| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Advances in Brief |
Cancer Research Campaign Department of Medical Oncology, University of Glasgow, Cancer Research Campaign Beatson Laboratories, Bearsden, Glasgow G61 1BD, United Kingdom [S. F. H., L. A. B., S. B., W. N. K.], Department of Gynaecology, University Hospital, 9700 RB, Groningen, the Netherlands [G. B. A. W., A. G. J. v. d. Z.], and Department of Pathology, G4 0SF, University of Glasgow, Scotland, United Kingdom [J. J. G.]
 |
ABSTRACT |
|---|
 Top ABSTRACT IntroductionMaterials and MethodsResultsDiscussionREFERENCES |
|---|
|
Introduction |
|---|
|
TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
|---|
85% of
malignant tumors (2)
suggests that telomerase activation
is the most common mechanism for telomere maintenance in cancer cells.
However, a number of immortal cell lines and tumors lack telomerase
activity yet have the ability to elongate their telomeres, which
suggests the existence of nontelomerase-dependent routes to
immortalization (3)
. These alternative mechanisms of
telomere length maintenance are termed ALT
(4)
.3
At present, the molecular basis for the ALT pathway is unknown, and it
is unclear what determines selection for the ALT pathway or telomerase
activation. Because of this lack of knowledge, it has not been possible
to induce ALT activity to identify the genes responsible. It would be
interesting to determine the status of the telomerase genes and their
expression in these ALT lines because this may provide clues as to why
ALT has been activated rather than telomerase. Bryan et al.
(5)
showed that the sequence of hTERC was
normal in all of the seven ALT lines looked at, which proved that
hTERC mutation was not involved. Whether methylation of the
hTERC promoter was influential or not was thought to be an
interesting question. Both hTERT and hTERC
contain CpG islands (6, 7, 8, 9)
, areas defined as having a
G + C content of >60% with an observed:expected ratio of
CpGs of at least 0.6 (10)
. CpG islands are generally
unmethylated in normal cells (11)
; however, cytosines
within CpG dinucleotides are targets for DNA methyltransferases, so
there exists the potential for methylation which would cause
transcriptional silencing of an associated gene by impeding the binding
of essential transcription factors. Abnormal methylation patterns are a
feature of both cultured cells (12)
and cancer cells (for
recent reviews, see Refs. 13, 14, 15, 16
), and CpG islands within
the promoters of several tumor suppressor genes (e.g.,
VHL, p16, RB1, MLH1; see
Ref. 13
for a review) are subject to hypermethylation and,
therefore, transcriptional silencing during carcinogenesis. Therefore,
it seemed possible that the CpG islands of hTERC and/or
hTERT might be subject to methylation during immortalization
by the ALT pathway either in culture or in tumors. In the current
study, we investigate hTERC expression levels in relation to
methylation status of the promoter in ALT cells, tumors, and normal
cells to determine whether methylation regulates expression of this
gene. |
Materials and Methods |
|---|
|
TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
|---|
Northern Blot Analysis.
Normal tissue Northern blots were Northern Territory Total RNA Human
Normal Tissue Blot III from Invitrogen (Groningen, the Netherlands) and
Multiple Choice Human Total RNA Blot 5 from Cambridge Bioscience
(Cambridge, United Kingdom). Northern blots of RNA from cell lines were
made using the NorthernMax kit (Ambion, Austin, TX). Briefly, total RNA
was extracted from cell lines using the RNeasy Midi Kit (Qiagen), and
30 µg resolved on a 1% formaldehyde agarose gel and transferred onto
positively charged nylon membrane (Roche Diagnostics). The
hTERC probe was generated by PCR with genomic DNA as a
template and the following primers: TRC3F 5' CTA ACC CTA ACT GAG AAG
GGC GTA 3'; and TRC3R 5' GGC GAA CGG GCC AGC AGC TGA CAT T 3'
(amplifying a 154-bp portion of the hTERC transcribed region
from position +46 to +199). PCR was carried out using Qiagen reagents
and 30 cycles of 95°C for 30 s, 55°C for 30 s, and 72°C
for 30 s. The PCR product was purified using the QIAquick PCR
purification kit (Qiagen). The 18S rRNA probe came from Ambion. Probes
were labeled with [
-32P]dCTP using the
Rediprime II random prime labeling kit (Amersham). Hybridization and
washing of the filters were carried out using the NorthernMax solutions
according to manufacturer’s protocols (Ambion). Autoradiography was
performed using Fuji Super RX film with exposures of 2–18 h.
Autoradiographs were quantified using Quantity One (PDI)
gel scanner, the hTERC signal was divided by that for
18S to standardize for loading, and then all of the results were
compared with HeLa to allow comparisons between blots.
Southern Blot Analysis.
Genomic DNA was extracted from cell lines using a DNA Maxi kit (Blood
and Cell Culture; Qiagen). Genomic DNA (30µg) was digested with
XmaI enzyme (Promega) overnight at 37°C. This was then
divided into three portions; one portion was left untreated, and the
other two were digested further with either HpaII or
MspI (Life Technologies, Inc.). The digested DNA was
separated on a 0.8% agarose gel and transferred onto positively
charged nylon membrane (Roche Diagnostics). The probe was generated by
PCR amplification of a 412-bp region of the hTERC promoter
using Qiagen reagents, genomic DNA as a template, and 1
µM of each of the following primers: Meth1F 5'
ATC ACG CCA CTA GAC TCC ATC C 3' (position 68 in sequence AF047386);
and Meth1R 5' AGG GCC TAA GAC ACA GAA CAC TAA 3' (position 479 in
sequence AF047386). Cycling conditions were 94°C for 2 min, then 30
cycles of 94°C for 30 s, 58°C for 30 s, 72°C for
30 s, followed by 1 cycle of 94°C for 30 s, 58°C for 1
min, and 72°C for 5 min. The PCR product was purified using the
QIAquick PCR purification kit (Qiagen) and labeled as described above.
Hybridization was carried out at 67°C overnight in a solution
containing 5 x SSC, 5 x Denhardt’s
solution, 10% dextran sulfate, 0.1% Na PPi, 1%
SDS, and 0.1 mg/ml denatured sheared salmon sperm DNA (Sigma Chemical
Co.). Blots were then washed in 2 x SSC, 0.1% SDS for
15 min at room temperature, then 15 min at 67°C followed by
3 x 30 min washes in 0.1 x SSC, 0.1%
SDS at 67°C. Autoradiography was performed using Kodak Xomat film
with exposure of 1–14 days.
MSP.
Genomic DNA was extracted as it was for Southern blot analysis.
Bisulfite modification of 1 µg DNA was performed using the CpGenome
DNA Modification Kit following manufacturer’s protocol (Intergen Co.,
Purchase, NY). PCR amplifications were carried out using three
different sets of primers (5' nucleotide positions are given relative
to the cloned genomic sequence with GenBank accession no. AF047386,
shown in Fig. 2
). Primers that detected methylated genomic sequences
were mhtr10F 5' GAC GTA AAG TTT TTT TCG GAC G 3' (position 605); and
mhtr10R 5' ACC CGA TAC GCT ACC GAA CG 3' (position 802). Primers that
detected unmethylated genomic sequences were uhtr10F 5' GTA AAG ATG TAA
AGT TTT TTT TGG ATG 3' (position 600); and uhtr10R 5' CCA CAA CCC AAT
ACA CTA CCA 3' (position 807). Control primers that detected wild-type
unmodified genomic sequence were whtr10F 5' GTA AAG ACG CAA AGC CTT TCC
C 3' (position 600); and whtr10R 5' TGC GCT GCC GGG CGA GTC GG 3'
(position 796). MSP was carried out in a 50-µl reaction using
1 x reaction buffer (Qiagen), 1.25 units of HotStarTaq
polymerase (Qiagen), 10 mM deoxynucleotide triphosphate, 50
ng of modified/unmodified genomic DNA, and 1 µM of each
primer. Cycling conditions were as follows: an initial denaturation at
94°C for 15 min, followed by 35 cycles of 94°C for 30 s,
annealing temperature for 45 s, 72°C for 30 s, with a final
extension step at 72°C for 5 min. The annealing temperature was
68°C for the wild-type primers, 57°C for the methylated primers,
and 58°C for the unmethylated primers. Each DNA sample was included
in a reaction with the wild-type primers to check against incomplete
bisulfite modification, and the methylated- and unmethylated-specific
primer pairs were included in reactions with unmodified DNA to check
for specificity. A selection of products were cloned into pPCR-Script
(Stratagene) and subjected to Big Dye terminator sequencing on an ABI
373A using T7 and T3 primers.
|
RT-PCR.
RNA was extracted from 5-aza-C + TSA treated and untreated
cells using the RNeasy Midi Kit (Qiagen) that included the optional
DNase I treatment step, according to manufacturer’s protocol.
First-strand cDNA synthesis was carried out in a 40-µl reaction that
used 1 µg RNA and the Taqman Gold RT-PCR Kit (PE Applied Biosystems).
A control that lacked the reverse transcriptase was included for each
sample to check against DNA contamination. cDNA (2 µl) was included
in a 50-µl PCR reaction with TRC3F and TRC3R primers (as described
for the hTERC Northern probe). Cycling conditions were as
follows: 94°C for 15 min, followed by 38 cycles of 95°C for 30 s, 59.5°C for 45 s, 72°C for 30 s, with a final extension
step of 72°C for 5 min. PCR with glyceraldehyde-3-phosphate
dehydrogenase primers (Clontech) was also performed to confirm cDNA
synthesis.
|
Results |
|---|
|
TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
|---|
and 13 normal tissues (Fig. 1B)
. Among the
immortal cell lines, the expression levels of hTERC are
quite variable, as indicated in Table 1
. Interestingly, three of five ALT cell lines (KMST-6, SUSM-1, and
WI38-VA13/2RA) show a complete lack of hTERC RNA. This
confirms previously reported results (5)
for SUSM-1 and
WI38-VA13/2RA and extends the finding to KMST-6. All of the three
mortal cell lines studied (WI38, IMR90, and HEK121) have detectable
hTERC expression but at levels consistently lower than those
found in the telomerase-positive lines. COLO320-DM showed the lowest
hTERC RNA levels among the telomerase-positive immortal
lines (22% that of HeLa), and this is still roughly twice the levels
seen in the mortal lines; WI38, IMR90, and HEK121 have levels of 7%,
7%, and 12% that of HeLa, respectively.
|
|
, hTERC expression was
only detected in RNA extracted from the testis. These data are
consistent with the distribution of expression routinely detected by
RNA in situ hybridization. However, it is possible that the
sensitivity of Northern blots and RNA in situ hybridization
may not detect extremely low levels or small subpopulations of
hTERC expression. The Northern blot analysis presented above clearly demonstrates differential expression of hTERC between normal tissues, between mortal and immortal cell lines, and in ALT cell lines. Thus it is important to investigate the mechanisms that govern hTERC expression.
Methylation of the hTERC Promoter.
Previously, we have cloned and characterized a region of 1765nt that
contained the hTERC gene and its promoter sequence (Ref.
6
; GenBank accession no. AF047386). The entire transcribed
region of hTERC (position 799-1248 of AF047386) lies within
a CpG island (as identified by GRAIL, Ref. 6
). The
CpG island (depicted in Fig. 2
) has a G + C content of 66% and a CpG observed:expected
ratio of 0.89, thus fulfilling the criteria of Antequera and Bird
(10)
. Because CpG islands can be subject to
hypermethylation, which leads to transcriptional silencing of the
associated gene, it was of interest to determine whether methylation of
the hTERC promoter was involved in the lack of expression
seen in the three ALT cell lines, WI38-VA13/2RA, SUSM-1, and KMST-6, as
well as in normal somatic tissue. With this aim in mind, Southern
blotting and MSP assays were developed to assess the methylation status
of several CpG dinucleotides within the hTERC promoter.
Digestion of genomic DNA first with XmaI and then with
either HpaII or MspI enabled us to determine the
methylation status of one of four CpG dinucleotides within the
hTERC CpG island. XmaI digestion releases a
fragment of 963 bp that contained the hTERC promoter, which
can be detected by a probe, as shown in Fig. 2
. MspI further
digests this fragment, cutting at all of the four sites marked "H"
in Fig. 2
regardless of whether the cytosines within are methylated or
not. The probe will therefore detect a fragment of 582 bp in this
situation. HpaII, on the other hand, recognizes the same
sites as MspI but will only cut if the cytosine of the CpG
dinucleotide is unmethylated. If all of the four sites are methylated,
HpaII will not digest the fragment further so the probe
would detect the full-size 963-bp fragment. This fragment will be
shorter if any of the four sites are unmethylated, giving a 582-bp
fragment if the most 5' site is free from methylation. Using this
method, DNA was examined from 1 mortal and 18 immortal cell lines
(listed in Table 1
), 17 different normal tissues, and 176 tumor samples
(21 lung, 28 breast, 15 cervical, 10 colon, and 102 ovarian).
Representative results are shown in Fig. 3
. All of the normal tissues examined showed no evidence of methylation,
at least at the most 5' HpaII site, the probe that detected
the fully digested 582-bp HpaII fragment (Fig. 3A
, and data not shown). There was more evidence of
methylation in the cell lines. Several samples showed partial
methylation, e.g., A2780 and MCF7 in Fig. 3B
(additional cell lines showing a similar result are listed in Table 1
).
Of more interest were the ALT cell lines. Of the three that did not
express hTERC, two showed complete methylation
(WI38-VA13/2RA and SUSM-1) and the third (KMST-6) showed partial
methylation, but in addition to the 963-bp and 582-bp HpaII
fragments, a third fragment was detected of 620 bp (data not shown)
which indicated that the first and second HpaII-associated
CpG sites are not completely methylated in this cell line.
|
by sample
OV350. Of the 102 ovarian tumors examined, 67 were classed as
malignant, 12 borderline, and 23 benign. The nine samples showing
partial methylation were all from malignant tumors, possibly reflecting
the general increase in CpG island methylation associated with tumor
progression (reviewed in Refs. 13, 14, 15, 16
).
In addition to the panel of cell lines, 13 of the ovarian tumor
samples (including all of those showing partial methylation) were
investigated further for hTERC promoter methylation using
MSP. MSP (17)
makes use of the observation that sodium
bisulfite converts unmethylated cytosines (but not methylated
cytosines) to uracil. PCR primers can then be designed to amplify
specifically from methylated or unmethylated sequences. We designed
three pairs of primers: one pair specific for methylated
hTERC promoter sequence, one specific for unmethylated
sequence, and one to detect untreated DNA sequence. All of the three
pairs amplified approximately the same region, from around nt605 to
nt801 of AF047386 (as shown on Fig. 2
). Each primer covered three CpG
dinucleotides, thereby allowing us to examine six different sites.
These were CpGs 1–3 and 19–22 of the 73 CpGs present in the
hTERC CpG island (numbering from the 5' end). CpG 2 lies
within the most 5' HpaII site previously examined by
Southern blotting, and CpG 22 crosses the transcription start site of
hTERC. Cloning and sequencing of the PCR products from some
of the samples enabled us to also determine the methylation status of
the intervening CpG sites as well as to confirm that the
technique was sufficiently specific.
In general, the MSP results supported those obtained by Southern
analysis, e.g., Fig. 4C and D
show the results for unmethylated and
partially methylated ovarian tumor samples, respectively. The samples
predicted to be unmethylated on the basis of Southern blotting are only
amplified by the unmethylated DNA-specific primers (Fig. 4C)
, whereas those judged from the Southern analysis
to be partially methylated support amplification by both the
unmethylated DNA- and methylated DNA-specific primers (Fig. 4D)
. For the cell lines examined by MSP, only KMST-6
produced results that differed slightly from those obtained by Southern
analysis. MSP only amplified a band with the methylated
DNA-specific primers (Fig. 4A)
, whereas Southern analysis
had detected partial methylation (data not shown). Sequencing of the
MSP product demonstrated that 20 of the 22 CpG sites between nt605 and
nt801 were in fact methylated, which indicated that the
hTERC promoter in KMST-6 cells was highly methylated.
Sequencing of MSP products from SUSM-1 and WI38-VA13/2RA similarly
showed that 21 of the 22 CpG sites were methylated in each case. MSP
products from other cell lines were also sequenced to check the
specificity of the technique (Table 2)
. "Methylated" products consistently contained at least 20
methylated CpGs, whereas "unmethylated" products were generally
completely free of methylation, with only one product (from C-33A)
showing a single methylated site. This confirms that the MSP technique
is specific for detecting methylated and unmethylated regions of the
hTERC promoter and strengthens the association between total
lack of hTERC RNA and hypermethylation of the promoter as
seen in the three ALT lines KMST-6, SUSM-1, and WI38-VA13/2RA. To
further investigate this relationship, the three methylated ALT lines
were treated with 5-aza-C + TSA to see if hTERC
expression could be restored. 5-aza-C inhibits methylation once
incorporated into DNA, and TSA has been found to enhance its
gene-activating effect by inhibiting histone deacetylases
(18)
. Partial demethylation of the hTERC
promoter (as detected by MSP) was seen in all of the three cell lines.
The results for KMST-6 are shown in Fig. 5A
. Only in KMST-6 could expression of hTERC be
detected (Fig. 5B)
, and even then levels were low,
suggesting that either incomplete demethylation was preventing greater
expression or necessary transcription factors were limiting. The cells
remained negative for telomerase activity as measured by the telomeric
repeat amplification protocol assay (data not shown). These
results would indicate that methylation and associated histone
deacetylation are partially responsible for the lack of
hTERC expression at least in KMST-6 cells but that
additional factors are acting to limit expression. These additional, as
yet unknown, factors are important in inhibiting hTERC
expression in SUSM-1 and WI38-VA13/2RA because partial demethylation
fails to trigger expression of the gene.
|
|
|
|
Discussion |
|---|
|
TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
|---|
It has been observed that during tumorigenesis and during the culturing of cells in vitro there is a general increase in methylation at CpG islands (12) . This could explain the results seen here. Apart from the mortal and the ALT cell lines, the remaining cell lines and the vast majority of the tumor samples were positive for telomerase and presumably rely upon telomerase for their immortality. Therefore, there would be considerable selection pressure to retain expression of telomerase components including hTERC. Methylation at CpG islands would be increasing generally, but there would be strong pressure to prevent hypermethylation and consequent down-regulation of hTERC because the resulting cells would lose their immortality. Such cells may indeed arise, e.g., Bryan et al. (21) found that telomerase-negative subclones did occasionally arise from cultures of telomerase-positive 293 and HeLa cells, but presumably these cells would be lost from tumors and cultures because they would no longer be immortal. Partial methylation, however, does not appear to affect expression of hTERC and can therefore be tolerated; this was seen in 6 of 13 telomerase-positive immortal cell lines and in 9 of 102 ovarian tumor samples. Interestingly, methylation was only seen in the malignant tumor samples, not the benign or borderline ones, perhaps reflecting increasing levels of CpG island methylation during tumor progression. Perhaps a thorough study of highly advanced tumors would detect similar methylation patterns among other types of tumor, not just ovarian.
In 3 of 5 of the ALT cell lines looked at in this study, methylation at the hTERC-associated CpG island has progressed to the stage where at least 90% of CpG sites upstream of the transcription start site are methylated, and there is no hTERC expression. Such a relationship between extreme hypermethylation of the promoter and lack of expression was also found at the hTERT gene in three ALT lines: SUSM-1 (20) , GM847, and WI38-VA13/2RA (19) . It could be that one or both of the hTERC and hTERT genes are hypermethylated in all of the ALT lines. SUSM-1 and WI38-VA13/2RA are hypermethylated at both loci, GM847 is hypermethylated at the hTERT promoter, and KMST-6 is hypermethylated at hTERC; however, only a single locus has been looked at in other ALT lines. What this actually means in terms of ALT versus telomerase activation is difficult to assess. It is possible that heavy methylation at either of the telomerase gene loci causes loss of expression and, therefore, loss of telomerase activity leading to selection for ALT activation. However, it is also possible that telomerase has never been activated in these cells, and ALT has been activated first, thereby eliminating any selection pressure to keep the hTERC and hTERT promoters free from hypermethylation, so the trend for increased methylation in an in vitro culture system is free to target both loci. At present, the only evidence that could help decide this issue comes from demethylation experiments.
In this study and the recently published hTERT studies (19 , 20) , treatment with 5-aza-C either alone or in combination with the histone deacetylase inhibitor TSA has had mixed success in triggering expression of the target gene. Where expression has been achieved, the levels appear low in comparison with other immortal cell lines, and no telomerase activity has been detectable (this study, data not shown; Ref. 19 ). Although it is possible that the treatment is unable to fully restore the chromatin to an active state, there is also the possibility that necessary transcription factors are limiting for hTERC and/or hTERT in ALT cell lines, and even when expression is achieved, the levels may be insufficient for active telomerase. If this is the case, it may be that the ALT pathway has become activated before any activation of telomerase, thereby eliminating selection pressure to keep the hTERC and hTERT promoters free of hypermethylation, which increases the likelihood that they will become methylated during culturing in vitro.
Whatever the cause, there is certainly a strong association between hypermethylation of the hTERC promoter and total lack of the RNA. We have shown this to be the case in 3 of 3 ALT cell lines that do not express hTERC, thereby making hTERC methylation a marker for a significant subset of ALT lines.
|
FOOTNOTES |
|---|
1 Supported by the Cancer Research Campaign
(United Kingdom) and Glasgow University. 
2 To whom requests for reprints should be
addressed, at Cancer Research Campaign Department of Medical Oncology,
University of Glasgow, Cancer Research Campaign Beatson Laboratories,
Garscube Estate, Switchback Road, Bearsden, Glasgow G61 1BD, United
Kingdom. Phone: 44-141-330-4811; Fax: 44-141-330-4127; E-mail: n.keith{at}beatson.gla.ac.uk
3 The abbreviations used are: 5-aza-C,
5-azacytidine; ALT, alternative lengthening of telomeres; TSA,
trichostatin A; hTERC, telomerase RNA gene;
hTERT, telomerase reverse transcriptase gene; MSP,
methylation-specific PCR; RT-PCR, reverse transcription-PCR.
Received 8/14/00. Accepted 11/13/00.
|
REFERENCES |
|---|
|
TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
|---|
This article has been cited by other articles:
|
|
 |
|
  A. E. Bilsland, K. Stevenson, S. Atkinson, W. Kolch, and W. N. Keith Transcriptional Repression of Telomerase RNA Gene Expression by c-Jun-NH2-Kinase and Sp1/Sp3 Cancer Res., February 1, 2006; 66(3): 1363 - 1370. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 I. Horikawa, Y. J. Chiang, T. Patterson, L. Feigenbaum, S.-H. Leem, E. Michishita, V. Larionov, R. J. Hodes, and J. C. Barrett Differential cis-regulation of human versus mouse TERT gene expression in vivo: Identification of a human-specific repressive element PNAS, December 20, 2005; 102(51): 18437 - 18442. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. P. Atkinson, S. F. Hoare, R. M. Glasspool, and W. N. Keith Lack of Telomerase Gene Expression in Alternative Lengthening of Telomere Cells Is Associated with Chromatin Remodeling of the hTR and hTERT Gene Promoters Cancer Res., September 1, 2005; 65(17): 7585 - 7590. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S.-i. Kumakura, T. W. Tsutsui, J. Yagisawa, J. C. Barrett, and T. Tsutsui Reversible Conversion of Immortal Human Cells from Telomerase-Positive to Telomerase-Negative Cells Cancer Res., April 1, 2005; 65(7): 2778 - 2786. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 N. Ikeda, H. Uemura, H. Ishiguro, M. Hori, M. Hosaka, S. Kyo, K.-i. Miyamoto, E. Takeda, and Y. Kubota Combination Treatment with 1{alpha},25-Dihydroxyvitamin D3 and 9-cis-Retinoic Acid Directly Inhibits Human Telomerase Reverse Transcriptase Transcription in Prostate Cancer Cells Mol. Cancer Ther., August 1, 2003; 2(8): 739 - 746. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 I. Guilleret, P. Yan, L. Guillou, R. Braunschweig, J.-M. Coindre, and J. Benhattar The human telomerase RNA gene (hTERC) is regulated during carcinogenesis but is not dependent on DNA methylation Carcinogenesis, December 1, 2002; 23(12): 2025 - 2030. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J.-L. Mergny, J.-F. Riou, P. Mailliet, M.-P. Teulade-Fichou, and E. Gilson Natural and pharmacological regulation of telomerase Nucleic Acids Res., February 15, 2002; 30(4): 839 - 865. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Komata, Y. Kondo, T. Kanzawa, S. Hirohata, S. Koga, H. Sumiyoshi, S. M. Srinivasula, B. P. Barna, I. M. Germano, M. Takakura, et al. Treatment of Malignant Glioma Cells with the Transfer of Constitutively Active Caspase-6 Using the Human Telomerase Catalytic Subunit (Human Telomerase Reverse Transcriptase) Gene Promoter Cancer Res., August 1, 2001; 61(15): 5796 - 5802. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Cancer Research | Clinical Cancer Research |
| Cancer Epidemiology Biomarkers & Prevention | Molecular Cancer Therapeutics |
| Molecular Cancer Research | Cancer Prevention Research |
| Cancer Prevention Journals Portal | Cancer Reviews Online |
| Annual Meeting Education Book | Meeting Abstracts Online |
