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,25-Dihydroxyvitamin D3 and Its Analog EB1089 under Growth-Inhibitory Conditions in Squamous Carcinoma Cells
Departments of Physiology and Medicine (N.A., R.L., Y.B., A.B.,
J.H.W.) McGill University Montreal, Quebec H3G 1Y6, Canada
Department of Otolaryngology-Head and Neck Surgery (D.J.E.,
M.J.B.), and Montreal Center for Experimental Therapeutics in
Cancer (M.J.B., J.H.W.) Jewish General Hospital and McGill
University Montreal, Quebec, H3T 1E2, Canada
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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,25-dihydroxyvitamin
D3 (1,
25(OH)2D3)
inhibit growth in vitro and in vivo of
cells derived from a variety of tumors. Here, we examined the effects
of 1,25(OH)2D3 and
its analog EB1089 on proliferation and target gene regulation of human
head and neck squamous cell carcinoma (SCC) lines SCC4, SCC9, SCC15,
and SCC25. A range of sensitivities to
1,25(OH)2D3 and
EB1089 was observed, from complete
G0/G1 arrest of SCC25
cells to only 50% inhibition of SCC9 cell growth. All lines expressed
similar levels of vitamin D3 receptor (VDR)
mRNA and protein, and no significant variation was observed in
1,25(OH)2D3-dependent
induction of the endogenous 24-hydroxylase gene,
or of a transiently transfected
1,25(OH)2D3-sensitive
reporter gene. The antiproliferative effects of
1,25(OH)2D3 and
EB1089 in SCC25 cells were analyzed by screening more than 4,500 genes
on two cDNA microarrays, yielding 38 up-regulated targets, including
adhesion molecules, growth factors, kinases, and transcription factors.
Genes encoding factors implicated in cell cycle regulation were
induced, including the growth arrest and DNA damage gene, gadd45,
and the serum- and glucocorticoid-inducible kinase gene, sgk. Induction
of GADD45 protein in EB1089-treated cells was confirmed by Western
blotting. Moreover, while expression of proliferating cell
nuclear antigen (PCNA) was reduced in EB1089-treated cells,
coimmunoprecipitation studies revealed increased association between
GADD45 and PCNA in treated cells, consistent with the capacity of
GADD45 to stimulate DNA repair. While
1,25(OH)2D3 and
EB1089 modestly induced transcripts encoding the cyclin-dependent
kinase inhibitor p21waf1/cip1, no changes
in protein levels were observed, indicating that
p21waf1/cip1 induction does not contribute to
the antiproliferative effects of
1,25(OH)2D3 and
EB1089 in SCC cells. Finally, in partially resistant SCC9 cells, there
was extensive loss of target gene regulation (10 of 10 genes
tested), indicating that resistance arises from widespread loss
of
1,25(OH)2D3-dependent
gene regulation in the presence of normal levels of functional VDRs. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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,25-dihydroxyvitamin D3
[1,25(OH)2D3]
modulates gene expression by binding to the vitamin
D3 receptor (VDR), which is a member of the
nuclear receptor family of transcriptional regulators.
1,25(OH)2D3-bound VDR
heterodimerizes with retinoid X receptors (RXRs) and binds to specific
DNA sequences in target genes known as vitamin D3
response elements (VDREs) (1, 2). Apart from its well characterized
role in calcium homeostasis (3),
1,25(OH)2D3 also
inhibits growth and stimulates differentiation of cancer cells derived
from a variety of tissues, including breast, prostate, colon, lung,
endometrium, hematopoietic cells, and oral cavity (4, 5, 6, 7, 8, 9, 10). A side
chain analog of
1,25(OH)2D3,
EB1089, caused apoptotic regression of MCF-7 breast carcinoma
xenografts in nude mice (9), and animal studies and early clinical
testing have shown that therapeutic doses of EB1089 can be tolerated
without inducing hypercalcemia (10).
Analogs of 1,25(OH)2D3 are
potential candidates for chemoprevention of squamous cell
carcinomas (SCCs) of the oral cavity, where formation of second primary
carcinomas after surgical removal of tumors is a major concern (11, 12). Retinoids, such as 13-cis retinoic acid
(13-cis-RA; isotretinoin) have been used clinically in SCC
chemoprevention (13). 13-cis-RA functions by binding to
retinoic acid receptors (RARs), which, like the VDR, are nuclear
receptors and function as heterodimers with RXRs (1). However, SCC
progression is associated with reduced expression of RARs, particularly
RARs ß and 
, loss of retinoid-regulated differentiation markers,
and resistance to the antiproliferative effects of retinoids
(14, 15, 16, 17, 18, 19).
Here, we have examined the effect of
1,25(OH)2D3 and EB1089
on proliferation and target gene regulation of four human SCC lines,
SCC4, SCC9, SCC15, and SCC25, which were derived from the floor of
mouth/base of tongue lesions (14). SCC25 cells express near normal
levels of RARs ß and and retain retinoid regulation of keratin-19
(K-19) gene expression, whereas SCC4, SCC9, and SCC15 cells express
reduced levels of RAR, no RARß, and have lost regulated K-19
expression (14). The SCC lines display differing sensitivities to
1,25(OH)2D3 and EB1089.
SCC25 cell growth was completely blocked by
1,25(OH)2D3 and EB1089,
while the other lines were partially resistant. We have identified 38
1,25(OH)2D3 target genes
in SCC25 cells, which encode several components of signal transduction
pathways. Our results indicate that the antiproliferative effects of
1,25(OH)2D3 and its
analogs are mediated by multiple downstream components. Moreover,
resistance to
1,25(OH)2D3 in SCC9
cells was accompanied by widespread loss of target gene regulation in
spite of normal levels of functional VDRs.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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,25(OH)2D3 and
EB1089 on Growth of SCC Lines,25(OH)2D3,
EB1089, and 13-cis-RA were evaluated in human
lines SCC4, SCC9, SCC15, and SCC25, derived from SCCs of the oral
cavity. The four lines displayed different sensitivities to
1,25(OH)2D3 or EB1089
(Fig. 1
). Over 10 days, SCC25 cell growth
was completely inhibited by 100 nM
1,25(OH)2D3, and 1–100
nM EB1089 (Fig. 1, A and B), while SCC4, SCC9,
and SCC15 cells displayed partial resistance to both compounds (Fig. 1, D, E, G, H, J, and K). Similarly, SCC25 cell growth was strongly
inhibited by 13-cis-RA (100 nM; Fig. 1C), whereas growth of SCC4, SCC9, and SCC15 cells was partially
resistant (Fig. 1, F, I, and L). Flow cytometric analysis showed that
treatment of SCC25 cells with 100 nM EB1089
for 72 h reduced the number of cells in S phase by 2.5-fold and
significantly increased the percentage in
G0/G1 (Fig. 2A). No evidence for DNA fragmentation
was observed by terminal deoxynucleotidyltransferase
dUTP-biotin nick end-labeling (TUNEL) assays under these
conditions or over extended periods (Fig. 2B).
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,25(OH)2D3 in SCC4,
SCC9, and SCC15 cells Is Not Accompanied by Loss of Expression of
Functional VDR, respectively (14),
it was of interest to examine the levels of functional VDR in SCC
cells. Northern and Western blots showed that VDR transcript and
protein levels were essentially identical in all four lines (Fig. 3, A and B). Similarly, no evidence was
found for loss of expression of the two major RXRs expressed in SCC,
RXR and RXRß (data not shown). VDR function was tested by
transient transfection of a
1,25(OH)2D3-sensitive
reporter-promoter plasmid containing a bacterial lacZ gene under
control of a synthetic promoter containing three VDREs (20). High
levels of
1,25(OH)2D3-inducible
ß-galactosidase activity were detected in all cell extracts (Fig. 4A), suggesting that the lines expressed
similar levels of functional VDRs. Both
1,25(OH)2D3 and EB1089
induced similar levels of expression of the endogenous 24-hydroxylase
(24-OHase) gene (Fig. 4B), whose promoter contains VDREs (21).
Moreover, EB1089 induced 24-OHase expression with essentially identical
potencies in
1,25(OH)2D3-sensitive
SCC25 cells and the partially resistant lines SCC4 and SCC9 (Fig. 4C).
Taken together, the results of Figs. 3 and 4 suggest that
resistance to
1,25(OH)2D3 does not
arise through loss of expression of functional VDRs.
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,25(OH)2D3 and
EB1089 on Cell Cycle Regulators in SCC25 Cells,25(OH)2D3 and EB1089
in SCC25 cells. Previous work has shown that
1,25(OH)2D3 rapidly (4
h) and strongly stimulated expression of the cyclin-dependent kinase
inhibitor genes p21waf1/cip1 and
p27kip1 in myeloid leukemia cells under
conditions where it induced differentiation and inhibited cell growth
(4, 22). However, the magnitude of the effect of
1,25(OH)2D3 on
p21waf1/cip1 expression varies widely in
different cell lines (4, 6, 22, 23, 24). We found that
1,25(OH)2D3- or
EB1089-dependent induction of p21waf1/cip1
transcripts in SCC25 cells was gradual and modest (Fig. 5A and data not shown), whereas no
effect was observed on expression of p27kip1 or
p53 mRNA levels (Fig. 5B). The modest effect of
1,25(OH)2D3 and EB1089
on p21waf1/cip1 mRNA levels did not give rise to
significant changes in p21waf1/cip1 protein,
however. In addition, no effect of
1,25(OH)2D3 or EB1089
was observed on p27kip1 protein levels (Fig. 5C).
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,25(OH)2D3 and
EB1089 by Screening of cDNA Microarrays,25(OH)2D3 and EB1089
in SCC25 cells to identify factors mediating their
antiproliferative effects. More than 4,500 genes on two different gene
arrays [Atlas array, 588 genes; (CLONTECH Laboratories, Inc., Palo Alto, CA); Named Genes filter, 4,000+ genes
(Research Genetics, Inc., Huntsville, AL)] were screened
with probes derived from vehicle-treated cells or cells treated with
EB1089 for 24 h. Previous work has shown that there is
considerable variation in gene expression levels associated with
screening gene arrays (25, 26, 27, 28). Arrays were therefore screened multiple
times, and only reproducibly regulated genes were retained. Two rounds
of screening of Atlas arrays yielded 10 candidate genes, of which 6
were revealed by Northern blotting to be regulated by
1,25(OH)2D3 and EB1089
(Fig. 6A, and data not shown; Table 1). In addition to
p21waf1/cip1 (not shown), these included novel
target genes amphiregulin, a member of the epidermal growth factor
family, the transcription factor fos-related antigen-1 (fra-1), the
growth arrest and DNA damage (gadd45) gene, and integrin 7B. We
also found that the vascular endothelial growth factor (VEGF), which
has been shown to be a
1,25(OH)2D3 target gene
in osteoblast-like cells (29, 30), was regulated by EB1089 in SCC25
cells.
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). Screening under these conditions did not reveal any
reproducibly down-regulated genes. Up-regulated genes included
calmodulin, which has previously been shown to be a
1,25(OH)2D3 target gene
(31). Northern blotting, used to further test expression of 10 of these
genes, revealed EB1089-stimulated expression in all cases (Fig. 6B),
indicating that the data in Table 1 are highly reliable. Most of the
genes retained from phosphorimager analysis of the Research Genetics, Inc. arrays were up-regulated 2- to 4-fold (Table 1).
This range of induction agrees well with that of up-regulated targets
identified in a similar screen of thyroid hormone-regulated genes
(33).
Broad but Selective Loss of Target Gene Expression in
1,25(OH)2D3-Resistant
SCC Lines
Given the resistance of SCC9 cells to the inhibitory effects of
1,25(OH)2D3 and EB1089,
we analyzed the regulation of target genes in these cells. Remarkably,
in spite of apparently normal induction of 24-OHase expression (Fig. 4), regulation of all of the target genes tested in SCC9 cells was
either lost, or in the case of calmodulin and GAP SH3 binding protein,
attenuated (Fig. 6C). These results provide a strong correlation
between increased resistance to the antiproliferative effects of
1,25(OH)2D3 and a broad
but selective loss of
1,25(OH)2D3 target gene
regulation in the presence of apparently normal levels of functional
VDR.
EB1089 Treatment Induces Expression of GADD45 Protein and
Enhances Formation of GADD45-Proliferating Cell Nuclear Antigen
(PCNA) Complexes
One of the more intriguing genes identified
from the array screening presented above was gadd45 (Fig. 6 and
Table 1). Gadd45 is a p53 target gene induced by a variety of agents
that damage DNA and arrest cell growth (33, 34, 35, 36), and overexpression of
GADD45 inhibits cell proliferation (34). Ablation of the gadd45
gene provided evidence that GADD45 functions to maintain global
genomic stability (35). Peak expression of GADD45 occurs in
G1. DNA repair is enhanced at the
G1/S checkpoint, and several studies have
suggested that GADD45 enhances DNA repair, at least in part, through
its interaction with PCNA (36, 37, 38).
Induction of gadd45 mRNA by EB1089 was only partially blocked by
protein synthesis inhibitor cycloheximide (Fig. 7A, and data not shown), indicating that
the effect of EB1089 is at least partially direct. In related studies,
we found no effect of cycloheximide on induction of gadd45
transcripts by EB1089 in the mouse SCC line AT-84 (38A ).
Immunoprecipitations from control and treated SCC25 cells revealed that
EB1089 induced expression of GADD45 protein (Fig. 7B), consistent
with its effects on gadd45 mRNA levels. Previous studies have
demonstrated that and UV irradiation induce GADD45 and enhance
its interaction with PCNA (36, 37). It was therefore of interest to
determine whether a similar interaction was induced by EB1089, which is
not a DNA damaging agent. While EB1089 treatment of SCC25 cells
consistently reduced expression of PCNA protein (Fig. 7B and data not
shown), reciprocal coimmunoprecipitations revealed an increased
association between PCNA and GADD45 in EB1089-treated cells (Fig. 7B). Thus, 1,25(OH)2D3
analog EB1089 induces expression of GADD45, leading to increased
formation of GADD45-PCNA complexes. Taken together, our results
suggest that induction of GADD45 contributes to the
growth-inhibitory effects of
1,25(OH)2D3 and EB1089
in SCC25 cells.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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,25(OH)2D3 and EB1089
were as or more potent, respectively, than 13-cis-RA in
inhibiting growth of SCC25 cells in culture. SCC4, SCC9, and SCC15
cells were partially resistant to 13-cis-RA and to
1,25(OH)2D3 and EB1089
(Fig. 1), raising the possibility of a common underlying
mechanism of resistance. Expression of RARs ß and is lost or
reduced, respectively, in SCC4, SCC9, and SCC15 cells (14). However, no
evidence was found for loss of VDR expression or function in these
lines. No substantial differences were observed in induction of
endogenous 24-hydroxylase gene expression, the transcription of which
is controlled by a VDRE-containing promoter (21), or of a transiently
transfected VDRE3-hsp68/lacZ reporter plasmid. This is consistent with
other findings suggesting that VDR levels vary little among SCC lines,
including SCC4 (39, 40). Our results showed that VDRs expressed in all
four lines studied retained the capacity to activate transcription from
VDRE-containing promoters. We have also characterized a mouse SCC line,
AT-84, which is highly sensitive to
1,25(OH)2D3 and EB1089
but resistant to the growth- inhibitory effects of retinoids
(38A ), showing that resistance to
1,25(OH)2D3 and
retinoids is not necessarily coupled.
Several results suggest that many factors contribute to the
growth-inhibitory effects of
1,25(OH)2D3 in a
cell-specific manner. Transcripts encoding the cyclin-dependent kinase
inhibitors p21waf1/cip1 and
p27kip1 were strongly and rapidly
up-regulated by
1,25(OH)2D3 in
myeloid leukemia cells, and forced expression of
p21waf1/cip1 induced myeloid cell differentiation
(4, 22). Moreover, a VDRE that functioned in U937 cells was identified
in the p21 promoter (4). However, the effect of
1,25(OH)2D3 on
p21waf1/cip1 and p27kip1
expression is highly cell specific. The induction of
p21waf1/cip1 mRNA by EB1089 in SCC25 cells was
gradual and modest, but no effect on protein levels was observed (Fig. 5). 1,25(OH)2D3
treatment modestly increased p21waf1/cip1 protein
in LNCaP prostate cancer cells (23). However, no significant effect on
transcript levels and no
1,25(OH)2D3-dependent
induction of the p21waf1/cip1 promoter was
observed in gene transfer experiments in LNCaP cells (23). Hershberger
et al. (6) found that
1,25(OH)2D3 repressed
p21waf1/cip1 expression in the mouse SCCVII/SF
line, and we have observed a similar repression of
p21waf1/cip1 transcripts and protein in the mouse
SCC line AT-84 (38A ).
The lack of induction of cyclin-dependent kinase inhibitors in
1,25(OH)2D3- or
EB1089-treated SCC25 cells led us to screen gene arrays to identify
other regulated genes in SCC25 cells. A total of 38 target genes,
including p21waf1/cip1, were identified in two
screens of more than 4,500 genes (Table 1). The 32 targets identified
on the Research Genetics, Inc. filter were retained after
9 sets of cross- comparisons of data derived from screening with probe
derived from vehicle- and EB1089-treated SCC25 cells, using a minimum
induction of 1.5-fold as a cut-off. We confirmed that 10 of 10
candidates analyzed by Northern blotting showed
1,25(OH)2D3-regulated
expression (Fig. 6), indicating that the data obtained from the array
screening are highly reliable. Most genes were up-regulated 2- to
4-fold, a range in good agreement with that of up-regulated targets
identified in a similar screen of thyroid hormone- regulated genes
(33), and generally more modest than the levels of gene regulation
observed by forced overexpression of the tumor suppressor genes BRCA1
(41) and WT1 (42).
The genes identified in this study encode several different classes of
proteins, many of which are components of different signal transduction
pathways. They include cell adhesion proteins (e.g.
galectin-2, integrin 7B), growth factors (e.g.
amphiregulin, VEGF), cytoskeletal proteins (e.g.
actin-related protein 3), protein kinases (e.g. serum- and
glucocorticoid-regulated kinase, sgk), other intracellular signaling
molecules, and transcription factors (AP-4, STAT-3, FRA-1). Some of the
genes identified here have been implicated in regulation of the cell
cycle and growth arrest. One example is serum- and
glucocorticoid-inducible kinase, SGK, which is shuttled between the
nucleus and the cytoplasm during the cell cycle. Its forced retention
in either compartment suppressed serum-induced growth and DNA synthesis
in mammary tumor cells (43).
We also found that
1,25(OH)2D3 and EB1089
induced expression of gadd45, which like
p21WAF1/CIP1 is a p53 target gene. However,
neither compound affected p53 expression in SCC25 cells. A similar
induction of GADD45 expression by
1,25(OH)2D3 and EB1089
was observed in vitro and in vivo in the murine
SCC line AT-84 under conditions in which expression of p53 was
unaffected and p21WAF1/CIP1 was repressed.
In contrast, DNA damaging agents induced p53,
p21WAF1/CIP1, and GADD45 in AT-84 cells
(38A ). Taken together, these results suggest that
1,25(OH)2D3- and
EB1089-dependent induction of gadd45 occurs by a p53-independent
mechanism.
Consistent with its effects on gadd45 mRNA, EB1089 treatment of
SCC25 cells enhanced expression of GADD45 protein and stimulated
formation of GADD45-PCNA complexes. Previous studies have shown that
DNA damaging agents, such as or UV irradiation, induce formation of
GADD45-PCNA complexes (36, 37). Induction of GADD45-PCNA
complexes by EB1089, which is a growth inhibitor, but not a DNA
damaging agent, indicates that increased DNA damage is not
necessary to induce complex formation.
PCNA function is required for DNA replication in S phase, and for DNA
repair through its association with polymerases 
and 
(44).
Association of GADD45 with PCNA is considered to divert PCNA from
sites of DNA replication to sites of DNA repair. GADD45 modifies DNA
accessibility on damaged chromatin and can stimulate DNA repair
in vitro (36, 45, 46). In addition, DNA damaging agents
induce changes in the nuclear distribution of PCNA (47). It should be
noted, however, that PCNA also interacts with a number of other
regulatory proteins, including p21WAF1/CIP1 (48),
at sites that overlap those recognized by GADD45 (49). The relative
roles and importance of interactions of
p21WAF1/CIP1 and GADD45 with PCNA remain to be
fully elucidated. Nonetheless, the induction of GADD45 expression
and its central role in enhancing DNA repair suggest that treatment of
SCC cells with
1,25(OH)2D3 or EB1089
would provide a genoprotective effect. This would be an important
characteristic of a potential chemopreventive agent.
The observation that
1,25(OH)2D3 induced
expression of VEGF in SCC cells was surprising given that increased
VEGF levels are associated with tumor vascularization and tumor
progression (50). Elevated VEGF levels have been correlated with a
higher rate of disease recurrence and a shorter disease-free interval
in SCC of the oral cavity (51). These results highlight the complexity
of cellular responses to growth regulators such as
1,25(OH)2D3 and its
analogs, where a combination of regulatory signals is induced under
conditions in which the overall effect of
1,25(OH)2D3 is growth
inhibitory. It should also be noted that
1,25(OH)2D3-regulated
expression of VEGF is highly cell specific. We did not observe any
induction of VEGF expression in MCF-7 and MBA-MD231 breast cancer or
LNCaP prostate cancer cells (data not shown), whereas others have shown
that VEGF expression is regulated by
1,25(OH)2D3 in
osteoblast-like cells (29, 30).
The partial resistance of SCC9 cells to the growth-inhibitory effects
of 1,25(OH)2D3
correlated with broad deregulation of target gene expression (10 of 10
genes tested). It is unlikely that loss of regulation arises through
repressed expression due to target gene methylation, since transcripts
of all genes refractory to
1,25(OH)2D3 were
detected in vehicle-treated SCC9 cells (Fig. 6). It is possible that
1,25(OH)2D3-dependent
induction of these genes requires synergism of the VDR with other
transcription factor(s) or downstream regulators, the function of which
is defective in SCC9 cells. Such factors would not be required for
regulated expression of the endogenous 24-hydroxylase gene or the
synthetic VDRE3-hsp68 promoter. One possible candidate is AP1, whose
function is enhanced by
1,25(OH)2D3 signaling
(52, 53, 54). However, this enhancement apparently requires, at least in
part, up-regulation of expression of AP1 components, particularly
c-jun. We have also found here that
1,25(OH)2D3 modestly
up-regulates fra-1 mRNA levels. This suggests that if loss of induced
AP1 activity contributes to deregulation of
1,25(OH)2D3 target gene
expression in resistant SCC lines, it may not be a primary defect. It
should also be noted that we have tested the effect of cycloheximide on
the six target genes identified on the Atlas array,
p21waf1/cip1, amphiregulin, VEGF, fra-1,
gadd45, and integrin 7B, and found in each case there was no
effect on
1,25(OH)2D3-stimulated
expression (Fig. 7, and data not shown). Therefore, in these instances,
the stimulatory effect of
1,25(OH)2D3 did not
require protein synthesis. Moreover, with the exceptions of integrin
7B and fra-1, which were not tested, regulation of all of these
genes was lost in SCC9 cells (Fig. 6).
In summary, our studies have shown that
1,25(OH)2D3
analogs can be potent inhibitors of SCC proliferation and control the
expression of several regulators of cell proliferation. However,
partial resistance to
1,25(OH)2D3 can arise
even in the presence of apparently normal levels of functional VDR.
Resistance arises from a broad, but selective, loss in
1,25(OH)2D3-regulated
gene expression in the presence of normal levels of functional
VDRs.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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,25(OH)2D3 and EB1089 were
kindly supplied by Dr. Lise Binderup (Leo Laboratories, Ballerup,
Denmark). 13-cis-RA was purchased from ICN Biochemicals, Inc. (Costa Mesa, CA). All hormones were
dissolved in dimethylsulfoxide (DMSO), and stock solutions were stored
in the dark at -20 C.
Tissue Culture
The SCC lines, SCC4, SCC9, SCC15, and SCC25, obtained from the
American Type Culture Collection (ATCC,
Manassas, VA), were cultured under recommended conditions. Effects of
1,25(OH)2D3, EB1089, and
13-cis-RA on cell growth were analyzed by seeding cells in
6-well plates at 15,000 cells per well in 2 ml of culture medium
containing charcoal-stripped serum. Media were changed after 24 h
to charcoal-stripped medium containing vehicle or ligand as indicated.
Media were changed every 48 h and fresh ligand added. Cells were
harvested by washing with 2 ml of PBS and incubation with 0.7 ml of
0.25% trypsin-EDTA. Cell numbers were determined using a
hemacytometer. Four grid sections were counted for each well and the
results were averaged. All treatments were performed in triplicate.
Transient Transfections
SCC cells were grown to 60% confluency in six-well plates in
charcoal-stripped medium, washed with 2 ml of Opti-MEM I-reduced serum
media (Life Technologies, Inc., Burlington, Ontario,
Canada), and cultured in 1 ml of Opti-MEM I. Cells were transfected
with 500 ng of VDRE3-LacZ reporter plasmid and 500 ng of tk-LUC
internal control using Lipofectin (Life Technologies, Inc.) according to the manufacturer’s protocol. After 18 h
media were replaced with charcoal- stripped medium containing ligands
as indicated. Cells were lysed 24 h later using lysis buffer
(Promega Corp., Madison, WI), and ß-galactosidase assays
were performed as described (57). Transfections were performed in
triplicate and standardized using the Luciferase Assay System with
reporter lysis buffer (Promega Corp.).
RNA Isolation and Northern Blotting
Cells were grown in 100-mm dishes. Media were replaced with
charcoal-stripped medium containing ligand as indicated. Total RNA was
extracted with TRIZOL (Life Technologies, Inc.). PolyA+
RNAs were isolated using an Oligotex mRNA Kit (QIAGEN,
Valencia, CA). One microgram of polyA+ RNA was separated on a 1.0%
agarose gel containing 6.3% formaldehyde, 20 mM
3-(N-morpholino)propanesulfonic acid (pH 7.0), 15
mM sodium acetate, and 1 mM
EDTA. Separated RNAs were transferred to a Nylon membrane (Hybond-N+,
Amersham Pharmacia Biotech, Baie d’Urfe, Quebec), which
then was soaked in 3xsaline-sodium citrate (SSC) and 0.1% SDS
at 50 C, and prehybridized at 42 C in 50 mM
phosphate buffer, pH 6.5, 50% formamide, 5x SSC, 10% Denhardt’s
solution containing 250 µg/ml sheared, denatured salmon sperm DNA.
Hybridization was carried out in the same solution by the addition of
32P-labeled cDNA probes. Membranes were washed
four times in 2xSSC and 0.2% SDS for 5 min, three times in 0.1xSSC
and 0.2% SDS for 30 min at 50 C, dried, and autoradiographed. All
blots were performed at least three times with independent preparations
of RNA.
RT-PCR
Ten micrograms of total RNA were subjected to oligo dT priming
first-strand cDNA synthesis by SuperScript II (Life Technologies, Inc., Burlington, Ontario, Canada). Twenty microliter aliquots
were diluted 5-fold with water. For RT-PCR analysis of p53 and p27
kip1 mRNA, expression of 1 µl of RT reactions
was analyzed by PCR amplification as follows: 30 sec denaturation at 94
C, 45 sec elongation at 72 C, and 30 sec annealing starting at 60 C,
down 1 C per cycle to 55 C, and continuing 20 cycles of amplification
(94 C for 30 sec, 57.5 C for 30 sec, 72 C for 45 sec). Complementary
DNAs for p53 and p27 kip1 were
amplified using 5'-primer 5'-CAAGTCTGTGACTTGCACGTA-3' and 3'-primer
5'-TTCTTGCGGAGATTCTCTTCC-3' for p53, and 5'-primer
5'-CCGGAATTCATGTCAAACGTGCGAGTGTCT-3' and 3'-primer
5'-CCGGAATTCTTACGTTTGACGTCTTCTGAGGC-3' for
p27kip1. For ß-actin, 1 µl of RT reaction was
subjected to 18 cycles of amplification (95 C for 30 sec, 56 C for 1
min, 72 C for 25 sec) using 5'-primer 5'-GCTGTGCTATCCCTGTACGC-3' and
3'-primer 5'-CCAATGGTGATGACCTGGC-3'. All of the above reactions were
performed in 25 µl of 1.5 mM MgCl2,
50 mM KCl, and 10 mM Tris-Cl (pH 9.0) using 2.5
U of Taq DNA polymerase (Amersham Pharmacia Biotech, Baie d’Urfe, Quebec, Canada). PCR reactions were
loaded on a 2% agarose gel, transferred for Southern blotting to a
nylon membrane (Hybond N+, Amersham Pharmacia Biotech),
and fixed by UV cross-linker. The membrane was soaked in 3x SSC and
0.1% SDS at 50 C, and prehybridized at 42 C in 50
mM phosphate buffer, pH 6.5, 5x SSC, 10%
Denhardt’s solution containing 250 µg/ml sheared and denatured
salmon sperm DNA. Hybridization was carried out in the same solution by
the addition of 32P end-labeled oligonucleotides
5'-CTACAAGCAGTCACAGCACAT-3' for p53, 5'-CTAACTCTGAGGACACGCATT-3' for
p27kip1, and 5'-CGAGAAGCTGTGCTACGTCG-3' for
ß-actin. After hybridization, the membrane was washed four times in
2x SSC and 0.2% SDS for 5 min, three times in 0.1x SSC and 0.2% SDS
for 30 min at 50 C, dried, and autoradiographed. All experiments were
repeated at least three times.
Immunoprecipitation and Western Blotting
After incubation with ligands, cells were washed twice with PBS
and harvested by scraping in 1 ml of PBS and centrifuged at 4 C. The
pellet was resuspended in 500 µl of ice-cold lysis buffer (10
mM Tris-HCl, pH 8.0, 60 mM KCl, 1
mM EDTA, 1 mM dithiothreitol, 0.5%
NP40) containing protease inhibitor cocktail (Roche Molecular Biochemicals, Mannheim, Germany), incubated on ice for 10 min.
Lysates were centrifuged at 4 C (14,000 rpm, 10 min), and supernatants
were recovered. For p21WAF1/CIP1 and
p27KIP1 immunoprecipitations, protein extracts
(200 µg) were immunoprecipitated at 4 C overnight with 3 µg of F-5
and F-8 anti-p21WAF1/CIP1 and
-p27KIP1 monoclonal antibodies, respectively
(Santa Cruz Biotechnology, Inc., Santa Cruz, CA) using 30
µl of 50% slurry protein S-Sepharose (Amersham Pharmacia Biotech). Beads were centrifuged, and pellets were washed four
times each with lysis buffer and boiled for 3 min in 2x
SDS-polyacrylamide gel loading buffer. Immunoprecipitates were resolved
on 20% SDS-polyacrylamide gels and analyzed by Western blotting with
the same antibodies. Immunoprecipitations of GADD45 and PCNA were
performed with anti-GADD45 antibody 4T-27 or with anti-PCNA antibody
PC-10 (Santa Cruz Biotechnology, Inc.). Immunoprecipitates
were harvested, processed for Western blotting as above and probed with
anti-GADD45 antibody (H-165) (Santa Cruz Biotechnology, Inc.) or with anti-PCNA (PC-10) (Santa Cruz Biotechnology, Inc.).
Western analysis of VDR expression was performed with 30 µg of total cell protein resolved on a 15% SDS-polyacrylamide gel. VDRs were probed with 800 ng of a rabbit polyclonal anti-VDR antibody (Santa Cruz Biotechnology, Inc.). Proteins were detected by enhanced chemiluminescence (ECL; NEN Life Science Products, Boston, MA).
Flow Cytometry and TUNEL Assays
SCC25 cells treated with 100 nM EB1089 or DMSO for
72 h were harvested with 0.25% trypsin-EDTA, fixed with 70%
ethanol for 1 h at 4 C, treated with 200 µg/ml RNase A for 30
min, stained with 5 µg/ml propidium iodide for DNA, and analyzed for
cell cycle status by flow cytometry (Becton Dickinson and Co., Franklin Lakes, NJ). Experiments were repeated three times.
TUNEL assays were performed using an Apoptag kit
(Intergen, Purchase, NY) according to the manufacturer’s
instructions. Briefly, after incubation with vehicle or ligand, cells
were fixed for 15 min in 1% paraformaldehyde, washed twice with PBS,
and stored in 70% ethanol at -20 C. Cells (100 µl) were then
incubated for 30 min at 37 C with terminal deoxynucleotidyl transferase
and digoxigenin-dUTP. After two washes with 0.1% Triton X-100 in PBS,
cells were incubated with fluorescein-conjugated antidigoxigenin
antibody for 30 min at room temperature. After two washes with 0.1%
Triton X-100 in PBS, cells were treated with RNase A and processed for
flow cytometry as above.
Array Screening
SCC25 cells were treated for 24 h with DMSO or EB1089 (100
nM). Atlas cDNA Expression Arrays containing 588 genes
(CLONTECH Laboratories, Inc. Palo Alto, CA) were screened
with 100 ng of polyA+ RNA. GF211 Named Human Genes arrays containing
more than 4,000 genes (Research Genetics, Inc.) were
probed with 1 µg of total RNA. Probe preparation and array screening
were carried out according to manufacturers’ instructions. Duplicate
Atlas arrays were screened twice each with probe derived from control
or treated cells and arrays were visualized by autoradiography. Genes
that appeared reproducibly regulated were studied by Northern analysis.
GF211 filters were probed three times each with probe derived from
control cells and EB1089-treated cells, and visualized by
phosphorimaging. Relative expression levels were compared using
Pathways software (Research Genetics, Inc.). Genes that
were up-regulated at least 1.5-fold in nine sets of cross-comparisons
were retained. Of these, 10 were further analyzed by Northern analysis
using cDNA probes from Research Genetics, Inc.
|
ACKNOWLEDGMENTS |
|---|
|
FOOTNOTES |
|---|
This work was supported by an operating grant from the Canadian Institutes of Health Research (MT-15160) to J.H.W. Initial experiments were supported by funds from the Department of Otolaryngology of the Jewish General Hospital, Montreal. N.A. was supported by a postdoctoral fellowship from the Royal Victoria Research Institute. J.H.W is a chercheur-boursier of the Fonds de Recherche en Santé du Québec (FRSQ).
1 N.A. and R.L. should be considered as equal first authors. 
Received for publication September 19, 2000. Revision received February 26, 2001. Accepted for publication March 13, 2001.
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