PD 0332991 is a highly specific inhibitor of cyclin-dependent kinase 4 (Cdk4) (IC50, 0.011 μmol/L) and Cdk6 (IC50, 0.016 μmol/L), having no activity against a panel of 36 additional protein kinases. It is a potent antiproliferative agent against retinoblastoma (Rb)-positive tumor cells in vitro, inducing an exclusive G1 arrest, with a concomitant reduction of phospho-Ser780/Ser795 on the Rb protein. Oral administration of PD 0332991 to mice bearing the Colo-205 human colon carcinoma produces marked tumor regression. Therapeutic doses of PD 0332991 cause elimination of phospho-Rb and the proliferative marker Ki-67 in tumor tissue and down-regulation of genes under the transcriptional control of E2F. The results indicate that inhibition of Cdk4/6 alone is sufficient to cause tumor regression and a net reduction in tumor burden in some tumors.

The proliferation of eukaryotic cells typically involves an orderly progression through four distinct phases of the cell cycle: G1, S, G2, and M (1–3). Cyclin-dependent kinases (Cdks) play a key role in regulating cell cycle progression and to a large degree govern cellular transitions from growth phases (G1 and G2) into phases associated with DNA replication (S) and mitosis (M; refs. 4, 5). Regulation of Cdk catalytic activity occurs at multiple levels including phosphorylation and dephosphorylation of the Cdk itself; cyclin synthesis and degradation; and expression, degradation, and availability of naturally occurring protein inhibitors and subcellular localization of these various regulatory components (2, 5, 6).

Progression through the G1-S phase requires phosphorylation of the retinoblastoma (Rb) protein by Cdk4 (7, 8) or the highly homologous enzyme Cdk6 (9, 10) in complex with their activating subunits, the D-type cyclins, D1, D2, or D3 (11). Hyperphosphorylation of Rb diminishes its ability to repress gene transcription through the E2F family of transcription factors and consequently allows synthesis of several genes, the protein products of which are necessary for DNA replication (12–15). Thus, the catalytic activity of Cdk4 or Cdk6 regulates a critical checkpoint for the G1-S transition and the commitment to cell division (16).

More than 90% of human tumors abandon the control mechanisms for this transition point through a variety of genetic and biochemical adaptations (1, 17). Examples of these abnormalities include up-regulation of Cdk4 itself; amplification of the D-type cyclins; down-regulation of a naturally occurring inhibitor of Cdk4, called p16INK4A; mutations in Cdk4 that prevent p16INK4A binding to the enzyme; and deletion or mutation of Rb itself (17–21). All of these aberrations can lead to loss of proliferative controls either through elimination of the checkpoint altogether or through inappropriate or enhanced Cdk4 activity resulting in hyperphosphorylation of Rb. The frequency of these alterations alone clearly implies that abrogation of the G1 checkpoint or acceleration of the Cdk4/cyclin D pathway provides a distinct advantage to cancer cells in terms of proliferation and perhaps survival.

Based on these observations, cyclin D–dependent kinases have been considered for many years a prime target for cancer chemotherapy (22, 23). Experimental evidence suggests that inhibition of cyclin D–dependent kinase activity may prevent tumor growth and/or at least partially revert the transformed phenotype. For example, reduction of cyclin D expression through antisense technology causes a concomitant decline in cyclin D–dependent kinase activity and results in inhibition of tumor growth, abolition of tumorigenicity, or, in some instances, tumor cell death (24–27). Other reports indicate that exogenous expression of p16INK4A in tumor cells using adenoviral gene delivery systems or inducible promoters blocks proliferation and tumorigenic potency both in vitro and in vivo (28–31). These observations lend credence to Cdk4/6 as a target for cancer treatment and allow a reasonable expectation that a specific inhibitor of these enzymes would produce a meaningful therapeutic response.

Several drug discovery programs have produced potent small molecule Cdk inhibitors (22, 32–35) from a variety of chemical classes, which include purine analogues (36–40), pyrimidine analogues (41–43), indenopyrazoles (44, 45), pyridopyrimidines (46–48), pyrazolopyridines (49, 50), indolocarbazoles (51), pyrrolocarbazoles (52, 53), oxindoles (54, 55), and aminothiazoles (56). Several compounds are currently in clinical trials including flavopiridol, R-roscovitine (CYC202), UCN-01 (7-hydroxystaurosporine), and BMS-387032 (57, 58). Most of these compounds, however, inhibit multiple Cdks, with Cdk2 being a particularly common target in drug discovery programs because this enzyme is easily crystallized with inhibitors of varying molecular structure (59, 60). Interestingly, several recent reports have provided evidence that mammalian cells can continue to proliferate in the absence of Cdk2/cyclin E activity (61–63) possibly due to compensation by Cdk4 and/or Cdk6. These reports suggest that Cdk2 may be less attractive than Cdk4 as an anticancer target. Although some reports in the literature claim Cdk4-specific agents, these compounds are generally weakly selective for Cdk4 versus Cdk2 in isolated enzyme assays (10–20-fold), are weakly potent (64, 65), or produce a G2 cell cycle arrest at elevated concentrations, which is inconsistent with Cdk4/6 inhibition (42, 43, 47, 51–53, 66). Several groups have reported significant Cdk4 selectivity versus Cdk2 without supporting biological data (67). Thus, the biological effects of selective inhibition of Cdk4 by small, drug-like molecules have not been shown previously.

The present report discloses PD 0332991 as a potent inhibitor of Cdk4/6 and provides evidence that the sole mechanism of action for this compound is inhibition of these enzymes.6

6

The discovery and preparation of PD 0332991 is described separately. PL Toogood, PJ Harvey, JT Refine, et al. Discovery of PD 0332991, a potent and selective Cdk 4/6 inhibitor, submitted for publication.

Furthermore, we describe the antitumor activity of this agent and show significant efficacy in a broad spectrum of human tumor xenografts in vivo, resulting in complete regression in some tumors with no evidence of acquired resistance or ability to circumvent the growth inhibitory properties of this agent.

Cdk Assays and Other Protein Kinases

All Cdk-cyclin complexes were expressed in insect cells through baculovirus infection and purified as described previously (68). The substrate for the Cdk assays was amino acids 792 to 928 of pRb fused to glutathione S-transferase (10). Cdk assays and IC50 determinations were done as described previously (47). Enzyme assays for other protein kinases were done as described previously (69–71).

Cell Culture

All cell lines were obtained from American Type Culture Collection (Manassas, VA) and maintained at 37°C at 5% CO2 in DMEM containing 10% fetal bovine serum (FBS; Life Technologies, Inc., Rockville, MD).

Thymidine Incorporation into DNA

Cells were seeded at 2 × 104 per well in a 96-well Cytostar T plate (Amersham Biosciences, Piscataway, NJ) and incubated overnight to allow cells to attach. Varying concentrations of PD 0332991 were added to the wells and incubated for 24 hours at 37°C. [14C]thymidine (0.1 μCi) was added to each well and incorporation of the radiolabel was allowed to proceed for 72 hours. Incorporated radioactivity was determined with a β plate counter (Wallac, Inc., Gaithersburg, MD).

Western Blot Analysis

Cell or tissue samples were lysed and Western blot analysis was done as described previously (47). Rb phosphorylation status was assayed with Ser780 and Ser795 phosphospecific antibodies (Cell Signaling Technology, Beverly, MA) and total Rb expression was monitored using Rb 4H1 monoclonal antibody (Cell Signaling Technology).

Immunohistochemistry

Rb 4H1, phospho-Rb (Ser780), and Ki-67 immunohistochemical staining were done using the Ventana Discovery autostainer (Ventana Medical Systems, Tucson, AZ) with heat at 37°C to 46°C. Slides were deparaffinized using Ventana EZ Prep buffer and antigen retrieval was done using the Ventana CC1 mild reagent (containing EDTA) at 100°C for 20 minutes. Endogenous peroxidase was inactivated using the Ventana Enhanced Inhibitor solution and nonspecific antibody binding was blocked using a 3% blocking solution (Roche Diagnostics, Indianapolis, IN) for 30 minutes. Primary antibodies [Rb 4H1 (1:100 dilution, catalogue no. 9309, Cell Signaling Technology), phospho-Rb (Ser780, 1:100 dilution, catalogue no. 9307, Cell Signaling Technology), and Ki-67 (MIB-1, 1:50 dilution, catalogue no. M7240, DAKO, Carpinteria, CA)] were manually applied separately for 30-minute incubation(s) followed by application of Ventana Endogenous Blocker kit solutions (4 minutes each) to block endogenous biotin and avidin. Secondary antibodies [goat anti-rabbit IgG (for pRb) or horse anti-mouse IgG (for Rb 4H1 and Ki-67) from Vector Laboratories (Burlingame, CA)] were incubated for 30 minutes. Following SA-HRPO conjugate (18 minutes), Ventana enhanced 3,3′-diaminobenzidine tetrahydrochloride (8 minutes), and copper sulfate solution (4 minutes) incubations, sections were counterstained with hematoxylin and bluing reagent for 4 minutes each. Slides were manually rinsed in diluted detergent, dehydrated through graded alcohols, cleared in xylene, and mounted with Permount.

Flow Cytometry

Cells were harvested and washed in PBS containing EDTA (5 mmol/L). They were then washed in PBS containing 1% FBS (1% FBS/PBS), fixed in 85% ethanol, and stored at 4°C for at least 16 hours and up to 5 days. Cells were then washed again in 1% FBS/PBS and incubated at 37°C for 30 minutes in 1% FBS/PBS containing propidium iodide (40 mg/mL, Molecular Probes, Eugene, OR) and RNase A (250 mg/mL, Roche Diagnostics). Data were collected using a Coulter EPICS Elite ESP (Miami, FL) equipped with a Spectraphysics argon ion laser and analyzed using ModFit (Verity Software House, Inc., Topsham, ME). Results represent a minimum of 15,000 cells assayed for each sample.

Tumor Xenografts and Animals

Solid human tumor models Colo-205 colon, SW-620 colon, MDA-MB-435 breast, SF-295 glioblastoma, ZR-75-1 breast, PC-3 prostate, H125 non–small cell lung, H23 non–small cell lung, and MDA-MB-468 breast were developed from cell lines and maintained in severe combined immunodeficient mice. All tumor models were serially passaged as s.c. implants of tumor fragments (∼30 mg) from tumors weighing ∼1,000 mg. Tumor models were passaged ≤10 generations in vivo before returning to cryopreserved early passage stock material. The mice used in these studies were obtained from Charles River Breeding Laboratories (Wilmington, MA) and Taconic Farms (Germantown, NY; National Cancer Institute colonies). All animals were examined prior to the initiation of studies to ensure that they were healthy and acclimated to the laboratory environment. Mice were housed in barrier facilities with food and water provided ad libitum on a 12-hour light/dark cycle. Animal care was provided in accordance with Association for Assessment and Accreditation of Laboratory Animal Care International guidelines. All protocols involving animals were reviewed and approved by the Pfizer Laboratories animal care and use committee (Ann Arbor, MI).

In vivo Chemotherapy

Mice (18–22 g) were randomized and then implanted s.c. with tumor fragments (∼30 mg) into the region of the right axilla. Treatment was initiated when tumors reached 100 to 150 mg. PD 0332991 was given according to the schedule and dose indicated in the table and figure legends by gavage as a solution in sodium lactate buffer (50 mmol/L, pH 4.0) based on mean group body weight. In all experiments, there were 12 mice in the control group and 8 mice each in the treated groups. Additional details for each experiment are given in the table legends.

Isolation of Total RNA and Taqman Reverse Transcription-PCR

RNA was isolated from frozen tumor powders from three separate mice for each dose using the Trizol method (Invitrogen, Carlsbad, CA) and further purified with DNase treatment and Qiagen column according to Qiagen RNA cleanup protocols (Qiagen, Valencia, CA). Quantitative real-time PCR was carried out using the Taqman 5′ nuclease assay system with the signal from a gene-specific probe that is normalized to the signal for the reference gene of β2-microglobulin (72–74). Primers and probes were ordered from Assay-on-Demand Gene expression products (Applied Biosystems, Foster City, CA). Each assay consisted of two unlabeled PCR primers and a FAM dye-labeled Taqman MGB probe. The first-strand cDNA was generated using total RNA (5 μg) and High-Capacity cDNA Archive kit in a 100 μL reaction (Applied Biosystems). PCR was done in 384-well plates (20 μL) containing equivalent to cDNA (11 ng), primer/probe sets (1 μL) for the gene of interest, and 2× Taqman Universal PCR Master mix (10 μL, Applied Biosystems). Duplicates were done for each cDNA sample. Thermal cycling was carried out in ABI PRISM 7900 with default cycling conditions and real-time fluorescence detection. Expression fold change was calculated using the manufacturer's suggested ΔΔCt method.

Statistics

Statistical analysis was done on the times for individual tumors (both treated and control) to reach 750 mg. Where tumors did not reach 750 mg, the regrowth curves were extrapolated to 750 mg. Time to 750 mg was selected as the examined variable because it is independent of the evaluation size chosen after completion of therapy (75). All data from each experiment were analyzed by ANOVA and, where significant differences were identified, analyzed using the Bonferroni t test for multiple comparisons versus control. Levels of statistical significance are indicated for individual curves in each figure. Statistical analyses were done with SigmaStat 3.0 for Windows.

PD 0332991 Is a Potent and Selective Inhibitor of Purified Cdk4/6

Inhibition of Cdks by pyridopyrimidines has been reported previously (4648). Optimization of this class of compounds for selective inhibition of Cdk4 was achieved by testing compounds against a small panel of four enzymes, including Cdk4/cyclin D1, Cdk2/cyclin A, fibroblast growth factor receptor, and platelet-derived growth factor receptor. Compounds that possessed high selectivity for Cdk4 versus the other kinases were further evaluated in an expanded panel of kinases and assessed for antiproliferative potential against MDA-MB-435 human breast carcinoma cells. Those compounds with potent antiproliferative properties were evaluated by flow cytometry for their ability to produce a clean G1 arrest in tumor cells across a range of concentrations. This strategy led to the identification of a subset of pyridopyrimidines that displayed unprecedented levels of selectivity for Cdk4, and from these compounds, PD 0332991 (Fig. 1) was selected for its superior physical and pharmaceutical properties.

Figure 1.

Molecular structure of PD 0332991.

Figure 1.

Molecular structure of PD 0332991.

Close modal

PD 0332991 is a potent and highly selective inhibitor of Cdk4/cyclin D1 kinase activity; it exhibits an IC50 value of 0.011 μmol/L against this enzyme under the conditions described in Materials and Methods (Table 1). Another enzyme, Cdk6, which also complexes with cyclin D subunits and is highly homologous to Cdk4, can perform identical functions to Cdk4 by phosphorylating Rb at the same sites (9, 10). Consequently, inhibition of both Cdk4 and Cdk6 is necessary to ensure complete suppression of Rb phosphorylation and to produce the maximum therapeutic response with the greatest spectrum of antitumor activity. PD 0332991 inhibited Cdk6 with equivalent potency to Cdk4 (Table 1). Within a panel of enzymes, PD 0332991 exhibited absolute selectivity for Cdk4/6 with little or no activity against 36 additional protein kinases including other Cdks and a wide variety of tyrosine and serine, threonine kinases (Table 1). The structure of PD 0332991 and its relationship to PD 0183812 (47) suggest that PD 0332991 is likely to inhibit Cdk4 by binding to the ATP site; however, it was not possible to show competitive inhibition of Cdk4 by PD 0332991 using a kinetic analysis due to the near equivalence of the KI and the enzyme concentration required to perform this assay. Similarly, it has not been possible yet to obtain crystal structures of Cdk4 either with or without bound ligands.

Table 1.

Inhibitory activity of PD 0332991 against a panel of protein kinases

Protein kinaseIC50 (μmol/L)*
Cdk4/cyclin D1 0.011 
Cdk4/cyclin D3 0.009 
Cdk6/cyclin D2 0.015 
Cdk2/cyclin E2 >10 
Cdk2/cyclin A >10 
Cdk1/cyclin B >10 
Cdk5/p25 >10 
Epidermal growth factor receptor >10 
Fibroblast growth factor receptor >10 
Platelet-derived growth factor receptor >10 
Insulin receptor >10 
Lymphocyte kinase >10 
Vascular endothelial growth factor receptor >10 
AMP-activated protein kinase >10 
Checkpoint kinase-1 >10 
Casein kinase-1 >10 
Casein kinase-2 >10 
c-Src kinase >10 
C-terminal Src kinase >12 
Dual-specificity tyrosine phosphorylation-regulated kinase 1A 2.0 
Glycogen synthase kinase-3β >10 
c-Jun NH2-terminal kinase >10 
Mitogen-activated protein kinase 2/Erk2 >10 
Mitogen-activated protein kinase–activated protein kinase 1a 8.0 
Mitogen-activated protein kinase–activated protein kinase 2 >10 
Mitogen-activated protein kinase kinase >10 
Mitogen and stress-activated protein kinase 1 >10 
p70 Ribosomal protein S6 kinase >10 
3-Phosphoinositide-dependent protein kinase 1 >10 
Phosphorylase kinase >10 
Cyclic AMP-dependent protein kinase >10 
Protein kinase B >10 
Protein kinase C >10 
p38-Regulated/activated kinase >10 
Rho-dependent protein kinase >10 
Stress-activated protein kinase 2a >10 
Stress-activated protein kinase 3 >10 
Stress-activated protein kinase 4 >10 
Serum and glucocorticoid induced kinase >10 
Protein kinaseIC50 (μmol/L)*
Cdk4/cyclin D1 0.011 
Cdk4/cyclin D3 0.009 
Cdk6/cyclin D2 0.015 
Cdk2/cyclin E2 >10 
Cdk2/cyclin A >10 
Cdk1/cyclin B >10 
Cdk5/p25 >10 
Epidermal growth factor receptor >10 
Fibroblast growth factor receptor >10 
Platelet-derived growth factor receptor >10 
Insulin receptor >10 
Lymphocyte kinase >10 
Vascular endothelial growth factor receptor >10 
AMP-activated protein kinase >10 
Checkpoint kinase-1 >10 
Casein kinase-1 >10 
Casein kinase-2 >10 
c-Src kinase >10 
C-terminal Src kinase >12 
Dual-specificity tyrosine phosphorylation-regulated kinase 1A 2.0 
Glycogen synthase kinase-3β >10 
c-Jun NH2-terminal kinase >10 
Mitogen-activated protein kinase 2/Erk2 >10 
Mitogen-activated protein kinase–activated protein kinase 1a 8.0 
Mitogen-activated protein kinase–activated protein kinase 2 >10 
Mitogen-activated protein kinase kinase >10 
Mitogen and stress-activated protein kinase 1 >10 
p70 Ribosomal protein S6 kinase >10 
3-Phosphoinositide-dependent protein kinase 1 >10 
Phosphorylase kinase >10 
Cyclic AMP-dependent protein kinase >10 
Protein kinase B >10 
Protein kinase C >10 
p38-Regulated/activated kinase >10 
Rho-dependent protein kinase >10 
Stress-activated protein kinase 2a >10 
Stress-activated protein kinase 3 >10 
Stress-activated protein kinase 4 >10 
Serum and glucocorticoid induced kinase >10 
*

Concentration of PD 0332991 necessary to inhibit activity by 50%. Mean of at least two separate determinations.

PD 0332991 Inhibits Rb Phosphorylation in Tumor Cells on Cdk4-Specific Sites

The only known natural substrates for Cdk4/6 are the Rb family of gene products, p110(Rb), p107, and p130 (76). Of the 16 known phosphorylation sites on Rb, 2 are specifically phosphorylated by Cdk4/6: Ser780 and Ser795 (77–79). The phosphorylation status of Rb at these specific sites in treated tumors therefore represents an appropriate biomarker for inhibition of Cdk4/6 by PD 0332991 in tumor cells and tissue. The IC50 for reduction of Rb phosphorylation at Ser780 in MDA-MB-435 breast carcinoma cells was 0.066 μmol/L (Fig. 2A and B). PD 0332991 was equally effective at reducing Rb phosphorylation at Ser795 in this tumor with an IC50 of 0.063 μmol/L, and similar effects on both Ser780 and Ser795 phosphorylation were obtained in the Colo-205 colon carcinoma (data not shown). In vitro time course experiments indicated that the reduction of Rb phosphorylation began to occur as soon as 4 hours after exposure to PD 0332991 and reached a maximum at 16 hours. The inhibition was completely reversible because phosphorylation on Ser780 and Ser795 began to return 2 hours after removal of the drug and was complete within 16 hours, by which time the cells were actively proliferating (data not shown).

Figure 2.

Inhibition of Rb phosphorylation at Ser780 by PD 0332991. A, MDA-MB-435 human breast carcinoma cells were treated for 24 hours with varying concentrations of PD 0332991. Extracts and Western blots were generated using Ser780 phosphospecific antibodies as described in Materials and Methods. B, scanning densitometry values of the results in A expressed as a percentage of the control. These data were used to generate IC50 values.

Figure 2.

Inhibition of Rb phosphorylation at Ser780 by PD 0332991. A, MDA-MB-435 human breast carcinoma cells were treated for 24 hours with varying concentrations of PD 0332991. Extracts and Western blots were generated using Ser780 phosphospecific antibodies as described in Materials and Methods. B, scanning densitometry values of the results in A expressed as a percentage of the control. These data were used to generate IC50 values.

Close modal

PD 0332991 Is a Potent Antiproliferative Agent That Arrests Rb-Positive Tumors Exclusively in G1

PD 0332991 is a potent inhibitor of cell growth and suppresses DNA replication by preventing cells from entering S phase. The compound inhibited thymidine incorporation into the DNA of Rb-positive human breast, colon, and lung carcinomas as well as human leukemias, with IC50 values ranging from 0.040 to 0.17 μmol/L (Table 2). PD 0332991 had no activity against Rb-negative cells, indicating that it does not interact with antiproliferative targets other than Cdk4/6. The compound was tested against the MDA-MB-468 human breast carcinoma and the H2009 human non–small cell lung carcinoma, both of which have deleted Rb and exhibited no antiproliferative activity in these cells at concentrations as high as 3 μmol/L (Table 2), which is 1 to 2 orders of magnitude higher than the concentration necessary to inhibit proliferation in Rb-positive tumor cells.

Table 2.

Inhibition of thymidine incorporation into DNA in human tumor cell lines treated with PD 0332991

Cell lineCell typeRb statusIC50 (μmol/L)*
MDA-MB-435 Breast carcinoma Positive 0.16 
ZR-75-1 Breast carcinoma Positive 0.17 
T-47D Breast carcinoma Positive 0.04 
MCF-7 Breast carcinoma Positive 0.10 
H1299 Lung carcinoma Positive 0.12 
Colo-205 Colon carcinoma Positive 0.13 
MDA-MB-468 Breast carcinoma Negative >3 
H2009 Lung carcinoma Negative >3 
CRRF-CEM Acute lymphoblastic leukemia Positive 0.25 
K562 Chronic myelogenous leukemia Positive 0.40 
Cell lineCell typeRb statusIC50 (μmol/L)*
MDA-MB-435 Breast carcinoma Positive 0.16 
ZR-75-1 Breast carcinoma Positive 0.17 
T-47D Breast carcinoma Positive 0.04 
MCF-7 Breast carcinoma Positive 0.10 
H1299 Lung carcinoma Positive 0.12 
Colo-205 Colon carcinoma Positive 0.13 
MDA-MB-468 Breast carcinoma Negative >3 
H2009 Lung carcinoma Negative >3 
CRRF-CEM Acute lymphoblastic leukemia Positive 0.25 
K562 Chronic myelogenous leukemia Positive 0.40 
*

Concentration of PD 0332991 necessary to inhibit cell proliferation by 50%. Mean of at least two separate determinations.

A selective Cdk4/6 inhibitor should cause a specific accumulation of cells in G1 but have no effect on other phases of the cell cycle in which cells should continue to progress and eventually decline in number. MDA-MB-453 breast carcinoma cells exposed to varying concentrations of PD 0332991 for 24 hours showed a significant increase in the percentage of cells in G1 in the presence of as little as 0.04 μmol/L PD 0332991 with a concomitant decline in other phases of the cell cycle (Fig. 3). Maximum effects were attained at 0.08 μmol/L and an exclusive G1 arrest was maintained even at concentrations as high as 10 μmol/L, consistent with the complete absence of any other effects on the cell cycle. Similar results were obtained with Colo-205 cells (data not shown).

Figure 3.

PD 0332991 causes an exclusive G1 arrest. MDA-MB-453 human breast carcinoma cells were exposed to varying concentrations of PD 0332991 for 24 hours. Cells were harvested and fixed as described in Materials and Methods. The DNA histograms were generated by flow cytometry and the percentage of cells in each phase of the cell cycle was determined using ModFit. Additional details are given in Materials and Methods.

Figure 3.

PD 0332991 causes an exclusive G1 arrest. MDA-MB-453 human breast carcinoma cells were exposed to varying concentrations of PD 0332991 for 24 hours. Cells were harvested and fixed as described in Materials and Methods. The DNA histograms were generated by flow cytometry and the percentage of cells in each phase of the cell cycle was determined using ModFit. Additional details are given in Materials and Methods.

Close modal

PD 0332991 Has Broad-Spectrum Anticancer Activity and Can Cause Regression of Certain Tumors

PD 0332991 exhibits significant antitumor efficacy against multiple human tumor xenograft models. In mice bearing Colo-205 colon carcinoma xenografts (p16 deleted), daily p.o. dosing for 14 days with PD 0332991 (150 or 75 mg/kg) produced rapid tumor regressions (Fig. 4A) and a corresponding tumor growth delay of ∼50 days with >1 log of tumor cell kill at the highest dose tested (Table 3). At 37.5 mg/kg, the tumor slowly regressed during treatment. Even at doses as low as 12.5 mg/kg, a 13-day growth delay was obtained indicating a 90% inhibition of tumor growth rate. Likewise, robust antitumor activity was seen in the MDA-MB-435 breast carcinoma (p16 deleted) where complete tumor stasis was apparent at 150 mg/kg (Fig. 4B) and some cell kill was evident at the highest dose (Table 3).

Figure 4.

In vivo antitumor activity of PD 0332991 given p.o. A, regression of the Colo-205 human colon carcinoma. Tumor fragments (∼30 mg) were implanted s.c. in SCID mice and allowed to grow to at least 100 mg. PD 0332991 was given daily p.o. for 14 days by gavage at the indicated drug doses in lactate buffer (50 mmol/L) at pH 4.0. Untreated control (•), 12.5 mg/kg (○), 37.5 mg/kg (▴), 75 mg/kg (▵), and 150 mg/kg (▪). B, growth suppression of the MDA-MB-435 human breast carcinoma. Tumor implantation and administration of PD 0332991 was done as in A, except drug was given daily for 28 days. Untreated control (•), 36 mg/kg (○), 58 mg/kg (▴), 93 mg/kg (▵), and 150 mg/kg (▪). C, tumors retain sensitivity to PD 0332991 after extended treatment. Colo-205 tumors in A that grew back after regression during treatment were harvested and implanted into naïve mice. Details of drug treatment are similar to A. Untreated control of tumors treated previously in A with 150 mg/kg (•), tumors treated previously with 150 mg/kg and then retreated with 150 mg/kg (▵), untreated control of tumors treated previously in A with 37.5 mg/kg (▴), and tumors treated previously with 37.5 mg/kg retreated with 150 mg/kg (○). Data in A to C are shown starting from the first day of dosing. Statistical analysis was done as described in Materials and Methods. Ps are relative to the appropriate control in each case.

Figure 4.

In vivo antitumor activity of PD 0332991 given p.o. A, regression of the Colo-205 human colon carcinoma. Tumor fragments (∼30 mg) were implanted s.c. in SCID mice and allowed to grow to at least 100 mg. PD 0332991 was given daily p.o. for 14 days by gavage at the indicated drug doses in lactate buffer (50 mmol/L) at pH 4.0. Untreated control (•), 12.5 mg/kg (○), 37.5 mg/kg (▴), 75 mg/kg (▵), and 150 mg/kg (▪). B, growth suppression of the MDA-MB-435 human breast carcinoma. Tumor implantation and administration of PD 0332991 was done as in A, except drug was given daily for 28 days. Untreated control (•), 36 mg/kg (○), 58 mg/kg (▴), 93 mg/kg (▵), and 150 mg/kg (▪). C, tumors retain sensitivity to PD 0332991 after extended treatment. Colo-205 tumors in A that grew back after regression during treatment were harvested and implanted into naïve mice. Details of drug treatment are similar to A. Untreated control of tumors treated previously in A with 150 mg/kg (•), tumors treated previously with 150 mg/kg and then retreated with 150 mg/kg (▵), untreated control of tumors treated previously in A with 37.5 mg/kg (▴), and tumors treated previously with 37.5 mg/kg retreated with 150 mg/kg (○). Data in A to C are shown starting from the first day of dosing. Statistical analysis was done as described in Materials and Methods. Ps are relative to the appropriate control in each case.

Close modal
Table 3.

Spectrum of antitumor activity for PD 0322991 against a panel of advanced stage human tumor xenografts

Tumor*Dose (mg/kg), MTD or highest dose testedDosing schedule% Weight change§T-CNet log10 kill
Colo-205 colon 150 Days 18–31 −6 48.4 +1.20 
MDA-MB-435 breast 150 12–39 32.7 +0.25 
SF-295 glioblastoma 150 7–20 −11 30.1 +2.24 
ZR-75-1 breast 150 7–20 −4 12.4 −0.06 
PC-3 prostate 130 8–21 −13 12.8 +0.02 
H125 lung 150 19–32 −12 7.3 −0.31 
SW-620 colon 150 10–23 −3 5.9 −0.55 
H23 lung 130 9–22 4.8 −1.07 
MDA-MB-468 breast (Rb negative) 150 22–35 −2 −4.3 — 
Tumor*Dose (mg/kg), MTD or highest dose testedDosing schedule% Weight change§T-CNet log10 kill
Colo-205 colon 150 Days 18–31 −6 48.4 +1.20 
MDA-MB-435 breast 150 12–39 32.7 +0.25 
SF-295 glioblastoma 150 7–20 −11 30.1 +2.24 
ZR-75-1 breast 150 7–20 −4 12.4 −0.06 
PC-3 prostate 130 8–21 −13 12.8 +0.02 
H125 lung 150 19–32 −12 7.3 −0.31 
SW-620 colon 150 10–23 −3 5.9 −0.55 
H23 lung 130 9–22 4.8 −1.07 
MDA-MB-468 breast (Rb negative) 150 22–35 −2 −4.3 — 
*

All tumors are Rb positive, except MDA-MB-468 in which Rb is deleted.

The vehicle was 50 mmol/L lactate buffer at pH 4.0. PD 0332991 was given daily p.o. for 14 days, except for MDA-MB-435, which was given for 28 days. Treatments were started when the tumor weights were between 100 and 150 mg.

MTD, maximum tolerated dose.

§

A negative number is the percentage weight loss seen during treatment. A plus indicates a weight gain through treatment.

T-C is the difference (in days) for the treated and control tumors to reach an evaluation size of 750 mg. A T-C of 0 would indicate no effect.

Net log10 tumor cell kill represents the change in tumor burden during therapy. A negative value indicates a net increase in tumor mass during therapy, whereas a positive value indicates a net reduction in tumor burden. Values near 0 indicate tumor stasis during therapy.

PD 0332991 was efficacious against a variety of human tumor xenografts (Table 3). Similar to the Colo-205, significant tumor regression was observed in mice bearing the SF-295 glioblastoma xenografts at doses of 150 mg/kg, and responses nearing stasis (complete suppression of tumor growth) were attained in the ZR-75-1 breast and PC-3 prostate tumor models (Table 3). Modest activity was achieved in H125 non–small cell lung carcinoma. The compound was completely inactive, however, against the Rb-negative MDA-MB-468 breast carcinoma, which is consistent with inhibition of Cdk4/6 as the sole mechanism by which PD 0332991 produces antitumor activity. All doses reported in Table 3 were well tolerated by the animals, with no obvious clinical signs and either minimal weight loss or some weight gain. Treated animals were maintained for up to 50 days after dosing to facilitate the observation of any delayed clinical toxicities. At all doses reported, the animals succumbed to tumor-related death or were sacrificed once their tumor burden reached ∼1,000 mg. Based on the data shown, 150 mg/kg is defined as a maximum tolerated dose of PD 0332991 given to mice daily for 14 or 28 days.

Despite complete regression of certain tumors in response to PD 0332991, the tumors eventually grew back after these short treatment periods. Since in recent years some in vitro evidence has suggested that tumors may have the potential to circumvent a Cdk4 block through activation or elevation of downstream control elements of the cell cycle (80–82), we wanted to determine if the new tumors that appeared in treated animals remained sensitive to further treatment with PD 0332991 or whether a tumor variant had evolved exhibiting acquired resistance to the compound. To address this possibility, Colo-205 colon tumors that had emerged after substantial regression from initial treatment with PD 0332991 in the experiment described in Fig. 4A were harvested and reimplanted into naïve mice. After the tumors grew to 100 to 150 mg, these tumor-bearing mice were treated with PD 0332991 using a schedule identical to the original experiment. The tumors responded with equal sensitivity to the drug and fully regressed, indicating that no resistance had developed during the initial treatment (Fig. 4C). Similar results were obtained with MDA-MB-435 tumors (data not shown).

PD 0332991 Causes a Sustained Suppression of Tumor Rb Phosphorylation In vivo

In parallel with the in vivo efficacy tests, additional tumors were harvested for pharmacodynamic analysis to test if antitumor activity correlated with modulation of the target and to investigate whether the proposed biomarker might predict for efficacy. Efficacious and nonefficacious doses of PD 0332991 were given to mice bearing the MDA-MB-435 breast carcinoma and the phosphorylation status of Ser780 on Rb in tumor tissue was monitored over time. The results show that all doses caused a reduction in the biomarker shortly after drug administration but that phosphorylation returned at nonefficacious doses (12.5 and 37.5 mg/kg) over the 24-hour interval before the next dose (Fig. 5A). The highly efficacious dose of 150 mg/kg suppressed Rb Ser780 phosphorylation over the full 24-hour period, implying that, with this particular breast tumor, complete suppression of the biomarker needs to be maintained between drug doses to achieve maximum efficacy. Target modulation in this tumor by PD 0332991 given p.o. was also established by immunohistochemistry where animals treated with an efficacious dose of PD 0332991 were characterized by complete elimination of phospho-Ser780 from their tumor (Fig. 5B). This effect also was associated with a substantial reduction in staining for Ki-67, a well-known marker of proliferation (83). Similar experiments with the highly sensitive Colo-205 colon carcinoma showed that complete suppression of the biomarker between doses is not necessary to produce growth inhibition in this tumor. However, total inhibition must be maintained between doses to achieve tumor regression (Fig. 5C). Comparing the results of efficacy experiments using the highly sensitive colon tumor and the moderately sensitive breast tumor reveals a 7- to 8-fold difference in the dose necessary to produce comparable efficacy. The pharmacodynamic results show a similar difference between the two tumors in the dose required to suppress Rb Ser780 phosphorylation, indicating that modulation of this biomarker is strongly associated with efficacy.

Figure 5.

Therapeutically active doses of PD 0332991 in vivo cause down-regulation of proteins and genes in tumor tissue that are consistent with inhibition of Cdk4/6. A to D, designated tumors were implanted into nude mice as described in Fig. 4 and allowed to grow to ∼200 mg. Mice were dosed daily for 2 days and tumor tissue was harvested at the designated times after the last dose and processed as described in the individual panels. A, inhibition of Rb Ser780 phosphorylation in MDA-MB-435 human breast carcinomas in mice treated with PD 0332991. Tumors were excised at the designated times after the last dose of drug and snap frozen in liquid nitrogen. Extracts of the tissue and Western blots were done as described in Materials and Methods. B, inhibition of Rb Ser780 phosphorylation in MDA-MB-435 human breast carcinoma and suppression of Ki-67 as visualized by immunohistochemistry. Tumors were identical to A, except that part of the tumor was fixed in formaldehyde and processed for staining as described in Materials and Methods. C, inhibition of Rb Ser780 phosphorylation in Colo-205 human colon carcinoma in mice treated with PD 0332991. Tumors were excised at the designated times after the last dose of drug and snap frozen in liquid nitrogen. Extracts of the tissue and Western blots were done as described in Materials and Methods. D, down-regulation of selected genes under the transcriptional control of E2F in the Colo-205 human colon carcinoma. Tumors were excised 3 hours after the last dose of drug and snap frozen in liquid nitrogen. RNA was isolated and relative gene expression levels were determined by reverse transcription-PCR as described in Materials and Methods. TK1, thymidine kinase (•); CDC2, Cdk1 (▴); TOP2A, topoisomerase 2A (▪); and CCNE2, cyclin E2 (▾).

Figure 5.

Therapeutically active doses of PD 0332991 in vivo cause down-regulation of proteins and genes in tumor tissue that are consistent with inhibition of Cdk4/6. A to D, designated tumors were implanted into nude mice as described in Fig. 4 and allowed to grow to ∼200 mg. Mice were dosed daily for 2 days and tumor tissue was harvested at the designated times after the last dose and processed as described in the individual panels. A, inhibition of Rb Ser780 phosphorylation in MDA-MB-435 human breast carcinomas in mice treated with PD 0332991. Tumors were excised at the designated times after the last dose of drug and snap frozen in liquid nitrogen. Extracts of the tissue and Western blots were done as described in Materials and Methods. B, inhibition of Rb Ser780 phosphorylation in MDA-MB-435 human breast carcinoma and suppression of Ki-67 as visualized by immunohistochemistry. Tumors were identical to A, except that part of the tumor was fixed in formaldehyde and processed for staining as described in Materials and Methods. C, inhibition of Rb Ser780 phosphorylation in Colo-205 human colon carcinoma in mice treated with PD 0332991. Tumors were excised at the designated times after the last dose of drug and snap frozen in liquid nitrogen. Extracts of the tissue and Western blots were done as described in Materials and Methods. D, down-regulation of selected genes under the transcriptional control of E2F in the Colo-205 human colon carcinoma. Tumors were excised 3 hours after the last dose of drug and snap frozen in liquid nitrogen. RNA was isolated and relative gene expression levels were determined by reverse transcription-PCR as described in Materials and Methods. TK1, thymidine kinase (•); CDC2, Cdk1 (▴); TOP2A, topoisomerase 2A (▪); and CCNE2, cyclin E2 (▾).

Close modal

Down-Regulation of Genes under the Control of E2F in Tumors from Mice Treated with PD 0332991

Hypophosphorylated Rb has been proposed to inhibit cellular proliferation partly by suppressing the transcription of genes under the control of the E2F transcription factors (12–15). Four E2F-regulated genes were selected and their expression levels were monitored by reverse transcription-PCR in Colo-205 tumors from mice treated p.o. with PD 0332991. The genes include CDC2 that codes for Cdk1, CCNE2 that codes for cyclin E2, TK1 that codes for thymidine kinase, and TOP2A that codes for topoisomerase 2A. All four genes were down-regulated in a dose-dependent manner, with maximum reductions ranging from 13- to 87-fold (Fig. 5D). The extent of the gene changes paralleled the magnitude of the therapeutic response in this tumor, providing additional evidence that PD 0332991 functions through inhibition of Cdk4/6.

In this report, we describe PD 0332991 as a potent and highly selective inhibitor of Cdk4 and Cdk6 and show that suppression of these enzymes in human tumor xenografts results in significant antitumor activity. Given that a major obstacle to establishing the usefulness of a Cdk4/6 inhibitor has been the difficulty in obtaining a molecule with complete specificity for these enzymes versus other Cdks and protein kinases, considerable effort was taken to establish the selectivity of this compound. PD 0332991 was tested against 39 individual serine, threonine, and tyrosine kinases, representing most of the primary protein kinase families (84). Other than Cdk4 and Cdk6, the compound had little or no activity against any of these enzymes. Based on the understood role of Cdk4/6 in cell cycle progression, a specific Cdk4/6 inhibitor is predicted to produce an exclusive G1 arrest. Consistent with this expectation, cells treated with concentrations of PD 0332991 as high as 200-fold above the IC50 for growth inhibition maintained an unequivocal G1 block as the sole perturbation in their DNA histograms. In contrast, previously reported Cdk4/D inhibitors have produced a G2-M block at high concentrations (47, 52). PD 0332991 had no effect on proliferation in tumor cells that were Rb deficient at concentrations >50 times the levels that cause growth inhibition in Rb-positive cells, consistent with selective inhibition of Cdk4/6. Initiation of the G1 block, emergence of antiproliferative activity, and dephosphorylation of Rb at the Cdk4/6-specific site of Ser780 all occurred at similar concentrations of PD 0332991. Collectively, these data show that the biochemical activities and biological effects of PD 0332991 are in accord with exclusive inhibition of its target enzymes and suggest a high degree of selectivity in the cellular environment.

Oral administration of PD 0332991 to mice bearing human tumor xenografts clearly showed that inhibition of Cdk4/6 can result in a significant antitumor effect. The spectrum of responses ranged from partial growth inhibition and tumor stasis to complete regressions, depending on the tumor, with a therapeutic index as high as 12 in the Colo-205 model. [The therapeutic index is defined as the maximum tolerated dose divided by the lowest dose to give a tumor growth delay of >50%.] The anticancer activity was associated with biochemical changes that are consistent with the mechanism of action for PD 0332991. Thus, significant antitumor activity was detected only at doses where there was a sustained and significant reduction of phospho-Ser780 on Rb. The decline in Rb Ser780 phosphorylation in tumor tissue was confirmed by immunohistochemistry techniques, which showed substantial reduction of this marker in tumors from treated animals. Moreover, there was a concomitant elimination of staining for Ki-67, a commonly measured marker of proliferation (83). Furthermore, reverse transcription-PCR of selected genes in tumor tissue known to be under the control of E2F and down-regulated in cells expressing a nonphosphorylatable Rb (85) showed a 13- to 87-fold reduction in expression in tumors from animals treated with therapeutically active doses of PD 0332991. Finally, PD 0332991 was completely inactive against Rb-negative tumor xenografts, which is expected based on its proposed mechanism of action. Taken together, these in vivo data are consistent with inhibition of Cdk4/6 as the sole basis for the observed antitumor activity.

The results of this study address two issues that have been raised in the past concerning inhibitors of Cdk4/6 as therapeutic agents for oncology, the first being an assumption that inhibition of Cdk4/6 may result in a cytostatic phenotype, causing little more than temporary tumor growth arrest. Indeed, cytostasis was the phenotype observed in tissue culture experiments in vitro, wherein cells exposed to PD 0332991 ceased proliferating but did not die even after prolonged exposure to the compound. In vivo, however, we show that inhibition of Cdk4/6 not only produces a robust growth suppressive effect in many human tumor xenografts but also is capable of causing complete regressions in some tumors. Secondly, based on results with in vitro culture models, it has been suggested that tumors might be able to circumvent the antiproliferative effects of Cdk4/6 inhibition through activation or elevation of downstream control elements of the cell cycle such as Cdk2/cyclin E activity or c-myc (80–82). The results of this study not only show a robust and sustained therapeutic response to Cdk4/6 inhibition but also show that tumors do not readily develop an ability to overcome a cell cycle block incurred through inhibition of Cdk4/6. Colo-205 human tumor xenografts treated with PD 0332991 for 14 days and then allowed to regrow over several weeks do not develop resistance and remain sensitive to inhibition of Cdk4/6. These results complement recent data suggesting that Cdk2 may not be generally required for proliferation in mammalian cells and that Cdk4/6 alone might be sufficient to regulate the G1-S phase transition (61). In fact, Cdk2 knockout mice have been reported to be entirely viable (86). Taken together, the results support Cdk4/6 as a relevant target for cancer chemotherapy.

We can only speculate on how an inhibitor that appears cytostatic in vitro can cause tumor regressions in vivo. One possibility is that Cdk4/6 activity is required for tumor survival. The fact that Cdk4 disruption renders MEF cells resistant to transformation by certain oncogenes (87) implies that the transformed phenotype requires Cdk4 activity. Furthermore, cyclin D1 is a true oncogene capable of transforming cells itself, and p16, a natural inhibitor of Cdk4 that is commonly absent or inactivated in tumors, has all the characteristics of a tumor suppressor. Both of these observations imply that Cdk4 activity is a critical component of oncogenesis in certain tumors. Another potential explanation for the observed tumor regressions might be that most solid tumors contain a significant population of spontaneously dying cells in addition to a proliferating fraction, with the latter having a slight advantage in an expanding tumor mass. If proliferation were slowed to the point that the naturally dying population was dominant, the net result would be regression of the tumor. Because Rb interacts with well over 100 proteins of various function (88) and because the response to PD 0332991 is tumor specific, sensitivity is likely governed by multiple factors.

In conclusion, the striking therapeutic response to inhibition of Cdk4/6 observed in the current study makes this approach attractive for clinical application because an inhibitor of Cdk4/6 could have the potential to cause tumor regressions or cures as a single agent. Pragmatically, conventional clinical means of assessing antitumor activity should be appropriate for PD 0332991 and mechanistically related compounds. Furthermore, we have shown that several robust biomarkers are measurable by immunohistochemistry and could possibly be used to detect drug-related target modulation in patients or perhaps eventually to predict therapeutic response. PD 0332991 is scheduled for clinical trials in 2004 and should help provide an answer to whether selective inhibitors of Cdk4/6 can provide a therapeutic benefit in cancer patients.

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.

We thank Joseph Repine, Hairong Zhou, Dennis McNamara, John Quin, Cathlin Flamme, Michael Waldo, and Vladimir Beylin for contributions and Alexander Bridges, Seth Sadis, and Ellen Dobrusin for support.

1
Malumbres M, Barbacid M. To cycle or not to cycle: a decision in cancer.
Nat Rev Cancer
2001
;
1
:
222
–31.
2
Obaya AJ, Sedivy JM. Regulation of cyclin-Cdk activity in mammalian cells.
Cell Mol Life Sci
2002
;
59
:
126
–42.
3
Sherr CJ. Cancer cell cycles.
Science
1996
;
274
:
1672
–7.
4
Morgan DO, Fisher RP, Espinoza FH, et al. Control of eukaryotic cell cycle progression by phosphorylation of cyclin-dependent kinases.
Cancer J Sci Am
1998
;
4
:
S77
–83.
5
Morgan DO. Cyclin-dependent kinases: engines, clocks, and microprocessors.
Annu Rev Cell Dev Biol
1997
;
13
:
261
–91.
6
Ekholm SV, Reed SI. Regulation of G(1) cyclin dependent kinases in the mammalian cell cycle.
Curr Opin Cell Biol
2000
;
12
:
676
–84.
7
Harbour JW, Luo RX, Santi AD, Postigo AA, Dean DC. Cdk phosphorylation triggers sequential intramolecular interactions that progressively block Rb functions as cells move through G1.
Cell
1999
;
98
:
859
–69.
8
Lundberg AS, Weinberg RA. Functional inactivation of the retinoblastoma protein requires sequential modification by at least two distinct cyclin-cdk complexes.
Mol Cell Biol
1998
;
18
:
753
–61.
9
Chen Q, Lin J, Jinno S, Okayama H. Overexpression of Cdk6-cyclin D3 highly sensitizes cells to physical and chemical transformation.
Oncogene
2003
;
22
:
992
–1001.
10
Meyerson M, Harlow E. Identification of G1 kinase activity for cdk6, a novel cyclin D partner.
Mol Cell Biol
1994
;
14
:
2077
–86.
11
Sherr CJ. D-type cyclins.
Trends Biochem Sci
1995
;
20
:
187
–90.
12
Trimarchi JM, Lees JA. Sibling rivalry in the E2F family.
Nat Rev Mol Cell Biol
2002
;
3
:
11
–20.
13
Mundle SD, Saberwal G. Evolving intricacies and implications of E2F1 regulation.
FASEB J
2003
;
17
:
569
–74.
14
Harbour JW, Dean DC. The Rb/E2F pathway: expanding roles and emerging paradigms.
Genes Dev
2000
;
14
:
2393
–409.
15
Nevins JR. The Rb/E2F pathway and cancer.
Hum Mol Genet
2001
;
10
:
699
–703.
16
Ho A, Dowdy SF. Regulation of G(1) cell-cycle progression by oncogenes and tumor suppressor genes.
Curr Opin Genet Dev
2002
;
12
:
47
–52.
17
Sellers WR, Kaelin WG. Role of the retinoblastoma protein in the pathogenesis of human cancer.
J Clin Oncol
1997
;
15
:
3301
–12.
18
Hall M, Peters G. Genetic alterations of cyclins, cyclin-dependent kinases, and Cdk inhibitors in human cancer.
Adv Cancer Res
1996
;
68
:
67
–108.
19
Kaelin WG Jr. Alterations in G1-S cell-cycle control contributing to carcinogenesis.
Ann N Y Acad Sci
1997
;
833
:
29
–33.
20
Bartkova J, Lukas J, Bartek J. Aberrations of the G1- and G1-S-regulating genes in human cancer.
Prog Cell Cycle Res
1997
;
3
:
211
–20.
21
Bartek J, Lukas J, Bartkova J. Perspective: defects in cell cycle control and cancer.
J Pathol
1999
;
187
:
95
–9.
22
Fry DW, Garrett MD. Inhibitors of cyclin-dependent kinases as therapeutic agents for the treatment of cancer.
Curr Opin Oncol Endocr Metabol Invest Drugs
2000
;
2
:
40
–59.
23
Garrett MD, Fattaey A. CDK inhibition and cancer therapy.
Curr Opin Genet Dev
1999
;
9
:
104
–11.
24
Arber N, Doki Y, Han EK, et al. Antisense to cyclin D1 inhibits the growth and tumorigenicity of human colon cancer cells.
Cancer Res
1997
;
57
:
1569
–74.
25
Kornmann M, Arber N, Korc M. Inhibition of basal and mitogen-stimulated pancreatic cancer cell growth by cyclin D1 antisense is associated with loss of tumorigenicity and potentiation of cytotoxicity to cisplatinum.
J Clin Invest
1998
;
101
:
344
–52.
26
Sauter ER, Nesbit M, Litwin S, Klein Szanto AJP, Cheffetz S, Herlyn M. Antisense cyclin D1 induces apoptosis and tumor shrinkage in human squamous carcinomas.
Cancer Res
1999
;
59
:
4876
–81.
27
Zhou P, Jiang W, Zhang YJ, et al. Antisense to cyclin D1 inhibits growth and reverses the transformed phenotype of human esophageal cancer cells.
Oncogene
1995
;
11
:
571
–80.
28
Serrano M, Gomez-Lahoz E, DePinho RA, Beach D, Bar-Sagi D. Inhibition of ras-induced proliferation and cellular transformation by p16ink4.
Science
1995
;
267
:
249
–52.
29
Wolf JK, Kim TE, Fightmaster D, et al. Growth suppression of human ovarian cancer cell lines by the introduction of a p16 gene via a recombinant adenovirus.
Gynecol Oncol
1999
;
73
:
27
–34.
30
Sumitomo K, Shimizu E, Shinohara A, Yokota J, Sone S. Activation of RB tumor suppressor protein and growth suppression of small cell lung carcinoma cells by reintroduction of p16(INK4A) gene.
Int J Oncol
1999
;
14
:
1075
–80.
31
Sandig V, Brand K, Herwig S, Lukas J, Bartek J, Strauss M. Adenovirally transferred p16INK4/CDKN2 and p53 genes cooperate to induce apoptotic tumor cell death.
Nat Med
1997
;
3
:
313
–9.
32
Toogood PL. Progress toward the development of agents to modulate the cell cycle.
Curr Opin Chem Biol
2002
;
6
:
472
–8.
33
Knockaert M, Greengard P, Meijer L. Pharmacological inhibitors of cyclin-dependent kinases.
Trends Pharmacol Sci
2002
;
23
:
417
–25.
34
Senderowicz AM. Small-molecule cyclin-dependent kinase modulators.
Oncogene
2003
;
22
:
6609
–20.
35
Fischer PM, Endicott J, Meijer L. Cyclin-dependent kinase inhibitors.
Prog Cell Cycle Res
2003
;
5
:
235
–48.
36
McClue SJ, Blake D, Clarke R, et al. In vitro and in vivo antitumor properties of the cyclin dependent kinase inhibitor CYC202 (R-roscovitine).
Int J Cancer
2002
;
102
:
463
–8.
37
Haesslein JL, Jullian N. Recent advances in cyclin-dependent kinase inhibition. Purine-based derivatives as anti-cancer agents. Roles and perspectives for the future.
Curr Top Med Chem
2002
;
2
:
1037
–50.
38
Davies TG, Bentley J, Arris CE, et al. Structure-based design of a potent purine-based cyclin-dependent kinase inhibitor.
Nat Struct Biol
2002
;
9
:
745
–9.
39
Chang Y-T, Gray NS, Rosania GR, et al. Synthesis and application of functionally diverse 2,6,9-trisubstituted purine libraries as CDK inhibitors.
Chem Biol
1999
;
6
:
361
–75.
40
Villerbu N, Gaben AM, Redeuilh G, Mester J. Cellular effects of purvalanol A: a specific inhibitor of cyclin-dependent kinase activities.
Int J Cancer
2002
;
97
:
761
–9.
41
Mesguiche V, Parsons RJ, Arris CE, et al. 4-Alkoxy-2,6-diaminopyrimidine derivatives: inhibitors of cyclin dependent kinases 1 and 2.
Bioorg Med Chem Lett
2003
;
13
:
217
–22.
42
Breault GA, Ellston RP, Green S, et al. Cyclin-dependent kinase 4 inhibitors as a treatment for cancer. Part 2: identification and optimization of substituted 2,4-bis anilino pyrimidines.
Bioorg Med Chem Lett
2003
;
13
:
2961
–6.
43
Beattie JF, Breault GA, Ellston RP, et al. Cyclin-dependent kinase 4 inhibitors as a treatment for cancer. Part 1: identification and optimization of substituted 4,6-bis anilino pyrimidines.
Bioorg Med Chem Lett
2003
;
13
:
2955
–60.
44
Nugiel DA, Vidwans A, Etzkorn AM, et al. Synthesis and evaluation of indenopyrazoles as cyclin-dependent kinase inhibitors. 2. Probing the indeno ring substituent pattern.
J Med Chem
2002
;
45
:
5224
–32.
45
Yue EW, Higley CA, DiMeo SV, et al. Synthesis and evaluation of indenopyrazoles as cyclin-dependent kinase inhibitors. 3. Structure activity relationships at C3.
J Med Chem
2002
;
45
:
5233
–48.
46
Toogood PL. Cyclin-dependent kinase inhibitors for treating cancer.
Med Res Rev
2001
;
21
:
487
–98.
47
Fry DW, Bedford DC, Harvey PH, et al. Cell cycle and biochemical effects of PD 0183812. A potent inhibitor of the cyclin D-dependent kinases CDK4 and CDK6.
J Biol Chem
2001
;
276
:
16617
–23.
48
Barvian M, Boschelli DH, Cossrow J, et al. Pyrido-[2,3-d]pyrimidin-7-one inhibitors of cyclin-dependent kinases.
J Med Chem
2000
;
43
:
4606
–16.
49
Misra RN, Xiao H, Rawlins DB, et al. 1H-Pyrazolo[3,4-b]pyridine inhibitors of cyclin-dependent kinases: highly potent 2,6-difluorophenacyl analogues.
Bioorg Med Chem Lett
2003
;
13
:
2405
–8.
50
Misra RN, Rawlins DB, Xiao HY, et al. 1H-Pyrazolo[3,4-b]pyridine inhibitors of cyclin-dependent kinases.
Bioorg Med Chem Lett
2003
;
13
:
1133
–6.
51
Zhu G, Conner SE, Zhou X, et al. Synthesis, structure-activity relationship, and biological studies of indolocarbazoles as potent cyclin D1-CDK4 inhibitors.
J Med Chem
2003
;
46
:
2027
–30.
52
Sanchez-Martinez C, Shih C, Faul MM, et al. Aryl[a]pyrrolo[3,4-c]carbazoles as selective cyclin D1-CDK4 inhibitors.
Bioorg Med Chem Lett
2003
;
13
:
3835
–9.
53
Sanchez-Martinez C, Shih C, Zhu G, et al. Studies on cyclin-dependent kinase inhibitors: indolo-[2,3-a]pyrrolo[3,4-c]carbazoles versus bis-indolylmaleimides.
Bioorg Med Chem Lett
2003
;
13
:
3841
–6.
54
Dermatakis A, Luk KC, DePinto W. Synthesis of potent oxindole CDK2 inhibitors.
Bioorg Med Chem
2003
;
11
:
1873
–81.
55
Bramson HN, Corona J, Davis ST, et al. Oxindole-based inhibitors of cyclin-dependent kinase 2 (CDK2): design, synthesis, enzymatic activities, and X-ray crystallographic analysis.
J Med Chem
2001
;
44
:
4339
–58.
56
Kim KS, Kimball SD, Misra RN, et al. Discovery of aminothiazole inhibitors of cyclin-dependent kinase 2: synthesis, X-ray crystallographic analysis, and biological activities.
J Med Chem
2002
;
45
:
3905
–27.
57
Sausville EA. Cyclin-dependent kinase modulators studied at the NCI: pre-clinical and clinical studies.
Curr Med Chem Anti-Canc Agents
2003
;
3
:
47
–56.
58
Senderowicz AM. Novel small molecule cyclin-dependent kinase modulators in human clinical trials.
Cancer Biol Ther
2003
;
2
:
S84
–95.
59
Davies TG, Pratt DJ, Endicott JA, Johnson LN, Noble ME. Structure-based design of cyclin-dependent kinase inhibitors.
Pharmacol Ther
2002
;
93
:
125
–33.
60
Noble MEM, Endicott JA. Chemical inhibitors of cyclin-dependent kinases: insights into design from X-ray crystallographic studies.
Pharmacol Ther
1999
;
82
:
269
–78.
61
Tetsu O, McCormick F. Proliferation of cancer cells despite CDK2 inhibition.
Cancer Cell
2003
;
3
:
233
–45.
62
Ortega SO, Prieto I, Odajima J, et al. Cyclin-dependent kinase 2 is essential for meiosis but not for mitotic cell division in mice.
Nat Genet
2003
;
35
:
25
–31.
63
Geng Y, Yu Q, Sicinska E, et al. Cyclin E ablation in the mouse.
Cell
2003
;
114
:
431
–43.
64
Soni R, Muller L, Furet PSJ, et al. Inhibition of cyclin-dependent kinase 4 (Cdk4) by fascaplysin, a marine natural product.
Biochem Biophys Res Commun
2000
;
275
:
877
–84.
65
Soni R, O'Reilly T, Furet P, et al. Selective in vivo and in vitro effects of a small molecule inhibitor of cyclin-dependent kinase 4.
J Natl Cancer Inst
2001
;
93
:
436
–46.
66
Zhu G, Conner S, Zhou X, et al. Synthesis of quinolinyl/isoquinolinyl[a]pyrrolo[3,4-c]carbazoles as cyclin D1/CDK4 inhibitors.
Bioorg Med Chem Lett
2003
;
13
:
1231
–5.
67
Honma T, Yoshizumi T, Hashimoto N, et al. A novel approach for the development of selective Cdk4 inhibitors: library design based on locations of Cdk4 specific amino acid residues.
J Med Chem
2001
;
44
:
4628
–40.
68
Booher RN, Holman PS, Fattaey A. Human Myt1 is a cell cycle-regulated kinase that inhibits Cdc2 but not Cdk2 activity.
J Biol Chem
1997
;
272
:
22300
–6.
69
Bain J, McLauchlan H, Elliott M, Cohen P. The specificities of protein kinase inhibitors: an update.
Biochem J
2003
;
371
:
199
–204.
70
Fry DW, Kraker AJ, Connors RC, et al. Strategies for the discovery of novel tyrosine kinase inhibitors with anticancer activity.
Anticancer Drug Des
1994
;
9
:
331
–51.
71
Fry DW, Nelson JM, Slintak V, et al. Biochemical and antiproliferative properties of 4-[ar(alk)ylamino]pyridopyrimidines, a new chemical class of potent and specific epidermal growth factor receptor tyrosine kinase inhibitor.
Biochem Pharmacol
1997
;
54
:
877
–87.
72
Holland PM, Abramson RD, Watson R, Gelfand DH. Detection of specific polymerase chain reaction product by utilizing the 5′→3′ exonuclease activity of Thermus aquaticus DNA polymerase.
Proc Natl Acad Sci U S A
1991
;
88
:
7276
–80.
73
Livak KJ, Flood SJ, Marmaro J, Giusti W, Deetz K. Oligonucleotides with fluorescent dyes at opposite ends provide a quenched probe system useful for detecting PCR product and nucleic acid hybridization.
PCR Methods Appl
1995
;
4
:
357
–62.
74
Heid CA, Stevens J, Livak KJ, Williams PM. Real time quantitative PCR.
Genome Res
1996
;
6
:
986
–94.
75
Schabel JFM, Griswold JDP, Laster JWR, Corbett TH, Lloyd HH. Quantitative evaluation of anticancer agent activity in experimental animals.
Pharm Ther
1977
;
1
:
411
–35.
76
Stiegler P, Giordano A. The family of retinoblastoma proteins.
Crit Rev Eukaryot Gene Expr
2001
;
11
:
59
–76.
77
Connell-Crowley L, Harper JW, Goodrich DW. Cyclin D1/Cdk4 regulates retinoblastoma protein-mediated cell cycle arrest by site-specific phosphorylation.
Mol Biol Cell
1997
;
8
:
287
–301.
78
Kitagawa M, Higashi H, Jung HK, et al. The consensus motif for phosphorylation by cyclin D1-Cdk4 is different from that for phosphorylation by cyclin A/E-Cdk2.
EMBO J
1996
;
15
:
7060
–9.
79
Zarkowska T, Mittnacht S. Differential phosphorylation of the retinoblastoma protein by G(1)/S cyclin-dependent kinases.
J Biol Chem
1997
;
272
:
12738
–46.
80
Lukas J, Herzinger T, Hansen K, et al. Cyclin E-induced S phase without activation of the pRb/E2F pathway.
Genes Dev
1997
;
11
:
1479
–92.
81
Leone G, DeGregori J, Sears R, Jakoi L, Nevins JR. Myc and Ras collaborate in inducing accumulation of active cyclin E/Cdk2 and E2F.
Nature
1997
;
387
:
422
–6.
82
Alevizopoulos K, Vlach J, Hennecke S, Amati B. Cyclin E and c-Myc promote cell proliferation in the presence of p16(INK4a) and of hypophosphorylated retinoblastoma family proteins.
EMBO J
1997
;
16
:
5322
–33.
83
Scholzen T, Gerdes J. The Ki-67 protein: from the known and the unknown.
J Cell Physiol
2000
;
182
:
311
–22.
84
Hanks SK, Hunter T. Protein kinases 6—the eukaryotic protein kinase superfamily: kinase (catalytic) domain structure and classification.
FASEB J
1995
;
9
:
576
–96.
85
Markey MP, Angus SP, Strobeck MW, et al. Unbiased analysis of RB-mediated transcriptional repression identifies novel targets and distinctions from E2F action.
Cancer Res
2002
;
62
:
6587
–97.
86
Berthet CB, Aleem E, Vincenzo C, Tessarollo L, Kaldis P. Cdk2 knockout mice are viable.
Curr Biol
2003
;
13
:
1775
–85.
87
Zou X, Ray D, Aziyu A, et al. Cdk4 disruption renders primary mouse cells resistant to oncogenic transformation, leading to Arf/p53-independent senescence.
Genes Dev
2002
;
16
:
2923
–34.
88
Morris EJ, Dyson NJ. Retinoblastoma protein partners.
Adv Cancer Res
2001
;
82
:
1
–54.