CD40-ligand down-regulates inhibitor of differentiation 3 expression in WEHI-231 B lymphoma cells
- VernacularTitle:CD40配体诱导小鼠B淋巴瘤细胞分化抑制因子3下调表达的研究
- Author:
Xiaojun LI
;
Junchuan QIN
- Publication Type:Journal Article
- Keywords:
Inhibitor of differentiation;
CD40;
CD40 ligand;
WEHI-231 cells;
Retrovirus
- From:
Journal of Medical Postgraduates
2004;0(02):-
- CountryChina
- Language:Chinese
-
Abstract:
Objective: To study mouse Id3(mId3) protein expression in WEHI-231 B lymphoma cells after CD40 ligand(CD40L) treatment. Methods: The mId3 cDNA was amplified from total RNA of WEHI-231 cells by RT-PCR and introduced into the p3FLAG-CMV-10 vector.The 3FLAG-Id3 fusion gene was amplified from p3FLAG-Id3-CMV-10 vector and subcloned into retroviral vector pMX-CITE/IRES-EGFP.Recombinant retrovirus was produced by transient transfection of the Phoenix-ecotropic packaging cell line with pMX-3FLAG-Id3-EGFP vector using FuGene-6 transfection reagent.(WEHI)-231 cells were transduced with recombinant retrovirus.EGFP expression was monitored by flow cytometry.Two cell lines stably expressing 3FLAG-Id3 fusion protein were generated. Results: The proportion of EGFP-positive cells in the population transduced with pMX-3FLAG-EGFP remained constant up to 2 days after transduction,whereas the proportion in the population of pMX-3FLAG-Id3-EGFP-transduced cells decreased with time.After coculturing with NIH3T3 cells or medium alone,the proportion of EGFP-positive cells in pMX-3FLAG-Id3-EGFP-transduced WEHI-231 cells decreased with time,whereas after coculturing with CD40L-NIH3T3 cells,the proportion of EGFP-positive cells in the transduced cells remained constant up to 2 days,suggesting that CD40L could rescue WEHI-231 from Id3 overexpressioninduced growth arrest.After coculturing with CD40L/NIH3T3 or NIH3T3 cells,3FLAG-Id3 fusion protein stably expressing WEHI-231 cells were harvested,lysed,and immunoblotted by using anti-FLAG antibody.The results showed that treatment via CD40 ligation induced significant downregulation of Id3 expression in pMX3FLAG-Id3-EGFP tansduced WEHI 231 cells. Conclusion:CD40 ligation pvevents the Id3 induced cell growth avvest by the dousn-regulation of Id3 expression.
