SUMO-1 Enhances Apoptosis Induced by Wild-Type p53 Plasmid Transfection in HepG2 Cells_CHINESE JOURNAL OF BASES AND CLINICS IN GENERAL SURGERY

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LU Xingrong , YI Jilin.

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Small ubiquitin-like modifier-1 p53 gene Murine double minute gene 2 HepG2 cell Apoptosis

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【Abstract】Objective To investigate whether SUMO-1 enhances the apoptosis induced by wild-type p53 plasmid transfection in HepG2 cells.
Methods The HepG2 cells were transfected respectively or simultaneouly with the following expressional plasmids as pcDNA3-wtp53
（pwtp53，including human wild-type p53 gene），pCMV-HDM1B（pMDM2，including HDM2 gene， homologous gene as murine double minute gene 2），pcDNA3-His6-SUMO-1（pSUMO-1 ，including small ubiquitin-like modifier1 gene）and plasmid pcDNA3.The proteins expressed in cells were detected by means of Western blotting and the apoptosis rates of cells were measured by flow cytometry.
Results The protein bands of p53 and MDM2 could be seen in cells transfected with pwtp53 and pMDM2. Meanwhile，the relative larger molecular weight bands were also seen in cells transfected with pSUMO-1 which represented the p53 and MDM2 protein modification by SUMO-1. Merely the trace of p53 protein was detected in cells not transfected with any plasmid or only transfected with empty plasmid and pSUMO-1. In cells transfected with pwtp53 and pwtp53+pSUMO-1，the apoptosis rates were (16.79±1.62)%and (18.15±1.36)%. When transfected with pwtp53+pMDM2，the rate decreased to (5.17±1.23)%. The apoptosis rate would come up again to (14.06±1.84)% after transfected with pwtp53+pMDM2+pSUMO-1 and the difference of rates were significant compared to the cells transfected with pwtp53+pMDM2 (PH＜0.01）. The apoptosis rates in other cells were less than 2% and had no significant difference.
Conclusion SUMO-1 could increase the apoptosis induced by wild-type p53 plasmid transfection in HepG2 cells through combining to p53 protein or its post-translational modification and inhibiting p53 degradation by MDM2.

Citation： LU Xingrong,YI Jilin.. SUMO-1 Enhances Apoptosis Induced by Wild-Type p53 Plasmid Transfection in HepG2 Cells. CHINESE JOURNAL OF BASES AND CLINICS IN GENERAL SURGERY, 2005, 12(4): 371-374. doi: Copy

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8.	Melchior F. SUMO——nonclassical ubiquitin [Ｊ]. Annu Rev Cell Dev Biol, 2000; 16∶591.
9.	Rodriguez MS, Desterro JM, Lain S, et al. SUMO1 modification activates the transcriptional response of p53 [Ｊ]. EMBO J, 1999; 18(22)∶6455.
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11.	Sambrook J, Ftitsch EF, Mauitis T. Molecular cloning.A laboratory manual [M]. 2nd. New York: Cold Spring Harbor Laboratory Press, 1988∶362-371,792-793.
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13.	Kurepa J, Walker JM, Smalle J, et al. The small ubiquitinlike modifier (SUMO) protein modification system in Arabidopsis. Accumulation of SUMO1 and 2 conjugates is increased by stress [Ｊ]. J Biol Chem, 2003; 278(9)∶6862.

1. Ko LJ, Prives C. p53: puzzle and paradigm [Ｊ]. Genes Dev, 1996; 10(9)∶1054.
2. Rosenfeld MR, Meneses P, Dalmau J, et al. Gene transfer of wildtype p53 results in restoration of tumorsuppressor function in a medulloblastoma cell line [Ｊ]. Neurology, 1995; 45(8)∶1533.
3. Honda R, Tanaka H, Yasuda H. Oncoprotein MDM2 is a ubiquitin ligase E3 for tumor suppressor p53 [Ｊ]. FEBS Lett, 1997; 420(1)∶25.
4. Haupt Y, Maya R, Kazaz A, et al. Mdm2 promotes the rapid degradation of p53 [Ｊ]. Nature, 1997; 387(6630)∶296.
5. Saitoh H,Hinchey J. Functional heterogeneity of small ubiquitinrelated protein modifiers SUMO1 versus SUMO2/3 [Ｊ]. J Biol Chem, 2000; 275(9)∶6252.
6. Bayer P, Arndt A, Metzger S, et al. Structure determination of the small ubiquitinrelated modifier SUMO1 [Ｊ]. J Mol Biol, 1998; 280(2)∶275.
7. Muller S, Hoege C, Pyrowolakis G, et al. SUMO, ubiquitin’s mysterious cousin [Ｊ]. Nat Rev Mol Cell Biol, 2001; 2(3)∶202.
8. Melchior F. SUMO——nonclassical ubiquitin [Ｊ]. Annu Rev Cell Dev Biol, 2000; 16∶591.
9. Rodriguez MS, Desterro JM, Lain S, et al. SUMO1 modification activates the transcriptional response of p53 [Ｊ]. EMBO J, 1999; 18(22)∶6455.
10. Gostissa M, Hengstermann A, Fogal V, et al. Activation of p53 by conjugation to the ubiquitinlike protein SUMO1 [Ｊ]. EMBO J, 1999; 18(22)∶6462.
11. Sambrook J, Ftitsch EF, Mauitis T. Molecular cloning.A laboratory manual [M]. 2nd. New York: Cold Spring Harbor Laboratory Press, 1988∶362-371,792-793.
12. Zheleva DI, Lane DP, Fischer PM. The p53Mdm2 pathway: targets for the development of new anticancer therapeutics [Ｊ]. Mini Rev Med Chem, 2003; 3(3)∶257.
13. Kurepa J, Walker JM, Smalle J, et al. The small ubiquitinlike modifier (SUMO) protein modification system in Arabidopsis. Accumulation of SUMO1 and 2 conjugates is increased by stress [Ｊ]. J Biol Chem, 2003; 278(9)∶6862.

CHINESE JOURNAL OF BASES AND CLINICS IN GENERAL SURGERY

SUMO-1 Enhances Apoptosis Induced by Wild-Type p53 Plasmid Transfection in HepG2 Cells

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