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Signal transduction and the regulation of actin conformation during myeloid
maturation: studies in HL60 cells
RL Sham, CH Packman, CN Abboud and MA Lichtman
Department of Medicine, University of Rochester Medical Center, NY.
Maturation of human myeloid cells is associated with quantitative and
qualitative changes in protein kinase C (PKC) and increases in N-formyl-
L-methionyl-L-leucyl-L-phenylalanine (FMLP) receptors, actin, and actin
regulatory proteins. We have studied the actin responses and cell shape
changes caused by FMLP and its second messenger pathways in HL60 cells
undergoing neutrophilic maturation. In uninduced cells, the PKC activators
12-O-tetradecanoyl phorbol-13-acetate (TPA), bryostatin, and
1-oleyl-2-acetylglycerol (OAG) resulted in 15% to 30% decreases in F-
actin, whereas FMLP had no effect. Ionomycin had no effect on actin but did
cause a 10-fold increase in intracellular calcium. Cells grown for 24 hours
in 1% dimethyl sulfoxide (DMSO) acquired the ability to polymerize actin in
response to FMLP and ionomycin. TPA continued to cause a decrease in
F-actin at 24 hours, but caused an increase in F- actin at 48 to 72 hours
of maturation. The PKC inhibitor 1-5- isoquinolinesulfonyl
2-methylpiperazine (H7) partially blocked the F- actin increase caused by
TPA in induced cells, but had no effect on the decrease in F-actin caused
by TPA in uninduced cells or the increase in F-actin seen in FMLP-treated
neutrophils. F-actin rich pseudopods developed following TPA or FMLP
stimulation of induced HL60 cells; in uninduced cells neither agent caused
pseudopod formation but TPA caused a dramatic loss of surface ruffles. The
ability of FMLP and ionomycin to elicit a neutrophil-like actin response in
HL60 cells within 24 hours after DMSO treatment shows that the actin
regulatory mechanism is mature by that time. The inability of ionomycin to
increase F-actin in uninduced cells supports the view that calcium
increases alone are insufficient for actin polymerization. The longer
maturation time required for HL60 cells to develop an actin polymerization
response to TPA compared with FMLP, coupled with the inability of H7 to
block the FMLP-mediated F-actin increase in neutrophils, suggests that the
F- actin increase caused by FMLP is not mediated solely by PKC. Lastly, the
TPA-induced F-actin decrease and shape changes in uninduced HL60 cells, and
the longer time required for a "mature" response to TPA, may reflect
immaturity in the PKC isoenzyme pattern rather than immaturity of the actin
regulatory mechanism.
Volume 77,
Issue 2,
pp. 363-370,
01/15/1991
Copyright © 1991 by The American Society of Hematology
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