Changes in antigen expression on differentiating HL60 cells treated with dimethylsulphoxide, all-trans retinoic acid, α1,25-dihydroxyvitamin D3 or 12-O-tetradecanoyl phorbol-13-acetate
Introduction
HL60 cells have been used extensively as a model of leukaemic myeloid cell differentiation, and they can be induced to differentiate into granulocyte, monocyte or macrophage-like cells by a number of physiological and non-physiological agents (for review see Ref. [1]). HL60 cells were originally isolated from a female patient diagnosed as having acute promyelocytic leukaemia (APL) [2], but they do not have a t(15;17) chromosomal translocation of the type that is characteristic of typical APL [3]. The majority of HL60 cells have a promyelocytic morphology, and a proportion differentiate spontaneously into more mature cells of the granulocytic and monocytic lineages. Previously described changes in antigen expression by differentiating cells include the induction of CD11b by ATRA, DMSO, D3 and TPA, the induction of CD14 by D3, the induction of CD38 and CD40 by ATRA, and the down-regulation of CD13 by ATRA 2, 3, 4, 5, 6, 7, 8. CD71 (transferrin receptor) down-regulation has been extensively characterised in cells treated with DMSO, ATRA and TPA 9, 10, 11. HL60 cells differentiate into cells that are not identical to normal mature cells, and they do not always express the same antigens as their mature counterparts. For example it has been reported that ATRA induces the expression of c-fms (M-CSF receptor), which would normally be expressed by monocytes and macrophages but not neutrophils [12].
Many leukocyte differentiation antigens are not fully characterised with respect to their patterns of expression or functional significance, and the signalling pathways that regulate their expression are not fully elucidated. Whereas many changes in antigen expression are consequences of differentiation, others may play a role in the regulation of differentiation. Also, some changes in expression may be direct effects of inducing agents that are independent of differentiation. It would be particularly useful if some of the pathways that regulate the expression of cell surface antigens and other differentiation markers could be dissociated from each other. In any case, these antigens are useful for characterizing differentiation-resistant HL60 cells and studying the signalling pathways that regulate the expression of differentiation-regulated molecules.
We describe a detailed analysis of changes in antigen expression that occur on HL60 cells that have been incubated with differentiation inducers, using the myeloid and CD66 panels of monoclonal antibodies (mAb) from the Fifth International Workshop on Human Leukocyte Differentiation Antigens 13, 14. Many of these mAb were assigned novel cluster designation (CD) numbers at the workshop. We have identified four distinct panels of antigenic markers that will be useful for studying the pathways which regulate the responses of HL60 cells to ATRA, DMSO, D3 and TPA.
Section snippets
Materials
Unless stated otherwise, all chemicals were obtained from Sigma (Poole, UK). The myeloid and CD66/67 panels of mAb were supplied as part of the Fifth International Workshop and Conference on Human Leukocyte Differentiation Antigens. Microbeads labelled with fluorescein isothiocyanate (FITC) were from Flow Cytometry Standards Corp (San Juan, PR, USA), FlTC-conjugated goat F(ab′)2 anti-mouse IgG/IgM (H+L) and FlTC-conjugated goat F(ab′)2 anti-rat IgG/IgM (H+L) were from Caltag Laboratories (San
Results
The mAb used in this study are listed in Table 1, along with their CD numbers (if assigned) and antigenic specificities (if known). Also listed are MESF values obtained for the binding of these mAb to exponentially growing HL60 cells, and their previously established reactivities with normal human granulocytes and monocytes. All reactivities were comparable with those previously reported for the binding of these mAb to undifferentiated HL60 cells [17].
We investigated the binding of these mAb to
Discussion and conclusion
The aims of this study were to identify novel HL60 cell response antigens, and to determine how changes in expression correlate with morphological differentiation. Our results indicate that most changes in antigen expression do correspond with morphological differentiation, and no examples of expression being regulated in an inappropriate direction were detected. However, the expression of several antigens was not modulated in a manner that might have been predicted from known expression
Acknowledgements
This work was funded by the Leukaemia Research Fund, and the authors are grateful to the organisers of the Fifth International Workshop on Leukocyte Differentiation Antigens for providing antibodies.
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Present address: Departamento de Genetica Humana, Instituto Nacional de Saude Dr. Ricardo Jorge, Av. Padre Cruz, 1600 Lisboa, Portugal.