HL-60 / A. Relationship between Expression of Cell Regulatory Factor SKP2 and Drug Resistance of HL-60 / A Cells

Μέγεθος: px

Εμφάνιση ξεκινά από τη σελίδα:

Download "HL-60 / A. Relationship between Expression of Cell Regulatory Factor SKP2 and Drug Resistance of HL-60 / A Cells"

Λυσιστράτη Σαμαράς
7 χρόνια πριν
Προβολές:

1 ( ) JOURNAL OF SUN YAT-SEN UNIVERSITY(MEDICAL SCIENCES) Vol.30 No.2 Mar SKP2 HL-60 / A, *,,,,,, (, ) : SKP2 P27 kip1 HL-60 / A HL-60, RT-PCR Western blot SKP2 P27 kip1 MRP mrna,, HL- 60 / A ;HL-60 / A MRP HL-60; HL-60 / A HL-60 G0 / G1 (35.10 ± 0.81)%(43.96 ± 1.12)%, P < 0.05;HL-60 / A SKP2 HL-60 ;P27 kip1 HL-60 HL-60 / A MRP HL-60 / A, : ; S 2; ; : R733.7 : A : (2009) Relationship between Expression of Cell Regulatory Factor SKP2 and Drug Resistance of HL-60 / A Cells XIAO Jie, YIN Song-mei *, XIE Shuang-feng, NIE Da-nian, LI Yi-qing, MA Li-ping, WANG Xiu-ju, FENG Jian-hong (Department of Hematology, The Second Affiliated Hospital, SUN Yat-sen University, Guangzhou , China) Abstract: Objective To observe the proportion of cell cycle and the expression of cell cycle regulators S-phase kinaseassociated protein 2 (SKP2) and P27 kip1 in drug-resistant leukemia cell line HL-60 / A and drug non-resistant cell line HL-60, and to investigate the relationship between proliferation and multi-drug resistance of leukemia cells. Methods The expression of SKP2, P27 kip1, and multi-drug resistance associated protein (MRP) mrna were determined by RT-PCR and Western blot analysis. The cell cycle was analyzed by flow cytometry. Drug toxicity analysis was used to determine the sensitivity of the cells to chemotherapeutic drugs. Results Drug-resistant leukemia cell line HL-60 / A showed fold resistance to Adriamycin, fold resistance to Daunorubicin, and 6.43-fold resistance to Arabinosylcytosine, respectively. MRP expression of HL-60 / A cell was higher than that of HL-60 cell. The proportions of HL-60 / A cells and HL-60 cells in the G0 / G1 phase of the cell cycle were (35.10 ± 0.81)% and (43.96 ± 1.12)%, respectively (P < 0.05). The expression of SKP2 was significantly higher in HL-60 / A cells than that in HL-60 cells, but the expression of P27 kip1 in HL-60 / A cells was lower. Conclusions The mechanism of multidrug resistance of HL-60 / A cell line is related to up-regulated expression of MRP. The proportion of drug resistant cell line HL- 60 / A at the proliferation stage is high which indicates that drug-resistant cells have higher proliferative activity. Key words: cell cycle; S-phase kinase-associated protein 2; multi-drug resistance-associated protein; leukemia [J SUN Yat-sen Univ(Med Sci),2009,30(2): ], (acute leukemia, AL), 20% ~ 30% AL [1-2],, 60% ~ 70% : : ( ) (2006J1-C0071) :,, ; * :,, yinsongmei@21cn.com

2 138 ( ) 30,, 2 ml 700 ml / L 4,, r / min(r = 10 cm) 5 min,,pbs HL-60 HL-60 / A 2, 50 mg / LRNA 50 μl,50 mg / L SKP2 P27 kip1, SKP2 P27 kip1 (propidium iodide, PI) 450 μl,37 1 h, HL-60, :5 -CTT TTC ACT TCG GGC TGT GT-, 100 ml / L 3, :5 -CAC AAA ACA TGC CAC TTT ( ) 30 g / L,100 IU / ml,100 μg / ml RPMI1640 CTT CGT TCT CAG GCA CAT CA-3, :5 ( Gibco), 5% CO GCC TCA TCC AAC ACA AGG AT-3,,2 1 HL-60 / A 447 bp;β-actin :5 -GTC CAC CTT CCA GCA GAT GT-3, :5 -CAC CTT P27 kip1 (IgG )( R&D system ); MRP (multi-drug resistance associated protein, MRP) RT-PCR SKP2 P27 kip1 MRP β- (IgG ) ( CHEMICON ); actin mrna HL-60 SKP2 (IgG ) ( Zymed Laboratories ); GAPDH (IgG) ( CHEMICON );HRP IgG ( );MMuLV, RNA RNA Taq DNA ( MBI ), ( 0.5 μg / μl 10 μl RNA, Biowest );Phototope-HRP detection kit ( cell sigaling);cell counting kit-8( 5 min, 5 4 μl, ); ( ); RNA (20 U / μl) 1 μl,10 mmol dntp ( ); 2 μl, 37 5 min M-MuLV ) 96, , 100 μl, min,70 10 min,, HL- cdna HL-60 / A,1 000 r / min(r = 10 cm) 5 PCR 2 μl, 10 min,, 5 μl,10 mmol dntp 1 μl,mgcl 2 3 μl, 2.5 μl,taq DNA (1 U / μl)1.5 μl, 24 h 37 CCK-8,32.5 μl, 50 SKP h,450 nm 30 s,94 30 s,54 30 s, 6, min,30 P27 kip HL s,94 30 s,54 30 s 60 HL-60 / A,PBS 2, PBS,72 1 min,30 MRP, / ml,0.5 ml RT-PCR SKP2,P27 kip1, MRP,β-actin primer premier 5.0 SKP2 :5 -GCC CCA ATC TTG TCC ATC TA-3, :5 -CTG CGA TTC CAA AAA CT-3, 192 bp; P27 kip1 GG-3, 368 bp;mrp :5 - CAC CGT TCC AGT TT-3, 245 bp HL-60 / A,PBS 2, trizol (invitrogen ) RNA, RNA (A260 / A280 > 1.8), 1 μl, (200 U / μl) 1 μl, 20 μl,42 (20 μmol / L) 2.5 μl, (20 μmol / L) s,94 30 s,55.5

3 2,. SKP2 HL-60 / A s,72 1 min,30 β-actin s,94 30 s, s,72 1 min,30 PCR 20 g / L, Western-blot SKP2 P27 kip1 MRP GAPDH HL-60 HL-60 / A,PBS 2, RIPA (), PMSF, 1 mmol / L μl,, min, g 15 min, -80 BCA, 5 5 min, SDS-PAGE, SKP2 P27 kip1 β-actin 12%,MRP 6%, 45 min,200 V SKP2 P27 kip1 GAPDH 90min,100 V,MRP 180 min,80 V PVDF,TBS / T 1 h TBS / T 3,,4,TBS / T 3, 2 h,tbs / T 3, 1 HL-60 HL-60 / A, 10 ml LumiGLO, 1 min X 1 ~ 3 min, 30 ~ 60 min, 10 min 1.3 ± (x ± s), 2.2 HL-60 / A MRP HL-60 t, P < HL-60 / A (t = -4.4, P = 0.004,2) Western blot ADR HL-60 (Inh MRP, GAPDH ibitory concentration 50, IC50) μg / ml, MRP (0.20 ± HL-60 / A IC μg / ml, 0.02)%(0.60 ± 0.04)%, DNR HL-60 IC μg / ml,hl-60 / A IC μg / ml, Ara-C HL-60 IC μg / ml, HL-60 / A IC μg / ml, 6.43 (1) DNR ADR Ara-c Fig.1 Drug sensitivity curves of HL-60 and HL-60 / A cells to DNR, ADR, and Ara-c at different drug concentration(n = 4) RT-PCR HL-60 HL-60 / A MRP mrna, β-actin HL-60 HL-60 / A MRP mrna (0.34 ± 0.12)%(0.60 ± 0.07)%, (t = -16.4, P = 0.000,3) 2.3 HL-60 / A SKP2,P27 kip1 RT-PCR HL-60 HL-60 / A SKP2 P27 kip1 mrna, β-actin

4 140 ( ) 30 2 Fig.2 HL-60 HL-60 / A SKP2 P27 kip1 MRP mrna SKP2, P27 kip1, and MRP mrna expression in HL60 and HL-60 / A cells A: HL-60 cell; B: HL-60 / A cell Lane 5 8: marker; Lane 4 10: MRP; Lane 3 9: P27 kip1 ; Lane 2 7: SKP2; Lane 1 6: β-actin 3 Fig.3 HL-60 HL-60 / A SKP2 P27 kip1 MRP Protein expression of SKP2, P27 kip1, and MRP in HL-60 and HL-60 / A cells A: GAPDH protein; B: SKP2 protein; C: P27 kip1 protein; D: MRP protein HL60 HL-60 / A SKP2 mrna (0.52 ± 0.01)% (0.83 ± 0.03)%, (t = -19.1, P = 0.000) P27 kip1 mrna (0.75 ± 0.04)% (0.41 ± 0.05)%, (t = 11.8, P = 0.000,2)Western blot HL60 HL-60 / A SKP2 P27 kip1, GAPDH HL60 HL-60 / A SKP2 (0.27 ± 0.045)% (0.58 ± 0.06)%,,(t = -7.4, P = 0.002);P27 kip1 (0.63 ± 0.13)%(0.30 ± 0.13)%, (t = 3.1, P = 0.037,3) 2.4 HL-60 / A G1 HL-60 HL-60 / A G0 / G1 : (43.96 ± 1.12)% (35.10 ± 0.81)%, (t = 11.1, P = 0.001) S :(49.85 ± 0.91)% (55.56 ± 0.95)%, (t = -7.5, P = 0.002) G2-M :(6.19 ± 1.52)% (9.34 ± 0.25)%, (t = -3.5, P = 0.066) S G2-M (4) A. B. Fig.4 4 Determination of Cell cycles of the different cells by flow cytometry A: HL-60 cells; B: HL-60 / A cells 3 MRP,HL-60 / DOX (multi-drug resistance, MRD) HL-60 / A (multi-drug resistance associated protein, MRP) 190 ku, ATP (ATP-binding cassette,, HL-60 / DOX, HL-60 / DOX,,HL-60 / A, [3] ABC) Wada [4]

5 2,. SKP2 HL-60 / A 141,RT-PCR Western blot HL-60 / A MRP HL-60 SKP2 MRP,SKP2, Wada HL-60 / A S G2-M, HL-60 / A,,,, : A549 / DDP A549 [1] Kim DH, Lee NY, Sung WJ, et al. Multidrug :A549 / DDP G1 resistance as a potential prognostic indicator in acute myeloid leukemia with normal karyotypes [J]. Acta A549, [5], HL-60 / A HL- [2] 60, G0 / G1,S G2-M, HL-60 / A, HL-60 [3] SKP2 HL-60 / A, P27 kip1 ; SKP2 P27 kip1 Haematol, 2005,114(2): Del Poeta G, Stasi R, Aronica G, et al. Clinical relevance of P-glycoprotein expression in de novo acute myeloid leukemia [J]. Blood, 1996,87 (5): Kruh GD, Chan A, Myers K, et al. Expression complementary DNA library transfer establishes mrp as S 2 (S-phase kinase- a multidrug resistance gene [J]. Cancer Res, 1994,54 associated protein 2, SKP2) (7): [4] Wada H, Saikawa Y, Niida Y, et al. Selectively, induced high MRP gene expression in multidrugresistant human HL60 leukemia cells [J]. Exp [6],P27 kip1 SKP2 [7] P27 kip1 27 KD Hematol, 1999,27(1): , [5],,,. A549 / DDP, G0 / G1, S [J]., 2000,27(6): Agarwal A [8 ] SKP2 [6] Schulman BA, Carrano AC, Jeffrey PD, et al. Insights, SKP2, into SCF ubiquitin ligases from the structure of the P27 kip1, skp1-skp2 complex [J]. Nature, 2000,408 (6810):,P27 kip1 SKP [7] Sutterluty H, Chatelain E, Marti A, et al. p45skp2, promotes p27kip1 degradation and induces S phase in [9] quiescent cells [J]. Nat Cell Biol, 1999,1(4): Yokozawa [10] P27 kip1 [8] Agarwal A, Bumm TG, Corbin AS, et al. Absence of AL P27 kip1 SKP2 expression attenuates BCR-ABL-induced myeloproliferative disease [J]. Blood, 2008,112 (5):1960- P27 kip1 Min [11] SKP2 [9] Kataqiri Y, Hozumi Y, Kondo S. Knockdown of Skp2,SKP2 by sirna inhibits melanoma cell growth in vitro and in, HL- vivo [J]. J Dermatol Sci, 2006,42(3): [10] Yokozawa T, Towatari M, Iida H, et al. Prognostic 60 HL-60 / A, HL-60 significance of the cell cycle inhibitor p27 Kip1 in acute myeloid leukemia [J]. Leukemia, 2000,14(1):28-33., [11] Min YH, Cheong JW, Lee MH, et al. Elevated S-, RT-PCR Western phase kinase-associated protein 2 protein expression in blot HL-60 HL-60 / A SKP2 acute myelogenous leukemia: its association with constitutive phosphorylation [J]. Clin Cancer Res, P27 kip1 : 2004,10(15): SKP2, P27 kip1 ( )

Σχετικά έγγραφα

Supplemental file 3. All 306 mapped IDs collected by IPA program. Supplemental file 6. The functions and main focused genes in each network.

LIST OF SUPPLEMENTAL FILES Supplemental file 1. Primer sets used for qrt-pcr. Supplemental file 2. All 1305 differentially expressed genes. Supplemental file 3. All 306 mapped IDs collected by IPA program.