Granulocytic differentiation of HL-60 promyelocytic leukemia cells is associated with increased expression of Cul5.
The human HL-60 promyelocytic leukemia cell line has been widely used as a model for studying granulocytic differentiation. All-trans retinoic acid (ATRA) treatment of HL-60 cells promotes granulocytic differentiation and is effective as differentiation therapy for patients with acute promyelocytic leukemia. The identification of genes that are transcriptionally regulated by ATRA has provided insight into granulocytic differentiation and differentiation therapy. The Asb-2 (ankyrin repeat SOCS box 2) gene has previously been identified as a transcriptional target in ATRA-treated HL-60 cells. The ASB-2 protein forms an E3 ubiquitin ligase complex with the proteins, Cul5, regulator of cullin 2 (ROC2), and elongin B and C. The purpose of this study was to determine if there is increased expression of Cul5 during granulocytic differentiation of HL-60 cells. To induce granulocytic differentiation, HL-60 cells were treated for 5 d with ATRA and differentiation was confirmed by examining superoxide anion production, nuclear morphology, and changes in the expression of CD11b, CD13, and CD15. Quantitative real-time RT-PCR was used to measure Cul5 mRNA expression and also the expression of other components of the E3 ubiquitin ligase (ASB-2, ROC2, elongin B and C). Granulocytic differentiation of HL-60 cells was associated with a 1.6-, 1.7-, and 23-fold statistically significant (P <or= 0.05) increase in mRNA expression for Cul5, ROC2, and ASB-2, respectively. No significant change was found in elongin B and C mRNA expression. Using Western blot analysis, the expression of Cul5 protein was increased 6.5-fold with granulocytic differentiation of the HL-60 cells. Increased expression of multiple components of the Cul5-containing E3 ubiquitin ligase complex with ATRA treatment of HL-60 cells indicates that this complex may play an important role in granulocytic differentiation.
Baxter
Shaneen S
SS
Department of Pharmacology, Chicago College of Osteopathic Medicine, Midwestern University, 555 31st Street, Downers Grove, IL 60515, USA.
Carlson
Lauren A
LA
Mayer
Alejandro M S
AM
Hall
Mary L
ML
Fay
Michael J
MJ
D015153:Blotting, Western; D002452:Cell Count; D002454:Cell Differentiation / Q000187:drug effects; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D002467:Cell Nucleus / Q000187:drug effects / Q000378:metabolism; D002470:Cell Survival / Q000187:drug effects; D044842:Cullin Proteins / Q000378:metabolism*; D000075065:Elongin; D015973:Gene Expression Regulation, Leukemic / Q000187:drug effects; D006098:Granulocytes / Q000166:cytology* / Q000187:drug effects; D018922:HL-60 Cells; D006801:Humans; D015473:Leukemia, Promyelocytic, Acute / Q000235:genetics / Q000473:pathology*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013481:Superoxides / Q000378:metabolism; D050826:Suppressor of Cytokine Signaling Proteins / Q000235:genetics / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism; D014212:Tretinoin / Q000494:pharmacology; D044767:Ubiquitin-Protein Ligases / Q000235:genetics / Q000378:metabolism
C437987:ASB2 protein, human; C496871:CUL5 protein, human; D044842:Cullin Proteins; C000616396:ELOB protein, human; D000075065:Elongin; D012333:RNA, Messenger; D050826:Suppressor of Cytokine Signaling Proteins; D014157:Transcription Factors; D013481:Superoxides; D014212:Tretinoin; C118307:RNF7 protein, human; D044767:Ubiquitin-Protein Ligases; D013755:Tetradecanoylphorbol Acetate