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Journal of Peking University (Health Sciences) ›› 2020, Vol. 52 ›› Issue (2): 214-220. doi: 10.19723/j.issn.1671-167X.2020.02.004

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Interaction between PSF and cytokeratin 18 mediates PSF relocation to cell membrane and maintains chemosensitivity of myeloid leukemia

Si-mei REN1,2,△(),Lu-yao LONG1,2,Cheng-shan XU1   

  1. 1. National Center for Clinical Laboratories, Beijing Hospital, National Center of Gerontology; Institute of Geriatric Medicine, Chinese Academy of Medical Sciences, Beijing 100730, China
    2. Graduate School of Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100730, China
  • Received:2019-12-09 Online:2020-04-18 Published:2020-04-18
  • Contact: Si-mei REN E-mail:rensimei4162@bjhmoh.cn
  • Supported by:
    Supported by the National Natural Science Foundation of China(81670161);Research Projects of Beijing Hospital(BJ-2015-105)

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Abstract:

Objective: To identify the chaperone of polypyrimidine tractor-binding protein-associated splicing factor (PSF) in myeloid leukemia cells, and to explore the mechanism and redistributive pattern to cell surface of PSF in chemo-sensitive HL60 cells and resistant HL60/DOX cells.Methods: The eukaryotic expression vector of PSF was transfected with liposomes transiently, then flow cytometry was used to detect the expression level of PSF on the cell surface 24 h, 48 h and 72 h after vector transfections. We constructed a chimeric expression vector, streptavidin binding peptide (SBP)-PSF, meanwhile this vector was transfected and made SBP-PSF fusion protein overexpress. In addition, we used streptavidin magnetic beads to precipitate the cellular chaperonin of PSF and then identified its chaperonin by mass spectrometry (MS). Lentiviral vectors containing cytokeratin18 (K18) interference sequences were transfected into 293T cells to prepare lentivirus. HL60 and HL60/DOX cells were infected with lentivirus to obtain stable interfering K18 cell lines. Next, flow cytometry was used to test the membrane relocation level of PSF. Together, these methods confirmed the similar or different mechanisms of the PSF redistributing to membrane synergistically mediated by K18 in HL60 and HL60/DOX cells.Results: The expression of membrane relocated PSF was detected every day for three days(at the end of 24 h,48 h and 72 h) after transient overexpression. The expressing rate of PSF on the cell surface was 22.4%±3.5%, 37.9%±6.0%, 58.3%±8.8%, respectively in sensitive HL60 cells, while that was 4.7%±0.5%, 3.9%±0.6%, 2.9%±0.6% , respectively in resistant HL60/DOX cells. The difference of expressing rate on each day was significant, P<0.01. We identified K18 detected by co-immunoprecipitation and mass spectrum assay which was the cellular chaperone of PSF. We found that K18 knockdown decreased the PSF expression level which redistributed on cell surface from 48.9%±5.4% to 6.2%±1.0% in sensitive HL60 cells, and from 9.11%±1.2% to 2.21%±0.51% in resistant HL60/DOX cells, respectively.Conclusion: K18 is the intracellular chaperonin of PSF. The interaction of PSF and K18 mediates its redistribution to cell membrane in sensitive cells. While in resistant cells, PSF failed to relocate at the cell surface and accumulated in cells, which mediated resistance to chemotherapeutics.

Key words: Tumor multidrug resistance, Polypyrimidine tract-binding protein-associated splicing factor, Cytokeratin 18

CLC Number: 

  • R557.1

Figure 1

Characteristics of HL60 and HL60/DOX A, cell viability of HL60 and HL60/DOX cells were detected by MTS assay; B, transcriptional and protein expression level of PSF in HL60 and HL60/DOX cells."

Figure 2

Ability of PSF to migrate to the cell membrane A, fragment inserted at a polyclonal site of pcDNA3.0 vector and working panel of experiment; B, HL60 and HL60/DOX were transfected with pcDNA3 vector containing HA-PSF fusion fragment, PSF expression on cell surface was detected by flow cytometry (△P<0.01). PSF, polypyrimidine tract-binding protein-associated splicing factor; PSF-FL, PSF full length."

Figure 3

K18 is the intracellular chaperone of PSF A, HL60 cells were transfected with SBP-PSF-pcDNA3.0 vector. Avidin magnetic beads were used for immunoprecipitation from whole lysates of HL60 cells. The precipitated proteins (arrow) were separated by 12% SDS-PAGE and visualized by Commassie blue staining; B, protein precipitation complexes were identified by mass spectrometry; C, reciprocal experiment between CO-IP and Western blot. CO-IP, co-immunoprecipitation; PSF, polypyrimidine tract-binding protein-associated splicing factor; SBP, streptavidin binding peptide; K18, cytokeratin 18."

Figure 4

PSF membrane expression assay after K18 interference A, transcriptional expression levels of K18 in sensitive and resistant HL60 cells, *P<0.05; B, K18 transcriptional expression levels after K18 knockdown in HL60 cells,# P<0.001; C, PSF expression on cell surface after K18 knockdown were detected by flow cytometry. PSF, polypyrimidine tract-binding protein-associated splicing factor; K18, cytokeratin 18."

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