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From the Program in Cancer Biology, Fred Hutchinson Cancer Research Center, Seattle, Washington
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Telomerase activation has been demonstrated to be a rather late event during multistage carcinogenesis of various cancers.6,8,11-13 In cervical cancer, human papillomavirus (HPV) infection is considered to be the initiating event of carcinogenesis. Although the HPV-16 E6 gene product induces the activation of telomerase during immortalization of skin or cervical epithelial cells,14 a recent study indicated that cervical carcinomas have high telomerase activity irrespective of the detection of HPV;15 however, it is unclear when telomerase is activated during the progression of cervical neoplasia. Based on the evidence that expression of the hTERT gene reflects the activation of telomerase,9,10 the in situ hybridization technique, using tissues of widely distributed stages of cervical carcinoma, was employed in this study to address this question. Furthermore, because a recent study16 has shown an association between telomerase activity and proliferative activity, we also estimated proliferative activity by immunohistochemistry in the tissues and compared the level of hTERT expression.
The in situ results demonstrate that expression of hTR and hTERT is focally up-regulated at the regions of invasive and in situ carcinoma of cervical tissues. We also show that both genes are expressed in normal epidermal layers and cultured keratinocytes and that the levels of these RNAs, as measured by RNase protection assay, do not reflect the levels of telomerase activity in cervical tissues and cultured cells.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Paraffin-embedded tissues were obtained from patients with cervical cancer. Forty-eight cases of squamous cell carcinoma (40 cases of invasive carcinoma and 8 cases of microinvasive carcinoma), 24 cases of adenocarcinoma (9 cases of invasive carcinoma and 15 cases of carcinoma in situ), and 1 case of adenosquamous carcinoma were tested. Among these samples, there were 9 cases with regions of cervical intraepithelial neoplasia (CIN3) and 19 cases with cervical intraepithelial neoplasia 1 or 2 (dysplasia) regions. Dysplastic or metaplastic changes of glands were accompanied by 15 cases of in situ adenocarcinoma. Normal cervical tissues were collected from patients who underwent hysterectomy due to leiomyoma of uteri (3 cases), adenomyosis of uteri (1 case), and ovarian cancer (1 case).
We obtained matched cervical cancer and adjacent nontumor tissues from seven patients with invasive cervical cancer (6 cases of squamous cell carcinoma and 1 case of adenosquamous carcinoma) and normal cervix from four patients with leiomyoma of uteri, who gave informed consent for this study. The samples were immediately frozen and stored at -80°C until use for telomeric repeat amplification protocol (TRAP), reverse transcription-polymerase chain reaction (RT-PCR), and RNase protection assays. Primary (mortal) cervical keratinocytes (from two different sources: passages 5 and 6), foreskin keratinocytes (from five different sources: passages 2, 2, 2, 4, and 5), immortalized cervical keratinocytes transfected with HPV-16 E6/E7 genes (E6/E7: passage 57), maintained as previously described,17 and HeLa cervical cancer cells were passaged and harvested simultaneously under subconfluent conditions to analyze the correlation between telomerase activity and its RNA expression.
hTR and hTERT Clonings
The hTR and hTERT clones were generated as follows: cDNA was synthesized with a SuperScript II kit (Life Technologies Inc., Grand Island, NY) using 3 µg of total RNA isolated from HeLa cells. The cDNA was subjected to PCR amplification using primers for the hTR gene (20-bp published sequence7 from 1 to 20 and 19-bp sequence from 176 to 194; GenBank accession no. U86046) and primers for the hTERT gene (23-bp published sequence9 from 1682 to 1704; 23-bp sequence from 2036 to 2058; GenBank accession no. AF015950). The resulting cDNA fragments were inserted into pBlueScript SK+ (Stratagene, La Jolla, CA) and pGEM-T Easy Vectors (Promega, Madison, WI), respectively. Sequences were confirmed using cycle sequencing with dye-labeled terminators.
In Situ Hybridization
Digoxigenin-labeled hTR and hTERT anti-sense probes were generated from HindIII- and SpeI-linearized plasmids according to the protocol of Riboprobe in vitro Transcription Systems (Promega) using digoxigenin RNA labeling mixture (Boehringer Mannheim, Indianapolis, IN) and sense-probes from BamHI- and NcoI-linearized plasmids using T7 and SP6 RNA polymerase, respectively. Formalin-fixed, paraffin-embedded tissue sections (5-µm thickness) were deparaffinized in xylene and graded alcohol series and pretreated with Target Retrieval Solution (DAKO, Carpinteria, CA) at 95°C for 20 minutes and 0.01% pepsin in 0.2 N HCl solution at room temperature for 20 minutes. The tissues were then fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS) at 4°C for 5 minutes, acetylated, dehydrated through graded alcohol series and air-dried. Digoxigenin-labeled anti-sense and sense probes were denaturated at 85°C for 3 minutes and placed over the tissues. After hybridization at 47°C overnight, the slides were washed in 50% formamide/2x standard saline citrate at 52°C for 30 minutes, treated with RNase A in TNE solution at 37°C for 30 minutes, and washed in 2x standard saline citrate and 0.2x standard saline citrate solution twice at 50°C for 20 minutes each. After the incubation in 2% blocking solution (Boehringer Mannheim), the tissues were incubated with a sheep monoclonal anti-digoxigenin antibody (Boehringer Mannheim) diluted 1:500 in 0.1 mol/L Tris-HCl, pH 7.5/0.15 mol/L NaCl buffer at room temperature for 30 minutes. After washing with 0.1 mol/L Tris-HCl, pH 7.5/0.15 mol/L NaCl buffer, the color detection was carried out by incubating with 5-bromo-4-chloro-3-indolyl phosphate/nitroblue tetrazolium solution containing levamisole (Vector Laboratories, Burlingame, CA) overnight. The sections were counterstained with fast green FCF (Aldrich Chemical Corp., Milwaukee, WI) and then mounted with Crystal Mount (Biomeda Corp., Foster City, CA). Differential diagnosis of the stages in adjacent regions and evaluation of signal intensity were independently confirmed by Dr. Ming-Gang Lin (Fred Hutchinson Cancer Research Center).
Immunohistochemistry
Formalin-fixed, paraffin-embedded sections (4-µm thickness) were deparaffinized and rehydrated through xylenes and a series of graded alcohols. Immunohistochemistry was carried out with an automated processor (TechMate 1000, Ventana Medical Systems, Inc., Tucson, AZ). The tissue sections were subjected to steaming in 10 mmol/L citrate buffer, pH 6.0 for 20 minutes and allowed to cool at room temperature. Anti-Ki-67 clone MIB-1 (Immunotech, Westbrook, ME) was applied for 1 hour at room temperature at a concentration of 1:200 in PBS containing 1% bovine serum albumin and 0.03% Tween 20 in the automated stainer, followed by an incubation with a secondary biotinylated antibody, avidin-biotinylated peroxidase complex, and 3,3-diaminobenzidine with NiCl2 enhancement as a substrate. The slides were counterstained with methyl green and mounted with Histmount (National Diagnostics, Atlanta, GA).
RT-PCR
Total RNA isolated from frozen tissues and cultured cells using RNeasy total RNA kit (Qiagen, Chatsworth, CA) was reverse transcribed by random primer and Super-Script II reverse transcriptase. The resulting cDNA was subjected to PCR with the primers as follows: hTERT, 5'-CGGAAGAGTGTCTGGAGCAA and 5'-GGATGAAG-CGGAGTCTGGA,10 hTR, 5'-GGGTTGCGGAGGGTGGG-CCT and 5'-ACGGGCCAGCAGCTGACAT, and 36B4 mRNA18 as an internal control, 5'-TGGCAGATGGATCAGC and 5'-AGTGTCTCTCTGCA. The amplification reactions were performed with an initial incubation step at 94°C for 3 minutes followed by 31 cycles at 94°C for 45 seconds, 57°C for 1 minute, and 72°C for 1 minute for hTERT; 30 cycles at 94°C for 1 minute, 60°C for 45 seconds, and 72°C for 45 seconds for hTR; and 30 cycles at 94°C for 1 minute, 50°C for 1 minute, and 72°C for 1 minute for 36B4, with a final incubation at 72°C for 7 minutes for all cDNAs. The reaction products were subjected to electrophoresis in 1.8% agarose gel and visualized by ethidium bromide staining.
RNase Protection Assay
Radiolabeled hTERT (345-bp length) and ß-actin
(340-bp length) probes that protect 285- and 200-bp fragments in RNase
protection assay were synthesized from AspI- and
HindIII-linearized plasmids (ß-actin, a gift from Dr.
Scott Foster, Fred Hutchinson Cancer Research Center) according to the
protocol of Riboprobe in vitro Transcription Systems
(Promega) using [
-32P]UTP and T7 RNA polymerase,
respectively. The RNase protection assay was carried out using the RPA
II kit (Ambion, Austin, Texas) according to the manufacturer's
protocol. Briefly, total RNA samples (20 µg from tissues and 30 µg
from cultured keratinocytes) and radiolabeled hTERT
(specific radioactivity, 0.9 to 1.0 x 109
cpm/µg)
and internal control ß-actin (1.6 to 2.2 x 107
cpm/µg) probes were coprecipitated in ethanol, resuspended in
hybridization buffer, hybridized at 42°C overnight, and then treated
with RNase A and T1. Samples were precipitated, resuspended in loading
buffer and analyzed on 5% denaturating polyacrylamide gel, after which
the gel was dried and exposed to X-OMAT film (Kodak, Rochester, NY)
at -80°C.
TRAP Assay
Frozen tissues were homogenized on ice using sterile dounce
homogenizer with TRAP lysis buffer as described.3
The
homogenized solution was centrifuged at 14,000 x g for
30 minutes at 4°C, and the supernatant was collected and stored at
-80°C until used for the TRAP assay. The TRAP assay was performed as
described.3
Telomerase synthesizes telomeric repeats onto a
nontelomeric [
-32P]ATP end-labeled repeat TS
(5'-AATCCGTCGAGCAGAGTT-3'). The reaction was carried out using 5 µg
of protein extractions in 50 µL of reaction mixture, to which an
internal telomerase assay standard was added for estimation of the
levels of telomerase activity and identification of any false-negative
samples that contain Taq polymerase inhibitors. These products are
specifically amplified by PCR with the downstream primer CX
(5'-(CCCTTA)3CCCTAA-3') and upstream labeled primer TS. An
aliquot of the reaction was run on a 10% nondenaturating acrylamide
gel, which was dried and exposed to X-OMAT film overnight.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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As shown in Figure 1
, expression of
hTR RNA and hTERT mRNA was co-localized not only
in carcinoma regions but also in normal to dysplastic epidermal layers
and dysplastic/metaplastic glandular epithelia in almost all cases. In
histologically normal epidermal layers, hTR and
hTERT expression was limited to basal and a couple of
suprabasal cell layers. Dysplastic epidermis with expanded cell layers
expressed both signals, and the level of expression appeared to be
higher than in histologically normal epidermis. In some cases of
squamous cell carcinoma, higher levels of expression of both signals
were detected compared with adjacent noncancerous epidermal layers,
whereas there was no apparent difference in signal intensity in other
cases between the regions. In histologically normal glands, in
contrast, there was a faint signal or no signal of hTR and
hTERT. Both signals were detected in dysplastic/metaplastic
glands, and in in situ and invasive adenocarcinomas. The
level of expression was heterogenous within the carcinoma regions in
the same tissue. Among stromal cells, both signals were mainly present
in lymphocytes of normal and cancer tissues, in which signal intensity
was similar, although a precise quantitative comparison of in
situ signal was difficult. An hTR but not
hTERT signal was detected in endothelial cells.
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High proliferative activity estimated by positivity of Ki-67
>50% was seen in carcinoma regions (74.6%; 44 of 59 cases). A
similar result (Ki-67 >50%) was observed in dysplastic/metaplastic
epidermal and glandular regions (67.6%; 23 of 34 cases), where
hTERT signal was increased compared with normal tissues.
Even in histologically normal epidermal layers, the intense signal was
detected in the regions with high proliferative activity, as
represented in Figure 2
, whereas the
incidence of Ki-67 >50% was fewer (21.1%; 4 of 19 cases).
Up-regulation of hTR expression was also accompanied by the
high proliferative activity in many cases (data not shown).
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We also examined hTERT and hTR expression
using frozen cervical cancer and adjacent nontumor tissues and cultured
keratinocytes by RT-PCR. Some of the cancer tissues showed an increased
expression of hTERT and hTR compared with the
control tissues, but almost all nontumor tissues and some primary
keratinocytes expressed both RNAs (Figure 3)
. To quantitate hTERT
expression more accurately, RNase protection assay was carried out
(Figure 4A)
. Nontumor control tissues and
primary cultured keratinocytes (four of seven) expressed levels of
hTERT similar to those of cancer tissues, HPV-16 E6/E7
immortalized keratinocytes, and HeLa cells. Although it was not
possible to detect hTERT expression in tissues by Northern
blot analysis because of limited tissue availability and the low level
of expression, the same level of hTERT-specific transcript
was identified in primary and immortal keratinocytes (data not shown).
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Telomerase enzyme activity was detected in cancer tissues and
immortal keratinocytes by TRAP assay but was either not detectable or
significantly lower in noncancer tissues and primary keratinocytes, as
depicted in Figure 4B
. Comparison of the results in RNase protection
and TRAP assays demonstrated no consistent association between the
enzyme activity and total level of hTERT expression in
cervical tissues and cultured keratinocytes.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Activation of telomerase has been shown to be an important event for immortalization and carcinogenesis. Up-regulation of hTR expression has been shown to be an early event in various cancers.6,8,13 In cervical carcinoma, the relation between up-regulation of hTR and hTERT expression and the progression of disease has not been investigated, although Soder et al5 have demonstrated amplification of the hTR gene on chromosome 3q in cervical cancer. In this study, signals of the both RNAs were focally increased in carcinoma regions, dysplastic/metaplastic epidermis, and glands. These results indicate that not only hTR but also hTERT up-regulation are early events during cervical carcinogenesis. hTR expression has previously been demonstrated to correlate with proliferation index in astrocytomas19 and ependymomas.20 The expression of hTERT seems to be coordinated with proliferation activity, because dysplastic/metaplastic epidermal and glandular cells as well as carcinoma cells possess high proliferative activity, whereas normal epidermal epithelia exhibit less activity. On the other hand, correlation between Ki-67 staining and hTERT signal was not 100% in vivo, and not all primary keratinocytes expressed detectable hTERT when proliferating under subconfluent conditions in vitro, suggesting possible inhibitory factors or rapid degradation of hTERT. Recently, telomerase catalytic subunit gene (hTERT) expression was reported to reflect the enzyme activity in cancer and immortalized cells.9,10 RNase protection and RT-PCR assays in this study, however, showed that some of control tissues expressed similar levels of hTERT mRNA to those in cancer tissues, which may indicate that some portion of these tissues might contain dysplastic or hyperplastic epithelia in which hTERT expression is up-regulated. Nevertheless, high telomerase activity is specific in cancer tissues, and up-regulation of hTERT expression in the control tissues does not indicate a high level of enzyme activity. Increased signals of hTERT detected by the in situ hybridization method were focally distributed within carcinoma regions. An increase in the proportion of lymphocytes to epithelial cells could mask a difference in hTERT levels between subsets of carcinoma and normal epithelial cells, resulting in a discrepancy between the mRNA level and telomerase activity. However, it is difficult to explain the cultured cell result in which primary (mortal) keratinocytes have no or weak telomerase activity despite a similar level of hTERT expression as immortal keratinocytes. The intensity of the in situ hybridization signal was not obviously different between primary and immortal keratinocytes, although heterogeneity of the signal was present in both, because keratinocytes are difficult to synchronize (data not shown). Primary keratinocytes are highly proliferative in vitro until senescence or terminal differentiation, as are most dysplastic epidermal cells in vivo. Proliferation activity seems to be more tightly correlated with hTERT expression than is telomerase activity, at least in keratinocytes, although this enzyme activity was recently reported to be a proliferation biomarker.16 These results suggest a second level of regulation in telomerase activity besides hTERT gene expression.
Among normal tissues, except germline tissues, colon and small intestine tissues express hTERT mRNA, as shown by Meyerson et al.9 A recent study21 has also shown that its expression is detected by RT-PCR in many kinds of normal tissues in addition to the above tissues. Telomerase is not activated in the majority of normal somatic tissues,3 suggesting the possibility that the hTERT gene product may be modulated during the process of translation and posttranslation in these tissues. It is also possible that splicing of hTERT mRNA differs between normal and cancer tissues as evidenced by Kilian et al,22 or a difference in minor nucleotide sequence exists between them, given that a mutation of hTERT abrogates its activity in vitro.23 The Ro60 protein as recently reported by Ramakrishnan et al24 possibly forms a complex with telomerase and modulates its function. Overexpression of the anti-apoptosis protein bcl-225 and phosphorylated status26 have been shown to activate telomerase in cancer cell lines, factors that might contribute to the difference between cervical cancer and normal tissues. Epidermal cells in the suprabasal layers of normal cervical tissues are differentiated; therefore, the enzyme activity could be inhibited, even though hTERT mRNA is expressed, because differentiation has an inhibitory effect on telomerase activity.27 The relation between telomerase activation and deregulation of cell cycle regulator proteins has been demonstrated in breast cancer.28 Although direct interaction between the p53 pathway and telomerase activation has not been noted in HPV-16 E6/E7-immortalized cervical keratinocytes,14 we have confirmed aberrant staining of other cell cycle regulator proteins in the cervical cancer tissues (data not shown).
We conclude that up-regulation of hTR and hTERT are early events during carcinogenesis and that additional regulation, such as modulation of hTERT gene product or coordination with other proteins that currently are not identified, may participate in the activation of telomerase in cervical cancer.
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Acknowledgements |
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Footnotes |
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Supported by National Institutes of Health Grant CA 42792 (to JKM). KN was partly supported by a fellowship of Sankyo Science Foundation, Japan.
Accepted for publication June 5, 1998.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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