Oral Oncology 36 (2000) 3±7

www.elsevier.com/locate/oraloncology

Commentary

An overview of the cell cycle arrest protein, p21WAF1

K. Harada*, G.R. Ogden
Oral Diseases Group, Unit of Oral Surgery and Medicine, Dundee Dental Hospital and School, Park Place, Dundee DD1 4HN, UK

Received 16 June 1999; accepted 22 June 1999

Abstract
p21, also known as WAF1, Cip1, Sdi1, Mda 6 and Cap20 is a cell cycle protein that regulates and can arrest the cell cycle in G1
or S phase (either dependent or independent of p53). Its role may be pivotal in many cell processes including di€erentiation and
apoptosis. This brief overview provides a summary of its presently known functions and indicates areas for further research, par-
ticularly in relation to oral malignant disease. Greater understanding of its role may lead to therapeutic advances in the manage-
ment of malignant disease. # 2000 Elsevier Science Ltd. All rights reserved.
Keywords: p21WAF1; Cell cycle; Arrest protein; Functions

1. Introduction ®broblasts associated tightly with a 20 K ptrotein) was

p21WAF1 is a 21-kDa cell cycle regulatory protein
encoded by the p21WAF1 gene which is located on chro-
mosome 6p21.2 [1]. p21WAF1 is also known as Cip1,
Sdi1, Mda6 and Cap20 according to the di€erent func-
tions assigned to it by various groups since it has been
cloned independently by a number of di€erent routes.
These are outlined below. WAF1 (wild-type p53-
activated fragment 1) was cloned by subtractive hybridi-
zation for genes activated by wild-type p53 [1]. Cip1
[cyclin-dependent kinase (CDK) interacting protein]
was found to encode a protein that interacted with the
cyclin/cdk2 complex and inhibited CDK that drove
the cell division cycle [2]. p21 was discovered by two-
hybrid screening in yeast for human proteins that inter-
acted with Cdk2, or on peptide sequencing and subse-
quent cloning of a protein co-immunoprecipitating with
cyclin D [3]. Sdi1 (senescent cell-derived inhibitor-1) was
found to encode an inhibitory factor of DNA synthesis
in human senescent cells [4]. Mda6 (melanoma dif-
ferentiation-associated gene 6) was identi®ed using
subtraction hybridization by virtue of its enhanced
expression in human melanoma cells induced to dif-
ferentiate terminally by treatment with human ®bro-
blast interferon and the anti-leukaemic compound
mezerein [5]. Cap20 (CDK2/cyclin complexes in mouse
* Corresponding author. Tel.: +44-1382-635989; fax: +44-1382-
635989.
E-mail address: kharada@bad.dundee.ac.uk (K. Harada)
1368-8375/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S1368-8375(99)00049-4
found as an ubiquitous inhibitor of cyclin kinase and an
integral component of cell cycle control [6].
WAF1 attracted a great deal of attention because it
was expected to shed further light on the function of p53.
However, p21WAF1 is synthesized in order to arrest the
cell cycle (G1 or S phase) through both p53-dependent
and -independent pathways when both normal and can-
cerous cells are stressed, e.g. following serum starvation,
exposure to DNA-damaging agents and di€erentiation-
inducing agents [7±9]. Moreover, the p53-related pro-
teins p73 [10] and p51 [11] were recently found to be
capable of inducing p21WAF1. It is believed that the
inhibitory e€ects on cell division by p21WAF1 are closely
related to cell di€erentiation, senescence, cancerous
change of normal cells, regulation and induction of
apoptosis involving p53 and Rb [12].
2. Cell cycle arrest
Overexpression of p21WAF1 induces cell cycle arrest
[1,2]. Also, expression levels of p21WAF1 are elevated in
cells which are induced to arrest in G0±G1 phase by
factors such as serum starvation, di€erentiation and
senescence [8,13,14]. Moreover, p21WAF1 mRNA
and protein levels are rapidly induced in response to X-
ray, UV or DNA-damaging agents, which induce a
transient G1 arrest [15±17]. Furthermore, cell cycle
arrest is not induced by radiation in p21WAF1 (ÿ/ÿ)
cells [18]. It would, therefore, appear that p21WAF1
4 K. Harada, G.R. Ogden / Oral Oncology 36 (2000) 3±7
plays an important role in inhibiting the progress of a
cell through the G1 checkpoint in response to various
exogenous and endogenous factors.
It is thought that p21WAF1 mediates cell cycle arrest
by inhibiting the CDKs that are required to drive the
cell cycle [1±3]. p21WAF1 may also promote cyclin-cdk
assembly [19,20]. p21WAF1 is localised in the nucleus,
and forms a quaternary complex with cyclin A (or B, D
or E), cdk2 (or 4) and proliferating cell nuclear antigen
(PCNA) [13,19,21].
A quaternary complex seems to be important for
restraining the cell cycle in normal cells. When CDKs
complex with p21WAF1, their kinase activity is inhibited
[22]. It is thought that the proliferating signal which
drives the cell cycle, such as phosphorylation of Rb,
activation of E2F, synthesis of DNA polymerase and
CDKs are not induced [22]. Interestingly, the qua-
ternary complexes have not always been detected in
cancer cells [23]. Their loss may contribute to progres-
sion of cancer cells.
p21WAF1 expression can be increased by serum star-
vation, contact inhibition and senescence without being
accompanied by an increase in p53 expression [8], and
p21WAF1 can be expressed in cells which come from p53-
de®cient transgenic mice [24]. These observations sug-
gest that there are other pathways which induce
p21WAF1 independently of p53 [25,26]. Recent evidence
has shown that p21WAF1 can be induced through p53-
independent pathways by such various factors as TGF-
b [24,27], TNF-a [28,29], IFN-g [30,31], vitamin D [32],
TPA [33], MyoD [34], nerve growth factor [35] and
okadaic acid [8].
Since p21WAF1 plays an important role in arresting
the cell cycle, loss of function in p21WAF1 may favour
tumour growth. Mutations in the p21WAF1 gene have
been detected in lung cancer [36], bladder cancer [37]
and Burkitt's lymphoma [38], although not in oral can-
cer. We have failed to detect such mutations in several
oral squamous cell carcinoma cell lines examined (data
not shown).
The very low frequency of p21WAF1 gene mutations in
human neoplastic disease was originally thought to
re¯ect the essential role for the gene product in cell divi-
sion and the maintenance of essential cellular activities.
However, mice null for p21WAF1 gene develop normally
with no increased incidence of neoplasia, although they
are de®cient in G1 cell cycle arrest in response to DNA
damage or nucleotide pool disruption [39]. Cells lacking
the p21WAF1 gene are defective in DNA repair, and this
defect can be compensated for by transfection of an
exogenous full-length p21WAF1 gene, or fragments
encoding the PCNA binding domain [40]. This suggests
that p21WAF1, when complexed with PCNA, plays an
important modulatory role in DNA repair [41±43]. It is
thought that the binding of p21WAF1 to PCNA can
directly inhibit DNA synthesis [21]. PCNA forms a
trimeric toroid structure that serves as a sliding clamp for
DNA polymerase delta and DNA polymerase epsilon
holoenzymes on DNA. This binding of p21WAF1 to
PCNA can inhibit DNA synthesis by blocking associa-
tion of DNA polymerase with PCNA [44]. Binding of
p21WAF1 to PCNA can also interfere with replication by
preventing binding of the Fenl 50 ±30 replicative exonu-
clease to the replicative complex [43,45]. It is possible
that the p21±PCNA interaction is important for ecient
DNA repair, and indeed it has been suggested that
p21WAF1 is involved in the disassembly of PCNA from
DNA repair sites in cells [46]. However, the mechanismes
of DNA repair and replication involved are still unclear.
3. Di€erentiation
Expression of p21WAF1 protein would seem to be
related to terminal di€erentiation because cell cycle
arrest is a ®rst step towards this state. In monocytic
di€entiation from HL-60 cells and in hepatoma cell
lines treated by di€erentiation inducers, expression of
p21WAF1 mRNA is detected [14]. Also, it has been
found that the Mda6 gene product is identical to
p21WAF1, and plays a role in the di€erentiation of nor-
mal melanocytes [47]. It is also thought that p21WAF1 is
involved in the di€erentiation of muscle cells because
induction of p21WAF1 by the myogenic basic helix±
loop±helix factor MyoD leads to cell cycle arrest [34]. It
seems that p21WAF1 is also involved in the di€erentia-
tion of peripheral nervous system neurones in response
to nerve growth factor mediated by protein p300 [35].
Expression of p21WAF1 has also been detected in nor-
mal oral epithelium and epithelial dysplasia as well as in
oral cancer [48±54]. Recently, it has been reported that
p21WAF1 was frequently expressed in oral squamous cell
carcinoma (about 70±80%), in oral epithelial dysplasia
(about 60±90%), and in normal oral epithelium (below
60%) as determined by immunohistochemistry [48,51,
52,56]. p21WAF1 expression was detected in the di€er-
entiating layers of normal oral epithelium and was not
detected in the proliferating cells [48]. This may suggest
that p21WAF1 expression is associated with the initiation
of di€erentiation in normal oral mucosa.
In oral cancer, some researchers have reported that
p21WAF1 expression is associated with tumour di€er-
entiation [48,54], while others have failed to show such a
link [53,56]. Further studies are indicated since the rela-
tionship between p21WAF1 expression and tumour dif-
ferentiation remains unclear in oral cancer.
4. Apoptosis
p21WAF1 is induced in a p53-dependent manner in the
pathway by which p53 promotes apoptosis and cell arrest
 K. Harada, G.R. Ogden / Oral Oncology 36 (2000) 3±7
[57]. However, p53-dependent apoptosis occurs nor-
mally in cells lacking p21WAF1 and cells null for p21WAF1
gene show very high apoptosis after g-irradiation [39,
58]. Therefore, p21WAF1 may not be essential for
apoptosis.
In p21WAF1-de®cient cells, apoptosis can be induced
by the chemotherapeutic agent doxorubicin [59],
p21WAF1-de®cient colon tumours are signi®cantly more
sensitive to radiation [60], and induction of p21WAF1
renders glioma cells resistant to cisplatin-dependent cell
death [61]. These ®ndings suggest that p21WAF1 may
protect cells from apoptosis induced by radiation and
chemotherapy through imposing a cell cycle arrest.
Interestingly, it has been recently reported that the
cleavage of p21WAF1 by caspase-3-like proteases is
observed in endothelial cell apoptosis [62], and caspase-
mediated cleavage of p21WAF1 converts cancer cells
from growth arrest to undergoing apoptosis [63]. As
caspase-3 may overcome the apoptosis-inhibiting func-
tion of p21WAF1, therapies aimed at activating caspase-3
might be e€ective against growth-arrested cancer cells
[64].
The p53 gene is one of the most frequently altered
genes in oral cancers [64,65], so apoptosis may not
always be induced in a p53-dependent manner. Inter-
estingly, it has been reported that 5-¯uorouracil and
radiation induce apoptosis of squamous cell carcinoma
cells in a p53- and p21-independent manner, in S and
G2/M, respectively [55]. Though the relationship
between p21WAF1 and apoptosis is unclear in oral can-
cer, p21WAF1 does not seem to play an essential role.
5. Role in malignant disease
The mutation or overexpression of the p53 tumour
suppressor gene is frequently found in many human
cancer cells [66], particularly in oral squamous cell car-
cinoma [64,65]. Since p21WAF1 is one of the critical
downstream mediators of wild-type p53, the relation-
ship between p21WAF1 expression and p53 status may
in¯uence the patient's prognosis.
The overexpression of p21WAF1 suppresses tumour
growth in several experimental models [67±69] including
the oral cancer model [70]. Expression of p21WAF1 in
human tumours in vivo and its possible role in progres-
sion and tumour cell di€erentiation have been examined
in some tumour models. p21WAF1 is expressed indepen-
dently of p53 status in pancreatic carcinoma [71], lung
carcinoma [72], ovarian carcinoma [73,74], stomach car-
cinoma [75], larynx carcinoma [76] and breast carci-
noma [77±79]. In the same way, it is reported that
p21WAF1 expression in oral cancer is unrelated to p53
functionally [80]. Moreover, it has recently been report-
ed that the cumulative survival rate of oral cancer
patients with p53-negative and p21WAF1-positive
5
tumours is higher compared to those with other combi-
nations of p53 and p21WAF1 status, though p21WAF1
expression did not correlate with p53 status [51]. Inter-
estingly, patients with p21WAF1 expression tend to have
a better prognosis than those without in endometrial
carcinoma [81].
Levels of p21WAF1 expression are dependent upon the
form of malignancy. It has been reported that p21WAF1
expression is increased in non-small cell lung carcinoma
[72], hepato-cellular carcinoma [82], cutaneous squa-
mous cell carcinoma [83] and head and neck cancer [84]
but decreased in colorectal carcinoma [85] and ovarian
carcinoma [79,86].
The ®nding of elevated p21WAF1 expression in some
cancers may be discordant with initial observations that
p21WAF1 can arrest cell cycle. p21WAF1 alone may not
be sucient to arrest cell cycle and inhibit tumour cell
growth.
In the future, p21WAF1 may be a useful prognostic
marker when combined with other markers such as p53,
PCNA, LeY and apoptosis index by TUNEL method
[87], because p21WAF1 is closely related to all of these.
Since p21WAF1 can arrest the cell cycle in G1 phase, in
order to allow repair of damaged DNA, it may have a
valuable role to play in increasing an understanding of
the induction of terminal di€erentiation and inhibition
of carcinogenesis. Furthermore, p21WAF1 may have
potential applications in gene therapy because it plays a
key role in regulating the progression of the cell cycle,
either in inducing arrest or possibly abrogating normal
function. It would appear that p21WAF1 is yet another
marker within the team that cannot be ignored.
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