Incomplete functional differentiation of HL‐60 leukemic cells by synthetic lipopetides

In human neutrophils, the synthetic lipopeptide, N ‐palmitoyl‐ S ‐[2,3‐bis(palmitoyoxy‐(2 RS )‐propyl]‐ R )‐cysteinyl‐( S )‐seryl‐( S )‐lysyl‐( S )‐lysyl‐ S )‐lysyl‐( S )‐lysine [Pam 3 CysSer(Lys) 4 ], activates NADPH‐oxidase catalyzed superoxide (O 2 − ) formation through pertussis‐toxin‐sensitive and per‐tussis‐toxin‐insensitive mechanisms (Seifert, R., Schultz, G., Richter‐Freund, M., Metzger, J., Wiesmüller, K.‐H., Jung, G., Bessler, W. G. & Hauschildt, S. (1990) Biochem. J. 267 , 795–802). We studied the effects of lipopeptides on differentiation of HL‐60 leukemic cells. Pam 3 CysSer(Lys) 4 enhanced phorbol‐12‐myristate‐13‐acetate‐induced O 2 − formation (presumably through the expression of components of NADPH oxidase) in a concentration‐dependent manner with a half‐maximal effect at 100 ng/ml and a maximum at 1 μg/ml. The effect of the lipopeptide was evident after 24 h and reached a plateau after 48 h. (2 S ,6 S )‐2‐Palmitoylamino‐6,7‐bis(palmitoyloxy)heptanoyl‐ ( S )‐seryl‐( S )‐lysyl‐( S )‐lysyl‐( S )‐lysyl‐ ( S )‐lysine enhanced O 2 − formation as well. The effects of Pam 3 CysSer(Lys) 4 were potentiated by dibutyryl cAMP, dimethyl sulfoxide, retinoic acid, 1,25‐dihydroxyvitamin D 3 , interferon‐γ and tumor‐necrosis‐factor‐α. Pertussis toxin, but not its B‐oligomer, partially inhibited enhanced O 2 − formation induced by Pam 3 CysSer(Lys) 4 . O 2 − formation induced by arachidonic acid and γ‐hexachlorocyclohexane were more sensitive to inhibition by pertussis toxin than O 2 − formation induced by phorbol 12‐myristate 13‐acetate. Enhanced O 2 − formation induced by dibutyryl cAMP was not affected by pertussis toxin. Unlike ATP, histamine, prostaglandin E 1 and the β‐adrenergic agonist, isoproterenol, Pam 3 CysSer(Lys) 4 did not increase cytosolic Ca 2+ ([Ca 2+ ] i ) in undifferentiated HL‐60 cells. Histamine but not lipopeptides stimulated high‐affinity GTPase of guanine‐nucleotide‐binding proteins in membranes of undifferentiated HL‐60 cells. In Pam 3 CysSer(Lys) 4 ‐differentiated HL‐60 cells, the responsiveness to the [Ca 2+ ] i ‐increasing agonists, N ‐formyl‐ l ‐methionyl‐ l ‐leucyl‐ l ‐phenylalanine, C5a and leukotriene B 4 , was increased, whilst the responsiveness to prostaglandin E 1 and isoproterenol was decreased. Pam 3 CysSer(Lys) 4 did not inhibit proliferation of HL‐60 cells but decreased transferrin receptor expression and increased C3bi receptor expression. Pertussis toxin did not affect proliferation and expression of transferrin and C3bi receptors. Dibutyryl cAMP was considerably more effective than Pam 3 CysSer(Lys) 4 at inducing alterations in the above parameters. Our results suggest that (a) Pam 3 CysSer(Lys) 4 induces incomplete functional differentiation of HL‐60 cells through a mechanism which does not depend on a rise in [Ca 2+ ] i and is different from that of other differentiation‐inducing substances and (b) the mechanism by which Pam 3 CysSer(Lys) 4 induces differentiation involves pertussis‐toxin‐sensitive and pertussis‐toxin‐insensitive mechanisms.