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[The effect of small ubiquitin-like modifier-1 modification on the formation of Lewy body-like inclusions in cytoplasm and apoptosis of HEK293 cell induced by overexpression and mutation of alpha-synuclein].
Zhonghua Yi Xue Yi Chuan Xue Za Zhi. 2011 Oct; 28(5):511-6.ZY

Abstract

OBJECTIVE

To investigate the effect of small ubiquitin-like modifier (SUMO-1) modification on the formation of Lewy body like inclusions in cytoplasm and apoptosis of HEK293 cell induced by overexpression and mutation of alpha-synuclein.

METHODS

cDNA encoding the human alpha-synuclein without the stop codon was cloned into a pGEM T-easy vector. Restriction enzyme mapping and DNA sequencing were performed to analyze the plasmid, which was then subcloned into a pEGFP-N1 vector. The recombinant plasmid alpha-synuclein-pEGFP was transfected into HEK293 cells by lipofectamin method. Inclusions in the cultured cells were identified with HE staining. Apoptosis of the HEK293 cell was measured by Hoechst 33258 staining, MTT and Annexin V-PE flow cytometry.

RESULTS

The Lewy-body like inclusions were found in cytoplasm of cultured cells. Hoechst staining showed that the nuclei of cells were enlarged in the wild-type and A53T mutation groups 48 h after transfection, chromatin were accumulated and appeared spot-like. The nucleus stain was equitable in the K96R and K96R-A53T groups. MTT assay showed that the viability of cells transfected with empty plasmid was 96.2%, but it dropped to 53.4% and 56.1% in cells transfected with wild-type alpha-synuclein-pEGFP and A53T mutant group, respectively. The viability was 72.3% and 69.8% in cells transfected with K96R and K96R-A53T, respectively (P<0.05). Forty eight hours after transfection, the apoptosis rate was 3.9% in empty plasmid group, 32.2% and 34.1% in cells transfected with wild-type and mutant alpha-synuclein-pEGFP, 19.4% and 20.3% in the K96R and K96R-A53T transfected cells. There was significant difference between the two groups (P<0.05).

CONCLUSION

SUMO-1 modification did not have influence on the Lewy body-like inclusions formation in cytoplasm of HEK293 cell in vitro, but had a toxic effect which could increase the apoptosis induced by wild type overexpression and mutation of alpha-synuclein.

Links

Publisher Full Text (DOI)

Authors+Show Affiliations

Chen T
Department of Neurology, Hainan Provincial People's Hospital, Haikou, Hainan, People's Republic of China. ctxwyc@163.com
Liao XP
No affiliation info available
Wen GQ
No affiliation info available
Long ZG
No affiliation info available
Ouyang F
No affiliation info available
Deng YD
No affiliation info available
Guo M
No affiliation info available

MeSH

ApoptosisCytoplasmGene ExpressionGene Expression RegulationGenetic VectorsHEK293 CellsHumansLewy BodiesMutationParkinson DiseaseRNA, MessengerSUMO-1 Proteinalpha-Synuclein

Pub Type(s)

English Abstract
Journal Article

Language

chi

PubMed ID

21983723

Citation

Chen, Tao, et al. "[The Effect of Small Ubiquitin-like Modifier-1 Modification On the Formation of Lewy Body-like Inclusions in Cytoplasm and Apoptosis of HEK293 Cell Induced By Overexpression and Mutation of Alpha-synuclein]." Zhonghua Yi Xue Yi Chuan Xue Za Zhi = Zhonghua Yixue Yichuanxue Zazhi = Chinese Journal of Medical Genetics, vol. 28, no. 5, 2011, pp. 511-6.
Chen T, Liao XP, Wen GQ, et al. [The effect of small ubiquitin-like modifier-1 modification on the formation of Lewy body-like inclusions in cytoplasm and apoptosis of HEK293 cell induced by overexpression and mutation of alpha-synuclein]. Zhonghua Yi Xue Yi Chuan Xue Za Zhi. 2011;28(5):511-6.
Chen, T., Liao, X. P., Wen, G. Q., Long, Z. G., Ouyang, F., Deng, Y. D., & Guo, M. (2011). [The effect of small ubiquitin-like modifier-1 modification on the formation of Lewy body-like inclusions in cytoplasm and apoptosis of HEK293 cell induced by overexpression and mutation of alpha-synuclein]. Zhonghua Yi Xue Yi Chuan Xue Za Zhi = Zhonghua Yixue Yichuanxue Zazhi = Chinese Journal of Medical Genetics, 28(5), 511-6. https://doi.org/10.3760/cma.j.issn.1003-9406.2011.05.008
Chen T, et al. [The Effect of Small Ubiquitin-like Modifier-1 Modification On the Formation of Lewy Body-like Inclusions in Cytoplasm and Apoptosis of HEK293 Cell Induced By Overexpression and Mutation of Alpha-synuclein]. Zhonghua Yi Xue Yi Chuan Xue Za Zhi. 2011;28(5):511-6. PubMed PMID: 21983723.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - [The effect of small ubiquitin-like modifier-1 modification on the formation of Lewy body-like inclusions in cytoplasm and apoptosis of HEK293 cell induced by overexpression and mutation of alpha-synuclein]. AU - Chen,Tao, AU - Liao,Xiao-ping, AU - Wen,Guo-qiang, AU - Long,Zhi-gang, AU - Ouyang,Feng, AU - Deng,Yi-dong, AU - Guo,Min, PY - 2011/10/11/entrez PY - 2011/10/11/pubmed PY - 2011/12/14/medline SP - 511 EP - 6 JF - Zhonghua yi xue yi chuan xue za zhi = Zhonghua yixue yichuanxue zazhi = Chinese journal of medical genetics JO - Zhonghua Yi Xue Yi Chuan Xue Za Zhi VL - 28 IS - 5 N2 - OBJECTIVE: To investigate the effect of small ubiquitin-like modifier (SUMO-1) modification on the formation of Lewy body like inclusions in cytoplasm and apoptosis of HEK293 cell induced by overexpression and mutation of alpha-synuclein. METHODS: cDNA encoding the human alpha-synuclein without the stop codon was cloned into a pGEM T-easy vector. Restriction enzyme mapping and DNA sequencing were performed to analyze the plasmid, which was then subcloned into a pEGFP-N1 vector. The recombinant plasmid alpha-synuclein-pEGFP was transfected into HEK293 cells by lipofectamin method. Inclusions in the cultured cells were identified with HE staining. Apoptosis of the HEK293 cell was measured by Hoechst 33258 staining, MTT and Annexin V-PE flow cytometry. RESULTS: The Lewy-body like inclusions were found in cytoplasm of cultured cells. Hoechst staining showed that the nuclei of cells were enlarged in the wild-type and A53T mutation groups 48 h after transfection, chromatin were accumulated and appeared spot-like. The nucleus stain was equitable in the K96R and K96R-A53T groups. MTT assay showed that the viability of cells transfected with empty plasmid was 96.2%, but it dropped to 53.4% and 56.1% in cells transfected with wild-type alpha-synuclein-pEGFP and A53T mutant group, respectively. The viability was 72.3% and 69.8% in cells transfected with K96R and K96R-A53T, respectively (P<0.05). Forty eight hours after transfection, the apoptosis rate was 3.9% in empty plasmid group, 32.2% and 34.1% in cells transfected with wild-type and mutant alpha-synuclein-pEGFP, 19.4% and 20.3% in the K96R and K96R-A53T transfected cells. There was significant difference between the two groups (P<0.05). CONCLUSION: SUMO-1 modification did not have influence on the Lewy body-like inclusions formation in cytoplasm of HEK293 cell in vitro, but had a toxic effect which could increase the apoptosis induced by wild type overexpression and mutation of alpha-synuclein. SN - 1003-9406 UR - https://www.unboundmedicine.com/medline/citation/21983723/[The_effect_of_small_ubiquitin_like_modifier_1_modification_on_the_formation_of_Lewy_body_like_inclusions_in_cytoplasm_and_apoptosis_of_HEK293_cell_induced_by_overexpression_and_mutation_of_alpha_synuclein]_ DB - PRIME DP - Unbound Medicine ER -