[Screening proteins related to retinoic acid resistance by proteomic analysis].
OBJECTIVE
To compare the expression profiles of differential proteins between retinoic acid resistant and sensitive cells and screen the proteins related to retinoic acid (RA) resistance by proteomic analysis.


METHODS
The total cellular proteins from the RA sensitive cells of the line NB4 and the RA resistant cells of the line MR2 obtained from a patient with acute promyelocytic leukemia (APL) were separated by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and analyzed by PDQuest v7.1 analysis software to screen the differential protein spots. Differentially expressed spots were analyzed by mass spectrometry for peptide mass finger (PMF) data and identified by Mascot software and SWISS-PROT protein database. The differentially expressed proteins were verified by Western blotting assay and semi-quantification RT-PCR.


RESULTS
2-DE patterns of APL cell lines with high-resolution and reproducibility were obtained. The average spots of the RA resistant cell line MR2 and RA sensitive cell line NB4 were 890 +/- 45 and 912 +/- 56 respectively. 57 significantly differentially expressed protein spots were screened, among which 23 protein spots were founded to be upregulated and 34 protein spots down regulated in the RA resistant cell line MR2. 25 differential protein spots were identified by mass spectrometry and 17 proteins were successfully assigned to 13 gene-reading frames, of which one was unknown function protein (FLJ00279) and the others were included in the categories of oncoprotein (DJ-1), transcription factor (MYC promoter-binding protein 1), molecule chaperone (HSP70, HSP60 and protein disulfide isomerase), metabolism protein (prohibitin, triosephosphate isomerase 1, and calreticulin), signal transduction (Rho GDP dissociation inhibitor), and cytoskeleton (ACTG1 protein, Beta 5-tubulin, and keratin 10). The results of Western blotting were similar to those of 2D-PAGE and showed differential expression of DJ-1 and calreticulin in several isoforms. Semi-quantification RT-PCR analysis showed that there was no correlation between the protein expression changes and mRNA levels of HSP70 or HSP60. Those results indicated that post-translational events might modify or shear the protein content of the specific spots.


CONCLUSIONS
The utilization of two-dimensional polyacrylamide gel electrophoresis coupled with mass spectrometry is effective in screening the all-trans RA resistance-associated proteins and could provide novel clue for study of elucidating all-trans RA resistance mechanisms.
Qin
Hui
H
Department of Hematology, West China Hospital, Sichuan University, Chengdu 610041, China. qinhui6957@sina.com
Liu
Ting
T
Yang
Jin-liang
JL
Huang
Xin
X
Liu
Bin
B
Song
Xin
X
Zhao
Xia
X
Wei
Yu-quan
YQ
D000970:Antineoplastic Agents / Q000494:pharmacology; D037282:Calreticulin / Q000032:analysis / Q000235:genetics; D045744:Cell Line, Tumor; D019008:Drug Resistance, Neoplasm; D015180:Electrophoresis, Gel, Two-Dimensional; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000032:analysis / Q000235:genetics; D040901:Proteomics / Q000379:methods*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014212:Tretinoin / Q000494:pharmacology*
D000970:Antineoplastic Agents; D037282:Calreticulin; D047908:Intracellular Signaling Peptides and Proteins; D014212:Tretinoin