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From the Department of Medicine,*
Mount Sinai Hospital,Toronto; and the Department of Pathology,
University Health Network, University of Toronto, Toronto,Ontario, Canada
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Abstract |
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 Top Abstract Materials and MethodsResultsDiscussionReferences |
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In addition to its role in calcium homeostasis, the hormonal form of
vitamin D, 1
,25-dihydroxy vitamin D3 (VD), has
been recognized to play a role in the modulation of the proliferation
and differentiation of several cell types.3-6
VD has been
reported to induce apoptosis in human breast cancer7
and
leukemic cell lines.8
Several VD analogs that lack
unwanted side-effects of hypercalcemia, hypercalciuria, and soft tissue
calcification have been shown to have anti-proliferative or apoptotic
effects, and their promise as an important therapeutic tool has been
recognized.6
However, the regulatory mechanism(s) by which
these agents exert their influence on the cell cycle remains to be
elucidated. It has been suggested that vitamin D compounds act by
inducing apoptosis, but the mechanism(s) of an anti-proliferative
action through cyclin-dependent kinases (CDKs) and/or their inhibitors
(CDKIs) remains to be elucidated.
In this study we examined whether VD and its noncalciomimetic analog EB1089 can inhibit growth of several thyroid cancer cell lines either by inhibition of proliferation, induction of differentiation, or induction of apoptosis. Our data indicate that the CDK inhibitor p27kip1 (p27) is an important cellular target for the action of VD and its analog on thyroid cancer.
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Materials and Methods |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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The expression level of p27 was investigated during a 72-hour
incubation of the cells with VD or EB1089. VD and its analog EB1089
(22,24-diene-24a,26a,27a-trihomo-1,25-dihydroxyvitamin
D3) were provided by Dr. L. Binderup of LEO
Pharmaceutical Products (Ballerup, Denmark). To demonstrate the
mechanism of increased p27 expression by VD and EB1089, NPA and WRO
cells were incubated with VD and EB1089 for 72 hours and in the
presence or absence of 50 µmol/L of the proteasome inhibitor LLnL
(Sigma, St. Louis, MO) for 5 hours. To demonstrate the role of PI-3
kinase-mediated pathways in VD/EB1089-mediated reduction of p27
degradation, we incubated cells with the PI-3 kinase inhibitors
wortmannin 0.2 µmol/L (Sigma) or LY900402 20 µmol/L (New England
Biolabs, Beverly, MA) for 12 hours. To evaluate the requirement
for phosphatase action in mediating VD or EB 1089 action on p27, we
incubated cells with the phosphatase inhibitor pervanadate (Sigma) at
0.1 µmol/L for 15 to 60 minutes. PTEN expression was performed using
a full-length wild-type pGZ21
xZ vector kindly provided by Dr. K.
Yamada (National Institute of Dental and Craniofacial Research,
National Institutes of Health, Bethesda, MD).
Cell Culture
To determine VD effects on cells with variable degrees of differentiation, several human thyroid carcinoma cell lines were studied. TPC-1 cells (a gift from Dr. S. M. Jhiang, Ohio State University, Columbus, OH), a well-differentiated papillary thyroid carcinoma line with a ret/PTC-1 gene rearrangement and intact p53, were cultured in Dulbecco’s modified Eagle’s medium supplemented with 5% FCS, 2 mmol/L L-glutamine. The NPA cell line is a poorly differentiated papillary carcinoma cell line with mutant p53, WRO cells are a well-differentiated follicular carcinoma cell line with mutant p53, and FRO is an anaplastic carcinoma cell line with wild-type p53 status. These cells were obtained from Dr. J. Fagin (University of Cincinnati, Cincinnati, OH) and cultured in RPMI 1640 supplemented with 10% FCS, 2 mmol/L L-glutamine, 1 mmol/L sodium pyruvate, and 1 x nonessential amino acid (Sigma-Aldrich Co. Ltd., Irvine, UK). Cell number and viability was assessed at the beginning and end of each experiment using trypan blue.
Proliferation Assay
[3H]Thymidine incorporation was used to assess effects of VD and EB1089 on proliferation of the above cell lines. Briefly, the cells were cultured in 6-well plates. After overnight recovery, the cells were incubated with VD or EB1089 at graded concentrations ranging from 10-6 to 10-10 mol/L for 72 hours. After refeeding the cells with the same medium and compound as above, the cells were exposed to 1 µCi [3H]thymidine/well for 5 hours. The medium was discarded and the cells were washed three times with Ca2+- and Mg2+-free phosphate-buffered saline (PBS). Incorporated [3H]thymidine was measured with a ß-counter. Inhibition of cell proliferation was expressed as the percentage of mean [3H]thymidine incorporation relative to that incorporated by vehicle-treated control cells.
Cell-Cycle Analysis
Cells were incubated with VD or EB1089 for 72 hours. After trypsinization, 1 to 3 x 106 cells were washed with PBS and fixed with cold 80% ethanol for 1 hour on ice. The fixed cells were washed with staining buffer (0.2% Triton X-100 and 1 mmol/L ethylenediaminetetraacetic acid, pH 8.0, in PBS) and resuspended in the staining buffer containing 50 µg/ml RNase A (Sigma) and 50 µg/ml propidium iodide for 1 hour. Cell-cycle analysis was done by fluorescence-activated cell sorting (FACScan; Becton Dickinson, San Jose, CA) using Cellquest Analysis and specific S phase was analyzed using Modfit DNA Analysis (Verity Software House Inc., Topsham, ME) programs.
Preparation of Cell Pellets
The cells were cultured in 10-cm plates. After overnight recovery, the medium was changed and the cells were exposed to VD or EB1089 at graded concentrations for 72 hours. The cells were trypsinized, washed, and centrifuged into pellets that were coated in 2% bactoagar until solidified, fixed in 10% formalin, and embedded in paraffin.
Immunocytochemistry
Sections of cell pellets were cut at 4 µm. Endogenous peroxidase activity was blocked with 3% aqueous hydrogen peroxidase and nonspecific binding was blocked with 20% protein blocker (Signet Laboratories, Inc., Dedham, MA). For cell proliferation analysis, the monoclonal anti-MIB-1 antibody (Immunotech, Marseilles, France) was used at 1:600 dilution for 60 minutes. For cell differentiation, thyroglobulin content was determined with a polyclonal antibody (DAKO Corporation, Carpinteria, CA) applied at 1:8000 dilution for 30 minutes after pepsin pretreatment. For localization of p27, a monoclonal antibody (Transduction Laboratories, Lexington, KY) was used at 1:1000 for 60 minutes after microwave antigen retrieval. The immunological reactions were visualized with the UltraStreptavidin detection system level 2 (Signet) and 3,3,-diaminobenzidine tetrahydrochloride as the chromogen. As negative controls, the primary antibody was replaced with normal mouse ascites and omission of primary antibody alone was performed.
Apoptosis Analysis
To determine whether DNA fragmentation characteristic of apoptosis occurred, we stained 4-µm sections of cell pellets using the terminal dUTP nick-end labeling (TUNEL) technique (ApopTag kit; Oncor). Briefly, sections were treated with 2% hydrogen peroxide to quench endogenous peroxide for 30 minutes and exposed to 5 µg/ml of proteinase K for 15 minutes at room temperature. The sections were washed extensively and exposed to equilibration buffer for 10 minutes. Each slide was then incubated with terminal deoxytransferase and digoxigenin-labeled TdT at 4°C overnight followed by horseradish peroxidase-conjugated anti-digoxigenin antiserum for 1 hour. The peroxidase reaction was visualized using 3,3,-diaminobenzidine tetrahydrochloride. Control sections were similarly stained but in the absence of terminal deoxytransferase, digoxigenin-conjugated TdT, or anti-digoxigenin antiserum.
Western Blotting
Cells were lysed in lysis buffer (150 mmol/L NaCl, 50 mmol/L Tris-HCl, pH 8.0, 5 mmol/L ethylenediaminetetraacetic acid, 1% Nonidet P-40) containing 1 mmol/L phenylmethyl sulfonyl fluoride, 10 µg/ml leupeptin, and 10 µg/ml aprotinin. Total cell lysates were quantified by the Lowry method, equal amounts (50 µg) were separated on 12% sodium dodecyl sulfate (SDS)-polyacrylamide gel by electrophoresis, and transferred onto nitrocellulose membranes. Nonspecific binding was blocked with 5% nonfat milk and 0.1% Tween-20 in Tris-buffered saline. Primary antibodies were directed against total p27 (1:1000, Transduction Laboratories), phosphatase, and tensin homologue deleted on chromosome 10 (PTEN) (1:100; Chemicon International, Inc., Temecula, CA), Akt/PKB or phosphorylated Akt/PKB-Ser 473 (pAkt/PKB)(1:1000, New England Biolabs), or actin (1:500, Sigma). Protein bands were visualized using chemiluminescence (Amersham, Oakville, Ontario, Canada) and band intensities were quantified by densitometry.
Immunoprecipitation and Immunoblotting
After incubation of VD and EB1089 for 72 hours, cells were lysed in RIPA buffer (1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 100 µg/ml phenylmethyl sulfonyl fluoride, aprotinin, and sodium orthovanadate in PBS). Total cell lysates were quantified and equal amounts of protein were immunoprecipitated with monoclonal p27 antibody (Transduction Laboratories), divided into equal parts and analyzed by Western blotting with antisera to phosphothreonine (Santa Cruz Biotechnology Inc., Santa Cruz, CA), pThr187-p27 (1:100; Zymed, San Francisco, CA), Skp2 (1:200, Santa Cruz), or total p27.
p27 Phosphorylation Assay
After 72 hours of VD or EB1089 treatment, cells were trypsinized and 5 x 106 cells were incubated with ortho-32 P (0.25 mCi/ml) in RPMI 1640 without sodium phosphate (Life Technologies, Inc., Grand Island, NY) for 1.5 hours at 37°C. This procedure was also performed with simultaneous labeling and treatment with VD or EB1089 for 8 hours. After washing with PBS four times, cells were lysed with RIPA buffer and equal amounts of lysate were immunoprecipitated with either p27 antibody (Santa Cruz) or actin and separated on a 12% SDS-polyacrylamide gel, followed by drying and autoradiography.
Statistical Analysis
Data are presented as mean ± SE. Differences were assessed by Student’s paired t-test. Significance level was assigned at P < 0.05.
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Results |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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Effects of VD and EB1089 on cell proliferation were analyzed by
measurement of [3H]thymidine incorporation.
Both compounds exerted a parallel growth inhibition of 
50% in all
cell lines. Representative experiments are shown in Figure 1
in WRO and NPA cells. These cell lines
showed a dose-dependent response to both compounds at concentrations
from 10-10 to 10-6mol/L
The combined use of both compounds did not result in an additive effect
(data not shown). Trypan blue exclusion confirmed that there was no
toxicity attributable to these agents (>95%, data not shown).
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Cell-cycle analysis from three independent experiments revealed that treatment with VD or EB1089 (10-7 mol/L) caused a decrease in the percentage of NPA cells in S phase (7.9 ± 0.3, or 7.7 ± 0.3 versus control 19.4 ± 0.1, P < 0.001), respectively. Similar results were found with the other cell lines (data not shown). The reductions in S-phase fractions were associated with corresponding accumulations of cells in G1 phase and no significant change was observed in the percentage of cells in G2 + M.
VD and EB1089 Do Not Induce Differentiation of Thyroid Carcinoma Cell Lines
To examine whether VD and EB1089 induced differentiation of human thyroid cancer cells, thyroglobulin immunostaining was used as a differentiation marker. Neither compound, however, resulted in thyroglobulin induction in any of the cell lines examined at concentrations ranging from 10-10 to 10-6 mol/L (data not shown).
VD and EB1089 Do Not Induce Apoptosis in Thyroid Carcinoma Cell Lines
It has been reported that VD and its analogs can induce apoptosis in several tumor cell lines. We examined the effects of EB1089 or VD on the various thyroid carcinoma cell lines using the TUNEL technique. Neither compound showed any sign of enhanced apoptosis at concentrations ranging from 10-10 to 10-6 mol/L (data not shown).
VD and EB1089 Increase p27 Protein Accumulation
p27, a cyclin-cdk inhibitor, plays a key role in
G1-S phase transition via binding cyclin/CDK
complexes, decreasing kinase activity, and arresting
G1/S phase transition in the cell cycle. To
assess the possible effects of VD and EB1089, p27 expression was
evaluated by immunoblotting. Both compounds increased p27 protein
levels in thyroid cancer cell lines in a dose-dependent manner in
concentrations ranging from 10-10 to
10-6 mol/L(Figure 3, a and b)
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It has been reported that thyroid tumor cell lines exhibit
cytoplasmic p27 staining and that expression of cyclin D3 induces
cytoplasmic retention of p27.9
In our system,
immunolocalization of p27 was primarily nuclear with the exception of
NPA cells that had both cytoplasmic and nuclear staining. Treatment
with VD or EB1089 increased nuclear staining in all cell lines (Figure 3c)
; no translocation of p27 into the cytoplasm was seen.
VD and EB1089 Interfere with Proteasome-Mediated p27 Degradation
p27 expression is regulated at the posttranscriptional level via
ubiquitin-dependent and nonubiquitin-dependent degradation
pathways.10
To determine the mechanism of increased p27
expression by VD and EB1089, NPA and WRO cells were treated with these
VD compounds (10-7 mol/L) alone or in
combination with the proteasome inhibitor LLnL (50 µmol/L) for up to
8 hours. Both VD and EB1089 increased p27 expression, an effect that
was similar to but not significantly additive to that achieved by LLnL
alone (Figure 4)
. As p27 degradation is
dependent on the activity of the SCFSkp2 (Skp2) protein complex that
targets phosphorylated proteins for ubiquitin-dependent
proteolysis,11
we demonstrated that VD treatment results
in diminished Skp2 expression (Figure 4b)
as well as reduced p27
association with Skp2 (Figure 4c)
. Taken together, these data suggest
that increased p27 expression by VD and EB1089 is, at least partly,
mediated by diminished targeting by Skp2 of p27 to the
ubiquitin-dependent degradation pathway.
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p27 is phosphorylated at threonine 187 and this phosphorylation
contributes significantly to enhanced ubiquitination and degradation by
the proteasome.12
To further elucidate the mechanism of
p27 accumulation induced by VD and EB1089, we examined the effect of
these compounds on p27 phosphorylation. Lysates from VD/EB1089-treated
cells were immunoprecipitated with anti-p27 antibody, followed by
immunoblotting with anti-phosphothreonine or anti-p27-Thr187
antibodies. Despite an increase in total p27, pThr content of p27 was
not increased in response to VD/EB1089 treatment (Figure 5a)
. To further demonstrate the effect of
VD/EB1089 on p27 phosphorylation, cells were metabolically labeled in
the presence or absence of VD or EB1089 for 8 hours (Figure 5b)
.
Diminished p27 phosphorylation was noted under such conditions (Figure 5b)
and also when cells were pretreated with VD or EB1089 for 72 hours
before metabolic labeling (not shown).
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PTEN is a tumor suppressor that has been shown to mediate
G1 cell-cycle arrest via the PI3-kinase/Akt/PKB
pathway.13-15
Moreover, multiple lines of evidence
support the significance of PTEN in thyroid carcinogenesis. Loss of
heterozygosity of PTEN has been documented in thyroid follicular
carcinoma16-18
and some thyroid carcinoma cell
lines.19,20
Moreover, germline mutations of PTEN result in
follicular thyroid adenoma and carcinoma.21
Thus, to begin
to test the potential of the PTEN/PI3-kinase/Akt/PKB pathway, cells
were treated with the phosphatase inhibitor pervanadate that we and
others have previously shown to activate this pathway.22
Treatment with this inhibitor diminished basal p27 levels and abrogated
VD/EB1089 induction of p27, consistent with a phosphatase role in
mediating this VD/EB1089 effect (Figure 6a)
. To specifically investigate whether
VD or EB1089 can regulate p27 through PTEN in thyroid carcinoma cells,
WRO and NPA cells were treated with these compounds and cell lysates
were analyzed by immunoblotting for PTEN and its downstream target
pAkt/PKB.15
Interestingly, despite the similar effects of
VD/EB1089 on p27 accumulation in both cell lines, only WRO but not NPA
cells showed an increase in PTEN expression (Figure 6b)
or associated
reduction in pAKT/PKB responses (Figure 6d)
. We also examined the
effect of VD/EB1089 treatment in the presence or absence of PTEN
transfection. Transfection of PTEN resulted in increased p27
accumulation an effect that was modestly additive in the presence of
VD/EB1089 (Figure 6c)
. Taken together these findings suggest that PTEN
is at least partially involved in mediating the effect of VD/EB1089 on
p27.
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with p27
accumulation in WRO and NPA cells, further highlighting the potential
importance of the Akt pathway in the regulation of p27 in both thyroid
cancer cell lines. |
Discussion |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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The mechanisms by which VD and its analogs exert their anti-proliferative effects are not well established. VD is known to induce the mRNA and protein expression of Bcl-2 but has no effect on Bcl-x or Bax expression. The increase in Bcl-2 expression correlates with protection of thyrocytes against the induction of apoptosis by either staurosporine or UV irradiation.25 Moreover, VD-induced increases in the expression of Bcl-2 could be mimicked by analogs with high nuclear VDR affinity, but not by analogs with only nongenomic actions.26 Our data indicate that VD and it’s analog with nongenomic action EB1089 display anti-proliferative actions in thyroid carcinoma cell lines. These effects were associated with induced p27 protein expression without a direct increase in cell apoptosis.
Progression of dividing mammalian cells is governed by a series of cyclin proteins that positively regulate a family of CDKs. The sequential activation/inactivation of cyclin/CDK complexes in turn is modulated by the interaction between constitutively expressed CDKs and oscillating cyclin levels. CDK activity is positively regulated by phosphorylation at a conserved threonine by a CDK-activating kinase (CAK). Conversely, CDK inhibitors (CDI) are classified into two major families. The INK4 (p15, p18, p19) proteins bind and inhibit CDK4 and CDK6 preventing cyclin D association.27 The Cip/Kip family includes p21 and p27 that inhibit a broader group of CDKs by binding to several cyclin/CDK complexes that are essential for G1 progression and S-phase entry. The cyclin E/CDK2 complex represents a major target of p27 inhibitory action. Whereas p21 expression is diminished in quiescent cells, p27 is induced on serum starvation and is required for G1 arrest.28 Moreover, increased p27 expression has been shown to mediate G1 arrest resulting from contact inhibition, interferon, or interleukin treatment.27 Increased p27 expression has also been reported to be associated with increased susceptibility to apoptosis in some systems29 but not others.30 Indeed, cleavage and inactivation of p27 have been suggested to be important steps in the induction of apoptosis in some cell types.27 Here we demonstrate, however, that VD and its analog EB1089 increase p27 expression, an effect that was associated with G1 arrest in thyroid carcinoma cells without concomitant induction of apoptosis. Our findings are consistent, however, with the effects of VD analogs on growth arrest of MCF-7 breast cancer cells without increased apoptosis.30
p27 is considered to be a tumor-suppressor protein playing a critical role in the pathogenesis and possibly prognosis of several human malignancies.31,32 In particular, thyroid neoplasms exhibit significantly lower p27 protein levels than normal thyroid tissue with poorly differentiated carcinomas having the lowest p27 immunohistochemical staining.31,33,34 Others have suggested that the localization of p27 may be of importance in the thyroid where cyclin D3 expression has been reported to be associated with cytoplasmic localization of p27. Expression of cyclin D3 in thyroid carcinoma cell lines induced cytoplasmic retention of co-transfected p27, an effect considered to remove p27 from intranuclear complexes that contain cyclins A to E and Cdk2.9 In our study, we noted that the accumulation of p27 remained within the nuclear compartment with no evidence of cytoplasmic translocation.
Recent evidence suggests that VD may stimulate the transcription of the p27 gene although the latter lacks a VD response element.35 Using gel shift assays, VD was shown to stimulate the transcription factors Sp1 and NF-Y DNA binding activities in inducing the p27 gene,35 indicating that VD may indirectly modulate p27 at the transcriptional level. Nevertheless, p27 expression is primarily regulated at the posttranscriptional level. On mitogen stimulation, p27 undergoes rapid degradation by the ubiquitin-proteasome pathway.36 Multiple lines of evidence support the notion that VD-induced p27 accumulation is the result of diminished degradation. We show here that under the same treatment conditions, the proteasome inhibitor LLnL mimics the effects of VD or EB1089 on p27 accumulation. Moreover, phosphorylation of p27 by cyclin E/CDK2 has been shown to trigger ubiquitin-mediated degradation of p27.37 Degradation of p27 is also dependent on the activity of the SCFSkp2 a protein complex that targets phosphorylated proteins for ubiquitin-dependent proteolysis.11 SCFSkp2 specifically associates with phosphorylated Thr-187 in targeting p27 degradation.11 We show here that VD and its analog EB1089 abrogate the rise of phosphothreonine content of p27 inasmuch as these compounds’ effects were uncoupled from their parallel effects on total p27. These findings were corroborated by direct p27 phosphorylation assays. We also show diminished Skp2 expression as well as association with p27 in response to VD treatment. These data suggest that VD-induced p27 accumulation and reduced degradation are mediated, at least partly, through diminished p27 phosphorylation and association with Skp2.
Mutations and/or deletions of PTEN have been implicated in various malignancies, including thyroid carcinoma.38,39 Moreover, mice heterozygous for PTEN deletion (+/-) develop papillary thyroid cancer40-42 further highlighting the relevance of intact PTEN function in normal thyroid cell growth. Indeed down-regulation of PTEN and p27 have been noted in a subset of human sporadic thyroid tumors16-18 as well as thyroid carcinoma cell lines.19,20 WRO cells express low levels of PTEN mRNA and protein compared to NPA cells that express low levels of mRNA but normal to high levels of PTEN protein. We show here that despite the comparable effects of VD/EB1089 on p27 in WRO and NPA cells, PTEN/pAkt/PKB protein levels were regulated by VD/EB1089 in WRO but not NPA cells. Transfection of PTEN also resulted in p27 accumulation an effect that was only modestly enhanced by subsequent treatment with VD. Taken together, these findings suggest that although PTEN may participate in mediating VD action in some thyroid cells, it is not an essential component for the effect of VD on p27 in thyroid carcinoma cells.
Loss of PTEN function leads to the constitutive activation of Akt/PKB via enhanced phosphorylation of threonine 308 and serine 473.43,44 Consistent with these findings, we demonstrate that VD/EB1089-induced PTEN expression in WRO cells is associated with downstream reduction in pAkt/PKB. NPA cells that failed to demonstrate an increase in PTEN immunoreactivity in response to VD/EB1089 treatment also demonstrated no change in pAkt/PKB. In both cell lines, however, treatment with PI-3 kinase inhibitors, wortmannin, and LY294002 regulated p27 and pAkt/PKB consistent with a potential PTEN/Akt/PKB-dependent but not essential role for this pathway in regulating p27 in thyroid cancer cells. Our findings of the lack of VD/EB1089-induced apoptosis in our model further supports the Akt-independent pathway in VD/EB1089 action, because Akt is recognized to play an important role in triggering apoptotic pathways.19
The active form of VD has long been known to have anti-proliferative actions. The hypercalcemic potential of VD limits its clinical use, however, noncalciomimetic analogs such as EB1089 may be clinically useful. In our studies, VD and EB1089 resulted in similar effects on p27 accumulation and cell-cycle arrest. The common inhibitory effects on cell-cycle progression and p27 protein accumulation lend themselves to the potential use of noncalciomimetic VD analogs in the treatment of thyroid cancer irrespective of PTEN/Akt status.
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Acknowledgements |
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Footnotes |
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Supported in part by grants from the Thyroid Foundation of Canada and the United States Pharmacopeia.
Accepted for publication October 25, 2001.
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References |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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