title: [Expression of ICAT and Wnt signaling-related proteins in the monocytic differentiation of HL-60 cells induced by a new steroidal drug NSC67657].
abstract: OBJECTIVE
  To investigate the expression of mRNA and proteins of β-catenin, TCF-4 (ICAT) and Wnt signaling pathway-related genes in the monocytic differentiation of acute myeloid leukemia HL-60 cells induced by a new steroidal drug NSC67657.
  
  
  METHODS
  Wright's staining and α-NBE staining were used to observe the differentiation of HL-60 cells after 5 days of 10 μmol/L NSC67657 treatment. Flow cytometry (FCM) was used to detect the differentiation and cell cycles. The expressions of mRNA and proteins of ICAT and Wnt signaling pathway-related factors, including β-catenin, TCF-4, c-myc, cyclin D1 and TCF-1 before and after differentiation, were detected by RT-PCR and Western blot.
  
  
  RESULTS
  Morphological observation showed that NSC67657 induced monocytic differentiation of HL-60 cells. At 5 days after 10 μmol/L NSC67657 treatment, the number of CD14(+) HL-60 cells was (94.37±2.84)%, significantly higher than the (1.31±0.09)% in control group (P<0.01). The flow cytometry assay revealed that NSC67657 induced (76.46±2.83)% of G1/G0 phase arrest, significantly higher than that of (59.40±5.42)% in the control group (P<0.05), while the S phase cells were of (18.76±0.98)%, significantly lower than that of (34.38±2.61) % in the control group (P<0.05). The NSC67657 treatment also up-regulated the expression of ICAT mRNA and protein, and down-regulated the expression of β-catenin mRNA and protin (P<0.01 for all). However, the nuclear expression of β-catenin was down-regulated (P<0.01). The NSC67657 treatment induced nonsignificant alterations of TCF-4 mRNA, total protein and nuclear protein in the HL-60 cells (P>0.05 for all). The target genes of Wnt signaling pathway, including c-myc, cyclinD1 and TCF-1 mRNA and proteins in the HL-60 cells were significantly down-regulated after NSC67657 treatment (P<0.05).
  
  
  CONCLUSIONS
  The new steroidal drug NSC67657 induces monocytic differentiation of HL-60 cells, and down-regulates the expression of β-catenin and target genes of Wnt signaling pathway. These results indicate that Wnt signaling pathway may be directly or indirectly involved in the monocytic differentiation process of HL-60 cells.
authors.affiliation: Key Laboratory of Clinical Laboratory Diagnostics of Ministry of Education, School of Laboratory Medicine, Chongqing Medical University, Chongqing 400016, China.
authors.forename: J S
authors.initials: JS
authors.lastname: Wang
authors.affiliation: Zhongshan People's Hospital Medical Laboratory Center, Zhongshan, Guangdong 528403, China.
authors.forename: W J
authors.initials: WJ
authors.lastname: Wang
authors.affiliation: Key Laboratory of Clinical Laboratory Diagnostics of Ministry of Education, School of Laboratory Medicine, Chongqing Medical University, Chongqing 400016, China.
authors.forename: T
authors.initials: T
authors.lastname: Wang
authors.affiliation: Key Laboratory of Clinical Laboratory Diagnostics of Ministry of Education, School of Laboratory Medicine, Chongqing Medical University, Chongqing 400016, China.
authors.forename: Y
authors.initials: Y
authors.lastname: Zhang
mesh_terms: D048868:Adaptor Proteins, Signal Transducing; D002453:Cell Cycle; D002454:Cell Differentiation; D019938:Cyclin D1 / Q000235:genetics / Q000378:metabolism; D015536:Down-Regulation; D018922:HL-60 Cells / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics / Q000378:metabolism*; D008698:Mesylates / Q000494:pharmacology*; D012333:RNA, Messenger / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects; D013256:Steroids / Q000494:pharmacology*; D051822:T Cell Transcription Factor 1 / Q000235:genetics / Q000378:metabolism; D014162:Transfection; D051153:Wnt Proteins / Q000378:metabolism*; D051176:beta Catenin / Q000235:genetics / Q000378:metabolism*
chemical_list: C544140:(4-(10, 13-dimethyl-3-methylsulfonyloxy-2,3,4,5,6,7,8,9,11,12,14,15,16, 17-tetradecahydro-1H-cyclopenta(a)phenanthren-17-yl)pentyl) methanesulfonate; D048868:Adaptor Proteins, Signal Transducing; C411982:CTNNBIP1 protein, human; D047908:Intracellular Signaling Peptides and Proteins; D008698:Mesylates; D012333:RNA, Messenger; D013256:Steroids; D051822:T Cell Transcription Factor 1; C497576:TCF7 protein, human; D051153:Wnt Proteins; D051176:beta Catenin; D019938:Cyclin D1