Biology Reference
In-Depth Information
Classic differentiation protocols for HL-60 cells to a myeloid cell phenotype
involve treatment with either DMSO, phorbol-12-myristate-13-acetate (PMA),
or all-
trans
-
b
-retinoic acid (RA), and this effect can be greatly potentiated by
cAMP-increasing agents (Chaplinski and Niedel
1982
). One group observed an
effect of RA priming with pretreatment for cAMP-dependent induction of differen-
tiation, which was not dependent on new protein synthesis (Olsson et al.
1982
).
Therefore, they postulated that RA priming was likely a more direct activation of
existing signaling components in the cell by cAMP, presumably through PKA or
Epac, or a decrease in production of an inhibitory polypeptide.
Along these lines, it was found that PMA-stimulated, cAMP-induced growth
regulation is dependent on the regulatory PKA subunits PKA RI
a
and PKA RII
b
.
Knockdown of the RII
b
subunit with an antisense oligodeoxynucleotide resulted
in a decrease in cAMP-induced growth inhibition and differentiation, without
affecting phorbol ester-induced differentiation (Tortora et al.
1990
). Conversely,
when the RI
a
subunit is knocked down, cell differentiation and growth arrest are
increased, even in the absence of a cAMP-inducing stimulus (Tortora et al.
1991
).
Differentiation can also occur through stimulation of G
a
s-coupled receptors
and increases in cAMP alone, without concomitant phorbol ester administration.
Given these data, it seems that phorbol ester and cAMP-induced pathways are
complementary pathways for inducing differentiation that can act synergistically
given the appropriate conditions.
The differentiation of monocytes can also be affected through stimulation of
G
a
s-coupled receptors, thereby raising intracellular cAMP. ATP can exert its
effects on differentiation through stimulation of the G
a
s-coupled P2Y11 receptor
in monocytes (Boeynaems et al.
2000
). The downstream effects of cAMP appeared
to be mediated through PKAI, as the authors observed a decrease in differentiation
using a PKA inhibitor, Rp-8-Br-cAMPS, which is more selective for PKAI than for
PKAII (Jiang et al.
1997
).
Cyclic nucleotide analogs are often used to dissect signaling pathways. These
analogs have differing potencies and specificities, and some of the more common
include hydrolysis-resistant analogs (8-Br-cAMP, 8-Br-cGMP), PKA-specific ana-
logs (N
6
-Benzoyl cAMP) and Epac-specific analogs (8-CPT-2
0
-O-Me-cAMPS)
(Poppe et al.
2008
). These analogs can be used to stimulate downstream effectors
directly. Some advantages of using analogs are that they are cell permeable,
therefore easily administered to cells in culture, often nonhydrolyzable by PDEs,
and the concentration can be carefully controlled. A major disadvantage is that most
can stimulate all cyclic nucleotide downstream effectors and are not limited to
endogenous signaling microdomains within the cell. Cho and colleagues differen-
tiated HL-60 cells for 48 h in the presence of the cAMP analog 8-Br-cAMP and
identified mature cells by their ability to produce superoxide in response to PMA
(Cho et al.
2003
). They concluded that cAMP induces ERK activation, and this
activation is essential for differentiation. ERK activation appears to be PKA
mediated as it was also not dependent on B-Raf, a downstream kinase of Rap1
and Epac. In fact, B-Raf activation was negatively regulated through PKA, and the
differentiation could be inhibited by using an MEK inhibitor. While these results
