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 realtime 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=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.
|Original language||English (US)|
|Number of pages||11|
|Journal||In Vitro Cellular and Developmental Biology - Animal|
|State||Published - 2009|
Bibliographical noteFunding Information:
Acknowledgments We thank the Robert H. Lurie Comprehensive Cancer Center Flow Cytometry Core Facility at Northwestern University and Dr. Alice Givan of the Englert Cell Analysis Laboratory at Dartmouth Medical School for assistance with the collection and analysis of the flow cytometry data. We also thank Dr. Nalini Chandar of the Biochemistry Department at Midwestern University for reviewing this manuscript prior to submission. This research was supported in part by the Biomedical Sciences Department and the Office of Research and Sponsored Programs at Midwestern University and by grant number R15 CA 122003-01 from the National Cancer Institute of the NIH.
- All-trans retinoic acid
- Elongin B/C
- Granulocytic differentiation