Knocking-Down Cyclin A2by siRNA Suppresses
Apoptosis and Switches Differentiation Pathways in
K562 Cells upon Administration with Doxorubicin
Xiaohui Wang, Yujun Song, Jinsong Ren, Xiaogang Qu*
Division of Biological Inorganic Chemistry, State Key Laboratory of Rare Earth Resources Utilization, Changchun Institute of Applied Chemistry, Graduate School of the
Chinese Academy of Sciences, Chinese Academy of Sciences, Changchun, Jilin, China
Cyclin A2is critical for the initiation of DNA replication, transcription and cell cycle regulation. Cumulative evidences indicate
that the deregulation of cyclin A2is tightly linked to the chromosomal instability, neoplastic transformation and tumor
proliferation. Here we report that treatment of chronic myelogenous leukaemia K562 cells with doxorubicin results in an
accumulation of cyclin A2and follows by induction of apoptotic cell death. To investigate the potential preclinical relevance,
K562 cells were transiently transfected with the siRNA targeting cyclin A2by functionalized single wall carbon nanotubes.
Knocking down the expression of cyclin A2in K562 cells suppressed doxorubicin-induced growth arrest and cell apoptosis.
Upon administration with doxorubicin, K562 cells with reduced cyclin A2showed a significant decrease in erythroid
differentiation, and a small fraction of cells were differentiated along megakaryocytic and monocyte-macrophage pathways.
The results demonstrate the pro-apoptotic role of cyclin A2 and suggest that cyclin A2 is a key regulator of cell
differentiation. To the best of our knowledge, this is the first report that knocking down expression of one gene switches
differentiation pathways of human myeloid leukemia K562 cells.
Citation: Wang X, Song Y, Ren J, Qu X (2009) Knocking-Down Cyclin A2by siRNA Suppresses Apoptosis and Switches Differentiation Pathways in K562 Cells upon
Administration with Doxorubicin. PLoS ONE 4(8): e6665. doi:10.1371/journal.pone.0006665
Editor: Hany A. El-Shemy, Cairo University, Egypt
Received April 27, 2009; Accepted July 7, 2009; Published August 17, 2009
Copyright: ? 2009 Wang et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by NSFC (20831003, 90813001, 20833006) and funds from the Chinese Academy of Sciences and Jilin Province. The funders
had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: firstname.lastname@example.org
Tumor cells are characterized by deregulation of cell cycle
checkpoints, leading to uncontrolled cell division and proliferation
under conditions where non-transformed cells cannot enter and
pass through the cell cycle. All these may come from over-
expression of cyclins and the abnormal activation of cyclin-
dependent kinases (CDKs) . Cyclins are a superfamily of
proteins whose levels vary in a cyclical fashion during the cell cycle
to activate specific CDK required for the proper progression
through the cell cycle. Cyclin A2, which is essential for initiation
and progression of DNA replication as well as for cell cycle
progression through G1/S and G2/M transitions [2–5], is over-
expressed in a variety of human cancers compared with normal
cells and tissues [6–15]. Deregulated expression of cyclin A2seems
to be closely associated with early events in tumor transformation
[6,15]. In addition, its expression level in many types of cancers
appears to be of prognostic value such as prediction of
aggressiveness, survival or early relapse [9–14].
Until recently it has been held that CDK2, presumed master of
the known CDK isoforms, is a promising anticancer target for
developing small molecule inhibitors. The first-generation CDK
inhibitors, flavopiridol and CY-202, are in late-stage clinical trials,
and have only modest activity . Recent findings , however,
suggest that CDK2 may not be a key cell cycle player and question
whether selective CDK2 inhibition is a useful cancer therapy
strategy. No cell cycle abnormalities are observed in either a CDK2
null mouse or following acute ablation of CDK2 in primary cells,
indicating that this gene is not strictly required for cell proliferation
. In contrast, deletion of cyclinA2inknockout miceis associated
with an embryonic lethal phenotype . Moreover, Fine et al.
demonstrated that cyclin A2and/or cyclin A2- CDK2 complex but
not CDK2 is a promising anticancer target with a high therapeutic
index . Therefore, inhibitors of CDK2 may not be appropriate
for cancer therapy and more efforts are focused on inhibition of
cyclin A2and/or cyclin A2- CDK2 complex activity. We have
shown that reduction of cyclin A2in human chronic myelogenous
leukaemia K562 cells using small interfering RNA significantly
inhibits cell proliferation , further supporting the notion that
cyclin A2can serve as a novel therapeutic target.
Apoptosis and differentiation are the predominant two mech-
anisms by which chemotherapeutic agents kill tumor cells. Low
dose of doxorubicin (DOX) induces erythroid differentiation in
K562 cells, while high concentration of DOX promotes apoptosis
. Although many molecular pathways are involved in the
apoptosis-regulatory mechanism, evidences suggest that the cell
cycle and apoptosis may be interconnected [22–28]. Several
studies have shown that increased expression of cyclin A2is found
in cells in response to several apoptotic stimuli, but very few studies
have dealt with this issue directly [29–33]. So it is important to
clarify whether the decreased expression of cyclin A2is a cause of
cell differentiation or a result of differentiation response.
PLoS ONE | www.plosone.org1 August 2009 | Volume 4 | Issue 8 | e6665
Carbon nanotubes possess the unique features of being able to
enter a living cell without causing its death or without inflicting
other damage and can shuttle biological molecules into mamma-
lian cells, indicating their potential application as a vector for the
delivery of therapeutic molecules [34–36]. Recently we have
reported that single wall carbon nanotubes (SWNTs) can induce a
sequence-dependent B-A DNA transition , selectively induce
human telomeric i-motif DNA formation , accelerate S1
nuclease cleavage rate , cause single-stranded poly(rA) to form
a duplex structure and bind to human telomeric i-motif DNA
under molecular-crowding conditions [40,41]. Herein, we explore
whether altering the levels of cyclin A2in K562 cells using RNAi
delivered by SWNTs can influence cell apoptosis and differenti-
ation induced by chemotherapeutic agent DOX. K562 cells with
reduced cyclin A2 showed a significant decrease in growth
suppression, apoptosis and erythroid differentiation, and were
differentiated along megakaryocytic and macrophage-monocytic
pathways upon administration with DOX. These findings indicate
a positive correlation between cyclin A2and apoptosis induced by
DOX and suggest that cyclin A2 is a key regulator of cell
differentiation, supporting the notion that cyclin A2 is an
important regulator for cell cycle as well as for cell apoptosis
and differentiation. To the best of our knowledge, this is the first
report that knocking down expression of one gene can switch
K562 cells differentiation pathways.
Upregulation of cyclin A2during apoptosis of K562 cells
induced by DOX
DOX can inhibit growth of a variety of cancer cells . To
measure apoptosis rates induced by varying concentrations of DOX
in K562 cells, we use acridine orange (AO)/ethidium bromide (EB)
staining assay (Fig. 1). The percentage of apoptotic cells increased
with time in a dose dependent fashion. RT-PCR and western
blotting were used for studying the effect of DOX on the expression
of cyclin A2in K562 cells. As shown in Fig. 2, cyclin A2expression
levelsincreasedwiththeincrease ofDOX,and apositivecorrelation
was observed. These results show that expression of cyclin A2was
significantly up-regulated in DOX-treated K562 cells at time points
when DOX caused a significant amount of apoptosis, indicating
that the levels of cyclin A2are correlated with the ability of DOX to
induce apoptosis in K562 cells.
Suppression of DOX-induced growth inhibition by down-
regulation of cyclin A2with siRNA delivered by SWNTs
We have previously demonstrated that functionalized single wall
carbon nanotubes (SWNTs) can efficiently deliver siRNA targeting
cyclin A2into K562 cells, resulting in specific suppression of cyclin
A2expression . Here, we use SWNTs to transfect cyclin A2
siRNA into K562 cells and evaluate the effect of cyclin A2on
growth inhibition induced by DOX. Two hours after transfection,
DOX was added. Cell proliferation was examined by trypan blue
exclusion method and methylthiazolyldiphenyl-tetrazolium bro-
mide (MTT) assay. Fig. 3 showed cell growth curves of K562 cells
after various treatments. It can be seen that depletion of cyclin A2
inhibited cell proliferation while carbon nanotubes vector had no
apparent effect on growth inhibition as well as no additive or
synergetic effect on cell toxicology of DOX. DOX (0.4 mM)
significantly suppressed cell growth, nevertheless, much less
growth inhibition was observed in cells incubated with both cyclin
Figure 1. Dose- and time-dependent apoptosis of K562 cells
upon administration with DOX. Apoptotic cells were determined
by AO/EB staining. Tests were done in triplicate, counting a minimum of
300 total cells from at least three random microscope fields each.
Figure 2. Expression of cyclin A2in K562 cells administered
with varying concentrations of DOX. RT-PCR (A) and western
blotting (B) were performed 32 hours and 60 hours after drug
Figure 3. Growth curves of K562 cells in response to cyclin A2
siRNA delivered by SWNTs and DOX. The viable cells were counted
by trypan blue exclusion at indicated time points. The data shown here
represent the average of two independent experiments.
Cyclin A2, Dox, K562 Cells
PLoS ONE | www.plosone.org2August 2009 | Volume 4 | Issue 8 | e6665
A2siRNA and DOX, supported by microscopic results (Fig. S1).
In order to further quantitively investigate the effect of down-
regulation of cyclin A2on the growth inhibition, MTT assays were
carried out 24 h after incubation with DOX. Interaction of DOX
with MTT was also checked in a cell free system and the results
indicated that DOX showed no inference to MTT assay (Fig. S2).
As shown in Fig. 4, IC50 of non-transfected cells was about
2.5 mM. For siRNA transfected K562 cells, IC50 was about
5.0 mM. The results clearly demonstrate that down-regulation of
cellular cyclin A2level suppress DOX-induced growth inhibition.
Suppression of DOX-induced apoptotic cell death by
down-regulation of cyclin A2with siRNA delivered by
To investigate whether cyclin A2participates in cell apoptosis
death and determine if reduction of cyclin A2has an effect on
apoptosis induced by DOX, siRNA specific for cyclin A2 was
transfected into K562 cells by SWNTs, and low dose of DOX
(0.4 mM) was administered. As shown in Fig. 5, carbon nanotubes
vector showed no apparent cell toxicology, while numerous larger
cells and typical lobular nuclei were observed in cells incubated with
DOX. For cells coadministered with cyclin A2siRNA and DOX,
much less apoptotic nuclei were observed, and the size of live cells
was much bigger than that of the control cells. Statistics analysis
showed that down-regulation of cyclin A2significantly reduced the
apoptosis rate from more than 60% to about 20%. Similar results
were obtained by annexin V-PI double staining flow cytometric
technique (as shown in Figure S3). These clearly indicated lowering
cyclin A2level in K562 cells led to suppression of apoptosis, thus a
marked decrease in DOX susceptibility, which provide direct
evidence that cyclin A2is involved in cellular responses to apoptosis.
The cytoplasmic subcellular distribution of cyclin A2
correlates with apoptosis of K562 cells induced by DOX
Early reports have demonstrated that there is a link between
cyclin A2subcellular localization and its cell function, and the level
of cyclin A2is correlated to cell apoptosis [22,23,26]. For clarifying
whether the subcellular distribution of cyclin A2in K562 cells
correlates with apoptosis induced by DOX, indirect immunoflu-
orescence detection of cyclin A2was performed. As shown in
Fig. 6A, a significant fraction of cells underwent apoptosis and
orange nuclei were observed, where DOX was mostly located.
The immunofluorescence labeling of cyclin A2showed its presence
predominantly in the nucleus of control K562 cells (Fig. S4),
whereas in cells administered with DOX, cyclin A2was mainly
located at the cytoplasm of early and late phases of apoptotic cells
(Fig. 6B). Cyclin A2labeling was not found in the K562 cells
incubated with non-immune serum (Fig. 6C). The results indicated
that translocation of cyclin A2from the nucleus to cytoplasm was
connected with its role in apoptosis.
Suppression of cyclin A2by siRNA can switch
differentiation pathways of K562 cells induced by DOX
As mentioned above, cells treated with both cyclin A2siRNA and
DOX were much bigger than the control. Since enlarged phenotype
may suggest cell differentiation, we performed the benzidine staining
to assess erythroid differentiation, which was the differentiation
pathway of K562 cells upon treatment with anthracycline antibiotics
including DOX [42,43]. Representative microscopy images of the
benzidine staining in K562 cells after various treatments were shown
in Fig. S5. For untreated cultures and cells administered with
SWNTs, the percentages of benzidine positive cells were very low
(less than 2%). Forty hours after incubation with DOX, around 14%
benzidine positive cells were observed. However, down-regulation of
cyclin A2by siRNA in K562 cells substantially suppressed erythroid
differentiation upon administration with DOX (less than 1%
benzidine positive cells, as shown in Fig. 7).
To determine whether K562 cells with reduced cyclin A2upon
treatment with DOX underwent megakaryocytic pathway and
monocyte-macrophage differentiation, which are the other two
differentiation pathways of K562 cells, flow cytometric measure-
ment of megakaryocytic specific surface antigen CD61 (GPIIIa)
and nitro blue tetrazolium (NBT) reduction assay were carried out,
respectively. As shown in Fig. S6, in comparison with the control,
cells co-administered with cyclin A2siRNA and DOX showed
significant increase in granularity measured by side scatter (side
scatter, on Y-axis) and cell size measured by forward scatter
(forward scatter, on X-axis), which was in good agreement with our
AO/EB staining observation. Cells treated with DOX or SWNTs
showed no detectable expression of CD61 (GPIIIa) (shown in Fig.
S7), whereas in K562 cells co-administered with cyclin A2siRNA
and DOX, the fraction of CD61 (GPIIIa) positive cells (,10%)
was clearly observed (Fig. 8). For NBT reduction assay,
representative microscopy images of K562 cells after various
treatments were shown in Fig. S8. In cells treated with DOX or
SWNTs, the percentage of NBT positive cells was very low (1%),
whereas a small fraction NBT positive cells (,6%) was observed in
K562 cells co-administered with cyclin A2 siRNA and DOX
(Fig. 9). Although the fractions of cells undergoing megakaryocytic
(,6%) were small, the difference is statistically significant
compared to the control group. Considering that DOX is an
erythroid differentiation-inducing agent for K562 cells [21,42,43],
it is not surprising that only a small fraction of K562 cells with
reduced cyclin A2 underwent megakaryocytic pathway and
Taken together, these results indicate that knocking down the
expression of cyclin A2 suppressed DOX-induced erythroid
differentiation and a small fraction of K562 cells with reduced
cyclin A2were differentiated along megakaryocytic and monocyte-
Figure 4. Effect of depletion of cyclin A2on the survival of
K562 cells treated with DOX. Cells were transfected with cyclin A2
siRNA (#) or not (N) two hours prior to the addition of drug. The
viability was assessed by MTT assay as specified in Materials and
Methods. The data shown here were means of two separate
experiments performed in triplicate.
Cyclin A2, Dox, K562 Cells
PLoS ONE | www.plosone.org3 August 2009 | Volume 4 | Issue 8 | e6665
macrophage pathways upon treatment with DOX. These findings
suggest that cyclinA2is an important regulatorofcelldifferentiation.
Cyclin A2is particularly interesting among the cyclin family
because it can activate two different CDKs and functions in both S
phase and mitosis. In S phase, phosphorylated cyclin A2-CDK2
complexes are suggested to play an important role in the initiation
of DNA replication. In mitosis, cyclin A2may contribute to the
control of cyclin B stability. Consistent with its role as a key cell
cycle regulator, overexpression of cyclin A2 is associated with
transformed cells [6–15]. However, it is difficult to determine
whether elevation of cyclin A2is a contributing factor or a mere
Materials and Methods section. Shown here were the representative images of three separate experiments. (A): upper left, untreated control cells; upper
right, cells treated with transfection vector SWNTs for 32 h; lower left, cells were incubated with 0.4 mM DOX for 32 h, many cells appeared typical
apoptotic bleb phenomenon; lower right, two hours after cyclin A2siRNA transfection, cells were then administered with 0.4 mM DOX for additional
32 h. B: quantification of the live, apoptotic, and necrotic cells. Tests were done in triplicate, counting a minimum of 300 total cells each.
Cyclin A2, Dox, K562 Cells
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consequence of the increased cell proliferation. In haematological
malignancies, cyclin A2is associated with proliferation rate of these
disorders and can be used for molecular diagnostics as a
proliferation marker [13,14].
Single-walled carbon nanotubes (SWNTs) have been considered
as the leading candidate for nanodevice applications ranging from
gene therapy and novel drug delivery to membrane separations.
We have previously showed that siRNA transfection efficiency of
lipofectamine 2000 in K562 cells was low (28%) and some cells
underwent apoptosis and necrosis during the process .
However, SWNTs could efficiently facilitate the coupling of
siRNA to form siRNA:SWNTs complexes and carry siRNA into
K562 cells, significantly knocking down the expression of target
gene. No apparent cell toxicology was observed. Hence, in order
to directly probe whether cyclin A2participates in cell apoptosis
and differentiation, we employed SWNTs as transfection vector to
deliver cyclin A2siRNA into K562 cells to specifically knock down
the expression of cyclin A2 and found that carbon nanotubes
vector showed no additive or synergetic effect on cell toxicology of
DOX, which was consistent with our previous report .
DOX, a prominent member of anthracycline antibiotics, has
been extensively used for treatment of solid tumors and leukemia.
It exerts its cytotoxic activity against cancer cells mainly by
intercalation into DNA, inhibition of topoisomerase II and
helicase activity, leading to cell-cycle arrest at the G2/M phase
and apoptosis . In clinical applications, doses of DOX are
strictly limited by its cardiotoxicity . It should be noted that the
dose of DOX administrated here (0.4 mM) is pharmacological
relevant compared to the initial or steady-state plasma concentra-
tions observed in patients after standard bolus infusions (5 mM and
25–250 nM, respectively).
It has been reported that there is a link between cyclin A2and
apoptosis[22–28]. Hoang etal.showed that inc-mycoverexpressing
serum deprived rat 1A fibroblasts undergoing apoptosis, cyclin A2
mRNA expression was increased, in contrast to the invariant
expression for cyclin B, C, D1 and E . Moreover, serum-
deprived rat1Afibroblaststably transfected withcyclinA2exhibited
Figure 6. The sub-cellular distribution of cyclin A2in K562 cells
correlated with cell apoptosis. Cells were administered with 0.4 mM
DOX for 32 h. A significant fraction of cells underwent apoptosis.
Immunofluorescence detection of cyclin A2was performed as described
in Materials and Methods section. A: representative fluorescence
microscopy image of K562 cells after treatment. Orange nuclei were
observed, where DOX was mostly located; B: representative immuno-
fluorescence microscopy image of K562 cells. Cyclin A2, normally
located at nucleus, can be observed in cytoplasm of early and late
phases of apoptotic cells. C: representative microscopy image of
negative control immunofluorescence in K562 cells. No significant non-
specific signal was observed.
Figure 7. Knocking–down the expression of cyclin A2in K562
cells significantly inhibited erythroid differentiation induced
by low concentration DOX. Cells were transfected with cyclin A2
siRNA or not two hours prior to the addition of 0.4 mM DOX. Forty hours
later, erythroid differentiation was scored by the benzidine staining
method to determine the percentage of hemoglobin-positive K562
cells. Tests were done four times, counting a minimum of 300 total cells
from at least three random microscope fields each. *p,0.05, **p,0.001
vs. control untreated cells by Student’s t-test.
Cyclin A2, Dox, K562 Cells
PLoS ONE | www.plosone.org5 August 2009 | Volume 4 | Issue 8 | e6665
increased apoptosis following stimulation of cyclin A2expression.
Meikrantz et al. reported that induction of apoptosis was uniformly
associated with activation of cyclin A2-dependent kinases but not
associated with cyclins E or B, and overexpression of the cyclin A2
could circumvent the anti-apoptosis activity of the oncogene BCL-2
inhuman Hela cells[25,26]. Furthermore,Hiromuraetal.indicated
that apoptosis was associated with an increase in cytoplasmic cyclin
A2-CDK2activity followingUV irradiation,under theseconditions,
nuclear cyclin A2-Cdk2 activity decreased significantly . Our
results showed knocking down the expression of cyclin A2in K562
cells significantly suppressed the apoptosis induced by DOX and a
positive correlation between the levels of cyclin A2and apoptosis
was observed. The findings also indicate that the cytoplasmic
subcellular distribution of cyclin A2correlates with its pro-apoptotic
role. We speculate that cyclin A2associated kinases are involved in
DOX-induced apoptotic cell death pathways in K562 cells,
although the exact downstream mechanisms are not known.
We have demonstrated that SWNTs could effectively deliver
cyclin A2 siRNA into K562 cells, significantly suppressing the
expression of cyclin A2with specificity and cell proliferation, and
cells with reduced cyclin A2showed a decrease in the percentage
of cells in S phase . Several studies have indicated that cancer
cells with a high S-phase fraction/high proliferative activity are
more sensitive to apoptosis induced by chemotherapy [44,45]. As
for DOX, it is active throughout the cell cycle, but the effect is
most pronounced for cells in S phase-G2 phase, especially in S
phase, where it interferes with the DNA replication and
transcription . Therefore, it was not surprising that K562
cells with reduced cyclin A2showed a marked decrease in DOX
Most of chemotherapeutic agents show significant side effects
and not all patients benefit from aggressive chemotherapy.
Therefore, searching for tumor biological factors which can
predict patient prognosis and chemotherapy response would be of
most importance. Several studies have indicated that a high level
of cyclin A2expression may be a marker of poor prognosis in
cancers [10–13]. Besides, previous studies have shown that cancer
patients with high level of cyclin A2had better chemotherapy
response and survival than those with reduced cyclin A2and low
expression of cyclin A2, indicating that the patients with high
expression of cyclin A2are more suitable for chemotherapy [46–
48]. Our results demonstrate the pro-apoptotic role of cyclin A2in
human myeloid leukemia K562 cells, and indicate that cells with
low level of cyclin A2were more resistant to chemotherapeutic
agent DOX. Poon et al. have suggested that a decrease of cyclin
A2, rather than increase, promotes tumorigenesis, and once the
tumor has developed, high levels of cyclin A2simply reflect a high
proliferation rate, which can explain this inconsistency .
Hence, despite its association with transformed cells, evaluating
cyclin A2level in patients will be an important prognostic marker
for use of chemotherapy. Patients with high level of cyclin A2may
be more responsive to anticancer drugs through the induction of
apoptotic cell death. Moreover, it should be cautious to combine
doxorubicin chemotherapy with any small molecule drug targeting
cyclin A2/cyclin A2 associated kinases since it can enhance
potential drug resistance.
In several systems, it has been reported that down-regulation of
cyclin A2and its associated CDK 2 activity are important for
successful differentiation [29–33]. Ito et al. have suggested cyclin
A2overexpression is directly related with poor differentiation .
Kiyokawa et al. have indicated that differentiation of murine
Figure 8. Flow cytometric measurement of megakaryocytic
specific surface antigen CD61 (GPIIIa) in K562 cells co-
administered with cyclin A2 siRNA and DOX. Cells were
transfected with siRNA by SWNTs two hours prior to the administration
of 0.4 mM DOX. Sixty hours later, expressions of CD61 (GPIIIa) were
evaluated by using FITC-conjugated isotype control immunoglobulin
and specific anti-CD61 (GPIIIa) FITC-conjugated monoclonal antibody.
The marker placed to the right of histogram designates positive events.
Figure 9. Knocking-down expression of cyclin A2in K562 cells
induced monocyte-macrophage differentiation upon adminis-
tration of DOX. Cells were transfected with siRNA by SWNTs two
hours prior to the treatment of 0.4 mM DOX. Ninety six hours later, NBT
dye reduction was used to qualitatively monitor monocyte-macrophage
differentiation. Tests were done twice, counting a minimum of 300 total
cells from at least three random microscope fields each. **p,0.001 vs.
control untreated cells by Student’s t-test.
Cyclin A2, Dox, K562 Cells
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erythroleukemia cells induced by hexamethylene is accompanied
by a decrease in the level of cyclin A2and CDK2 proteins and the
persistent suppression of cyclin A2expression may play a role in
HMBA-induced commitment to terminal differentiation .
Yoshizumi et al. showed down-regulation of cyclin A2 gene
expression in vivo at both the RNA and protein levels appears to
be important in the permanent withdrawal of human and rat
cardiomyocytes from the cell cycle during development .
Moreover, Rieber et al. demonstrated that the interaction of cyclin
A2with E2F is the target for tyrosine induction of B16 melanoma
terminal differentiation . In this work, we found that knocking
down the expression of cyclin A2 in K562 cells significantly
suppressed DOX-induced erythroid differentiation and a small
fraction of cells with reduced cyclin A2were differentiated along
megakaryocytic and monocyte-macrophage pathways upon treat-
ment with DOX. To the best of our knowledge, this is the first
report that knocking down expression of one gene can switch
K562 cells differentiation pathways. The results suggested that
cyclin A2 is directly involved in the checkpoint of cell
differentiation pathways and is a key regulator of this process,
although the detail downstream mechanisms are not known. For
cancer cells with low level of cyclin A2, which are less responsive to
chemotherapeutic agents, induction of differentiation might be an
alternative strategy. Combination of cyclin A2siRNA and DOX
may provide a novel option of such therapeutic strategy.
In conclusion, knocking down the expression of cyclin A2by
siRNA delivered by SWNTs suppresses apoptosis and erythroid
differentiation, and promotes megakaryocytic and monocyte-
macrophage differentiation in human chronic myelogenous
leukaemia K562 cells upon administration with DOX. The results
demonstrate the pro-apoptotic role of cyclin A2and suggest that
cyclin A2is a key regulator of cell differentiation, supporting the
notion that cyclin A2is an important regulator for cell cycle as well
as for cell apoptosis and differentiation.
Materials and Methods
K562  was used in this study.
The human erythroleukemic cell line
Doxorubicin (DOX), Acridine Orange (AO), Ethidium Bromide
(EB), Methylthiazolyldiphenyl-tetrazolium bromide (MTT), DAPI,
Nitro blue tetrazolium (NBT), benzidine and SWNTs (w=1.1 nm,
purity.90%) were purchased from Sigma-Adrich (St. Louis, MO,
USA). Annexin V apoptosis detection kit was obtained from
Keygentec (Nanjing, China). The dye mix for the EB/AO staining
was 100 mg/mL acridine orange and 100 mg/mL ethidium
bromide in phosphate buffered saline (PBS).
The human erythroleukemic cell line K562  was grown in
Iscove’s modified Dulbecco’s medium (Gibco BRL) supplemented
with 10% fetal calf serum in a humidified 37uC incubator with 5%
CO2. Cells were passed three times per week. DNA fluorochrome
staining (DAPI or Hoechst 33258) is used as our routine
mycoplasma detection and the cells used for experiments are free
of contamination. Exponentially growing cells were used for all
experiments described below. Cell viability was determined by
trypan blue exclusion in a haemocytometer chamber.
The siRNA oligonucleotides were synthesized by Genepharma
Corporation (Shanghai, China). The sequence used for targeting
silencing of cyclin A2 was 59-CCAUUGGUC CCUCUU-
GAUUTT-39. The nonsilencing control siRNA is an irrelevant
siRNA with random nucleotides UUCUCCGAACGUGUCAC-
GUTT. Functionalized single wall nanotubes (f-SWNTs) were
prepared according to method described in our previous work
. Cyclin A2siRNA: f-SWNTs complexes (wf-SWNTs/wsiRNA
=40) were added at 25 n mol/L (siRNA concentration) to culture
Reverse transcriptase-polymerase chain reaction (RT-PCR)
Total RNA was extracted using Trizol reagent (Invitrogen)
according to the manufacturer’s instructions. The primers and
conditions for cyclin A2 were TCCATGTCAGTGCTGA-
GAGGA (59), GAAGGTCCATGAGACAAGGC (39); 94uC for
30 seconds , 60uC for 30 seconds, 72uC for 1 minute for 25 cycles.
Three introns are present in this pair of primer so that any
contaminating genomic DNA would not be amplified. Primers
(GAPDH) were ACCTGACCTGCCGTCTAGAA (59), TCCAC-
CACCCTGTTGCTGTA (39). Two sets of primers were used for
each sample, including primers specific for the gene of cyclin A2
and primers for GAPDH as an internal control. All PCR products
were visualized on 1.5% agarose gel with 0.5 mg mL21EB.
For Western blot analysis, cells were lysed in radio-immuno-
precipitation assay (RIPA) buffer (150 mmol/L NaCl, 50 mmol/L
Tris-HCl, 0.5% sodium deoxycholate, 1% NP-40, 0.1% SDS,
pH 7.6) containing protease inhibitors for protein extraction.
Protein concentrations were determined using the Bradford assay.
20 mg cell samples were denatured by addition of 26 reducing
sample buffer (100 mmol/L Tris, 4% SDS, 25% glycerol, 10% b-
mercaptoethanol, 0.01% bromphenol blue, pH 6.8), incubated for
10 minutes at 95uC, and separated on a 12% SDS-PAGE. The
proteins were electroblotted to PVDF membrane. After blocking
with Tris-buffered saline containing 0.05% Tween 20 (TTBS) and
2% BSA, the membranes were incubated overnight at 4uC with
appropriate primary antibody diluted in TTBS. Working dilutions
were: 1/500 rabbit polyclonal anti-cyclin A2primary antibody
(Lab Vision Corporation, CA, USA); 1/400 anti-GAPDH primary
antibody (Santa Cruz, CA, USA). The membranes were washed
three times in TTBS for 5 min each and then incubated for 1 h at
room temperature with goat anti-rabbit IgG conjugated to
UK). After extensive washing in TTBS, the protein–antibody
complexes were visualized by CN/DAB substrate according to
method described by Yong . Images were photographed using
a UVP gel documentation system (Ultraviolet Products, Upland,
K562 cells were plated in 96-well culture plate at a
concentration of 0.56104cells/well, and were transfected with
cyclin A2siRNA or not by SWNTs. Two hours later, the cells were
treated with various concentrations of DOX while the blank
control wells were added medium without drug. Cells were then
cultured for another 24 hours and 20 mL MTT (5 mg/mL) was
added in each well, followed by additional four hour incubation.
The supernatants were then discarded carefully and 150 mL
dimethylsulphoxide was added and shaken vigorously to dissolve
the purple precipitation formation. Optical density (OD) of each
well was tested using Bio-Rad model-680 microplate reader with a
wavelength of 490 nm.
Cyclin A2, Dox, K562 Cells
PLoS ONE | www.plosone.org7August 2009 | Volume 4 | Issue 8 | e6665
Cell fluorescence staining
Cells were collected by centrifugation at 2006g for 5 minutes,
and then washed twice with PBS. Cell concentration was adjusted
as 26106256106cells/mL. 1 mL EB/AO dye mix was added in
10 mL cell suspension, followed by 10 minutes of incubation in
dark. Stained cells suspension were placed on a clean microscope
slide and covered with a cover-slip. Cells were viewed and counted
using an Olympus BX-51 optical system microscope (Tokyo,
Japan) at 4006 magnification with a blue filter. Pictures were
taken with an Olympus digital camera. We note that the definition
is sharper by eye through the microscope than in the photo. Tests
were done in triplicate, counting a minimum of 300 total cells from
at least three random microscope fields each.
Indirect immunofluorescence detection
Cells were harvested, washed three times with PBS, fixed with
4% paraformaldehyde in PBS for 30 min at 4uC and permeabi-
lized with 0.5% Triton X-100 in PBS for 5 min at 4uC. After
blocking in 10% goat serum, primary cyclin A2antibody (rabbit,
polyclonal; Lab Vision Corporation, CA) was diluted to 1:400.
Goat anti-rabbit FITC-conjugated secondary antibody (Jackson
Immunoresearch, Stratech, UK) was used at a 1:200 dilution. If
necessary, DAPI was used to visualize cell nuclei. Cells were
observed and photographed by fluorescence microscopy with oil
immersion objective and appropriate filters.
Erythroid differentiation was scored by the benzidine staining
method for the determination of the percentage of hemoglobin-
positive K562 cells induced by low concentration DOX. Briefly,
cells were washed twice and then resuspended in 20 mL PBS.
10 mL of benzidine solution (0.2% benzidine, 0.6% H2O2, 0.5 M
acetic acid) was added and incubated for 20 min at room
temperature in the dark. Benzidine-positive cells (blue-black
staining) were quantitated by light microscopy. At least 300 cells
were counted in triplicate for each condition.
Flow cytometry analysis
Expression of GPIIIa is considered the most selective marker of
the megakaryocyte lineage since it is not expressed on cells of other
hematopoietic lineages from normal human bone marrow. Hence,
staining of cells for surface CD61 (GPIIIa) was used to evaluate
megakaryocytic differentiation. It employed a mouse monoclonal
antibody fluorescein isothiocyanate (FITC)-conjugated anti-CD61
(eBioscience) or isotype-matched immunoglobulin (IgG1-FITC,
eBioscience) at a concentration of 0.6 mg/mL. Cells were
harvested, washed, resuspended in PBS containing 10% fetus calf
serum and 0.1% NaN3and incubated for 30 min on ice with
antibodies in the dark. After washing three times, cells were
resuspended in PBS containing 0.5% formaldehyde and 0.1%
NaN3, and then analyzed on a FACS Arial cytometer (Becton-
Dickinson, San Diego, CA, USA). Viable cells were gated using
forward and side scatter characteristics. Fluorescence intensity
data were acquired using the BD FACSDIVATMsoftware.
NBT reduction assay
NBT dye reduction was used to qualitatively monitor monocyte-
macrophage differentiation. Briefly, cells were collected, washed
with PBS and resuspended in IMDM medium without serum.
Cells suspension were mixed with an equal volume of 0.1% NBT
dissolved in PBS and incubated at 37uC for 40 min. NBT was
reduced to insoluble formazan because of the intracellular oxygen
radical release in the cells differentiated to monocytes-macro-
phages. The percentage of cells containing intracellular reduced
blue-black formazan was determined by light microscopy. At least
300 cells per preparation were observed.
Data are expressed as mean6s.d. and analysis of variance was
carried out using Student’s t test with Origin 7.5 (OriginLab
Corporation, Northampton, MA, USA), where p,0.05 was
cells significantly suppressed growth inhibition and apoptosis
induced by DOX. Cells were plated in 6-well plate at a density of
0.86105 cells/mL and transfected with cyclin A2 siRNA (A, B) or
not (C, D) by SWNTs two hours prior to the administration of
0.4 mM DOX. 96 hours later, cells were viewed using an inverted
microscope with objectives610 (A, C) and 640 (B, D). Pictures
were taken with an Olympus digital camera. Shown here are the
representative images of three independent experiments.
Found at: doi:10.1371/journal.pone.0006665.s001 (0.72 MB TIF)
Knocking-down the expression of cyclin A2 in K562
assay was performed as follows: 200 mL of medium containing
DOX at different concentrations was placed in a 96-well plate;
20 mL of MTT solution (5 mg/mL) was added to each well; After
incubation for 4 h at 37uC, 150 mL of DMSO was added to each
well and absorbance at 490 nm was measured in a Bio-Rad
model-680 microplate reader. DOX-free complete medium was
used as control and was treated in the same way as the DOX-
containing media. Variation (%)=(absorbance of DOX contain-
ing medium - absorbance of control)/absorbance of control6100.
Found at: doi:10.1371/journal.pone.0006665.s002 (3.08 MB TIF)
Chemical interaction of DOX with MTT assay. The
treatments. Upper left, untreated control; upper right, cells treated
with transfection vector SWNTs; lower left, cells treated with
0.4 mM DOX for 32 h; lower right, two hours after cyclin A2
siRNA transfection, cells were then administered with 0.4 mM
DOX for additional 32 h.
Found at: doi:10.1371/journal.pone.0006665.s003 (0.24 MB TIF)
Annexin V-PI double staining of cells after various
in control K562 cells. DAPI was used to visualize cell nuclei. Cells
were viewed using an Olympus BX-51 optical system microscope
(Tokyo, Japan) with oil lens and appropriate filters. Representative
stained fields are shown: (A), DAPI staining (blue); (B), immuno-
fluorescence detection of cyclin A2 (FITC, green); (C) merged
image. As indicated, cyclin A2 was located at the nucleus of K562
cells without DOX treatment.
Found at: doi:10.1371/journal.pone.0006665.s004 (0.43 MB TIF)
Indirect immunofluorescence detection of cyclin A2
staining of K562 cells after various treatments. Cells were
transfected with cyclin A2 siRNA or not two hours prior to the
addition of 0.4 mM DOX. Forty hours later, erythroid differen-
tiation was scored by the benzidine staining method as described
in Materials and Methods section. Cells were viewed and counted
using an Olympus BX-51 optical system microscope (Tokyo,
Japan) at 2006 magnification. Four independent tests were
performed. Pictures were taken with an Olympus digital camera.
Found at: doi:10.1371/journal.pone.0006665.s005 (0.65 MB TIF)
Representative microscopy images of the benzidine
treatments. Cells were transfected with cyclin A2 siRNA or not
two hours prior to the administration of 0.4 mM DOX. Cells were
Morphological changes of K562 cells after various
Cyclin A2, Dox, K562 Cells
PLoS ONE | www.plosone.org8 August 2009 | Volume 4 | Issue 8 | e6665
then cultured for another 32 hours. Flow cytometry was used to
show changes in size (forward scatter, on X-axis) and granularity
(side scatter, on Y-axis). No significant morphological changes of
K562 cells were observed upon RNAi mediated by SWNTs
compared to the control. Cells administered with DOX underwent
G2/M arrest and changes in granularity and cell size were clearly
observed. Although knocking-down the expression of cyclin A2 by
RNAi significantly inhibited growth suppression and apoptosis
induced by DOX, cells co-administered with siRNA targeting for
cyclin A2 and DOX showed similar increases in cell size and
granularity compared to those treated with DOX alone.
Found at: doi:10.1371/journal.pone.0006665.s006 (2.11 MB TIF)
specific surface antigen CD61 (GPIIIa) in K562 cells. Cells were
incubated with 0.4 mM DOX (A, B) or SWNTs (C, D) for 60 h.
Expressions of CD61 (GPIIIa) were evaluated by using FITC-
conjugated isotype control immunoglobulin (A, C) and specific
anti-CD61 (GPIIIa) FITC-conjugated monoclonal antibody (B,
D). The marker placed to the right of histogram designates positive
events. K562 cells administrated with low concentration of DOX
or SWNTs did not undergo apparent megakaryocytic differenti-
Flow cytometric measurement of megakaryocytic
Found at: doi:10.1371/journal.pone.0006665.s007 (1.96 MB TIF)
reduction assay of K562 cells after various treatments. Cells were
transfected with cyclin A2 siRNA or not two hours prior to the
addition of 0.4 mM DOX. Ninety six hours later, NBT dye
reduction was used to qualitatively monitor monocyte-macro-
phage differentiation. Cells were viewed and counted using an
Olympus BX-51 optical system microscope (Tokyo, Japan) at
2006 magnification. Two independent tests were performed.
Pictures were taken with an Olympus digital camera.
Found at: doi:10.1371/journal.pone.0006665.s008 (0.34 MB TIF)
Representative microscopy images of the NBT
We thank Yong Chen (College of Life Science, Jilin University) and
Professor Yongchen Zheng (Central Laboratory of the 2nd Hospital, Jilin
University) for their technical assistance.
Conceived and designed the experiments: JR XQ. Performed the
experiments: XW. Analyzed the data: XW YS JR. Contributed
reagents/materials/analysis tools: YS. Wrote the paper: XW JR XQ.
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