Decrease in equilibrative uridine transport during monocytic differentiation of HL-60 leukaemia: involvement of...

Area of Science:

• Cell Biology
• Molecular Biology
• Biochemistry

Background:

• Equilibrative nucleoside transporters (ENTs) facilitate nucleoside uptake in various cell types.
• HL-60 cells are a human promyelocytic leukemia cell line commonly used to study differentiation.
• Nitrobenzylthioinosine (NBMPR) is a well-characterized inhibitor of certain nucleoside transporters.

Purpose of the Study:

• To investigate the effect of monocytic differentiation on equilibrative uridine transport in HL-60 cells.
• To elucidate the role of protein kinase C (PKC) in regulating nucleoside transporter activity during differentiation.

Main Methods:

• Dose-response inhibition studies using dilazep, dipyridamole, and NBMPR.
• Induction of monocytic differentiation using phorbol 12-myristate 13-acetate (PMA).
• Measurement of NBMPR binding sites and nucleoside transport kinetics (Vmax).
• In vitro experiments with purified plasma membranes and exogenous PKC.

Main Results:

• Dose-response curves for uridine transport inhibition were biphasic, with distinct sensitive and insensitive components.
• PMA-induced differentiation caused a 20-fold decrease in Vmax for both NBMPR-sensitive and insensitive transport.
• NBMPR-sensitive transport reduction correlated with decreased NBMPR binding sites, suggesting fewer transporters.
• PKC activation, in the presence of ATP and PMA, reduced NBMPR binding sites in purified membranes.

Conclusions:

• Monocytic differentiation of HL-60 cells significantly impairs equilibrative uridine transport.
• PKC activation plays a crucial role in downregulating NBMPR-sensitive nucleoside transport during differentiation.
• A PKC-induced conformational change in the transporter may explain the observed reduction in transport activity.