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Down-Regulation of Cyclin D1 by Transcriptional Repression in MCF-7 Human Breast Carcinoma Cells Induced by Flavopiridol¹

Developmental Therapeutics Program (DTP), DTP Clinical Trials Unit, Division of Cancer Treatment and Diagnosis, National Cancer Institute, Bethesda, Maryland 20892 [B. C., T. L. S. S., A. L-P., E. A. S., A. M. S.]; Department of Medicine and Developmental and Molecular Biology, The Albert Einstein Cancer Center, Bronx, New York 10461 [R. P., C. A.]; and Mitotix Inc., Cambridge, Massachusetts 02139 [P. J. W.]

	ABSTRACT

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Flavopiridol is a novel flavonoid that induces cell cycle arrest at different stages of the cell cycle because of the inhibition of cyclin-dependent kinases (cdks). In previous studies from our laboratory, (B. A. Carlson et al., Cancer Res., 56: 2973–2978, 1996), we observed that exposure of the MCF-7 breast carcinoma cell line to flavopiridol resulted in G₁-S arrest, which was associated with the loss of cdk4 and cdk2 activity by 24 h of exposure. Along with this inhibition, flavopiridol decreased total cyclin-D protein levels in this cell line. In this work, we demonstrate that using isoform-specific antibodies, flavopiridol induces an early (by 6 h) decrease in cyclin D1 protein levels. This decline is followed by a decline in cyclin D3 with no effect on cyclin D2 or cyclin E levels by 10 h. Furthermore, at early time points (up to 8 h), the activity of cdk4 and the expression of endogenous phosphorylated retinoblastoma species from intact cells exposed to flavopiridol are unchanged. Thus, the decline in cdk4 activity and the induction of retinoblastoma hypophosphorylation follows cyclin D1 decline. Turnover studies demonstrate that the half-life of cyclin D1 (30 min) is not shortened in flavopiridol-exposed cells, and that the turnover of cdk4-bound cyclin D1 is unaltered. However, steady-state levels of cyclin D1 mRNA display a significant decrease by 4 h of flavopiridol treatment, with total disappearance by 8 h. This mRNA decline is not abrogated by the presence of cycloheximide. Furthermore, we have found that flavopiridol specifically represses the activity of the full-length cyclin D1 promoter linked to a luciferase reporter gene. In summary, we have found that the flavopiridol-induced decline in cyclin D1 is an early event, specific and, at least in part, due to the transcriptional repression of the cyclin D1 promoter. These results extend our understanding of flavopiridol’s action to include regulation of cyclin D1 transcription.

	INTRODUCTION

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Enzymes responsible for the coordinated progression through the cell cycle include the cdks.³ Thus far, nine cdks have been discovered (1, 2, 3) . Cdks 4 and 6 are responsible for progression through G₁ phase. These cdks are active on association with D-type cyclins (cyclin D1, D2, and D3; Ref. 4 ). This association promotes the activation of cdk4 and/or cdk6 "G₁ kinases" by cdk7 (also known as CAK "cdk-activating kinase"). Activated G₁ kinases together with cyclin E/cdk2 phosphorylate the product of the Rb gene, Rb, resulting in the derepression of E2F/DP-dependent transcription and passage through S phase of the cell cycle (5 , 6) . Cyclin D1 has an important role in G₁ phase cell cycle progression. Forced ectopic expression of cyclin D1 causes a reduction in cell size, reduces requirements for growth factors, and shortens the G₁ cell cycle phase (7) . Moreover, microinjection of cyclin D1 antibodies or antisense cyclin D1 plasmids block cells at the early mid-G₁ phase (8) . Cyclin D1 is important for both cell cycle progression and neoplastic transformation: forced ectopic expression of cyclin D1 contributes to the oncogenic transformation of cells in vitro and in vivo (9 , 10) ; these effects have been observed when cyclin D1 is cotransfected with other oncogenes, such as activated Ha-ras and adenovirus E1A (9 , 11, 12, 13) . Overexpression of cyclin D1 in the mammary gland of transgenic mice induces mammary carcinoma (14) . Furthermore, cyclin D1 overexpression regulates DNA repair/DNA replicative synthesis, and overexpression of cyclin D1 is associated with genomic instability, thus increasing the frequency of genetic defects and promoting gene amplification (15 , 16) .

The isolation of the cyclin D1 cDNA was the result of the independent work of many laboratories with different biochemical and genetic strategies (17, 18, 19, 20) . Initially, the PRAD1 (cyclin D1, bcl-1) gene was found rearranged and overexpressed in benign monoclonal parathyroid tumors (19) . Furthermore, overexpression or deregulation of PRAD1/cyclin D1 was observed in mantle cell lymphoma and in head and neck, breast, and esophageal carcinomas, among others (21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32) . Because of the frequent deregulation of cyclin D1 found in human neoplasias and the prominent role that it plays in cell cycle progression, it is believed that compounds that modulate cyclin D1 expression could have a role in the prevention and/or treatment of human neoplasias (33) .

Flavopiridol, also known as L86–8275, is a protein kinase inhibitor that potently inhibits cell cycle progression in many different cell lines (34) . Moreover, flavopiridol induces cell cycle arrest, in part because of its capacity to inhibit most cdks including cdk1, cdk2 and cdk4, cdk7 (35, 36, 37, 38) , and cdk6.⁴ In previous studies, purified cdk4 enzyme activity was potently inhibited by flavopiridol. However, the loss of cdk4 activity observed in intact cells occurred when MCF-7 cells were exposed to flavopiridol for longer than 10 h, a time when total cyclin D levels were already significantly diminished (35) . The mechanism(s) and specificity for this decline have not been defined. This circumstance also leads to the question of whether flavopiridol-induced loss of cdk4 activity in living cells arises from direct inhibition of the cyclin D/cdk4 complex, or indirectly, as a result of decreased cyclin D levels thereby preventing the normal formation of cyclin D/cdk4 (or putative cyclin D/cdk6) complexes.

In this report, we focused on the mechanism by which flavopiridol inhibits cyclin D1 abundance, and we demonstrate that the initial effect of flavopiridol is to inhibit the expression of cyclin D1. This decline in protein expression is rapid (6 h), precedes the loss of cdk 4 activity induced by flavopiridol, and is not associated with increased cyclin D1 turnover. Furthermore, the effects of flavopiridol are associated with an early decline in cyclin D1 mRNA levels despite protein synthesis inhibition, and accompanied by a concomitant specific decline in cyclin D1 promoter activity.

	MATERIALS AND METHODS

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Tissue Culture.
The MCF-7 breast cancer cell line and U2OS human osteosarcoma cell line were obtained from American Type Culture Collection (Rockville, MD). Both cell lines were maintained in RPMI 1640 containing 10% (v/v) heat-inactivated fetal bovine serum, 100 units/ml penicillin G, 100 µg/ml streptomycin, and 2 mM glutamine (complete medium). Flavopiridol was obtained from Drug Synthesis and Chemistry Branch, Developmental Therapeutics Program, National Cancer Institute. All chemicals were from Sigma (St. Louis, MO) unless specified otherwise. Twenty µg of the -1745 CD1luc reporter (39) was cotransfected with 5 µg of pcDNA3, and stable clones were selected with 500 µg/ml genetecin, Life Technologies, Inc. (Gaithersburg, MD). The estrogen-responsive reporter element linked to the TK promoter (40) , subcloned in the vector pA₃ LUC was stably integrated into MCF-7 cells as described above.

Antibodies.
Antisera against cyclin D1 (clone H-295), D2 (clone C-17), and D3 (clone C-16) and against Cyclin E (clone C-19) were purchased from Santa Cruz Laboratories (Santa Cruz, CA). Polyclonal cdk4 antiserum has been described previously(35) . Specific polyclonal antisera that recognized phosphorylated serine 780 and threonine 356 Rb species were based on Taya et al. (41) .⁵

Pulse Labeling.
Exponentially growing MCF-7 cells were incubated with cysteine/methionine-free RPMI with 5% dialyzed serum for 30 min and then labeled with 110 µCi/ml [³⁵S]methionine from NEN (Boston, MA) for an additional 30 min. After this period of incubation, the labeling media were removed and plates were washed twice with PBS and then chased with complete media in the presence of 300 nM flavopiridol or vehicle only for the times described in "Results." At each time point, cells were lysed with buffer containing 10 mM phosphate, 100 mM NaCl, 1 mM Na₃VO₄, 0.5% sodium deoxycholate, 1.0% Triton X-100, 20 µg/ml aprotinin and leupeptin, and 2 mM phenyl methyl sulfonyl fluoride, and the lysates were clarified by centrifugation (15,000 x g for 15 min) before immunoprecipitation with either anti-cyclin D1 or anti-Cdk4 from 400 µg of soluble protein. The immune complexes were pelleted with Protein A-Sepharose from Oncogene Science (Cambridge, MA), washed twice with lysis buffer, electrophoresed in a 4–20% gradient gel (Novex, San Diego, CA), and dried, and radioactive bands were quantitated by PhosphorImager. Parallel samples were transferred to a polyvinylidene fluoride membrane as described below, exposed to film to assess equal protein incorporation of ³⁵S, and immunoblotted with cyclin-D1 antibodies to confirm the specific band as cyclin D1.

Immunoblot Analysis.
Twenty-five µg of protein lysate were fractionated in 4–20% SDS PAGE gradient gels, transferred to polyvinylidene fluoride membranes from Immobilon (Bedford, MA), blocked with 5% nonfat milk/TNE containing 10 mM Tris (pH 7.5), 2.5 mM ethylene diaminetetraacetic acid, and 0.05 M NaCl. Primary antibodies against cyclin D1 and cyclin E from Santa Cruz Laboratories and cdk4 (see above) were incubated for 1 h, and secondary antibody conjugated with horseradish peroxidase (Amersham, Piscataway, NJ) was added for 45 min; ECL chemiluminescence (Amersham) was used for protein detection. X-ray films were quantified using a densitometer from Molecular Dynamics (Sunnyvale, CA).

In Vitro Kinase Assays.
After incubation with vehicle or 300 nM flavopiridol for increasing time periods, MCF-7 were lysed with buffer containing 50 mM HEPES (pH 7.5), 20 mM EDTA, 0.5% NP40, 1 mM p-aminoethylbenze sulfonyl fluoride, 5 µg/ml aprotinin, 5 µg/ml leupeptin, 10 mM ß-glycerophosphate, 0.5 mM NaF, and 0.4 mM Na₃VO₄. The lysate was centrifuged at 15,000 x g for 30 min at 4°C. Four hundred µg of protein were incubated with 2 µl of cdk4 antiserum (35) for 1 h in a shaker at 4°C followed by the addition of 20 µl Gammabind G Sepharose from Pharmacia (Piscataway, NJ) suspension (50%). After a further incubation for 1 h at 4°C, the immunocomplex was centrifugated at 800 x g for 1 min and was washed three times with lysing buffer containing 0.5 M NaCl and once with kinase buffer [50 mM HEPES (pH 7.5), 10 mM MgCl₂, 5 mM MnCl, 1 mM DTT, 10 mM ß-glycerophosphate, 2.5 mM EGTA, 0.5 mM NaF, and 0.4 mM Na₃VO₄]. The kinase reaction was started by the addition of 30 µl of kinase buffer containing 5 µM ATP, 1 µCi of [³²P]ATP, and 1 µg of GST-Rb from Santa Cruz Laboratories. The reaction mixture was incubated for 30 min at 30°C with constant mixing and was stopped by adding 6 µl of 6x SDS sample buffer followed by heating for 5 min at 95°C. After a quick centrifugation, proteins (30 µl) were separated using a 12% SDS gel. The gels were dried and quantified in a Storm PhosphorImager (Molecular Dynamics).

Northern Blot Analysis.
Total cellular RNA was isolated from MCF-7 cells using Trizol from Life Technologies, Inc. Total RNA (15 µg) was resolved by formaldehyde gel electrophoresis (42) and was transferred to Zeta Probe GT Membrane (Bio-Rad Laboratories, Hercules, CA) and cross-linked with Stratalinker from Stratagene (La Jolla, CA), using the protocol provided by the manufacturer. Cyclin D1 and cyclin E probes were kindly provided by Steven Dowdy (Washington University School of Medicine, St. Louis, MO), cyclin D3 plasmid was kindly provided by Michele Pagano (New York University, New York, NY), and GAPDH was purchased from Clontech (Palo Alto, CA). Probes were labeled by random priming using a Random Primers DNA Labeling kit from Life Technologies, Inc. Probes were hybridized overnight at 65°C, washed twice at room temperature with 2x SSC/0.1% SDS, washed once with 0.1x SSC/0.1% SDS at 50°C, and quantified by Phos-phorImager (Molecular Dynamics).

Luciferase Reporter Assays.
MCF-7 cell lines—stably transfected with either the full-length cyclin D1 promoter subcloned in the vector pA₃ LUC "1745CD1LUC" (39) or estrogen-responsive element linked to the TK promoter (40) subcloned in the vector pA₃LUC—were plated overnight into 24-well plates in a medium containing 350 µg/ml G418. After the exposure of cells to 300 nM flavopiridol for increasing time periods, cells were washed with PBS and lysed using a lysis reporter buffer from Promega (Madison, WI). The protein lysate was centrifuged at 13,000 rpm for 10 min, and 20 µl of lysate was added to 100 µl of luciferase substrate (Promega). Arbitrary units of light were detected using a Lumicount luminometer from Packard (Meriden, CT). Total protein concentration was determined as described above. Promoter activity was expressed as arbitrary units of light by µg of total protein.

	RESULTS

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Flavopiridol Depletes Cyclin D1 Followed by Cyclin D3 Protein Levels in MCF7 Cells.
Our prior report demonstrated that exposure of MCF-7 cells to flavopiridol reduced cyclin D protein levels, as detected by a pan D-cyclin antibody, by 6 h (35) . To address the mechanism by which flavopiridol reduced cyclin D protein levels, exponentially growing MCF-7 cells were exposed to flavopiridol for increasing time periods. Cyclin D1, cyclin D3, and cyclin E steady-state protein levels were determined by immunoblotting. As observed in Fig. 1A and quantified in Fig. 1B , there was a notable decrease (50%) in the steady-state levels of cyclin D1 by 6 h, with almost total disappearance by 10 h. Cyclin D3 showed a delayed decline in protein expression, particularly at 8 h. In contrast, cyclin E levels were maintained during the same period (Fig. 1, A and B) . In parallel experiments, cdk4 and cdk2 levels were unaltered (data not shown), which underscored the rapid effect of flavopiridol treatment in the decline of cyclin D1. This decline in cyclin D1 levels was observed in all of the tumor cells lines tested thus far (43) .⁶

View larger version (36K): [in this window] [in a new window]   Fig. 1. Time-exposure analysis of G1 cyclins in the MCF-7 cell line after exposure to flavopiridol. Exponentially growing MCF-7 cells were exposed to 300 nM flavopiridol for the periods indicated in the figure. Protein (25 µg) was fractionated by SDS PAGE. A, immmunoblotting with cyclin D1, cyclin D3, and cyclin E were performed in parallel samples as described in "Materials and Methods." B, densitometric quantitation of each G1 cyclin comparing each time point with vehicle-treated control (100%); (), cyclin D1; (), cyclin D3; (), cyclin E.

View larger version (25K): [in this window] [in a new window]   Fig. 2. Immunoblot analysis of cyclin D1, cyclin D2, and cyclin D3 species in U2OS cell line after flavopiridol treatment. Twenty-five µg of protein extracted from cells exposed to 300 nM flavopiridol were immunoblotted with cyclin D1-, cyclin D2-, or cyclin D3-specific antibodies as described in "Materials and Methods."

Fig. 3A shows that, shortly after the addition of flavopiridol (2, 4, and 6 h), there is an apparent increase in immunoprecipitated cdk4 activity, a phenomenon previously associated in other cdks with flavopiridol-induced loss of the inhibitory cdk tyrosine phosphorylation (35 , 46) . Of significance to the current investigation, up to 6 h after the addition of the drug, at a time when cyclin D levels have begun to decrease, cdk 4 activity is maintained above control levels. However, by 8 h (2 h after the initial reduction in cyclin D1 levels), cdk4 activity begins to decline. Thus, a decrease in cyclin D1 induced by flavopiridol (Fig. 1) precedes the loss of cdk4 activity in MCF7 cells (Fig. 3A) .

View larger version (36K): [in this window] [in a new window]   Fig. 3. Cdk4 activity and endogenous Rb phosphorylation from MCF-7 cells exposed to flavopiridol. A, cdk4 activity from intact cells. Exponentially growing cells were treated with 300 nM flavopiridol as shown in Fig. 1 and immunoprecipitated with cdk4 specific antisera; immune complex assays were performed, and kinase activities were determined and quantified by phosphorimager. The "extra-band" appearing above the GST-Rb band at 4 and 6 h is observed in immunoprecipitates with cdk4 but is not suppressed by immunizing peptide or by the addition of GST-p16INK4A. It is thus a "nonspecific" species which was not considered in the quantitation of GST-Rb kinase activity. B, immunoblots against specific Rb-phosphorylated species. Protein lysates from cells described in A were electrophoresed and immunoblotted with Rb serine 780- and Rb threonine 356-specific antisera.

Cyclin D1 Half-Life Is Not Altered by Flavopiridol.
As part of the normal regulation of cell cycle progression, steady-state levels of cyclins are regulated by transcriptional, translational, and degradation processes in a timely manner (47, 48, 49) . To determine whether flavopiridol induces a decline in the steady-state cyclin D1 protein levels due to increased protein degradation, we determined cyclin D1 protein turnover using pulse-chase experiments in the MCF-7 cell line. MCF-7 cells were pulse-labeled for 30 min with [³⁵S]methionine and then chased in the presence of the unlabeled precursor for various times in the presence or absence of 300 nM flavopiridol. Protein lysates were subjected to immunoprecipitation with either specific cyclin D1 or specific cdk4 antisera and resolved on polyacrylamide gels, and the gels were analyzed in a phosphorimager. Consistent with a previous study (50) , in the vehicle-treated samples (Fig. 4A , upper panel), cyclin D1 protein half-life was 30 min. Cyclin D1 protein half-life from flavopiridol-treated cells showed similar results (half-life, 33 min; Fig. 4A , lower panel), allowing the conclusion that flavopiridol does not alter the turnover of cyclin D1 protein. Total cell lysates taken from the same samples showed equal ³⁵S incorporation in the control and the treated groups (data not shown). In parallel experiments, pulsed ³⁵S-labeled MCF-7 cells were immunoprecipitated with cdk4 antiserum and analyzed by phosphorimager (Fig. 5) . Again, cyclin D1 associated to cdk4 in vehicle-treated (control) or flavopiridol-treated cells showed a similar decline in cyclin D1 associated to cdk4, with the total disappearance of cyclin D1-associated cdk4 species by 120 min, again confirming that flavopiridol does not shorten the half-life of either unbound or cdk4-associated cyclin D1. The position of cyclin D1 in these lysates was confirmed by Western blotting (data not shown). These data, therefore, demonstrate that the effects of flavopiridol on cyclin D1 abundance are not the result of flavopiridol-induced cyclin D1 dissociation from cdk4 complexes.

View larger version (37K): [in this window] [in a new window]   Fig. 4. Cyclin D1 protein turnover of MCF-7 cells after flavopiridol exposure. A, exponentially growing cells were pulsed with [35S]methionine for 30 min and chased with complete media in the presence (lower panel) or absence (vehicle-treated, upper panel) of 300 nM flavopiridol. Lysates were immunoprecipitated with cyclin D1 monoclonal antibody (Santa Cruz Laboratories), fractionated in a 4–20% gradient gel, dried, and exposed to PhosphorImager. B, quantitation of cyclin D1 protein levels over time.

View larger version (70K): [in this window] [in a new window]   Fig. 5. Cyclin D1 associated with cdk4 turnover of MCF-7 cells after flavopiridol exposure. Exponentially growing cells were pulsed with [35S] methionine for 30 min and chased with complete media in the presence (lower panel) or absence (vehicle-treated, upper panel) of 300 nM flavopiridol. Lysates were immunoprecipitated with cdk4 polyclonal-specific antisera (see "Materials and Methods"), fractionated in a 4–20% gradient gel, dried, and exposed to a phosphorimager.

View larger version (45K): [in this window] [in a new window]   Fig. 6. Cyclin D1 mRNA steady-state levels in MCF-7 cells treated with flavopiridol. Exponentially growing cells were exposed to either vehicle or 300 nM flavopiridol for the times indicated in the figure. Fifteen µg of total RNA were resolved by formaldehyde gel electrophoresis, transferred to a nylon membrane, and hybridized with cyclin D1, cyclin D3, cyclin E, and GAPDH. C, vehicle (control); F, 300 nM flavopiridol.

View larger version (12K): [in this window] [in a new window]   Fig. 7. Cyclin D1 promoter activity from stably transfected MCF-7–1745 CD1 luciferase cells. Exponentially growing stable MCF-7 cells, stably transfected with -1745 CD1 pA3LUC reporter construct, were exposed to 300 nM flavopiridol for the times indicated in the figure. The arbitrary units of light measured from duplicate samples were normalized per µg of total protein and were plotted relative to vehicle control sample (100% activity). This experiment is the representative of four independent experiments.

	DISCUSSION

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Cyclin D1 (also known as bcl-1, PRAD1) is a proto-oncogene that is overexpressed and/or amplified in numerous neoplasias (21 , 24 , 30 , 51, 52, 53) . Ectopic expression of cyclin D1 and microinjection studies with neutralizing cyclin D1 antibodies and/or cyclin D1 antisense constructs support the notion that cyclin D1 is an important determinant in the progression through the G₁ phase of the cell cycle (8 , 54) . Moreover, when oncogenes such as activated Ha-ras or adenovirus E1A are cotransfected with cyclin D1 into human fibroblasts, the development of transformed clones is evident, underscoring the role of this cyclin in malignant transformation (9 , 12) . When cyclin D1 associates with either cdk4 or cdk6 in early G₁ phase, the complex is phosphorylated by cdk7 with the consequent activation of the holoenzyme (55) . Cyclin D2 and cyclin D3 also associate with cdk4 or cdk6 and can phosphorylate the Rb protein, thereby promoting entry into S-phase (55) . Although several laboratories have demonstrated that the D-type cyclins could have redundant cell cycle functions, the tissue expression and regulation of these cyclins are very different: cyclin D1 is more prevalent in epithelial tissues; in contrast, cyclins D2 and D3 are more prevalent in hematopoietic tissues (56) . In the case of tumor cells that lack Rb, D-type cyclins are probably dispensable (57 , 58) . Nevertheless, the possibility of specific down-regulation of cyclin D1 in tumor types that depend on this oncogene for proliferation is an interesting strategy for antitumor therapy (33) .

The expression of cyclin D1 in normal cells varies with the phase of the cell cycle. A peak induction of cyclin D1 in early G₁ of normal cells is followed by a decline during S and G₂-M Phases. This periodicity is lost in several tumor cell lines (59 , 60) . The expression of most cyclins (i.e., cyclin B1, A, E) are regulated by degradation processes (61) . In contrast, the expression of cyclin D1 is regulated by transcriptional induction and by posttranscriptional and translational mechanisms (48 , 62 , 63) . It is clear that the multiple signaling pathways that are necessary to induce cells to proliferate and to progress to S phase are important in the regulation of cyclin D1 expression. Several laboratories demonstrated that mitogen-activated protein kinase family members such as extracellular signal-regulated kinase and Jun-N kinases are involved in the transcriptional regulation of cyclin D1 (39 , 64, 65, 66) . Moreover, cyclin D1 is directly activated by oncogenic ras (11 , 39 , 67) and by src kinase in MCF-7 cells (68) . Serum, growth factors, and cytokines also induce the cyclin D1 gene directly (68 , 69) . Ectopic expression of E2F1 inhibited the cyclin D1 protein by transcriptional mechanisms (70) , thus providing a "negative" feedback for cells already in S phase. Cyclin D1 abundance could be also regulated by posttranscriptional and posttranslational mechanisms (49) . In some systems, overexpression of cyclin D1 failed to increase cyclin D1 protein levels despite a proportional increase in cyclin D1 mRNA (49) . Muise-Helmericks et al. demonstrated that, in MCF-7 cells, posttranscriptional mechanisms regulated by the proto-oncogene AKT and the mammalian homologue of TOR are important for regulating cyclin D1 production (71) . Ras-, PI3K-, and AKT-dependent pathways activate GSK3ß, causing phosphorylation of threonine 286 on the COOH terminus of cyclin D1, which leads to its degradation (63) . Experiments evaluating the capacity of flavopiridol to alter posttranscriptional or translational aspects of cyclin D1 regulation are being undertaken. The current experiments raise the question of how flavopiridol selectively alters the activation state of the cyclin D1 promoter.

Flavopiridol has the capacity to inhibit all of the cdks thus far tested in a competitive manner with ATP (35, 36, 37 , 46) . In our previous report, we demonstrated that cdk2 and cdk4 kinase activity can be potently and directly inhibited in vitro (100 nM) but the loss of cdk4 activity with G₁ arrest in MCF-7 cells, with Rb hypophosphorylation, was evident between 12 and 24 h (35) . Furthermore, at earlier time points, cyclin D protein levels diminished (35) . In this report, we determine that this early decline in cyclin D levels, observed previously, is mainly the initial decline in cyclin D1 followed by cyclin D3 levels. Moreover, the remaining G₁ cyclins, cyclin D2 and cyclin E, were not altered at the time points tested. Experiments from our group have also demonstrated that head and neck tumor cell lines exposed to flavopiridol show apoptosis and cyclin D1 depletion (43) . Although other antitumor agents, such as bleomycin and - irradiation, induced apoptosis, neither agent induced cyclin D1 depletion, underscoring the specificity of flavopiridol’s effect on down-regulating cyclin D1 (43) . To determine the mechanism by which flavopiridol reduced cyclin D1 levels, we first asked whether cyclin D1 could be depleted as a result of the loss of cdk4 activity by flavopiridol. Clearly, as shown in Fig. 1 , cyclin D1 depletion occurs at least 2 h before the decline in cdk4 activity (8–10 h; Fig. 3 ). This result, indeed, demonstrates clearly that the decreased cdk4 activity caused by flavopiridol with the concomitant loss of Rb dephosphorylation is at least in part due to the decline in cyclin D1 levels.

We then studied cyclin D1 protein turnover and demonstrated that cyclin D1 half-life in the presence of flavopiridol (33 min) was not shortened, thus excluding altered turnover as responsible for the decline in the steady-state protein levels. Similar results were obtained with respect to the half-life of cyclin D1 bound to cdk4, demonstrating that the decreased total cyclin D1 is not caused by selective increase of cdk4-cyclin D1 dissociation. To assess the possible effects of flavopiridol on cyclin D1 transcription, Northern blots from MCF-7 cell lines exposed to flavopiridol demonstrated a significant decline in steady-state cyclin D1 mRNA by 4 h, with total disappearance by 10 h. This depletion was not inhibited by the presence of cycloheximide. Furthermore, studies with the full-length cyclin D1 promoter in contiguity to a luciferase reporter gene revealed a specific decrease in cyclin D1 promoter activity, at times similar to those when cyclin D1 protein and mRNA diminished. These results indicate that the decrease in cyclin D1 mRNA can be attributed to diminished cyclin D1 promoter activity. Additional studies with cyclin D1 promoter deletions are being undertaken to investigate the nature of the molecular signaling pathways abrogated by flavopiridol, responsible for cyclin D1 decline.

Recent studies have shown that cdk9 and cdk8 participate in transcriptional activation control mechanisms of, for example, HIV transcription and RNA polymerase activity, respectively (72 , 73) . Thus, if flavopiridol inhibits an RNA-polymerase or a transcription factor-related cdk—important for cyclin D1 promoter activity—the present results could be understood from the known kinase specificity of flavopiridol. On the other hand, the basis for this effect may reflect the action of flavopiridol on some other targets that may govern cyclin D1 promoter activity.

In vitro experiments using a variety of tumor cell lines have demonstrated that unrelated compounds with antiproliferative effects provoke altered expression of several cell cycle-related proteins. One example is the nonspecific protein kinase inhibitor staurosporine, which down-regulates cyclin D1 in MCF-7 cells (74) . Another example are the antiestrogens, such as tamoxifen, that have been able to inhibit the expression of cyclin D and other cell cycle-related proteins in breast carcinoma cell lines with a decline in cdk activity (75) . Retinoids, such as trans-retinoic acid and 9-cis- retinoic acid, showed a decrease in multiple cell cycle parameters including cyclin D1, cyclin D3, cdk2, and cdk4 when breast carcinoma cell lines were exposed for 72 h (76) . Furthermore, rapamycin, an inhibitor of the FK506-binding-protein/mammalian-homologue-of-TOR pathways, was also associated in some models systems with a decline in cyclin D1 protein by a translational mechanism (77) . Although these compounds may down-regulate cyclin D along with perturbation in other cell cycle-related proteins, it is unclear whether this cyclin D1 depletion reflects a direct effect on the regulatory pathways necessary for cyclin D1 production or is the consequence of G₁ arrest and/or Rb dephosphorylation observed with these compounds. In the case of flavopiridol, cyclin D1 depletion precedes the cdk4 inhibition, Rb hypophosphorylation, and G₁-S arrest induced by this agent.

Cyclin D1 depletion by flavopiridol is followed by a decline in cyclin D3, which is associated with a decline in cyclin D3 mRNA. Although much less is known about cyclin D3 regulation, it is clear that cyclin D3 gene expression and promoter regulation are quite different from the other D-type cyclins (78 , 79) . The expression of cyclin D3 is prevalent in hematopoietic tissues. Although some investigators have suggested that the D-type cyclins have a redundant function, others have pointed out that each D-type cyclin has specific roles in different biological processes (80, 81, 82, 83, 84) . On the basis of the possible nonredundant effects of each D-type cyclin on biological processes beyond cell cycle regulation, it is likely that flavopiridol may be able to affect pathways that normally require cyclin D1 and cyclin D3. Further work in the regulation of cyclin D1 and D3 by flavopiridol is being undertaken. Interestingly, in contrast to cyclin D1 and cyclin D3, cyclin D2 is not affected by flavopiridol.

Although the data presented here strongly suggest that flavopiridol exerts its regulatory action on cyclin D1 expression at the level of transcription, we cannot exclude a possible additional posttranscriptional or translational regulation by flavopiridol. Regardless of the underlying mechanism(s), the present study provides the first evidence that flavopiridol induces an early decline in cyclin D1 protein before a decrease of cdk4 activity. This early decline was followed by a decline in cyclin D3 but was not followed by cyclin D2 or cyclin E down-regulation. The decline in cyclin D1 protein expression is not due to the loss of cdk4 activity or Rb phosphorylation but the loss of cdk4 activity could result from down-regulation of cyclin D1 mRNA with a decline in cyclin D1 promoter activity. This novel cell cycle regulatory aspect of flavopiridol may have clinical significance because many human neoplasias exhibit cyclin D1 deregulation. How this effect may be related to the effect of flavopiridol on the cdk family of cell cycle and transcriptional regulators or whether the consideration of additional target molecules is necessary is of present interest to explore.

	ACKNOWLEDGMENTS

 
We thank Steven Dowdy and Michele Pagano for the kind gifts of cyclin D1/cyclin E and cyclin D3 probes, respectively; Eduardo Sainz for expert technical assistance; and Silvio Gutkind for advice and for critically reviewing the manuscript.

	FOOTNOTES

 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ This work was supported in part by R29CA70897 and RO1CA75503 from NIH (to R. G. P.). R. G. P. is a recipient of the Ira T. Hirschl award and an award from the Susan G. Komen Breast Cancer Foundation. Work conducted at the Albert Einstein College of Medicine was supported by Cancer Center Core NIH Grant 5-P30-CA13330--26.

² To whom requests for reprints should be addressed, at DTP Clinical Trials Unit, National Cancer Institute, 10 Center Drive, Building 10, Room 6N113, Bethesda, MD 20892. Phone: (301) 496-4119; Fax: (301) 480-7456; E-mail: sendero{at}helix.nih.gov

³ The abbreviations used are: cdk, cyclin-dependent kinase; TOR, target of rapamycin; Rb, retinoblastoma; TK, thymidine kinase; GST, glutathione S-transferase; GAPDH, glyceraldehye-3-phosphate-dehydrogenase.

⁴ S. Singh, A. Logiza-Perez, T. Lahusen, J. Trepel, C. Albanese, R. Pestell, E. A. Sausville, and A. M. Senderowicz. G₁ arrest induced by flavopiridol is mediated by downregulation of cyclin D1/cdk6 and is rescued by cyclin E/cdk2, manuscript in preparation.

⁵ T. Lahusen, L. Guedez, A. Loaiza-Perez, E. A. Sausville, and A. M. Senderowicz. Novel reporters for in vivo cdk activity: development of phospho-specific Rb antibodies, manuscript in preparation.

⁶ T. Lahusen, L. Guedez, E. A. Sausville, and A. M. Senderowicz, unpublished results.

Received 4/22/99. Accepted 7/16/99.

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