ABSTRACT
DNA topoisomerase
II[alpha]
is an essential enzyme for chromosome segregation during mitosis. Consistent
with a cell division-specific role, the expression of the topoisomerase
II[alpha]
gene is strongly influenced by the proliferation status of cells. The p53
protein is one of the most important regulators of cell cycle progression in
mammals, with an apparent dual role in the induction of cell cycle arrest
following cytotoxic insults and in the regulation of the apoptotic cell death
pathway. We have analysed whether p53 plays a role in regulating expression of
the human topoisomerase
II[alpha]
gene. We show that wild-type, but not mutant, p53 is able to decrease substantially the activity
of the full length topoisomerase
II[alpha]
gene promoter. Using a series of constructs comprising various deleted or
mutated versions of the promoter lacking critical
cis
-acting elements, we show that this p53-specific regulation of the topoisomerase
II[alpha]
promoter is independent of all characterised transcription factor binding sites
and is directed at the minimal gene promoter. We conclude that expression of
wild-type p53 induces downregulation of the human topoisomerase
II[alpha]
promoter by acting on the basal transcription machinery. These findings
implicate topoisomerase II as one of the downstream targets for p53-dependent regulation of cell cycle progression in human cells.
Topoisomerase II is an essential nuclear enzyme that catalyses changes in the
topological state of DNA (for reviews see
1
-
4
). Genetic studies in yeast have indicated that topoisomerase II plays a
critical role during mitosis, being required for the disentanglement of newly-replicated sister chromatids (
5
-
8
). As a consequence of this role, chromosome segregation during cell division is
prevented in the absence of active topoisomerase II, leading to chromosome
breakage during an abortive anaphase (for a review see
9
). Other roles for topoisomerase II include relief of torsional stress during
DNA replication and transcription, and the suppression of hyperrecombination
within repetitive DNA sequences (
2
,
10
-
12
). In humans, topoisomerase II is also the primary cellular target for many of the most effective antineoplastic drugs, including etoposide, doxorubicin, mitoxantrone and epirubicin (for
reviews see
13
-
15
). The relationship between the cytotoxicity of these drugs and the
intracellular level of the target protein (topoisomerase II) is unusual, in
that high levels of topoisomerase II confer relative drug sensitivity, because
the enzyme participates in the formation of the cytotoxic DNA lesions.
There are two closely related isoforms of topoisomerase II in mammalian cells
that are designated topoisomerase II[alpha] (170 kDa form) and topoisomerase II[beta] (180 kDa form) (
16
-
20
). The respective functions of the two isoforms remain to be identified,
although the [alpha] isoform is a well-established marker of proliferation both in cultured cell lines and
in tissues
in vivo
, while the [beta] isoform appears to be expressed in both proliferating and quiescent cells
(
21
-
24
).
The factors that regulate topoisomerase II gene expression have not been defined in detail. The level of expression of topoisomerase II[alpha] mRNA is low in quiescent cells, but accumulates to a much
higher level as cells traverse the cell division cycle with peak levels in late
S phase (
21
,
25
). Levels of topoisomerase II[alpha] protein are very low in G
1
phase cells, reflecting the degradation of this protein that occurs during late
M phase. Previous studies using constructs of the human topoisomerase II[alpha] gene promoter fused to a chloramphenicol acetyl transferase (CAT)
reporter gene have localised the elements required for maximal expression of
the gene to the 350 bp region immediately upstream of the major transcription
start site (
26
). A similar core region required for high level expression of the hamster
topoisomerase II[alpha] promoter has been identified (
27
). However, it is known that a minimal region of ~100 bp still retains substantial promoter activity (
26
) suggesting that many of the
trans
-acting factors required for basal expression of human topoisomerase II[alpha] mRNA interact with a short region of the promoter immediately 5' to the CAP site.
The p53 protein is a sequence-specific DNA binding protein capable of activating transcription from a
set of genes that contain a consensus p53 binding element in either their
promoter region or elsewhere in the gene (
28
-
30
). For example, p53 positively regulates expression of the cyclin-dependent kinase inhibitor p21 (also known as CIP1/WAF1) (
31
) as well as the growth arrest and DNA damage-inducible gene, GADD45 (
32
,
33
). In this latter case, the p53 binding element is located within one of the
introns of the gene, not directly in the promoter region (
32
). Conversely, wild-type p53 protein has been shown to repress the activity of certain
cellular and viral promoters that do not contain p53 binding sites (
34
-
36
). In this paper, we have investigated whether the p53 protein is able to
regulate expression of the topoisomerase II[alpha] gene. We show that wild-type p53, but not mutant p53, represses transcription
from the topoisomerase II[alpha] promoter by targeting the minimal sequences required for
promoter activity.
The human ovarian cancer cell line SKOV3, which does not express p53 mRNA or
protein (
37
), was grown in RPMI-1640 medium supplemented with 10% foetal calf serum (FCS). The SK23a and
SKN cell clones (
38
) were both derived from SKOV3 cells. To generate the SK23a derivative, SKOV3
cells were co-transfected with a plasmid encoding murine temperature-sensitive mutant p53 (
39
) and the pSV2neo vector containing the neomycin selectable marker genes. The
SKN cell line was derived by transfection of SKOV3 cells with the neomycin
expression vector alone, and served as a negative control. The human
osteosarcoma cell line Saos-2, which expresses no p53 (
40
) and no functional pRb protein (
41
) as well as a derivative (designated Saos-2 ts p53) that stably expresses a temperature-sensitive human p53 protein due to a valine to alanine substitution
at amino acid 138, were maintained in RPMI-1640 supplemented with 10% FCS. Where required for transfection using
calcium phosphate (see below), cells were grown in Dulbecco's modified Eagles's
Medium (DMEM) for 24 h prior to addition of the precipitate. All cells were
grown in a humidified atmosphere in the presence of 5% CO
2
, and were regularly screened for the presence of mycoplasma.
The construct encoding the wild-type human p53, designated pLSVhp53c62 (
42
) utilises the SV40 early promoter in the vector pLSV. The construct for
expression of mutant murine p53 (pLTRp53cGVal 135; kindly supplied by Dr M.
Oren) has been described in detail elsewhere (
39
), and encodes a temperature-sensitive p53 that adopts a wild-type conformation at 32oC, but a mutant conformation at 37oC due to a mutation at position 135 (valine to alanine).
The construct (designated pCMVtsp53Val 138) for expression of a human p53 that
adopts a mutant conformation at 37oC and a wild-type conformation at 32oC, was kindly provided by Dr J. Jenkins and is essentially
identical to that described previously (
43
). pnlsLacZ is an expression plasmid encoding the bacterial [beta]-galactosidase gene under control of SV40 promoter. Plasmids encoding
the
Escherichia coli
CAT gene under the control of different fragments of the human topoisomerase II[alpha] gene promoter, have been reported previously (
26
). Plasmids carrying the mouse ferritin (FGH) and the human phosphoglycerate
kinase 1 (PGKGH) gene promoters linked to the human growth hormone (HGH) coding
region (
44
,
45
) were kindly provided by Dr J. Firth (Oxford, UK). The pKV461/CD2 (kindly
provided by Dr C. J. Norbury) contains a truncated rat CD2 cDNA and was
generated by excising the CD2 cDNA from pERCD2-2 (
46
) and cloning it into the
Bgl
II site of pKV461 (kindly supplied by Dr M. Sowden;
47
).
The plasmid used for all of the HGH constructs was PGEM7Zf+ (PROMEGA),
containing the 1.8 kb HGH cDNA as a reporter gene. This plasmid, designated
pSVGH, was a generous gift from Dr J. Firth, Oxford, UK. The SV40 promoter in
pSVGH was replaced by the topoisomerase II[alpha] promoter to generate pHGH, as described previously (
48
). Deletion constructs containing various truncated forms of the topoisomerase
II[alpha] promoter were generated by PCR from the full length 2.5 kb fragment of
the promoter (
26
). PCR primers incorporated restriction sites to enable directional cloning via
the
Xba
I and
Hin
dIII, or the
Bam
HI sites in pHGH. Sequences for the 5' primers (with the 5' limit indicated on the left) used in generating the promoter
truncations were as follows. Numbering begins at +1, the transcription start
site.
5'-GATCTCTAGAGCCACCGCACACAGCCTACTT-3'
5'-GATCTCTAGATTTGAAGCCTCTCTAGTCC-3'
5'-GATCTCTAGAAGCCGTTCATAGGTGGATAT-3'
5'-GATCTCTAGACTTCTGGACGGAGACGGTGA-3'
5'
-
GATCTCTAGAGCTTCGGGCGGGCT-3'
In each case, the 3' primer, which ran up to the translation start site, was as below:
ATG
5'
-
GGATCAAGCTTATGGTGACGGTCGTGAAGG-3'
After directional cloning of the PCR products into pHGH, the orientation and
sequence of all promoter fragments was confirmed.
Exponentially growing cells were transfected with 1-15 [mu]g of the p53 expression vectors and 10 [mu]g topoisomerase II[alpha] promoter/reporter gene constructs using the CaPO
4
co-precipitation procedure of Graham and van der Eb (
49
). Forty-eight hours after transfection, cells were harvested and lysed by three
successive cycles of freezing and thawing. Where indicated, an expression plasmid encoding the bacterial [beta]-galactosidase gene was included in the transfection, both to monitor transfection efficiency and to
standardize the amount of extract to be used in subsequent CAT assays. [beta]-galactosidase assays were performed as described by Herbomel
et al
. (
50
). An equivalent concentration of DNA was used in all transfections by adjusting
the level of control vector DNA.
Exponentially growing Saos-2 or Saos-2 ts p53 cells were transfected by the CaPO
4
co-precipitation method of Graham and van der Eb (
49
). Precipitates (1.5 ml) which contained 30 [mu]g of the appropriate pGEM plasmid carrying the topoisomerase II[alpha] promoter linked to the HGH reporter gene, and, where required, 15 [mu]g pKV461/CD2 (
51
) or 15 [mu]g of a control plasmid, were applied to subconfluent cells for 16 h. The
cells were then washed in phosphate buffered saline, trypsinized, and seeded at
a ratio of 1:2 in RPMI-1640 medium. Cultures were then maintained either at 32 or 37oC for up to 120 h.
CAT activity was measured by the conversion of
14
C-labelled chloramphenicol to its acetylated forms using standard
techniques. Briefly, cells were harvested and lysed by three cycles of freezing
and thawing. Aliquots of extract were incubated for 2 h at 37oC with 4 mM acetyl co-enzyme A and [
14
C]deoxychloramphenicol. The acetylated reaction products were separated from the substrate by thin layer chromatography. The percent conversion was determined by excising radioactive spots from the thin layer chromatography
plates and measuring the level of radioactivity in each sample in a
scintillation counter.
Promoter activity was determined by measuring the level of the HGH gene product
in the media of transfected cells in culture, as described previously (
48
). All assays were kindly performed by Gillian Campling at Littlemore Hospital,
Oxford, UK.
This was performed using the Muta-gene
in vitro
mutagenesis system (BioRad), which utilizes oligonucleotide primers containing the appropriate point mutations, and a single-stranded template into which uracil residues have been incorporated to
permit selection against the template strand of DNA. This protocol was based on
the method of Kunkel
et al
. (
52
). The pHGH derivative containing the -617 promoter fragment was used to generate single-stranded DNA templates. Primers used for mutating the first
inverted CCAAT box (ICB1) and the first GC box consensus (GC1) elements (Fig.
4
a), were as follows:
Mutant GC1: 5'-GGTCTGCTTCGTGCGTGCTAAAGG-3'
Mutant ICB1: 5'-AGTCAGGGATTCCCTGGTCTGCTT-3'
Nucleotide sequencing was performed on double-stranded plasmid templates using the dideoxy chain termination method and Sequenase
enzyme, as recommended by the suppliers (US Biochemical Corp.).
The detection of the CD2 cell surface antigen was achieved using a FITC-conjugated anti-rat CD2 monoclonal antibody (OX-34; SeroTec), as described by O'Connell
et al
. (
51
).
In order to determine whether p53 has a role in regulating expression of the
topoisomerase II[alpha] gene, we analysed the ability of wild-type p53, expressed from pLSVhp53c62, to modulate expression of a
CAT reporter gene that was linked to the full-length, 2.5 kb, topoisomerase II[alpha] gene promoter (
26
). Following transient transfection of SKOV3 cells with different concentrations
of the human wild-type p53 expression construct, together with a constant amount of the full
length topoisomerase II[alpha] promoter/CAT plasmid, a concentration-dependent repression of promoter activity was observed. A reduction in CAT
activity >90% was found in the presence of 15 [mu]g of the p53 expression construct (Fig.
1
). Co-transfection of the pLSV vector alone did not influence CAT expression
from the topoisomerase II[alpha] promoter (data not shown).
In the same SKOV3 cell line, we also tested whether the observed repression of
topoisomerase II[alpha] promoter activity showed any specificity for wild-type p53. In co-transfection experiments using expression constructs
containing either wild-type p53 (human and murine) or mutated versions of human and murine p53
containing amino acid substitutions in the DNA binding domain of the protein,
significant repression of CAT activity was observed only in cells transfected
with the constructs encoding wild-type p53 (data not shown). This suggested that the repression of
topoisomerase II[alpha] promoter activity was specific for wild-type p53. However, a number of alternative explanations for these
data were possible. For example, p53 might be directly influencing transfection
efficiency, or the expression of the [beta]-galactosidase reporter gene used to control for differences in
transfection efficiency between experiments.
To circumvent these potential problems, we took advantage of an expression
construct, designated pLTRp53cGVal 135, encoding a murine p53 protein that
assumes a wild-type conformation at 32oC, but a mutant conformation at 37oC (
39
). The construct containing the CAT reporter gene under the control of the 2.5
kb fragment of the topoisomerase II[alpha] promoter was transfected into a clone of SKOV3 cells (designated SK23a),
into which the construct encoding the temperature-sensitive p53 had been stably integrated. Following transfection, the
cells were cultured at either 32 or 37oC, and the level of CAT expression was quantified. A clone of SKOV3 cells
stably transfected with the neomycin-containing vector alone (SKN) was studied in parallel as a control. Figure
2
shows that the temperature shift had little or no effect on expression of CAT
in the control SKN cells. In contrast, while CAT activity was readily
detectable in extracts of SK23a cells grown at 37oC, a dramatic reduction in CAT activity was seen in the cells maintained at
32oC. Because of the potential regulation of [beta]-galactosidase gene expression from the SV40 promoter in pnlsLacZ by p53, we excluded this reporter as a
means of quantifying transfection efficiencies. Instead, in these experiments,
a single population of cells was divided equally and incubated at the different
temperatures only after transfection, and therefore the effects of wild-type p53 on promoter activity could not be explained by differences in the
ability of DNA constructs to transfect the host cell line. We conclude that p53
downregulates expression from the topoisomerase II[alpha] gene promoter and that this effect is specific for p53 in its
wild-type conformation.
We next addressed the possibility that the wild-type p53 protein was influencing the cell cycle distribution of the
transfected cells and that this was indirectly affecting the activity of the
topoisomerase II[alpha] promoter. To study this, Saos-2 ts p53 cells were co-transfected with the pKV461/CD2 expression vector
encoding the CD2 cell surface protein (to act as an antigenic tag for
transfected cells) and the 101 bp topoisomerase II[alpha] promoter fragment linked to HGH. The control promoters, FGH and PGKGH,
were also co-transfected with the pKV461/CD2 vector into Saos-2 ts p53 cells. Following transfection, the cells were cultured at
either 32 or 37oC. The cell cycle distribution of the cells cultured at 32oC (when p53 would be wild-type) and at 37oC (when p53 would be mutant) was found to be similar (data
not shown). Thus, cell cycle perturbations are unlikely to account for the
dramatic downregulation of promoter activity by p53 wild-type.
In order to eliminate the possibility that wild-type p53 was having a general negative influence over gene transcription
in the transfected cells, we analyzed the effect of p53 on expression of the
HGH reporter gene from two control promoters. Figure
5
shows that expression of HGH from the phosphoglycerate kinase and ferritin gene
promoters in Saos-2 ts p53 cells was not substantially downregulated by temperature shift
from 37 to 32oC, indicating that the activity of these promoters was not negatively
regulated by co-expression of wild-type p53 protein. Indeed, the activity of the phosphoglycerate
kinase gene promoter was somewhat higher in cells grown at 32 than at 37oC.
We have shown that wild-type p53 is a negative regulator of the activity of the human
topoisomerase II[alpha] promoter and that this effect is mediated through the minimal sequences
required for topoisomerase II[alpha] promoter activity. This regulation is apparently independent of a
perturbation in the cell cycle distribution of the transfected cells, and has
been demonstrated in different cell lines using different constructs encoding
either human or murine p53. Moreover, we have shown that p53 has some apparent
specificity for the topoisomerase II[alpha] promoter, in that the activity of two control gene promoters
was not downregulated by expression of wild-type p53.
The p53 protein is a key regulator of gene expression in mammalian cells. In its
wild-type conformation, p53 activates the transcription of those genes that
contain a consensus p53 binding element in their promoter or other regulatory
sequences (
28
-
30
). Conversely, wild-type p53 is able to repress the transcription of certain genes that lack a
consensus binding element in their regulatory sequences (
34
-
36
). Many studies have focused on the ability of p53 to stimulate or repress the
activity of genes important either for the control of cell proliferation, or
for the response of cells to DNA damaging agents. For example, p53 has been
reported to activate the transcription of both the p21
CIP1/WAF1
gene, which encodes an inhibitor of cyclin-dependent kinases (
53
-
55
), and the GADD45 gene, which encodes a 18 kDa protein whose expression is
induced by DNA damaging agents, and which may play a role in regulating cell
cycle progression and/or DNA repair through its interactions with the
proliferating cell nuclear antigen (
56
,
57
). Similarly, wild-type p53 has been shown to activate the transcription of the
BAX
gene, which heterodimerises with Bcl-2, and consequently antagonises the anti-apoptotic role of Bcl-2 (
58
). In contrast, wild-type p53 represses the activity of the interleukin 6 gene promoter,
apparently in combination with the retinoblastoma susceptibility gene product (
35
).
p53 interacts with DNA in a sequence-specific fashion binding to DNA containing two contiguous monomers of the
sequence 5'-PuPuPuC (T/A) (T/A) GPyPyPy. These two elements are generally
separated by between 0 and 13 bp of non-conserved sequence (
59
). The topoisomerase II[alpha] promoter does not contain a precise match for this consensus
p53 binding element. However, a similar sequence (AAGCTT
Because of the cell cycle regulatory role of p53, we addressed whether the
topoisomerase II[alpha] promoter could be responding to cell cycle perturbation induced by p53,
and not to a direct effect of p53 on the transcription machinery. No evidence
was obtained for a substantial accumulation of cells in any particular cell
cycle phase, in those cells in which wild-type p53 was expressed, at least over the time course of these
experiments. Clearly, the other genetic changes that accompany the acquisition
of a transformed state, such as alteration in the retinoblastoma susceptibility
gene (which is not expressed in Saos-2 cells) or cyclin-dependent kinase inhibitors, could be influencing the efficiency
with which wild-type p53 can mediate cell cycle arrest in the cell lines chosen for study
here. Yamato
et al.
(
43
) showed that expression of wild-type p53 caused some accumulation of Saos-2 cells in the G
1
and G
2
phases of the cell cycle 20 h after initiating protein expression. In our
study, cell cycle distribution was analysed at both 96 and 120 h after
temperature shift and in neither case did we observe an obvious cell cycle
perturbation, suggesting that any arrest that might have occurred was transient
in nature. However, it is extremely unlikely that cell cycle perturbations
induced by expression of p53 are responsible for altering the activity of the
topoisomerase II[alpha] promoter, since we have shown that promoter activity varies <2-fold during cell cycle traverse in human cells (unpublished
observations). Indeed, recent data indicate that the variation in topoisomerase
II[alpha] mRNA expression that occurs during the cell cycle traverse is almost
exclusively due to changes in transcript stability, not promoter activity (
25
). We have shown elsewhere that the topoisomerase II[alpha] promoter responds to growth arrest signals (
48
). However, the regulatory elements in the promoter that respond to changes in
growth state are distinct from those responding to p53, and are located
upstream of the minimal topoisomerase II[alpha] gene promoter defined in this work. In particular, the 101 bp
minimal promoter, which we have shown to be regulated by p53, lacks normal
negative regulation brought about by inhibition of proliferation (
48
). Topoisomerase II protein is a key target for many clinically used anticancer
drugs, with the cellular level of topoisomerase II being an important
determinant of the response of tumour cells to these agents. As a consequence,
the identification of factors that control topoisomerase II[alpha] gene expression may be important in determining the clinical efficacy of several classes of
antineoplastic agents. We would suggest that the differential sensitivity of tumour cells to
killing by topoisomerase II-targeting drugs may be dependent upon the p53 status of individual tumour
cells. Since p53 is frequently mutated in human cancers, it is possible that a
dysregulation of topoisomerase II[alpha] gene expression will be evident in those tumour cells that
lack expression of wild-type p53. If stable overexpression of topoisomerase II[alpha] were to occur under these circumstances, it might be
expected that this would lead to an increased susceptibility of these cells to
killing by topoisomerase II-targeting drugs. This may be one explanation for the differential
sensitivity of tumour and normal tissues to topoisomerase II-targeting drugs. Further analysis of this phenomenon is now warranted.
At least one action of p53 as a negative regulator of gene transcription appears
to be directed towards components of the basal transcription machinery. An
important feature of this effect may be the formation of complexes between p53
and TATA box-binding protein (TBP)-associated factors, possibly TBP itself. Moreover, Liu and Berk (
60
) have shown recently that p53 may act through direct or indirect interactions
with both TFIIB and TFIID, which act as basal transcription factor complexes.
Our data are consistent with the hypothesis that wild-type p53 acts via negatively regulating the basal transcription machinery
required to effect expression of the topoisomerase II[alpha] gene. In other cases, such as in the regulation of SV40 and hsp70 gene
transcription, p53 appears to disrupt the ability of a protein complex to bind
to DNA that includes either the transcription factor Sp1 (GC box binding
factor) (
61
), or CBF (CCAAT box binding factor) (
62
). Although consensus Sp1 and CCAAT boxes lie close to the CAP site in the
topoisomerase II[alpha] gene promoter, mutation of these sites did not prevent p53 from
negatively-regulating promoter activity. This suggests that Sp1 and CBF are unlikely
to be important targets for p53 action in regulating the expression of the
topoisomerase II[alpha] gene.
In summary, we have shown that wild-type p53 can specifically down-regulate the activity of the human topoisomerase II[alpha] gene promoter. The challenge is now to delineate the precise
downstream consequences of this regulation and to ascertain whether
topoisomerase II[alpha] gene expression is an important target for other regulators of cell
cycle progression in human cells.
We would like to acknowledge Dr M. Ines Sandri's scholarship funding from the
Brazilian Government through CNPq (Conselho Nacional de Desenvolvimento
Cientifico e Tecnologico). We also thank Drs C. Redwood and C. Norbury for
reading the manuscript, Dr J. Firth for the growth hormone constructs, Dr J.
Jenkins for the human ts p53 construct,
and Mrs E. Clemson for typing the manuscript. This work was supported by the
Imperial Cancer Research Fund and the Italian Association for Cancer Research.
REFERENCES
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