Download Telomerase Activity Is Associated with Cell Cycle

(CANCERRESEARCH
57, 549-554,February1, 19971
Telomerase Activity Is Associated with Cell Cycle Deregulation in Human
Breast
Cancer1
GöranLandberg, Niels Hilmer Nielsen, Na Nilsson, Stefan 0. Emdin, Jenny Cajander, and GöranRoos2
Departments ofPa:hology
(G. L, N. H. N., P. N., I. C., G. RI, Oncology (N. H. N.], and Surgety (S. 0. El, Umed University. 5-90187 Umed, Sweden
ABSTRACT
Deregulation
of the cell cycle by abnormal
expression
of one or several
cell cycle regulatory proteins is a common finding In malignant tumors
and might be a prerequisite for cancer development. Telomerase activity
Is an immortalization marker that is found in most cancers and for which
an association with an active cell cycle has been Implicated. In the tissue
of 106 human breast cardnemas, we analyzed the relationship between
telomerase activity levels and defects In the cell cycle machinery with a
focus en the retinoblastoma
protein (pRB) pathway(s). The fraction ef
telemerase-posltlve
tumors was 85%, and large differences in telomerase
activity were found. Overexpression of cydlin Dl and/er cydlin E, in
combination
with a normal pRB, was a typical feature oftumors
with high
telomerase activity levels. Down-regulation ofpl6@'4 was not related per
se to telomerase activity, but tumors with low p16'@4 j@combination with
cydlin Dl or E overexpresslon
demonstrated
high activity.
Tumor
cell
proliferation, determined by Kl-67 expression, correlated significantly to
telemerase activity levels. There was, however, not a strict association
between
proliferation
rate and telemerase
activity, because tumors
with
Inactivated pRB had the highest Kl-67 fractions but Intermediate telom
erase activity. Also, cyclin Dl everexpresslon was associated with high
telomerase levels without an increase in tumor cell proliferation. The
present study indicates that telomerase activation occurs preferentially in
breast cancers
with certain
cell cycle regulatory
defects and that telom
types (25, 26). There is also an association between telomerase
erase activity levels may depend on the specific defect(s).
INTRODUCTION
After a certain number of cell divisions, normal cells activate a
senescence program characterized by morphological changes, growth
arrest in G1, and shortened telomeres. Telomeres, which consist of
TTAGGG repeats with associated proteins, shorten 50—100bp after
each cell cycle round due to incomplete replication of the 3'-ends. At
a critically short telomere length, cells enter the senescence stage and
eventually they will die. Thus, the life span of a normal cell popula
tion is limited, and a strong association between the replicative ca
pacity of normal cells and telomere length has been demonstrated,
which forms the basis for the telomere hypothesis of senescence
(1—4).The telomere loss may lead to DNA breaks with activation of
p53-dependent or -independent DNA damage pathways, induction of
CDK3 inhibitors (e.g., p21 and p2'l) and the final G@ block of
senescence (5). In support of this hypothesis, a temporary rescue from
the senescence program can be achieved by inactivation of the pRB or
p53 tumor suppressor genes, leading to a prolonged life span but
usually not to immortalization (6—9).During this period of prolonged
life, a further decrease in telomere length takes place until a second
stage of crisis characterized by genomic instability is reached. Rare
cells will survive this state and become immortalized, a process most
Received 8/12/96; accepted 12/3/96.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance with
18 U.S.C. Section 1734 solely to indicate this fact.
@
I
was
supported
by grants
from
the Swedish
Cancer
Foundation,
Medical
Faculty of UmeA, Sahlbergs Foundation, Lions Cancer Research Foundation (UmeA,
Sweden), and a special grant from the VSsterbotten County Council.
2 To
whom
requests
for
reprints
should
be addressed.
Fax:
+46-90-7852829;
E-mail:
[email protected].
3 The
abbreviations
used
are:
CDK,
cyclin-dependent
kinase;
RB,
likely due to loss of normal gene function, because hybrids between
normal and immortalized cells usually have a limited life span (10).
One typical feature of inunortalized cells in vitro is the presence of
active telomerase, which can synthesize telomeric DNA repeats using
an internal RNA template, compensating for the loss of telomeric
DNA after each cell division cycle (11, 12). Thus, telomerase activity
has been demonstrated in the vast majority ofpermanent cell lines, but
immortal cell lines without detectable activity have been described (7,
13—16).Active telomerase was first shown in vivo in advanced ovar
ian carcinoma and hematopoietic B-cell neoplasias (17, 18) with the
conventional primer-extension telomerase assay (1 1). Using a more
sensitive method, the TRAP assay, telomerase activity has been
detected in high frequency in various tumors (19, 20—24). Thus,
telomerase activity appears to be a frequently appearing marker in
human cancer.
The regulation of telomerase activity is poorly understood, but cell
line data indicate that a telomere length-dependent mechanism is the
major pathway. However, normal lymphocytes up-regulate a notable
telomerase activity upon antigen and mitogen stimulation in vitro as
well as in vivo, indicating that telomere length-independent mecha
nisms for telomerase activation can be important for specific cell
retinoblastoma;
pRB, RB protein; TRAP, telomeric repeat amplification protocol; ITAS, internal TRAP
assay standard.
activity and cell proliferation, because terminal differentiation and cell
cycle exit is followed by down-regulation of the telomerase activity
(27, 28).
The pRB has a central role in the control of the cell cycle by
suppressing important transcription factors, such as E2F, which is
released after inactivation of pRB by phosphorylation (29). CDKS and
associated cyclins are responsible for the phosphorylation of pRB, and
the coordinate expression and association of CDKS, cyclins, and CDK
inhibitors drives cells through the cell cycle (30). In human tumors,
deregulation of the cell cycle machinery is common, and abnormal
expression of one or several cell cycle-regulatory proteins might be a
prerequisite for all cancer development (31). In a fraction of breast
cancers, the cyclin Dl gene is amplified and the protein overex
pressed, potentially inducing inactivation of pRB by phosphorylation
and S-phase progression (32, 33). RB inactivation, caused by muta
tions and deletions, is found in breast cancer and represents the most
severe cell cycle-regulatory defect with a complete lack of cell cycle
control (30, 3 1). Cyclin E is also often overexpressed in various
malignancies (34, 35), including 25% of breast cancers (36). The
functional consequences of a deregulated cyclin E is not fully under
stood, but apart from an abnormal phosphorylation of pRB and release
of E2F, other yet undefined targets might be affected (37, 38).
Only a few studies have been directed toward the relationship
between telomerase activity in tumors and the status of different cell
cycle regulators. In lung cancer, an association between telomere
length alterations and loss of heterozygosity in p53 and RB was
suggested, but no data on telomerase activity were presented (39). In
cell lines from four immortalization complementation groups, no
correlation between telomerase activity and abnormalities in RB, p53
and pl6'@4 could be demonstrated, but all cell lines were defective
in at least one of these components (40). In the present study, we
demonstrate an association between telomerase activity levels and the
status of pRB, cyclin Dl, cyclin E, and p16IN@@in a material of breast
549
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TELOMERASE
ACTIVITYANDCELLCYCLEREGULATION
cancer, a tumor type with a high frequency of telomerase-positive
cells (20, 41). Our data indicate that telomerase activation occurs
preferentially in tumors with certain deregulations in the cell cycle
machinery.
(Amersham International).
Membranes
were blocked in PBS containing 5%
dried milk and 0.1% Tween-20 and probed with monoclonal mouse antibodies
against cyclin E (HE 12, Santa Cruz Biotechnology,
Inc.), cyclin Dl (DCS-6),
and pl6@4 (@55Ø; DCS antibodies were kind gifts from Dr. Jiri Bartek,
Copenhagen, Denmark), and mouse anti-actin antibodies (Boehringer Mann
heim) for 1 h. After a second incubation with peroxidase-conjugated
MATERIALS
AND METHODS
Samples. Diagnostic tumor samples were collected from 106 patients with
antimouse
antibodies (Amersham Corp.), proteins were detected using an enhanced
chemiluminescent detection system (Amersham Corp.).
Relative protein concentrations were determined as described earlier (36).
breast carcinoma. According to the classification from The International Union
Briefly, different cell line standards were loaded on each gel with the tumor
Against Cancer, 28 patients had stage 1, 60 patients stage II, two patients stage
stage.
samples, and after densitometric quantification (Molecular Dynamics, Inc.) of
the enhanced chemiluminescence films, relative protein concentrations were
before sampling.
calculated by dividing optical densities from tumor specimens by that of the
ifi, and eight patients stage IV cancer. Eight patients had an unknown
No patient had been treated with radiation or chemotherapy
All tumors were taken care of immediately after excision, and at least two
pieces were snap frozen in liquid nitrogen and stored in —
80°Cuntil extracts
for immunoblotting and telomerase activity analysis were prepared. Adjacent
tissue pieces were formalin fixed, paraffin embedded, and used for routine
morphological
examination and immunohistochemical
cell line standard.
Immunohistochemistry. Breast cancer specimens were fixed in formalin
and embedded in paraffin according to the clinical routine. Fixation time in
formalin varied slightly between different samples but were in general between
15 and 24 h. Three-pm
staining. The presence
paraffin
sections
were
prepared
from
73 available
of tumor cells in samples used for telomerase assay and immunoblouing
studies was verified by Giemsa-stained imprints from each sample.
Telomerase Assay. Preparationof tumorextracts,theTRAPassay,andthe
quantifications of telomerase activity were performed as detailed previously
(13, 42, 43). Briefly, frozen tumor tissue was, depending on the sample size,
cases, and the slides were dried and thereafter deparaffinized in xylene and
rehydrated in alcohol and water according to standard procedures. To obtain
adequate pRB and Ki-67 staining, slides were boiled (96°C)in a microwave
oven for a total of 15 mm (5 mm X 3) using a citrate buffer at pH 6.0.
homogenized in 50—200 @l
of ice-cold lysis buffer, which was composed of 10
mM Tris-HC1 (pH 7.5), 1 mM MgCl2, 1 mxi EGTA, 0.5% 3-[(3-cholamido
istry
antibodies (C15; Santa Cruz Biotechnology, Inc.), and antigen-antibody visu
propyl)dimethylammonio]-l-propanesulfate (Pierce Chemical Co.), 10% glyc
erol, 5 mM f3-mercaptoethanol (Sigma Chemical Co.), and 0.1 mi@iphenyl
alization as well as H&E counterstaining were performed according to the
Ventana program. Ki-67 stainings were processed manually using MIB-l
methylsulfonyl
antibodies (Immunotech) and avidin-biotin complex method kits (Vector Lab
oratories, Inc.).
fluoride (Boehringer
Mannheim).
After a 30-mm incubation
on
ice, the lysate was centrifuged for 20 mm at 13,000 X g, and the supernatant
was snap frozen in liquid nitrogen and stored at —80°C.
Protein concentration
was measured using the bicinchoninic acid protein assay kit (Pierce Chemical
lyophilized at the bottom and
sequestered by a wax barrier (Ampliwax; Perkin-Elmer Corp.). Protein extracts
were diluted appropriately, and an aliquot of 0.005—5@g
protein was assayed
in 50
@lof reaction mixture composed of 50
staining
Microscopic
Co.). PCR tubes for the TRAP assay contained 0.1 @agof the CX primer
(5'-CCCTFACCC'VFA-CCCTFACCCTAA-3')
Microwave-treated
@M
of each deoxynucleotide
triphosphate, 0.1 @gof TS primer (5'-AATCCGTCGAGCAGAGTT-3'), 5
attogram of a l50-bp ITAS (43), 0.5 @M
T4 gene 32 protein (Boebringer
Mannheim), 2 pCi each of [a32P]dCTP and [a@2P]dUP, 3000 Ci/mmol
slides were processed in an automatic immunohistochem
machine
(Ventana
Evaluation.
320—202; Ventana,
Stained
sections
were
Inc.)
using
anti-pRB
microscopically
exam
mcd, and the staining intensity of pRB in tumor cells was compared to that in
adjacent normal cells. A tumor was considered to be pRB abnormal if there
was no or very weak nuclear pRB staining in all tumor cells. The percentage
of Ki-67-positive cells was determined by calculating at least 300 tumor cells
crossing randomly distributed lines.
Statistical Methods. Comparison between groups was performed using
log-rank and x@ (Pearson) correlation analysis. All calculations were per
formed using SPSS version 6.0 (SPSS, Inc., Chicago, IL).
(Amersham Corp.), and 2 units of Taq polymerase (Boehringer Mannheim).
RESULTS
Following a 30-rain incubation at room temperature, the samples were sub
jected to 31 PCR cycles of94°C for 30 s, 50°Cfor 30 s, and 72°Cfor 45 s. The
PCR products were examined by electrophoresis on 10% nondenaturing poly
acrylamide gels. The gels were fixed in 0.5 M NaC1, 40 mM sodium acetate (pH
4.2), and 50% ethanol for 25 mm and exposed without drying to phosphor
screens. Evaluations and calculations were done with a Molecular Imager and
the Molecular
Analyst
software
(Bio-Rad
Laboratories,
Inc.). To control the
telomerase activity origin of the six-nucleotide ladder, protein extracts were
incubated with RNase (Boebringer Mannheim) for 20 mm at 37°Cprior to
mixing with the reaction mixture. To make semiquantitative assessments of
relative telomerase activity levels, only extract dilutions in which the intensity
of the ITAS was approximately the same as the intensity of the ITAS in the
lysis buffer alone were quantitated. The relative telomerase activity in a sample
was derived from the ratio in intensity between the ITAS and the telomerase
ladder in a lane. The activity value assigned for a sample was the mean of at
least two experiments and was calculated for 0.5 @.tg
protein.
Western
Blotting and Cyclin Dl, Cyclist E, and p16@4
PrOtein Deter
mination. Tissue samples of approximately50 mg were homogenized and
sonicated for 2 x 15 s in lysis buffer [0.5% NP4O,0.5% sodium deoxycholate,
0.1% SDS, 50 mM Tris-HC1 (pH 7.5), 150 mM NaCl, with the following
protease and phosphatase inhibitors: 20 mg/mi leupeptin; 20 mg/mi aprotinin;
10 mg/mi pepstatin; 1 mM phenylmethylsulfonyl fluoride; 1 mM sodium
fluoride; and 1 nmi EDTAJ with adequate cooling during the procedure.
Samples were centrifuged at 14,000 X g for 30 mm at 4°C,and aliquots of the
supernatant were stored at —80°Cuntil further analysis. Total protein concen
tration was determined by a bicinchoninic acid protein assay (Pierce Chemical
Co.). Electrophoresiswas performedon 11%SDS-polyacrylamidegels using
40-mg protein samples per lane (Bio-Rad Minigel System, Bio-Rad Labora
tories, Inc.), and proteins were then transferred to mtrocellulose membranes
Telomerase Activity. Taq polymerase-inhibitory activity was re
vealed in a significant number of samples demonstrated as no PCR
amplification product of the internal standard. In all cases with Taq
polymerase inhibition, the internal standard was amplifiable after
dilution of the cell extracts and estimation of activity levels could be
achieved. Fig. 1 shows examples of tumors with differences in telom
erase activity and with Taq inhibition. By the semiquantitative deter
mination large differences in telomerase activity were demonstrated,
with values ranging from 0 to 153 as illustrated in Fig. 2. The mean
telomerase activity level for all tumors was 10.2 (median, 1.6). Below
an activity of 0.2, no telomere repeat ladder was visible by the eye,
and therefore, a cutoff at 0.2 was used to discriminate telomerase
positive from -negative tumors, giving 90 (84.9%) positive and 16
(15.1%) negative tumors at 0.5 @g
tumor extract. The highest telom
erase activity levels in the tumors were comparable to the activity
found in permanent cell lines (data not shown).
Telomerase Activity in Relation to Cydlin Dl and Cyclin E
Expression. Cylin Dl and cyclin E expression varied considerably
between individual tumors, as described separately (36). In summary,
both cycin Dl and cyclin E showed biphasic distributions, and the
majority of the tumors expressed low protein levels, whereas a frac
tion of the tumors expressed higher and more heterogeneous levels of
one of the cycins or both in combination. The majority of tumors with
high cycin E levels expressed low cydin Dl . Tumors defined to
overexpress cyclin E or Dl were selected with cutoff points based on
550
Downloaded from cancerres.aacrjournals.org on August 3, 2017. © 1997 American Association for Cancer Research.
@
@
@
-@ In
.
C4
In
V)
0
@q
0
0
IL,
TELOMERASE
ACflVITY
AND CELL CYCLE REGULATION
their protein distribution in the tumor samples. Examples of cyclin E,
cydlin Dl, and actin immunoblots of five breast tumors and a control
cell line are presented in Fig. 3A. Correlation analysis revealed a
significant association between cycin Dl protein content and telom
erase activity level (r = 0.3416; P = 0.001; n = 100; not shown in
figures). No such association was found for cyclin E (r = 0.13 15;
P = 0.196; n = 100).
To evaluate telomerase activity in relation to the expression of cell
@
@
@
‘@
,,
‘-4
—
—
e
A
— e@
_______
Cydin E
@-—.
Cydlln Dl
Actin
—@1
—@
—
—
—
B
@
____________
p16Th11@@
@
11
@
.p
i@I
Fig. 3. Western blots of selected breast cancer samples and control cell lines. A, cyclin
E, cyclin Dl, and actin expression in five tumors (the same samples as shown in Fig. 1),
demonstrating obvious differences in protein expression of the two cyclins. Cyclin E
demonstrates several bands shown previously to represent biologically active isoforms
(35), all of which were included in the densitometric quantification. B. pl6@4
seven tumors showing large differences in protein expression.
Fig. 1. TRAP assay data on five breast cancer samples. U210, telomerase negative.
U210 and U214 indicate Taq polymerase inhibition at a protein concentration of 5 .sg (but
U214 is clearly positive) and show amplification of the internal standard after dilution of
the extract.U208,U211,and U212demonstratevariabletelomeraseactivity.
A
L.
.@
=
z
o@
—,@
50
Relative
@
100
telomerase
activity
B
I.
E
=
z
Relative telomerase
activity
and
pRB
Staining.
DV0W, and p16LNK4 high demonstrated
Fig. 2. Histograms showing relative telomerase activity in 106 breast cancer samples.
A, disalbutin
Activity
Seven
out of 73 tumors
(9.6%) studied for pRB expression by immunohistochemistry lacked
pRB, whereas nontumor cells stained positive (Fig. 5). pRB-negative
tumors, which were regarded as
had intermediate telom
erase levels (Table 1), and the telomerase activity was considerably
lower compared to tumors with cyclin D or E defects (Table 2).
Combination of Various Cell Cycle Defects and Telomerase
Activity. Table 2 shows relative telomerase activity in different tu
mor groups subdivided according to the expression of cyclin Dl,
cycin E, pl6'@4, and pRB. Tumors lacking detectable cell cycle
defects, i.e., tumors with cyclin DV0@@,
cyclin
and
@norma1
had
the lowest telomerase activity levels (Table 2). The highest activity
was demonstrated in p@normaltumors with high cyclin Dl or/and
cyclin E expression. For tumors with high cyclin Dl or cycin E
levels, a low expression of pl6tN@@seemed to increase the telomerase
activity. p@abnoflTUL1
tumors, all of which were cyclin Elu5t@,
cycin
4)
@
cycle regulators, the tumors were subdivided into four groups, with
cutoffs at relative telomerase activity levels of 0.2, 0.8, and 8. Sub
dividing the tumors according to cyclin Dl and cyclin E levels
demonstrated that overexpression of each cyclin was associated
strongly with intermediate to high telomerase activity levels (Table 1).
All tumors (n = 5) with high levels of both cyclin Dl and E had high
telomerase activity.
Telomerase Activity and p16'@4 Expression. pl6tN@@protein
levels were estimated by densitometry of immunoblots (Fig. 3B), and
the tumors were divided in three groups (low, intermediate, and high)
according to protein levels. pl6'@―expression did not correlate with
telomerase activity (r = —0.1256; P = 0.211; n = 101), and tumors
with intermediate or high p161N@@
expression did not deviate sigmf
icantly from low p16IN@@expressers regarding telomerase activity
(Table 1 and 2). Interestingly, the highest telomerase activities were
found in tumors with low expression of pl6tN@@(Fig. 4), indicating
that down-regulation of pl6@?4@@
in combination with other cell cycle
defects (Table 2) might contribute to maximum telomerase activity in
tumors.
Telomerase
150
levels in
ofall samples; B, distribution ofsamples with a relative telomerase activity
between 0 and 3.5.
intermediate
telomerase
activ
ity levels (Table 2). A summary of the data is given in Fig. 6,
indicating various cell cycle defects in relation to telomerase when the
tumors were subdivided using a cutoff at 0.8 of relative activity.
551
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TELOMERASE
ACTIVITYANDCELLCYCLEREGULATION
@Statistical
Table I Telomerase activity in breast cancer extracts in relation to the expression of cyclin DI, cyclin E, pI6―@4,
cross-table analysis (x@according to Pearson) comparing tumor distribution within the different subgroups defined by relative telomerase activity (@0.8 and >0.8) and
expression
showeda of the cell cycle regulators showed a significant difference for cyclin Dl (P = 0.021) and cyclin E (P < 0.001) but not for @16@N1@
(P = 0.698), whereas pRB
borderline value (P = 0.059).Cydin
Dla
AlteredRelative
Telomerase activity
Low
Cydin E―
High
Low
p16INK4a
High
Low
@p@b
Intermediate and high
Normal
activitycO.2
telomerase
15133
181
>0.2—0.8
0>0.8—
@
@
@8
1Tumors
>8
100Totalnumber
with telomerase activity >0.8 (%)
a Determined
by
densitometry
of
Western
ll@
4J
15136
2lJ
112
lJ
14126
l2J
2112
101
10123
l3J
0
25
l8J
6
27 1
18J
11 1
12123
28
16J
10 1 24
l4J
23
2lJ
15
9J
24
58
82
55
92
63
67
65
78
28
80
26
70
36
66
o
7
blots.
b Evaluated by immunohistochemistry.
C T'@'@
assay;
relative
activity
in
relation
to
internal
standard,
given
as
number
of
tumors
activityMean telomerase
Median
Eb0w, @[email protected]
37p16b0@v
pl6)@i5I@
Dl@―,
Dlhigh,
@@@norma1
panflOITh@l, pl6bow
D1―@―,
panflonnal, pl6intcrmediatc
E―@',
p@nOflflaI
@
pannormal, p16'°―
EhI5I@, p@flOflfl@1, @16intcrmed1ate
Dlhi5I@,E―@―,
p@flOflfl@l
@
@p@abnorn@aI (all
7All
106Dl,
tumors10.2
and
D1b0w)12.1
0.7
1.5
70
6.3
7.0
1.3
2.5
23
13
18.8
22.3
11.7
13.4
15
11
9.1
8.5
4
24.9
21.6
13
26.6
23.3
9
22.1
20.1
5.2
11.7
21.6
2.5
1.6
4
5
cyclin Dl; E, cyclin E.
@
each
Telomerase Activity and Tumor Cell Proliferation. Expression
of the nuclear antigen Ki-67 is characteristic for cycling cells, and in
the present material, the fraction of Ki-67-positive cells ranged from
4 to 84%, with a mean of 25. 1%. In the total tumor material tested for
Ki-67, a significant and positive correlation to telomerase activity
level was found (r = 0.39; P = 0.001; not shown in figures).
However, regarding tumor subgroups with different cell cycle defects,
another picture emerged as illustrated in Fig. 6, because cycin D1to@
tumors had higher telomerase activity than cydlin DV0―tumors with
a comparable Ki-67 index. More strikingly,
cases dem
onstrated the highest proliferation rate but a telomerase activity lower
than cyclin Dihigh expressers (Fig. 7). Thus, it was concluded that
telomerase activity levels in breast cancer could be dissociated from
the tumor cell proliferation rate and seemed to be connected to the
expression of specific cell cycle regulators.
@‘l4O
-@e
120
@lO0
DISCUSSION
@
category.
cell extract dilutions, potential Taq polymerase-inhibitory activities
can be revealed, and by using an internal standard, the importance of
such inhibition could be verified as in the study of Hiyama et a!. (20).
Our telomerase activity data were calculated for a protein content of
0.5 ,ag, which means that if an extract was diluted 10 times to achieve
amplification of the internal standard, the activity value obtained was
multiplied by 10. Further analysis supported the assumption that
semiquantitative determinations can be useful and of biological sig
nificance, because specific tumor subgroups with defined defects in
the cell cycle machinery had clearly different telomerase activity
levels.
The M1/M2 model proposed by Shay et a!. (9) states that a cell must
bypass the M1 (senescence stage) and M2 checkpoints before immor
talization and that activation of telomerase can occur at M2. The
senescence stage (M @)
can be overcome by inactivation of the pRB or
p53 tumor suppressor proteins, leading to a prolonged life span but
usually not immortalization (6—8).This means that inactivation of the
p53 or pRB pathways could provide a tumor with the capability for
many extra cell divisions and a higher chance for activation of
telomerase. Our data gave some support for this scenario concerning
pRB inactivation.
Regarding p53, it is well recognized that breast cancers frequently
harbor p53 gene mutations and that these also might be of prognostic
significance. Previous studies on human mammary epitheial cells
have indicated that p53, but notably not RB, is involved in the M1
mechanism (45). In the present tumor material, the p53 gene and
protein status were unknown, but studies have been initiated in our
Table 2 Relative
subgroup?Tumor
telomerase activity levels in different breast cancer
NumberDlb0@@@,
groupRelative
in
I.
4)
E
In the present study of 106 breast cancers, semiquantitative deter
mination of telomerase activity levels revealed a spectrum from
negative to strongly positive tumors, and the activity levels were
demonstrated to be associated with defective cell cycle regulation.
The finding of 85% telomerase-positive tumors is in agreement with
recent data on breast cancer showing 73—93%positive cases (20, 41).
Regarding the fraction of telomerase-positive cases, breast cancer is
similar to other epithelial malignancies analyzed with the TRAP assay
(19, 20, 22—24).
Semiquantitative determinations of telomerase activity, as per
formed in the present study, have recently been published for malig
nant and nonmalignant lymphoid cells and skin disorders (26, 44). By
80
060
4)40
.#;
@20
..:
0
.•
...
0.2
...
0.4
0.6
0.8
1.0
p16@4protein
Fig. 4. Dot plot showing
1.2
1.4
1.6
1.8
2.0
level
@l6tN@@4
levels in relation to relative telomerase activity.
Samples with the highest telomerase activity values were found in tumors with low
expression of pl6@4.
552
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TELOMERASE
ACI1VITY
AND CELL CYCLE REGULATION
suggests that down-regulation of pl6@4 alone, which represents a
rather moderate cell cycle defect with preserved cycin Dl-CDK4/6
regulation, is insufficient to reach M2 crisis and activation of telom
erase. There was nevertheless an additive effect of low @165N@@
and
overexpression of cyclins E or Dl on telomerase activity, suggesting
that the @16JNK4
level is important together with other cell cycle
defects. This was highlighted by the fact that all four tumors with a
relative telomerase activity above 60 expressed low pl6@@4 protein
levels.
We have shown recently that cyclin E is overexpressed in a fraction
of breast cancers (36). Cyclin E could potentially have functions
similar to cycin Dl and be involved in the transformation of cells
(54), even though no experimental evidence for cyclin E as an
oncogene has been published. The highest telomerase activity levels
were found in tumors with cycin E overexpression, suggesting that
cycin E deregulation represents a cell cycle defect of importance for
telomerase activity.
The present study is the first demonstrating an association between
telomerase activity and defects in the cell cycle machinery in a human
tumor type, and our data also indicate that such defects might influ
ence the expression levels of telomerase. We found that this associ
ation for breast cancer and the relevance of the findings for other
tumor types remains to be established. Tumors of diverse origin have
different patterns concerning their spectrum of cell cycle defects, and
it is conceivable that differences will be found regarding the effects on
telomerase activity. The data presented here are compatible with the
M11M2 model, but there is a possibility that certain cell cycle proteins
@
.
Cyclin E@―
Fig. 5. Immunohistochemical staining of pRB in breast cancer. A, tumor with pRB
expression in tumor cells as well as in benign cells. B, pan-negative
tumor, with positive
. Cycli
Cyclin Dl―1ti'
reaction in surrounding benign cells.
-#----—
@-
@
pRBnormaI
laboratory to explore its possible impact on activation and regulation
of telomerase.
The fraction of p@thnoflfl@1tumors was somewhat lower than found
previously in breast cancer (46, 47), and all tumors were telomerase
positive but with intermediate activity, indicating that other factors are
important for maintenance of high activity levels. To define the RB
status, we used immunohistochemistry, which is considered to be a
reliable method because of difficulties for mutated pRB to enter the
nucleus as well as a complete lack of staining in tumors with ho
mozygous RB deletions. In support of our immunohistochemical data,
all
tumors expressed high pl6@4 levels, indicating a
stabilization of pl6@4 through association with CDK4/6 due to the
inactivation of RB and down-regulation of cycin Dl (48, 49). We
have also observed a low fraction of hyperphosphorylated pRB as
well as frequent loss of heterozygosity at two intragenic RB polymor
phisms in pRBa@@@@0rmM
tumors supporting the immunohistochemical
approach (50).
In the pRB-regulatory pathway(s), both cydin Dl and pl6@4 are
key factors, and a potential deregulated expression might cause func
tional inactivation of pRB by phosphorylation and unordered entrance
into S phase (31). There are several clinical and experimental data
indicating that cyclin Dl is an oncogene (51—53),and the protein is
overexpressed in a substantial part of breast tumors (33, 53). In the
present study, the expression of cycin Dl was associated with telom
erase activity. @16INK4
down-regulation was in our material the most
common cell cycle defect (65%), giving an increased phosphoryla
tion/inactivation of pRB with enhanced proliferation pressure (31),
but we found no significant correlation to telomerase activity. This
•
Cyclin E@@W
Cyclin D11w
Cyclin
DIM@@
—k.
.
Cyclin
or Cyclin D1―@'
+ :II::::::::@.c:zz::::I
p16b0@@
•
.
pRba@@@@
% = fraction of tumors with relative telomerase activity > 0.8
Fig. 6. A model for acquisition of telomerase activity in breast cancer in relation to
various defects in the expression of the cell cycle regulators cyclin Dl, cyclin E, pl6'@4,
and pRB. The percentages given represent the fraction of tumors with relative telomerase
activity levels >0.8 (see also Table 1).
80
UKI-67(%)—0
activityi—L@@1I-Lr-@
Tdomerue
60
S
@40
20
0
C,dMElsw
CydINEIIIØI C@tIInEISW Cyd1EW@I pRBâsssm@
CyduiDl I.w
C@ Dl low
CydinDl hi@i CydkiDl hid.
Fig. 7. Histogram exploring the relationship between tumor cell proliferation, defined
as Ki-67 antigen-positive cells, and relative telomerase activity in subgroups of breast
cancer with different cell cycle defects.
553
Downloaded from cancerres.aacrjournals.org on August 3, 2017. © 1997 American Association for Cancer Research.
TELOMERASE
ACTIVITY
AND CELL CYCLE REGULATION
can have specific effects on telomerase regulation, an important issue
for future experimental studies on telomerase regulation.
C. A., Nochols, G., Khaled, Z., Telang, N. T, Narayanan, R. Differentiation of
immortal cells inhibits telomerase activity. Proc. Natl. Acad. Sci. USA, 92: 12343—
12346, 1995.
28. Holt, S. E., Wright, W. E., and Shay, J. W. Regulation of telomerase activity in
immortal cell lines. Mol. Cell Biol., 16: 2932—2939,1996.
ACKNOWLEDGMENTS
We thank Bodil Backlund and Ulla-Brin Westman for skillful technical
assistance.
29. Weinberg, R. A. The retinoblastoma protein and cell cycle control. Cell, 81: 323—330,
1995.
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31. Lukas, J., Aagaard, L., Strauss, M., and Bartek, 3. Oncogenic aberrations of pl6―@
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Telomerase Activity Is Associated with Cell Cycle Deregulation
in Human Breast Cancer
Göran Landberg, Niels Hilmer Nielsen, Pia Nilsson, et al.
Cancer Res 1997;57:549-554.
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