|
|
|
Original Paper |
|
|||
|
Acta Biochim |
||||
|
doi: 10.1093/abbs/ gmp058. |
SKP2 siRNA inhibits the degradation of P27kip1
and down-regulates the expression of MRP in HL-60/A cells
Jie Xiao, Songmei Yin*, Yiqing
Li, Shuangfeng Xie, Danian Nie, Liping
Ma, Xiuju Wang, Yudan Wu, and
Jianhong Feng
Department of Hematology, Second Afflicted
S-phase kinase-associated
protein 2 (SKP2) gene is a tumor suppressor gene, and
is involved in the ubiquitinmediated degradation of
P27kip1. SKP2 and P27kip1 affect the proceeding and prognosis of leukemia
through regulating the proliferation, apoptosis and differentiation of leukemia
cells. In this study, we explored the mechanism of reversing of HL-60/A drug
resistance through SKP2 down-regulation. HL-60/A cells were nucleofected
by Amaxa Nucleofector
System with SKP2 siRNA. The gene and protein
expression levels of Skp2, P27kip1, and multi-drug resistance associated
protein (MRP) were determined by reverse transcription-polymerase chain
reaction and western blot analysis, respectively. The cell cycle was analyzed
by flow cytometry. The 50% inhibitory concentration
value was calculated using cytotoxic analysis
according to the death rate of these two kinds of cells under different
concentrations of chemotherapeutics to compare the sensitivity of the cells.
HL-60/A cells showed multi-drug resistance phenotype characteristic by
cross-resistance to adriamycin,
daunorubicin, and arabinosylcytosine,
due to the expression of MRP. We found that the expression of SKP2 was higher
in HL-60/A cells than in HL-60 cells, but the expression of P27kip1 was lower.
The expression of SKP
Keywords leukemia; RNA
interference; S-phase kinase-associated protein 2;
multi-drug resistanceassociated protein
Received: February 17, 2009 Accepted: April 27, 2009
Introduction
Acute myeloid leukemia (AML) is a malignant disease in hematology, which
shows frequent recurrence and a poor outcome despite combined chemotherapy
because of the multi-drug resistance of leukemia cells [1,2].
Therefore, reversion multi-drug resistance of leukemic cells is important to
effectively cure AML patients. The cyclin-dependent kinase inhibitor P27kip1 is a key regulator of the cell
cycle in mammalian cells, which negatively regulates cell cycle progression by
directly inhibiting cyclin/Cdk2 complexes [3,4].
S-phase kinase-associated protein 2 (SKP2) is
involved in the ubiquitin-mediated degradation of
P27kip
sequence-specific post-transcription gene silencing by short small interfering
RNA (siRNA), which is thought to be a powerful
approach for studying gene function and gene therapy. Using the HL-60/A cells
as a model of multi-drug resistance leukemia cells that were resistant to adriamycin, daunorubicin,
and arabinosylcytosine, we found that the expression
of SKP2 or MRP in HL-60/A cells was higher than that in HL-60 cells, but the
expression of P27kip1 was lower. Moreover, we designed and synthesized a pair
of siRNA based on SKP2 sequence, which had very good
effect. The expression of P27kip
Materials and Methods
Cell lines and culture conditions
HL-60 cells were provided by Department of Pharmaceutical Science, Sun Yat-Sen University (Guangzhou, China), maintained in RPMI
1640 medium containing 10% fetal bovine serum, 100 U/ml penicillin, and 100 mg/ml streptomycin at
37ºC in a humidified incubator containing 5% CO2 (Gibco,
Gaithersburg, USA). HL-60/A was constructed using a stepwise increase in
concentration gradient of adriamycin.
It was obtained from Pharmacology Department of Institute of Hematology and
SKP2 siRNA and primer
The design and synthesis of SKP2 siRNA were
completed by Amaxa (
Cell transfection
One nucleofection sample contained 2 ´ 106 cells,
10 ml of 20 mM siRNA and 100 ml Nucleofector
Solution V (Amaxa). The supplemented Cell Line Nucleofector Solution V was pre-warmed to room temperature.
The 6-well plates filled with culture medium containing supplements and serum
were pre-incubated in a humidified 5% CO2 incubator at 37ºC. The cells were resuspended in Cell Line Nucleofector
Solution V to a final concentration of 2 ´ 106 cells/100 ml nucleofection.
The transfection program was started by mixing the nucleofection sample with 10 ml siRNA
and then transferred to an Amaxa certified cuvette. The sample was transferred to a
prepared 6-well plates after the program was finished. Cells were
incubated in a humidified incubator at 37ºC with 5% CO2.
Reverse transcription-polymerase chain reaction
Total RNA was isolated from each group cells using Trizol
regent (Invitrogen, Carlsbad, USA) according to the
manufacturer’s instruction. Total cellular RNA was used for reverse transcription (RT)
of cDNA by a standardized technique (MBI Fermentas,
Western blot analysis
About 1 ´ 106–107 cells
(HL-60, HL-60/A, HL-60/A cells transient nucleofected
with SKP2 siRNA and negative control siRNA for 24, 48, 72 h, respectively) were lysed at 4ºC for 30 min with 100–200 ml RIPA lysis buffer containing
In vitro analysis for cell growth
HL-60 and HL-60/A cells (1.0 ´ 105 cells/well) were seeded in 96-well plates in serum-containing
medium and treated with different chemotherapeutics (adriamycin,
daunorubicin, and arabinosylcytosine)
in different concentrations for 24 h. Cell counting kit-8 (DOJINDO
Laboratories, Tokyo, Japan) was used in each well (10 ml/well). After incubation
for 4 h at 37ºC, the coloring reaction was quantified by an automatic plate reader Wellscan MK3 (Labsystem Dragon,
were resuspended in complete medium, and seeded in
96-well plates (1 ´ 105 cells/ well). After being incubated for 6 h, the cells were
treated with different chemotherapeutics for 24 h, and then used to assess the
cell viability using CCK-8 assay.
Flow cytometry
For cell cycle analysis, 1 ´ 106 cells were stained with propidium
iodide. Briefly, different cells (HL-60, HL-60/A, HL-60/A cells transient nucleofected with SKP2 siRNA and
negative control siRNA for 24, 48, 72 h,
respectively) were washed twice in cold PBS and fixed in ice-cold 70% ethanol
overnight. After two more washes in PBS, propidium
iodide and RNase A (Sigma,
Statistical analysis
Results were presented as the mean±standard deviation. Associations
between the variables were tested by Student’s t-test. All statistical differences were deemed significant at the level of
P< 0.05.
Results
HL-60/A cells had the characteristic of multi-drug resistance
with SKP2, MRP high expression and P27kip1 low expression
The drug resistance of HL-60 and HL-60/A cells was summarized in Fig. 1. According to the
result of CCK-8 assay, IC50 of HL-60 cells to adriamycin, daunorubicin,
and arabinosylcytosine were 0.044, 0.035, 0.04 mg/ml, respectively.
IC50 of HL-60/A cells to adriamycin,
daunorubicin, and arabinosylcytosine
were 2.162, 1.388, and 0.244 mg/ml, respectively. HL-60/A cells were
49.14 fold more resistant to adriamycin
and show multidrug resistance phenotype characteristic by 39.20- and 6.43 fold
cross-resistance to daunorubicin and arabinosylcytosine, respectively.
RT-PCR and western blot analysis were used to detect the transcription and
protein levels of SKP2, P27kip1, and MRP, respectively (Figs. 2 and 3). The transcription level of SKP2 was higher in HL-60/A cells than that
in HL-60 cells. SKP2/b-actin ratios in these two different cell lines were 0.83±0.03% and 0.52±0.01%, respectively.
The difference was statistically significant (P< 0.01). The protein level of SKP2 was also higher in HL-60/A cells, and the
SKP2/GAPDH ratios were 0.58±0.06% and 0.27±0.05% (P< 0.01). The transcription and protein levels of MRP in HL-60/A cells were
higher than those in HL-60 cells. The MRP/b–actin
ratios in HL-60/A and HL-60 cells were 0.61±0.07% and 0.34±0.12%, respectively (P< 0.01). The MRP/GAPDH ratios were 0.60±0.04% and 0.20±0.02%, respectively (P< 0.05). The P27kip1/b-actin and P27kip1/GAPDH ratios in HL-60/ A cells were 0.41±0.05% and 0.30±0.13%, lower than
those in HL-60 cells (P< 0.05) (Figs. 2–5). HL-60/A cells had
larger proportions of S phase and G2/M phase than HL-60 cells HL-60/A cells in the
G0/G1 phase were 35.10±0.81%. HL-60 cells in the G0/G1 phase were 43.96±1.12%. HL-60 cells in
the G0/G1 phase were more than in the HL-60/A cells (Fig. 6). The proportion of S phase and G2/M phase
in HL-60 cells was 56.04%±1.12%, the proportion in HL-60/A cells was
64.90%±0.81%, and the difference was significant (t = – 11.104, P = 0.001). HL-60/A cells had higher ability of proliferation because cell
kinetic analysis could be based on counting of tritiated
thymidine labeled S phase cells and mitotic phase
(G2/M phase) cells.
SKP2 siRNA suppressed the
expressions of SKP2
and the subsequent ubiquitin-mediated
degradation
of p27kip
SKP
SKP2 siRNA induced cell cycle
arrest in G0/G1 phase
The proportion of HL-60/A cells nucleofected by
SKP2 siRNA for 24 h in the G0/G1 phase was increased
comparing with the control cells nucleofected by
negative control siRNA. Our study showed that 48.25±1.67% of HL-60/A cells
accumulated in the G0/G1 phase after nucleofected by
SKP2 siRNA for 24 h, in contrast, 39.67±1.73% of the cells nucleofected by negative control siRNA.
The proportion of S phase and G2/M phase in HL-60/A cells nucleofected
by SKP2 siRNA was 51.73±1.68%. The proportion
of S phase and G2/M phase in HL-60/A cells nucleofected
by negative control siRNA was 60.33±1.73%. The difference
was significant (P< 0.001). After HL-60/A cells were nucleofected by
SKP2 siRNA, the proliferation ability of the cells
was suppressed [Fig. 10(A,B)].
SKP2 siRNA improved the
chemotherapeutic
sensitivity of HL-60/A cells with MRP
down-regulation
After HL-60/A cells were nucleofected by SKP2 siRNA for 24 h, the IC50 values of the cells to adriamycin, daunorubicin,
and arabinosylcytosine were 2.17, 0.33, and 0.06 mg/ml. In the control
groups, the IC50 values of the cells to adriamycin,
daunorubicin, and arabinosylcytosine
were 2.49, 1.59, and 0.22 mg/ml, respectively. The cells nucleofected by SKP2 siRNA were
more sensitive to adriamycin,
daunorubicin, and arabinosylcytosine
by 1.15, 4.79 and 3.76 fold, respectively (Fig. 11). With the down-regulation of SKP2
expression, the transcription and protein levels of MRP were down-regulated.
After the cells were nucleofected by SKP2 siRNA for 24 h, the MRP/b–actin
and MRP/GAPDH ratios were 40±7% and 29±7%, respectively.
Comparing with the cells nucleofected by negative
control siRNA, the transcription and protein levels
of MRP were down-regulated by 55 and 62%, respectively
(Figs. 8 and 12).
Discussion
Leukemia is one of the most common diseases in hematology. After
chemotherapy, about 20% of incipient patients cannot achieve complete remission
(CR) [11,12]. Improving sensitivity of leukemia cells
to chemotherapy is crucial for curing leukemia. SKP2, a kind of oncogenes,
relates to cell cycle regulation closely by regulating the expression of
P27kip1. SKP2 gene
locating within 5p13 amplicon encodes the protein of
436 amino acids, containing an F-box, 10 leucine rich
repeat (LRR) domain and a C-terminal tail. During the ubiquitin-proteasome
pathway, LRR domain directly binds to the substrates, and P27kip1 is one of the
most frequent substrates [13,14]. P27kip1 gene is located at
12p13 encoding a protein with a molecular weight of 27 kDa
[3]. It is one of the members in CDK-interacting protein/kinase
inhibition protein (CIP/KIP) family, inducing G1 cell cycle arrest [4].
Down-regulation of the negative cell cycle regulators results in cell division
[15]. Some researches have demonstrated that the expressions of SKP2 and
P27kip1 were inversely correlated in tumor tissue and both of them were
independent prognostic marker of patients. The expression level of P27kip1could
be one of the useful prognostic molecular markers for
AML. Yokozawa et al. [16]. found
that patients with high P27kip1 expression had a significantly increased
disease-free survival. The SKP2 overexpression was
significantly associated with shorter disease-free survival and overall
survival. SKP2 expression was an independent marker for a poor prognosis in AML
[17].
In this study, HL-60/A cells were 49.14 fold more resistant to adriamycin and showed multi-drug
resistance phenotype characteristic by 39.20- and 6.43 fold crossresistance
to daunorubicin and arabinosylcytosine,
respectively. Wada et al. [18] showed that the multi-drug resistance of HL-60/DOX was attributable
to the high levels of MRP expression in the HL60/DOX cells, and high expressing
MRP induced the alteration of the drug accumulation from enhanced efflux. We
used RT-PCR and western blot analysis to detect the MRP expression and found
that MRP expression was remarkably increased in HL-60/A cells. High MRP
expression might be the reason for the multi-drug resistance of HL-60/A cells.
We found that SKP2 expression in HL-60 cells was lower than that in HL-60/A
cells, but the P27kip1 expression was higher. Results demonstrated that SKP2
and P27kip1 might be new targets for molecular therapy of tumor. Kataqiri et al. [19] found that SKP2 protein was decreased and the P27kip1 protein was
accumulated in SKP2 siRNA-transfected melanoma cells.
SKP2 siRNA inhibited the cell growth of melanoma
cells. Agarwal et al. [20] identified SKP2 as a crucial
mediator of BCR-ABL-induced leukemogenesis and
provided in vivo evidence that SKP2 promoted oncogenesis. Hence
stabilization of P27 by inhibiting its recognition by SCFSkp2 may be
therapeutically useful. Meanwhile, cell cycles of these two kinds of cells were
analyzed by flow cytometry in this study. Results
showed that the G0/G1 phase proportion of HL-60/A cells was less than that of
HL-60 cells, but the number of HL-60/A cells staying in the S phase and G2/M
phase were more than that of HL-60 cells. Results showed that drug-resistance
leukemia cells had greater ability of proliferation than normal leukemia cells.
The results were consistent with the report in which GLC-82 cells were treated
with adriamycin at a final concentration of 0.05 mg/ml. Cells in the S
phase of the cell cycle were increased. MRP expression was significantly
increased in a time-dependent manner, and was positively correlated with
changes in the S phase of the cell cycle [21]. Resent studies demonstrated that
SKP2 and P27kip1 played an important role in the control of tumor cell
proliferation, apoptosis, and differentiation, and affected the development of
tumor eventually. Fu et al. [22] demonstrated that integrins gave rise to
growth inhibition of SMMC-7721 cells through suppressing the proteasomeand calpain-mediated
P27 degradation in SMMC-7721 cells. But there are two different views in the
relationship between SKP2/P27kip1 and drug resistance of leukemia cells. Some
showed that P27kip1 inducing G1 arrested of the cell cycle could make leukemia
cells resistant to those cell cycle-dependent chemotherapeutics. Others
demonstrated that SKP2 and P27kip1 played a major role in drug sensitivity of
leukemia by detecting bone marrow samples from leukemia patients. Patients with
low P27kip1 had poor prognosis, whereas patients with higher P27kip1 were more
sensitive to chemotherapeutics [23]. Sengupta et al. [24] demonstrated
that expression of doubly modified P27kip1 (T
Funding
This work was supported by the grants from the Nature
Science Foundation of
References
1 Kim DH, Lee NY, Sung WJ, Baek JH, Kim JG, Sohn SK and Suh JS, et al. Multidrug resistance
as a potential prognostic indicator in acute myeloid leukemia with normal karyotypes. Acta Haematol 2005, 114: 78–83.
2 Del Poeta G, Stasi R, Aronica
G, Venditti A, Cox MC, Bruno A and Buccisano F, et al. Clinical relevance of P-glycoprotein expression in de novo acute myeloid
leukemia. Blood 1996, 87: 1997–2004.
3 Polyak K, Lee MH, Erdjument
BH, Koff A, Roberts JM, Tempst
P and Massagué J, et al. Cloning
of p27Kip1, a cyclin-dependent kinase
inhibitor and a potential mediator of extracellular antimitogenic
signals. Cell 1994, 78: 59–66.
4 Sherr CJ and Roberts JM. CDK inhibitors:
positive and negative regulators of G1-phase progression. Gene 1999, 13: 1501–1512.
5 Qu Z, Weiss JN and Maclellan
WR.
Regulation of the mammalian cell cycle: a model of the G1-to-S transition. Am J Physiol Cell Physiol 2003, 284: C349–C364.
6 Andren EJ, Lledo E, Poch E, Ivorra C, Albero MP, Martínez-Climent JA and Montiel-Duarte C, et al. BCR-ABL induces the expression of Skp2 through the PI3K pathway to
promote p27Kip1 degradation and proliferation of chronic myelogenous
leukemia cells. Cancer Res 2005, 65: 3264–3272.
7 Wang QH, Zhang M, Fang HH, Nie XX, Gao L, Liu Y and Xie Y, et al. Effect of
adenovirus-mediated p27 gene expression on the proliferation and apoptosis of
HL-60 and Raji cell lines. Hematol
J 2004, 5: 519–523.
8 Seo YJ, Jeon MH, Lee
JH, Lee YJ, Youn HJ, Ko JH
and Lee JH, et al. Bis induces growth inhibition and
differentiation of HL-60 cells via up-regulation of p27. Exp Mol Med 2005, 37:
624–630.
9 Munoz-Alonso MJ, Acosta JC, Richard C, Delgado MD, Sedivy
J and Leo′n J. p21Cip1 and p27Kip1 induce distinct
cell cycle effects and differentiation programs in myeloid leukemia cells. J Biol Chem 2005, 280: 18120–18129.
10 Barata JT, Cardoso AA, Nadler LM and Boussiotis VA. Interleukin-7 promotes survival and cell
cycle progression of T-cell acute lymphoblastic leukemia cells by down-regulating
the cyclin-dependent kinase
inhibitor p27kip1. Blood 2001, 98: 1524–1531.
intensive chemotherapy for adults with de novo acute myeloid leukaemia
(AML): the nationwide AML-92 study by the Finnish Leukaemia
Group. Eur J Haematol 2007,
78: 477–486.
13 Schulman BA, Carrano AC, Jeffrey PD, Bowen Z,
Kinnucan ER, Finnin MS and Elledge SJ, et al. Insights into SCF ubiquin ligases
from the structure of the skp1-skp2 complex. Nature 2000, 408: 381–386.
14 Bornstein G, Bloom J, Sitry-Shevah D,
Nakayama K, Pagano M and Hershko
A. Role of the SCFSkp2 ubiquitin ligase
in the degradation of p21-Cip
LZ and He J, et al. Simultaneous knockdown of p18INK
M, Iida H, Takeyama K, Tanimoto M, Kiyoi H and Motoji T, et al. Prognostic
significance of the cell cycle inhibitor p27Kip
protein expression in acute myelogenous leukemia: its
association with constitutive phosphorylation. Clin Cancer Res 2004, 10: 5123–5130.
18 Wada H, Saikawa Y, Niida
Y, Nishimura R, Noguchi T, Matsukawa H and Ichihara T, et al. Selectively
induced high MRP gene expression in multidrug-resistant human HL60 leukemia cells. Exp Hematol 1999, 27: 99–109.
19 Kataqiri Y, Hozumi
Y and Kondo S. Knockdown of Skp2 by siRNA inhibits
melanoma cell growth in vitro and in vivo. J Dermatol Sci 2006,
42: 215–224.
20 Agarwal A, Bumm TG,
Corbin AS, O’Hare T, Loriaux M, VanDyke
J and Willis SG, et al. Absence of SKP2 expression attenuates BCRABL- induced myeloproliferative disease. Blood 2008, 112: 1960–1970.
21 Xu M and Li J. Development of drug resistance
and alteration of cell cycle in lung cancer: a flow cytometric
study. Zhonghua Zhong Liu Za Zhi 2000, 22: 385–388.
22 Fu Y, Wang LY, Liang YL, Jin JW, Fang ZY and Zha
XL. Integrin
A, Tang R, Marie JP and Ajchenbaum-Cymbalista F. Cell
cycle regulatory protein expression in fresh acute myeloid leukemia cells and
after drug exposure. Leukemia 2001, 15: 559–566.
24 Sengupta A, Banerjee
D, Chandra S and Banerjee S. Gene therapy for BCR-ABL+ human CML with dual phosphorylation resistant
p27Kip1 and stable RNA interference using an EBV vector. J Gene Med 2006, 8:
1251–1261.
25 Yang Q, Sakurai T, Yoshimura G, Takashi Y, Suzuma
T, Tamaki T and Umemura T, et al. Overexpression
of p27 protein in human breast cancer correlates with in vitro resistance to
doxorubicin and mitomycin C. Anticancer Res 2000, 20:
4319–4322.
26 Ishii T, Matsuse T, Masuda M and Teramoto S. The effects of S-phase kinase-associated
protein 2 (SKP2) on cell cycle status, viability, and chemoresistance
in A549 lung adenocarcinoma cells. Exp Lung
Res 2004, 30: 687–703.