[Study on the mechanism of THP-1 cell differentiation imduced by a new steroidal drug NSC67657].
OBJECTIVE
To study the potential mechanism of the new steroidal drug NSC67657 induced leukemic cells differentiation.


METHODS
Cell proliferation was assayed by MTT assay. Surface antigen CD14 on THP-1 cells treated by NSC67657 at different time different concentration, was detected by flow cytometry (FCM). The expression of beta-catenin- interacting protein 1 (ICAT) gene and protein were detected by RT-PCR and Western blot. Eukaryotic expressing vector pDsRed-ICAT was constructed and transfected into HL60 cell line. FCM, Wright's staining and electronmicroscope were employed to analyse the differentiation of transfected THP-1 cells after they were treated with NSC67657 for 24 hours.


RESULTS
The proliferation of THP-1 cells was significantly inhibited by NSC67657 treatment. The level of CD14 expression was elevated in line with the increasing drug concentration and treatment time. 10 µmol/L NSC67657 treatment for five days was the optimal condition for the induction of THP-1 cells differentiation, when the CD14(+) THP-1 cells were more than 90%. Morphological study indentified the THP-1 cells of monocytic differentiation. The eukaryotic expressing vector pDSRed-ICAT was successfully constructed, and almost 90% positive clone could be obtained after G418 screening. Electro-transfection was employed for transfecting the vector into THP-1 cells. After the transfection the expression of ICAT gene and protein was increased. On the NSC67657 treatment, there was not significant difference in CD14 expression on transfected THP-1 cells compared to that on the control groups. After 24 h treatment, the transfected THP-1 cells remained in early differentiated stage.


CONCLUSIONS
NSC67657 can induce THP-1 cell to monocytic differentiation and activate the expression of ICAT gene, but overexpression of ICAT itself is not sufficient to induce such differentiation.
Zhongshan People's Hospital, Nanfang Medical University, Guangzhou 528402, China.
Wei-Jia
WJ
Wang
Xiu-Ming
XM
Zhang
Dong-Mei
DM
Wen
D002454:Cell Differentiation / Q000235:genetics; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D018922:HL-60 Cells; D006801:Humans; D012333:RNA, Messenger / Q000235:genetics; D014162:Transfection; D051176:beta Catenin / Q000235:genetics
D012333:RNA, Messenger; D051176:beta Catenin