CDK2 Is a Target for Retinoic Acid-Mediated Growth Inhibition in MCF-7 Human Breast Cancer Cells

Christine Teixeira and M. A. Christine Pratt

Department of Pharmacology University of Ottawa Ottawa, Ontario, Canada, K1H 8M5


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Retinoic acid (RA) inhibition of breast cancer cell growth is associated with an accumulation of cells in G1 phase of the cell cycle. We have investigated the effects of RA on the expression and activity of cell cycle-regulatory proteins in MCF-7 human breast cancer cells. Flow cytometry analysis of MCF-7 cells treated with RA revealed a decrease in the percentage of cells in S phase by 48 h, which was maximal by 72 h. Phosphorylation of the retinoblastoma protein (pRb) was partially reduced in RA-treated cells accompanied by a decrease in the level of retinoblastoma protein. Expression of the cyclin D1 transcript was reduced by 48 h and cyclin-dependent kinase 2 (cdk2) mRNA levels declined within 8 h posttreatment followed by a decrease in cyclin D1 and cdk2 protein levels. Message and protein levels of cdk4 and cdc2 were not affected by RA. While cdk4 activity was similar in control and RA-treated cells, cdk2 activity began to decrease within 48 h of exposure to RA and was profoundly reduced after 72 h. This reduced activity was associated with decreased phosphorylation of cdk2. The decrease in cdk2 activity occurred in the absence of RA-mediated increases in the levels of the cdk inhibitors p21and p27. However, assays of cdk2 from pooled lysates from RA-treated and control cells showed that RA-treated cells contain a cdk2-inhibitory activity. Our results show that RA inhibits cell cycle progression of MCF-7 cells by inhibiting cdk2 mRNA and protein production and by decreasing cdk2 activity.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The two retinoid isomers, all-trans-retinoic acid (RA) and 9-cis-RA signal through nuclear receptors [retinoic acid receptors (RARs) and retinoid X receptors] that act as ligand-inducible transcription factors on target genes containing RA-response elements in their regulatory regions (1, 2). Retinoids influence growth and differentiation in a wide variety of cell types (3, 4). Among those cells that are growth inhibited by RA are a subset of breast cancer cells that are estrogen receptor (ER) positive. In most instances, ER-positive breast cancer cells such as MCF-7 cells also express RARs whereas ER-negative cells do not (5, 6). We (7) and others (8, 9) have shown that RA inhibits activation of the ER in breast cancer cells. Using transient transfection of MCF-7 cells, we have shown that this inhibition involves transcriptional interference through the RAR AF-2 activation domain (7). However, this is unlikely to be the sole mechanism by which RA inhibits the growth of breast cancer cells since this interference is incomplete, and ER-negative cells transfected with the RAR become sensitive to RA-induced growth inhibition (10, 11).

Past studies have shown that RA causes breast cancer cells to accumulate in G1 of the cell cycle (12). Cyclins and cyclin-dependent kinases (cdks) play a central role in regulation of the cell cycle in eukaryotic cells (13, 14). Cyclins are positive regulatory subunits for cdks, and together they form active complexes that phosphorylate substrates involved in cell cycle progression. The D-type cyclins and cyclin E, in association with cdk4, cdk6, and cdk2, govern progression through G1 (15, 16). The activity of cyclin:cdk complexes is subject to positive and negative regulation. Both cyclin binding and phosphorylation by the cdk-activating kinase (cak) are required for activation whereas inhibitory phosphates are removed by members of the cdc25 phosphatase family (17, 18, 19). Recently, several new inhibitor proteins have been identified that bind to and negatively regulate either cyclin D-cdk4 complexes (p15,p16,p18,p19) (20, 21, 22, 23) or inhibit both cdk2 and cdk4-cyclin complexes (p27Kip1,p21,p57) (24, 25, 26, 27, 28, 29, 30, 31). A major function of cdk4 complexed with D cyclins is the phosphorylation of the tumor suppressor protein, retinoblastoma (pRb) (32, 33, 34, 35). Phosphorylation of pRb prevents the binding of members of the E2F/DP family of transcription factors (36, 37, 38, 39). Free E2F/DP proteins act by promoting the transcription of genes whose products facilitate progression through G1 (40). Cdk2 has also been implicated in the phosphorylation of pRb (41) and is required for the G1 to S phase transition (42) and DNA synthesis (43).

In this report we demonstrate that RA induces accumulation of MCF-7 cells in G1 of the cell cycle. These cell cycle effects are accompanied by a decrease in pRb phosphorylation. Both cdk2 mRNA and protein levels were decreased by RA treatment of MCF-7 cells associated with a profound decrease in cdk2 activity. This occurred in the absence of alterations in p27 and p21 levels although cdk2-inhibitory activity was present in extracts from RA-treated cells. We conclude that RA induces accumulation of breast cancer cells in G1 phase, at least in part, as a result of decreased expression and inhibition of cdk2 activity.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Effect of RA on Cell Cycle Phase Distribution
While numerous studies have shown that RA inhibits the growth of MCF-7 cells (44, 45), it has been suggested that there is some clonal variation in responsiveness (9, 46). To establish the effects of RA on our MCF-7 cells, flow cytometric analysis was performed on control and RA-treated populations. The cells were partially synchronized by contact inhibition to help clarify RA effects on specific phases of the cell cycle. After release from contact inhibition, cells were plated in the presence or absence of 1 µM RA and harvested at the indicated intervals. Table 1Go shows that almost 70% of contact-inhibited (CI) cells were in G0/G1 phase. Both RA-treated and control cells showed similar phase distribution over the initial 24-h period after release. However, by 48 h the percentage of RA-treated cells in S phase began to decline compared with control cells accompanied by a proportional transient increase in G2/M phase cells. At 72 h after release there was a clear accumulation of RA-treated cells in G0/G1 accompanied by a decline in the S phase population, which persisted to the 96-h time point.


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Table 1. Cell Cycle Distribution of CI MCF-7 Cells and Cells Released from Contact Inhibition in the Presence or Absence of 1 µM RA

 
RA Decreases Rb Phosphorylation and Protein Levels in MCF-7 Cells
Since the Rb protein is central to regulation of the cell cycle we examined the effects of RA on pRb phosphorylation (Fig. 1AGo). pRb was present in CI cells in various states of phosphorylation. At 24 h following release, no difference was observed between control and RA-treated cells; however, by 48 h, hypophosphorylated pRb was observed in the RA-treated cells. By 72 h hypophosphorylated pRb was present in both populations, which may indicate that a subpopulation of untreated cells were leaving M phase and entering G1. At 96 h post-RA, no hyperphosphorylated forms of pRb were evident in contrast to control cells, which only contained hyperphosphorylated forms of pRb. Additionally, there appeared to be less total pRb at the final time point compared with earlier times. To determine whether RA acted to simply prevent the semi-synchronized cells from exiting G1, we performed a similar analysis on asynchronous cultures of both MCF-7 and T47-D cells. Like MCF-7 cells, this breast cancer cell line also expresses the ER. The results in Fig. 1BGo show a profound reduction in hyperphosphorylated pRb after RA treatment in both cell lines accompanied by a decrease in total pRb levels. The kinetics of this reduction in phosphorylated forms of pRb were more rapid than in the semi-synchronized cells. Reactivity of the blots with {alpha}-actin is shown below to control for total protein levels.



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Figure 1. RA Decreases pRb Phosphorylation and Protein Levels

A, Western blot analysis of pRb was performed on lysates from CI cells and at the indicated times after release in the presence or absence of RA. Immunoreactive protein was detected by chemiluminescence as described in Materials and Methods. Multiple phosphorylated species of pRb are indicated by the open box, and hypophosphorylated pRb is indicated by the arrow. B, Western blot analysis of pRb was performed on exponentially growing MCF-7 and T47-D cells after treatment with RA. Phosphorylated forms of pRb are indicated. Immunoreactivity with an {alpha}-actin antibody was used to control for loading of protein.

 
RA Decreases Cyclin D1 and cdk2 Gene Expression
To determine whether changes in the gene expression for cell cycle proteins might account for the RA-induced changes in cell cycle progression, we performed Northern blot analysis of RNA from MCF-7 cells released from contact inhibition in the presence or absence of RA. Figure 2AGo shows that cyclin A mRNA was present in CI cells, increased transiently in control cells, was reduced at 24 h, and underwent a second increase at 48 h, which persisted through 96 h. The transient decrease in cyclin A appeared earlier in RA-treated cells (8 h), and the second increase was also accelerated, beginning at 24 h. This early decrease in cyclin A appeared to be without major effects on cell growth since both control and RA-treated cells were equally distributed in the cell cycle by 24 h after release. However, the oscillation in cyclin A levels in both control and RA cells may indicate a differential cell cycle phase distribution of the cells. Consistent with this, cyclin A levels in RA and control cells were again similar by 48 h, but by 96 h, cyclin A was again lower in RA-treated MCF-7 cells. Cyclin D1 mRNA levels were high in CI cells and were similar in control and RA-treated cells until 48 h, at which point there was a reduction in the 4.5-kb transcript in the RA-treated cells compared with the control. The lower (1.5 kb) transcript did not decrease significantly in these cells.



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Figure 2. Effect of RA on Expression of mRNA for Cyclins and cdks

Twenty micrograms of total RNA from CI cells and cells released from contact inhibition in the presence or absence of RA were electrophoresed and transferred to nylon as described in Materials and Methods. Two separate RNA blots were hybridized with cDNA probes for: cyclin A and cyclin D1 (A); cdk2 and cdk4 (B). C, Twenty micrograms of RNA from exponentially growing MCF-7 cells treated for the indicated times with RA were subjected to Northern blot analysis of cdk2 mRNA as described above. All blots were reacted with GAPDH to control for equivalency of loading. D, Densitometric scan of cdk2 mRNA. The graph depicts the ratio of the relative density of bands in RA-treated samples in panel B to the GAPDH control for the same time point.

 
Northern blot analysis of the expression of the G1 kinases, cdk2 and cdk4, in Fig. 2BGo indicated that while cdk4 transcript levels did not change over the course of the cell cycle in either control or RA-treated MCF-7 cells, a decrease in the expression of cdk2 mRNA was evident within 8 h after RA addition, which persisted over the course of culture. A densitometric analysis of cdk2 mRNA levels in treated vs. control cells showed initial oscillations in mRNA levels that fell to 20% of control levels by 48 h (Fig. 2CGo). To ensure that the effect of RA on cdk2 mRNA was not due to prevention of exit from G1, we performed Northern blot analysis of RNA from exponentially growing MCF-7 cells treated with RA for various times. The result in Fig. 2DGo shows that RA produces a similar rapid decrease in cdk2 mRNA under these conditions. RA had no effect on cyclin E or D3 mRNA expression in MCF-7 cells (data not shown).

RA Decreases Levels of Cyclin D1 and cdk2 Protein in MCF-7 Cells
Since we observed decreases in the mRNA levels for both cyclin D1 and cdk2 in RA-treated cells, we looked for associated changes in protein levels. Cyclin D1 protein levels correlated well with expression of the 4.5-kb transcript decreasing within 48 h after release from contact inhibition in the presence of RA (Fig. 3AGo). The p33cdk2 protein levels remained stable for several hours after RA treatment of MCF-7 cells, but by 48 h the protein levels decreased by 30% and were further reduced by 80% at 72 h and 96 h after treatment with RA (Fig. 3BGo), as determined densitometrically. By contrast, RA did not alter cdk4 protein levels (Fig. 3CGo).



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Figure 3. RA Decreases cdk2 and Cyclin D1 Protein Levels

Fifty micrograms of MCF-7 cell extract from CI cells and cells released in the presence or absence of RA as described in Materials and Methods were used for Western blot analysis of cyclin D1 (A); Western blot analysis of cdk2 using 5 µg cell extract (B); Western blot analysis of cdk4 using 5 µg cell lysate (C). An antibody to {alpha}-actin was used to control for loading equivalency.

 
RA Decreases the Phosphorylation of cdk2
To determine whether RA affects the phosphorylation of cdk2, we metabolically labeled cdk2 with 32Pi in the presence or absence of RA and analyzed the cdk2 immunoprecpitates by electrophoresis followed by transfer to a nylon membrane and autoradiography. Figure 4Go is an autoradiogram of metabolically labeled cdk2, which shows a marked decrease in phosphorylation of p33cdk2 after a 72-h exposure to RA. Western blot analysis with cdk2 antibody (shown below) confirmed that the difference in labeling was not due to differential amounts of immunoprecipitated cdk2 protein in the treated and untreated lanes since the RA-treated lysate has been overloaded with respect to the control. Two other labeled bands, one at 34 kDa and the other at 30 kDa, that coimmunoprecipitated with cdk2 are evident in the immunoprecipitate. The 30-kDa band also cross-reacts with the cdk2 antibody on Western blot analysis.



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Figure 4. Metabolic Labeling of cdk2 with 32Pi

Extracts were prepared from MCF-7 cells 72 h after release from contact inhibition in the presence or absence of RA. The cells were metabolically labeled for the last 16 h of culture in the presence of 32Pi, and the extracts were immunoprecipitated with a cdk2 antibody, subjected to SDS-PAGE, and transferred to a PVDF membrane as described in Materials and Methods. The membrane was exposed to film to detect 32P-labeled cdk2 then subjected to Western blot analysis with cdk2 antibody to determine total cdk2 protein in the immunoprecipitate.

 
Cdk2 Activity but Not cdk4 Activity Is Inhibited by RA
The decrease in cdk2 protein and phosphorylation predicted that the activity of cdk2 would be decreased by RA. Therefore, cultures of CI MCF-7 cells were released in the presence or absence of RA. The results in Fig. 5AGo show that CI MCF-7 cells contain high levels of cdk2 activity measured by histone H1 phosphorylation. Twenty-four hours after release from CI, cdk2 activity decreased in both control and RA-treated cells. By 48 h both control and RA-treated cells contained increased cdk2 activity, although this activity was significantly lower in RA-treated cells. Within 72 h, cdk2 activity was inhibited by 40% in RA-treated cells and dropped to less than 10% of control levels after 96 h. The decrease in cyclin D1 protein levels in RA-treated MCF-7 cells suggested that RA might also inhibit cdk4 activity. We therefore assayed cdk4 activity in control and RA-treated cells using a glutathione-S-transferase (GST)-Rb fusion protein as substrate. The lower panel in Fig. 5AGo shows the results of this assay and indicates that there was no change in cdk4 activity in RA-treated cells compared with control cells over the 96-h treatment period. Figure 5BGo is a graph of the densitometric analysis of the ratio of phosphorylated histone H1 in RA-treated cells to that in control cells.



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Figure 5. RA Inhibits cdk2 Activity in MCF-7 Cells

A, MCF-7 cell extracts from CI cells or after release for the indicated times in the presence or absence of RA were prepared as described in Materials and Methods. For assays of cdk2 and cdk4 kinase activities, cdk2 and cdk4 immunoprecipitates were mixed with histone H1 or a GST-Rb fusion protein, respectively, in the presence of {gamma}-32P-ATP as described in Materials and Methods. Kinase reactions were subjected to SDS-PAGE followed by autoradiography. Upper panel, cdk2 phosphorylation of H1 histone; lower panel, cdk4 phosphorylation of GST-Rb. B, Densitometric scan of histone H1 phosphorylation expressed as a ratio of activity in RA-treated to untreated control lysate at each time point.

 
RA Does Not Decrease Levels or Activity of the cdk-Activating Kinase, cak
To investigate whether the profound decrease in levels of phosphorylated cdk2 is due to RA modulation of cak (cdk7), the kinase responsible for the activating phosphorylation of cdk2 (47), we assessed cak protein levels. Figure 6AGo shows that cak exists primarily as a 37 kDa protein in CI MCF-7 cells, and increasing amounts of a faster migrating species appeared after release in both control and RA-treated cells. We then wished to determine whether decreased cdk2 activity might be due to reduced cak activity in RA-treated cells. We therefore assayed cak activity in anti-cak immunoprecipitates using GST-cdk2 fusion protein as a substrate. Figure 6BGo shows that CI MCF-7 and released cells grown in the absence or presence of RA all contained equivalent levels of cak activity. Thus, a decrease in cak protein levels or activity is not responsible for the decreased levels of cdk2 phosphorylation in RA-treated cells.



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Figure 6. Effect of RA on cak Protein and Activity

A, Western blot analysis of cak. Extracts from CI MCF-7 cells and after release in the presence or absence of RA for the indicated times were used for Western blot analysis of cak. Two immunoreactive bands are evident in all samples except in CI cells. Equivalency of loading was assessed by reactivity with an {alpha}-actin antibody. B, Assay of cak activity. Extracts from MCF-7 cells cultured as in panel A were immunoprecipitated with an anti-cak antibody. Immunoprecipitates were mixed with a GST-cdk2 fusion protein in the presence of {gamma}-32P-ATP as described in Materials and Methods and reactions subjected to SDS-PAGE followed by autoradiography.

 
RA Does Not Alter Levels of Cyclin Inhibitor Proteins
Analysis of TGFß-treated mink lung epithelial cells has shown that cdk inhibitors play a role in mediating growth inhibition induced by this factor (48). We have studied the effects of RA on two of these inhibitors, p21 and p27Kip1, in MCF-7 cells by Western blot analysis of CI and released cells (Fig. 7Go, A and B). p21 and p27Kip1 were both present in CI MCF-7 cells. While the levels of p27Kip1 did not change in either control or RA-treated released cells over the 6-day study period, p21 protein levels were reduced compared with control by 96 h of treatment. Next we determined whether p21 or p27 levels were altered in asynchronous cells treated with RA. The results in Fig. 7CGo show that neither p21 nor p27 were increased after exposure to RA. Both p21 and p27Kip1 can form complexes with cdks to inhibit their activity. To determine whether RA inhibits cdk2 activity by increasing the level of these inhibitors complexed with cdk2, we immunoprecipitated cdk2 protein and performed Western blot analysis of p21 and p27Kip1 in the immunoprecipitate. The results in Fig. 7DGo indicate that cdk2-associated p21 does not increase but instead decreases by 72 h in RA compared with control cells, while p27Kip1 levels remain relatively constant in control and RA-treated cells. Since a decrease in cdk2-associated cyclin E could also have an impact on cdk2 activity, we also performed Western blot analysis of cdk2-associated cyclin E. The third panel in Fig. 7DGo shows that cyclin E levels are unchanged in RA-treated cells compared with controls. Western blot analysis of immunoprecipitated cdk2 was performed to control for cdk2 levels in the complexes. The decreased level of cdk2 immunoprecipitated after 96 h likely reflects the overall decrease in cdk2.



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Figure 7. Western Blot Analysis of cdk Inhibitors

Extracts from semi-synchronized MCF-7 cells were subjected to Western blot analysis as described in Materials and Methods. Blots were incubated with anti-p21 antibody (A) and anti-p27Kip1 antibody (B). Extracts from exponentially growing MCF-7 cells were subjected to Western blot analysis of p21 and p27Kip1 (C). All blots were reacted with an anti-{alpha} actin antibody to control for loading equivalency. Western blot analysis of cdk2-associated p21, p27, and cyclin E (D). Semi-synchronized MCF-7 cells were released in the presence or absence of RA. At the indicated times, extracts were immunoprecipitated with cdk2 antibody and the complexes subjected to SDS-PAGE followed by immunoblot analysis with anti-p21, anti-p27, and anti-cyclin E. The blot was reacted with anti-cdk2 to control for efficiency of the immunoprecipitation.

 
RA-Treated MCF-7 Cells Contain a cdk2-Inhibitory Activity
Cdk-inhibitory activity induced by TGFß has been characterized by the ability of treated cell lysates to inhibit cdk kinase activity in control lysates (49, 50). We have thus performed a similar experiment using RA-treated cell lysates to determine whether an inhibitor of cdk2 activity is present in these cells. Figure 8Go shows that immunoprecipitation of cdk2 from two pooled control lysates (100 µg each) yielded a high level of histone H1 kinase activity while immunoprecipitation of 100 µg of protein from a single control lysate produced about half of this activity. As expected, the activity in RA-treated lysates was much reduced compared with the control lysate. Strikingly, rather than producing an additive effect, the mixing of control lysate with RA-treated lysate resulted in levels of kinase activity similar to those in the RA lysate consistent with the presence of a cdk2-inhibitory activity in the RA-treated cells.



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Figure 8. RA-Treated MCF-7 Cells Contain a cdk2-Inhibitory Activity

Control (C) or RA-treated MCF-7 cell lysates (RA) were immunoprecipitated with cdk2 antibody (C or RA) or premixed for 1 h at 37 C before immunoprecipitation with cdk2 antibody (C+C or C+RA). Immunoprecipitates were used to phosphorylate histone H1 in the presence of 32Pi as described in Materials and Methods. The reaction mixture was then subjected to PAGE after which the gel was dried and subjected to autoradiography.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Studies of factors that either promote or inhibit cell growth have shown that individual agents may target specific components of the cell cycle-regulatory apparatus. Several years ago it was shown that treatment of MCF-7 cells with RA results in an accumulation of these cells in G1 of the cell cycle (12). In this study we have attempted to identify the cell cycle-regulatory targets associated with RA-induced changes in cell cycle progression in MCF-7 breast cancer cells. RA treatment of MCF-7 cells results in a decrease in the S phase population after 48 h, which continues to decrease accompanied by an increase in the G1 population. Thus the first cell cycle alterations associated with RA exposure begin at 48 h after treatment and peak by 72 h. Of the cell cycle components studied, levels of cdk2 and cyclin D1 mRNA and protein were reduced in this time frame. In addition, cdk2 activity decreased in RA-treated cells within 48 h following RA treatment coincident with the initial decrease in the S phase cell population. These results contrast with the effects of an antiestrogen on MCF-7 cells, which causes a decrease in the S phase fraction associated with repression of cdk4 activity (51). The relative lack of effect on cdk4 activity was somewhat surprising given that cyclin D1, a G1 partner for cdk4 (52), was reduced in RA-treated cells. However, cyclin D3 was abundant in both RA-treated and control cells, and it is possible that it might substitute for cyclin D1 in an active kinase complex. To this end, cyclin D1 and D3 have both been shown to elevate cdk4 activity (35).

The observation that RA results in accumulation of underphosphorylated pRb in both synchronous and asynchronous cells suggests that RA is not acting to simply prevent semi-synchronized cells from exiting G0/G1 but rather to cause the accumulation of cells in G1. Furthermore, the profound effects of RA on pRb phosphorylation in T47-D cells supports the notion that this is not a cell line-specific effect of RA. There do appear, however, to be some differences between the cell lines. Wilcken et al. (53) showed that RA treatment of T47-D cells results in an inhibition of cdk4 activity and a small decrease in pRb phosphorylation that was not associated with alterations of cyclin D1 or cdk enzyme levels. The kinetics of RA reduction of the S phase population in these cells were faster than what we observed in MCF-7 cells with decreases in S phase being evident by 24 h after RA treatment compared with 48 h in this study. The reasons for the differences between responses to RA in T47-D and MCF-7 cells are not clear but may reflect cell-specific responses to RA.

Unlike what has been observed for prostaglandin A2 (54), an agent that reduces G1 cdk activity in MCF-7 breast cancer cells without alteration of pRb levels, both antiestrogen (51) and RA treatment (this study) cause a decrease in pRb that may reflect a lack of requirement for the Rb protein in maintaining growth inhibition.

The recent discovery of several cdk inhibitors has established another level of regulation of cdk activity. These proteins have been shown to function as targets of certain growth inhibitors. For instance TGFß-treated Mv1Lu mink lung epithelial cells exhibit decreased phosphorylation and activity of cdk2-cyclin E complexes (49) mediated by the heat-stable inhibitor, p27Kip1 (50). TGFß appears to act by increasing the levels of the cdk4 inhibitor, p15Ink4B, resulting in the release of p27Kip1, thereby freeing it to bind to cdk2 and inhibit its activity (48). The response varies with cell type since keratinocytes respond to TGFß by increasing both p15 and p21Cip1 (51). Although these inhibitors can prevent cdk activation by cak, they do not inhibit the activity of cak (30). Kato et al. (55) have shown that p27Kip1 association with cdk4-cyclin D complexes prevents cak from phosphorylating cdk4 to activate the enzyme complex in macrophages arrested in G1 after treatment with cAMP. Extracts from quiescent mouse mammary epithelial cells also contain p27Kip1 inhibitory activity (56) while prostaglandin A2- treated MCF-7 cells undergo growth arrest associated with marked increases in p21 levels (54). Since p21 can block the phosphorylation of cdks by cak (57), it was also a possible mediator of RA effects on cdk2. Surprisingly, RA did not increase levels of either p21 or p27Kip1 in MCF-7 cells. These effects are unlike those of an antiestrogen wherein both p21 and p27Kip1 are weakly increased in MCF-7 cells (51, 58) in apparent antagonism of the effects of estrogen, which decreases the level of p27Kip1 in MCF-7 cells (58). In contrast, we observed a decrease in p21 and cdk2-associated p21 in RA-treated cells.

Our data show that a major downstream target for RA inhibition in MCF-7 breast cancer cells is the cell cycle protein, cdk2, through a decrease in mRNA and protein levels that apparently does not involve increases in the levels of cdk2-associated cell cycle inhibitors, p21 and p27Kip1. It is not clear whether RA acts directly or indirectly to decrease cdk2 mRNA levels. The cdk2 promoter has recently been cloned and contains regulatory sequences corresponding to sites of interaction for E2F, AP-1, and several other factors (59). Interestingly the ligand-bound RA receptor has been shown to interfere with transcription from AP-1 sites through binding of a common transcriptional coactivator protein, the cAMP response element binding protein (CBP) (60). Future studies will determine the mechanism of retinoid inhibition of this gene.

Results of metabolic labeling in the presence of RA showed that the phosphorylation of cdk2 was decreased, suggesting that RA may induce the activity of a phosphatase. Several phosphatases, including cdc25 (61) and protein phosphatases 1 and 2a [PP-1 and PP2A (62)], are known to play a role in regulation of cdk activity. While cdc25 removes the inhibitory phosphates at residues Y15 and T14 (61), PP1 and PP2A have been implicated in the inactivation of a cdk (cdc2) through the removal of an activating phosphate on T160 (62). Since RA treatment results in decreased total phosphorylation of cdk2, it is possible that both types of phosphatase activity are increased by RA. The other possibility is that the phosphorylation of cdk2 by cak is inhibited by RA. Although our results show that the intrinsic activity of cak has not been decreased by RA treatment, it is possible that the phosphorylation of cdk2 is blocked in RA-treated cells.

The results of the cdk2-inhibitory assay show that a putative RA-inducible inhibitor can block cdk2 activity in control lysates containing preactivated cdk2. This may result from one or more mechanistic possibilities. The first is that the decrease in cdk2 protein in RA-treated cells in the absence of a change in cellular levels of p27Kip1 results in an increase in free p27Kip1 relative to cdk2. The free p27Kip1 would then be available to interact with cdk2 complexes from control lysates to inhibit their activity. Alternatively, a novel inhibitor may be induced by RA in these cells, resulting in a decrease in cdk2 activity.

In combination with cyclin E, cdk2 is necessary for the G1 to S phase transition (42). The cyclin A-cdk2 complex binds to E2F-1 (63, 64) and inactivates E2F-1 DNA-binding activity by phosphorylation (65), an activity that is necessary for orderly progression through S phase (66). In addition, recent evidence shows that cdk2 activity is also necessary for entry into mitosis since it activates the mitotic cyclin-cdc2 kinase activity (43). In light of the expansive role of cdk2 in cell cycle regulation, RA-mediated decreases in cdk2 levels and activity are likely to have profound effects on several aspects of proliferation.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Cell Culture
MCF-7 ER-positive breast cancer cells (obtained from Dr. Leigh Murphy, Manitoba) and T47-D ER-positive breast cancer cells (ATCC, Rockville, MD) were maintained in DMEM (GIBCO BRL, Burlington, Ontario) containing phenol red and supplemented with nonessential amino acids, 5% FBS (GIBCO BRL), 0.3% glucose, and 2 µg/ml gentamicin sulfate at 37 C/5%CO2. In some experiments cells were semi-synchronized by contact inhibition achieved by maintaining the cells at confluence for 48 h without a change of medium. This routinely resulted in a blockage of approximately 70% of the cells in G1 of the cell cycle. Cells were released by trypsinization and plated at low density in the presence or absence of 1 µM RA (Sigma Chemical Co., St. Louis, MO) added from a 1 mM stock in ethanol.

Flow Cytometry
Cells were fixed in 100% ethanol for 1 h at 4 C and washed twice with PBS before staining with 32 µM propidium iodide containing 500 µg/ml RNase A. DNA content was analyzed on a Coulter Epics V FACscan (Coulter Corp., Hialeah, FL), and cell cycle phase distributions were estimated by computer fit using the Multicycle analysis program.

RNA Isolation and Northern Blot Analysis
Total RNA was isolated from LiCl-urea as described (67). Twenty micrograms of total RNA were electrophoresed in denaturing agarose gels and transferred to Hybond N (Amersham, Oakville, Ontario). Membranes were hybridized with multiprime-labeled probes, washed, and autoradiographed. Equivalency of RNA loading was monitored by hybridization of the blot with a glyceraldehyde phosphate dehydrogenase (GAPDH) probe whose level is not altered in RA-treated MCF-7 cells. The cDNAs encoding human cyclins and cdks were obtained from Dr. Paul Hamel (Toronto, Ontario, Canada).

Western Blot Analysis
Protein extracts were prepared from cultures washed twice with PBS and lysed with 1 ml per 107 cells of RIPA buffer (1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 10 mg/ml phenymethylsulfonyl fluoride (PMSF), 3 µg/ml aprotinin, 10 mg/ml sodium orthvanadate in PBS). Insoluble material was removed by centrifugation at 4000 rpm for 15 min at 4 C. Protein concentrations were determined using a Bio-Rad (Mississauga, Ontario) protein assay kit. Samples were resolved on 7.5% (for detection of pRb) or 12% (for all other proteins) SDS porous polyacrylamide gels (68) or the Laemmli gel system (cdk2 only) and transferred electrophoretically to PVDF polyscreen membranes (DuPont NEN, Boston, MA). Blots were incubated with primary and secondary antibodies according to the supplier’s directions. Antibodies were obtained from the following sources: pRb (Pharmingen, Cedarlane Labs, Hornby, Ontario); cyclin A, cyclin D1, cyclin E, cdk2, cdk4, cak, p21, and p27 (Santa Cruz Biotechnology, Santa Cruz, CA); {alpha}-actin (Sigma); goat anti-mouse IgG-horseradish peroxidase (Jackson Labs, BioCan Sci, Mississauga, Ontario). Bound antibodies were detected using the ECL chemiluminescence system (Dupont NEN). In some experiments densitometry was performed on Northern and Western blots using the microcomputer imaging densitometry (MCID) software system (Imaging Research, Brock University, Ontario, Canada).

In Vitro Kinase Assays
For cdk2 and cak assays, 400 µg of cell lysed as described (69) were precleared by incubation with protein A Sepharose (Pharmacia, Baie d’Or, Quebec) and incubated with antibody to cdk2 or cak adsorbed to protein A Sepharose at 4 C overnight. Immune complexes were washed four times with RIPA buffer. Cdk2 activity was assayed by resuspending the beads in 40 mM Tris (pH 7.5), 10 mM MgCl2, 5 µM ATP, 0.5 mM dithiothreitol (DTT), 0.5 mM EGTA, 400 µg/ml histone H1 (Sigma), 50 µCi [{gamma}-32P]ATP (Amersham) and incubation at room temperature for 20 min. To assay cak activity, beads were resuspended in 50 mM HEPES (pH 7.5), 10 mM MgCl2, 4 mM ATP, 1 mM DTT, 400 µg/ml of a glutathione-S-transferase-cdk2 fusion protein (GST-cdk2) and 10 µCi [{gamma}32P]ATP and incubated for 30 min at 30 C. To assay cdk4 activity, cells were lysed by sonication at 4 C in 50 mM HEPES (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1 mM DTT, 0.1% Tween 20 containing 10% glycerol, 0.1 mM PMSF, 10 mM ß-glycerophosphate, 1 mM NaF, 0.1 mM sodium orthovandate, and 3 µg/ml aprotinin as described (35). Lysates were cleared by centrifugation, precleared with protein A-Sepharose, and incubated for 2 h at 4 C with protein A-Sepharose precoated with anti-cdk4. Immunoprecipitates were washed four times with lysis buffer and twice with 50 mM HEPES (pH 7.5), 1 mM DTT. Beads were resuspended in 50 mM HEPES (pH 7.5), 1 mM NaF, 2.5 mM EGTA, 1 mM DTT, 10 mM ß-glycerophosphate, 0.1 mM sodium orthovanadate, 10 mM MgCl2, 20 µM ATP, and 10 µCi[{gamma}-32P]ATP and incubated for 30 min at 30 C with 400 µg/ml GST-pRB fusion protein. All kinase reactions were resolved by SDS-PAGE and subjected to autoradiography. GST-pRb was obtained from Dr. Paul Hamel (Toronto, Ontario, Canada); GST-cdk2 was obtained from Dr. Tim Hunt (Oxford, U.K.).

Metabolic Labeling
MCF-7 cells released from contact inhibition were plated in the presence or absence of 1 µM RA. After 56 h, cells were washed twice with DMEM without phosphate then preincubated in the same medium for 30 min. Cells were then cultured for 16 h in 3 ml phosphate-free DMEM containing 1 mCi of 32Pi (Amersham). Cells were lysed in RIPA buffer and immunoprecipitated with cdk2 antibody as described above. Precipitated complexes were subjected to SDS-PAGE and transferred to a PVDF membrane for autoradiography followed by Western blot analysis with cdk2 antibody.

Cdk2 Inhibitor Assay
Cells incubated with or without 1 µM RA for 96 h were lysed in a buffer containing 20 mM Tris, pH 7.5, 250 mM NaCl, 0.1% NP-40, 1 mM Na orthovanadate, 10 mM NaF, and 1 mM PMSF. One hundred micrograms of lysates were mixed where indicated for 1 h, preadsorbed with protein A Sepharose, and incubated with antibody against cdk2 overnight at 4 C. Immunoprecipitates were formed after incubation with protein A Sepharose for 1 h at 4 C, pelleted, washed four times with lysis buffer, and assayed for cdk2 kinase activity as described above. The reaction was allowed to proceed for 20 min at room temperature, stopped by the addition of SDS-PAGE sample buffer, boiled, and loaded on a 10% SDS polyacrylamide gel. The fixed, dried gel was subjected to autoradiography at -80 C.


    FOOTNOTES
 
Address requests for reprints to: M. A. Christine Pratt, Department of Pharmacology, University of Ottawa, 451 Smyth Road, Ottawa, Ontario, Canada, K1H 8M5.

This work was supported by grants from the Medical Research Council of Canada and the American Institute for Cancer Research (No. 96A020) to M.A.C.P.

Received for publication August 2, 1996. Revision received December 23, 1996. Revision received April 30, 1997. Accepted for publication May 14, 1997.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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