Page 174 - Vitamin D and Cancer

7 Induction of Differentiation in Cancer Cells by Vitamin D 161

the monocytic lineage, but not in lymphocytes, or cells induced to granulocytic
differentiation by retinoic acid. The activator p35 is present in a complex with Cdk5
that has protein kinase activity, and when ectopically expressed together with Cdk5
in undifferentiated HL60 cells it induces the expression of CD14 and NSE markers
of the monocytic phenotype [188]. These observations not only indicate a func-
tional relationship between Cdk5 and p35, but also support a role for this complex
in monocytic differentiation.
A likely link to the diminution of ERK MAPK pathway activity at the onset of
phase 2 of 1,25D-induced differentiation is provided by the EGR-1 → p35/Cdk5
---| MEK 1/2 pathway, that was elucidated in leukemia cells by this laboratory
[189]. The schematic representation is shown in Fig. 7.4, and the supporting data
can be summarized as follows.

7.3.2.1 Control of p35 Expression by the EGR-1 Transcription Factor

The evidence that supports a role of EGR-1 in regulating the expression of p35
includes the coordinate expression of EGR-1 along with Cdk5, and the co-inhibition
of the 1,25D-induced upregulation of these proteins by PD 98059, an inhibitor of the
MEK/ERK1/2 pathway [171, 190]. Further, the promoter region of human p35 has
an EGR-1 binding site that overlaps with an Sp1 site, and a gel shift assay showed
that a double-stranded oligonucleotide that contained this sequence bound proteins
in nuclear extracts from 1,25D-treated HL60 cells. The EGR-1-site binding proteins
were competed most efficiently by an anti-EGR-1 antibody, though some competi-
tion was also observed with an anti-Sp1 antibody, but no competition was observed
with an irrelevant antibody, e.g., anti-VDR. The data suggested that EGR-1, and
perhaps Sp1 proteins, regulate the expression of p35 and contribute to induction of
the monocytic phenotype. A “decoy” EGR-1 response element oligonucleotide inhib-
ited both 1,25D-induced p35 expression and monocytic differentiation [189].

7.3.2.2 The Cdk5/p35 Complex Phosphorylates MEK

We also found that the Cdk5/p35 can phosphorylate MEK in cell extracts [189]. If
this can be demonstrated to occur in leukemia cells, it will provide a potential
mechanism for the inhibition of the MAPK/ERK pathway seen in the later stages of
differentiation (48 h after the addition of 1,25D to the cultures), since phosphoryla-
tion of MEK by p35/Cdk5 inhibits its kinase activity. Intriguingly, upregulation of
p35 (which activates Cdk5) is observed pari passu as ERK 1/2 phosphorylation is
waning, consistent with a cause–effect relationship. We have thus proposed a
mechanism that can shut down cell proliferation, possibly by allowing p27 Kip1 to
accumulate in the cell nucleus due to a decline in ERK 1/2 activity, since it has been
reported that the ERK pathway can increase nuclear export of p27 [191].