Study On Effects Of P33～(ING1b) In Proliferation And Apoptosis Of Glioma Cells

Objective:To construct the recombinant eukaryotic expression plasmid of p33^ING1b and itsNLS peptide, observe the subcellular location of the target proteins and assess theireffects in proliferation inhibition and apoptosis induction in human ghoblastoma cellline TJ905.Methods:The reading frame of p33^ING1b was amplified by RT-PCR, and then subcloned topBS-T vector to construct the recombinant plasmid pBS-T-p33^ING1b. After propagation,the insert was subcloned into the multiple clone sites of the eukaryotic expression vectorpcDNA3.1/V5-His to construct eukarytic fusion protein expression plasmid pcDNA3.1-p33^ING1b-V5-His. The coding sequence of NLS was also subcloned into the same vector toobtain recombinant plasmid pcDNA3.1-NLS-V5-His. Both plasmids were used totransfect the human glioblastoma cell line TJ905. Stable transfectants were obtained byG418 screening. The subcellular localization of the exogenous p33^ING1b and NLS peptidewere detected by immunocytochemistry. The effects of both exogenous proteins inproliferative inhibition and apoptotic induction in human glioblastoma cell line TJ905were assessed by means of Ki-67 immunocytochemical stain, colony formation assay, flowcytometry and comet assay.Results:Agarose gel electrophoresis showed that the molecular weight of the p33^ING1breading frame corresponded well to the therotical value, DNA sequencing result showed that thecDNA fragments we cloned was integrated without any mutation and it lied in the same reading frame with the DNA fragments encoding the V5 and the 6×His tag in the recombinant plasmidpcDNA3.1-p33^ING1b-V5-His. Similar results were also obtained for recombinant plasmidpcDNA3.1-NLS-V5-His. DNA sequencing results also showed that the sequence and thereading frame of both recombinant eukaryotic fusion protein expression plasmids werecompletely correct. With immunocytochemical examination, we confirmed that both fusionproteins expressed by the recombinant plasmids were completely located in nucleus. Compared tothe results seen with the empty vector transfectants and the control, the colony numbers werestrongly reduced in the recombinate plasmids transfectants. Otherwise, the numbers ofKi-67 positive cells in both recombinate plasmids transfected cell groups were reduced.Utilized the flow cytometry to detect the apoptosis, we observed that no differencebetween the vector-transfected clones and control, but both recombinant eukarytic fusionprotein expression plasmids pcDNA3.1/V5-His-NLS and pcDNA3.1/V5-His-p33^ING1btransfected clones had more apoptosis cells(p＜0.001) than the vector-transfectedclones and control. The comet assay also came to the same conclusion that the cellsfrom the recombinate plasmids transfected clones form comet more easier than thevector-transfected clones and control(p＜0.001).Conclusions:From the present study, we prove that p33^ING1b locates absolutely in nucleusphysiologically. Its NLS plays decisive role in the transporting process of subcellularlocalization. In addition, the sequence around NLS may have the function of inducingapoptosis in cells with DNA damage. The peptide of p33^ING1b from amino acid residue 171 to196 may plays important role in transcriptional silencing function of p33^ING1b. We also confirmthat transfecting exogenous gene to increase the expression of p33^ING1b can inhibitproliferation and promote apoptosis in the cells of glioblastoma cell line TJ905.