For personal use. Only reproduce with permission from The Lancet.
THE LANCET Oncology Vol 5 January 2004 http://oncology.thelancet.com 27
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.
Lancet Oncol 2004; 5: 27–36
Eukaryotic cell division occurs in four phases of the cell cycle
(figure 1). The cell is prepared for DNA replication in G1
phase, then chromosomes are replicated during S phase. A
gap period, G2, allows preparation for mitosis, before
chromosome segregation and cytokinesis in M phase
(mitosis). During development, differentiation, or growth-
factor withdrawal, cells can enter an inactive period G0,
before returning to G1.
Cell-cycle checkpoints ensure faithful chromosome
replication and separation, thereby maintaining genetic
stability. Failure of these checkpoints to arrest the cell after
appropriate stimuli is a hallmark of cancer.
1
One check-
point, the restriction point, occurs in mid-G1; after this
point, cells become independent of growth factors and
commit to cell division.
2
Genetic aberrations of regulators of
restriction-point passage occur at high frequency in human
tumours.
This review discusses the cyclin/cyclin-dependent kinase
(cdk) complex and the cdk inhibitors (CKIs) that coordinate
restriction-point passage. The discussion covers phase I and
II data on agents that target cyclin/cdk activity, such as
flavopiridol, UCN-01, and inhibitors of proteosomes and
histone deacetylase.
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 amount of DNA, they are not
arrested at any defined point in the cell cycle. When growth-
factor restimulation occurs, cells do not enter S phase
synchronously, as might be expected from the restriction-
point model, but in a sequence determined by their order
before growth-factor withdrawal.
3
For the purposes of this
review, however, discussion is based on the widely accepted
restriction-point model.
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). Restriction-
point 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
Review
Cell-cycle targeted therapies
CS is a clinical scientist and specialist registrar at the Royal
Marsden Hospital Breast Unit, London, UK.
Correspondence: Dr Charles Swanton, Royal Marsden Hospital
NHS Trust, Fulham Road, London SW3 6JJ, UK. Tel: +44 (0) 207
352 8171. Email: RobertCharles.Swanton@rmh.nthames.nhs.uk
Cell-cycle targeted therapies
Charles Swanton
Figure 1. Fluorescent light micrograph of human HeLa cells in M phase of
the cell cycle.

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Rights were not granted to include this
image in electronic media. Please refer
to the printed journal.

For personal use. Only reproduce with permission from The Lancet.
THE LANCET Oncology Vol 5 January 2004 http://oncology.thelancet.com28
activities of both cyclins D and E are required for regulated
progression from G1 into S phase mediated, partly, through
the coordinated phosphorylation of the retinoblastoma
substrate (Rb). Unphosphorylated Rb represses E2F-
mediated transcription of genes necessary for entry to
S phase and DNA replication. Progressive phosphorylation
of Rb through G1 phase leads to the release of E2F from Rb-
mediated repression and transcription of genes necessary for
entry to S phase. The regulation of cyclin/cdk activity is
therefore central to restriction-point passage.
Regulation of cdk
The cdks are regulated predominantly at the post-
translational level, because protein concentrations remain
constant throughout the cell cycle. Inhibitory phos-
phorylation 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 cdk-
activating kinase (cyclin H-cdk7). Further regulation of
cyclin/cdk activity is mediated by the CKI of the p16
ink4
and
p21 families.
The p16
ink4a
family
There are four members of the p16 family named according
to their molecular weights, p16
ink4a
and p15
ink4b
, which share a
single genomic locus (9p21), p18
ink4c
, and p19
ink4d
. 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 role in the inhibition of
cyclin D activity, growth arrest depends on functional Rb.
The Ink4a locus (9p21) encodes three proteins, p15, p16,
and alternative reading frame (ARF) protein.
4
p16 is
encoded by three exons (1 alpha, 2, and 3). An alternative
exon 1 beta, 5 to exon 1 alpha, is spliced to the identical
acceptor sites of Ink4a exon 2 encoding the p14
ARF
protein, of
132 aminoacids (figure 2). There is no sequence homology
between p14
ARF
and p16.
Mouse deletion studies show that both ARF and p16 are
candidate tumour suppressors. Mice nullizygous for ARF
develop both lymphomas and fibrosarcomas.
5
ARF function
depends on p53 for growth arrest, associated with increased
p21 expression. Mice nullizygous for p16 have a higher than
normal frequency of spontaneous tumours (soft-tissue
sarcomas, splenic lymphomas, and metastatic melanoma)
and an increased propensity to carcinogen-induced
tumorigenesis.
6,7
These studies confirm the importance of
this extraordinary locus in tumorigenesis and indicate that
both p16
ink4a
and ARF are tumour-suppressor proteins.
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 p14
ARF
). 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. Tumour-
specific alterations in p16 can affect protein function by
impairing the interaction with cdk4/6, reducing inhibitory
activity of p16—a tumour-derived mutation of p16, P114L,
causes the protein not to bind cdk4 in vitro and also leads to
failure to induce a G1 arrest.
8
Conversely, cdk4 mutations
have been identified in melanoma that reduce the ability of
cdk4 to interact with p16.
9
Mutations that do not seem to
affect the interaction with cdk4/6 could lead to reduced
stability of the protein or affect its folding directly.
10
Finally,
methylation of CpG residues adjacent to and within exon 1a
may lead to silencing of p16 expression in tumours.
p21
cip1
family
The p21 family consists of three members, p21
cip1
, p27
kip1
, and
p57
kip2
. 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-E/cdk2-kinase activity. p27 concen-
trations increase with cell–cell contact, serum deprivation,
and transforming growth factor , and decline on mitogenic
stimulation.
Members of the p21 family interact with both cyclin D
and cyclin E complexes in G1 phase but preferentially inhibit
cdk2 activity. p21 proteins promote assembly of cyclin-D-
family/cdk4 complexes both in vivo and in vitro.
11
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
Kaposi’s sarcoma herpesvirus (HHV-8) evade inhibition by
Review
Cell-cycle targeted therapies
p15
ink4b
Ex1 Ex2 Ex1 Ex1 Ex2 Ex3
Ex1 Ex2
Ex1 Ex2 Ex3
Ex1 Ex2 Ex3
p16
ink4a
p14
ARF
Figure 2. The 9p21 locus. Three genes are encoded on locus 9p21:
p15
ink4b
, p16
ink4a
, and p14
ARF
.