a role for p53 in the frequency and mechanism of mutation, 2002

Mutation Research 511 (2002) 45–62

Review

A role for p53 in the frequency and mechanism of mutation

Suzanne M. Morris∗

Division of Genetic and Reproductive Toxicology, National Center for Toxicological Research, 3900 NCTR Road, Jefferson, AR 72079, USA

Received 6 August 2001; received in revised form 3 December 2001; accepted 3 December 2001

Abstract

The tumor suppressor protein, p53, is often referred to as the guardian of the genome. When p53 function is impaired, its

ability to preserve genomic integrity is compromised. This may result in an increase in mutation on both a molecular and

chromosomal level and contribute to the progression to a malignant phenotype. In order to study the effect of p53 function

on the acquisition of mutation, in vitro and in vivo models have been developed in which both the frequency and mechanism

of mutation can be analyzed. In human lymphoblastoid cells in which p53 function was impaired, both the spontaneous and

induced mutant frequency increased at the autosomal thymidine kinase (TK) locus. The mutant frequency increased to a

greater extent in cell lines in which p53 harbored a point mutation than in those lines in which a “null” mutation had been

introduced by molecular targeting or by viral degradation indicating a possible “gain-of-function” associated with the mutant

protein. Further, molecular analysis revealed that the loss of p53 function was associated with a greater tendency towards

loss-of-heterozygosity (LOH) within the TK gene that was due to non-homologous recombination than that found in wild-type

cells. Most data obtained from the in vivo models uses the LacI reporter gene that does not efficiently detect mutation that

results in LOH. However, studies that have examined the effect of p53 status on mutation in the adenine phosphoribosyl

transferase (APRT) gene in transgenic mice also suggest that loss of p53 function results in an increase in mutation resulting

from non-homologous recombination. The results of these studies provide clear and convincing evidence that p53 plays a

role in modulating the mutant frequency and the mechanism of mutation. In addition, the types of mutation that occur within

the p53 gene are also of importance in determining the mutant frequency and the pathways leading to mutation. Published by

Elsevier Science B.V.

Keywords: p53; Mutant p53; Loss-of-function; p53 knockout

Abbreviations: IR, ionizing radiation; topo-I, topoisomerase-I;

topo-II, topoisomerase-II; HPV16E6, human papillomavirus Type

16 open reading frame E6; DDB2, DNA damage binding pro-

tein 2; ARF, alternate reading frame; TK, thymidine kinase; LOH,

loss-of-heterozygosity; HPRT, hypoxanthine-guanine phosphoribo-

syl transferase; 4-NQO, 4-nitroquinoline oxide; B[a]P, benzo(a)-

pyrene; DAP, 2,6-diaminopurine; APRT, adenine phosphoribosyl

transferase; pun, pink-eyed unstable mutation; NER, nucleotide

excision repair; 2-AAF, 2-acetylaminofluorene∗ Tel.: +1-870-543-7580; fax: +1-870-543-7136.

E-mail address: [email protected] (S.M. Morris).

1. Introduction

It is becoming increasingly accepted that the pro-

gression of mammalian cells towards malignancy is

an evolutionary process that involves an accumulation

of mutations on both the molecular and chromosomal

level. Inherent in models for malignant progression

is the concept that an initial mutation in an important

regulatory gene (protein) may be pivotal in this pro-

cess. Once the initial mutation has been introduced,

loss of normal gene function or the acquisition of

1383-5742/02/$ – see front matter. Published by Elsevier Science B.V.

PII: S1383 -5742 (01 )00075 -8

46 S.M. Morris /Mutation Research 511 (2002) 45–62

deleterious functions may lead to additional mutations

furthering the malignant transformation of the cell.

A candidate for involvement in this process is the

tumor suppressor, p53. The p53 protein provides one

of the key regulatory elements monitoring genomic

integrity in mammalian cells and is involved in a

multiplicity of cellular functions. The p53 gene is

also recognized as the most commonly mutated gene

in human malignancies. Taken together, these factors

suggest that the role of “guardian of the genome” as-

cribed to p53 [1] may reflect its ability to reduce the

accumulation of mutation in the genome and inhibit

progression towards malignancy. An approach to test-

ing this hypothesis is to examine the role of mutations

in the p53 gene on the frequency and mechanism of

mutations in other genes.

2. Structure of p53

Knowledge of the structure of the p53 gene

provides some insight into the mechanism by which

mutations in p53 affect pathways important in the

mutational process. The human gene contains 11 ex-

ons that code for a protein 393 amino acids in length.

The p53 protein contains several functional domains

including the amino terminal transactivation domain

(amino acids 20–42), a proline-rich sequence con-

taining multiple copies of the PXXP sequence (amino

acids 60–97), a central DNA binding domain (amino

acids 100–293), a flexible linker region (amino acids

300–325), an oligomerization domain (amino acids

319–360) and a highly basic region of the C-terminus

(amino acids 363–393, reviewed in [2,3]). Sequences

found within the central DNA binding domain (exons

5–8) have been evolutionarily conserved across

species (reviewed in [2]).

The p53 protein undergoes extensive post-trans-

lational modification during an “activation” process.

Specific phosphorylation sites have been identified

and are located primarily in the amino and carboxy

terminal regions of the protein (reviewed in [3–12]).

The N-terminal domain functions primarily in the

transcriptional control activity of p53 whereas the C-

terminus affects the specific DNA binding of the

protein [4]. The amino terminal domain undergoes

phosphorylation by kinases that include casein kinase,

checkpoint kinase 1 and checkpoint kinase 2, DNA-

dependent protein kinase, ataxia telangiectasia mutant,

jun kinase, and mitogen-activated protein kinases.

The C-terminus undergoes phosphorylation by casein

kinase II, various cyclin-dependent kinases, and pro-

tein kinase C. Acetylation sites that also contribute

to the activation process have recently been identi-

fied (reviewed in [4,7,12]). Phosphorylation of the

N-terminus sets in motion a conformational change

that enables transcription to proceed, has a minor

effect on DNA binding, and contributes to the stabi-

lization of p53 by decreasing the interaction of p53

with mdm2 (reviewed in [6]). Changes in the phos-

phorylation of the carboxy terminus alter the con-

formation of the protein to modulate DNA binding

activity (reviewed in [4]). For example, dephospho-

rylation of serine 376 creates a binding site for the

14-3-3s protein, which when bound to p53, increases

the binding capacity of p53 [13]. In addition to phos-

phorylation and dephosphorylation, the C-terminus

undergoes acetylation at lysine 320 and 382 [14] and

binding to the small ubiquitin-like protein, SUMO-1,

at lysine 386 [15,16] as part of the activation process.

Because mutations in the p53 gene are so common

in human tumors and have been widely reported in the

literature, extensive databases exist such as that main-

tained by IARC [17]. Walker et al. [18] have utilized

the IARC database to define 73 “hotspots” for muta-

tion in p53 and related the mutations to changes in

protein structure and function. Most “hotspots” were

found at CpG dinucleotides within exons 5–8 [18].

Methylation of CpG sites lends itself to the induction

of mutation due to the tendency for deamination of

cytosine to uracil and the insertion of an inappropri-

ate thymine during repair (reviewed in [19,20]). It has

also been suggested that the binding of certain carcino-

gens is targeted to methylated CpG sites (reviewed in

[20]). Although the mechanism is not fully defined,

six CpG “hotspots” have been identified within the

DNA binding domain of human p53 and are found at

codons 175, 213, 245, 248, 273 and 282. An additional

“hotspot” has been found at codon 249 in aflatoxin

B1-induced hepatocellular carcinoma [21]. The effect

of these mutations on the structure of p53 has been ex-

amined by Cho et al. [22] utilizing X-ray crystallogra-

phy and provides convincing data that these mutations

alter the ability of p53 to bind to the promoter regions

of genes under its regulatory influence. Additional

“hotspots” have been identified outside of the DNA

S.M. Morris /Mutation Research 511 (2002) 45–62 47

binding domain, but the mutation spectrum is much

broader than that observed in exons 5–8. Nonsense,

frame-shifts and a small proportion of missense mu-

tations have been found outside of exons 5–8 [18,23].

3. Function of the p53 protein

Models have been proposed in which p53 functions

directly in DNA replication, repair and recombination

in order to eliminate spontaneous and chemically-

induced DNA damage (see Fig. 1). Once damage

occurs, p53 undergoes phosphorylation, dephospho-

rylation, acetylation and sumoylation at specific sites

in order to serve as a transcription factor. In this role,

p53 regulates the synthesis of proteins that participate

in cellular functions important in the elimination of

cells with DNA damage or preventing the replication

of damaged cells [7,24,25].

The association between loss of p53 function and

malignant progression is partially due to the role p53

plays in the response to DNA damage as “guardian

of the genome”. Initial studies on the cell-cycle with

ionizing radiation (IR)-exposed cells led to the finding

that the G1 checkpoint is absent in cells with mutant

p53 [26]. Subsequently, it was found that p21waf−1,

an integral component of the G1 checkpoint, is under

the transcriptional control of p53 and that synthesis

of p21 increases after exposure to either IR or chem-

ical mutagens in cells with wild-type p53 [27]. A G2

checkpoint is intact in many cell lines with mutations

in p53 [28], but evidence exists that a p53-regulated

checkpoint also operates during the G2 transition and

that loss of this checkpoint contributes to an increase

in polyploid cells [29].

Apoptosis or programmed cell death is an ordered

event under genetic control [30] with multiple path-

ways leading to the final destruction of the cell. The

p53 protein is an integral component of one apopto-

sis pathway that responds to DNA damage such as

IR, topoisomerase inhibitors, and mutagens that form

covalent DNA adducts. P53 responds to DNA damage

signals, which may be the blockage of RNA poly-

merase II [31], by upregulating the synthesis of bax,

an apoptosis-promoting protein. The p53 protein also

down-regulates bcl-2, an apoptosis-inhibitory protein

[32–34], by interacting with the TATA binding pro-

moter to repress bcl-2 expression [35]. The Bax and

bcl-2 form homo- and heterodimers and function in the

control of the mitochondrial permeability transition

and the progression to the execution phase of apop-

tosis (reviewed in [36]). Interestingly, recent studies

have demonstrated that p53 translocates to the mito-

chondria and is sensitive to the levels of bcl-2 and bax

in the mitochondria [37,38]. A feedback loop for p53

and bcl-2 has recently been described in which bcl-2

inhibits the transactivation of the p53-regulated pro-

teins bax, p21waf−1, mdm2, cyclin G, and GADD45

promoters [39,40]. The p53 also functions as a tran-

scriptional regulator in the mitochondrial-independent

forms of apoptosis in which signals converge from

death receptors such as FAS/APO-1, killer/DR5, and

DR4 and the p53-inducible genes, or PIG’s which

respond to oxidative damage at the level of caspase-3

(reviewed in [41,42]).

Evidence is accumulating that p53 plays an

important role in DNA excision repair, both as a

transcription factor and as a component of nucleotide

and base excision repair complexes. It has recently

been shown that p53 functions in base excision repair

[43], to upregulate the synthesis of the repair enzyme,

O6-alkylguanine transferase [44], and to upregulate

the p48 subunit of the DNA damage binding protein

2 (DDBP2) which is involved in the binding of the

“global genomic repair” complex to DNA damaged

by UV [45]. Loss of p53 function in UV-exposed

Li–Fraumeni p53 mutant cells resulted in a decrease

in global genomic repair, but did not affect the rate

or efficiency of transcription-coupled repair [46,47].

Transfection of wild-type p53 human fibroblasts with

human papillomavirus Type 16 open reading frame

E6 (HPV16E6) and the subsequent loss of wild-type

p53 activity also resulted in the loss of global genomic

repair [48–50]. In addition to regulating the synthesis

of DBBP2 or p48, p53 physically interacts with XPB

and XPD, components of the nucleotide excision

repair (NER)-associated TFIIH transcription/repair

complex with helicase activity [51]. Through its

C-terminus, p53 binds to single-stranded DNA ends

[52] and participates in strand exchange activity

[53,54]. Sequence-specific binding by the C-terminus

of p53 is postulated to be a step in damage recog-

nition as part of its repair function [54]. However,

Liu and Kulesz-Martin [55] have proposed that the

DNA binding function of p53 may serve as a sensor

for directing damaged cells to the repair pathways or


Fig. 1. Normal p53 response to DNA damage. Cellular DNA is damaged by chemical or physical insult (1); p53 is upregulated (2)

and undergoes phosphorylation, dephosphorylation and acetylation (3) to active isoforms; p53 acts as a transcription factor (4a) and as

a structural component (4b) of protein complexes; the proteins under the regulatory influence of p53 and the p53 protein complexes all

modulate cellular functions that influence the mutant frequency (5); cells are halted at the various p53-regulated checkpoints (6) to allow

the removal (repair) of DNA damage (7); when DNA damage is not repaired, the cells are then targeted for death (8); these factors

combine to reduce the accumulation of mutations within the genome (9). When mutation in p53 leads either to the loss of the protein or to

aberrant function of the protein, the p53-mediated response to DNA damage is abrogated, leading to an increase in the mutant frequency.


toward the apoptosis pathways, rather than serve a

direct repair function.

The recombinational activity of p53 is separate from

its transactivational properties [24,56] and mutations

in p53 increase the rate of recombination [24,56–58].

The p53 protein interacts with the proteins, RAD51,

topoisomerase-I (topo-I) and topoisomerase-II

(topo-II)a, each of which plays a role in recombi-

nation, DNA repair and DNA replication. The p53

protein interacts with RAD51 through two binding

sites on p53 (amino acids 94–160 and amino acids

264–315). Binding was reduced, but not eliminated,

when the binding efficiency of p53 with specific mu-

tations (135Y, 249S, and 273H) was compared to that

of wild-type p53. The presence of a mutation in p53

resulted in a decrease in the efficiency of binding to

RAD51 [59]. Wild-type p53 recognizes three-stranded

DNA junctions that structurally resemble early re-

combination intermediates [60]. Further, the 3′→ 5′

exonuclease activity of p53 [61] appears to target the

destruction of these intermediates [62]. The p53 mu-

tants, especially the 273H mutant, are defective in this

ability [24] contributing to the lack of recombinational

control in certain p53 mutants [24]. A more indirect

role for p53 in recombination involves its transcrip-

tional control of bcl-2. In a recent study with exciting

implications for the understanding of recombination,

Saintigny et al. [63] have demonstrated that bcl-2 in-

hibits RAD51-mediated conservative, or homologous,

recombination. RAD51 undergoes post-translational

modification by bcl-2 which then shifts the processing

of double strand breaks to an error-prone pathway and

produces an increase in the mutant frequency. Further,

the effects of bcl-2 on recombination are separate and

distinct from its effects on the cell-cycle and apopto-

sis. These factors may contribute to the genomic insta-

bility observed in p53 mutant cells (reviewed in [64]).

The interaction of p53 with topo-I is mediated

through a binding site that is localized to the carboxy

terminus of p53 [65–67]. The p53/topo-I complex is

found at the site of the “cleavable complex” which is

the site of the breakage and religation that occurs as

part of topo-I activity [68]. The 273H mutation trans-

fected into HT 29 cells and the 239S, 245S and 273H

mutations transfected into Sf-9 insect cells all result

in a constitutive, rather than transient, association be-

tween p53 and topo-I [65,66] and possibly contribut-

ing to the stabilization of the “cleavable complex”.

The observations that both p53 and topo-II are

involved in the DNA breakage–ligation mechanisms

associated with recombination and replication led

Wang et al. [69] to study the relationship between the

two proteins. The p53 protein was found to serve as

a transcriptional repressor of topo-II, reducing syn-

thesis through an interaction with an ICE element

in the topo-II promoter. Neither a mutation at codon

175 nor the double mutation at codons 22 and 23

repressed the synthesis of topo-II. This may account

for the overexpression of topo-II observed in hepato-

cellular carcinoma [70]. However, Yuwen et al. [70]

also described an interaction between p53 and topo-II

that was detected by co-immunoprecipitation which

was later confirmed by others [71,72]. The interaction

occurs via the C-terminal region of p53 and similar

binding efficiencies were found for wild-type p53 and

mutant p53 (M237R, M237I and R273C) [72].

4. Mechanisms for loss-of-function

of wild-type p53

The p53 protein exists primarily as a tetramer,

forming the complex through the oligomerization

domain at the C-terminus of the protein [73]. When

mutant and wild-type subunits form a heterotetramer,

the mutant subunit drives the tetramer into the mu-

tant conformation in a dominant-negative manner.

Loss-of-function that is associated with heterozy-

gosity in the p53 gene is thought to be due to this

proposed dominant-negative mode of action [74,75].

The role of base-pair substitution mutations on

dominant-negative interactions was evaluated utilizing

wild-type p53 colon adenocarcinoma cells transfected

with plasmids carrying different point mutations in

p53 [76]. Loss-of-function, as determined by the lack

of mdm2 synthesis and G1 arrest after IR exposure

and dominant-negative interaction, were dependent

upon mutation that resulted in a sequence change

in the protein [76]. Further, the mutant protein may

interfere with normal cellular processes and possess

a “gain-of-function” phenotype (reviewed in [77]).

Recently, p53 has been described as a member

of a “supergene” family with members p63 and

p73 exhibiting structural and functional similarity

to p53. In contrast to p53, however, both p63 and

p73 encode multiple splicing variants that encompass


the C-terminal region (reviewed in [78]). P73,

described independently by Jost et al. [79] and by

Kaghad et al. [80], is characterized by the presence

of DNA binding domain CpG “hot spots” for muta-

tion, contains 14 exons and acts a transcription factor

for several p53-regulated genes, including those that

regulate apoptosis [81]. The p73 protein responds to

DNA damage by undergoing post-translational mod-

ification by the tyrosine kinase activity of c-abl and

inducing the apoptotic cascade [82–84]. In one study

[85], it was suggested that neither wild-type p53 nor

mutant p53 (R273H) formed heteroligomers with

p73. DiComo et al. [86], however, found that while

wild-type p53 did not oligomerize with wild-type

p73, mutant p53 (R175H and 248W) was found to

co-immunoprecipitate with p73, indicative of an inter-

action. Marin et al. [87], however, have demonstrated

that the strength of the interaction between certain

mutant forms of p53 and p73 may be determined by

the presence of a polymorphism at codon 72 of p53.

This polymorphism does not appear to affect p53–p53

interaction, but does demonstrate that certain muta-

tions in p53 proteins can lead to an interaction with

p73. P63 is composed of 15 exons and bears amino

acid homology to p53 in the transactivation domain,

DNA binding domain, and oligomerization domain

(reviewed in [78]). Neither the full length nor the

truncated splicing variants of p63 have been shown

to interact with p53 in a dominant-negative manner

[88].

Nuclear and cytoplasmic p53 levels are modulated

in part by the p53/mdm2/ARF pathway which serves

as a point of convergence for oncogenic signals such

as ras, E2F1, and c-myc (reviewed in [89,90]). The

p53 protein undergoes ubiquitination by the E3 ligase

activity of mdm2 and is shuttled from the nucleus to

the cytoplasm for degradation by the 26S proteosome

(reviewed in [91]). Mutation in p53 that results in

conformational alteration may inhibit mdm2’s ability

to bind p53 and target the protein for destruction,

thus, leading to increased nuclear levels of the mutant

protein [91]. The mdm2 ligase activity is under the

control of the alternate reading frame (ARF) proteins,

p14ARF (human) and p19ARF (mouse) which are

encoded in the INK4A/ARF locus [92]. In addition

to ligase activity, ARFs are also involved in the se-

questration of mdm2 in the nucleolus [93,94]. The

ARF-mediated decrease in mdm2 levels is paralleled

by an increase in the nuclear p53 levels which allows

the p53-directed transcription of the genes important

in cell proliferation, apoptosis, and perhaps, DNA

repair, to proceed. Mutation in the INK4A/ARF lo-

cus is quite common in malignancies and the loss of

ARF-mediated control of mdm2 activity can result in

biological responses similar to those observed in cells

with mutation in p53 (reviewed in [95]).

Another mechanism for the loss of wild-type func-

tion is the interaction with viral proteins. The SV40

protein, whose interaction with p53 led to its dis-

covery, binds to the DNA binding domain of p53

eliminating its functional ability [96]. HPV16E6 on-

coprotein binds to the p53 protein and transports it to

the ubiquitin pathway resulting in premature degra-

dation and a functionally p53 “null” state in the cell

[97]. A recent study also demonstrates that HPV16E6

down-regulates p53 by binding to CBP/p300 [98,99].

The adenovirus E1B-55KD protein abrogates p53

function, possibly by interacting with the RNA poly-

merase II transcription complex [100,101].

5. Cell line families that differ in p53 status

One of the original studies to suggest a role for p53

in modulating the mutant frequency was conducted in

TK6 human lymphoblastoid cells ([102]; see Tables 1

and 2). TK6 was derived from the human lymphoblas-

toid cell line, WIL2, as was the “closely-related” cell

line, WTK1 [102]. Both lines are heterozygous for the

thymidine kinase (TK) gene with frame-shift muta-

tions in exon 4 of the mutant TK allele and exon 7 of

the wild-type allele [103]. IR produces a much greater

frequency of TK mutants in WTK1 cells than in TK6

cells [102]. Molecular analysis of the TK gene in mu-

tant clones [104] revealed that the difference in mutant

frequency was due to an increase in the frequency of

a specific class of mutant recovered in WTK1 cells.

Loss-of-heterozygosity (LOH), due either to a deletion

mechanism or to errors in recombinational repair, was

detected at a much higher frequency in WTK1. Subse-

quent studies revealed that the two lines differed in p53

status with a homozygous G → A mutation present at

codon 237 of exon 7 ofWTK1 (andWIL2-NS, another

TK6-derived cell line) [105,106]. The codon 237 mu-

tation lies in the DNA binding region of p53 and the

homozygous mutation would be expected to interfere


Table 1

Cell lines, their p53 status and reporter genes used for mutant frequency evaluationa

Parent cell line Cell line p53 status Loss of p53 function due to Reporter gene

WIL2 (H) TK6 Wild-type p53 function intact TK, HPRT

TK6-E6 Wild-type Targeted degradation of p53 by HPV16E6 TK, HPRT

TK6-NH32 Mutant (null) Homozygous deletion TK, HPRT

WTK1 Mutant (point) Homozygous C → T at codon 237 (exon 7) TK, HPRT

AHH-1 (H) L3 Wild-type p53 function intact TK, HPRT

MCL-5 Wild-type p53 function intact TK, HPRT

AHH-1 TK+/− Mutant (point) Heterozygous C → T at codon 282 (exon 8) TK, HPRT

SAOS-2 (H) SAOS-2 Mutant (null) Homozygous deletion of exons 2–11 HPRT

RKO (H) RKO Wild-type p53 function intact HPRT

RKO-E6 Wild-type Targeted degradation of p53 by HPV16E6 HPRT

LN12 (M) LN12 Mutant Deletion supF

a H: human origin; M: mouse origin.

with the transcription factor properties associated with

p53 as well as affecting p53-mediated recombination.

In a series of studies, Honma et al. [107,108] have

elegantly demonstrated that although spontaneous and

IR-induced TK mutations in both TK6 and WTK1

cells resulted from LOH, the mechanism leading to

the mutation differed as a function of p53 status. TK

mutants were identified as LOH by Southern blot pat-

terns and then classified as homozygous (two copies

of the non-functional allele) or hemizygous (one copy

of the non-functional allele) by densitometric analysis.

In TK6 cells, both hemizygous and homozygous LOH

was detected, in contrast to WTK1 cells, in which

two non-functional alleles were detected in the major-

ity of clones [107]. When representative clones were

analyzed by chromosome painting, it was revealed

that the LOH in TK6 cells could not be accounted

for by chromosome rearrangements. In WTK1 cells,

however, a high percentage of translocations was

found in both the spontaneous and IR-induced clones

[108]. These findings led to a model in which the

cells with mutant p53, WTK1, had a predisposition

towards non-homologous recombination that could be

followed by mitotic non-disjunction. In contrast, ho-

mologous recombination predominated in TK6 cells

with wild-type p53 [108].

When the same mutation in p53 found in WTK1

cells, H237I, was transfected into wild-type TK6

cells, both the spontaneous and the IR-induced mutant

frequencies increased to the level observed in WTK1

cells [109]. These findings led to the question of

whether the increase in the mutant frequency was due

to the presence of the mutant protein or to the absence

of the wild-type protein. Thus, TK6 cells were trans-

fected with high-risk, HPV16E6, effectively creating a

p53 “null” cell line (TK6-E6), due to the rapid degra-

dation of the p53 protein. The spontaneous mutant fre-

quency in TK6-E6 cells was elevated in comparison to

TK6 cells, but not to the degree observed in the WTK1

line [110,111]. Exposure of TK-E6 cells to IR resulted

in a TK mutant frequency intermediate to IR-exposed

TK6 and WTK1 cells [111]. Molecular and cytoge-

netic analyses were performed on spontaneous mu-

tants recovered from the TK6, the TK6-E6, and the

vector control, TK6-20C lines [110]. Quantitative PCR

revealed the loss of the functional TK allele in mutants

derived from each of the cell lines. The mutations re-

covered from TK6-E6 cells in which p53 underwent

viral degradation were predominately hemizygous

and the loss of the terminal portion of chromosome

17q was confirmed by microsatellite analysis. Sub-

sequent chromosome painting analysis revealed that

allele loss and the formation of deletions and translo-

cations could be attributed to errors in end-rejoining

and non-homologous recombination. In contrast, both

hemizygous and homozygous TK mutants were de-

rived from both the TK6 and TK6-20C cell lines in

which p53 remains functionally intact [110].

A confounding factor in the experiments that uti-

lize TK6-E6 cells is the observation that the effects

of HPV16E6 may not be limited to the destruction

of p53 and that those effects may contribute to the


differences in the TK mutant frequency. To address

this question, Chuang et al. [112] developed a TK6

derivative, TK6-NH32, in which both alleles of p53

were disrupted by molecular targeting resulting in a

p53 “null” genotype with neither wild-type nor mu-

tant p53. When cells from the TK6, TK6-NH32, and

WTK1 cell lines were exposed to IR and the spon-

taneous and IR-induced TK mutant frequencies mea-

sured, the increase in the mutant frequency in cultures

derived from WTK1 was substantially greater than

that observed in either TK6 or TK6-NH32 cells. These

data led to the suggestion that the mutation in p53 in

the WTK1 line led to a “gain-of-function” which con-

tributed to the mutant frequency to a greater extent

than the “loss-of-function” mutation in the TK6-NH32

line [112].

Evidence indicating a role for p53 in determining

the mutant frequency also has come from studies

with the AHH-1 cell line family. AHH-1 TK+/− and

MCL-5 (L3) cells were derived from the AHH-1 cell

line described by Crespi and Thilly [113]. Both are

heterozygous for the TK gene with a frame-shift mu-

tation in exon 4 of the mutant allele [114]. Sequence

analysis of the p53 gene in these cells revealed that

a C → T transition had occurred at codon 282 of

exon 8 of AHH-1 TK+/− cells. This is a reported

“hot spot” for C → T mutations and results in an

amino acid substitution in the DNA binding domain

of the protein [115]. Several classes of compounds

produce a five-fold greater TK mutant frequency in

AHH-1 TK+/− cells than in MCL-5 (L3) cells. When

AHH-1 TK+/− and L3 cells were exposed to the pu-

tative topo-II inhibitors and phytoestrogens, genistein

and coumestrol [116,117], a five-fold increase in the

percentage of TK mutant clones with the slow-growth

phenotype was found. When a screening assay for

LOH was utilized to evaluate the coumestrol-induced

TK mutant clones, wild-type exon 4 was not detected.

Mutagen-induced LOH was also detected in the ex-

periments of Dobo et al. [118] in which a higher

frequency of TK mutants was found in the AHH-1

TK+/− cell line. Deletion analysis by microsatellite

loci revealed that the length of the LOH tracts were

substantially longer in the AHH-1 TK+/− cell line.

The increased mutant frequency in the TK gene of

AHH-1 TK+/− cells was accompanied by a decreased

rate of apoptosis and the loss of the G1 checkpoint,

factors consistent with the mutation in p53 [116,118].

Studies addressing the role of p53 on mutant fre-

quency have not been limited to the TK gene. Sev-

eral studies have examined the effect of p53 status

on hypoxanthine-guanine phosphoribosyl transferase

(HPRT) mutant frequency and on the frequency of

mutations in the bacterial reporter gene, supF, which

has been integrated into mouse LN12 cells. Wild-type

p53, under the control of an inducible promoter, was

transfected into SAOS-2 cells which are derived from

a human osteosarcoma and deficient in p53 expression

due to a deletion encompassing exons 2–11 [119].

HPRT mutant frequencies, measured after exposure to

X-rays [120] or to UV [121], were markedly reduced

in the clones that expressed wild-type p53 compared

to the p53 null cells. UV exposure of RKO cells trans-

fected with HPV16E6 resulted in mutant frequencies

in the HPRT gene greater than non-transfected RKO

cells [122]. A similar approach, the transfection and

expression of wild-type p53 into p53-deficient cells,

was utilized by Yuan et al. [123] in mouse LN12

cells. Mutations in the supF reporter gene decreased

four-fold in the wild-type p53-expressing cell line.

Molecular analysis was not performed in the exper-

iments with SAOS-2 or RKO cells. Finally, IR pro-

duced a higher mutant frequency in the HPRT gene

in WTK1 cells than in TK6 cells [124]. However, no

differences in the mutation spectra were detected.

6. In vivo mutation detection systems

Although cell culture models have increased our

knowledge of p53 function in maintaining genomic in-

tegrity, understanding the p53-modulated response to

DNA damaging agents in an animal or human model

involves a greater level of complexity than in a cell

culture system. A major advance in modeling the role

of p53 has been the production of three strains of trans-

genic mice in which a major deletion in p53 was in-

troduced by recombinant DNA technology [125–127].

Differences among the strains exist in that intron 4 and

exon 5 were deleted in the Donehower strain [125] in

contrast to the deletion of exons 2 through 6 in the

models developed by Jacks et al. [126] and by Purdie

et al. [127]. In addition, the genetic background differs

among the strains with both the Donehower and Jacks

strains being derived on a C57Bl/6 × 129/SV back-

ground and the Purdie strain on a 129/Ola background.


However, common to each of these strains is a propen-

sity to develop thymic lymphoma at an early age.

The tumor spectrum of the p53 heterozygous mouse

differs from that found in the null mouse in that there

is a predisposition towards the induction of sarcomas

rather than lymphoma. As discussed in Purdie et al.

[127] and MacLeod and Jacks [128], the difference

in the spectrum between the “knockout” and the het-

erozygote may reflect the susceptibility of the tissue

for the loss of the second p53 allele. Differences in the

characteristics of the heterozygotes exist between the

strains. In the Donehower strain [125], p53 levels are

reduced below what would be expected on the basis of

allele dosage and the lowered level of p53 may con-

tribute to the phenotype of this p53+/− mouse [129].

Table 3

The p53 status, reporter locus, and mutant frequency (MF) in transgenic mice

Reporter locus Tissue Mutagen Mutant frequency Reference

LacI Liver None p53+/+= p53−/− [130]

Spleen p53+/+= p53−/−

Brain p53+/+= p53−/−

LacI Thymic

lymphoma

None Increase of 3× in one of the four tumors (2.3 × 10−5 in p53+/+

mice vs. 6.8 × 10−5 in one tumor from p53−/− mouse)

[132]

LacI Embryonic

fibroblast

2mM 4-NQO No p53-mediated difference in spontaneous MF; no

p53-mediated difference in MF after exposure to 2mM 4-NQO

although the LacI MF increased in response to 4-NQO exposure

[133]

Thymus None p53+/+= p53−/−

Thymic

lymphoma

None p53+/+= p53−/−

APRT Fibroblasts None Increase of 3× in spontaneous MF (10−5 for p53+/− mice and

33.5 × 10−5 for p53−/− mice); molecular analysis revealed

mutation due to mitotic recombination which results in LOH

[136]

T-lymphocytes p53+/−= p53−/−

pun Mouse pups B[a]P 30mg/kg Control litters were 12–16% “spotted” or mutant pups; 40–50%

of the pups from exposed dams of each genotype were “spotted”

[140]

B[a]P 150mg/kg Control litters were 12–16% “spotted” pups; 77–100% of the

pups from exposed dams were “spotted”

IR 1Gy Reduced litter sizes in all genotypes, but most predominately in

the IR-exposed p53−/− litters. No “spotted” pups were present

at 10 days post-partum in p53−/− litters

LacZ Lung 13mg/kg of B[a]P

for 13 weeks

p53+/+= p53+/− [142]

Liver p53+/+= p53+/−

Spleen p53+/+= p53+/−

LacZ Spleen 300 ppm of AAF

for 12 weeks

p53+/+= p53+/− [143]

Liver p53+/+= p53+/−

Bladder p53+/+ < p53+/−

Many of the tumors in the Donehower heterozygote re-

tain the wild-type allele and tumor induction is linked

to the lowered gene dosage [129]. P53 levels are at ap-

proximately 50% of that found in the wild-type mouse

in the Jacks strain [126]. Further, LOH of the p53 gene

occurs in a high proportion of the tumors found in the

Jacks strain apparently by a chromosomal mechanism.

Each of the p53 transgenic mouse models has been

utilized to explore the effect of mutations in the p53

gene on the frequency and spectrum of mutations at

reporter genes (see Table 3). Utilizing the Donehower

model [125], animals of each of the p53 genotypes

(p53+/+; p53+/−; p53−/−) and bearing the LacI

transgene (TSG-p53/Big Blue©) were created. Spon-

taneous mutant frequencies were measured in liver,


spleen and brain [130,131], and after correcting for

clonal expansion of single mutations, no difference

in the LacI mutant frequency as a function of p53

genotype was observed. The spontaneous LacI mutant

frequency was also determined in thymic lymphomas

derived from p53−/−/LacI+/− mice. A 2.3-fold in-

crease in the mutant frequency (6.8 × 10−5 versus

2.9 × 10−5 in p53−/− thymus cells) was detected in

one of four tumors with an increase in A : T → G : C

transition mutations. Two tumors demonstrated a

trend towards an increase in the mutant frequency

(3.7 × 10−5 versus 2.9 × 10−5 in p53−/− thymus

cells) and no increase in the mutant frequency was

found in the other tumor [132].

The p53+/−/LacI mouse has also been utilized

to examine the relationship between p53 status

and both spontaneous and 4-nitroquinoline oxide

(4-NQO)-induced mutations. The spontaneous LacI

mutant frequencies were measured in embryonic

fibroblasts, thymocytes derived from 2–3 months

old animals and thymomas detected in animals 5–8

months old. In addition, embryonic fibroblasts derived

from p53+/+/LacI and p53−/−/LacI animals were ex-

posed to 2mM 4-NQO. Although a dose-responsive

increase in the LacI mutant frequency was found in

the 4-NQO-treated fibroblasts, no effect of p53 status

was found with any of the three genotypes [133].

Studies with the LacI reporter gene may be limited

since the LacI is a transgene, rather than an endoge-

nous gene, and does not efficiently detect chromo-

some damage and recombination events [130]. In

contrast, adenine phosphoribosyl transferase (APRT)

is an endogenous, autosomal gene, capable of de-

tecting these types of events. A mouse model, de-

veloped by Stambrook and co-workers [134,135],

is heterozygous for the APRT gene and responds to

mutagen exposure with an increase in APRT mutant

frequency. Thus, the APRT+/− mouse was crossed

with p53+/− mice to create animals with the geno-

types of p53+/+/APRT+/−, p53+/−/APRT+/−, and

p53−/−/APRT+/−. Mutant frequencies in the APRT

gene, measured by resistance to 2,6-diaminopurine

(DAP) were similar in the T-lymphocytes of the

p53+/+ and p53−/− mice. However, a three-fold

increase in the frequency of DAP-resistant variants

derived from fibroblasts was found in p53−/− mice

compared to p53+/− mice. The APRT mutant fre-

quency in the fibroblasts of p53+/− mice did not differ

significantly from that of the p53+/+ animals. Mitotic

recombination accounted for the majority of mutations

in the wild-type, the heterozygous and the “knockout”

mice; however, deletion and gene conversion ac-

counted for 5 out of 47 variants in the p53+/− mice

and 6 of 106 variants in the p53−/− mice. An increased

number of variants (7/106) resulting from chromo-

some damage (non-homologous recombination) were

encountered in the p53−/− fibroblast clones [136].

Another locus which has been utilized to examine

the effect of p53 status on mutant frequency is the

pink-eyed dilution unstable (pun) mutation which is

maintained in the C57Bl/6J mouse strain. This mu-

tation results from the tandem duplication of 70 kb

internal to the “p” gene that is expressed in embryonic

melanocytes [137,138]. Reversion to wild-type occurs

by the deletion of one of the duplicated sequences

and can be measured phenotypically by the presence

of black spots on the grey coat of the mouse. The

reversion frequency of the pun mutation in mice with

wild-type p53 increases with exposure to IR [139].

When mice of each of the p53 genotypes were crossed

with the pun mouse and pregnant dams exposed to IR,

an increase in the number of “spotted” offspring was

found in the p53+/+ and p53+/− offspring, but not

in the p53−/− pups. It was suggested that IR-induced

damage was processed differently in the p53−/− pups

and that this could account for the lack of “spotted”

or mutant pups [140]. When pregnant dams were ex-

posed to the point mutagen, benzo(a)pyrene (B[a]P),

p53 status did not affect the reversion frequency [140].

NER-defective mice, Xpa [141], were crossed with

the Jacks strain [126] to create a transgenic mouse with

the Xpa−/−/p53+/− genotype. In order to study the ef-

fect of the loss of NER and p53 heterozygosity on mu-

tant frequency, a triple transgenic mouse was created

by crossing this animal to a strain harboring a plasmid

vector containing the LacZ transgene [142,143]. When

exposed to B[a]P, the LacZ mutant frequencies in the

lungs, liver and spleens of the p53+/− mice were not

significantly elevated compared to those observed in

the p53+/+ animals. However, when exposed to the

carcinogen, 2-acetylaminofluorene (2-AAF), the LacZ

mutant frequency in the bladder, but not the spleen

or liver, increased significantly in the p53+/− animals

compared to the wild-type. Thus, the effect of p53 het-

erozgosity on LacZ mutation induction appears to be

restricted to specific tissues [143].


The strains developed by Jacks et al. [126] were uti-

lized to examine the effect of IR on the HPRT mutant

frequency in preB cells that are sensitive to IR [144].

Although IR-induced 6-thioguanine-resistant clones

were recovered from p53−/− mice, none was recov-

ered from the wild-type mice due to an extremely low

cloning efficiency. Southern analysis confirmed the

loss of multiple exons within the HPRT gene.

7. Summary

Clear and consistent evidence is emerging that

mutations in p53 affect both the frequency and the

pathway leading to mutation. In models in which loss

of p53 function occurs due to point mutation, targeted

deletion, or loss of protein due to viral degradation,

there is a consistent increase in the TK mutant fre-

quency, and the increase is due, in most instances, to

mechanisms that result in LOH. Loss of p53 func-

tion leads to an increase in the frequency of mutation

due to non-homologous recombination that may be

followed by mitotic non-disjunction and result in the

formation of chromosome rearrangements. In cells

with wild-type p53, mutation that results in LOH

seems to occur preferentially through a homologous

recombination pathway that results in the loss of the

wild-type allele.

Further, the type of mutation in p53 may play a

major role in determining the mutant frequency in

the reporter gene. Most point mutations in p53 that

have been extensively studied are those that reside

in the DNA binding domain and in the classic CpG

“hotspots”. Studies with the representative DNA

binding domain mutant, 273H, indicate that recombi-

nation activities are hindered by the altered binding

of mutant p53 to recombination pathway proteins

such as RAD51. The down-regulation of bcl-2 by p53

would be reduced in p53 mutant cells, resulting in an

increase in bcl-2-induced modification of RAD51 and

a shift to error-prone pathways. Thus, recombination

in cells with mutant p53 would more likely result

in damage being processed through “error-prone”

pathways and a high mutant frequency. In addition,

mutant p53 protein interferes with other cellular

processes that may affect the mutant frequency, in-

cluding apoptosis [145]. The importance of apoptosis

in determining mutant frequency is suggested by

experiments in which a decreased rate of apoptosis is

accompanied by an increase in TK mutant frequency

[116,118]. Additional support for this hypothesis

comes from the studies of Cherbonnel-Lassere et al.

[146,147] in which ectopic overexpression of the

p53-regulated apoptosis-inhibitory protein, bcl-2, re-

sulted in an increase in spontaneous and induced TK

mutant frequency. Although the increased expression

of bcl-2 may result in a shift to error-prone pathways,

apoptosis is also inhibited by high levels of bcl-2

and is manifested in an increase in clonogenic sur-

vival. Cells that would normally be targeted for death

contribute to an increase in the mutant frequency.

A different pattern emerges in cells in which p53

function has been abrogated as a result of gene

deletion or viral infection. Recombination-mediated

mutations occur at a much lower frequency in p53

“null” cells, cells subjected to viral infection, and

wild-type cells than in p53 mutant cells. These results

are consistent with the hypothesis that mutant protein

interferes with the pathways involved in the repair

and removal of cells with DNA damage. The lack of

wild-type p53 protein may affect the recombination

processes, perhaps through monitoring and degrading

early recombination intermediates, or may shift the

DNA damage response to an alternate pathway, such

as apoptosis. Mutant p53 protein may interfere with

recombination, apoptosis, and other cellular processes

and contribute to the high levels of mutations resulting

in LOH. The absence of mutant protein may possibly

account for the minimal increase in the mutant fre-

quency not only in cell lines, but also in the in vivo

model systems that have a p53 “null” background. An

approach to testing this hypothesis would be to cross

a transgenic mouse that is heterozygous at a reporter

gene loci, e.g. the TK+/− mouse of Doborovolsky

et al. [148] or the APRT+/− mouse of Stambrook and

co-workers [134,135], with a mouse that carries a

point mutation in p53 such as that recently described

by Liu et al [149].

A body of evidence exists that mutation leading

to either the absence of the wild-type protein or the

presence of the mutant protein results in the decreased

ability of p53 to function both as a transcription fac-

tor and to properly serve as a structural component

of protein complexes involved in the response to

DNA damage. Experiments that more fully define the

mechanism(s) by which the presence of mutant p53


results in a substantially greater increase in the mu-

tant frequency than does the absence of the wild-type

protein will be of considerable interest. An approach

which may provide insight into the effect of mutation

on the ability of p53 to serve as a transcription factor

would include genome-wide expression analysis of

cells with mutations in critical sites of the gene. The

effect of critical site mutations on the interaction of

p53 with other cellular proteins, as well as the iden-

tification of additional proteins that interact with p53,

could be characterized by protein “chip” analysis

combined with two-dimensional gel electrophoresis.

This knowledge would contribute substantially to our

understanding of the pathways leading to malignancy.

It is clear, however, that with the failure of the G1

checkpoint, the inability of cells to repair damaged

DNA before replication and the abrogation of the tar-

geted removal of cells with damaged DNA from the

population by apoptosis, damaged or mutant cells can

survive, proliferate and contribute to the progression

to malignancy. Further, p53 serves as a convergence

point for the integration of signals from oncogenic

stimuli as well as those from the DNA damage path-

ways and both pathways contribute to the cell’s abil-

ity to maintain genomic integrity. Through its ability

to modulate the acquisition of additional mutation,

p53 plays a central role in the guardianship of the

genome and loss of p53 contributes significantly to

the progression of cells to a malignant state.

Acknowledgements

The author would like to thank Dr. William Tolleson

and Dr. Greg Akerman for helpful discussions and Dr.

Robert Heflich for critical review of the manuscript.

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