactions. Thus, the apparent
level of hTERT mRNA increased with successive passages. To accurately
measure this progressive increase in hTERT mRNA, we used a quantitative
PCR methodology. Cellular RNA was reverse-transcribed as described in
Materials and Methods to generate a first-strand cDNA preparation.
Subsequent multiplex amplifications were performed with the Taqman
assay system and analyzed with the ABI Prism 7700 sequence detector
(Figure 2B)
. 18S RNA was also amplified as a control housekeeping gene
and used to normalize the hTERT data. Similar to the initial PCR
results shown in Figure 2A
, the quantitative PCR experiments
demonstrated that cells transduced with the HPV-16 E6/E7 genes
exhibited a 25-fold increase in hTERT mRNA at late passages (passage
20) when compared to the same cells at passage 1. Intermediate passage
cells (passage 6) showed an intermediate level of hTERT increase
(threefold increase compared to passage 1). Thus, two independent
assays have verified that hTERT expression increases progressively with
cell passaging. Similar results were obtained from a second series of
transduced cervical cells.
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To evaluate whether the increased levels of hTERT mRNA were
reflected in corresponding increases in telomerase activity, we
performed a highly sensitive PCR-based assay referred to as the
telomeric repeat amplification protocol (TRAP assay). Five µg of
protein was used to measure telomerase at the same time points at which
we measured hTERT mRNA (Figure 3A)
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Telomerase activity was clearly not detectable at any time in LXSN
control cells or in ectocervical cell lines transduced with low-risk
HPV-6 E6/E7. These findings parallel the lack of hTERT mRNA levels in
these cells. In contrast, viral transduction with high-risk HPV-16
E6/E7 oncogenes immediately induced telomerase activity in ectocervical
cells. This activity increased progressively throughout cell passaging,
similar to the increases in hTERT mRNA.
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, 1.5 µg (Figure 3B)
, 0.15
µg (Figure 3C)
, 0.015 µg (Figure 3D)
. Cells with the highest
telomerase activity will exhibit a positive TRAP assay even at the
higher dilutions. Because we observed similar TRAP assay signals for
early-passage cells (passage 4) at high protein concentration (5
µg/reaction) compared to late-passage cells (passage 30) at low
protein concentration (0.015 µg/reaction), we can estimate that
late-passage cells have 
300-fold more telomerase activity than
early-passage cells. These assays have been repeated at least three
times for each cell line and passage number with virtually identical
results. |
Discussion |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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As anticipated from previous studies,15,16,23 our results show that the E6 oncoprotein rapidly induces hTERT expression and telomerase activity. In this investigation, we have used real-time PCR to obtain reliable quantitation of hTERT mRNA levels. Our studies demonstrate clearly that, despite stable expression of the E6 and E7 proteins, there is a progressive and dramatic increase in hTERT mRNA expression and telomerase activity during cell passaging in vitro. The level of hTERT and telomerase activity in cervical cells, therefore, is not directly proportional to the level of E6 expression. This observation could result from either of three distinct mechanisms:
First, telomerase expression, induced uniformly in all cells by E6 at early times, might be further amplified by cellular mechanisms during the process of immortalization. In the current in vitro system in which we do not observe a distinct crisis phase of cell proliferation, this would suggest that cellular immortalization is a nonclonal event with many different cells becoming immortal, and with all exhibiting higher telomerase activity at late times. The increase of telomerase activity observed in late-passage or immortalized cells would therefore be a composite of E6-dependent and E6-independent mechanisms.
Second, telomerase activity might be imprinted or established at an early time by E6, with some cells expressing very high levels of telomerase and other cells expressing lower levels. Cell passaging would then select for the subpopulation of transduced cells that had high telomerase activity. This second proposed mechanism is consistent with the experimental findings of Kiyono and colleagues12 who found that clones of E6/E7-transduced cells varied considerably in their telomerase activity. They hypothesized that only cells with higher telomerase activity, and consequently longer average telomeres, were selected for continuous proliferation.
Third, early-passage keratinocytes represent a mixed population of stem cells, replicative cells, and differentiating cells. The stem cells, although representing only a very small percentage of the total number of cervical cells, are believed to be responsible for the detectable levels of telomerase observed in early-passage cervical cells.24 Later passage cells lack this activity. Thus, it is possible that E6 induces telomerase in all cervical cells, but that the induction in stem cells results in higher levels of telomerase (because of the combined action of E6 and existing cellular factors) and the selective outgrowth of these cells produces the progressive increase in hTERT and telomerase.
Although one study has reported that telomerase activity can only be detected in late-passage HPV-immortalized keratinocytes,25 several laboratories, including our own, have shown that telomerase is rapidly induced in nonimmortalized keratinocytes by E6.15-17,26 Our analysis of hTERT expression by real-time PCR further substantiates this early induction in nonimmortal cells. Indeed, this sensitive quantitative PCR technique can detect low-level hTERT mRNA expression at the earliest time after transduction with E6.
Although hTERT mRNA and telomerase activity are increased in the large majority of cervical cancers,10,11,27,28 telomerase activity is probably not applicable as a prognostic factor in early-stage cervical cancer.28 For example, telomerase activity has also been detected in normal cervical tissues29,30 as well as in benign cervical lesions30-32 and is independent of the presence of HPV.33 It is possible, however, that quantitative detection of hTERT mRNA by real-time PCR could be useful diagnostically. The absolute levels of hTERT mRNA in normal cervical tissue and low-risk HPV-induced neoplasms would be expected to be much lower than those found in high-risk HPV-expressing lesions.
In summary, the mechanism responsible for the progressive increase of hTERT gene expression (and concomitant telomerase activity) during cellular immortalization is unknown. Identifying the relevant cellular changes involved in this progression should assist in understanding and diagnosing the tumorigenic conversion of HPV-induced lesions.
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Acknowledgements |
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Footnotes |
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Supported by the National Institutes of Health (grant no. R01CA53371 to R. S).
Accepted for publication January 18, 2002.
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References |
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