TOXICOLOGICAL SCIENCES 91(1), 132–139 (2006)
doi:10.1093/toxsci/kfj146
Advance Access publication March 1, 2006
Mechanisms of Induction of Cell Cycle Arrest and Cell Death by
Cryptolepine in Human Lung Adenocarcinoma A549 Cells
Huijun Zhu1 and Nigel J. Gooderham
Biological Chemistry (Molecular Toxicology), Faculty of Natural Sciences, Imperial College London, London SW7 2AZ, United Kingdom
Received November 17, 2005; accepted February 2, 2006
We investigated p53-dependent and -independent molecular
events associated with cell cycle alteration and cell death in
human lung adenocarcinoma A549 cells using cryptolepine,
a DNA-damaging agent. After a 24-h treatment, cryptolepine
caused an accumulation of p53 at concentrations of 1.25–10mM
and induction of p21Cip1/WAF1 but only at concentrations up to
5mM. p21Cip1/WAF1 was also strongly induced by cryptolepine
(2.5–5mM) in cells with p53 largely ablated via small interfering
RNA–mediated gene silencing. Cryptolepine induced G1-phase
block at 1.25–2.5mM, S-phase and G2/M-phase block at 2.5–5mM,
and cell death at 10mM. The dead cells displayed condensed and
fragmented nuclei, features of apoptosis. Wortmannin, an inhibitor of ataxia telangiectasia–mutated and DNA-dependent
protein kinase (DNA-PK), caused cell cycle arrest at G1 phase
without inducing p53 and p21Cip1/WAF1 expression and cell death.
The addition of wortmannin partially prevented cryptolepineinduced expression of p53 and p21Cip1/WAF1 together with the Sphase block and sensitized cells to induction of cell death.
NU7026, a DNA-PK–specific inhibitor, showed neither induction
of cell cycle arrest and apoptosis nor the expression of p53 and
p21Cip1/WAF1. The presence of NU7026 caused further reduction of
cells in G1 phase induced by cryptolepine at 5mM without
affecting the induction of p53 and p21Cip1/WAF1 and cell death.
This study using the A549 cell as a model demonstrated that
cryptolepine selects different molecular pathways to cell cycle
checkpoint activation in a dose-specific manner and evokes
a wortmannin-sensitive antiapoptosis response.
Key Words: ATM; DNA-PK; A549 cells; p21Cip1/WAF1; p53;
cryptolepine.
The role of p53 in mediating the cellular effects of DNAdamaging agents has been a major focus for investigation for
more than 20 years. p53 functions as a transcriptional activator
or repressor, depending upon the promoter context (Ginsberg
et al., 1991; Ludes-Meyers et al., 1996). p53 can be stabilized
and modified by phosphorylation in response to cellular
genotoxic stress. The phosphorylation is often mediated by
1
To whom correspondence should be addressed at Institute of Environment
and Health, Cranfield University, Silsoe, Bedfordshire MK45 4DT, United
Kingdom. Fax: þ44 01525 86 3420. E-mail: [email protected].
the phosphatidylinositol 3-kinase (PI3K)–related proteins,
including ataxia telangiectasia–mutated (ATM) and ataxia
telangiectasia and Rad3 (ATR)–related kinase (Banin et al.,
1998; Canman et al., 1998; Tibbetts et al., 1999), of which
ATM plays a central role in sensing DNA double-strand breaks
and repairs of the damage via homologous recombination
(Abraham, 2004). The phosphorylation promotes p53 accumulation and enables p53 to bind to DNA in a sequence-specific
manner to induce or repress the expression of a large number of
genes (Bourdon et al., 1997; el-Deiry et al., 1992; Funk et al.,
1992; Shieh et al., 1997).
Among the p53 target genes, p21Cip1/WAF1 is a cyclindependent kinase (CDK) inhibitor, which binds to cyclinkinase complexes, resulting in the inhibition of CDK activity
during all phases of cell cycle (Xiong et al., 1993). CDKmediated hyperphosphorylation of pRb causes release of E2F
from pRb-mediated inhibition and transactivates genes required for the S-phase entry (Johnson et al., 1993; Kato et al.,
1993). The expression of p21Cip1/WAF1 maintains pRb in a
hypophosphorylated state that sequesters E2F, thereby causing
G1 arrest. p21Cip1/WAF1 also plays a role in p53-mediated G2
arrest through binding directly to and inhibiting CDKs,
including CDK1, which drives cells from G2 into mitosis
(Xiong et al., 1993). In addition, the ability of p21Cip1/WAF1 to
interact with proliferating cell nuclear antigen (Chen et al.,
1995; Luo et al., 1995), a protein functioning to provide replicative polymerases with the high processivity required to
duplicate an entire genome (Johnson and O’Donnell, 2005;
Majka and Burgers, 2004), and sequester E2F, which is
required for cyclin A gene transcription (Yam et al., 2002),
leads to the inhibition of DNA replication. More importantly,
there is increasing evidence suggesting that the universal CDK
inhibitor p21Cip1/WAF1 can be induced via a p53-independent
pathway (Aguero et al., 2005; Aliouat-Denis et al., 2005;
Sheikh et al., 1994).
It is now well established that multiple cascade pathways are
evoked in response to DNA damage. Inhibition of CDK CDC2
by phosphorylation is essential for p53-independent G2 arrest.
ATM can phosphorylate and activate checkpoint kinase (CHK)
1 and CHK2 (Liu et al., 2000; Matsuoka et al., 2000), which in
turn phosphorylates CDK CDC25, causing it to bind to and
Ó The Author 2006. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved.
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CRYPTOLEPINE INDUCES CELL CYCLE ARREST AND APOPTOSIS
therefore to activate 14-3-3 proteins (Chaturvedi et al., 1999;
Furnari et al., 1997; Sanchez et al., 1997). The 14-3-3 proteins
anchor CDC25 in the cytoplasm, preventing the activation of
nuclear CDC2 (Kumagai and Dunphy, 1999; Lopez-Girona
et al., 1999), which is essential for entry into mitosis (Nurse,
1990). DNA-dependent protein kinase (DNA-PK) is another
PI3K-related protein, which plays a key role in the repair of
double-strand DNA breaks via nonhomologous end joining
(Critchlow et al., 1997). It can also phosphorylate many
proteins in vitro, including p53 and CHK2, though its role
in DNA damage signaling and apoptosis is controversial
(Abraham, 2004; Li and Stern, 2005).
Apart from being the regulator of DNA damage–initiated cell
cycle checkpoint, p53 is an important regulator of apoptosis.
Many of its transcriptional targets function at different points
in the apoptosis program (Schuler and Green, 2005). For
example, p53-upregulated modulator of apoptosis initiates
apoptotic cell death by modulating the activity of Bax, another
p53 transcriptional target, to facilitate cytochrome c release
from the mitochondron (Yee and Vousden, 2005). In response
to DNA-damaging stimulus, p53 also rapidly translocates to
mitochondria, where it interacts with Bcl2 and Bcl-xL to
antagonize their antiapoptotic stabilization of the outer mitochondrial membrane (Erster et al., 2004; Mihara et al., 2003).
Clarification of the molecular mechanisms of cell cycle arrest
and cell death induced by different DNA-damaging chemicals
has substantial implications for molecule-targeted cancer
therapy. In this study, we used human lung adenocarcinoma
A549 cells, which express wild-type p53 or have been transfected with small interfering RNA (siRNA) to silence the expression of p53, and inhibitors of different PI3K-related proteins to
discriminate the mechanisms of p53-dependent and -independent cellular events induced by cryptolepine. A plant-derived
alkaloid of a herbal remedy that has traditionally been used for
treating malaria in Central and Western Africa, cryptolepine has
recently been shown to interfere with topoisomerase II and inhibit DNA synthesis (Bonjean et al., 1998). Its inhibitory effects
on cell cycle and potent toxicity to a number of tumor cells
make it a promising antitumor agent (Ansah and Gooderham,
2002; Ansah et al., 2005; Dassonneville et al., 2000).
Delineated through dose-related effects, we report that three
independent pathways lead to cell cycle arrest in response to
cryptolepine treatment. A wortmannin-sensitive target(s) plays
a key role in preventing A549 cells from succumbing to
cryptolepine-induced cell death. Due to its effectiveness in
inducing cell cycle arrest and apoptosis-like cell death via
multiple pathways, cryptolepine deserves further study on its
potential to treat cancer.
MATERIALS AND METHODS
All reagents were purchased from Sigma-Aldrich Chemical Co. (Poole,
United Kingdom), unless otherwise stated.
133
Cell culture. The human lung adenocarcinoma cell line A549 was
purchased from the European Collection of Cell Culture (Wiltshire, United
Kingdom). Cells were cultured in Ham’s F12 medium supplemented with
L-glutamine (20lM), 10% fetal bovine serum, and 10 lg/ml gentamycin (all
from Invitrogen, Paisley, UK) in a humidified incubator at 37°C with 5% CO2.
Treatment of cells. All experiments were performed when cells reached
70% confluence. Different concentrations of stock solutions were made with
culture medium for cryptolepine (gift from Dr. Addae-Kyeremeh, University of
Science and Technology, Kumasi, Ghana) and with dimethyl sulfoxide
(DMSO) for wortmannin, caffeine, and NU7026. The stock solutions were
added to cell culture flasks at a dilution of 1:1000 to give desired final concentrations. In all experiments, the concentration of DMSO was maintained
at 0.1% (vol/vol). Cells were treated with cryptolepine for 24 h in the absence
of inhibitor. When an inhibitor was included, cells were preincubated with
the inhibitor (wortmannin, caffeine, or NU7026) for 30 min and then were
treated with cryptolepine for further 24 h in the presence of the inhibitor.
DMSO was used at 0.1% (vol/vol) as control. All experiments were repeated at
least three times.
Phase contrast and fluorescence microscopy. After treatment, cells in
culture flasks were examined under light microscope for morphology. Floating
cells were collected by centrifugation at 400 3 g for 5 min and incubated with
0.5 ml of phosphate-buffered saline (PBS; containing 100 lg/ml RNase and
5 lg/ml propidium iodide) at 37°C for 30 min. The images of cell in culture
flasks were taken using a Nikon digital camera (COOLPIX 950) at 3100
magnification.
Flow cytometry analysis. The adherent cells were washed with PBS once,
trypsinized, and collected by centrifugation at 400 3 g for 5 min. The cells (106
cells per sample) were fixed in 0.5 ml of cold 70% ethanol at 20°C overnight.
After centrifugation at 1000 3 g for 10 min, cell pellets were incubated with
0.5 ml of PBS containing 100 lg/ml RNase and 5 lg/ml propidium iodide at
37°C for 30 min. Cell cycle distribution was examined by measuring DNA
content using a flow cytometer (FACScan, Becton Dickinson, San Jose, CA) as
described previously (Zhu et al., 2000). A minimum of 104 cells per data point
was examined. The regions marked M1, M2, M3, and M4 represent sub-G1,
G1, S, and G2/M phase, respectively, of the cell cycle.
Design and transfection of RNA oligonucleotides. The siRNAs duplex
with two thymidine residues (dTdT) at the 3# end of the sequence were designed for targeting the p53 gene using the program Sidesign, as recommended
by the siRNA supplier (Dharmacon, Lafayette, CO; sense, 5#-ACUCCAGUGGUAAUCUAC-3#).
Approximately 2.5 3 106 cells were plated per 100-mm plate in media
containing 10% fetal bovine serum, incubated overnight to give 30–50%
confluence. Just before transfection, the medium was exchanged for 3.6 ml
Opti-MEM (Invitrogen). Transfection of the RNA oligonucleotides was
performed using Oligofectamine reagent (Invitrogen) to give a final RNA
concentration of 200nM. The cells were transfected for 4–6 h at 37°C in
a humidified CO2 incubator. One milliliter of growth medium containing 30%
serum was added, and the transfection was carried out for a total of 48 h. Cells
incubated with Oligofectamine reagent alone (no siRNAs) were used as control
(sham transfection). In preliminary transfection experiments, we determined
siRNA dose and temporal aspects of p53 knockdown. Under conditions
described herein, p53 knockdown was > 95% as estimated by the levels of
immunoreactive p53 protein. After a 48-h transfection, cells were treated with
cryptolepine for 24 h and harvested for further analysis.
Immunoblotting. After treatment, cells were trypsinized and collected as
described before. Cell pellets were resuspended in PBS. After one cycle of
freeze and thaw, whole-cell lysates were analyzed for protein concentrations
using the Protein Assay Dye Reagent Concentrate (Bio-Rad Laboratories Ltd.,
Hemel Hempstead, UK, Cat. 5000–6000). Equal volume of 2X Laemmli
buffer was added to each lysate sample prior to boiling the samples at 95°C for
10 min. Ten micrograms of protein from each sample was resolved over 12%
sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to
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ZHU AND GOODERHAM
nitrocellulose membranes. The antibodies and dilutions used included the
following: anti-p53 (FL-393, sc-6243, 1:1000), anti-p21 (CP74, 1:2000,
Neomakers, Fremont, CA), anti-b-actin (A-5441, 1:10,000), anti-rabbit IgGHRP (A4914, 1:1000, Santa Cruz, CA), and anti-mouse horseradish peroxidase
(HRP)–conjugated antibody IgG-HRP (A-5441, 1:10,000, Santa Cruz, CA).
The membranes were incubated with the respective antibodies at 4°C overnight.
After three washes with PBS containing 0.015% (vol/vol) Tween 20 for 10 min
each, the membranes were incubated with anti-mouse or anti-rabbit IgG-HRP
for 1 h at room temperature. The membranes were washed three times again
and developed using enhanced chemiluminescence (Amersham Biosciences,
Uppsala, Sweden).
RESULTS
Effect of Wortmannin, NU7026, and Caffeine on the
Induction of Cell Cycle Arrest and Cell Death
by Cryptolepine
After 24 h, cells treated with DMSO (a) formed a monolayer
(Fig. 1a). It was observed that cell density across the whole
culture surface was generally lower in the flasks treated with
wortmannin (10lM, Fig. 1b) or cryptolepine (5lM, Fig. 1c)
than those treated with DMSO, suggesting that these two agents
inhibited cell growth. Cells treated with wortmannin displayed
elongated shape, while cells treated with cryptolepine looked
more irregularly shaped. The difference in morphology between
cells treated with wortmannin and cryptolepine may reflect the
stages of cell cycle arrest induced by these two agents as seen in
Figures 2b and 2g. Treatment with cryptolepine alone at 10lM
(Fig. 1d) or at 5lM in the presence of wortmannin (Fig. 1e)
resulted in an increase in the number of floating cells (floaters,
indicated by arrows), which were rarely seen in DMSO-treated
cells (Fig. 1a). The floaters displayed condensed and fragmented nuclei (Fig. 1f), distinctive features of apoptosis.
In the DMSO (0.1% vol/vol)-treated sample, 57% cells
distributed in G1 phase of the cell cycle (Fig. 2a). There were
more than 80% of cells in G1 phase in samples treated with
wortmannin alone (Fig. 2b) or with cryptolepine at 1.25–
2.5lM in the absence and presence of wortmannin (Figs. 2c–
2f). At 5lM, cryptolepine-induced S-phase and G2/M-phase
block (M3 þ M4, Fig. 2g), with the S-phase blocking being
abrogated in the presence of wortmannin (compare M3 in
Fig. 2h with that in Fig. 2g). The diminished S-phase blocking
was associated with the occurrence of apoptosis-like cell death
(Fig. 1f). At 10lM, cryptolepine alone or together with wortmannin induced an increase of cells in the sub-G1 phase (Figs. 2i
and 2j), indicating the induction of apoptosis (Darzynkiewicz
et al., 1992; Nicoletti et al., 1991). To further understand the
molecular mechanism of cryptolepine-induced cell cycle
arrest, we used NU7026 (inhibitor of DNA-PK) and caffeine
(inhibitor of ATM and ATR). NU7026 and caffeine alone had
no distinctive phase-specific effect on cell cycle compared to
control (Figs. 3a, 3b, and 3c). NU7026 at 10lM apparently
depleted G1-phase cells treated with cryptolepine at 5lM
(Fig. 3e), suggesting that DNA-PK is involved in cryptolepineinduced G1 checkpoint. On the other hand, caffeine slightly
attenuated cryptolepine-induced S-phase and G2-phase block
(Fig. 3f).
Effects of Wortmannin and NU7026 on the Expression of
p53/p21 Induced by Cryptolepine
After 24 h, cells treated with DMSO (0.1% vol/vol) or wortmannin (10lM) expressed low levels of p53 and p21Cip1/WAF1
(Fig. 4A, lanes 1 and 2). Cryptolepine induced an accumulation
of p53 at doses 1.25–10lM (Fig. 4A, lanes 3, 5, 7, and 9). The
upregulation of p21Cip1/WAF1 was seen in cells treated with
cryptolepine at 1.25–5lM (Fig. 4, lanes 3, 5, and 7), but not at
10lM (Fig. 4A, lane 9). Wortmannin largely prevented the
accumulation of p53 and the upregulation of p21Cip1/WAF1
induced by cryptolepine at 1.25lM (Fig. 4A, compare lane
4 with lane 3) and to a lesser degree with cryptolepine at
FIG. 1. Effect of cryptolepine on cell morphology changes in the presence and absence of wortmannin in A549 cells. Cells were treated with 0.1% (vol/vol)
(a), wortmannin alone (b), and cryptolepine (CLP) at 5 and 10lM (c and d, respectively) for 24 h. Cells were also preincubated with wortmannin and further treated
with cryptolepine at 5lM for 24 h (e). The floating cells were stained with propidium iodide (f). Images were taken as described in the ‘‘Materials and Methods’’
section. The data are representative of at least three consistent experiments.
CRYPTOLEPINE INDUCES CELL CYCLE ARREST AND APOPTOSIS
135
FIG. 2. Effect of cryptolepine on cell cycle progression in the presence and absence of wortmannin. A549 cells were treated with cryptolepine (CLP) for 24 h
at concentrations of 0–10lM with (b, d, f, h, and j) or without (a, c, e, g, and i) preincubation with wortmannin (10lM, WM) for 30 min. The figures represent the
percentage of cells distributed in each phase of the cell cycle with each treatment.
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FIG. 3. Effects of NU7026 and caffeine on cryptolepine-induced cell cycle alterations in A549 cells. Cells were treated with 0.1% (vol/vol) DMSO (a),
NU7026 (10lM, b), caffeine (4mM, c), or cryptolepine (CLP, 5lM, d) for 24 h. In cotreatment, cells were preincubated with NU7026 (10lM, e) or caffeine
(4mM, f) for 30 min and further treated with cryptolepine for 24 h.
2.5–10lM (Fig. 4A, compare lanes 6, 8, and 10 with lanes 5, 7,
and 9), although cryptolepine induced much less accumulation
of p21Cip1/WAF1 at 10lM. These results suggest that a wortmannin-sensitive target(s) is in part responsible for cryptolepine-induced accumulation and activation of p53. NU7026, on
the other hand, was not effective in preventing cryptolepineinduced accumulation of p53 and p21Cip1/WAF1 (Fig. 4B),
though it effectively depleted G1-phase cells (Fig. 3e).
Caffeine induced a substantial amount of expression of p53
FIG. 4. Effect of wortmannin and NU7026 on cryptolepine-induced
expression of p53 and p21Cip1/WAF1 in A549 cells. Cells treated with
cryptolepine in the presence or absence of wortmannin or NU7026 were
subjected to immunoblotting assay. (A) Cells were preincubated with
wortmannin (WM) for 30 min and treated with cryptolepine (CLP) for 24 h.
(B) Cells were preincubated with NU7026 for 30 min and treated with
cryptolepine (CLP) for further 24 h. b-Actin was use as a loading control.
and p21Cip1/WAF1 (data not shown), and therefore, was not
informative.
Effect of p53 on Cell Cycle Progression in
Cryptolepine-Treated Cells
In order to understand the role of p53 in the regulation of cell
cycle progression in cells treated with cryptolepine, siRNAmediated p53 gene silencing was conducted. Compared with
sham-transfected cells, p53-specific siRNA-transfected cells
displayed no increase in the expression of p53 after the
treatment with cryptolepine at 0.6–2.5lM (Fig. 5A, compare
lanes 4, 6, and 8 with 3, 5, and 7). However, p21Cip1/WAF1 was
induced by cryptolepine even in the absence of the induction of
p53 (Fig. 5A, lanes 6 and 8), although cryptolepine-induced
induction of p21 was more pronounced in sham-transfected
cells. This suggests that both p53-dependent and -independent
pathways contribute to the upregulation of p21Cip1/WAF1 by
cryptolepine. The transfection of p53-siRNA only weakly reduced induction of p53 by cryptolepine at 5lM. It is likely that
a higher concentration of siRNA is needed to effectively
downregulate the expression of p53 induced by cryptolepine
at concentrations of 5lM or higher. G1-phase cells were largely
diminished in samples transfected with p53-siRNA and treated
with cryptolepine at 2.5 and 5lM (Fig. 5B, i and j), compared
with cells subjected to the same treatment in the absence of
p53-siRNA transfection (Fig. 5B, d and e). This diminishment
of G1 phase appeared to be associated with a decreased
expression of p21Cip1/WAF1 (Fig. 5A, compare lanes 8 and 10
with 7 and 9).
CRYPTOLEPINE INDUCES CELL CYCLE ARREST AND APOPTOSIS
137
FIG. 5. Effect of p53 on cryptolepine-induced cell cycle alteration in A549 cells. Cells with or without p53-siRNA transfection were treated with cryptolepine
(0–5lM, CLP) for 24 h. Cells were subjected to immunoblotting assay (A) and cell cycle profiling (B).
DISCUSSION
In this study we observed that cryptolepine inhibited cell
growth through the induction of cycle arrest at either G1 phase
or S and G2/M phase, dependent on the concentration of the
agent. Although cryptolepine induced the upregulation of
p21Cip1/WAF1 to a substantial level at 0.6lM, an effect on cell
cycle was only observed at higher concentrations, suggesting
that upregulation at low dose was insufficient to induce cycle
arrest. This is compatible with the suggestion that other mechanisms, such as posttranscriptional modification may contribute to the control of p21Cip1/WAF1 activity (Scott et al., 2000).
By using inhibitors of PI3K-related proteins, we investigated
the role of these proteins in cryptolepine-induced cell cycle
arrest and cell death. Unexpectedly, wortmannin had effect on
cell cycle progression in A549 cells, suggesting that the
target(s) of this inhibitor is required for maintaining normal
cell growth status in A549 cells. Wortmannin prevented Sphase arrest and potentiated apoptotic cell death induced by
cryptolepine, suggesting that ATM and DNA-PK are involved
in these events. To examine whether DNA-PK plays a role in
cryptolepine-induced cell cycle arrest and cell death, we
employed NU7026, which has been recently developed as
a specific DNA-PK inhibitor (Veuger et al., 2003; Willmore
et al., 2004). NU7026 had no effect on G1 arrest induced by
cryptolepine at 1.25lM (data not shown), but it reduced cells in
the G1 phase induced by cryptolepine at 5lM without affecting
the level of p53 expression and apoptosis. Our study is
consistent with the report that p53 response to DNA damage
in vivo is independent of DNA-PK (Jhappan et al., 2000). Our
results suggest that DNA-PK contributes to the control of G1phase checkpoint, while another wortmannin target other than
DNA-PK is responsible for resistance to cryptolepine-induced
cell death. Indeed, it has been reported by others that
wortmannin converts cellular response from growth arrest to
apoptosis in cells with wild-type p53 (Ren et al., 2003) via
blocking PI3K/AKT signaling pathway.
Depletion of p53 by siRNA reduced the level of p21Cip1/WAF1
protein and abrogated the G1-phase arrest induced by cryptolepine at 2.5lM but not at 1.25lM, suggesting that the G1
arrest induced by cryptolepine at these two concentrations involves p53-independent and -dependent mechanisms, respectively. It is evident that the depletion of p53 expression only
partially reduced the induction of p21Cip1/WAF1 by cryptolepine, suggesting a p53-independent mechanism involved in
the regulation of p21Cip1/WAF1. In this regard, estrogen receptor
alpha and mitogen-activated protein kinases (MAPK) dependent upregulation of p21Cip1/WAF1 have been reported
(Margueron et al., 2003; Park et al., 2000), though we have
not further investigated the mechanism of p53-independent
induction of p21Cip1/WAF1 by cryptolepine. Abrogation of G1
checkpoint renders cell cycle arrest at S phase and G2/M phase
upon cryptolepine treatment. It is likely that p21Cip1/WAF1,
which is only partially reduced by depleting p53 or wortmannin
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ZHU AND GOODERHAM
treatment, is responsible for cryptolepine-induced activation of
these cell cycle checkpoints.
By using a DNA-PK–specific inhibitor and siRNA-mediated
p53 silencing, we are able to distinguish multiple pathways
involved in cell cycle arrest at the G1 phase. It appears that
cryptolepine activates this cell cycle checkpoint via p21Cip1/
WAF1
-dependent, p53-dependent, and DNA-PK–dependent
pathways. The p21Cip1/WAF1-dependent pathway appears to
be the most sensitive one responding to 1.25lM of cryptolepine
followed by the p53-dependent pathway responding to 2.5lM
of cryptolepine. The DNA-PK–dependent pathway is the least
sensitive one requiring 5lM of cryptolepine. It has been
reported that cryptolepine induces DNA double-strand breaks
in vivo, and the primary cellular target of cryptolepine is DNA
(Ansah et al., 2005); perhaps, the different activation of these
pathways reflects the different degree of DNA damage. It
appears that a wortmannin-sensitive target(s) plays a role in
protecting cells from cryptolepine-induced cell death in A549
cells, although the nature of the death needs to be further
characterized.
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