Page 171 - Vitamin D and Cancer

158 E. Gocek and G.P. Studzinski

beginning of the transition to the macrophage phenotype. The first phase is
characterized by high levels of ERKs activated by phosphorylation, and these levels
decrease as the cells enter the second phase, while the levels of the cell cycle inhibitor
p27 KIp1 increase at that time. Serum-starved HL60 cells or cells treated with the
MAPK inhibitor PD 98059 have reduced growth rate and a slower rate of differen-
tiation, but the G1 block under these conditions also coincides with decreased levels
of activated ERK1/2 [152]. Our data suggested that the MEK/ERK pathway main-
tains cell proliferation during the early stages of differentiation, and the consequent
G1 block leads to “terminal” differentiation. Using a different experimental design
similar results were obtained by Marcinkowska [153].
We also demonstrated that the JNK pathway, as shown by the increased phos-
phorylation of c-jun, plays a role in the induction of differentiation of HL60 cells
by 1,25D. The data showed that 1,25D-induced differentiation of a stable clone of
U937 cells transfected with a dominant negative construct of JNK-1 was reduced,
as compared to cells transfected with a control construct [154], and potentiation of
1,25D-induced differentiation by the plant antioxidants curcumin and silibinin
increased the phosphorylation of c-jun [155]. This suggested that the JNK-jun
pathway is involved in 1,25D-induced differentiation, and was further established
in experiments which showed that the AP-1 transcription factor complex is required
for this process, since c-jun, together with ATF-2, is the principal component of this
complex [140]. This appears to be of wider significance, as c-jun expression was
also reported to enhance macrophage differentiation of U937 cells [156].
However, it seems clear that the ERK and JNK MAPK pathways are not the
only ones involved in signaling of 1,25D-induced differentiation. For instance,
compounds SB203580 and SB2902190, reported to be specific inhibitors of the
signaling protein p38 MAP kinase [157], were found to markedly accelerate
monocytic differentiation of HL60 cells induced by low concentrations of 1,25D
[158]. Paradoxically, these compounds also induced a sustained enhancement of
p38 phosphorylation and of its activity in cell extracts in the absence of added
inhibitor, which raised the possibility of a lack of specificity of SB compounds in
this cell system, or of an up-regulation of the upstream components of the p38
pathway. In addition, SB 203580 or SB 202190 treatment of HL60 cells resulted
in a prolonged activation of the JNK and the ERK MAPK pathways [158]. Honma
and colleagues also found that SB203580 treatment of HL60, HT93 and ML-1
human myeloid leukemia cell lines increased cellular ERK activity [159]. These
data are consistent with the hypothesis that in HL60 cells an interruption of a nega-
tive feedback loop from a p38 target activates a common regulator of multiple
MAPK pathways, but it is also possible that SB203580 has an additional,
unknown, action.
Another signaling cascade known to be activated by 1,25D in human AML cells
is the PI3K-AKT pathway, which is often envisaged to signal from the cell mem-
brane to the intracellular regulators in parallel with the MAPK pathways, e.g.,
[160]. As first noted by Reiner and colleagues [161], monocytic leukemia cells
THP-1 exposed to 1,25D in serum-free medium show a rapid and transient increase
in PI3K activity, which was attributed to the formation of a VDR-PI3K protein