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Introduction
The mammalian target of rapamycin (mTOR) pathway
plays an important role in energy metabolism, the regulation
of cell proliferation, and promoting cancer cell
survival[1,2]. The critical functions of mTOR have led to the development
of mTOR inhibitors (MTI), including rapamycin and its
analogs as novel anticancer agents. The anticancer effects of
MTI are presently evaluated in phase 1/2 clinical trials in
various solid tumors. Moreover, the use of MTI is
increasingly documented in hematological malignancies, such
as chronic myeloid leukemia (CML), myelodysplastic syndromes
(MDS), and acute myelogenous leukemia
(AML)[3_6]. However, the antileukemic mechanism of rapamycin has not
been extensively evaluated in acute T lymphoblastic
leukemia (ATLL).
Telomerase is a ribonucleoprotein enzyme, which is
necessary to compensate for telomere shortening during cell
division by synthesizing telomere DNA. In most normal
somatic cell types, telomerase is usually undetectable; however,
the activation of telomerase is seen in cancer cells and is
thought to be a critical element in cancer pathogenesis.
Considering that telomerase activities were documented in some
types of leukemia[7], and in addition, high telomerase
activity and shortened telomere length were associated with
poorer prognosis in adult T-cell leukemia[8]
, antitelomerase strategies in leukemia might be of considerable interest. As
reported in an in vitro reconstitution assay, the human
telomerase reverse transcriptase (hTERT), the rate-limiting
factor for telomerase activity, could be phosphorylated at
serine/threonine residues by protein kinase C and
recombinant protein kinase B/Akt[9], but it remains unknown whether
mTOR, a downstream of Akt, also affects telomerase activity.
Thus, we used Jurkat cells, a cell line established from acute
T lymphoblastic leukemia patient, to investigate the
relationship between AKT/mTOR signaling and telomerase
activity, and to explore the antileukemic mechanism of
rapamycin in Jurkat cells.
In this work, we found that rapamycin displayed potent,
antiproliferative activity against Jurkat cells, probably by
inducing G1 phase arrest other than apoptosis. Moreover,
exposure to rapamycin could reduce telomerase activity in
Jurkat cells via the mTOR-mediated pathway, suggesting that
rapamycin might be a novel potential treatment agent against
acute T lymphoblastic leukemia.
Materials and methods
Cells culture and reagents Human T cell leukemia
Jurkat cells were obtained from the Shanghai Institute for
Biological Sciences, Chinese Academy of Science (Shanghai,
China). The Jurkat cells were routinely cultured in
RPMI-1640 medium (Gibco, Grand Island, NY, USA) supplemented
with 10% fetal bovine serum (Gibco, USA) at 37 °C in a
humidified atmosphere with 5% CO2. Exponentially-growing
cells in a suspension were used in the experiments.
Rapamycin and LY294002 were purchased from Sigma (St
Louis, MO, USA), dissolved in Me2SO, and stored frozen at
_20 °C. The antibodies against cyclin D1, cyclin D2, cyclin
D3, cyclin-dependent kinase (CDK)4, CDK6, p27Kip1,
p21waf1 and phospho-Akt (Ser473), phospho-p70S6K (Thr389), phospho-S6 (Ser235/236), as well as their
non-phosphorylated antibodies were purchased from Cell Signaling
Technology (Beverly, MA, USA). Mouse anti-hTERT 2C4
and peroxidase-conjugated goat antimouse
immunoglobulin M (IgM) were purchased from Abcam (Cambridge, MA,
USA). Rabbit anti-actin antibody, goat antirabbit or
antimouse immunoglobulin G (IgG) conjugated with
horseradish peroxidase were purchased from Santa Cruz
Biotechnology (Santa Cruz, CA, USA).
Assays of cell growth and apoptosis Cell growth was
measured using the
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric reduction
method as previously described. Measurements were taken
in quadruple at 24, 48, and 72 h after drug incubation at the
indicated concentrations. Absorbance at 570 nm was
measured in a BIO-RAD microplate reader (Hercules, California,
USA). Apoptosis was measured by a flow cytometric
analysis of cells stained with Annexin V-fluorescein-isothiocyanate
(FITC) and propidium iodide (PI; BD PharMingen, San Diego,
CA, USA) according to the manufacturer's instructions.
After incubation in Me2SO alone or rapamycin at the
indicated concentrations for 72 h, the cells were pelleted by
centrifugation and incubated with Annexin V-FITC and PI.
Single-cell suspensions were analyzed by FACScan (Becton
Dickinson, San Jose, CA, USA). Early apoptotic cells were
scored as Annexin V+, PI-, whereas late apoptotic cells scored
as Annexin V+, PI+ to exclude necrotic cells (Annexin V-,
PI+).
Cell cycle analysis For the cell cycle analysis,
2×105 Jurkat cells were harvested, washed with ice-cold
phosphate-buffered saline (PBS), fixed in 70% ethanol, and incubated at
4 ºC overnight. The cells were then resuspended in 1 mL
PBS containing 50 μg/mL PI and 100 U/mL RNAseA and
incubated for 30 min at room temperature. The DNA content
was monitored by flow cytometry. Cell cycle was analyzed
with CellQuest software (Becton Dickinson, USA).
Western blot analysis The protein lysates were prepared
from the control and drug-treated cells and separated by
electrophoresis on 12% SDS-PAGE. The proteins were then
transferred to polyvinylidene difluoride membranes.
Subsequently, they were probed for molecules of interest
with specific primary antibodies and then with goat antirabbit
or antimouse IgG or IgM conjugated with horseradish
peroxidase. The protein bands were visualized by a Western
blot chemiluminescence reagent (KPL, Gaithersburg, MD,
USA) according to the instructions of the manufacturer. For
reprobing, the membranes were stripped (0.2 mmol/L
NaOH) and reprobed with the desired antibodies. The
molecular sizes of the protein bands were determined by
comparison with prestained protein markers (M-0671; Sigma,
USA).
Telomeric repeat amplication protocol assay
A PCR-ELISA-based assay (Roche Molecular Biochemicals,
Mannheim, Germany) was used to measure the telomerase
activity, as described in detail by the manufacturer. In total,
2×105 cells were lysed with 200 μL lysis buffer on ice for 30
min. After the pelleting of cellular debris by centrifugation,
the supernatant was stored at -80 °C until further use. Each
telomeric repeat amplication protocol (TRAP) reaction
contained 1 mg of total protein. The telomerase substrate primer
was elongated by telomerase at 25 ºC for 30 min, and then
incubated at 94 ºC for 5 min to induce the telomerase
inactivation. The reaction mixture was then amplified by 30
cycles of PCR at 94 ºC for 30 s, 50 ºC for 30 s, 72 ºC for 90 s,
and 72 ºC for 10 min. The 5 μL PCR products were denatured,
hybridized to a digoxigenin-labeled probe, and detected by
ELISA. Relative telomerase activity (RTA) was calculated as
follows:
RTA=(ODexp-ODneg)/(ODpos-OD
neg)×100%. As a positive control, the human epithelial carcinoma cell
line HeLa was used; as a negative control, a positive sample
that had been subjected to heating (85 °C) for 20 min prior
to the TRAP assay was used.
RT-PCR analysis Total cellular RNA was extracted
using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) and
isolated with the single-step acid
guanidinium-isothiocyanate/phenol-chloroform extraction method. In total, 1
μL total RNA (approximately 1 ng) was used to synthesize cDNA
with random primers and Moloney murine leukemia virus RT
(Promega, Madison, WI, USA). hTERT mRNA (619 bp) was
amplified with the following primers: sense,
5´-CGGAAGAGTGTCTGGAGCAA-3´; antisense,
5´-GGATGAAGCGGAGTCTGGA-3´. β-Actin mRNA (144 bp)
was amplified with the following primers: sense,
5´-CGCTGCGCTGGTCGTCGACA-3´; antisense,
5´-GTCACGCACGATTTCCCGCT-3´. After an initial
denaturation for 3 min at 94 °C, cDNA was amplified in a final volume
of 25 μL with1.25 units Taq DNA polymerase (Promega, USA).
The amplification consisted of 36 cycles (hTERT) or 28 cycles
(β-actin) for 45 s at 94°C, 60 s at 60°C, and 60 s at 72°C. The
PCR product (10 μL) was subjected to electrophoresis on
1.5% agarose containing ethidium bromide and visualized by UV
absorption. The gels were photographed, and the bands
were analyzed by computerized densitometry. β-Actin mRNA
was used as the internal control.
Statistical analysis The data were presented as
mean±SD and analyzed by SPSS 11.0 software (SPSS,
Chicago, IL, USA) using Student's t-test and ANOVA.
Differences were considered statistically significant at
P<0.05.
Results
Effect of rapamycin on growth, cell cycle, and apoptosis in Jurkat cells
Exposure to rapamycin (0.1_100 nmol/L) resulted in a significant inhibitory effect on
the proliferative activity of Jurkat cells: an effect that was
dose- and time-dependent (Figure 1A). The 50 % inhibitory
concentrations (IC50) values for 24, 48, and 72 h were 344, 92,
and 16 nmol/L, respectively. To determine the mechanism of
growth inhibition by rapamycin in more detail, we analyzed
the cell cycle profile after treatment with rapamycin. As
expected, G1 phase arrest was induced by rapamycin (100
nmol/L) after 16 h, and the ratio of G1 continued to grow as
treatment was prolonged to 32 h (Figure 1B). Furthermore,
we evaluated the effect of rapamycin on the cell cycles of
synchronized cells by serum starvation. Serum starvation
resulted in the accumulation of cells in the
G1 phase. Serum stimulation resulted in the transition of cells from the
G1 phase to the S phase with a concomitant decrease in the
G1 phase. Rapamycin significantly blocked serum-induced entry to the
S phase in a dose-dependent manner in Jurkat cells (Figure
1C). In addition, rapamycin did not increase the amount of
cells in the Annexin V+ fraction 72 h after treatment,
confirming no increase in apoptosis (Figure 1D). These results
suggest that rapamycin inhibits cell growth via cell cycle arrest
and not by the induction of apoptosis in Jurkat cells.
Expression of p27Kip1, p21waf1, CDK4, CDK6, cyclin D1, cyclin D2, and cyclin D3 in Jurkat cells treated
with rapamycin We investigated the effects of rapamycin
on G1 phase proteins, specifically p27Kip1, p21waf1, CDK4,
CDK6, cyclin D1, cyclin D2, and cyclin D3 (Figure 2). The
Jurkat cells were serum deprived for 16 h, and then
stimulated with 10% serum and either rapamycin or ME2SO as the
control for 12, 24, and 48 h. First, the levels of p27Kip1 and
p21waf1 were high in serum-starved Jurkat cells (0 h), but
underwent rapid elimination upon the addition of serum.
Rapamycin treatment maintained elevated levels of p27Kip1
and p21waf1. Cyclin D1 was not detected in the Jurkat cells
under any conditions tested, which is in agreement with
previously published observations[10]. Rapamycin did not
affect the protein levels of either CDK4 or CDK6; however,
rapamycin downregulated cyclin D3 without affecting the
cyclin D2 protein levels. This decrease in cyclin D3 protein
levels could be observed as early as 12 h after release from
serum starvation, and it persisted throughout the 48 h
experiment period.
Effect of rapamycin on the PI3K/Akt/mTOR signal
transduction pathway Much is already known about the
PI3K/Akt/mTOR signaling pathway and its modulation of
cell growth. To ascertain it, we characterized the effect of
rapamycin on the cell signaling pathways on Jurkat cells.
The cells were treated with 10 nmol/L rapamycin or 50
μmol/L LY294002 for 0_24 h. As a PI3K inhibitor, LY294002 inhibited
Akt phosphorylation at Ser473. Inconsistent with a
previous model that rapamycin functioned downstream of
Akt[11], rapamycin treatment was found to slightly downregulate the
phosphorylation of Akt (Figure 3A). Previous studies
suggested that p70S6K is a downstream target of
mTOR[12]. The p70S6K kinase directly phosphorylates the 40S ribosomal
protein S6, which results in the enhanced translation of
proteins that contain a polypyrimidine tract in the 5´-untranslated
region[13]. Therefore, the effect of rapamycin on the
phosphorylation of p70S6K and S6 was examined. As a result,
rapamycin markedly reduced the phosphorylation of p70S6K
and S6. This effect was seen as early as 12 h after exposure to
rapamycin, and the total proteins of the pan-S6 and p70S6K
maintained stable levels (Figure 3B). These experimental
data suggest that rapamycin exerts its effect through the
PI3K/Akt/mTOR/p70S6K pathway by regulating the
phosphorylation of p70S6K and S6 proteins.
Rapamycin inhibits telomerase activity in Jurkat
cells To date, it remains unclear how telomerase activity is
differentially regulated by mTOR signaling in various cell systems,
including the human T-cell leukemia cell line. Thus, we
were interested in the regulation of telomerase by rapamycin in
Jurkat cells. First, telomerase activity was measured by the
TRAP assay. When the cells were treated with rapamycin,
telomerase activity was downregulated within 24 h and had
a reduced tendency that was dose- and time-dependent
(Figure 4A). Because telomerase activity correlates with the
expression of hTERT mRNA[14], we used RT-PCR to examine
the expression of hTERT mRNA in Jurkat cells treated with
either rapamycin or Me2SO as the control for 48 h. We found
that treatment with varying concentrations of rapamycin
significantly reduced the hTERT mRNA level in a
dose-dependent manner (Figure 4B). Furthermore, by Western blotting
with the 2C4 monoclonal antibody specific for hTERT, we
noted that the hTERT protein level decreased and correlated
with these changes in the expression of hTERT mRNA
induced by rapamycin (Figure 4C). These data suggest that
rapamycin may inhibit telomerase activity by rapidly
decreasing the hTERT mRNA levels.
Discussion
Acute lymphoblastic leukemia (ALL) is the most
common type of leukemia in young children. This disease also
affects adults, especially those aged 65 years or older. While
the outcome for children with ALL has improved
dramatically with current therapy, children with relapsed ALL and
adults with ALL often develop resistance to standard
chemotherapy. Even under aggressive therapy, these
patient groups have 5 year disease-free survival rates of only
28%_39%[15]. Evidence accumulated over recent years has
indicated the phosphoinositide 3-kinase/Akt/mTOR signal
transduction pathways as one of the major factors
implicated in human leukemia resistance to conventional
chemotherapy[16]. Thus, the development of novel therapeutic
agents override the resistance, and directly targeting this
signaling network is crucial. One potential class of novel
therapeutics is MTI, such as rapamycin.
Rapamycin, a macrocyclic lactone produced by
Streptomyces hygroscopicus, was the first MTI to be used clinically.
Initially developed as an immunosuppressive agent,
rapamycin is well tolerated in humans. Preclinical studies on
human cancer cell lines and human tumor xenograft models
have shown that rapamycin may be effective for the
treatment of prostate, small cell lung, glioblastoma, renal cell, and
breast cancer[17]. Moreover, the use of MTI is increasingly
documented in hematological malignancies, such as CML,
MDS, and AML[3_6]. To data, however, the antileukemic
mechanism of rapamycin has not been extensively
evaluated in ATLL. In this work, we used the human T-cell
leukemia cell line Jurkat to study the antileukemic effect of
rapamycin.
First, we observed that rapamycin could greatly inhibit
the proliferation of Jurkat cells in dose- and time-dependent
manners in vitro. To further determine the mechanism of
growth inhibition by rapamycin, we performed an apoptosis
assay with flow cytometry. As previously demonstrated in
other cancer cell lines, rapamycin did not significantly
induce apoptosis in the Jurkat cells, indicating that it may exert
antiproliferative effects via other mechanisms. It is now well
established that besides apoptosis, cell cycle regulation in
the G1 phase may be a promising target for the development
of new chemotherapeutic anticancer agents. Various
molecules, including cyclins, CDK, and CDK inhibitors play
important roles in controlling major checkpoints in the cell
cycle. Their alterations could lead to tumorgenesis. Cyclin
D acts as a sensor of extracellular mitogenic signals and
plays a critical, rate-limiting role in cell cycle progression
during mid G1 by initiating the multistep process that leads
to pRb inactivation[18,19]. p27Kip1 and p21waf1 act as a
negative regulator of cyclins and CDK activity, and appear to be
essential to arrest cells before the late
G1 restriction point[20,21].
We performed a cell cycle analysis in our work and found
that rapamycin significantly induced G1 arrest and blocked
serum-induced entry to the S phase in a dose-dependent
manner in Jurkat cells. Rapamycin downregulated cyclin D3
without affecting the cyclin D2, CDK4 or CDK6 protein levels,
and maintained elevated levels of p27Kip1and p21waf1.
Therefore, we concluded that rapamycin potently suppressed
proliferation in Jurkat cells partly via the induction of
G1 cell cycle arrest, which was mediated by affecting cyclin D3,
p27Kip1,and p21waf1. To understand the mechanisms by
which rapamycin functions, we characterized the signaling
pathways affected by rapamycin. A previous model showed
that rapamycin functioned downstream of
Akt[11]; unexpectedly, we found that rapamycin treatment also slightly
downregulated the phosphorylation of Akt. This could be explained by data
obtained by others showing that rapamycin inhibits the
assembly of mTORC2, which phosphorylates and activates
Akt[22]. Furthermore, we found that rapamycin reduced the
phosphorylation of mTOR downstream cascade targets p70S6K
and S6 in Jurkat cells. As it is known, p70S6K modulates the
initiation of translation and cell cycle progression through
downstream effectors, such as cyclin D, CDK4 and
CDK6[23], so it is possible to assert that rapamycin inhibits
G1 cell cycle progression through affecting the mTOR/p70S6K-signaling
pathway.
The major novel finding from this work is that rapamycin
potently suppresses telomerase activity in Jurkat cells.
Information about the regulation of telomerase by rapamycin
is presently incomplete, even if some data have been
documented in endometrial cancer
cells[24]. Telomerase, the ribonucleoprotein complex involved in telomere maintenance, is
composed of 2 main components: the telomerase RNA
template[25] and hTERT[26]. In most normal somatic cell types,
telomerase activity is usually
repressed[27]. The activation of telomerase is seen in cancer cells, including leukemia cells,
and is thought to be a critical element in oncology
pathogenesis[28]. Thus, telomerase expression and activity can
serve as a molecular marker of the clinical progression and
prognosis of most leukemias. Antitelomerase strategies have
been proven to be promising in some types of
leukemias[7]. With regards to hTERT, the rate-limiting factor for telomerase
activity[29], we determined the hTERT mRNA and protein
levels in Jurkat cells treated with rapamycin and found that
rapamycin exposure resulted in significantly decreased
levels of hTERT mRNA and protein. This implies that rapamycin
may exert its effect on telomerase through the regulation of
hTERT gene transcription. In mammalian cells, the
expression of various transcription factors, such as E2F,
hypoxia-inducible factor 1a (HIF-1a), and signal transducer and
activator of transcription 3 (STAT3) seems to be modulated by
the mammalian target of
rapamycin[30_32]. Hirotaka et al
reported that the hTERT promoter region between _165 and
+51 contains 2 HIF-1 consensus
motifs[33]. However, the exact transcription factors, which modulate hTERT gene
transcription and are targeted by the mTOR pathway, need to be
further identified.
Although the exact mechanisms are not clear, there may
be some alternative pathways involved in telomerase
downregulation by rapamycin, other than the direct
regulation of hTERT gene transcription. A potential connection
between rapamycin and telomerase regulation is through
rapamycin's effect on the cell cycle. Zhu et
al reported that telomerase activity was high during the S phase, but
undetectable in the G2-M
phase[34]. Our group also found that the
expression of hTERT and telomerase activity were both
upregulated in normal mesenchymal stem cells during their
transit through the S phase (data not shown). If hTERT
transcription is cell cycle dependent, then the effect of
rapamycin on hTERT expression could be the indirect
consequence of cell cycle arrest. As earlier mentioned, p27Kip
is essential in blocking the G1-S transition. Recent studies
have shown that the overexpression of p27 suppresses hTERT
promoter activity and telomerase activity in mouse
embryonic fibroblasts (MEF) deficient in p27 (p27-/-
MEF)[35], which implies that hTERT transcription is cell cycle dependent. We
also found that rapamycin upregulates the p27 protein level
in Jurkat cells, which might correlate with the downregulated
hTERT expression. In addition, as reported in
an in vitro reconstitution assay, hTERT was an Akt kinase substrate
protein, and human telomerase activity was enhanced
through the phosphorylation of the hTERT protein by
recombinant protein kinase B/Akt[9]. In our work, we
demonstrated that rapamycin treatment reduced the
phosphorylation of Akt. Therefore, rapamycin might downregulate hTERT
peptide phosphorylation and telomerase activity together
through suppressing the phosphorylation of Akt.
Taken together, our findings suggest that rapamycin
displays potent antileukemic effects in the human T-cell
leukemia cell line by the inhibition of cell proliferation through
G1 cell cycle arrest and also through the suppression of
telomerase activity. Since telomerase activity appears to
play a critical role in leukemiagenesis, the use of a mTOR
inhibitor, such as rapamycin, is a promising targeted
therapeutic strategy and may have significant clinical
implications in the treatment of some leukemias.
Acknowledgements
We thank Dr Jian-ping LAN from Zhejiang Provincial
People's Hospital for insightful discussion during the course
of this study. We also gratefully acknowledge the generous
help from members of the Institute of Hematology at The
First Affiliated Hospital of Zhejiang University Medical
School.
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