Journal of General Virology (1993),74, 803 810. Printed in Great Britain
803
Biological activities of p53 mutants in Burkitt's lymphoma cells
Karen H. V o u s d e n , * T i m C r o o k and P a u l J. Farrell
Ludwig Institute for Cancer Research, St Mary's Hospital Medical School, Norfolk Place, London W2 1PG, U.K.
Wild-type human p53 and a series of p53 point mutants
isolated from Burkitt's lymphoma (BL) cell lines were
tested for their ability to inhibit D N A synthesis in a p53negative BL cell line and to bind and be degraded by the
human papillomavirus type 16 E6 protein. All the
mutants lost the wild-type ability to inhibit DNA
synthesis, demonstrating that they are all functionally
altered. Binding to E6 and consequent degradation of
the p53 mutants frequently correlated with changed
suppressor properties in BL cells.
Introduction
interaction with each viral protein may lead to loss of
p53 function, the consequence of the virus-p53 protein
interaction for p53 stability differs considerably. SV40
large T antigen interaction results in the stabilization of
p53 (Yewdell et al., 1986) and binding to E6 of HPV
leads to the rapid proteolytic degradation of p53
(Scheffner et al., 1990). E6-directed degradation of p53
requires at least one other cell protein (Huibregtse et al.,
1991) and involves the ubiquitin-directed proteolytic
pathway, a mechanism by which p53 may normally be
turned over in an uninfected cell (Ciechanover et al.,
1991). We have shown recently that some mutant p53
proteins identified in anogenital cancers have a reduced
ability to bind E6 and therefore display some resistance
to E6-directed degradation (Crook & Vousden, 1992).
This may reflect a change in the interactions of p53 with
other cell proteins, although no correlation between
transforming activity in rodent cells and ability to be
targeted for degradation by E6 in an in vitro assay could
be identified.
We and others recently found that p53 is frequently
mutated in Burkitt's lymphoma (BL) cell lines (Farrell
et al., 1991; Gaidano et al., 1991 ; Wiman et al., 1991).
Analysis of several of these mutant p53s in primary
rodent epithelial cells revealed a range of activities from
suppression of transformation, through loss of suppressor activity to acquisition of dominant transforming
activity (Farrell et al., 1991). In this study we have
assayed the activity of these mutant p53 proteins in
Akata ceils, a p53-negative BL cell line representative of
the cell type from which the mutants were isolated. We
have also examined the ability of the mutant p53 proteins
to form a complex with the HPV-16 E6 protein and
subsequently be degraded, to assess whether this activity
correlates with altered biological activity in the relevant
cell type.
Mutations within the p53 tumour suppressor gene have
been detected in a wide range of different human tumours
and are the most common genetic change detected in
human cancers (Hollstein et aI., 1991). The majority of
mutations identified are single base substitutions resulting in the alteration of one amino acid within one of the
evolutionarily conserved regions of the p53 protein. The
observation that mutation usually results in the expression of an altered protein, rather than complete loss
of p53, suggests that mutant p53 proteins have an
activity other than loss of wild-type p53 function.
Support for this hypothesis has been provided by
experimental studies showing transforming activities of
mutant p53 genes in p53-negative cells (Shaulsky et al.,
1991). Transfection studies in rodent cells have demonstrated positive transforming activities of many p53
mutants which appear to be separable from the loss of
wild-type ability to suppress transformation in the same
cell systems (Hinds et al., 1992; Farrell et al., 1991).
Expression studies have shown that in many cases
point mutation within p53 results in the stabilization of
the protein leading to an overexpression of p53 in
tumour cells (Lane & Benchimol, 1990). The basis for
this stabilization is not understood but it is of interest
that p53 is targeted by the transforming proteins encoded
by small DNA tumour viruses and that these interactions
also affect p53 stability (Levine, 1990). Adenovirus,
simian virus 40 (SV40) and oncogenic human papillomavirus (HPV) types such as HPV-16 and -18 all encode
oncoproteins which form a complex with wild-type p53
(Lane & Crawford, 1979; Sarnow et al., 1982; Werness
et al., 1990). This complex formation is thought to
prevent wild-type p53 function and so relieve the negative
control of cell growth normally exerted by p53. Although
0001-1425 © 1993SGM
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804
K. H. Vousden, T. Crook and P. J. Farrell
Results
Methods
Construction o f A k a t a / p 5 3 transfectants. The p53 cDNAs described
earlier (Farrell et al., 1991) were subcloned into pMEP4 from the pJ4f~
constructs using the BglII and HindIII polylinker sites in pJ4f~ and the
BamHI and HindIII polylinker sites in pMEP4 (Invitrogen). This
leaves the p53 correctly oriented in pMEP4 for expression from the
metallothionein promoter in that vector. Plasmid DNAs were electroporated into Akata cells (Takada & Ono, 1989) and hygromycinresistant lines were selected as described elsewhere (Lau et al., 1992).
The cells were maintained in RPMI-1640 containing 10 % fetal calf
serum and 0.3 mg/ml hygromycin B.
Western blotting. Cells were lysed in SDS sample buffer, electrophoresed on SDS-polyacrylamide gels and blotted to nitrocellulose.
p53 was detected using PAbl801 (see below) and the Amersham ECL
system. Typically about 8 x 10~ cells were resuspended in 200 gl of SDS
sample buffer, sonicated, boiled and 50 lal was loaded per lane.
[3H]Thymidine incorporation assays. Cells were maintained in
exponential growth by dilution every 1 to 2 days for at least 7 days prior
to labelling. Normally 75 lal of cell culture (about 4 x 104 cells) was
mixed in a well of a 96-well microtitre plate with 75 gl of complete
medium with or without CdC1e to give a final CdC12 concentration of
5 gM. After incubation for 24 h at 37 °C, 50 gl of medium containing
0.4 gCi of [aH]thymidine was added and incubation continued for a
further 4 h at 37 °C. Cells were then harvested onto filters using the
Skatron semiautomatic harvester and the dried filters were scintillationcounted. Duplicate wells were sampled for each cell line in two
experiments and the mean ratios of thymidine incorporation after 24 h
incubation with ( + CdC12) and without ( - CdC12) CdC12 and standard
deviations are shown in Fig. 2. Typical results for BL2 were about
250000 d.p.m, for -CdC12 and about 70000 d.p.m, for +CdCI~.
Transfections o f primary rat cells. Baby rat kidney (BRK) cells were
prepared and transfected with plasmids encoding mutant p53 sequences
using calcium phosphate coprecipitation as previously described
(Crook et al., 1991 a). Cells were cotransfected with 5 gg of each test
plasmid, 5 gg pJ4~E7, 5 lag pEJ6.6 and 1.5 gg pSV2neo. Selection for
transfected cells was carried out using 0"3 mg/ml G418 and colonies of
transformed cells were scored after 2 to 3 weeks.
E6 binding and degradation. Wild-type and mutant p53 and HPV-16
E6 protein were synthesized following in vitro transcription and
translation in a rabbit reticulocyte lysate as previously described
(Crook et al., 1991b). For binding experiments, the E6 protein was
translated in the presence of [35S]cysteine and the p53 proteins were
labelled with [35S]methionine. Equal amounts of each p53 protein were
mixed with E6 in 100 mM-NaC1, 0-1 M-Tris HC1 pH 8-0, 1% NP40 and
allowed to incubate on ice for 1 to 2 h. The p53 E6 complexes were
immunoprecipitated using the monoclonal antibody (MAb) PAbl801
(Banks et al., 1986), an antibody recognizing an N-terminal epitope of
p53 distant from the position of any of the mutations, and Protein A
or Protein ~ S e p h a r o s e as previously described (Crook et al., 1991 b).
Coprecipitated proteins were resolved by PAGE and levels of binding
quantified by densitometric scanning, making allowance for variation
in p53 input. Assays for E6-directed degradation were carried out by
mixing in vitro translated E6 and p53 in 25 mM-Tris-HC1 pH 7.5,
100 mM-NaC1, 3 mM-DTT and incubating at 30 °C or 37 °C for 0 to
3 h. For studies at 37 °C, proteins were equilibrated for 10 min at 37 °C
prior to E6 addition. The p53 protein remaining after incubation with
E6 was resolved by PAGE either with or without prior immunoprecipitation with PAbl801, equivalent results being obtained using
either protocol. The extent of p53 degradation was assessed by
densitometric scanning of the autoradiograms.
Inducible expression of p53 in a p53-negative B L cell
line
Although the mutant p53s detected in BL cell lines have
previously been assayed for transformation and suppressor activities in rodent cells (Farrell et al., 1991), it
would be more appropriate to determine the effect of
expressing these proteins in human B lymphocytes. Most
of the cell lines in which the mutations were originally
identified showed deletion of the second p53 allele and so
did not express any wild-type p53 protein. These assays
were therefore carried out in Akata cells, a BL cell line
previously shown not to express p53 (Farrell et al., 1991),
to avoid any influence of endogenous wild-type p53
protein. Expression of wild-type p53 in normal or
transformed cells has been shown to arrest progress
through the cell cycle and is incompatible with continued
cell growth (Baker et aI., 1990; Diller et al., 1990; Mercer
et al., 1990). To examine the effects of expressing wildtype and mutant p53 proteins in Akata cells, we therefore
used a system which allowed inducible expression of the
p53 proteins following transfection and isolation of cell
lines. The p53 sequences were placed under the control of
the metallothionein promoter in a vector containing the
Epstein-Barr virus origin of replication and a dominant
selectable marker. Transfections and subsequent drug
selection were carried out without induction of p53
expression and several independently derived pools of
transfected ceils were isolated for each p53 plasmid. Hirt
supernatants (Hirt, 1967) were assayed by Southern
blotting and revealed approximately the same copy
number of episomally maintained plasmid in each of the
lines (data not shown). Induction of p53 proteins was
achieved by adding 5 [tM-cadmium chloride to the culture
medium and protein expression was normally assayed by
Western blotting 24 h after addition of cadmium chloride. Five independently derived pools of cells transfected
with wild-type p53 were shown to induce about the same
level of p53, whereas uninduced cells expressed no
detectable p53 protein (Fig. 1a). A truncated form of p53
was also observed in all the inductions. We are unsure
whether this is a degradation product of the full-length
p53 or a result of erroneous initiation or termination of
translation from the cDNA, which lacks some of the
normal 5' and 3' untranslated sequences of p53 mRNA.
A similar band has been detected in other cell lines
overexpressing p53 with several different anti-p53 antibodies (Rodrigues et al., 1990; Gannon et al., 1990).
Levels of p53 protein expression following cadmium
induction were similar to the high levels seen in Ramos
cells (Fig. 1b), one of the BL cell lines in which a mutant
p53 was originally detected. Increased levels of p53
protein were detected by Western blotting 4 h after
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p53 mutants in Burkitt's lymphoma cells
(b)
(a)
Akata
--
+
--
+
--
+
Akata + p53 wt
+
_
÷
--
Akata
+ p53 wt
+ Ramos
4-
-
-53
53
(d)
(c)
0
P3
805
WT
2
P3 WT
4
P3 WT
8
P3 WT
p53 Ramos
24
P3 wT
-
-
+
_
p53 Raji
+
-
+
-
+
-53
53
(e)
p53 BL41
-I-
p53 J~oye
+
--
+
p53 BL37
+
+
--
p53 P3HR1
+
--
+
--
+
53
- 53
Fig. 1. Western blotting of p53 with PAb 1801 MAb. The position of p53 is marked. CdCl~-treated (+), not treated ( - ) . (a) Akata cells
or Akata + MEP4p53 BL2 (wild-type) cells were treated for 24 h with 5 gM-CdC12.(b) Akata + MEP4p53 BL2 (wild-type) cells induced
( + ) with 5 gM-CdC12for 24 h or uninduced cells ( - ) as a control were compared with a similar quantity of Ramos cells. (c) Time course
of p53 accumulation after 0, 2, 4, 8 or 24 h of 5 gM-CdCI~treatment in (P3) Akata + MEP4p53 P3HR1 cells or (WT) Akata + MEP4p53
BL2 (wild-type) cells. (d) Induction of p53 by 5 gM-CdC12for 24 h in separate pools ofAkata + MEP4p53 Ramos and Akata + MEP4p53
Raji cell lines. (e) Induction of p53 by 5 gM-CdC12 for 24 h in separate pools of Akata + MEP4p53 BL41, Akata + MEP4p53 Jijoye,
Akata + MEP4p53 BL37 and Akata + MEP4p53 P3HR1 cell lines.
i n d u c t i o n with c a d m i u m a n d m a x i m u m expression was
achieved b y 8 h (Fig. 1 c). Similar studies with cell lines
transfected with the m u t a n t p53 p l a s m i d s s h o w e d t h a t
b r o a d l y c o m p a r a b l e levels o f p53 p r o t e i n were expressed
after i n d u c t i o n (Fig. 1 a, d a n d e) a l t h o u g h levels o f
P 3 H R 1 p53 were r e p r o d u c i b l y slightly lower. N o
c o r r e l a t i o n b e t w e e n p h e n o t y p e a n d level o f p53 exp r e s s i o n was o b s e r v e d between different lines expressing
the same p53 m u t a n t so it a p p e a r s t h a t the a m o u n t o f
p53 being p r o d u c e d is n o t limiting. C o m p a r i s o n o f
m u t a n t p53 p r o t e i n s revealed slight differences in
m o b i l i t y reflecting the p o l y m o r p h i s m at a m i n o acid 72.
Transfections of primary rat cells
O u r p r e v i o u s studies s h o w e d t h a t expression o f w i l d - t y p e
p53 c o u l d suppress t r a n s f o r m a t i o n b y a d e n o v i r u s E l a o r
H P V - 1 6 E7 in c o o p e r a t i o n w i t h ras in B R K cells,
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806
K. H. Vousden, T. Crook and P. J. Farrell
Table 1. Transfection of p53 mutant genes in B R K cells
Number of transformed colonies
o
1.2
o
1.0
Transfected clone
(+E7+ras)
pMo*"
Ramos
J~oye
BL2 (wild-type)
Expt. 1
Expt. 2
Expt. 3
Mean
18
32
46
1
10
18
17
1
19
14
49
0
16
21
37
1
* Moloney routine leukaemia virus long terminal region vector
lacking p53 sequences.
whereas transfection with mutant p53s derived from BL
cell lines showed a range of phenotype from suppression
to enhancement of transformation (Farrell et al., 1991).
In this original study, Ramos mutant p53 was found to
suppress transformation by both HPV-16 E7 and
adenovirus Ela and was thus indistinguishable from
wild-type p53 in this assay. Subsequent analyses have
revealed, however, that the p53 clone used for those
studies had acquired a frameshift mutation during PCR
cloning which resulted in the expression of a truncated
protein. The authentic Ramos p53 mutant carrying only
the Ile to Asp substitution at position 254 has now been
tested in the BRK assay and shown to have lost
suppressor activity and possibly gained a slight positive
transforming function as measured by the ability to
enhance E7 plus ras transformation (Table 1). This
corrected version of the Ramos p53 mutant has been
used in all further experiments. The p53 mutant derived
from Jijoye cells was also tested in this assay and
similarly shown to have lost suppressor and gained some
transforming activity (Table 1).
Activities of mutant and wild-type p53 in Akata cells
The effect of expressing p53 protein on growth of Akata
cells was assessed by measuring [aH]thymidine incorporation following cadmium induction. Initial experiments showed that expression of wild-type p53 resulted
in a decrease in DNA synthesis which was seen most
clearly after a 24 h induction with cadmium. Longer
exposure to cadmium (48 to 72 h) resulted in a nonspecific inhibition of growth characterized by a reduction
in aH incorporation in all cells including those transfected
by vector sequences only (data not shown). Comparisons
of DNA synthesis in cells expressing wild-type and
mutant p53 proteins were therefore made following a
24 h exposure to cadmium. Expression of wild-type p53
reduced [aH]thymidine incorporation to approximately
20 to 50 % of that seen in cells transfected by vector
sequences only (Fig. 2), presumably reflecting the
negative effect of wild-type p53 on cell growth. The
ability to cause this substantial arrest of cell growth was
";~
~3 ..~ 0 6
.=r.) 0.4
O
0.2
0
,, i iiii Iil
Fig. 2. Ratios of thymidine incorporation with and without 24 h CdCI~
induction of p53 in Akata cells. Each bar represents the value for an
independent pool of Akata cells containing the indicated p53 construct.
lost by all the mutant p53s tested and no consistent
difference could be detected between them (Fig. 2). None
of the mutant p53s increased the rate of DNA synthesis
above that seen for control Akata cells.
The microscopic appearance of the Akata cell line
containing the inducible wild-type p53 construct was
studied over a 3 day period of induction by CdC12. By the
third day there was extensive cell death. A myeloid
leukaemia cell line growth-arrested by p53 has been
reported to die by apoptosis (Yonish-Rouach et al.,
1991). However, 24 h after addition of CdC12, when
DNA synthesis has been arrested, there was no visible
difference between the treated and control Akata cells
containing inducible wild-type p53. In addition, agarose
gel electrophoresis of DNA from the cells treated with
CaC12 for 24 h showed no evidence of the nucleosomal
ladders of cleaved DNA characteristic of apoptosis (data
not shown). It therefore appears that the inhibition of
DNA synthesis by p53 in these BL cells occurs before any
apoptotic or other cell death. The uniform phenotype
displayed by the different mutant p53s in the Akata cell
assay is in contrast to the variable activities displayed in
the rodent cell transformation assay, where the Ramos
and P3HR1 mutants showed evidence of dominant
transforming activity. In BRK cells, the BL41 mutant
lost suppressor activity without gaining transforming
activity but Raji and BL37 retained suppressor activity
and were indistinguishable from wild-type p53.
HPV-16 E6-directed degradation of p53
Wild-type p53 is able to form a complex with the HPV16 E6 protein (Werness et al., 1990), resulting in the
rapid degradation of p53 (Scheffner et al., 1990). This
association is thought to be one of the mechanisms by
which HPV-16 interferes with normal cell growth control
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p53 mutants in Burkitt's lymphoma cells
807
Table 2. Interactions between p53 and E6
(a) Binding of p53 to E6
Binding to E6
Plasmid
Wild-type p53
Ramos
P3HR1
BL41
BL37
Raji
Expt. 1
Expt. 2
Expt. 3
1.0
0.23
0.25
0.24
0.55
1.05
1.0
0
0'16
0.25
0.71
1.06
1.0
0
0
0.17
NI~*
0.93
(b) E6-directed degradation o f mutant p53 at 30 °C for 3 h
Percentage of input p53 degraded
Mutant
Wild-type p53
Ramos
P3HR1
BL41
BL37
R@
Expt. 1
Expt. 2
Expt. 3
Expt. 4
Expt. 5
Mean-I-S.D,
98
13
69
73
26
96
100
39
65
61
51
100
100
18
62
ND
ND
ND
100
36
65
ND
ND
97
100
15
41
82
57
86
100±1
24±12
60 ± 11
72 ± 10
45 ± 16
95±6
(e) E6-directed degradation after 1 h at 30 °C or 37 °C
Percentage ofinput p53 degraded
Expt. 1
Plasmid
Wild-type p53
BL41
BL37
Raji
Expt. 2
30 °C
37 °C
30 °C
37 °C
96
70
48
82
47
23
6
0
86
40
20
65
44
36
5
7
* ND, Not determined.
and the ability to interact with E6 may reflect an
important normal function of p53, perhaps related to the
rapid turnover of the wild-type protein. The mutant p53
proteins described in this study were assayed for their
ability to be degraded following complex formation with
E6 to determine whether this activity correlated with
phenotypes displayed by these mutants when expressed
in rodent epithelial or human B cells. All the mutant p53s
tested, with the exception of Raji, showed a reduction in
E6 binding compared to wild-type protein (Table 2).
BL37 p53 displayed a modest reduction in binding
whereas BL41, Ramos and P3HR1 p53 showed a more
severe loss of the ability to complex E6. These binding
activities were essentially reflected in the ability of these
mutant p53s to be targeted for degradation by E6 (Table
2, Fig. 3) where only the Raji protein is degraded like
wild-type p53. Other p53 mutants showed varying
degrees of resistance to degradation, Ramos p53 being
the most clearly resistant. These assays were carried out
at 30 °C because previous studies suggested that degradation of p53 is most efficient at this temperature
Ramos
0
P3HR 1
3
0
3
Raji
0
Wild-type
3
0
3
.,4.- p53
Fig. 3. Degradation of in vitro translated p53 proteins at 30 °C
following incubation with equal amounts of in vitro translated HPV-16
E6 for 0 or 3 h.
(Scheffner et al., 1990). Recent reports, however, have
suggested that some human mutant p53 proteins may be
temperature-sensitive, displaying a wild-type conformation at 30 °C and a mutant conformation at 37 °C
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808
K. H. Vousden, T. Crook and P. J. Farrell
Table 3. Summary of p53 activity in human and rodent cells, E6 binding and degradation
Clone
Mutation
BRK assay
pMEP4 vector
Wild-type p53
None
Suppressing
Ramos
P3HR1
Jijoye
BL41
BL37
Raji
Ile (254) Asp
Tyr (163)-His
Lys (132~Gln
Arg (248)-Gln
Met (237)-Ile
Arg (213) Gln
Transforming
Transforming
Transforming
Null
Suppressing
Suppressing
Akata assay
[~H]incorporation
1.01 _+0.09
0.22 +- 0.10
0.48_+0.12
0'81 + 0.11
0.95+_0.06
0.76+_0.14
0.93_+0.14
0.72_+0.15
0.91 +_0.06
E6 binding
E6 degradation
+ + +
+ + +
+
+
ND*
+
+ +
+ + +
+
+ +
ND
+ +
+ +
+ + + 30 °C
+ 37 °C
* ND, Not determined.
(Medcalf et al., 1992). We therefore also performed
degradation assays at 37°C to test Raji p53 for
temperature-dependent sensitivity to E6-directed degradation. As reported by Scheffner et al. (1990), degradation at 37 °C was less efficient in these in vitro assays
than that measured at 30 °C and more consistent
differences between degradation at the two temperatures
were seen when degradation was assayed after 1 h, before
complete degradation of wild-type p53 at 30 °C (Table
2). Although the wild-type degradation of Raji p53 was
confirmed at 30 °C, this mutant showed significantly
enhanced resistance to degradation at 37 °C.
Discussion
Sequence analyses have demonstrated the presence of
mutations within the p53 gene of many different human
cancers and it is generally assumed that expression of
these mutant p53 proteins contributes to malignant
development. Although many different missense mutations in p53 have been identified, relatively few have been
examined for alterations in biological activities such as
the ability to suppress or enhance transformation. A high
proportion of BL cell lines had previously been shown to
carry mutations within the p53 gene but transfection of
these mutant p53s into primary rodent epithelial cells
showed that few of them exhibited a dominant transforming activity. Some of the BL-derived p53 mutants
lost suppressor activity without gaining transforming
activity and others retained the ability to suppress
transformation, behaving indistinguishably from wildtype p53 in this assay. To examine this apparent range of
activities more closely in a relevant cell type we have
determined the effect of expressing wild-type and mutant
p53 proteins on DNA synthesis in Akata cells, a p53negative BL cell line. In contrast to the activities assayed
in rodent cells, all the mutant p53s showed a similar loss
of the wild-type ability to depress thymidine incor-
poration into DNA. The ability of wild-type p53 to
reduce DNA synthesis is consistent with previous studies
showing arrest of cell growth following expression of
p53. Although the levels of p53 protein induced in these
studies are clearly much higher than seen for wild-type
p53 during the normal cell cycle, our studies suggest that
they are comparable with levels of mutant p53 protein
seen in BL cell lines and a recent proposal for the normal
role of p53 (Lane, 1992) is based on the high level of
expression of wild-type p53 in response to DNA damage.
None of the p53 mutants tested showed any ability to
increase DNA synthesis compared to control Akata cells
and no evidence for a positive growth-enhancing activity
could be detected in this assay. The variation in activities
in the rodent and human cell assays could reflect the
difference in cell type used or differences in phenotype
being assayed. Akata cells do not express p53 and may
have acquired compensatory mutations in other genes
controlling cell growth, masking potentially growthpromoting effects of the mutant p53 proteins. It will
therefore be worthwhile to assess the activity of these
mutant p53 proteins in untransformed lymphoblastoid
cell lines expressing endogenous wild-type p53. Our
results also show for the first time that inhibition of
DNA synthesis by wild-type human p53 occurs in cells
carrying the translocated myc locus characteristic of BL,
which may be of interest since one potential function of
p53 is in transcriptional control of growth regulatory
proteins (Fields & Jang, 1990; Raycroft et al., 1990;
Ginsberg et al., 1991).
It is clear from our studies that mutant p53s identified
in human cancer cell lines show alterations in growth
suppressing or promoting activities when compared to
wild-type p53, but that these differences depend on the
cell type in which the assay is carried out as well as the
specific type of mutation involved. We have therefore
used an in vitro assay to examine another property of
p53, the ability to be targeted for degradation by HPV16 E6, to determine whether this activity could be
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p53 mutants in Burkitt's lymphoma cells
correlated with any of the effects on cell growth. In
general, the acquisition of resistance to degradation
correlated well with the loss of ability to depress DNA
synthesis in Akata cells, whereas the more complex range
of phenotypes assayed in primary rodent cells was not
clearly reflected in the degradation assay. The Raji p53
mutant (Arg to Gln at residue 213) showed some
temperature sensitivity for resistance to degradation and
it may be of interest to determine whether expression of
Raji p53 at 30 °C in Akata cells results in a reduction of
DNA synthesis consistent with a wild-type activity at this
temperature.
We have used several different assays to measure the
function of mutant p53s, summarized in Table 3. It is
tempting to correlate intermediate phenotypes in some of
these assays, for example BL37 and Raji retain suppressor activity in BRK cells and retain the strongest E6
binding of all the mutants whereas Ramos is transforming in the BRK assay and most defective for E6
binding and degradation. This, however, is not reflected
in the Akata DNA synthesis assay where none of the p53
mutants, including Raji and Ramos, are distinguishably
different. At present we think that the assays are too
complex to allow much detailed interpretation beyond
demonstrating loss of wild-type function and offering
useful clues to the underlying biochemical changes in p53
function resulting from the mutations. So, in summary,
these studies indicate that all the BL p53 mutants are
functionally altered and that the in vitro assays may prove
useful in the rapid screening for functional alterations of
p53 mutants.
We would like to thank Frances Shanahan and Chris Fisher for
excellent technical assistance and Fritz Propst and Roger Watson for
reading the manuscript.
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(Received 21 October 1992; Accepted 15 December 1992)
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