Elsevier

The Lancet Oncology

Volume 5, Issue 1, January 2004, Pages 27-36
The Lancet Oncology

Review
Cell-cycle targeted therapies

https://doi.org/10.1016/S1470-2045(03)01321-4Get rights and content

Summary

Eukaryotic organisms depend on an intricate and evolutionary conserved cell cycle to control cell division. The cell cycle is regulated by a number of important protein families which are common targets for mutational inactivation or overexpression in human tumours. The cyclin D and E families and their cyclin-dependent kinase partners initiate the phosphorylation of the retinoblastoma tumour suppressor protein and subsequent transition through the cell cycle. Cyclin/cdk activity and therefore control of cell division is restrained by two families of cyclin dependent kinase inhibitors. A greater understanding of the cell cycle has led to the development of a number of compounds with the potential to restore control of cell division in human cancers. This review will introduce the protein families that regulate the cell cycle, their aberrations in malignant progression and pharmacological strategies targeting this important process.

Section snippets

Cell-cycle controversies and the continuum model

Although majority opinion accepts the restriction point as an important model on which to base the interpretation of current cell-cycle data, challenges have been raised to the concepts of restriction point, G0 phase, and cellular checkpoints, based on criticisms of experimental methods used to synchronise cell cultures. Rather than proposing that cells arrest at the restriction point on withdrawal of growth factor, the continuum model predicts that, although cells are arrested with a G1 phase

Cell-cycle regulators and restriction-point control

Disruption of restriction-point control is a common biological feature in human cancer. Cell-cycle progression is regulated by two protein classes, the cyclins and their kinase partners, the cyclin-dependent kinases (cdks). Restrictionpoint passsage is coordinated by two families of cyclins, the cyclin D family (D1, D2, and D3) and the cyclin E family (E1 and E2). The D-type cyclins bind to and activate cdks 4 and 6, and cyclins E1 and E2 interact with and activate cdk2. The activities of both

Regulation of cdk

The cdks are regulated predominantly at the posttranslational level, because protein concentrations remain constant throughout the cell cycle. Inhibitory phosphorylation on N-terminal threonine and tyrosine residues of cdk maintains kinase complexes in an inactive state. Positive regulation of cdk activity occurs in two steps: dephosphorylation of the threonine and tyrosine residues by the cdc25 phosphatase family (cdc25 A, B, and C) and phosphorylation of a central threonine residue by

The p16ink4a family

There are four members of the p16 family named according to their molecular weights, p16ink4a and p15ink4b, which share a single genomic locus (9p21), p18ink4c, and p19ink4d. p16 interacts with and inhibits cdk4 and cdk6, forming binary complexes with cdk4 in vitro (hence its name, Ink4a, inhibitor of cdk4). Addition of extracts containing proteins of the Ink4 family to active cyclin-D/cdk4 and cyclin-D/cdk6 complexes inhibits their ability to phosphorylate Rb substrate. In keeping with their

p16 mutations

The 9p21 locus is commonly disrupted in human tumours, resulting in aminoacid mutations or premature terminations that affect each of the three encoded proteins alone or in combination (homozygous deletion of this locus is common in both glioblastoma and melanoma affecting both p16 and p14ARF). The table on page 29 documents the primary cancers in which mutations or deletions at the p16 locus are found in over 15% of tumours studied. Tumourspecific alterations in p16 can affect protein function

p21cip1 family

The p21 family consists of three members, p21cip1, p27kip1, and p57kip2. Unlike the p16 proteins, which interact with and inhibit cdk4 or cdk6, the p21 family can interact with both the cyclin and cdk subunits. p21 was identified as a cdk2-interacting protein; subsequently Waf1, the same protein, was found to be expressed in diverse cell types depending on p53 expression. Gamma or ultraviolet irradiation leads to a p53-dependent G1 arrest with increased p21 protein and inhibition of

Cyclin-binding motifs

The p21/p27 interaction with the cyclin is important for cdk inhibition. Deletion of a cyclin-binding motif in p21 results in loss of binding to cyclin E and abolishes the ability of p21 to inhibit cyclin-D1/cdk4 activity. There are at least three classes of protein with similar cyclin-binding motifs: those that inhibit cyclin/cdk activity (p21 family); those that increase cdk activation (cdc25A); and the substrates themselves (E2F). The viral cyclins encoded by gammaherpesviruses including

p21/p27 localisation and degradation

Protein turnover through the ubiquitin/ proteosome is an essential means of p27 regulation. Phosphorylation of p27 on threonine 187 by cyclin-E/cdk2 leads directly to a decline in p27 protein concentrations.15 Recognition of the phosphorylated form of p27 by the ubiquitin ligase SCF(Skp2) targets p27 for degradation. Colorectal tumours with low or absent expression of p27 protein show increased proteolytic activity specific for p27.16 Patients with colon cancer and low or absent p27 protein had

G1-phase restriction point and Rb gene product

The Rb tumour suppressor is one of the mammalian regulators of passage through the restriction point. Rb deletions are frequent in retinoblastomas and osteosarcomas, and gene mutations have been found in a diverse subset of tumours, including many sarcomas, small-cell lung carcinomas, and bladder carcinomas. Changes in regulators of the Rb pathway occur in 90% of human cancers.24

Rb exists in various states of phosphorylation, progressing from an unphosphorylated transcriptionally repressive

Cyclin D family

Growth-factor and mitogenic signals converge on the cell cycle to stimulate expression of the cyclin D family (figure 6). Cyclin D is the prime integrator of these cellular signals to initiate progression through the early phase of the G1 period. The cyclin D family acts in two ways, first by cdk4/6-dependent phosphorylation and partial inactivation of the repressive activity of Rb (figure 4), and second by titrating the p27 molecule from inactive cyclin-E/cdk2 complexes.

The signalling pathways

Cyclin E family

Cyclin-D/cdk complexes regulate the activity of cyclin-E/cdk2 in two ways. First, by partly inactivating the repressive activity of Rb, the E2F transcription factor can initiate transcription of cyclin E. Second, cyclin-D/cdk complexes sequester the cdk2 inhibitor p27Kip1 from previously inactive cyclin-E/cdk2/p27Kip1x complexes.

Cyclin E, like cyclin D, is also targeted for degradation by phosphorylation. The active cyclin-E/cdk2 complex itself phosphorylates the C-terminal threonine 380. The

Therapeutic strategies

Interventions to mimic physiological cdk inhibitors have targeted the ATP-binding site of the cdk molecule. Clinical trials of one such agent, flavopiridol, have begun. The cellcycle modulator UCN-01 has a complex mode of action and can both promote and inhibit cdk activity. Pharmacological agents targeting the proteosome or histone deacetylases interfere with the degradation and expression of molecules that police the restriction point. Promising phase I and II trial data have emerged for

Kinase inhibition with flavopiridol

Flavopiridol interacts with the ATP-binding pocket of cdk2. The drug inhibits all cdks including cdk7, leads to a reduction in cyclin D1 mRNA transcription, and arrests cells in G1 or at the G2/M transition. Blockade of cdk7 can interfere with activation of cdks mediated by cdk-activating kinase, providing an alternative route for cdk inhibition.

In the earliest dose-escalation study at the US National Cancer Institute with 76 patients receiving a 72 h infusion of flavopiridol every 2 weeks,

Kinase inhibition with UCN-01

UCN-01 was first isolated from the culture broth of a Streptomyces and found to be an inhibitor of the calciumdependent protein kinase C isoenzymes. The compound was subsequently discovered to have many direct and indirect effects on cell-cycle targets. UCN-01 induces arrest with a G1-phase amount of DNA in normal cells dependent on functional Rb, attributable to a decline in cdk2 activity through increased p27/cdk2 interaction.51 In non-small-cell lung cancer cell lines expressing Rb, UCN-01

Proteosome inhibition with bortezomib

Manipulation of ubiquitination and subsequent proteosome-mediated degradation of specific cell-cycle proteins could prove advantageous in cancer therapy. Proteosome inhibition is particularly compelling, because it might exploit the genetic aberrations found in tumour cells similar to a situation when an increase in p27 protein, despite constant c-myc concentrations after proteasome inhibition, is seen to accompany apoptosis of human leukaemic HL60 cells.59 Proteosome inhibition arrests normal

Unclassified cell-cycle inhibitors

E7070 (ER-35744) was synthesised during the search for sulphonamide-related compounds with cell-cycle inhibitory activity. E7070 inhibits the activation of cyclin E/cdk2 and has cytotoxic activity against human HCT116 colon carcinoma and LX-1 non-small-cell lung carcinoma xenografts.63, 64 In the HCT116 xenograft, E7070 treatment improved activity over fluorouracil, mitomycin c, and irinotecan.64 The exact mechanism of cyclin-E/cdk2 inactivation is unclear.

Four different treatment schedules

Histone deactetylase inhibition

Histone acetylation and deacetylation is central to the control of gene transcription via modification of chromatin structure. The repression of E2F-dependent genes occurs partly by the Rb-mediated recruitment of a histone deacetylase to E2F promoter sites; removal of the acetyl group from lysine residues restores the positive charge, limiting chromatin transcriptional activity.

Paradoxically, inhibition of histone deacetylase leads to a gene expression profile associated with differentiation

Conclusion

There are many unanswered questions about cell-cycletargeted therapies. The challenges of how to assess treatment response to cytostatic agents and their sequence of administration when used in combination with conventional cytotoxic agents will have to be confronted in more advanced clinical studies. Nevertheless, with improvement in our understanding of both the mechanism of action of these agents and the intricate regulation of the cell cycle, the opportunity to offer treatment directed

Search strategy and selection criteria

PubMed was used to search for relevant publications in the past 20 years with bias to more recent publications. The search terms used were: “cyclin D”, “cyclin D1”, “cyclin D2”, “cyclin D3”, “cyclin E”, “cyclin E1”, “cyclin E2”, “retinoblastoma”, “pRB”, “cdk2”, “cdk4”, “cdk6”, “CKI”, “cdk inhibitor”, “p16ink4”, “p21cip1”, “p27kip1”, “p57kip2”, “familial melanoma”, “flavopiridol”, “UCN-01”, “staurosporine”, “E7070”, “proteosome inhibitor”, “PS-341”, “histone deacetylase inhibitor”, and

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    CS is a clinical scientist and specialist registrar at the Royal Marsden Hospital Breast Unit, London, UK.

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