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Experimental Therapeutics |
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.]
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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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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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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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.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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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)
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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.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, 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)
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, there was a rapid decline in cyclin D1 levels by 6 h, as already demonstrated in Fig. 1
for the MCF-7 cell line, followed by a decline in cyclin D3, as observed in Fig. 1
. However, cyclin D2 was unaltered during these time periods. Taken together with the results from Fig. 1
, we see that flavopiridol selectively depletes cyclin D1 and then cyclin D3 without altering the levels of cyclin D2 or cyclin E.
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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)
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. Again, minimal changes (up to 8 h) were observed in the phosphorylated Rb at serine 780 (a site that is specific for cdk4 phosphorylation) or at threonine 356 [a site that is specific for cdk2 phosphorylation (41)
] or in total Rb (not shown). These results are consistent with our previous report in which Rb phosphorylation declined by 12 h after treatment with flavopiridol (35)
. We can conclude, therefore, that the specific decline in cyclin D1 levels does not result from the loss of cdk4 kinase activity. Instead, cyclin D1 depletion precedes, and most probably leads to, the loss of cdk4 activity.
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.
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. A significant decline in cyclin D1 mRNA levels was observed by 4 h of exposure to flavopiridol with total disappearance by 8 h. To test whether this decline in cyclin D1 mRNA was dependent on protein synthesis, MCF-7 cells were coincubated with flavopiridol and cycloheximide (10 µg/ml) for 6 h, a time period that is associated with an 50% decline in cyclin D1 protein levels (Fig. 1)
and in mRNA levels (Fig. 6)
. No changes in the cyclin D1 depletion were observed in cycloheximide-treated cells, which demonstrated that the decline is not dependent on new protein synthesis (data not shown). The same blot was stripped and hybridized with either cyclin D3 or cyclin E probes. A similar decline in cyclin D3 mRNA species was observed. However, no decline in cyclin E mRNA was observed, consistent with the results observed for cyclin E protein levels (Fig. 1)
; GAPDH hybridization showed equal loading of the gel (Fig. 6)
. Therefore, the decrease in the steady-state cyclin D1 levels occurs at the mRNA level.
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) at 6 h after the addition of the drug, a time point that corresponds to the down-regulation of cyclin D1 mRNA and protein levels. In contrast, an MCF-7 cell line, stably transfected with an estrogen-responsive reporter element that was linked to the TK promoter and the luciferase reporter, showed an increase by 6% at 6 h (data not shown), underscoring the specific inhibitory activity of flavopiridol on the cyclin D1 promoter. Taken together, these results indicates that flavopiridol depletes cyclin D1 with a prominent decrease in cyclin D1 mRNA levels. This decline is not abrogated by cycloheximide and is associated with a decrease in cyclin D1 promoter activity.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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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.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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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L. A. Gomez, A. de las Pozas, and C. Perez-Stable Sequential combination of flavopiridol and docetaxel reduces the levels of X-linked inhibitor of apoptosis and AKT proteins and stimulates apoptosis in human LNCaP prostate cancer cells Mol. Cancer Ther., May 1, 2006; 5(5): 1216 - 1226. [Abstract] [Full Text] [PDF]
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 M. Sengottuvelan, P. Viswanathan, and N. Nalini Chemopreventive effect of trans-resveratrol - a phytoalexin against colonic aberrant crypt foci and cell proliferation in 1,2-dimethylhydrazine induced colon carcinogenesis Carcinogenesis, May 1, 2006; 27(5): 1038 - 1046. [Abstract] [Full Text] [PDF]
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 G. I. Shapiro Cyclin-Dependent Kinase Pathways As Targets for Cancer Treatment J. Clin. Oncol., April 10, 2006; 24(11): 1770 - 1783. [Abstract] [Full Text] [PDF]
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Y. K. Lee, C. R. Isham, S. H. Kaufman, and K. C. Bible Flavopiridol disrupts STAT3/DNA interactions, attenuates STAT3-directed transcription, and combines with the Jak kinase inhibitor AG490 to achieve cytotoxic synergy Mol. Cancer Ther., January 1, 2006; 5(1): 138 - 148. [Abstract] [Full Text] [PDF]
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J. A. Whitlock, M. Krailo, J. M. Reid, S. L. Ruben, M. M. Ames, W. Owen, and G. Reaman Phase I Clinical and Pharmacokinetic Study of Flavopiridol in Children With Refractory Solid Tumors: A Children's Oncology Group Study J. Clin. Oncol., December 20, 2005; 23(36): 9179 - 9186. [Abstract] [Full Text] [PDF]
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G. K. Schwartz and M. A. Shah Targeting the Cell Cycle: A New Approach to Cancer Therapy J. Clin. Oncol., December 20, 2005; 23(36): 9408 - 9421. [Abstract] [Full Text] [PDF]
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J. E. Karp, A. Passaniti, I. Gojo, S. Kaufmann, K. Bible, T. S. Garimella, J. Greer, J. Briel, B. D. Smith, S. D. Gore, et al. Phase I and Pharmacokinetic Study of Flavopiridol followed by 1-{beta}-D-Arabinofuranosylcytosine and Mitoxantrone in Relapsed and Refractory Adult Acute Leukemias Clin. Cancer Res., December 1, 2005; 11(23): 8403 - 8412. [Abstract] [Full Text] [PDF]
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G. K. Schwartz Development of Cell Cycle Active Drugs for the Treatment of Gastrointestinal Cancers: A New Approach to Cancer Therapy J. Clin. Oncol., July 10, 2005; 23(20): 4499 - 4508. [Abstract] [Full Text] [PDF]
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 K. Lacrima, A. Valentini, C. Lambertini, M. Taborelli, A. Rinaldi, E. Zucca, C. Catapano, F. Cavalli, A. Gianella-Borradori, D. E. MacCallum, et al. In vitro activity of cyclin-dependent kinase inhibitor CYC202 (Seliciclib, R-roscovitine) in mantle cell lymphomas Ann. Onc., July 1, 2005; 16(7): 1169 - 1176. [Abstract] [Full Text] [PDF]
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J. R. Brown Chronic Lymphocytic Leukemia: A Niche for Flavopiridol? Clin. Cancer Res., June 1, 2005; 11(11): 3971 - 3973. [Full Text] [PDF]
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M. F. Aguero, M. M. Facchinetti, Z. Sheleg, and A. M. Senderowicz Phenoxodiol, a Novel Isoflavone, Induces G1 Arrest by Specific Loss in Cyclin-Dependent Kinase 2 Activity by p53-Independent Induction of p21WAF1/CIP1 Cancer Res., April 15, 2005; 65(8): 3364 - 3373. [Abstract] [Full Text] [PDF]
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 J.-W. Huang, C.-W. Shiau, Y.-T. Yang, S. K. Kulp, K.-F. Chen, R. W. Brueggemeier, C. L. Shapiro, and C.-S. Chen Peroxisome Proliferator-Activated Receptor {gamma}-Independent Ablation of Cyclin D1 by Thiazolidinediones and Their Derivatives in Breast Cancer Cells Mol. Pharmacol., April 1, 2005; 67(4): 1342 - 1348. [Abstract] [Full Text] [PDF]
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 A. Skildum, E. Faivre, and C. A. Lange Progesterone Receptors Induce Cell Cycle Progression via Activation of Mitogen-Activated Protein Kinases Mol. Endocrinol., February 1, 2005; 19(2): 327 - 339. [Abstract] [Full Text] [PDF]
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X.-Y. Pei, Y. Dai, and S. Grant The small-molecule Bcl-2 inhibitor HA14-1 interacts synergistically with flavopiridol to induce mitochondrial injury and apoptosis in human myeloma cells through a free radical-dependent and Jun NH2-terminal kinase-dependent mechanism Mol. Cancer Ther., December 1, 2004; 3(12): 1513 - 1524. [Abstract] [Full Text] [PDF]
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J. Dupont, B. Bienvenu, C. Aghajanian, S. Pezzulli, P. Sabbatini, P. Vongphrachanh, C. Chang, C. Perkell, K. Ng, S. Passe, et al. Phase I and Pharmacokinetic Study of the Novel Oral Cell-Cycle Inhibitor Ro 31-7453 in Patients With Advanced Solid Tumors J. Clin. Oncol., August 15, 2004; 22(16): 3366 - 3374. [Abstract] [Full Text] [PDF]
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A. R. Tan, X. Yang, A. Berman, S. Zhai, A. Sparreboom, A. L. Parr, C. Chow, J. S. Brahim, S. M. Steinberg, W. D. Figg, et al. Phase I Trial of the Cyclin-Dependent Kinase Inhibitor Flavopiridol in Combination with Docetaxel in Patients with Metastatic Breast Cancer Clin. Cancer Res., August 1, 2004; 10(15): 5038 - 5047. [Abstract] [Full Text] [PDF]
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X. Lu, W. E. Burgan, M. A. Cerra, E. Y. Chuang, M.-H. Tsai, P. J. Tofilon, and K. Camphausen Transcriptional signature of flavopiridol-induced tumor cell death Mol. Cancer Ther., July 1, 2004; 3(7): 861 - 872. [Abstract] [Full Text] [PDF]
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S. Grant and P. Dent Gene profiling and the cyclin-dependent kinase inhibitor flavopiridol: What's in a name? Mol. Cancer Ther., July 1, 2004; 3(7): 873 - 875. [Full Text] [PDF]
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M. D'Amico, K. Wu, M. Fu, M. Rao, C. Albanese, R. G. Russell, H. Lian, D. Bregman, M. A. White, and R. G. Pestell The Inhibitor of Cyclin-Dependent Kinase 4a/Alternative Reading Frame (INK4a/ARF) Locus Encoded Proteins p16INK4a and p19ARF Repress Cyclin D1 Transcription through Distinct cis Elements Cancer Res., June 15, 2004; 64(12): 4122 - 4130. [Abstract] [Full Text] [PDF]
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G. I. Shapiro Preclinical and Clinical Development of the Cyclin-Dependent Kinase Inhibitor Flavopiridol Clin. Cancer Res., June 15, 2004; 10(12): 4270S - 4275S. [Abstract] [Full Text] [PDF]
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M. M. Facchinetti, A. De Siervi, D. Toskos, and A. M. Senderowicz UCN-01-Induced Cell Cycle Arrest Requires the Transcriptional Induction of p21waf1/cip1 by Activation of Mitogen-Activated Protein/Extracellular Signal-Regulated Kinase Kinase/Extracellular Signal-Regulated Kinase Pathway Cancer Res., May 15, 2004; 64(10): 3629 - 3637. [Abstract] [Full Text] [PDF]
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Z. N. Demidenko and M. V. Blagosklonny Flavopiridol Induces p53 via Initial Inhibition of Mdm2 and p21 and, Independently of p53, Sensitizes Apoptosis-Reluctant Cells to Tumor Necrosis Factor Cancer Res., May 15, 2004; 64(10): 3653 - 3660. [Abstract] [Full Text] [PDF]
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S. C. Maggio, R. R. Rosato, L. B. Kramer, Y. Dai, M. Rahmani, D. S. Paik, A. C. Czarnik, S. G. Payne, S. Spiegel, and S. Grant The Histone Deacetylase Inhibitor MS-275 Interacts Synergistically with Fludarabine to Induce Apoptosis in Human Leukemia Cells Cancer Res., April 1, 2004; 64(7): 2590 - 2600. [Abstract] [Full Text] [PDF]
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K. Camphausen, K. J. Brady, W. E. Burgan, M. A. Cerra, J. S. Russell, E. E.A. Bull, and P. J. Tofilon Flavopiridol enhances human tumor cell radiosensitivity and prolongs expression of {gamma}H2AX foci Mol. Cancer Ther., April 1, 2004; 3(4): 409 - 416. [Abstract] [Full Text] [PDF]
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R. R. Rosato, J. A. Almenara, C. Yu, and S. Grant Evidence of a Functional Role for p21WAF1/CIP1 Down-Regulation in Synergistic Antileukemic Interactions between the Histone Deacetylase Inhibitor Sodium Butyrate and Flavopiridol Mol. Pharmacol., March 1, 2004; 65(3): 571 - 581. [Abstract] [Full Text] [PDF]
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 Y. Takada and B. B. Aggarwal Flavopiridol Inhibits NF-{kappa}B Activation Induced by Various Carcinogens and Inflammatory Agents through Inhibition of I{kappa}B{alpha} Kinase and p65 Phosphorylation: ABROGATION OF CYCLIN D1, CYCLOOXYGENASE-2, AND MATRIX METALLOPROTEASE-9 J. Biol. Chem., February 6, 2004; 279(6): 4750 - 4759. [Abstract] [Full Text] [PDF]
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A. De Siervi, M. Marinissen, J. Diggs, X.-F. Wang, G. Pages, and A. Senderowicz Transcriptional Activation of p21waf1/cip1 by Alkylphospholipids: Role of the Mitogen-Activated Protein Kinase Pathway in the Transactivation of the Human p21waf1/cip1 Promoter by Sp1 Cancer Res., January 15, 2004; 64(2): 743 - 750. [Abstract] [Full Text] [PDF]
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U. Raju, E. Nakata, K. A. Mason, K. K. Ang, and L. Milas Flavopiridol, a Cyclin-dependent Kinase Inhibitor, Enhances Radiosensitivity of Ovarian Carcinoma Cells Cancer Res., June 15, 2003; 63(12): 3263 - 3267. [Abstract] [Full Text] [PDF]
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C. Yu, M. Rahmani, Y. Dai, D. Conrad, G. Krystal, P. Dent, and S. Grant The Lethal Effects of Pharmacological Cyclin-dependent Kinase Inhibitors in Human Leukemia Cells Proceed through a Phosphatidylinositol 3-Kinase/Akt-dependent Process Cancer Res., April 15, 2003; 63(8): 1822 - 1833. [Abstract] [Full Text] [PDF]
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C. Qin, R. Burghardt, R. Smith, M. Wormke, J. Stewart, and S. Safe Peroxisome Proliferator-activated Receptor {gamma} Agonists Induce Proteasome-dependent Degradation of Cyclin D1 and Estrogen Receptor {alpha} in MCF-7 Breast Cancer Cells Cancer Res., March 1, 2003; 63(5): 958 - 964. [Abstract] [Full Text] [PDF]
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D.-M. Kim, S.-Y. Koo, K. Jeon, M. H. Kim, J. Lee, C. Y. Hong, and S. Jeong Rapid Induction of Apoptosis by Combination of Flavopiridol and Tumor Necrosis Factor (TNF)-{alpha} or TNF-related Apoptosis-inducing Ligand in Human Cancer Cell Lines Cancer Res., February 1, 2003; 63(3): 621 - 626. [Abstract] [Full Text] [PDF]
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R. A. Messmann, C. D. Ullmann, T. Lahusen, A. Kalehua, J. Wasfy, G. Melillo, I. Ding, D. Headlee, W. D. Figg, E. A. Sausville, et al. Flavopiridol-related Proinflammatory Syndrome Is Associated with Induction of Interleukin-6 Clin. Cancer Res., February 1, 2003; 9(2): 562 - 570. [Abstract] [Full Text] [PDF]
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M. Alonso, C. Tamasdan, D. C. Miller, and E. W. Newcomb Flavopiridol Induces Apoptosis in Glioma Cell Lines Independent of Retinoblastoma and p53 Tumor Suppressor Pathway Alterations by a Caspase-independent Pathway Mol. Cancer Ther., February 1, 2003; 2(2): 139 - 150. [Abstract] [Full Text] [PDF]
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S. Wittmann, P. Bali, S. Donapaty, R. Nimmanapalli, F. Guo, H. Yamaguchi, M. Huang, R. Jove, H. G. Wang, and K. Bhalla Flavopiridol Down-Regulates Antiapoptotic Proteins and Sensitizes Human Breast Cancer Cells to Epothilone B-induced Apoptosis Cancer Res., January 1, 2003; 63(1): 93 - 99. [Abstract] [Full Text] [PDF]
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Y. Ma, W. D. Cress, and E. B. Haura Flavopiridol-induced Apoptosis Is Mediated through Up-Regulation of E2F1 and Repression of Mcl-1 Mol. Cancer Ther., January 1, 2003; 2(1): 73 - 81. [Abstract] [Full Text] [PDF]
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L. Cartee, S. C. Maggio, R. Smith, H. M. Sankala, P. Dent, and S. Grant Protein Kinase C-dependent Activation of the Tumor Necrosis Factor Receptor-mediated Extrinsic Cell Death Pathway Underlies Enhanced Apoptosis in Human Myeloid Leukemia Cells Exposed to Bryostatin 1 and Flavopiridol Mol. Cancer Ther., January 1, 2003; 2(1): 83 - 93. [Abstract] [Full Text] [PDF]
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J. E. Karp, D. D. Ross, W. Yang, M. L. Tidwell, Y. Wei, J. Greer, D. L. Mann, T. Nakanishi, J. J. Wright, and A. D. Colevas Timed Sequential Therapy of Acute Leukemia with Flavopiridol: In Vitro Model for a Phase I Clinical Trial Clin. Cancer Res., January 1, 2003; 9(1): 307 - 315. [Abstract] [Full Text] [PDF]
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R. S. DiPaola To Arrest or Not To G2-M Cell-Cycle Arrest : Commentary re: A. K. Tyagi et al., Silibinin Strongly Synergizes Human Prostate Carcinoma DU145 Cells to Doxorubicin-induced Growth Inhibition, G2-M Arrest, and Apoptosis. Clin. Cancer Res., 8: 3512-3519, 2002. Clin. Cancer Res., November 1, 2002; 8(11): 3311 - 3314. [Full Text] [PDF]
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I. Gojo, B. Zhang, and R. G. Fenton The Cyclin-dependent Kinase Inhibitor Flavopiridol Induces Apoptosis in Multiple Myeloma Cells through Transcriptional Repression and Down-Regulation of Mcl-1 Clin. Cancer Res., November 1, 2002; 8(11): 3527 - 3538. [Abstract] [Full Text] [PDF]
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V. Patel, T. Lahusen, C. Leethanakul, T. Igishi, M. Kremer, L. Quintanilla-Martinez, J. F. Ensley, E. A. Sausville, J. S. Gutkind, and A. M. Senderowicz Antitumor Activity of UCN-01 in Carcinomas of the Head and Neck Is Associated with Altered Expression of Cyclin D3 and p27KIP1 Clin. Cancer Res., November 1, 2002; 8(11): 3549 - 3560. [Abstract] [Full Text] [PDF]
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A. R. Tan, D. Headlee, R. Messmann, E. A. Sausville, S. G. Arbuck, A. J. Murgo, G. Melillo, S. Zhai, W. D. Figg, S. M. Swain, et al. Phase I Clinical and Pharmacokinetic Study of Flavopiridol Administered as a Daily 1-Hour Infusion in Patients With Advanced Neoplasms J. Clin. Oncol., October 1, 2002; 20(19): 4074 - 4082. [Abstract] [Full Text] [PDF]
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 A. M. Senderowicz The Cell Cycle as a Target for Cancer Therapy: Basic and Clinical Findings with the Small Molecule Inhibitors Flavopiridol and UCN-01 Oncologist, August 1, 2002; 7(90003): 12 - 19. [Abstract] [Full Text] [PDF]
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K. Wu, C. Wang, M. D'Amico, R. J. Lee, C. Albanese, R. G. Pestell, and S. Mani Flavopiridol and Trastuzumab Synergistically Inhibit Proliferation of Breast Cancer Cells: Association with Selective Cooperative Inhibition of Cyclin D1-dependent Kinase and Akt Signaling Pathways Mol. Cancer Ther., July 1, 2002; 1(9): 695 - 706. [Abstract] [Full Text] [PDF]
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L. Cartee, R. Smith, Y. Dai, M. Rahmani, R. Rosato, J. Almenara, P. Dent, and S. Grant Synergistic Induction of Apoptosis in Human Myeloid Leukemia Cells by Phorbol 12-Myristate 13-Acetate and Flavopiridol Proceeds via Activation of Both the Intrinsic and Tumor Necrosis Factor-Mediated Extrinsic Cell Death Pathways Mol. Pharmacol., June 1, 2002; 61(6): 1313 - 1321. [Abstract] [Full Text] [PDF]
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G. K. Schwartz, E. O'Reilly, D. Ilson, L. Saltz, S. Sharma, W. Tong, P. Maslak, M. Stoltz, L. Eden, P. Perkins, et al. Phase I Study of the Cyclin-Dependent Kinase Inhibitor Flavopiridol in Combination With Paclitaxel in Patients With Advanced Solid Tumors J. Clin. Oncol., April 15, 2002; 20(8): 2157 - 2170. [Abstract] [Full Text] [PDF]
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R. Nahta, J. D. Iglehart, B. Kempkes, and E. V. Schmidt Rate-limiting Effects of Cyclin D1 in Transformation by ErbB2 Predicts Synergy between Herceptin and Flavopiridol Cancer Res., April 1, 2002; 62(8): 2267 - 2271. [Abstract] [Full Text] [PDF]
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V. Patel, T. Lahusen, T. Sy, E. A. Sausville, J. S. Gutkind, and A. M. Senderowicz Perifosine, a Novel Alkylphospholipid, Induces p21WAF1 Expression in Squamous Carcinoma Cells through a p53-independent Pathway, Leading to Loss in Cyclin-dependent Kinase Activity and Cell Cycle Arrest Cancer Res., March 1, 2002; 62(5): 1401 - 1409. [Abstract] [Full Text] [PDF]
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C. B. Matranga and G. I. Shapiro Selective Sensitization of Transformed Cells to Flavopiridol-induced Apoptosis following Recruitment to S-Phase Cancer Res., March 1, 2002; 62(6): 1707 - 1717. [Abstract] [Full Text] [PDF]
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R. R. Rosato, J. A. Almenara, L. Cartee, V. Betts, S. P. Chellappan, and S. Grant The Cyclin-dependent Kinase Inhibitor Flavopiridol Disrupts Sodium Butyrate-induced p21WAF1/CIP1 Expression and Maturation while Reciprocally Potentiating Apoptosis in Human Leukemia Cells Mol. Cancer Ther., February 1, 2002; 1(4): 253 - 266. [Abstract] [Full Text] [PDF]
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Y. A. Elsayed and E. A. Sausville Selected Novel Anticancer Treatments Targeting Cell Signaling Proteins Oncologist, December 1, 2001; 6(6): 517 - 537. [Abstract] [Full Text] [PDF]
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S. Lin, H.-C. Huang, L.-L. Chen, C.-C. Lee, and T.-S. Huang GL331 Induces Down-Regulation of Cyclin D1 Expression via Enhanced Proteolysis and Repressed Transcription Mol. Pharmacol., October 1, 2001; 60(4): 768 - 775. [Abstract] [Full Text] [PDF]
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M. E. Lane, B. Yu, A. Rice, K. E. Lipson, C. Liang, L. Sun, C. Tang, G. McMahon, R. G. Pestell, and S. Wadler A Novel cdk2-selective Inhibitor, SU9516, Induces Apoptosis in Colon Carcinoma Cells Cancer Res., August 1, 2001; 61(16): 6170 - 6177. [Abstract] [Full Text] [PDF]
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C. P. Jung, M. V. Motwani, and G. K. Schwartz Flavopiridol Increases Sensitization to Gemcitabine in Human Gastrointestinal Cancer Cell Lines and Correlates with Down-Regulation of Ribonucleotide Reductase M2 Subunit Clin. Cancer Res., August 1, 2001; 7(8): 2527 - 2536. [Abstract] [Full Text] [PDF]
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G. I. Shapiro, J. G. Supko, A. Patterson, C. Lynch, J. Lucca, P. F. Zacarola, A. Muzikansky, J. J. Wright, T. J. Lynch Jr., and B. J. Rollins A Phase II Trial of the Cyclin-dependent Kinase Inhibitor Flavopiridol in Patients with Previously Untreated Stage IV Non-Small Cell Lung Cancer Clin. Cancer Res., June 1, 2001; 7(6): 1590 - 1599. [Abstract] [Full Text] [PDF]
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E. A. Sausville, S. G. Arbuck, R. Messmann, D. Headlee, K. S. Bauer, R. M. Lush, A. Murgo, W. D. Figg, T. Lahusen, S. Jaken, et al. Phase I Trial of 72-Hour Continuous Infusion UCN-01 in Patients With Refractory Neoplasms J. Clin. Oncol., April 15, 2001; 19(8): 2319 - 2333. [Abstract] [Full Text] [PDF]
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X.-K. Li, M. Motwani, W. Tong, W. Bornmann, and G. K. Schwartz Huanglian, A Chinese Herbal Extract, Inhibits Cell Growth by Suppressing the Expression of Cyclin B1 and Inhibiting CDC2 Kinase Activity in Human Cancer Cells Mol. Pharmacol., April 13, 2001; 58(6): 1287 - 1293. [Abstract] [Full Text]
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G. K. Schwartz, D. Ilson, L. Saltz, E. O'Reilly, W. Tong, P. Maslak, J. Werner, P. Perkins, M. Stoltz, and D. Kelsen Phase II Study of the Cyclin-Dependent Kinase Inhibitor Flavopiridol Administered to Patients With Advanced Gastric Carcinoma J. Clin. Oncol., April 1, 2001; 19(7): 1985 - 1992. [Abstract] [Full Text] [PDF]
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W. Li, J. Fan, and J. R. Bertino Selective Sensitization of Retinoblastoma Protein-deficient Sarcoma Cells to Doxorubicin by Flavopiridol-mediated Inhibition of Cyclin-dependent Kinase 2 Kinase Activity Cancer Res., March 1, 2001; 61(6): 2579 - 2582. [Abstract] [Full Text]
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L. Cartee, Z. Wang, R. H. Decker, S. P. Chellappan, G. Fusaro, K. G. Hirsch, H. M. Sankala, P. Dent, and S. Grant The Cyclin-dependent Kinase Inhibitor (CDKI) Flavopiridol Disrupts Phorbol 12-Myristate 13-Acetate-induced Differentiation and CDKI Expression while Enhancing Apoptosis in Human Myeloid Leukemia Cells Cancer Res., March 1, 2001; 61(6): 2583 - 2591. [Abstract] [Full Text]
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 A. M. Senderowicz and E. A. Sausville Preclinical and Clinical Development of Cyclin-Dependent Kinase Modulators J Natl Cancer Inst, March 1, 2000; 92(5): 376 - 387. [Abstract] [Full Text] [PDF]
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S. M. Lippman and P. H. Brown Tamoxifen Prevention of Breast Cancer: an Instance of the Fingerpost J Natl Cancer Inst, November 3, 1999; 91(21): 1809 - 1819. [Full Text] [PDF]
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S.-H. Chao, K. Fujinaga, J. E. Marion, R. Taube, E. A. Sausville, A. M. Senderowicz, B. M. Peterlin, and D. H. Price Flavopiridol Inhibits P-TEFb and Blocks HIV-1 Replication J. Biol. Chem., September 8, 2000; 275(37): 28345 - 28348. [Abstract] [Full Text] [PDF]
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S.-H. Chao and D. H. Price Flavopiridol Inactivates P-TEFb and Blocks Most RNA Polymerase II Transcription in Vivo J. Biol. Chem., August 17, 2001; 276(34): 31793 - 31799. [Abstract] [Full Text] [PDF]
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), cyclin D1; (
), cyclin D3; (
), cyclin E.
