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* Center for Radiological Research, Columbia University, New York, New York 10032, USA;
Radiation Oncology Department, St.-Luc University Hospital, Belgium; and
Johns Hopkins Oncology Center, Baltimore, Maryland 21205, USA
1Correspondence: Center for Radiological Research, VC11–213 College of Physicians and Surgeons, Columbia University, 630 West, 168th St., New York, NY 10032, USA. E-mail: tkp1{at}columbia.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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Key Words: cell kill • ionizing radiation • tumor growth
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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. The telomere is a
functional domain of the chromosome and is essential for normal
chromosome stability (2)
. Somatic cells progressively lose telomeric
repeats with each cell division due to incomplete replication (3,
4)
.
Immortal and cancer cells compensate for the obligatory loss of
terminal sequences by expressing the enzyme telomerase, a
telomere-specific DNA polymerase that resynthesizes telomeric DNA
de novo (5,
6)
.
Telomerase, a ribonucleoprotein enzyme comprised of protein components
and an internal RNA template, catalyzes telomere elongation through the
addition of TTAGGG repeats (2)
. This enzyme recognizes the G-rich
strand of an existing telomere repeat sequence and, in the absence of a
complementary DNA strand, synthesizes a new copy of the repeat using
the internal RNA template. The telomerase RNA component in human TERC
(hTERC)2
has been cloned (8)
and found
to have widespread expression. Telomerase activity has been shown to
correlate more closely with the expression status of the telomerase
catalytic subunit gene TERT (9,
10)
. Expression of human TERT (hTERT)
in telomerase-negative cells results in reconstitution of telomerase
activity, elongation of telomeres, and extension of cellular life span
(7,
11)
.
Using a standard biochemical method, detection of telomerase activity
in crude HeLa cell extract was first demonstrated by Morin (12)
. Kim et
al. (13)
described a sensitive technique, termed telomeric repeat
amplification protocol (TRAP), for detection of telomerase activity.
Subsequently, several modifications of the TRAP assay have lead to a
simplified, improved and rapid method for assaying telomerase activity.
With the TRAP assay, telomerase activity has been detected in ~90%
of human cancers (13
14
15)
, but not in most somatic cells, with a few
exceptions that include proliferative stem cells (16
17
18)
. The
difference in telomerase activity between normal and cancer cells
therefore makes telomerase a quantitatively reliable marker for cancer
detection (15,
19)
. For various types of tumors, telomerase activity
can be assayed using a variety of clinical specimens including fine
needle aspirates, cell sediments from urine, tissue brushes and washes,
and fresh and frozen tissue samples (20
21
22)
.
Since radiotherapy plays a key role in the treatment of many tumors, accurate estimation of telomerase activity after (or during) radiotherapy might be a useful indicator of tumor regression. To determine the link between the telomerase activity and cell kill, we studied in vitro and in vivo effects of gamma radiation on cell kill, tumor regression, and telomerase activity. A dose-dependent decrease in telomerase activity was proportional to cell kill and tumor regression after ionizing radiation treatment.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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. Telomerase-positive human colorectal
carcinoma (RKO) cells were maintained in McCoy's 5A medium
supplemented with 10% fetal bovine serum and antibiotics. The
SV40-transformed telomerase-positive human bronchial epithelial (BEP2D)
cells were grown in serum-free LHC-8 medium (Biofluids, Rockville,
Md.), supplemented with growth factors as described previously (24)
.
Cells were irradiated at room temperature with graded doses (0 to 40
Gy) of gamma rays at a dose rate of 1.1 Gy/min using a
137Cesium source.
Mice and tumor irradiation
Eight-week-old NMRI nu/nu male mice were maintained in a
pathogen-free mouse colony for the duration of experiments. Mice were
randomly distributed four per cage and each mouse was labeled with a
code on the ear. Two million exponentially growing RKO cells in a
volume of 20 µl were injected intramuscularly (i.m.) in the right
thigh and mice were checked daily for tumor appearance. Animals with a
tumor of 8 mm in mean diameter were locally irradiated with a single
dose of 25 Gy using a 8 MeV linac (dose rate of 2 Gy/min). For the
regrowth daily experiments, the tumors were measured daily up to a mean
diameter of 14–15 mm, at which time the mice were killed. For the
telomerase activity assay, mice were killed by cervical dislocation at
various time intervals after irradiation, and single cell suspensions
of the tumors were prepared. Briefly, the tumors were dissected,
minced, aspirated through 19 to 24 G needles, and washed with cold
phosphate-buffered saline (PBS). The tumor cells were stored frozen at
-80°C.
Cell viability
Cell viability was monitored by trypan blue exclusion, and cell
counts were made by both hemacytometer and electronic counting (Coulter
Electronic Inc., Hilaleah, Fla.).
Clonogenic assays
Cell survival after ionizing radiation treatment was determined
by clonogenic assay. Briefly, cells in exponential phase were plated
into 60 mm dishes in 5.0 ml medium. The number of cells per dish was
chosen to ensure that ~50 colonies would survive a particular
treatment. After 24 h incubation, cells were irradiated with gamma
rays. Cells were subsequently incubated for 12 days, fixed with
methanol:acetic acid (3:1), and stained by crystal violet. Only
colonies containing more than 50 cells were scored as derived from
viable, clonogenically competent cells.
Telomerase activity assay
Telomerase activity was determined using the telomerase
polymerase (PCR) enzyme-linked immunoassay (ELISA) kit (Boehringer
Mannheim, Mannheim,. Germany). Briefly, 1 million cells were washed
three times with cold PBS and lysed in 100 µl of precooled lysis
solution by incubating the suspension on ice for 30 min. Samples were
microfuged and protein concentrations of the supernatant were measured
using the Bio-Rad Protein Assay kit. Cell extracts were incubated at
25°C in the presence of biotin-labeled primers and the telomeric
repeats added onto the ends of the synthetic primers were amplified by
PCR. The denatured products were allowed to bind to a
streptavidin-coated 96-well plate and hybridized to a DIG-labeled,
telomeric repeat-specific probe. The biotin-labeled PCR products were
detected using peroxidase-conjugated antibody to DIG and subsequently
visualized by virtue of the enzyme's ability to metabolize TMB so as
to produce a colored reaction product. The sample absorbance at 450 nm
measured using an ELISA reader represents telomerase activity in the
sample. Telomerase activity was determined in triplicate and a negative
as well as a positive control was run each time. A negative control was
provided for each extract by heat inactivating the telomerase enzyme
present in cell lysate at 95°C for 10 min prior to the PCR step.
Semiquantitative RT-PCR of hTERT
To determine the mRNA levels of the hTERT, we treated HeLa cells
with 0 or 10 Gy of ionizing radiation and incubation for different
periods after irradiation. Cells were washed with PBS and collected in
Trizol (Life Technologies, Inc., Gaithersburg, Md.). RNA was isolated
by the procedure described previously (25)
. First-strand cDNA was
prepared from 4 µg of RNA using Superscript II reverse transcriptase
(Life Technologies, Inc.) in a 40 µl reaction according to the
manufacturer's instructions. The reverse-transcribed (RT) products
were amplified in paired PCR reactions using two sets of primers and
three 10-fold dilutions (1/20, 1/200 and 1/2000) of the RT products. A
157 bp fragment of the human acidic ribosomal phosphoprotein PO gene
cDNA (36B4, Gene Bank accession number M17885) was amplified using the
oligonucleotide primers, 5'-gat tgg cta ccc aac tgt tgc a- 3' (36B4FW)
and 5'-cag ggg cag cag cca caa agg c- 3' (36B4RV). A 182 bp fragment of
the hTERT cDNA (Gene Bank accession number AF0181167) was amplified
using the oligonucleotide primers, MS113 (5'-aga gtg tct gga gca agt
tgc- 3') and MS114 (5'-cga ttg tga aca tgg act acg- 3'). PCR reactions
were performed using Taq DNA polymerase (Perkin Elmer,
Foster City, Calif.), following assay conditions recommended by the
manufacturer. Buffer C (PCR Optimizer Kit, Invitrogen, Carlsbad,
Calif.) was used for hTERT amplification. The thermal cycling protocols
were: 94°C for 5 min, followed by 62°C/45 s, 72°C/45 s, and
94°C/30 s for 20 cycles for 36B4, then 94°C for 5 min, followed by
60°C/45 s, 72°C/45 s, and 94°C/30 s for 32 cycles for hTERT. PCR
products were separated in a 6% nondenaturing polyacrylamide gel in
0.5x TBE buffer using 12.5 µl from both paired reactions per lane
and imaged by PhosphorImager screen (Molecular Dynamics, Sunnyvale,
Calif.). PCR product bands were measured densitometrically using the
IPLabGel program (Signal Analytics, Vienna, Va.).
Mixing experiments
AG 1522 and HeLa cells were grown as described above. Cells were
washed in PBS and lysed with telomerase kit lysis solution. Cell
lysates were mixed in different concentrations in order to get serial
dilutions of each cell lysates.
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RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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27)
, there is no universal marker that could
be used for most types of tumor. Since telomerase activity is present
in almost all carcinomas (15)
and the level of telomerase activity
correlates with the stage of carcinogenesis (28)
, determination of
telomerase activity has a great potential for use in clinical diagnosis
and to monitor the recurrence of various malignancies after
radiotherapy. As telomerase activity is frequently proposed to be
associated with the malignant phenotype, we attempted to determine
whether a correlation exists between ionizing radiation-induced cell
death and telomerase activity. We selected four telomerase-positive
cell lines (human cervical carcinoma, HeLa; colorectal carcinoma, RKO;
ataxia-telangiectasia, GM5849; and SV40-transformed human
bronchial epithelial cells, BEP2D) and one telomerase-negative cell
strain (normal human fibroblast, AG1522) for our study. The survival of
different cell types after exposure to graded doses of radiation
(137Cesium gamma rays at 1.1 Gy/min) as
determined by clonogenic assay is shown in Fig. 1
. As expected, ataxia telangiectasia (GM5849) cells were the
most sensitive to cell kill by ionizing radiation, whereas the others
showed some variability, with relatively small differences between
them.
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To determine the range over which the telomerase assay products were
proportional to the protein concentration, cell lysates were titrated
between 0.001 and 2 µg protein per assay using a telomerase PCR ELISA
technique (Fig. 2
). The telomerase-positive cell lines (HeLa, RKO, GM5849, and BEP2D)
showed an increase in telomerase activity with the increase in protein
concentration, whereas untransformed human fibroblasts (AG1522) showed
no telomerase activity even at the concentration of 23 µg per assay
(data not shown). With this procedure, the telomerase activity was
found to plateau at 0.5 µg of protein used per assay. When telomerase
activity was compared among various telomerase-positive cell lines at
protein concentrations lower than 0.2 µg per assay, a significant
variation was found (Fig. 3
). Further, when telomerase activity was compared between exponentially
growing cells, S-phase synchronized cells, and serum-deprived cells, a
decrease in telomerase activity was observed in serum-deprived
noncycling cells, suggesting higher telomerase activity in the
metabolically active cells (data not shown).
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To determine whether a telomerase assay might be used to monitor
residual cancer cells that survive radiation treatment, we studied the
influence of ionizing radiation on telomerase activity and cell
viability in telomerase-positive cells. Serum-deprived, plateau phase
HeLa and RKO cells were exposed to 0 to 40 Gy of ionizing radiation
using a 137Cesium source and incubated at 37°C
for up to 168 h. Cells collected at different times
postirradiation were examined for cell viability and telomerase
activity. The cell viability remained almost unchanged in untreated
cells whereas the treated cells showed a decrease in the cell viability
with an increase in the radiation dose (Fig. 4
). Decrease in cell viability as assessed by trypan blue exclusion assay
correlates with radiation dose and the posttreatment period. When
telomerase activity was compared at 0 h postirradiation, no
differences in telomerase activity were found among treated and
untreated samples (Fig. 5
). With time, the irradiated cells had less activity compared with
untreated controls, and this difference became significant in samples
collected 72 h postirradiation (Fig. 5)
. We could not detect
telomerase activity in samples collected 168 h posttreatment with
20 Gy or higher doses of ionizing radiation. A strong correlation was
detected between telomerase activity and cell viability in HeLa cells
treated with ionizing radiation. A similar pattern of decrease in
telomerase activity (Fig. 6
) and cell viability (data not shown) with an increase in radiation dose
treatment was observed in the colorectal carcinoma (RKO) cell line.
Collectively, these in vitro findings suggest that the
determination of telomerase activity 96 h postirradiation provides
an accurate assessment of the residual telomerase-positive cells after
ionizing radiation.
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Telomerase activity is detectable in most human tumor tissue and in
tumor-derived cell lines, but not in normal cells adjacent to tumors or
mixed with tumor-derived cell lines (29)
. To detect the presence of a
minimum proportion of telomerase-positive cells in a
telomerase-negative cell environment, as occurs in vivo
after radiotherapy of tumors, we studied the sensitivity of the
TRAP-ELISA method for detecting telomerase activity. Serially diluted
extracts of telomerase-positive (HeLa or RKO or BEP2D) and
telomerase-negative (AG1522) cells were mixed and used to estimate
telomerase activity. Using the TRAP-ELISA technique, we were able to
detect telomerase activity in extract equivalent to a single
telomerase-positive HeLa or RKO or BEP2D cell when mixed with extract
from ~10,000 telomerase-negative AG1522 cells (Fig. 7
).
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Since cells in an actively growing tumor are distributed in different
cycle stages, we also attempted to determine whether the telomerase
activity in exponentially growing cells is affected by ionizing
radiation. We first evaluated cell cycle phase distributions in growing
HeLa cells by flow cytometry. The exponential HeLa population contains
~40% of G1-phase, 50% of S-phase, and ~10% of G2/M phase cells
(data not shown). Exponentially growing HeLa cells were treated with
graded doses of gamma rays, incubated at 37°C for different time
periods, and analyzed for telomerase activity. We found no difference
in telomerase activity among treated and untreated exponentially
growing cells immediately after radiation treatment (Fig. 8
) but, unlike plateau phase cells, there was a two- to threefold
increase in telomerase activity in cells collected 72 h
postirradiation. Samples collected 120 h and later, however,
showed a decrease in telomerase activity in treated cells compared with
the untreated cells (Fig. 8)
. In contrast to the initial increased
telomerase activity in irradiated HeLa cells, treatment of
exponentially growing RKO cells showed no such increase in telomerase
activity, but showed a dose time-dependent difference between
irradiated and control cells (data not shown).
|
To determine whether the increase in the telomerase activity in
exponentially growing HeLa cells after gamma ray treatment is due to an
increase in the transcriptional levels of the catalytic unit of
telomerase (hTERT), we quantitated the mRNA levels of hTERT in HeLa
cells after radiation treatment. Exponentially growing HeLa cells were
treated with 0 or 10 Gy of radiation and incubated for up to 96 h.
Levels of hTERT mRNA and telomerase activity were determined at defined
intervals. As seen in Fig. 9
, no differences in the levels of hTERT mRNA were observed. In view of
the correlation between the expression status of hTERT and telomerase
activity (9,
10)
, possible reasons for the increase of telomerase
activity (Fig. 8)
in the absence of corresponding increase in the hTERT
mRNA level (Fig. 9)
could be due to a reduced degradation of the
telomerase enzyme postirradiation or release of telomerase component
due to structural change(s) brought about by ionizing radiations. It is
known that ionizing radiation cause DNA-DNA as well as DNA protein
cross-links (30)
, and also release soluble proteins bound to the
chromatin. Thus, the increase in telomerase activity in HeLa cells
could be due to release of chromatin-bound telomerase by ionizing
radiation.
|
We studied the telomerase activity in tumors in nude mice before and
after irradiation to determine whether the in vitro data
correlates with the in vivo situation. The mice were
injected in the leg with RKO cells; when tumors reached 8 mm, they were
irradiated with a single dose of 25 Gy. As seen in Fig. 10
, untreated tumors grew rapidly at a relatively uniform rate whereas
radiation treatment caused temporary shrinkage of the tumors, followed
by regrowth in most of the tumors. After a single dose of 25 Gy,
~20% of the tumors were still below detectable levels at 2 months
posttreatment and were designated locally cured.
|
To determine the telomerase activity in tumors, mice were killed at
different times and the tumors were collected. As shown in Fig. 11
, unirradiated tumors did not show any change in the telomerase
activity, whereas the irradiated tumors showed a decrease in the
telomerase activity. In tumors destined not to recur, telomerase
activity was zero; in tumors that were to relapse, telomerase activity
never reached zero and by 20 days postirradiation was similar to that
of the untreated tumors. In the irradiated tumors that showed regrowth,
the telomerase activity paralleled the tumor size, with maximum
significant differences in both parameters occurring 10 days
postirradiation compared with the unirradiated samples.
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Both in vitro and in vivo studies showed a link between the levels of cell kill, tumor regression, and telomerase activity. Although HeLa exponential cell populations treated with radiation showed an initial increase in telomerase activity, samples collected after 120 h showed less enzyme activity than the untreated controls. From the in vivo studies, telomerase activity in tumor cells from irradiated mice decreased gradually over time, with tumor cells collected on day 5 postirradiation from treated animals exhibiting significantly lower levels of telomerase activity than tumor cells from unirradiated mice. Collectively, these in vitro and in vivo results suggest that important information regarding the viable residual tumor cell burden can be gained by comparing the telomerase activity in samples collected immediately and 7 days postirradiation. However, assessment of telomerase activity in samples collected up to 3 days posttreatment could give a false picture of the residual disease.
These data suggest that telomerase activity varies among different telomerase-positive cell lines. A link has been established between cell kill and telomerase activity after radiation treatment. The reduction in telomerase activity in in vivo and in vitro experiments seen after radiation treatment parallels cell kill. Tumor samples as early as day 7 postirradiation might be informative about residual disease. The assay we used is sensitive enough to detect a single telomerase-positive cancer cell in 10,000 telomerase-negative cells. These findings suggest the telomerase enzyme has clinical potential as a marker of efficacy of radiation therapy.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Received for publication September 14, 1998.
Revision received January 27, 1999.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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-particles simulating radon progeny. Oncogene 13,1423-1430[Medline]
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