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,25-Dihydroxyvitamin D3 on the G1–S Phase-Controlling Machinery
Institute of Cancer Biology (S.S.J., J.L., J.B.) The Danish Cancer Society, DK-2100 Copenhagen, Denmark; and LEO Pharmaceutical Products (S.S.J., M.W.M., L.B.), DK-21002750 Ballerup, Denmark
Address all correspondence and request for reprints to: Dr. Simon Skjode Jensen, Department of Molecular Biology and Biochemistry, Leo Pharmaceutical Products, Industriparken 55, Ballerup, Denmark 2750.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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,25-dihydroxyvitamin D3 induces
cell cycle arrest, differentiation, or apoptosis depending on target
cell type and state. Although the antiproliferative effect of
1,25-dihydroxyvitamin D3 has been known for years, the
molecular basis of the cell cycle blockade by 1,25-dihydroxyvitamin
D3 remains largely unknown. Here we have investigated the
mechanisms underlying the G1 arrest induced upon
1,25-dihydroxyvitamin D3 treatment of the human breast
cancer cell line MCF-7. Twenty-four-hour exposure of exponentially
growing MCF-7 cells to 1,25-dihydroxyvitamin D3 impeded
proliferation by preventing S phase entry, an effect that correlated
with appearance of the growth-suppressing, hypophosphorylated form of
the retinoblastoma protein (pRb), and modulation of cyclin-dependent
kinase (cdk) activities of cdk-4, -6, and -2. Time course
immunochemical and biochemical analyses of the cellular and molecular
effects of 1,25-dihydroxyvitamin D3 treatment for up to
6 d revealed a dynamic chain of events, preventing activation of cyclin
D1/cdk4, and loss of cyclin D3, which collectively lead to repression
of the E2F transcription factors and thus negatively affected cyclin A
protein expression. While the observed 10-fold inhibition of cyclin D1/cdk 4-associated kinase activity appeared independent of cdk inhibitors, the activity of cdk 2 decreased about 20-fold, reflecting joint effects of the lower abundance of its cyclin partners and a significant increase of the cdk inhibitor p21CIP1/WAF1, which blocked the remaining cyclin A(E)/cdk 2 complexes.
Together with a rapid down-modulation of the c-Myc oncoprotein in
response to 1,25-dihydroxyvitamin D3, these results
demonstrate that 1,25-dihydroxyvitamin D3 inhibits cell
proliferation by targeting several key regulators governing the
G1/S transition.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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,25-DIHYDROXYVITAMIN
D3 (1,25-VD3) and its
analogs represent candidate compounds for treatment of
hyperproliferative diseases including psoriasis and diverse types of
cancer. A major advantage of these reagents lies in the ability not
only to halt proliferation, but also to induce differentiation or cell
death [reviewed in (1, 2)]. 1,25-VD3 is the physiologically active ligand to the VDR. The VDR forms stable receptor complexes, preferably as heterodimers with the RXR. The receptor dimers regulate transcription in either a negative or positive fashion (3, 4, 5). The VDR-RXR dimer binds specific palindromic vitamin D response elements, located in the promoters of 1,25-VD3-regulated genes (6, 7). The 1,25-VD3-regulated genes are numerous and range from genes involved in bone mineralization and proliferation to transcription factors, interleukins, and structural proteins (reviewed in Ref. 8).
Over the last decade, many antiproliferative agents have been shown to interfere with the cell cycle machinery and arrest cells in G1 phase of the cell cycle. Cell cycle transitions are largely governed by a family of cyclin-dependent kinases (cdks), which target critical substrates such as pRb, the inactivation of which by cdk-mediated phosphorylation in mid-to-late G1 phase is a prerequisite for entry into S phase (reviewed in Refs. 9 and 10).
cdk Activity is regulated by association with its activating cyclin partner, physical interaction with cdk inhibitors, and by both positive and negative regulatory phosphorylations (11, 12). cdk Inhibitors (cdki) include first the INK4 family: p15INK4B, p16INK4A, p18INK4C, and p19INK4D, which bind and inactivate the major G1 phase kinases, cdk4 and cdk6, by forming inactive dimeric complexes (13, 14, 15). Another family of cdkis include p21CIP1/WAF1, p27KIP1, and p57KIP2, which inhibit a broader range of cdks, including cdk1, -2, -4, and -6 (16, 17, 18). Regulation at the level of phosphorylation is best characterized for the cyclin B-cdk1 complex, but the mechanisms are conserved in other cdk complexes acting in G1 and S phase. Stimulatory phosphorylations occur at Thr 160 in cdk2 and Thr 172 in cdk4. This activating phosphorylation is carried out by the cdk activating kinase (CAK), a complex consisting of cdk7, cyclin H, and the MAT1 assembly factor (19, 20). Cdk4/6 and cdk2 contribute to pRb phosphorylation in G1 and S phase, respectively, resulting in derepression/activation of the pRb-regulated E2F transcription factors required for further cell cycle progression (21, 22, 23).
The cell cycle arrest induced by 1,25-VD3 and its analogs has been investigated in tumor cells of leukemic (24, 25, 26, 27), prostate (28, 29, 30), pancreatic (31), and breast cancer origin (32, 33, 34) as well as in normal keratinocytes (35, 36). The consensus view emerging from these studies identifies G1 phase as the major target of the observed cell cycle blockade and points to the p21CIP1/WAF1 or p27KIP1 cdkis as candidate mediators of these cell cycle effects.
In MCF-7 breast cancer cells, the 1,25-VD3 analog EB1089 up-regulates p21CIP1/WAF1, which then targets and inactivates cdk2 complexes (32). BT-20 and ZR75 breast cancer cells respond in a similar manner, possibly including induction of p27KIP1 (32). The finding of a vitamin D response element in the p21CIP1/WAF1 promoter and the induction of p21CIP1/WAF1 transcript within 2 h after 1,25-VD3 addition suggested that p21CIP1/WAF1 represents an early mediator of the 1,25-VD3-induced cell cycle arrest (24).
The aim of this study was to further elucidate the mechanisms by which 1,25-VD3 exerts its growth-inhibitory activities. We chose the human breast cancer cell line MCF-7 as a model, which expresses VDR and responds to 1,25-VD3 in a growth-inhibitory manner, to investigate the effects of 1,25-VD3 on key cell cycle regulators, the cyclin-cdk complexes controlling the mammalian G1–S phase transition, and their cognate inhibitors.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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, proliferation of exponentially growing MCF-7 cells was inhibited after
only 2 d of 1,25-VD3 treatment, and the
cell number did not increase significantly after day 4. To determine
the cell cycle phase in which MCF-7 cells arrest when exposed to
1,25-VD3, we analyzed the DNA content of the
cells by flow cytometry. After 24 h treatment, the
G1 phase population increased significantly from
49 to 57% of the total cell population, correlating with a concomitant
S phase decrease (Fig. 1B). The G1 phase
accumulation in 1,25-VD3-treated cells remained
approximately 20–30% above control throughout the time course. After
6 d of treatment, approximately 80% of the treated cells were in
G1 phase, indicating that the MCF-7 cell
population does not respond with a complete G1
phase blockade. This incomplete response was caused by heterogeneity in
the MCF-7 cell line showing a subpopulation of nearly
1,25-VD3-resistant cells (data not shown).
Control cells increased in the G1 population at
later time points, reflecting the characteristic growth of MCF-7 cells
in "islands," in the center of which the cells become increasingly
contact inhibited during the time course, eventually arresting in
G1 phase in a
p27KIP1-dependent manner (39). Taken
together, these data show that MCF-7 cells respond to
1,25-VD3 and accumulate in
G1 phase of the cell cycle after only 24 h
of treatment.
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). At day 0, the bulk of pRb was in the
slower migrating, hyperphosphorylated form (marked
pRbpp) (40), but after only 24–36 h
treatment, the hypophosphorylated form started to accumulate when
compared with control cells (Fig. 2, A and D). Accumulation of the
hypophosphorylated form increased throughout the time course, whereas
the hyperphosphorylated form gradually decreased in
1,25-VD3 treated cells. The middle
panel shows a blot of less separated pRb, which indicates decrease
of pRb in lovastatin-arrested cells and in MCF-7 cells treated for 72
and 96 h, relative to loading control (cdk7-blot). Given the
ability of the hypophosphorylated pRb to repress the E2F transcription
factors (23, 42), we next examined mRNA and protein levels
of cyclin A and E, both known E2F targets, in
1,25-VD3 treated cells. Expression of cyclin A
and E were modulated at both transcript and protein levels by
1,25-VD3 treatment. Cyclin A and E mRNA in
1,25-VD3-treated cells remained at low levels as
seen at the zero time point, whereas the control cells increased
transcript levels as the cells entered an exponential growth phase and
reached a 2-fold higher level after 4 days of treatment (Fig. 2B).
Western blot of total cell extracts showed increased cyclin A protein
levels in control cells during the time course with highest levels
after 72–96 h, whereas 1,25-VD3 treatment
prevented this increase and retained cyclin A protein close to the
initial low levels at time zero. These data indicate that
1,25-VD3 prevents entry into exponential growth
phase by targeting the pRb pathway, resulting in hypophosphorylated,
active pRb, and consequently preclude E2F activation and transcription
of E2Fregulated genes such as cyclin A.
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). Cdk6
displayed a similar pattern, with significant difference between
control and treated cells from 72 h 1,25-VD3
treatment, whereas cdk4 was not significantly decreased.
Cyclin D1, which primarily activates cdk4 in MCF-7 cells, showed
only a minor change after 4 d of 1,25-VD3
treatment, whereas cyclin D3 was significantly decreased from 48 h
treatment (Fig. 2C). The cdk inhibitor
p21CIP1/WAF1 was reduced in control cells at 72
and 96 h of treatment, but remained at high levels in
1,25-VD3-treated cells, correlating with the
difference in growth properties previously identified in Fig. 1.
Analysis of p27KIP1 and the INK4 cdki proteins
revealed no significant change upon 1,25-VD3
treatment (data not shown).
Interestingly, protein levels of the c-Myc decreased significantly from
24 h of 1,25-VD3 treatment relative to the
zero time point, but also relative to control cells, which
showed increased c-myc protein levels, correlating to
the increased proliferation state (Fig. 1).
These data show that 1,25-VD3 prevents the cells from entering the exponential growth phase, as seen in control cells, by uncoupling expression of proteins required for cell proliferation, e.g. c-Myc, cdk, and cyclin proteins. Simultaneous high levels of the cdk inhibitor p21CIP1/WAF1 further sustain growth retardation.
The Effects of 1,25-VD3 on cdk Activity
The observed G1 phase arrest and the lack of
pRb phosphorylation strongly suggested that the activities of pRb
kinases, including cyclin D/cdk4 (6) and cyclin E(A)/cdk2,
may be affected upon treatment of cells with
1,25-VD3. Investigation of the cdk complexes
responsible for pRb phosphorylation in G1 and S
phase of the cell cycle showed a general inhibition of cdk activity,
when assayed by immunoprecipitation (ip) of the complexes followed by
in vitro kinase assays using
glutathione-S-transferase (GST)-pRb as substrate. The kinase
activity of cyclin D1-cdk4, cyclin D3-cdk4/6, and cdk6-cyclin D1/3 were
all strongly inhibited in MCF-7 cells treated with
1,25-VD3 for 4 d (Fig. 3A). The most abundant
G1 phase complex cyclin D1-cdk4 is inhibited more
than 6-fold, and the less abundant complexes containing cyclin D3
and/or cdk6 show somewhat weaker activity and are significantly
inhibited 2- to 3-fold relative to vehicle- treated controls. Cdk2 is
activated by cyclin E at G1–S phase transition
and in S phase by cyclin A (43, 44). To examine whether
1,25-VD3 leads to inhibition of cdk2 associated
with both cyclin E and A, we performed kinase assays with complexes
immunoprecipitated with antibodies to cyclin E, cyclin A, or cdk2. As
shown in Fig. 3B, 4-d exposure to 1,25-VD3 leads
to a 3-fold reduction of cyclin E-associated kinase activity, while
cyclin A-associated kinase activity was reduced 17-fold and cdk2-cyclin
E(A) activity was reduced 25-fold, showing an overall dramatic
inhibition of cdk2. Examination of the dynamics of the cdk2
(4) regulation showed increased cdk activity in control
cells as exponential growth progressed, but with cdk activity in
1,25-VD3-treated cells remaining at low levels
throughout the time course (Fig. 3, C and 3D). Surprisingly, activity
of both cdk2 and cdk4 kinase was weakly but significantly increased
after 8 h of treatment, effects that could be related to
nongenomic fast responses mediated by 1,25-VD3
(45).
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Inhibition of cdk2 Correlates with Increased Targeting of
p21CIP1/WAF1, Decreased Cyclin A and E Association, and
Lack of CAK-Mediated Phosphorylation
Since cyclin A was affected at the level of total protein (as
described above, Fig. 2C), lack of cdk-cyclin association could
contribute to failure of cdk2 activation. In addition, members of the
p21CIP1/WAF1/p27KIP1
family of cdk inhibitors were identified as important
mediators of glucocorticoid-, retinoic acid-, and
1,25-VD3-induced cdk inhibition (24, 32, 46, 47) and would therefore also be good candidates for
mediating the effects on cdk2 kinase activity in our model system.
Northern blot analysis showed an increase of
p21CIP1/WAF1 transcript in MCF-7 cells after
only 8 h of 1,25-VD3 treatment
(Fig. 4A). The increased
p21CIP1/WAF1 mRNA levels were seen throughout
the time course, with a maximal 3-fold induction after
72 h. Coimmunoprecipitation experiments from cells treated for
96 h revealed that the increase in
p21CIP1/WAF1 protein correlated with increased
binding of p21CIP1/WAF1 with cyclin A(E)-cdk2
complexes (Fig. 4B, lower panel, lanes 5–10). In whole-cell
extracts (Fig. 4B, lanes 1–2), cdk2 was largely shifted to the slower
migrating, inactive form, known to reflect the lack of CAK
activation/phosphorylation of cdk2 (see also Fig. 2C). When
p21CIP1/WAF1 ips were analyzed, both active and
inactive cdk2 were seen in treated cells, showing that
p21CIP1/WAF1 preferentially targets the active
cdk2 complexed to cyclins, despite the low amounts of active cdk2 after
4 days of treatment (Fig. 4B, upper panel, lanes 3–4). In
immunoprecipitates of cyclin E and A, mainly containing the
CAK-phosphorylated active cdk2, an overall significant decrease in the
levels of cyclin-associated cdk2 was seen after 4 days of treatment,
possibly reflecting the lower levels of cyclin A protein identified in
total cell extract (Fig. 4B, upper panel, lanes 5–8). These
data indicate that after 4 d of 1,25-VD3
treatment, cdk2 kinase activity is deregulated by a combination of
increased association with the cdk inhibitor
p21CIP1/WAF1 and decreased association/activation
with both cyclin A and cyclin E. Consequently, the overall proportion
of the active form of cdk2, phosphorylated at Thr 160 by CAK, is
reduced after 1,25-VD3 treatment.
Regulation of G1 Phase Complexes Independently of
p21CIP1/WAF1
The G1 phase complexes cyclin D1(3)-cdk4(6)
and their cognate inhibitors were also examined. Ips with antibodies
against p21CIP1/WAF1,
p27KIP1, cdk4, cdk6, cyclin D1, and cyclin D3 did
not show any dramatic increase in either
p21CIP1/WAF1 or p27KIP upon
1,25-VD3 treatment in any of these complexes
(Fig. 4C). The lack of cdk4-cyclin D1 activation in treated cells, as
seen in control cells in Fig. 3C, could not be explained by disruption
of the complex, since the stoichiometry between cdk4-cyclin D1 remained
largely preserved upon treatment (Fig. 4C, lanes 5–6 and 9–10). In
contrast to cdk4-cyclin D1, complexes containing cyclin D3 were
disrupted (Fig. 4C, lanes 11–12), showing decreased levels of cdk4
associated with cyclin D3 upon 1,25-VD3
treatment. This effect was probably due to decreased protein levels of
cyclin D3 following 4 d of 1,25-VD3
treatment (Fig. 2C). Furthermore, these ips show that cyclin D1
primarily associates with cdk4, rather than cdk6, strongly indicating
that the kinase complex assayed by the anticyclin D1 antibody in Fig. 3A, lanes 2 and 3, and in Fig. 3C, is mainly cyclin D1-cdk4.
These data suggest that 1,25-VD3 treatment prevents activation of the major G1 phase complex, cdk4-cyclin D1, by mechanisms distinct from targeting by cdkis and dissociation of the cyclin-cdk complex.
Cdk2 Kinase Activation Is Prevented by p21CIP1/WAF1
Targeting and Accompanied by Decreased Cyclin A Activation
As proliferation of the MCF-7 cells was affected by
1,25-VD3 after only 24 h treatment
(Fig. 1B), we next analyzed the dynamics of cdk2 activity in an attempt
to identify the events that may initiate the effects on cdk2 kinase
activity. cdk2 Was immunoprecipitated every 6 h between 18 and
48 h of 1,25-VD3 treatment, and the
complexes were analyzed for associated cyclin A and
p21CIP1/WAF1 proteins. Analysis of
cdk2-coprecipitated proteins (Fig. 5A)
showed a weak reduction in precipitated cdk2 relative to control from
24 h treatment, correlating to the different cdk2 protein levels
seen in total cell extract (Fig. 2C). When the coprecipitated
p21CIP1/WAF was quantitated relative to the
decreased levels of cdk2, the p21CIP1/WAF1-cdk2
ratio was significantly increased from 36 h treatment (Fig. 5, A
and C). Analysis of coprecipitated cyclin A revealed a slightly lower
cyclin A level in treated vs. control cells from 24 h,
but since cdk2 was reduced in a similar manner, the cdk2-cyclin A ratio
was not significantly reduced until after 48 h of treatment. The
cdk2-cyclin E ratio was affected even later in the time course (not
shown). Immunoblotting of p21CIP1/WAF1 in total
cell extracts prepared as above showed a significant increase in
p21CIP1/WAF1 protein levels relative to cdk7
(loading control) from 30 h of treatment and throughout the time
course (Fig. 5, B and D).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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), of 1,25-VD3
targets and effects that lead to cell cycle block in
G1 phase. 1) cdk4(6)-cyclin D1(3) kinase activity
is deregulated starting from 24 h after
1,25-VD3 treatment, preventing pRb
phosphorylation/inactivation concomitant with sequestration and
inactivation of E2F. Continued silencing of E2F by active pRb prevents
cyclin A and E protein expression (21, 22, 23), resulting in
deficient cdk2 activation. 2) Induction of
p21CIP1/WAF1 leads to failure of cdk2 activation
after 24–36 h. 3) c-Myc deregulation could possibly contribute to
silencing of cdk2 activity, since c-Myc has been shown to induce
cdk2 activity and concomitant S phase entry in a cyclin E- and
Cdc25A-dependent manner (48).
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The cdk4-cyclin D1 complex stoichiometry was not affected, despite a 7-fold difference in kinase activity after 96 h of treatment. This indicates that no INK4 proteins are involved, since this would result in disruption of the cdk4-cyclin D1 complex and sequestration of cdk4 in a dimeric complex with the INK4 proteins (17, 49, 50, 51). Sequestration of cdk4 into HSP90/cdc37 complexes is another known mechanism for cdk4 inactivation (52, 53, 54), but since this process apparently involves monomeric cdk4 only, it would also require cdk4-cyclin D1 disassembly and is therefore unlikely to play a role in our model system. A plausible mechanism for deregulation of cdk4 by 1,25-VD3 could therefore reflect regulation at the level of phosphorylation. Cdk4 was reported to be phosphorylated at the inhibitory Tyr 17 upon entry into quiescence induced by either contact inhibition or serum starvation in rat kidney fibroblasts (55, 56), or after TGFß treatment of MCF-10A cells, in the latter case due to decreased activity of the Cdc25A phosphatase. Thus, Cdc25A could be a good candidate for an upstream regulator of both cdk2 and cdk4 activity in the 1,25-VD3-mediated cell cycle arrest (48, 57).
In this context, 1,25-VD3-mediated regulation of
cdc25A activity could account for the difference in timing
between significant inhibition of cdk2 kinase activity
(Fig. 3D), at 24 h (60% of control) until
p21CIP1/WAF1 could be significantly identified in
cdk2 complexes at 36 h (Fig. 5, A and C).
Consistent with others we found induction of p21CIP1/WAF1 transcript within 8 h of 1,25-VD3 treatment (24, 28, 33). Induction of the p21CIP1/WAF1 transcript occurs very likely by a VDR-dependent mechanism, since a VDR response element has been identified in the p21CIP1/WAF1 promoter (24). These data suggest that p21CIP1/WAF1 induction is an event upstream of cdk2 inhibition, independent of the pathways leading to cdk4 inhibition and c-Myc down-regulation. Surprisingly, the early increase in p21CIP1/WAF1 transcript does not directly correspond to an increase in p21CIP1/WAF1 protein, which first occurs after 30 h treatment, where we identified a significant increase in p21CIP1/WAF1 protein, suggesting regulation at the level of p21CIP1/WAF1 translation or stability of p21CIP1/WAF1 mRNA or protein. The increased p21CIP1/WAF1 protein contributed to the failure of cdk2 activation and possibly prevented CAK activation of cdk2, by analogy with the effects of PGA2-induced p21CIP1/WAF1 (58). A similar phenomenon was reported for p27KIP1 in inhibition of cdk4 in response to contact inhibition (59).
The pathways of 1,25-VD3 signaling, leading either through VDR or nongenomic signaling through possible 1,25-VD3 membrane receptors, are poorly understood. Candidate molecules on the pathway leading to the 1,25-VD3-mediated cell cycle arrest remain elusive, but possibly transcription factors such as c-jun and c-Myc could play important roles, since they are affected early by 1,25-VD3 treatment (60, 61, 62, 63). AP1 activation and c-jun induction have been shown to occur within 30–60 min after 1,25-VD3 addition, in a process possibly involving PKC, JNK1, and ERK2 (63, 64, 65). Other studies report an increase in intracellular Ca2+, which possibly activates Ca2+-dependent PKC isoforms (63, 66). Collectively, 1,25-VD3 signaling through VDR-independent mechanisms involves activation of MAPK pathways, leading to activation of transcriptional complexes such as AP1. Since c-Myc is a downstream target of ERK2 (67), activation of this pathway could possibly play a role in the observed down-regulation of c-Myc in our model system.
Our data show that the strong antiproliferative effect of 1,25-VD3 in human breast cancer cells reflects targeting of several key cell cycle regulators and warrants further research and attempts to develop 1,25-VD3 analogs suitable for cancer treatment. One of the most promising analogs of 1,25-VD3 in terms of cancer treatment is EB1089, for which significant anticancer effects were reported from both in vitro and in vivo experiments [reviewed in (68, 69)]. The antiproliferative potency of this compound is greatly improved with an IC50 value 50–200 times below that of 1,25-VD3. In vivo, EB1089 causes significant inhibition of tumor progression in both rats (70) and mice (71, 72), and it lacks the serious hypercalcemic side effects characteristic for 1,25-VD3.
In conclusion, our data show that cdk2 and cdk4 kinase activities are deregulated within 24 h of 1,25-VD3 treatment. Prevention of cdk4 activation likely contributes to the G1 phase arrest by decreased phosphorylation of pRb, leading to sustained E2F sequestration, and thereby decreased cyclin A expression and subsequent repression of cdk2 kinase activity, which is essential for G1/S phase transition. The mechanism of initial cdk2 deregulation likely includes a combination of p21CIP1/WAF1 increase, followed by lack of cyclin A association and activation. Lack of cdk2 activation is further promoted via low cdk2 protein levels and decreased cdk2-cyclin E association.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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,25(OH)2vitamin D3
was synthesized in the Department of Chemical Research, Leo Pharmaceuticals Products, where purity and concentration were
determined. 1,25-VD3 was diluted in media from an
isopropanol stock at 4 x 10-4 M to a
final concentration of 10-7 M. In experiments
the media was changed every 24 h with fresh prewarmed
CO2-equilibrated media including either vehicle or
1,25-VD3. Vehicle concentration was kept below
0.0025%.
Antibodies
Western blot against pRb was performed with a monoclonal mouse
antibody (mMAb); G3–245 from PharMingen (San Diego, CA),
against p27KIP1 with a rabbit polyclonal antibody
(PC55), was from Calbiochem (La Jolla, CA). mMAbs against
cyclin D1 (DCS6), cyclin D3 (DCS28), cdk6 (DCS130), and cdk7 (MO-1.1)
were produced and used as either hybridoma supernatant or ascites. 5D4
was against cyclin D1/2 and donated by M. Sato; HE12 and HE172 are
mMAbs against cyclin E (73). Rabbit antisera against
p21CIP1/WAF1 (sc-397),
p27KIP1 (sc-776), cyclin A (sc-751), cdk2
(sc-163), cdk4 (sc-601), and cdk6 (sc-177) were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).
Cell Cycle Analysis and Proliferation Assay
Progression through the cell cycle was determined by flow
cytometry analysis of DNA content of cell populations stained with
propidium iodide as described previously (74).
Proliferation assays were done in six-well plates with initially 1
x 103 cells/cm2. Every
counting was done in triplicate as the average of two countings in a
Coulter counter. Before counting the cells were passaged through a
syringe to prevent formation of cell aggregates.
Kinase Assays
cdk2, cdk4, and cdk6 kinase assays were performed as described
previously (75, 76, 77), using a short C-terminal GST-pRb
substrate (amino acids 773–928) and the antibodies indicated in each
figure. The incubations were at 30 C for 30 min, and the kinase
reaction was stopped by adding 8 µl 4x Laemmli sample buffer
including 10 mM EDTA. The kinase reactions were separated
by SDS-PAGE, and the proteins were transferred to nitrocellulose
membranes by the semidry method. Membranes were exposed on a
phosphorimage screen to measure incorporation of
32P
ATP into the substrate using the STORM
analyzer from Molecular Dynamics, Inc. (Sunnyvale,
CA).
Immunochemical Analysis
Extraction of protein for ip, estimation of protein content, and
ip procedures were as described previously (76, 78). For
each ip in Figs. 4 and 5 the protein content of each lysate was
measured using the BCA protein assay reagent (Pierce Chemical Co., Rockford, IL) ensuring ip from equal amounts of lysate.
Cross-linking of cdk2 antibody was done as described previously
(79). Western blot was performed as in (80),
except for secondary antibodies (PI1000 and PI2000), which were from
Vector Laboratories, Inc. (Burlingame, CA). For
quantitation in Fig. 5, the ECF system was used from
Amersham Pharmacia Biotech (Arlington Heights, IL), on a
Storm analyzer from Molecular Dynamics, Inc.
Northern Blot
Total cellular RNA was extracted and purified using the
RNAqueous kit from Ambion, Inc. (Austin, TX, catalog no.
1912) according to the manufacturer’s instructions. Ten micrograms of
RNA were electrophoresed on a 1% agarose formaldehyde gel and
transferred onto a nylon membrane. Expression of the
p21CIP1/WAF1, cyclin A, and cyclin E transcripts
were monitored using the full-length cDNA from each gene as probes, cut
out from appropriate plasmids. The 36B4 cDNA probe encoding acidic
ribosomal phosphoprotein PO was used in parallel to control for
balanced loading.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Abbreviations: CAK, cyclin-dependent kinase-activating kinase;
cdk, cyclin-dependent kinase; cdki, cyclin-dependent kinase inhibitor;
GST, glutathione-S-transferase; ip, immunoprecipitation;
mMAb, monoclonal mouse antibody; pRb, retinoblastoma protein;
1,25-VD3,1,25-dihydroxyvitamin D3.
Received for publication June 22, 2000. Accepted for publication April 11, 2000.
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  C. KRIEBITZSCH, L. VERLINDEN, G. EELEN, B. K. TAN, M. VAN CAMP, R. BOUILLON, and A. VERSTUYF The Impact of 1,25(OH)2D3 and its Structural Analogs on Gene Expression in Cancer Cells - A Microarray Approach Anticancer Res, September 1, 2009; 29(9): 3471 - 3483. [Abstract] [Full Text] [PDF]
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 J. N. P. Rohan and N. L. Weigel 1{alpha},25-Dihydroxyvitamin D3 Reduces c-Myc Expression, Inhibiting Proliferation and Causing G1 Accumulation in C4-2 Prostate Cancer Cells Endocrinology, May 1, 2009; 150(5): 2046 - 2054. [Abstract] [Full Text] [PDF]
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 D. Nowak, D. Stewart, and H. P. Koeffler Differentiation therapy of leukemia: 3 decades of development Blood, April 16, 2009; 113(16): 3655 - 3665. [Abstract] [Full Text] [PDF]
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 I. H. Onen, A. Ekmekci, M. Eroglu, E. Konac, S. Yesil, and H. Biri Association of Genetic Polymorphisms in Vitamin D Receptor Gene and Susceptibility to Sporadic Prostate Cancer Experimental Biology and Medicine, December 1, 2008; 233(12): 1608 - 1614. [Abstract] [Full Text] [PDF]
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 R. Bouillon, G. Carmeliet, L. Verlinden, E. van Etten, A. Verstuyf, H. F. Luderer, L. Lieben, C. Mathieu, and M. Demay Vitamin D and Human Health: Lessons from Vitamin D Receptor Null Mice Endocr. Rev., October 1, 2008; 29(6): 726 - 776. [Abstract] [Full Text] [PDF]
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 M. L Neuhouser, B. Sorensen, B. W Hollis, A. Ambs, C. M Ulrich, A. McTiernan, L. Bernstein, S. Wayne, F. Gilliland, K. Baumgartner, et al. Vitamin D insufficiency in a multiethnic cohort of breast cancer survivors Am. J. Clinical Nutrition, July 1, 2008; 88(1): 133 - 139. [Abstract] [Full Text] [PDF]
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 S. Abbas, J. Linseisen, T. Slanger, S. Kropp, E. J. Mutschelknauss, D. Flesch-Janys, and J. Chang-Claude Serum 25-hydroxyvitamin D and risk of post-menopausal breast cancer--results of a large case-control study Carcinogenesis, January 1, 2008; 29(1): 93 - 99. [Abstract] [Full Text] [PDF]
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 L. J. Schedlich, A. Muthukaruppan, M. K. O'Han, and R. C. Baxter Insulin-Like Growth Factor Binding Protein-5 Interacts with the Vitamin D Receptor and Modulates the Vitamin D Response in Osteoblasts Mol. Endocrinol., October 1, 2007; 21(10): 2378 - 2390. [Abstract] [Full Text] [PDF]
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 A. Martirosyan, S. Leonard, X. Shi, B. Griffith, P. Gannett, and J. Strobl Actions of a Histone Deacetylase Inhibitor NSC3852 (5-Nitroso-8-quinolinol) Link Reactive Oxygen Species to Cell Differentiation and Apoptosis in MCF-7 Human Mammary Tumor Cells J. Pharmacol. Exp. Ther., May 1, 2006; 317(2): 546 - 552. [Abstract] [Full Text] [PDF]
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 L. Verlinden, G. Eelen, I. Beullens, M. Van Camp, P. Van Hummelen, K. Engelen, R. Van Hellemont, K. Marchal, B. De Moor, F. Foijer, et al. Characterization of the Condensin Component Cnap1 and Protein Kinase Melk as Novel E2F Target Genes Down-regulated by 1,25-Dihydroxyvitamin D3 J. Biol. Chem., November 11, 2005; 280(45): 37319 - 37330. [Abstract] [Full Text] [PDF]
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 E. T. Jacobs, M. R. Haussler, and M. E. Martinez Vitamin D Activity and Colorectal Neoplasia: A Pathway Approach to Epidemiologic Studies Cancer Epidemiol. Biomarkers Prev., September 1, 2005; 14(9): 2061 - 2063. [Full Text] [PDF]
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E. Kallay, G. Bises, E. Bajna, C. Bieglmayer, W. Gerdenitsch, I. Steffan, S. Kato, H.J. Armbrecht, and H. S. Cross Colon-specific regulation of vitamin D hydroxylases--a possible approach for tumor prevention Carcinogenesis, September 1, 2005; 26(9): 1581 - 1589. [Abstract] [Full Text] [PDF]
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S. Nagpal, S. Na, and R. Rathnachalam Noncalcemic Actions of Vitamin D Receptor Ligands Endocr. Rev., August 1, 2005; 26(5): 662 - 687. [Abstract] [Full Text] [PDF]
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 C. P. Sanchez and Y.-Z. He Daily or Intermittent Calcitriol Administration during Growth Hormone Therapy in Rats with Renal Failure and Advanced Secondary Hyperparathyroidism J. Am. Soc. Nephrol., April 1, 2005; 16(4): 929 - 938. [Abstract] [Full Text] [PDF]
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J. Welsh Vitamin D and breast cancer: insights from animal models Am. J. Clinical Nutrition, December 1, 2004; 80(6): 1721S - 1724S. [Abstract] [Full Text] [PDF]
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 D. M. Harris and V. L. W. Go Vitamin D and Colon Carcinogenesis J. Nutr., December 1, 2004; 134(12): 3463S - 3471S. [Abstract] [Full Text] [PDF]
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L. S. Adams and D. Teegarden 1,25-Dihydroxycholecalciferol Inhibits Apoptosis in C3H10T1/2 Murine Fibroblast Cells Through Activation of Nuclear Factor {kappa}B J. Nutr., November 1, 2004; 134(11): 2948 - 2952. [Abstract] [Full Text] [PDF]
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L. V. Stewart and N. L. Weigel Vitamin D and Prostate Cancer Experimental Biology and Medicine, April 1, 2004; 229(4): 277 - 284. [Abstract] [Full Text] [PDF]
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 T. M. Beer and A. Myrthue Calcitriol in cancer treatment: From the lab to the clinic Mol. Cancer Ther., March 1, 2004; 3(3): 373 - 381. [Abstract] [Full Text]
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O. Morales, M. K. R. Samuelsson, U. Lindgren, and L.-A. Haldosen Effects of 1{alpha},25-Dihydroxyvitamin D3 and Growth Hormone on Apoptosis and Proliferation in UMR 106 Osteoblast-Like Cells Endocrinology, January 1, 2004; 145(1): 87 - 94. [Abstract] [Full Text] [PDF]
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 I. Audo, S. R. Darjatmoko, C. L. Schlamp, J. M. Lokken, M. J. Lindstrom, D. M. Albert, and R. W. Nickells Vitamin D Analogues Increase p53, p21, and Apoptosis in a Xenograft Model of Human Retinoblastoma Invest. Ophthalmol. Vis. Sci., October 1, 2003; 44(10): 4192 - 4199. [Abstract] [Full Text] [PDF]
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J. Welsh, J. A. Wietzke, G. M. Zinser, B. Byrne, K. Smith, and C. J. Narvaez Vitamin D-3 Receptor as a Target for Breast Cancer Prevention J. Nutr., July 1, 2003; 133(7): 2425S - 2433. [Abstract] [Full Text] [PDF]
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J. B. Cordero, M. Cozzolino, Y. Lu, M. Vidal, E. Slatopolsky, P. D. Stahl, M. A. Barbieri, and A. Dusso 1,25-Dihydroxyvitamin D Down-regulates Cell Membrane Growth- and Nuclear Growth-promoting Signals by the Epidermal Growth Factor Receptor J. Biol. Chem., October 4, 2002; 277(41): 38965 - 38971. [Abstract] [Full Text] [PDF]
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D. J. Bettoun, D. W. Buck II, J. Lu, B. Khalifa, W. W. Chin, and S. Nagpal A Vitamin D Receptor-Ser/Thr Phosphatase-p70 S6 Kinase Complex and Modulation of Its Enzymatic Activities by the Ligand J. Biol. Chem., July 5, 2002; 277(28): 24847 - 24850. [Abstract] [Full Text] [PDF]
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K. Prufer, C. Schroder, K. Hegyi, and J. Barsony Degradation of RXRs Influences Sensitivity of Rat Osteosarcoma Cells to the Antiproliferative Effects of Calcitriol Mol. Endocrinol., May 1, 2002; 16(5): 961 - 976. [Abstract] [Full Text] [PDF]
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