REVIEW

Preclinical and Clinical Development of Cyclin-Dependent Kinase Modulators

Adrian M. Senderowicz, Edward A. Sausville

Affiliation of authors: DTP Clinical Trials Unit, Developmental Therapeutics Program, Division of Cancer Treatment and Diagnosis, National Cancer Institute, Bethesda, MD.

Correspondence to: Adrian M. Senderowicz, M.D., National Institutes of Health, Bldg. 10, Rm. 6N113, Bethesda, MD 20892 (e-mail: sendero{at}helix. nih.gov).


    ABSTRACT
 Top
 Abstract
 Introduction to Cell Cycle...
 Targets for Intervention in...
 Indirect cdk Modulators
 Direct cdk Modulators (Small...
 Consequences of cdk Inhibition
 Preclinical Pharmacology of...
 Human Clinical Trials of...
 Preclinical Pharmacology of UCN...
 Phase I Clinical Trials...
 Summary
 Notes
 References
 
In the last decade, the discovery and cloning of the cyclin-dependent kinases (cdks), key regulators of cell cycle progression, have led to the identification of novel modulators of cdk activity. Initial experimental results demonstrated that these cdk modulators are able to block cell cycle progression, induce apoptotic cell death, promote differentiation, inhibit angiogenesis, and modulate transcription. Alteration of cdk activity may occur indirectly by affecting upstream pathways that regulate cdk activity or directly by targeting the cdk holoenzyme. Two direct cdk modulators, flavopiridol and UCN-01, are showing promising results in early clinical trials, in which the drugs reach plasma concentrations that can alter cdk activity in vitro. Although modulation of cdk activity is a well-grounded concept and new cdk modulators are being assessed for clinical testing, important scientific questions remain to be addressed. These questions include whether one or more cdks should be inhibited, how cdk inhibitors should be combined with other chemotherapy agents, and which cdk substrates should be used to assess the biologic effects of these drugs in patients. Thus, modulation of cdk activity is an attractive target for cancer chemotherapy, and several agents that modulate cdk activity are in or are approaching entry into clinical trials.



    INTRODUCTION TO CELL CYCLE REGULATION
 Top
 Abstract
 Introduction to Cell Cycle...
 Targets for Intervention in...
 Indirect cdk Modulators
 Direct cdk Modulators (Small...
 Consequences of cdk Inhibition
 Preclinical Pharmacology of...
 Human Clinical Trials of...
 Preclinical Pharmacology of UCN...
 Phase I Clinical Trials...
 Summary
 Notes
 References
 
After activation of several mitogenic signaling cascades, cells traverse the cell cycle in several tightly controlled phases (Fig. 1). G1 phase separates M and S phases. In this period, cells commit to enter the cell cycle and prepare to duplicate their DNA (1). After G1 phase, cells enter S phase, the period of DNA synthesis (genome duplication). After S phase, cells enter G2 phase, the period in which cells can repair errors that might occur during DNA duplication and thus prevent passing these errors to daughter cells. During G2 phase, cells prepare to enter M phase, the period in which chromatids and then daughter cells separate. After M phase, cells can enter G1 phase again or enter G0 phase, a replicatively quiescent phase. In G0 phase, the cells usually have a diploid amount of DNA, which represents the differentiated functioning cell not committed to the cell cycle.



View larger version (60K):
[in this window]
[in a new window]
 
Fig. 1. Schematic representation of the cell cycle showing pathways for various cyclin-dependent kinases (cdks). PCNA = proliferating cell nuclear antigen.

 
The progression of cells from G1 to S phase is accompanied by the phosphorylation of the retinoblastoma gene product (Rb protein), a tumor suppressor gene active in the control of G1 phase (2,3). Phosphorylation of Rb protein by serine/threonine kinases known as cyclin-dependent kinases (cdks) inactivates Rb (4). The cdks, key regulators of the cell cycle, consist of catalytic subunits that form complexes with proteins known as cyclins. There are at least nine cdks (cdk1-cdk9) (4-7). The cdks that are clearly involved in cell cycle control are cdk1 through cdk7. Although structurally related to cdk1 through cdk7, cdk8 and cdk9 are important transcriptional regulators (5,6). There are at least 15 cyclins (cyclin A through cyclin T) (8-10). Cyclin expression varies during the cell cycle, and indeed the periodic expression of different cyclins defines the start of each phase of the cell cycle and also marks the transitions between the various phases. Cyclins and their cognate cdk catalytic subunits noncovalently form 1 : 1 complexes to produce the cdk holoenzyme. The holoenzyme is activated by the phosphorylation of specific residues in the cdk catalytic subunit. This phosphorylation can be catalyzed by cdk7/cyclin H, which is also known as the cdk-activating kinase (11,12).

Specific cdks operate in distinct phases of the cell cycle. Complexed with their respective D-type cyclin partners, cdk4 and cdk6 are responsible for the cell's progression through G1 phase (Fig. 1). A complex of cdk2 and cyclin E is responsible for the cell's progression from G1 phase to S phase. A complex of cdk2 and cyclin A is required for the cell's progression through S phase, and a complex of cdk1 (also known as cdc2) and cyclin B is required for mitosis (1). These complexes are in turn regulated by a stoichiometric combination with small inhibitory proteins called endogenous cdk inhibitors. The INK4 (inhibitor of cdk4) family includes p16ink4a, p15ink4b, p18ink4c, and p19ink4d, and its members specifically inhibit cyclin D-associated kinases. Members of the kinase inhibitor protein family p21waf1, p27Kip1, and p57kip2 bind and inhibit the activity of complexes of cyclin E and cdk2 and complexes of cyclin A and cdk2 (13-15). Although members of the kinase inhibitor protein family were initially thought to exclusively regulate G1 and S phases, several reports (16-18) demonstrated that these proteins can also regulate the G2/M-phase transition.

DNA synthesis (S phase) begins with the cdk4- and/or cdk6-mediated phosphorylation of Rb protein (which is complexed with the transcriptional factor E2F). Phosphorylated Rb is released from its complex with E2F. The released E2F then promotes the transcription of numerous genes required for the cell to progress through S phase, including thymidylate synthase and dihydrofolate reductase, among others (2,19,20). Additional information about cell cycle regulation can be found in several reviews (21-24).

The vast majority of human cancers have abnormalities in some component of the Rb pathway (Fig. 2) because of hyperactivation of cdks resulting from the overexpression of positive cofactors (cyclins/cdks) or a decrease in negative factors (endogenous cdk inhibitors) or Rb gene mutations (Fig. 2). Therefore, a pharmacologic cdk inhibitor that could be used in "mechanism-based therapy" would be of great theoretical interest as a treatment for many neoplasms (25). This possibility is intriguing because, for cancer patients, the loss of endogenous cdk inhibitors confers poor prognosis. For example, loss of p27kip1 protein predicts a poor outcome in patients with breast, prostate, lung, colon, or gastric carcinoma [reviewed in (26)]. Loss of p16ink4a is clearly associated with poor prognosis in patients with non-small-cell lung cancer or melanoma (26). However, the results with p21waf1 are inconclusive; loss of p21 may be prognostic in certain cancers, but inconsistent results were obtained for breast cancer (26). If validated in larger clinical studies, these markers could be incorporated in the routine pathologic examination of many tumors to determine prognosis.



View larger version (31K):
[in this window]
[in a new window]
 
Fig. 2. Inactivation of the retinoblastoma (Rb) pathway in human cancer. P = phosphate; cdk = cyclin-dependent kinase. The inactivation of the Rb pathway may occur by mutation of the Rb gene itself or by the inactivation of Rb by phosphorylation with the cdks. Cdks can be activated by an increase in the amount of the catalytic enzyme, by an increase in the amount of cofactor (cyclins), or by a decrease in the amount of endogenous cdk inhibitors (i.e., p16ink4a).

 

    TARGETS FOR INTERVENTION IN THE CELL CYCLE
 Top
 Abstract
 Introduction to Cell Cycle...
 Targets for Intervention in...
 Indirect cdk Modulators
 Direct cdk Modulators (Small...
 Consequences of cdk Inhibition
 Preclinical Pharmacology of...
 Human Clinical Trials of...
 Preclinical Pharmacology of UCN...
 Phase I Clinical Trials...
 Summary
 Notes
 References
 
Several strategies for therapeutic intervention could modulate cdk activity (Fig. 3). These strategies are divided into direct efforts that target the catalytic cdk subunit or indirect efforts that target the regulatory pathways that govern cdk activity. Compounds that directly target the catalytic cdk subunit are chemical inhibitors (small molecule cdk inhibitors); these compounds provide the opportunity for rational design of drugs that interact specifically with the adenosine 5'-triphosphate (ATP)-binding site of cdks (27-29). Compounds may inhibit cdk activity by targeting the regulatory pathways that modulate the activity of cdks; by altering the expression and synthesis of the cdk/cyclin subunits or the cdk inhibitory proteins; by modulating the phosphorylation of cdks; by targeting cdk-activating kinase (cdk7); by affecting cdc25 and wee1/myt1 (Fig. 3); or by manipulating the proteolytic machinery that regulates the catabolism of cdk/cyclin complexes or their regulators.



View larger version (29K):
[in this window]
[in a new window]
 
Fig. 3. Possible targets for cyclin-dependent kinase (cdk) inactivation. CKI = endogenous cdk inhibitor; ATP = adenosine 5'-triphosphate; PO4- = phosphate; T160 = threonine-160; T14 = threonine-14; Y15 = tyrosine-15. 1 = cyclin; 2 = CDK (cdk); 3 = CKI (cdk inhibitor); 4 = ATP; 5 = CDK7; 6 = Cdc25, a dual threonine-tyrosine phosphatase that removes phosphates from the negative phosphorylation sites, T14 and Y15; 7 = wee1/myt 1, protein kinases that phosphorylate the negative phosphorylation sites, T14 and Y15. Compounds can be designed to decrease the amount of cofactors (1) or of catalytic subunit (1). Moreover, compounds may increase the expression of endogenous cdk inhibitors (3). Activation of wee1/myt1 (7) or inhibition of cdk7 (5) and cdc25 (6) leads to loss of cdk activity. Direct competitors of ATP at the cdk-binding site (4) may specifically modulate cdk activity.

 

    INDIRECT CDK MODULATORS
 Top
 Abstract
 Introduction to Cell Cycle...
 Targets for Intervention in...
 Indirect cdk Modulators
 Direct cdk Modulators (Small...
 Consequences of cdk Inhibition
 Preclinical Pharmacology of...
 Human Clinical Trials of...
 Preclinical Pharmacology of UCN...
 Phase I Clinical Trials...
 Summary
 Notes
 References
 
cdk Inhibition by Overexpression of Endogenous cdk Inhibitors

When the cdk inhibitor p16ink4A was introduced into a lung cancer cell line with defective endogenous p16ink4A, the cells were arrested in G1 phase (30). This effect occurs only in cells with functional Rb (31). When both p53 and p16ink4a were introduced into cells, apoptotic cell death followed (32). When p21waf1 or p27kip1 were introduced, in vitro and in vivo antitumor effects and G1/S arrest were observed (33,34). When p27 was introduced in several preclinical tumor models, apoptotic cell death was induced, but the relationship between the putative cdk inhibition of p27kip1 and its induction of apoptotic cell death is unclear (35,36).

Several small molecule chemical inhibitors of cdks appear to modulate the expression of cdk inhibitors. For example, lovastatin blocks cells in G1 phase by induction of p21waf1and p27kip1, which leads to the loss of cdk activity (37,38). Rapamycin blocks lymphocytes in G1 phase by preventing the interleukin 2-stimulated degradation of p27kip1 (39). However, it is unclear whether the cell cycle inhibitory effects of these small molecule chemical inhibitors can be solely explained by the induction of p27kip1.

cdk Inhibition by Peptidomimetic-Based Approaches

Another strategy to block cell cycle progression by loss of cdk activity is to use small peptides that mimic the effects of endogenous cdk inhibitors. Several carriers have been tested that introduce peptides into cells, including a 16-residue segment derived from the Drosophila antenappedia protein. When this 16-residue transmembrane carrier was linked to the third ankyrin repeat of the p16ink4A protein and inserted into cells, Rb-dependent G1 arrest and cell senescence were observed (40). Hybrids containing the antenappedia peptide and different p21waf1 peptides were constructed. In a breast-derived cell line, the chimera containing the carboxyl-terminal peptide of p21, amino acids 141-160, had a higher specificity for cdk4/cyclin D than for cdk2/cyclin E and arrested the cells in G1 phase (41). In contrast, in vitro the chimera containing amino-terminal peptides of p21, amino acids 17-33 and 63-77, inhibited both cdk1 and cdk2, and cells transduced with this chimera were arrested in all phases of the cell cycle (42).

Another approach to inhibiting cdk activity is to develop peptides that bind to cdks and inhibit cdk kinase activity (43,44). Colas et al. (43) demonstrated that a 20-amino acid peptide, identified by use of a combinatorial library, specifically binds cdk2 and inhibits its activity at low nanomolar concentrations in vitro. This peptide could act by blocking the interaction of the catalytic subunit with substrates or cyclin cofactors (43). Chen et al. (44) have shown that 8-amino acid peptides derived from the putative cyclin-cdk2-binding region of p21waf1 (PVKRRLFG) and E2F1 (PVKRRLDL) linked to N-terminal residues derived from human immunodeficiency virus Tat protein or antennapedia protein can block cells in S phase. This effect was associated with a loss of cdk2 activity. Although all of the cells tested with these chimeras showed clear evidence of G1/S-phase arrest, immortalized/transformed cells were more prone to apoptotic cell death.

cdk Inhibition by Depletion in cdk/Cyclin Subunits

Antisense technology has been used to deplete messenger RNAs (mRNAs) for cdk and/or cyclins (45-47). When cyclin D1 was depleted from tumor cell lines, a substantial antiproliferative effect was observed that was synergistic with different standard chemotherapeutic agents (45).

Several compounds can inhibit tumor progression by the modulation of cdk/cyclin subunits. In breast carcinoma cell lines, antiestrogens, such as tamoxifen, inhibit the expression of cyclin D and other cell cycle-related proteins and inhibit cdk activity (48). In breast carcinoma cell lines, retinoids, such as all-trans-retinoic acid and 9-cis-retinoic acid, inhibit the expression of multiple cell cycle regulators, including cyclin D1, cyclin D3, cdk2, and cdk4 (49). In some model systems, rapamycin, an inhibitor of FKBP (FK-506-binding protein)/mTOR (mammalian target of rapamycin), was also associated with a decline in cyclin D1 protein (39). Although treatment of cells with any of these compounds may lead to the decline of cyclin proteins and the perturbation of other cell cycle-related proteins, it is unclear how these compounds act. Perhaps the changes result from a direct interaction between the drug and the pathways that regulate the production of cyclin/cdk or result from the G1-phase arrest and/or Rb dephosphorylation, which are observed with these compounds.

cdk Inhibition by Modulation of Proteasomal Machinery

Sequential turnover of certain cell cycle regulators, including cyclins and p27kip1, is mediated by the 20S proteasome, which promotes proteolytic degradation through the ubiquitin/proteasome pathway. Increased turnover of cyclins with the associated loss of cdk activity may lead to cell cycle arrest with or without apoptotic cell death. Inhibiting 20S proteasome-mediated degradation could lead to accumulation of cdk inhibitors, such as p27ink4a, and to cell cycle arrest with or without apoptotic cell death (50). An important unresolved issue is the net effect and/or specificity of modulating proteasomal pathways. Nonspecific proteasome modulation could alter many signaling pathways (by the accumulation of proteins that activate or inhibit cdks) and thus could have a final effect on cells that is difficult to predict.

cdk Activation by Modulation of Upstream Phosphatases/Kinases

The abrogation (overriding) of intact cell cycle checkpoints by upstream phosphatases and/or kinases could induce the "inappropriate acceleration" of certain phases of the cell cycle. For example, when the G2-phase checkpoint is activated by genotoxic stress (i.e., {gamma}-irradiation), the G2 period is extended to allow DNA repair. In the presence of agents that abrogate (override) this checkpoint, premature mitosis occurs, resulting in apoptotic cell death (51). Thus, abolishing the G2 checkpoint might sensitize cells to agents that would normally cause cells to pause or arrest in G2 phase (51,52).


    DIRECT CDK MODULATORS (SMALL MOLECULAR CDK INHIBITORS)
 Top
 Abstract
 Introduction to Cell Cycle...
 Targets for Intervention in...
 Indirect cdk Modulators
 Direct cdk Modulators (Small...
 Consequences of cdk Inhibition
 Preclinical Pharmacology of...
 Human Clinical Trials of...
 Preclinical Pharmacology of UCN...
 Phase I Clinical Trials...
 Summary
 Notes
 References
 
Chemical (small molecular) cdk inhibitors can be subdivided into the following eight families (Fig. 4): 1) purine derivatives (isopentenyladenine, 6-dimethylaminopurine, olomoucine, roscovitine, CVT-313, and purvalanol and its derivatives), 2) butyrolactone I, 3) flavopiridols (flavopiridol and deschloroflavopiridol), 4) staurosporines (staurosporine and UCN-01), 5) toyocamycin, 6) 9-hydroxyellipticine, 7) polysulfates (suramin), and 8) paullones. Not all small molecular cdk inhibitors are specific for cdks. In fact, staurosporine, UCN-01, suramin, 6-dimethylaminopurine, and isopentenyladenine are relatively nonspecific protein kinase inhibitors. In contrast, flavopiridol, butyrolactone I, olomoucine, roscovitine, CVT-313, paullones, and purvalanol derivatives are clearly more selective for cdks. Butyrolactone I, olomoucine, roscovitine, CVT-313, purvalanol, and paullone derivatives are relatively selective for cdk1 and cdk2 but are relatively inactive for cdk4 and cdk6. Flavopiridol can inhibit all cdks tested (53-55)



View larger version (24K):
[in this window]
[in a new window]
 
Fig. 4. Chemical structures of small molecular cdk inhibitors. R = residual group.

 
Olomoucine, Roscovitine, and Other Purine Derivatives

The first cdk inhibitor discovered was dimethylaminopurine (56). This compound was initially shown to inhibit mitosis in sea urchin embryos without inhibiting protein synthesis. Later, dimethylaminopurine was shown to inhibit cdk1 activity (IC50 [concentration that inhibits activity by 50%] = 120 µM) but to be relatively nonspecific (27). Isopentenyladenine, a derivative of dimethylaminopurine, was somewhat more potent and selective for the cdks (IC50 = 55 µM) (57). Other active purine derivatives have been identified in screening campaigns for more specific and potent cdk inhibitors. Olomoucine potently inhibited cdk1 and cdk2 activities (IC50 = 7 µM) (27,57). Roscovitine, a derivative of olomoucine, is a more potent cdk inhibitor (IC50 values for cdk1/cdk2 = 0.7 µM) (27).

The crystal structures of cdk2 complexed with isopentenyladenine, olomoucine, or roscovitine showed that all three inhibitors bind to the ATP site (29,58). CVT-313, another purine analogue, was identified by use of a combinatorial library strategy and the crystal structure of cdk2. Similar to previous analogues, CVT-313 was specific for cdk1 and cdk2 with IC50 values of 4.2 and 1.5 µM, respectively (59).

A combinatorial approach was then used to modify the purine scaffold of 2-fluoro-6-chloropurine, and several compounds that potently and specifically inhibited cdc2 and cdk2 were identified. Four novel compounds (purvalanol-A, purvalanol-B, compound 52, and compound 52E) were characterized through a battery of in vitro kinases experiments (60). The crystal structure of purvalanol-B complexed with cdk2 showed that purvalanol-B bound to the ATP-binding site resembles the binding of olomoucine to cdk2. The more membrane-permeable purvalanol-A was tested on the National Cancer Institute's (NCI's) 60-cell-line anticancer drug screen panel. The average IC50 value of purvalanol-A was 2 µM, demonstrating that it was a more active antiproliferative agent than purvalanol-B (60). Cell cycle studies of purvalanol-A on human fibroblasts showed that it arrested cells in G1/S phase and G2/M phase, compatible with the putative inhibitory properties in cdk1 and cdk2, respectively (60).

Paullones

With the use of the antiproliferative in vitro profile of flavopiridol in NCI's anticancer drug screen panel and the computational algorithm COMPARE, several members of the paullone family were identified (61). Kenpaullone (NSC 664704) potently inhibited cdk1/cyclin B (IC50 = 0.4 µM), cdk2/cyclin A (IC50 = 0.68 µM), cdk2/cyclin E (IC50 = 7.5 µM), and cdk5/p35 (IC50 = 0.85 µM) but had much lower activity toward other kinases (28). Kenpaullone competitively inhibits the binding of ATP, with an apparent Ki (i.e., inhibitory constant) for cdk1/cyclin B of about 2.5 µM. Molecular modeling studies demonstrated that kenpaullone may bind to the ATP-binding site with residue contacts similar to other cdk2 inhibitors (28). Cell cycle effects of kenpaullone were characterized with the MCF10A breast epithelial cell line. Cells were synchronized in G0/G1 phase by serum starvation and then stimulated to re-enter the cell cycle in the presence of vehicle or kenpaullone at its approximate IC50 concentration (30 µM). Twenty hours later, vehicle-treated cells entered S phase. However, cells exposed to kenpaullone were arrested at the G1/S boundary. A similar effect was obtained with another paullone analogue, 10-bromo-paullone (NSC 672234) (28).


    CONSEQUENCES OF CDK INHIBITION
 Top
 Abstract
 Introduction to Cell Cycle...
 Targets for Intervention in...
 Indirect cdk Modulators
 Direct cdk Modulators (Small...
 Consequences of cdk Inhibition
 Preclinical Pharmacology of...
 Human Clinical Trials of...
 Preclinical Pharmacology of UCN...
 Phase I Clinical Trials...
 Summary
 Notes
 References
 
Cell Cycle Arrest

Initial cell cycle studies by van den Heuvel and Harlow (62) demonstrated that ectopic expression of cdk1-dominant negative alleles was able to block U2OS osteosarcoma cell lines at the G2/M-phase boundary. In contrast, expression of dominant negative alleles of cdk2 or cdk3 blocked cells in S phase (62). Thus, roles for each cdk in human cell cycle began to be assigned.

Ectopic expression of endogenous cdk inhibitors (such as p16, p21, or p27) or peptidomimetics derived from p21, p16, or E2F1, as described above in detail, demonstrates the feasibility of using this method to arrest cells in the cell cycle.

As described above, several small molecular cdk inhibitors, including roscovitine, olomoucine, purvalanol, and flavopiridol, arrest cells at either the G1/S- or the G2/M-phase boundaries (53,59,60,63,64). It is unclear why these agents arrest some cells in G1/S phase and other cells in G2/M phase or both.

Apoptotic Cell Death

The antiproliferative effects of olomoucine, flavopiridol, and roscovitine were accompanied by the induction of apoptotic cell death in certain cell types (64-66). In one study (66), the ability of flavopiridol and olomoucine to induce apoptotic cell death varied, depending on the growth status of the cells. That is, flavopiridol or olomoucine protected postmitotic nondividing PC12 neuronal cells from apoptotic cell death after the withdrawal of nerve growth factor. However, flavopiridol did not protect cycling PC12 cells from apoptotic cell death after the withdrawal of nerve growth factor (66). Similarly, cdk4 and cdk6 proteins from dominant negative alleles, but not cdk2 or cdk3 proteins from dominant negative alleles, protected neurons from apoptotic cell death after the withdrawal of nerve growth factor (67). Thus, susceptibility of PC12 cells to flavopiridol- and olomucine-induced apoptotic cell death may vary, depending on the growth state of the cells.

HeLa cervical carcinoma cells treated with staurosporine and tumor necrosis factor-{alpha} were protected from apoptotic cell death by cdc2, cdk2, or cdk3 encoded by dominant negative alleles. However, only cdk2 encoded by a dominant negative allele protected cells from apoptotic cell death induced by ectopic expression of topoisomerase-II{alpha} (68). Finally, certain apoptotic stimuli induce the caspase-mediated cleavage in endogenous cdk inhibitors (p21/p27) or cdk-inhibitory proteins (wee1 and cdc27) leading to activation of cdks (69-71). Thus, cell cycle arrest and/or apoptosis induced by the inhibition of cdks depends on several factors, including the mechanism of inhibition, the type of cells, and the proliferation status of the cells.

Differentiation

During differentiation, cells exit the cell cycle and lose cdk2 activity. Lee et al. (72) tested whether the chemical cdk2 inhibitors flavopiridol and roscovitine could induce a differentiated phenotype by exposing NCI-H358 lung carcinoma cells to a cdk2 antisense construct, flavopiridol, or roscovitine. They observed that all three cdk2 inhibitors could induce mucinous differentiation with the loss of cdk2 activity.

When U937 myelomonocytic leukemia cells were treated with aminopurvalanol, the cells acquired a phenotype characteristic of differentiated macrophages. Moreover, aminopurvalanol, a potent inhibitor of cdk1 and cdk2, appeared to arrest cells at the G2/M boundary and then to induce apoptotic cell death (73). Other investigators (74) observed a similar phenomenon; ectopic expression of p21waf1 or p27kip1 resulted in a "differentiated phenotype" with cells arrested in G1 or G2 phases and a 4N amount of DNA.

Transcriptional Effects

To compare the effects of several chemical cdk inhibitors on the expression of complementary DNA in yeast cells, Gray et al. (60) incubated Saccharomyces cerevisiae with compound 52 and flavopiridol (each at 25 µM ) for 2 hours and measured mRNA by oligonucleotide array methods. Two percent to 3% of the 6200 yeast genes examined showed a greater than twofold change in transcript levels in the presence of these agents. Moreover, almost 50% of affected transcripts were affected by both compound 52 and flavopiridol. These genes fell into distinct groups, including genes that regulate progression of cell cycle, genes that regulate phosphate and cellular energy metabolism, and genes that regulate guanosine 5'-triphosphate (GTP)- or ATP-binding proteins. However, more than 40% of the changes in mRNA were not concordant between flavopiridol and compound 52. These discrepancies might be explained 1) by the broad cdk-inhibitory activity of flavopiridol compared with the selective cdk2/cdk1-inhibitory activities of compound 52, 2) by the different intracellular concentrations achieved by these inhibitors, 3) by the distinctive molecular structures of these inhibitors, or 4) from their putative effects on other cellular targets.


    PRECLINICAL PHARMACOLOGY OF FLAVOPIRIDOL
 Top
 Abstract
 Introduction to Cell Cycle...
 Targets for Intervention in...
 Indirect cdk Modulators
 Direct cdk Modulators (Small...
 Consequences of cdk Inhibition
 Preclinical Pharmacology of...
 Human Clinical Trials of...
 Preclinical Pharmacology of UCN...
 Phase I Clinical Trials...
 Summary
 Notes
 References
 
Of the cdk inhibitors, flavopiridol has advanced the farthest toward clinical applications.

Mechanism of Action

Flavopiridol, also known as L86-8275 or HMR 1275, is a semisynthetic flavonoid derived from rohitukine, an alkaloid isolated from a plant indigenous to India (Fig. 4). Table 1Go contains a summary of the most important preclinical in vitro effects of flavopiridol. Initially, flavopiridol displayed modest activity in vitro as an inhibitor of tyrosine kinase of the epidermal growth factor receptor and an inhibitor of protein kinase A (IC50 = 21 and 122 µM, respectively) (75). However, when flavopiridol was tested in the NCI's 60-cell-line anticancer drug screen panel, its IC50 was 66 nM. This concentration is about 1000 times lower than the concentration required to inhibit protein kinase A and the tyrosine kinase of the epidermal growth factor receptor (75). The antiproliferative effect was not associated with the presence of the epidermal growth factor receptor (75,76). Flavopiridol was shown to arrest cells in G1 phase or at the G2/M boundary, raising the possibility that flavopiridol may inhibit cdk2 and cdk1 (76). Studies using purified cdks showed that flavopiridol inhibits the activities of cdk1, cdk2, and cdk4; this inhibition is competitively blocked by ATP, with a Ki of 41 nM (53,54,76-78). The crystal structure of the complex of deschloroflavopiridol and cdk2 showed that flavopiridol binds to the ATP-binding pocket, with the benzopyran occupying the same region as the purine ring of ATP (79). This observation was concordant with our earlier biochemical studies with flavopiridol (54). Flavopiridol inhibits all cdks thus far examined (IC50 = approximately 100 nM), but it inhibits cdk7 (cdk-activating kinase) less potently (IC50 = approximately 300 nM) (53,54,77).


View this table:
[in this window]
[in a new window]
 
Table 1. Pharmacologic effects of flavopiridol*

 
In addition to directly inhibiting cdks, flavopiridol causes a decrease in the level of cyclin D1, an oncogene that is overexpressed in many human neoplasias. Neoplasms that overexpress cyclin D1 have a poor prognosis (80-82). When MCF-7 human breast carcinoma cells were incubated with flavopiridol, levels of cyclin D1 protein decreased within 3 hours (83). This effect was followed by a decline in the levels of cyclin D3 with no alteration in the levels of cyclin D2 and cyclin E, the remaining G1 cyclins, and then 2 hours later, loss of cdk4 activity occurred. Thus, depletion of cyclin D1 appears to lead to the loss of cdk4 activity (83). The depletion of cyclin D1 is caused by depletion of cyclin D1 mRNA, not by shortening the half-life of the protein. Depletion of cyclin D1 mRNA was associated with a specific decline in cyclin D1 promoter, measured by a luciferase reporter assay (83). The transcriptional repression of cyclin D1 observed after treatment with flavopiridol is consistent with the effects of flavopiridol on yeast cells (see above) and underscores the conserved effect of flavopiridol on eukaryotic cyclin transcription (60). In summary, flavopiridol can induce cell cycle arrest by at least three mechanisms: 1) by direct inhibition of cdk activities by binding to the ATP-binding site; 2) by prevention of the phosphorylation of cdks at threonine-160/161 by inhibition of cdk7/cyclin H (77,78); and 3) for G1-phase arrest, by a decrease in the amount of cyclin D1, an important cofactor for cdk4 and cdk6 activation.

Another important action of flavopiridol is the selective induction of apoptotic cell death. Hematopoietic cell lines are often quite sensitive to flavopiridol-induced apoptotic cell death (65,84-86), but the mechanism(s) by which flavopiridol induces apoptosis have not yet been elucidated. Flavopiridol does not modulate topoisomerase I/II activity (65). In certain hematopoietic cell lines, neither BCL-2/BAX nor p53 appeared to be affected (65,85), whereas, in other systems, BCL-2 may be inhibited (86). It is unclear whether the putative flavopiridol-induced inhibition of cdk activity is required for induction of apoptosis.

Cell cycle arrest, but again with clear evidence of apoptotic cell death, was observed with a panel of head and neck squamous cell carcinoma cell lines, including a cell line (HN30) that is refractory to several DNA-damaging agents, such as {gamma}-irradiation and bleomycin (87). The apoptotic effect was independent of p53 status and was associated with the depletion of cyclin D1 (87). These findings have been corroborated in other preclinical models (88-90). Efforts to understand flavopiridol-induced apoptosis are under way.

To determine whether flavopiridol has antiangiogenic properties, Brusselbach et al. (91) incubated human umbilical vein endothelial cells (HUVECs) with flavopiridol and observed apoptotic cell death even in cells that were not cycling. In another report, Kerr et al. (92) tested flavopiridol in an in vivo angiogenesis model and found that flavopiridol decreased blood vessel formation in the mouse Matrigel model of angiogenesis. Melillo et al. (93) demonstrated that, at low nanomolar concentrations, flavopiridol blunted the induction of vascular endothelial growth factor (VEGF) by hypoxia in human monocytes. This effect was caused by a decreased stability of VEGF mRNA, which paralleled the decline in VEGF protein. Thus, the antitumor activity of flavopiridol may be supplemented by antiangiogenic effects. Whether the various antiangiogenic actions of flavopiridol result from its interaction with a cdk target or other targets requires further study.

To test for synergistic effects with other compounds, cytotoxic assays of flavopiridol in combination with standard chemotherapeutic agents were performed (94,95). Synergistic effects in A549 lung carcinoma cells were demonstrated when treatment with flavopiridol followed treatment with paclitaxel, cytarabine, topotecan, doxorubicin, or etoposide. In contrast, a synergistic effect was observed with 5-fluorouracil only when cells were treated with flavopiridol for 24 hours before addition of 5-fluorouracil. Synergistic effects with cisplatin were not schedule dependent (95). However, Chien et al. (88) failed to demonstrate a synergistic effect between flavopiridol and cisplatin and/or {gamma}-irradiation in bladder carcinoma models.

Several questions about the antiproliferative activity of flavopiridol remain unanswered. Why does treatment with flavopiridol cause some cells to arrest at the G1/S-phase boundary and other cells to arrest at the G2/M-phase boundary? What role does the depletion of D-type cyclins play in flavopiridol-induced G1/S arrest? What is the relationship between cdk inhibition and apoptotic cell death, and what are the targets for flavopiridol-induced apoptotic cell death?

Antitumor Effect in Preclinical Models

Experiments using colorectal (Colo205) and prostate (LnCap/DU-145) carcinoma xenograft models in which flavopiridol was administered frequently over a protracted period demonstrated that flavopiridol is cytostatic (75). This demonstration led to clinical trials in humans of flavopiridol administered as a 72-hour continuous infusion every 2 weeks (96) (see below).

Subsequent studies in some models of human leukemia/lymphoma xenografts demonstrated that flavopiridol administered intravenously as a bolus rendered animals tumor free, whereas flavopiridol administered as an infusion only delayed tumor growth (84). In head and neck (HN-12) xenografts, when flavopiridol was administered as an intraperitoneal bolus daily at 5 mg/kg for 5 days, a substantial growth delay was observed (87). Again, apoptotic cell death and cyclin D1 depletion were observed in tissues from xenografts treated with flavopiridol at peak plasma concentrations of 5-8 µM (84). Based on these results, the feasibility of a phase I trial with the administration of flavopiridol as a 1-hour infusion is currently being explored at the NCI (see below).

Preclinical Pharmacokinetics and Toxicology

Murine plasma concentration-time profiles for flavopiridol exhibited biexponential behavior with mean {alpha} and ß half-lives of 16.4 and 201.0 minutes, respectively. The mean total-body plasma clearance was 22.6 mL/minute per kg, and the mean oral bioavailability after bolus intragavage was approximately 20%. Pharmacokinetic studies in dogs (75) had very similar results.

The metabolism of flavopiridol was investigated in isolated liver perfusion models. Flavopiridol was glucuronidated in the liver, and then this flavopiridol metabolite excreted in the biliary tract. This property underlies flavopiridol's propensity to undergo enterohepatic circulation (97,98). Preclinical pharmacologic and toxicologic evaluations have identified dose-limiting toxic effects as reversible hematopoietic and gastrointestinal effects.


    HUMAN CLINICAL TRIALS OF FLAVOPIRIDOL
 Top
 Abstract
 Introduction to Cell Cycle...
 Targets for Intervention in...
 Indirect cdk Modulators
 Direct cdk Modulators (Small...
 Consequences of cdk Inhibition
 Preclinical Pharmacology of...
 Human Clinical Trials of...
 Preclinical Pharmacology of UCN...
 Phase I Clinical Trials...
 Summary
 Notes
 References
 
Two clinical trials of flavopiridol given as a 72-hour continuous infusion every 2 weeks have been completed (96,99). In the NCI phase I trial of infusional flavopiridol, 76 patients were treated. Dose-limiting toxicity was secretory diarrhea with a maximal tolerated dose of 50 mg/m2 per day for 3 days.

In the presence of antidiarrheal prophylaxis (a combination of cholestyramine and loperamide), patients tolerated higher doses, defining a second maximal tolerated dose, 78 mg/m2 per day for 3 days. The dose-limiting toxicity observed at the higher dose level was reversible hypotension and a substantial proinflammatory syndrome (fever, fatigue, local tumor pain, and modulation of acute-phase reactants) (96).

Tumors in one patient with non-Hodgkin's lymphoma, one patient with colon cancer, and one patient with kidney cancer decreased in size (minor responses = shrinkage of <50%) for more than 6 months. Moreover, one patient with refractory renal cancer achieved a partial response (shrinkage of >50% of masses) (96). Of 14 patients who received flavopiridol for more than 6 months, five patients received flavopiridol for more than 1 year and one patient received flavopiridol for more than 2 years (96). This potential "disease stabilization," which may have been noted in this trial, is consistent with preclinical models, where tumor stasis is observed. Appropriate measurements of cytostatic effects are necessary to confirm that cdk inhibition might be related to this clinical outcome. Plasma concentrations of 300-500 nM flavopiridol, which inhibit cdk activity in vitro, were safely achieved during our trial (96).

In a complementary phase I trial also exploring the use of a 72-hour continuous infusion of flavopiridol every 2 weeks, Thomas et al. (99) found that the dose-limiting toxicity is diarrhea, corroborating the experience of the NCI. Moreover, plasma concentrations of 300-500 nM flavopiridol were also observed. It is interesting that there was one patient in this trial with refractory gastric cancer that had metastasized to the liver who was initially treated surgically and subsequently failed to respond to one treatment regimen of 5-fluorouracil. When treated with flavopiridol, this patient achieved a sustained complete response without any evidence of disease for more than 2 years after treatment was completed.

In September 1998, we began the first phase I trial of a daily 1-hour infusion of flavopiridol for 5 consecutive days every 3 weeks. This dose schedule was based on our antitumor results observed in leukemia/lymphoma and head and neck xenografts treated with flavopiridol (see above). At this time, 27 patients have been treated in this phase I trial. The recommended phase II dose is 37.5 mg/m2 per day for 5 consecutive days. Dose-limiting toxic effects observed at 52.5 mg/m2 per day are nausea/vomiting, neutropenia, fatigue, and diarrhea. Other (non-dose-limiting) side effects are "local tumor pain" and anorexia (Senderowicz AM: unpublished results). To reach higher flavopiridol concentrations, the protocol was amended to administer flavopiridol for 3 days only. Higher peak plasma flavopiridol concentrations (approximately 4 µM) may be obtained with this schedule (Senderowicz AM: unpublished results).

A phase I trial testing the combination of paclitaxel and flavopiridol demonstrated good tolerability with a dose-limiting pulmonary toxicity (100).

Phase II trials of flavopiridol given as a 72-hour continuous infusion to patients with chronic lymphocytic leukemia, non-small-cell lung cancer, non-Hodgkin's lymphoma, or colon, prostate, gastric, head and neck, or kidney cancer, etc., and phase I trials of flavopiridol administered on novel schedules and in combination with standard chemotherapeutic agents are being explored (101-104).

Several important clinical questions remain to be answered in these trials. Is flavopiridol an "effective" anticancer agent? Which is the best schedule for flavopiridol monotherapy? What is the best method to combine flavopiridol and other agents? Which is the most reliable pharmacodynamic parameter to follow in patients? How should "stable disease" be defined in phase II trials?


    PRECLINICAL PHARMACOLOGY OF UCN-01
 Top
 Abstract
 Introduction to Cell Cycle...
 Targets for Intervention in...
 Indirect cdk Modulators
 Direct cdk Modulators (Small...
 Consequences of cdk Inhibition
 Preclinical Pharmacology of...
 Human Clinical Trials of...
 Preclinical Pharmacology of UCN...
 Phase I Clinical Trials...
 Summary
 Notes
 References
 
Staurosporine is a nonspecific protein kinase inhibitor that arrests cell cycle progression in transformed and nontransformed cells at 1-100 nM (105). At similar concentrations, staurosporine inhibits many protein and tyrosine kinases (105). Several analogues of staurosporine have been evaluated to identify compounds with greater specificity for protein kinases.

Mechanism of Action

One staurosporine analogue, UCN-01 (7-hydroxystaurosporine; Fig. 4), has potent activity against several protein kinase C isoenzymes, particularly the Ca2+-dependent protein kinase C with an IC50 of about 30 nM. UCN-01 has lower potency against the novel Ca2+-independent protein kinases C (IC50 = approximately 500 nM) and no effect against the atypical protein kinases C (106-108), similar to the activity of staurosporine. In addition to its effects on protein kinase C, UCN-01 has antiproliferative activity in several human tumor cell lines (109-113). In contrast, another highly selective potent protein kinase C inhibitor, GF 109203X, has a modest antiproliferative activity, despite a similar capacity to inhibit protein kinase C in vitro (110). Thus, these results suggest that the antiproliferative activity of UCN-01 is probably not explained solely by inhibition of protein kinase C. UCN-01 moderately inhibited the activity of immunoprecipitated cdk1 (cdc2) and cdk2 (IC50 = 300-600 nM). However, when intact cells were exposed to UCN-01, "inappropriate activation" of the same kinases occurred (110).

Experimental evidence suggests that DNA damage leads to cell cycle arrest to allow DNA repair. In cells where the G1-phase checkpoint is not active because of p53 inactivation, irradiated cells accumulate in G2 phase because the G2 checkpoint is mediated by the inactivation of cyclin B/cdc2 by wee1 kinase (Fig. 5, A). In contrast, UCN-01 (Fig. 5, B) induces the activation of cdc2/cyclin B and thus promotes cells to enter early mitosis with the onset of apoptotic cell death. These effects could be partially explained by the inactivation of wee1, the kinase that negatively regulates the G2/M-phase transition or activation of cdc25 phosphatase (114). Thus, although UCN-01 at high concentrations can directly inhibit cdks in vitro, UCN-01 can modulate cellular upstream regulators at much lower concentration, leading to inappropriate cdc2 activation by acting on targets that remain to be defined. DNA-damaging agents not only can activate the G2-phase checkpoint but also can activate the S-phase checkpoint (115). Studies from other groups suggest that UCN-01 not only is able to abrogate the G2 checkpoint induced by DNA-damaging agents but also, in some circumstances, is able to abrogate the DNA damage-induced S-phase checkpoint (115).



View larger version (18K):
[in this window]
[in a new window]
 
Fig. 5. Abrogation (overriding) of the G2-phase checkpoint by UCN-01. Cells with wild-type p53 arrest in G1 phase. A) After exposure to genotoxic stress (i.e., {gamma}-irradiation), tumor cell lines with mutated p53 arrest in G2 phase of the cell cycle by inhibition of cyclin B/cdc2. B) After exposure to genotoxic stress (i.e., {gamma}-irradiation), tumor cell lines with mutated p53 incubated with UCN-01 do not arrest in G2 phase and progress to M phase as a result of inappropriate activation of cyclin B/cdc2, leading to premature mitosis and apoptosis.

 
Another pharmacologic feature of UCN-01 is the increased cytotoxicity in cells containing mutated p53 genes (51). In CA-46 Burkitt's lymphoma and HT-29 colon carcinoma cell lines carrying mutated p53 genes, cytotoxicity results when these cells are exposed to UCN-01. Compared with the isogenic wild-type MCF-7 cell line, the MCF-7 cell line with no endogenous p53 because of the ectopic expression of E6, a human papillomavirus type-16 protein, showed enhanced cytotoxicity when treated with a DNA-damaging agent, such as cisplatin, and UCN-01. The synergistic effect of UCN-01 is enhanced with many chemotherapeutic agents, including mitomycin C, 5-fluorouracil, carmustine, and camptothecin, among others (116-122). Therefore, it is possible that combining UCN-01 with these and other agents could improve its therapeutic index.

Another interesting aspect of UCN-01 is its ability to arrest cells in G1 phase of the cell cycle (109,112,113,123-125). Human epidermoid carcinoma A431 cells contain mutated p53. When incubated with UCN-01, these cells were arrested in G1 phase, Rb was hypophosphorylated, and p21waf1 and p27kip1 accumulated (113). However, in another report (125), the antiproliferative effect of UCN-01 was not dependent on the functional status of Rb. Thus, the G1 arrest observed with UCN-01 is apparently independent of the status of p53 and Rb. Further studies on the putative target(s) for UCN-01 in the G1-phase arrest of cells are under way.

Courage et al. (126) found that UCN-01-resistant A549 lung carcinoma cells were sensitive to two other protein kinase C inhibitors, CGP 41251 and Ro 31-8220, and were marginally resistant (about twofold) to etoposide. These UCN-01-resistant cells had lost several protein kinase C isoenzymes, protein kinase C {alpha}, {epsilon}, and |gv. However, the levels of these isoenzymes returned to baseline when these resistant cells were cultured in UCN-01-free medium. Thus, as described above, it is unlikely that the protein kinase C family of signaling proteins is the only target for UCN-01 cytotoxicity (110,126).

Although many important questions have been answered, several questions remain. What is the real target for UCN-01 in G1/S phase-arrested cells? Does protein kinase C play any role in UCN-01-induced cell cycle arrest and/or apoptotic cell death? What is the real target for the G2 checkpoint abrogation?

Spectrum of In Vivo Antitumor Activity

UCN-01 administered by an intravenous or intraperitoneal route displayed antitumor activity in xenograft model systems with breast carcinoma (MCF-7), renal carcinoma (A498), and leukemia (MOLT-4 and HL-60) cell lines (Senderowicz AM: unpublished results). The antitumor effect was greater when UCN-01 was given over a longer period. This requirement for a longer period of treatment was also observed in in vitro models, with highest antitumor activity observed when UCN-01 was present for 72 hours (109). Thus, a clinical trial using a 72-hour continuous infusion every 2 weeks was conducted (described below).

Preclinical Pharmacokinetics and Toxicology

Pharmacokinetics and toxicologic studies using several schedules were done in rats and dogs. When beagle dogs were given a 72-hour continuous infusion of UCN-01, local (site of injection) and gastrointestinal toxic effects were dose-limiting and a steady-state plasma concentration of 330 nM UCN-01 was achieved. Pharmacokinetic parameters were a volume of distribution (6.09 L/kg), a total clearance of 0.6 L/kg per hour with a ß half-life of about 12 hours (127).


    PHASE I CLINICAL TRIALS OF UCN-01
 Top
 Abstract
 Introduction to Cell Cycle...
 Targets for Intervention in...
 Indirect cdk Modulators
 Direct cdk Modulators (Small...
 Consequences of cdk Inhibition
 Preclinical Pharmacology of...
 Human Clinical Trials of...
 Preclinical Pharmacology of UCN...
 Phase I Clinical Trials...
 Summary
 Notes
 References
 
We recently completed the first phase I trial of UCN-01 in humans (128). Clinical features of UCN-01 observed included the unexpectedly long half-life (approximately 30 days). This half-life was 100 times longer than the half-life observed in preclinical models, which was probably caused by the avid binding of UCN-01 to {alpha}1-acid glycoprotein (129,130). Other clinical features were the relative lack of myelotoxicity or gastrointestinal toxicity (prominent side effects observed in animal models), despite very high plasma concentrations achieved (35-50 µM) (128-131). Dose-limiting toxic effects were nausea/vomiting (amenable to standard antiemetic treatments), symptomatic hyperglycemia associated with an insulin-resistance state, and pulmonary toxicity characterized by substantial hypoxemia without obvious radiologic changes. The recommended phase II dose of UCN-01 given on a 72-hour continuous infusion schedule was 42.5 mg/m2 per day (131). One patient with refractory metastatic melanoma developed a partial response that lasted 8 months. Tumors in a few patients with leiomyosarcoma, non-Hodgkin's lymphoma, or lung cancer were stabilized (>=6 months) (131). The concentration of "free" UCN-01 was assessed in saliva. At the recommended phase II dose, concentrations of free UCN-01 that may cause G2 checkpoint abrogation can be achieved.Table 2 compares important clinical and pharmacologic features of the cdk modulators flavopiridol and UCN-01. Future trials are being explored in which UCN-01 would be given by infusion for shorter periods (132) and/or in combination with DNA-damaging agents.


View this table:
[in this window]
[in a new window]
 
Table 2. Phase I trials with cdk modulators

 

    SUMMARY
 Top
 Abstract
 Introduction to Cell Cycle...
 Targets for Intervention in...
 Indirect cdk Modulators
 Direct cdk Modulators (Small...
 Consequences of cdk Inhibition
 Preclinical Pharmacology of...
 Human Clinical Trials of...
 Preclinical Pharmacology of UCN...
 Phase I Clinical Trials...
 Summary
 Notes
 References
 
With knowledge of the role of cdks in cell cycle regulation and the discovery that approximately 90% of all neoplasias are associated with "cdk hyperactivation" leading to the inactivation of the Rb pathway (2), novel cdk inhibitors are being developed. The first two modulators of cdk function tested in clinical trials, flavopiridol and UCN-01, have been observed to reach plasma concentrations that can modulate cdk-related functions. Future clinical trials should determine what is the best schedule for administering chemical cdk inhibitors, should determine what is the best combination of chemical cdk inhibitors and standard chemotherapeutic agents, and should demonstrate cdk modulation in tumor samples from patients treated with cdk inhibitors.


    NOTES
 
Editor's note: E. A. Sausville is a participant in the National Cancer Institute's Cooperative Research and Development Agreement with Hoechst Marian Rousell that manufactures flavopiridol.

We are indebted to colleagues from the Laboratory of Biological Chemistry and Developmental Therapeutics Program Clinical Trials Unit, National Cancer Institute, for all the encouragement and heavy work provided for these projects. We also want to acknowledge the National Cancer Institute and the National Institutes of Health staff for their contributions in these clinical trials. Finally, we want to acknowledge the patients and their relatives for their willingness to participate in our clinical trials and contribute to the advancement of medicine.


    REFERENCES
 Top
 Abstract
 Introduction to Cell Cycle...
 Targets for Intervention in...
 Indirect cdk Modulators
 Direct cdk Modulators (Small...
 Consequences of cdk Inhibition
 Preclinical Pharmacology of...
 Human Clinical Trials of...
 Preclinical Pharmacology of UCN...
 Phase I Clinical Trials...
 Summary
 Notes
 References
 

1 Sherr CJ. Cancer cell cycles. Science 1996;274:1672-7.[Abstract/Free Full Text]

2 Hatakeyama M, Weinberg RA. The role of RB in cell cycle control. Prog Cell Cycle Res 1995;1:9-19.[Medline]

3 Morgan DO, Fisher RP, Espinoza FH, Farrell A, Nourse J, Chamberlin H, et al. Control of eukaryotic cell cycle progression by phosphorylation of cyclin-dependent kinases. Cancer J Sci Am 1998;4 Suppl 1:S77-83.[Medline]

4 Morgan DO. Cyclin-dependent kinases: engines, clocks, and microprocessors. Annu Rev Cell Dev Biol 1997;13:261-91.[Medline]

5 Rickert P, Seghezzi W, Shanahan F, Cho H, Lees E. Cyclin C/CDK8 is a novel CTD kinase associated with RNA polymerase II. Oncogene 1996;12:2631-40.[Medline]

6 Wei P, Garber ME, Fang SM, Fischer WH, Jones KA. A novel CDK9-associated C-type cyclin interacts directly with HIV-1 Tat and mediates its high-affinity, loop-specific binding to TAR RNA. Cell 1998;92: 451-62.[Medline]

7 Grana X, De Luca A, Sang N, Fu Y, Claudio PP, Rosenblatt J, et al. PITALRE, a nuclear CDC2-related protein kinase that phosphorylates the retinoblastoma protein in vitro. Proc Natl Acad Sci U S A 1994;91:3834-8.

8 MacLachlan TK, Sang N, Giordano A. Cyclins, cyclin-dependent kinases and cdk inhibitors: implications in cell cycle control and cancer. Crit Rev Eukaryot Gene Expr 1995;5:127-56.[Medline]

9 Edwards MC, Wong C, Elledge SJ. Human cyclin K, a novel RNA polymerase II-associated cyclin possessing both carboxy-terminal domain kinase and Cdk-activating kinase activity. Mol Cell Biol 1998;18:4291-300.[Abstract/Free Full Text]

10 Peng J, Zhu Y, Milton JT, Price DH. Identification of multiple cyclin subunits of human P-TEFb. Genes Dev 1998;12:755-62.[Abstract/Free Full Text]

11 Kaldis P, Russo AA, Chou HS, Pavletich NP, Solomon MJ. Human and yeast Cdk-activating kinases (CAKs) display distinct substrate specificities. Mol Biol Cell 1998;9:2545-60.[Abstract/Free Full Text]

12 Tassan JP, Schultz SJ, Bartek J, Nigg EA. Cell cycle analysis of the activity, subcellular localization, and subunit composition of human CAK (CDK-activating kinase). J Cell Biol 1994;127:467-78.[Abstract]

13 Sherr CJ, Roberts JM. CDK inhibitors: positive and negative regulators of G1-phase progression. Genes Dev 1999;13:1501-12.[Free Full Text]

14 LaBaer J, Garrett MD, Stevenson LF, Slingerland JM, Sandhu C, Chou HS, et al. New functional activities for the p21 family of CDK inhibitors. Genes Dev 1997;11:847-62.[Abstract]

15 Cheng M, Olivier P, Diehl JA, Fero M, Roussel MF, Roberts JM, et al. The p21(Cip1) and p27(Kip1) CDK `inhibitors' are essential activators of cyclin D-dependent kinases in murine fibroblasts. EMBO J 1999;18:1571-83.[Abstract/Free Full Text]

16 Bates S, Ryan KM, Phillips AC, Vousden KH. Cell cycle arrest and DNA endoreduplication following p21Waf1/Cip1 expression. Oncogene 1998;17:1691-703.[Medline]

17 Dulic V, Stein GH, Far DF, Reed SI. Nuclear accumulation of p21Cip1 at the onset of mitosis: a role at the G2/M-phase transition. Mol Cell Biol 1998;18:546-57.[Abstract/Free Full Text]

18 Niculescu AB 3rd, Chen X, Smeets M, Hengst L, Prives C, Reed SI. Effects of p21(Cip1/Waf1) at both the G1/S and the G2/M cell cycle transitions: pRb is a critical determinant in blocking DNA replication and in preventing endoreduplication [published erratum appears in Mol Cell Biol 1998;18:1763]. Mol Cell Biol 1998;18:629-43.[Abstract/Free Full Text]

19 Zhang HS, Postigo AA, Dean DC. Active transcriptional repression by the Rb-E2F complex mediates G1 arrest triggered by p16INK4a, TGFbeta, and contact inhibition. Cell 1999;97:53-61.[Medline]

20 Dyson N. The regulation of E2F by pRB-family proteins. Genes Dev 1998;12:2245-62.[Free Full Text]

21 DelSal G, Loda M, Pagano M. Cell cycle and cancer: critical events at the G1 restriction point. Crit Rev Oncog 1996;7:127-42.[Medline]

22 Grana X, Reddy EP. Cell cycle control in mammalian cells: role of cyclins, cyclin dependent kinases (CDKs), growth suppressor genes and cyclin-dependent kinase inhibitors (CKIs). Oncogene 1995;11:211-9.[Medline]

23 Pardee AB. Multiple molecular levels of cell cycle regulation. J Cell Biochem 1994;54:375-8.[Medline]

24 Pines J. Cyclins and cyclin-dependent kinases: theme and variations. Adv Cancer Res 1995;66:181-212.[Medline]

25 Sausville EA, Zaharevitz D, Gussio R, Meijer L, Louarn-Leost M, Kunick C, et al. Cyclin-dependent kinases: initial approaches to exploit a novel therapeutic target. Pharmacol Ther 1999;82:285-92.[Medline]

26 Tsihlias J, Kapusta L, Slingerland J. The prognostic significance of altered cyclin-dependent kinase inhibitors in human cancer. Annu Rev Med 1999;50:401-23.[Medline]

27 Meijer L, Kim SH. Chemical inhibitors of cyclin-dependent kinases. Methods Enzymol 1997;283:113-28.[Medline]

28 Zaharevitz DW, Gussio R, Leost M, Senderowicz AM, Lahusen T, Kunick C, et al. Discovery and initial characterization of the paullones, a novel class of small-molecule inhibitors of cyclin-dependent kinases. Cancer Res 1999;59:2566-9.[Abstract/Free Full Text]

29 De Azevedo WF, Leclerc S, Meijer L, Havlicek L, Strnad M, Kim SH. Inhibition of cyclin-dependent kinases by purine analogues: crystal structure of human cdk2 complexed with roscovitine. Eur J Biochem 1997;243:518-26.[Abstract]

30 Jin X, Nguyen D, Zhang WW, Kyritsis AP, Roth JA. Cell cycle arrest and inhibition of tumor cell proliferation by the p16INK4 gene mediated by an adenovirus vector. Cancer Res 1995;55:3250-3.[Abstract]

31 Chintala SK, Fueyo J, Gomez-Manzano C, Venkaiah B, Bjerkvig R, Yung WK, et al. Adenovirus-mediated p16/CDKN2 gene transfer suppresses glioma invasion in vitro. Oncogene 1997;15:2049-57.

32 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.[Medline]

33 Craig C, Wersto R, Kim M, Ohri E, Li Z, Katayose D, et al. A recombinant adenovirus expressing p27Kip1 induces cell cycle arrest and loss of cyclin-Cdk activity in human breast cancer cells. Oncogene 1997;14:2283-9.[Medline]

34 Eastham JA, Hall SJ, Sehgal I, Wang J, Timme TL, Yang G, et al. In vivo gene therapy with p53 or p21 adenovirus for prostate cancer. Cancer Res 1995;55:5151-5.[Abstract]

35 Katayose Y, Kim M, Rakkar AN, Li Z, Cowan KH, Seth P. Promoting apoptosis: a novel activity associated with the cyclin-dependent kinase inhibitor p27. Cancer Res 1997;57:5441-5.[Abstract]

36 Wang X, Gorospe M, Huang Y, Holbrook NJ. p27Kip1 overexpression causes apoptotic death of mammalian cells. Oncogene 1997;15:2991-7.[Medline]

37 Lee SJ, Ha MJ, Lee J, Nguyen P, Choi YH, Pirnia F, et al. Inhibition of the 3-hydroxy-3-methylglutaryl-coenzyme A reductase pathway induces p53-independent transcriptional regulation of p21(WAF1/CIP1) in human prostate carcinoma cells. J Biol Chem 1998;273:10618-23.[Abstract/Free Full Text]

38 Gray-Bablin J, Rao S, Keyomarsi K. Lovastatin induction of cyclin-dependent kinase inhibitors in human breast cells occurs in a cell cycle-independent fashion. Cancer Res 1997;57:604-9.[Abstract]

39 Hashemolhosseini S, Nagamine Y, Morley SJ, Desrivieres S, Mercep L, Ferrari S. Rapamycin inhibition of the G1 to S transition is mediated by effects on cyclin D1 mRNA and protein stability. J Biol Chem 1998;273:14424-9.[Abstract/Free Full Text]

40 Fahraeus R, Paramio JM, Ball KL, Lain S, Lane DP. Inhibition of pRb phosphorylation and cell-cycle progression by a 20-residue peptide derived from p16CDKN2/INK4A. Curr Biol 1996;6:84-91.[Medline]

41 Ball KL, Lain S, Fahraeus R, Smythe C, Lane DP. Cell-cycle arrest and inhibition of Cdk4 activity by small peptides based on the carboxy-terminal domain of p21WAF1. Curr Biol 1997;7:71-80.[Medline]

42 Bonfanti M, Taverna S, Salmona M, D'Incalci M, Broggini M. p21WAF1-derived peptides linked to an internalization peptide inhibit human cancer cell growth. Cancer Res 1997;57:1442-6.[Abstract]

43 Colas P, Cohen B, Jessen T, Grishina I, McCoy J, Brent R. Genetic selection of peptide aptamers that recognize and inhibit cyclin-dependent kinase 2. Nature 1996;380:548-50.[Medline]

44 Chen YN, Sharma SK, Ramsey TM, Jiang L, Martin MS, Baker K, et al. Selective killing of transformed cells by cyclin/cyclin-dependent kinase 2 antagonists. Proc Natl Acad Sci U S A 1999;96:4325-9.[Abstract/Free Full Text]

45 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.[Abstract/Free Full Text]

46 Wang MB, Billings KR, Venkatesan N, Hall FL, Srivatsan ES. Inhibition of cell proliferation in head and neck squamous cell carcinoma cell lines with antisense cyclin D1. Otolaryngol Head Neck Surg 1998;119: 593-9.[Medline]

47 Driscoll B, Wu L, Buckley S, Hall FL, Anderson KD, Warburton D. Cyclin D1 antisense RNA destabilizes pRb and retards lung cancer cell growth. Am J Physiol 1997;273:L941-9.[Abstract/Free Full Text]

48 Watts CK, Sweeney KJ, Warlters A, Musgrove EA, Sutherland RL. Antiestrogen regulation of cell cycle progression and cyclin D1 gene expression in MCF-7 human breast cancer cells. Breast Cancer Res Treat 1994;31:95-105.[Medline]

49 Zhou Q, Stetler-Stevenson M, Steeg PS. Inhibition of cyclin D expression in human breast carcinoma cells by retinoids in vitro. Oncogene 1997;15:107-15.

50 Adams J, Palombella VJ, Sausville EA, Johnson J, Destree A, Lazarus DD, et al. Proteasome inhibitors: a novel class of potent and effective antitumor agents. Cancer Res 1999;59:2615-22.[Abstract/Free Full Text]

51 Wang Q, Fan S, Eastman A, Worland PJ, Sausville EA, O'Connor P. UCN-01: a potent abrogator of G2 checkpoint function in cancer cells with disrupted p53. J Natl Cancer Inst 1996;88:956-65.[Abstract/Free Full Text]

52 Roberge M, Berlinck RG, Xu L, Anderson HJ, Lim LY, Curman D, et al. High-throughput assay for G2 checkpoint inhibitors and identification of the structurally novel compound isogranulatimide. Cancer Res 1998;58:5701-6.[Abstract]

53 Carlson BA, Dubay MM, Sausville EA, Brizuela L, Worland PJ. Flavopiridol induces G1 arrest with inhibition of cyclin-dependent kinase (CDK) 2 and CDK4 in human breast carcinoma cells. Cancer Res 1996;56:2973-8.[Abstract]

54 Losiewicz MD, Carlson BA, Kaur G, Sausville EA, Worland PJ. Potent inhibition of CDC2 kinase activity by the flavonoid L86-8275. Biochem Biophys Res Commun 1994;201:589-95.[Medline]

55 Singh SS, Sausville EA, Senderowicz AM. Cyclin D1 and Cdk6 are the targets for flavopiridol-mediated G1 block in MCF10A breast epithelial cell line [abstract]. Proc Am Assoc Cancer Res 1999;40:28.

56 Meijer L, Pondaven P. Cyclic activation of histone H1 kinase during sea urchin egg mitotic divisions. Exp Cell Res 1988;174:116-29.[Medline]

57 Rialet V, Meijer L. A new screening test for antimitotic compounds using the universal M phase-specific protein kinase, p34cdc2/cyclin Bcdc13, affinity-immobilized on p13suc1-coated microtitration plates. Anticancer Res 1991;11:1581-90.[Medline]

58 Schulze-Gahmen U, Brandsen J, Jones HD, Morgan DO, Meijer L, Vesely J, et al. Multiple modes of ligand recognition: crystal structures of cyclin-dependent protein kinase 2 in complex with ATP and two inhibitors, olomoucine and isopentenyladenine. Proteins 1995;22:378-91.[Medline]

59 Brooks EE, Gray NS, Joly A, Kerwar SS, Lum R, Mackman RL, et al. CVT-313, a specific and potent inhibitor of CDK2 that prevents neointimal proliferation. J Biol Chem 1997;272:29207-11.[Abstract/Free Full Text]

60 Gray NS, Wodicka L, Thunnissen AM, Norman TC, Kwon S, Espinoza FH, et al. Exploiting chemical libraries, structure, and genomics in the search for kinase inhibitors. Science 1998;281:533-8.[Abstract/Free Full Text]

61 Paull KD, Shoemaker RH, Hodes L, Monks A, Scudiero DA, Rubinstein L, et al. Display and analysis of patterns of differential activity of drugs against human tumor cell lines: development of mean graph and COMPARE algorithm. J Natl Cancer Inst 1989;81:1088-92.[Abstract]

62 van den Heuvel S, Harlow E. Distinct roles for cyclin-dependent kinases in cell cycle control. Science 1993;262:2050-4.[Medline]

63 Buquet-Fagot C, Lallemand F, Montagne MN, Mester J. Effects of olomucine, a selective inhibitor of cyclin-dependent kinases, on cell cycle progression in human cancer cell lines. Anticancer Drugs 1997;8: 623-31.[Medline]

64 Meijer L, Borgne A, Mulner O, Chong JP, Blow JJ, Inagaki N, et al. Biochemical and cellular effects of roscovitine, a potent and selective inhibitor of the cyclin-dependent kinases cdc2, cdk2 and cdk5. Eur J Biochem 1997;243:527-36.[Abstract]

65 Parker BW, Kaur G, Nieves-Neira W, Taimi M, Kolhagen G, Shimizu T, et al. Early induction of apoptosis in hematopoietic cell lines after exposure to flavopiridol. Blood 1998;91:458-65.[Abstract/Free Full Text]

66 Park DS, Farinelli SE, Greene LA. Inhibitors of cyclin-dependent kinases promote survival of post-mitotic neuronally differentiated PC12 cells and sympathetic neurons. J Biol Chem 1996;271:8161-9.[Abstract/Free Full Text]

67 Park DS, Morris EJ, Greene LA, Geller HM. G1/S cell cycle blockers and inhibitors of cyclin-dependent kinases suppress camptothecin-induced neuronal apoptosis. J Neurosci 1997;17:1256-70.[Abstract/Free Full Text]

68 Meikrantz W, Schlegel R. Suppression of apoptosis by dominant negative mutants of cyclin-dependent protein kinases. J Biol Chem 1996;271:10205-9.[Abstract/Free Full Text]

69 Gervais JL, Seth P, Zhang H. Cleavage of CDK inhibitor p21(Cip1/Waf1) by caspases is an early event during DNA damage-induced apoptosis. J Biol Chem 1998;273:19207-12.[Abstract/Free Full Text]

70 Levkau B, Koyama H, Raines EW, Clurman BE, Herren B, Orth K, et al. Cleavage of p21Cip1/Waf1 and p27Kip1 mediates apoptosis in endothelial cells through activation of Cdk2: role of a caspase cascade. Mol Cell 1998;1:553-63.[Medline]

71 Zhou BB, Li H, Yuan J, Kirschner MW. Caspase-dependent activation of cyclin-dependent kinases during Fas-induced apoptosis in Jurkat cells. Proc Natl Acad Sci U S A 1998;95:6785-90.[Abstract/Free Full Text]

72 Lee HR, Chang TH, Tebalt MJ 3rd, Senderowicz AM, Szabo E. Induction of differentiation accompanies inhibition of cdk2 in a non-small cell lung cancer cell line. Int J Oncol 1999;15:161-6.[Medline]

73 Rosania GR, Merlie J Jr, Gray N, Chang YT, Schultz PG, Heald R. A cyclin-dependent kinase inhibitor inducing cancer cell differentiation: biochemical identification using Xenopus egg extracts. Proc Natl Acad Sci U S A 1999;96:4797-802.[Abstract/Free Full Text]

74 Liu M, Subramanyam YV, Baskaran N. Preparation and analysis of cDNA from a small number of hematopoietic cells. Methods Enzymol 1999;303:45-55.[Medline]

75 Sedlacek HH, Czech J, Naik R, Kaur G, Worland P, Losiewicz M, et al. Flavopiridol (L86-8275, NSC-649890), a new kinase inhibitor for tumor therapy. Int J Oncol 1996;9:1143-68.

76 Kaur G, Stetler-Stevenson M, Sebers S, Worland P, Sedlacek H, Myers C, et al. Growth inhibition with reversible cell cycle arrest of carcinoma cells by flavone L86-8275. J Natl Cancer Inst 1992;84:1736-40.[Abstract]

77 Carlson B, Pearlstein R, Naik R, Sedlacek H, Sausville E, Worland P. Inhibition of CDK2, CDK4 and CDK7 by flavopiridol and structural analogs [abstract]. Proc Am Assoc Cancer Res 1996;37:424.

78 Worland PJ, Kaur G, Stetler-Stevenson M, Sebers S, Sartor O, Sausville EA. Alteration of the phosphorylation state of p34cdc2 kinase by the flavone L86-8275 in breast carcinoma cells. Correlation with decreased H1 kinase activity. Biochem Pharmacol 1993;46:1831-40.

79 De Azevedo WF Jr, Mueller-Dieckmann HJ, Schulze-Gahmen U, Worland PJ, Sausville E, Kim SH. Structural basis for specificity and potency of a flavonoid inhibitor of human CDK2, a cell cycle kinase. Proc Natl Acad Sci U S A 1996;93:2735-40.[Abstract/Free Full Text]

80 Michalides R, van Veelen N, Hart A, Loftus B, Wientjens E, Balm A. Overexpression of cyclin D1 correlates with recurrence in a group of forty-seven operable squamous cell carcinomas of the head and neck. Cancer Res 1995;55:975-8.[Abstract]

81 Gansauge S, Gansauge F, Ramadani M, Stobbe H, Rau B, Harada N, et al. Overexpression of cyclin D1 in human pancreatic carcinoma is associated with poor prognosis. Cancer Res 1997;57:1634-7.[Abstract]

82 Fredersdorf S, Burns J, Milne AM, Packham G, Fallis L, Gillett CE, et al. High level expression of p27(kip1) and cyclin D1 in some human breast cancer cells: inverse correlation between the expression of p27(kip1) and degree of malignancy in human breast and colorectal cancers. Proc Natl Acad Sci U S A 1997;94:6380-5.[Abstract/Free Full Text]

83 Carlson B, Lahusen T, Singh S, Loaiza-Perez A, Worland PJ, Pestell R, et al. Downregulation of cyclin D1 by transcriptional repression in MCF-7 human breast carcinoma cells induced by flavopiridol. Cancer Res 1999;59:4634-41.[Abstract/Free Full Text]

84 Arguello F, Alexander M, Sterry JA, Tudor G, Smith EM, Kalavar NT, et al. Flavopiridol induces apoptosis of normal lymphoid cells, causes immunosuppression, and has potent antitumor activity in vivo against human leukemia and lymphoma xenografts. Blood 1998;91:2482-90.[Abstract/Free Full Text]

85 Byrd JC, Shinn C, Waselenko JK, Fuchs EJ, Lehman TA, Nguyen PL, et al. Flavopiridol induces apoptosis in chronic lymphocytic leukemia cells via activation of caspase-3 without evidence of bcl-2 modulation or dependence on functional p53. Blood 1998;92:3804-16.[Abstract/Free Full Text]

86 Konig A, Schwartz GK, Mohammad RM, Al-Katib A, Gabrilove JL. The novel cyclin-dependent kinase inhibitor flavopiridol downregulates Bcl-2 and induces growth arrest and apoptosis in chronic B-cell leukemia lines. Blood 1997;90:4307-12.[Abstract/Free Full Text]

87 Patel V, Senderowicz AM, Pinto D Jr, Igishi T, Raffeld M, Quintanilla-Martinez L, et al. Flavopiridol, a novel cyclin-dependent kinase inhibitor, suppresses the growth of head and neck squamous cell carcinomas by inducing apoptosis. J Clin Invest 1998;102:1674-81.[Abstract/Free Full Text]

88 Chien M, Astumian M, Liebowitz D, Rinker-Schaeffer C, Stadler W. In vitro evaluation of flavopiridol, a novel cell cycle inhibitor, in bladder cancer. Cancer Chemother Pharmacol 1999;44:81-7.[Medline]

89 Schrump DS, Matthews W, Chen GA, Mixon A, Altorki NK. Flavopiridol mediates cell cycle arrest and apoptosis in esophageal cancer cells. Clin Cancer Res 1998;4:2885-90.[Abstract]

90 Bible KC, Kaufmann SH. Flavopiridol: a cytotoxic flavone that induces cell death in noncycling A549 human lung carcinoma cells. Cancer Res 1996;56:4856-61.[Abstract]

91 Brusselbach S, Nettelbeck DM, Sedlacek HH, Muller R. Cell cycle-independent induction of apoptosis by the anti-tumor drug flavopiridol in endothelial cells. Int J Cancer 1998;77:146-52.[Medline]

92 Kerr JS, Wexler RS, Mousa SA, Robinson CS, Wexler EJ, Mohamed S, et al. Novel small molecule alpha v integrin antagonists: comparative anti-cancer efficacy with known angiogenesis inhibitors. Anticancer Res 1999;19:959-68.[Medline]

93 Melillo G, Sausville EA, Cloud K, Lahusen T, Varresio L, Senderowicz A. Flavopiridol, a protein kinase inhibitor, down-regulates hypoxic induction of vascular endothelial growth factor expression in human monocytes. Cancer Res 1999;59:5433-7.[Abstract/Free Full Text]

94 Schwartz G, Farsi K, Maslak P, Kelsen D, Spriggs D. Potentiation of apoptosis by flavopiridol in mitomycin-C-treated gastric and breast cancer cells. Clin Cancer Res 1997;3:1467-72.[Abstract]

95 Bible KC, Kaufmann SH. Cytotoxic synergy between flavopiridol (NSC 649890, L86-8275) and various antineoplastic agents: the importance of sequence of administration. Cancer Res 1997;57:3375-80.[Abstract]

96 Senderowicz AM, Headlee D, Stinson SF, Lush RM, Kalil N, Villalba L, et al. Phase I trial of continuous infusion flavopiridol, a novel cyclin-dependent kinase inhibitor, in patients with refractory neoplasms. J Clin Oncol 1998;16:2986-99.[Abstract]

97 Jager W, Zembsch B, Wolschann P, Pittenauer E, Senderowicz AM, Sausville E, et al. Metabolism of the anticancer drug flavopiridol, a new inhibitor of cyclin dependent kinases, in rat liver. Life Sci 1998;62:1861-73.[Medline]

98 Lush R, Stinson S, Senderowicz A, Hill K, Feuer J, Headlee D, et al. Flavopiridol pharmacokinetics suggest entoerohepatic circulation. Clin Pharmacol Ther 1997;61:145.

99 Thomas J, Cleary J, Tutsch K, Arzoomanian R, Alberti D, Simon K, et al. Phase I clinical and pharmacokinetic trial of flavopiridol [abstract]. Proc Am Assoc Cancer Res 1997;38:222.

100 Schwartz G, Kaubisch A, Saltz L, Ilson D, O'Reilly E, Barazzuol J, et al. Phase I trial of sequential paclitaxel and the cyclin-dependent kinase inhibitor flavopiridol [abstract]. Proc ASCO 1999;18:160a.

101 Wright J, Blatner GL, Cheson BD. Clinical trials referral resource. Clinical trials of flavopiridol. Oncology (Huntingt) 1998;12:1018, 23-4.

102 Werner J, Kelsen D, Karpeh M, Inzeo D, Barazzuol J, Sugarman A, et al. The cyclin-dependent kinase inhibitor flavopiridol is an active and unexpectedly toxic agent in advanced gastric cancer [abstract]. Proc ASCO 1998;17:234a.

103 Shapiro G, Patterson A, Lynch C, Lucca J, Anderson I, Boral A, et al. A phase II trial of flavopiridol in patients with stage IV non-small cell lung cancer [abstract]. Proc ASCO 1999;18:522a.

104 Bennett S, Mani S, O'Reilly S, Wright J, Schilsky R, Vokes E, et al. Phase II trial of flavopiridol in metastatic colorectal cancer: preliminary results [abstract]. Proc ASCO 1999;18:277a.

105 Tamaoki T. Use and specificity of staurosporine, UCN-01, and calphostin C as protein kinase inhibitors. Methods Enzymol 1991;201:340-7.[Medline]

106 Takahashi I, Kobayashi E, Asano K, Yoshida M, Nakano H. UCN-01, a selective inhibitor of protein kinase C from Streptomyces. J Antibiot (Tokyo) 1987;40:1782-4.

107 Takahashi I, Saitoh Y, Yoshida M, Sano H, Nakano H, Morimoto M, et al. UCN-01 and UCN-02, new selective inhibitors of protein kinase C. II. Purification, physico-chemical properties, structural determination and biological activities. J Antibiot (Tokyo) 1989;42:571-6.

108 Seynaeve CM, Kazanietz MG, Blumberg PM, Sausville EA, Worland PJ. Differential inhibition of protein kinase C isozymes by UCN-01, a staurosporine analogue. Mol Pharmacol 1994;45:1207-14.[Abstract]

109 Seynaeve CM, Stetler-Stevenson M, Sebers S, Kaur G, Sausville EA, Worland PJ. Cell cycle arrest and growth inhibition by the protein kinase antagonist UCN-01 in human breast carcinoma cells. Cancer Res 1993;53:2081-6.[Abstract]

110 Wang Q, Worland PJ, Clark JL, Carlson BA, Sausville EA. Apoptosis in 7-hydroxystaurosporine-treated T lymphoblasts correlates with activation of cyclin-dependent kinases 1 and 2. Cell Growth Differ 1995;6:927-36.[Abstract]

111 Akinaga S, Gomi K, Morimoto M, Tamaoki T, Okabe M. Antitumor activity of UCN-01, a selective inhibitor of protein kinase C, in murine and human tumor models. Cancer Res 1991;51:4888-92.[Abstract]

112 Akinaga S, Nomura K, Gomi K, Okabe M. Effect of UCN-01, a selective inhibitor of protein kinase C, on the cell-cycle distribution of human epidermoid carcinoma, A431 cells. Cancer Chemother Pharmacol 1994;33:273-80.[Medline]

113 Akiyama T, Yoshida T, Tsujita T, Shimizu M, Mizukami T, Okabe M, et al. G1 phase accumulation induced by UCN-01 is associated with dephosphorylation of Rb and CDK2 proteins as well as induction of CDK inhibitor p21/Cip1/WAF1/Sdi1 in p53-mutated human epidermoid carcinoma A431 cells. Cancer Res 1997;57:1495-501.[Abstract]

114 Yu L, Orlandi L, Wang P, Orr M, Senderowicz AM, Sausville EA, et al. UCN-01 abrogates G2 arrest through a cdc2-dependent pathway that is associated with inactivation of the Wee1Hu kinase and activation of the Cdc25C phosphatase. J Biol Chem 1998;273:33455-64.[Abstract/Free Full Text]

115 Bunch RT, Eastman A. 7-Hydroxystaurosporine (UCN-01) causes redistribution of proliferating cell nuclear antigen and abrogates cisplatin-induced S-phase arrest in Chinese hamster ovary cells. Cell Growth Differ 1997;8:779-88.[Abstract]

116 Akinaga S, Nomura K, Gomi K, Okabe M. Enhancement of antitumor activity of mitomycin C in vitro and in vivo by UCN-01, a selective inhibitor of protein kinase C. Cancer Chemother Pharmacol 1993;32:183-9.[Medline]

117 Bunch RT, Eastman A. Enhancement of cisplatin-induced cytotoxicity by 7-hydroxystaurosporine (UCN-01), a new G2-checkpoint inhibitor. Clin Cancer Res 1996;2:791-7.[Abstract]

118 Hsueh CT, Kelsen D, Schwartz GK. UCN-01 suppresses thymidylate synthase gene expression and enhances 5-fluorouracil-induced apoptosis in a sequence-dependent manner. Clin Cancer Res 1998;4:2201-6.[Abstract]

119 Husain A, Yan XJ, Rosales N, Aghajanian C, Schwartz GK, Spriggs DR. UCN-01 in ovary cancer cells: effective as a single agent and in combination with cis-diamminedichloroplatinum(II)independent of p53 status. Clin Cancer Res 1997;3:2089-97.[Abstract]

120 Pollack IF, Kawecki S, Lazo JS. Blocking of glioma proliferation in vitro and in vivo and potentiating the effects of BCNU and cisplatin: UCN-01, a selective protein kinase C inhibitor. J Neurosurg 1996;84:1024-32.[Medline]

121 Shao RG, Cao CX, Shimizu T, O'Connor PM, Kohn KW, Pommier Y. Abrogation of an S-phase checkpoint and potentiation of camptothecin cytotoxicity by 7-hydroxystaurosporine (UCN-01) in human cancer cell lines, possibly influenced by p53 function. Cancer Res 1997;57:4029-35.[Abstract]

122 Tsuchida E, Urano M. The effect of UCN-01 (7-hydroxystaurosporine), a potent inhibitor of protein kinase C, on fractionated radiotherapy or daily chemotherapy of a murine fibrosarcoma. Int J Radiat Oncol Biol Phys 1997;39:1153-61.[Medline]

123 Akiyama T, Shimizu M, Okabe M, Tamaoki T, Akinaga S. Differential effects of UCN-01, staurosporine and CGP 41 251 on cell cycle progression and CDC2/cyclin B1 regulation in A431 cells synchronized at M phase by nocodazole. Anticancer Drugs 1999;10:67-78.[Medline]

124 Kawakami K, Futami H, Takahara J, Yamaguchi K. UCN-01, 7-hydroxyl-staurosporine, inhibits kinase activity of cyclin-dependent kinases and reduces the phosphorylation of the retinoblastoma susceptibility gene product in A549 human lung cancer cell line. Biochem Biophys Res Commun 1996;219:778-83.[Medline]

125 Shimizu E, Zhao MR, Nakanishi H, Yamamoto A, Yoshida S, Takada M, et al. Differing effects of staurosporine and UCN-01 on RB protein phosphorylation and expression of lung cancer cell lines. Oncology 1996;53:494-504.[Medline]

126 Courage C, Bradder SM, Jones T, Schultze-Mosgau MH, Gescher A. Characterisation of novel human lung carcinoma cell lines selected for resistance to anti-neoplastic analogues of staurosporine. Int J Cancer 1997;73:763-8.[Medline]

127 Kurata N, Kuwabara T, Tanii H, Fuse E, Akiyama T, Akinaga S, et al. Pharmacokinetics and pharmacodynamics of a novel protein kinase inhibitors, UCN-01. Cancer Chemother Pharmacol 1999;44:12-8.[Medline]

128 Senderowicz AM, Headlee D, Lush R, Bauer K, Figg W, Murgo A, S, et al. Phase I trial of infusional UCN-01, a novel protein kinase inhibitor, in patients with refractory neoplasms [abstract]. In: 10th National Cancer Institute-European Organization for Research on Treatment of Cancer Symposium Proceedings. Dordrecht (The Netherlands): Kluwer Academic Publishers; 1998. p. 111.

129 Sausville EA, Lush RD, Headlee D, Smith AC, Figg WD, Arbuck SG, et al. Clinical pharmacology of UCN-01: initial observations and comparison to preclinical models. Cancer Chemother Pharmacol 1998;42 Suppl:S54-9.[Medline]

130 Fuse E, Tanii H, Kurata N, Kobayashi H, Shimada Y, Tamura T, et al. Unpredicted clinical pharmacology of UCN-01 caused by specific binding to human alpha1-acid glycoprotein. Cancer Res 1998;58:3248-53.[Abstract]

131 Senderowicz AM, Headlee D, Lush R, Bauer K, Figg W, Murgo AS, et al. Phase I trial of infusional UCN-01, a novel protein kinase inhibitor, in patients with refractory neoplasms [abstract]. Proc ASCO 1999;18:159a.

132 Tamura T, Sasaki Y, Minami H, Fujii H, Ito K, Igarashi T, et al. Phase I study of UCN-01 by 3-hour infusion [abstract]. Proc ASCO 1999;18:159a.

Manuscript received June 15, 1999; revised December 7, 1999; accepted December 15, 1999.


This article has been cited by other articles in HighWire Press-hosted journals:


             
Copyright © 2000 Oxford University Press (unless otherwise stated)
Oxford University Press Privacy Policy and Legal Statement