Inhibitory Effects of 1{alpha},25-Dihydroxyvitamin D3 on the G1–S Phase-Controlling Machinery

Simon Skjøde Jensen, Mogens Winkel Madsen, Jiri Lukas, Lise Binderup and Jiri Bartek

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.


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The nuclear hormone 1{alpha},25-dihydroxyvitamin D3 induces cell cycle arrest, differentiation, or apoptosis depending on target cell type and state. Although the antiproliferative effect of 1{alpha},25-dihydroxyvitamin D3 has been known for years, the molecular basis of the cell cycle blockade by 1{alpha},25-dihydroxyvitamin D3 remains largely unknown. Here we have investigated the mechanisms underlying the G1 arrest induced upon 1{alpha},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{alpha},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{alpha},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{alpha},25-dihydroxyvitamin D3, these results demonstrate that 1{alpha},25-dihydroxyvitamin D3 inhibits cell proliferation by targeting several key regulators governing the G1/S transition.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
1{alpha},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.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
1,25-VD3-Treated MCF-7 Cells Display Decreased Proliferation and Accumulation in G1 Phase
To determine the proliferation-inhibitory effect of 1,25-VD3 on the MCF-7 cells, proliferation assays with concentrations of 1,25-VD3 at 10-7 M were performed; these concentrations were shown by others to have antiproliferative, but not toxic effects, in several cell lines including MCF-7 (32, 37, 38). As seen in Fig. 1AGo, 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. 1BGo). 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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Figure 1. Antiproliferative Effect of 1,25-VD3 on MCF-7 Cells

A, A summary of growth curves from three experiments, in which day 0 corresponds to the time when 1,25-VD3 (10-7 M) was added (24 h after plating the cells). Error bars represent SD. B, Summary of analysis of cell cycle phase distribution of cells treated as in panel A, analyzed by flow cytometry over a 6-d period. The change in G1 phase cells was significant from 24 h treatment, and the decrease in S phase population was significant from 48 h treatment (P < 0.05, analyzed by Student’s t test, n = 3–5).

 
1,25-VD3-Treated MCF-7 Cells Exhibit Hypophosphorylated pRb and Impaired Expression of Cyclin A and E mRNA and Protein
The 1,25-VD3-induced accumulation of cells in G1 phase indicated a potential effect on pRb, whose growth-suppressive, hypophosphorylated form is characteristic for cells in early G1 (40, 41). In a 4-d time course experiment, we analyzed pRb in total cell lysates by gel electrophoresis and immunoblotting (Fig. 2AGo). 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. 2Go, 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. 2BGo). 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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Figure 2. Effects of 1,25-VD3 on Cell Cycle Regulators

A, Western blot analysis of pRb in total cell extracts from mock (-)- or 1,25-VD3 (+)-treated MCF-7 cells at the indicated time points. The upper panel allowed good separation of the pRb species in an 8% gel separated overnight; pRbpp marks the hyperphosphorylated form of pRb above the hypophosphorylated pRb, for which cells treated with lovastatin for 48 h served as control (marked L). The hypophosphorylated pRb was quantitated from several experiments and plotted as fold to control ± SEM in panel D (upper graph). The middle panel (A) shows a less separated blot of pRb, allowing quantitative interpretation of pRb amounts in each lysate. B, Northern blot analysis of cyclin A and cyclin E expression using 10 µg of total RNA from MCF-7 cells at the indicated times. The 36B4 probe was used as a loading control. C, Time course Western blot analysis of the indicated cell cycle-regulatory proteins in MCF-7 cell extracts, with antibodies specified in Materials and Methods. Statistical analyses of multiple experiments were performed. Protein levels of cyclin A, cyclin D3, cdk2, and the faster migrating CAK-activated cdk2 species marked Cdk2CAK were all significantly reduced from 48 h treatment. c-Myc was significantly reduced from 24 h treatment and cdk6 was reduced from 72 h treatment. p21 was significantly increased from 72 h treatment, whereas protein levels of p27, cyclin D1, and cdk4 were not significantly altered. Cdk7 served as a loading control. D, Representative diagram showing statistical analysis of selected proteins; bars indicate SEM (data evaluated using Student’s t test, n = 3–6, P < 0.05).

 
1,25-VD3 Modulates Protein Levels of Important G1–S Phase Regulators
Western blots performed with total cell extracts showed altered protein levels of cyclin and cdk regulators, controlling G1–S phase progression. Protein levels of cdk2 and the activated cdk2CAK migrating slightly faster than the inactive cdk2 increased in control cells as they entered exponential growth, whereas 1,25-VD3 treatment retained cdk2 at the low initial levels, with only a minor appearance of the activated cdk2CAK (Fig. 2CGo). 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. 2CGo). 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. 1Go. 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. 1Go).

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. 3AGo). 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. 3BGo, 4Go-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. 3Go, 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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Figure 3. 1,25-VD3 Treatment Modulates G1 and S Phase cdk Activities

A, Kinase activities of the indicated cyclin D-associated complexes toward GST-pRb substrate, assayed after 4 d of mock (-) or 1,25-VD3 (+) treatment in immunoprecipitates from MCF-7 cell extracts obtained with antibodies DCS6 (recognizing only cdk-free cyclin D1; negative control), 5D4 (recognizing cyclin D1/cdk4 complex), DCS28 (recognizing cyclin D3/cdk4(6 )), and DCS130 (against the C terminus of cdk6). B, Kinase activities assayed in cells treated as in panel A, in immunoprecipitates obtained with antibodies HE12 (recognizing only cdk-free cyclin E; negative control), HE172 (recognizing cyclin E complexed to cdk2), sc-751 (cyclin A/cdk complexes), and sc-163 (recognizing cdk2 complexed with cyclins E or A). All complexes were significantly inhibited relative to control and mean ± SEM indicated as percent of control below the panels (n = 2–3, P < 0.05). Panels C and D, Four to 6-d time course analysis of cdk4 (C)- and cdk2 (D)-associated kinase activities in extracts of mock (-)- or 1,25-VD3-treated (+) MCF-7 cells. The diagrams below the panels show a summary of three to five independent kinase assays indicating mean value ± SEM, evaluated by a one-sided, unpaired t test. After 8 h of treatment there was a significant increase in cdk2 (4 ) kinase activity relative to control (P < 0.05). After 24 h and throughout the time course, cdk2 and cdk4 were significantly inhibited relative to control (P < 0.005). A–D, The phosphorylation signals in GST-pRb were quantitated using a PhosphorImager (Molecular Dynamics, Inc.).

 


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Figure 4. 1,25-VD3-Induced p21CIP1/WAF1 Targets cdk2 but Not cdk4

A, Time course Northern blot analysis of p21CIP1/WAF1 mRNA in mock (-)- and 1,25-VD3-treated (+) MCF-7 cells (36B4 = loading control). B and C, MCF-7 cells treated for 96 h with 1,25-VD3 (+) or vehicle (-) were analyzed by ip of equal amount of lysates with antibodies against the proteins shown on the top, followed by Western blotting with antibodies against the proteins indicated on the left. B, WCE (whole-cell extract) marks direct immunoblots on WCEs; and cdk2CAK indicates the CAK-activated form of cdk2. Below the blots, analyses of independent experiments are shown as mean ± SEM and indicated as decrease in total cdk2/increase in associated p21 relative to control (n = 2–5, P < 0.05). C, Analysis of independent experiments of cdk4(6 )-cyclin D-p21-p27 complexes revealed no significant stoichiometric alteration in these complexes, except that the cdk4-cyclin D3 complex was significantly reduced to 46 ± 14% of untreated controls, after 96 h treatment (n = 4, P < 0.05).

 
The M phase-specific cdk1-cyclin B1 complex showed a 3-fold inhibition of kinase activity toward the histone H1 substrate after 4 d of 1,25-VD3 treatment (data not shown). Taken together, these data show that the known cdk complexes essential for G1 and S phase progression are not activated in 1,25-VD3-treated MCF-7 cells, which are consequently precluded from entering a productive exponential growth phase.

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. 2CGo), 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. 4AGo). 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. 4BGo, lower panel, lanes 5–10). In whole-cell extracts (Fig. 4BGo, 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. 2CGo). 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. 4BGo, 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. 4BGo, 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. 4CGo). The lack of cdk4-cyclin D1 activation in treated cells, as seen in control cells in Fig. 3CGo, could not be explained by disruption of the complex, since the stoichiometry between cdk4-cyclin D1 remained largely preserved upon treatment (Fig. 4CGo, lanes 5–6 and 9–10). In contrast to cdk4-cyclin D1, complexes containing cyclin D3 were disrupted (Fig. 4CGo, 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. 2CGo). 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. 3AGo, lanes 2 and 3, and in Fig. 3CGo, 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. 1BGo), 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. 5AGo) 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. 2CGo). 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. 5Go, 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. 5Go, B and D).



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Figure 5. Dynamics of 1,25-VD3-Induced Modulation on cdk2 Activity

A, cdk2-Associated proteins (coprecipitated with antibody sc-163) were analyzed at the indicated time points by Western blotting using antibodies to cyclin A, cdk2, and p21CIP1/WAF1. B, Detailed time course analysis of p21CIP1/WAF1 protein abundance by direct immunoblotting of total cell extracts. cdk7 served as loading control. C and D, Six independent experiments were evaluated by unpaired, one-sided t test, with the following values *, 0.05 > P > 0.025; **, 0.025 > P > 0.01; and ***, P < 0.01. Protein levels of p21CIP1/WAF1 were quantitated by the ECF system (Amersham Pharmacia Biotech) and correlated to the amount of cdk2 (A), or cdk7 (B) in each lane, and finally to mock (-)-treated cells standardized to 1. Bars indicate SEM.

 
These data suggest that the initial events responsible for deficient cdk2 kinase activation in 1,25-VD3-treated MCF-7 cells include increased association between cdk2 and p21CIP1/WAF1, accompanied by a lack of cdk2 activation by cyclin A association after 48 h of treatment.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Our current data show that 1,25-VD3 treatment of MCF-7 cells prevented activation of G1 phase complexes cdk4(6)-cyclin D1(3), and the G1-S governing complexes of cdk2-cyclin A(E). Based on these results we propose a model (Fig. 6Go), 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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Figure 6. Schematic Model of 1,25-VD3 Effects on Diverse Pathways Controlling G1/S Transition

1,25-VD3 affects at least three pathways of the cell cycle machinery: 1) Inhibition of cyclin D-dependent kinases leading to activation of pRb and thereby repression of E2F; 2) Down-regulation of c-Myc abundance; and 3) Induction of p21CIP1/WAF1; all of which contribute to regulation of cdk2 kinase activity and G1 phase blockade.

 
Deregulation of G1 phase complexes including cdk6 and cyclin D3 can be explained by lack of protein induction and/or decreased protein levels of cdk6 and cyclin D3 from approximately 48 h of 1,25-VD3 treatment. At present, we have no mechanistic explanation for the observed effects on cdk4-cyclin D1 kinase activity after 24 h treatment.

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. 3DGo), at 24 h (60% of control) until p21CIP1/WAF1 could be significantly identified in cdk2 complexes at 36 h (Fig. 5Go, 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.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Cell Culture
The human breast cancer cell line MCF-7 (obtained from DkFZ, TZB610030) was cultured in DMEM without phenol red (Life Technologies, Inc., Gaithersburg, MD, catalog no. 11880) supplemented with 5% FCS, 2 mM glutamine, penicillin (10 U/ml), and streptomycin (10 U/ml). The cells were routinely passaged every week, and media were changed every second to third day. The cell line was regularly tested for mycoplasma infection (PCR-primer set, Stratagene, La Jolla, CA), and found to be mycoplasma negative. Twenty-four hours before 1,25-VD3 addition, the cells were passaged with 1 mM EDTA, spun down, and plated as single cells at a density of 3–5 x 103 cells/cm2. 1{alpha},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{gamma}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. 4Go and 5Go 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. 5Go, 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.


    ACKNOWLEDGMENTS
 
We thank M. Sato, S. I. Reed, and E. Harlow for important reagents, and D. Hansen for help with fluorescence-activated cell sorting analysis.


    FOOTNOTES
 
This work was supported by the Danish Cancer Society and the Danish Medical Research Council.

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{alpha},25-dihydroxyvitamin D3.

Received for publication June 22, 2000. Accepted for publication April 11, 2000.


    REFERENCES
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 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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