Probing the requirement for CD38 in retinoic acid-induced HL-60 cell differentiation with a small molecule dimerizer and genetic knockout

Sci Rep. 2017 Dec 12;7(1):17406. doi: 10.1038/s41598-017-17720-4.

Abstract

CD38 is an ectoenzyme and receptor with key physiological roles. It metabolizes NAD+ to adenosine diphosphate ribose (ADPR) and cyclic ADPR, regulating several processes including calcium signalling. CD38 is both a positive and negative prognostic indicator in leukaemia. In all-trans retinoic acid (RA)-induced differentiation of acute promyelocytic leukaemia and HL-60 cells, CD38 is one of the earliest and most prominently upregulated proteins known. CD38 overexpression enhances differentiation, while morpholino- and siRNA-induced knockdown diminishes it. CD38, via Src family kinases and adapters, interacts with a MAPK signalling axis that propels differentiation. Motivated by evidence suggesting the importance of CD38, we sought to determine whether it functions via dimerization. We created a linker based on the suicide substrate arabinosyl-2'-fluoro-2'-deoxy NAD+ (F-araNAD+), dimeric F-araNAD+, to induce homodimerization. CD38 homodimerization did not affect RA-induced differentiation. Probing the importance of CD38 further, we created HL-60 cell lines with CRISPR/Cas9-mediated CD38 truncations. Deletion of its enzymatic domain did not affect differentiation. Apart from increased RA-induced CD11b expression, ablation of all but the first six amino acids of CD38 affected neither RA-induced differentiation nor associated signalling. Although we cannot discount the importance of this peptide, our study indicates that CD38 is not necessary for RA-induced differentiation.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • ADP-ribosyl Cyclase 1 / genetics
  • ADP-ribosyl Cyclase 1 / metabolism*
  • Antineoplastic Agents / pharmacology*
  • CD11b Antigen / metabolism
  • CRISPR-Cas Systems
  • Cell Differentiation / drug effects*
  • Cell Differentiation / physiology*
  • Gene Knockdown Techniques
  • HL-60 Cells
  • Humans
  • Membrane Glycoproteins / genetics
  • Membrane Glycoproteins / metabolism*
  • Protein Multimerization
  • RNA, Messenger / metabolism
  • Tretinoin / metabolism
  • Tretinoin / pharmacology*
  • src-Family Kinases / metabolism

Substances

  • Antineoplastic Agents
  • CD11b Antigen
  • ITGAM protein, human
  • Membrane Glycoproteins
  • RNA, Messenger
  • Tretinoin
  • src-Family Kinases
  • CD38 protein, human
  • ADP-ribosyl Cyclase 1