Page 173 - Vitamin D and Cancer

160 E. Gocek and G.P. Studzinski

A rather speculative mechanism describing how MEK/ERK signaling is diminished
in the later stages of differentiation, when cell proliferation becomes arrested, is
presented below.

7.3.2 p35/Cdk5, a Protein Kinase System That May Interface
Differentiation Processes with Cell Cycle Arrest

After 24–48 h of exposure of myeloid leukemia cells to moderate concentrations of
1,25D (1–10 nM), cell cycle progression becomes progressively arrested, princi-
pally due to a G1 to S phase block, though a G2 phase block can also be observed
[183]. Several mechanisms could explain these cell cycle effects, and these include
activation of cyclin-dependent kinase 5 (Cdk5).
Cdk5 is a proline-directed serine-threonine kinase with sequence homology to
the cyclin-activated kinases which regulate cell cycle progression, but its best
known function is participation in differentiation of neuronal cells [184]. When
combined with a “cyclin-like” neuronal Cdk5 activator (Nck5a) 35 kDa protein
(p35/Nck5a, or p35), the p35/Cdk5 complex functions in monocytic cells and has
an important role in the normal, and possibly abnormal development of this
hematopoietic lineage. Our initial observations were that in HL60 cells treated with
1,25D the monocytic phenotype and expression of Cdk5 appear in parallel. Both
active and inactive Cdk5 was associated with cyclin D1 protein, and the inhibition
of Cdk5 expression by an antisense oligonucleotide construct reduced the intensity
of 1,25D-induced expression of the monocytic marker CD14 [185]. This finding
demonstrated a novel (other than neuronal) cellular type for Cdk5 activity, and a
concomitant enhancement of monocytic differentiation.
The above study showed that protein levels and kinase activity of Cdk5 increase
in HL60 cells induced to monocytic differentiation by 1,25D, but did not establish
the specificity of the association of Cdk5 with the monocytic phenotype. Therefore,
we showed in a subsequent study that the upregulation of Cdk5 does not occur in
granulocytic differentiation, whereas an inhibition of Cdk5 activity by the pharma-
cological inhibitor olomoucine, or of its expression by a plasmid construct expressing
antisense Cdk5, switches the 1,25D-induced monocytic phenotype (a combination
of the positive NSE reaction, the expression of the CD14 marker, and morphology)
to a general myeloid phenotype (a positive NBT reaction, the CD11b marker, and
morphology) [186]. These findings showed that in human myeloid cells the up-
regulation of Cdk5 is specifically associated with the monocytic phenotype.
The Nck5a 35 kDa protein has hitherto been considered to be exclusively
expressed in neuronal cells, as its name implies [187]. However, since we had clear
evidence that Cdk5 is an active kinase in human leukemia cells HL60 and U937
induced to differentiate with 1,25D, and since the “classical” cyclins (e.g., cyclin
D1, cyclin E) are not known to activate Cdk5, we investigated whether p35 can be
detected in cells with active Cdk5. Indeed, we demonstrated that p35 is expressed
in normal human monocytes and in leukemic cells induced to differentiate toward