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Molecular Biology and Genetics |
Department of Molecular Genetics, The Ohio State University [Y. M., S. O., M. T. M.], and Department of Pediatrics, Children’s Hospital [L-S. C.], Columbus, Ohio 43210
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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and ß are not stimulated by DNA damage. We show that topo I and
p53 are able to associate at arrested topo I-genomic DNA covalent
complexes in vivo, suggesting that p53 directly
stimulates topo I activity and damage to the genome of the afflicted
cell. Moreover, cells that express wild-type p53 are most proficient at
recruiting topo I after DNA damage; however, the p53 dependence is
conditional because topo I recruitment after DNA damage can be restored
if p53 mutant cells (containing a single mutant allele) are
artificially held in G1. In contrast, p53 null mutants do
not recruit topo I after DNA damage under any conditions (although
camptothecin-dependent topo I/DNA complexes readily form in the nulls).
These results show that topo I activation after DNA damage depends on
the p53 status of the cell. It also depends upon the cell cycle in a
way that is very different from that observed with DNA
replication-dependent, camptothecin-mediated DNA breaks. The data
suggest a model where p53 activates topo I, which inflicts additional
genomic damage after the initial UV damage events. Topoisomerases
therefore contribute to the p53 commitment to apoptosis, and topo I
might assist in elimination of DNA-damaged cells as part of the
cellular proofreading function inherent in the p53 pathway. |
INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Eukaryotic topo I makes single strand breaks in DNA, followed by one or more cycles of controlled rotation, followed by resealing (13) . Topo I participates in a variety of DNA templating activities, such as transcription (14, 15, 16, 17) and DNA replication (18 , 19) , presumably to reduce torsional stress in the template. Topo I is also thought to influence genomic instability through illegitimate recombination (20, 21, 22, 23) . Although topo I is not an essential gene in yeast (24) , it is required for embryonic development in Drosophila melanogaster (25) and mice (26) ; therefore, topo I is essential in the context of a multicellular organism.
There have been hints that topo I participates in post-DNA damage response. Distortions in the helix, such as abasic sites and UV photoproducts inhibit the catalytic activity of topo I and/or trap topo I on DNA in vitro (27 , 28) . Previously, our group reported that DNA damage by UV irradiation specifically stimulates the formation of covalent complexes between topo I and DNA in vivo (29) . We refer to this phenomenon as the topo I/DNA damage response, and in the present work, evidence is presented that this response is strongly dependent upon cell cycle checkpoints activated by p53 after UV-damaged DNA. These findings suggest a model whereby topo I and p53 might cooperate to eliminate damaged genomes from the organism.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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antibody is a rabbit
polyclonal antibody directed against the
Mr 170,000 form of human topo
II. The topo IIß antibody is a mouse monoclonal antibody to the
Mr 180,000 form of human topo II. The
topo II antibody was donated by TopoGEN, Inc. (Columbus, OH), and
the topo IIß antibody was a kind gift from Dr. A. Kikuchi. The
anti-p53 antibody (Pab 421) was obtained from Oncogene Science
(Uniondale, NY). CPT and VP16 were also donated by TopoGEN, Inc. G418
was purchased from Life Technologies, Inc. (Rockville, MD). DMS and
cycloheximide were purchased from Sigma Chemical Co. (St. Louis, MO).
Cell Culture.
The MCF-7 and SK-BR-3 cell lines in this study are derived from human
mammary adenocarcinoma. MCF-7 cells express wild-type p53 (30
, 31)
, whereas SK-BR-3 cells carry a deletion in one of the
p53 alleles and a point mutation at amino acid 175 in the
remaining allele, encoding a nonfunctional p53 protein that is
defective in sequence-specific DNA binding (30
, 32)
. Both
cell lines are cultured in DMEM supplemented with 10% fetal bovine
serum (CellGro, Inc., Herndon, VA). SK-BR-3/hp53 cells are a clonal
isolate of SK-BR-3 cells containing the human p53
gene under control by the cytomegalovirus immediate-early
promoter. HL60 is a p53-null human leukemia cell line and cultured in
RPMI 1640 supplemented with 10% fetal bovine serum (Life Technologies,
Inc., Grand Island, NY).
Transfection.
Freshly plated MCF-7 and SK-BR-3 cells at 50–80% confluence were
transfected with 2 µg of DNAs per 35-mm dish, using SuperFect
transfection reagent (Qiagen, Valencia, CA), according to the
manufacturer’s instructions. For stable transfection of the wild-type
p53 gene into SK-BR-3 cells, cells were split into fresh
medium containing 400 µg/ml G418 (CellGro, Inc., Herndon, VA) until
resistant colonies formed. Colonies were recovered using Scienceware
cloning cylinders (Fisher Scientific) and then checked for p53
expression by Western blotting.
UV Irradiation of Cells.
For UV treatment of cells, culture medium was aspirated, and cell
monolayers were washed several times with room temperature PBS (140
mM NaCl, 2.5 mM KCl, 10 mM
Na2HPO4, and 1.75
mM KH2PO4). The
cells were then exposed to a germicidal lamp emitting at 254 nm light
at a fluence rate of 2 J/m2/s. Immediately after
irradiation, medium was replaced, and cells were incubated at 37°C.
At indicated times after UV treatment, cells were processed according
to the ICT bioassay protocol.
In Vivo Complex of Topo (ICT) Bioassay.
The method for detecting topo-DNA covalent complexes in vivo
is described in detail elsewhere (29
, 33
, 34)
. After DNA
purification on CsCl gradients, DNA concentrations were measured by
fluorometry, and fixed concentrations of DNA were blotted onto a slot
blot device that was subsequently probed with a topo I antibody.
Signals were illuminated using 125I-labeled
protein A (ICN, Costa Mesa, CA) and quantified using phosphorimage
analysis.
For analysis of p53-topo I complexes, the ICT bioassay was modified as follows. At 4 h after UV, MCF-7 cells were exposed to 10 mM DMS at room temperature for 1 h to induce protein/protein cross-linking. The ICT bioassay was then carried out, and the presence of p53 in the DNA peak of CsCl gradients was determined by Western slot blotting.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, within 30 min, the level of topo I-DNA complex increased 1.5–2-fold
over that of untreated controls, and the complexes continued to
increase through the time when nucleotide excision repair was maximal.
Within 5 h, topo I-DNA complexes reached peak level (about
3–4-fold over no incubation) and then gradually receded within one
cell cycle. The kinetics of complex formation closely paralleled the
p53 profile of response after UV irradiation (5
, 6)
. To
confirm that p53 accumulated in the nucleus after UV irradiation, MCF-7
cells were stained with an anti-p53 antibody and examined by indirect
immunofluorescence microscopy. UV-treated cells displayed strong
nuclear fluorescence; however, untreated controls presented minimal
background nuclear staining (data not shown).
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, and the DNA was isolated from the ICT analysis and probed with
antibody to topo I. The data show that blocking protein synthesis had
no effect on topo I-DNA complexes that were elevated after UV damage.
CPT-induced complexes were slightly reduced (<20%) by blocking
translation; however, this may be explained by concomitant arrest of
S-phase because it is known that CPT-mediated toxicity is greater in
S-phase (36
, 37) .
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demonstrated that the topo I/DNA damage response
parallels the nuclear accumulation of p53, suggesting a role for p53 in
the response. To test whether the topo I/DNA damage response is related
to p53, we evaluated the response in cell lines with different p53
status. MCF-7 cells express wild-type p53, whereas SK-BR-3 cells carry
a deletion in one p53 allele and a point mutation (amino
acid 175) in the remaining allele, encoding a nonfunctional p53 protein
that is defective in sequence specific DNA binding (30
, 32)
. The HL60 cell line is p53-null. As expected, MCF-7 cells
(wild-type p53) showed an elevation in topo I-DNA complex formation
after DNA damage (Fig. 3A
). In contrast, SK-BR-3 cells and HL60 cells that lack
functional p53 did not show UV-induced topo I-DNA complex formation
(Fig. 3 and C
). Western blotting data revealed
that these results cannot be attributable to differences in topo I
protein levels between these cell lines, because all cell lines contain
similar amounts of cellular topo I before and after UV treatment (data
not shown). Moreover, treating SK-BR-3 cells and HL60 cells with CPT
results in similar covalent complex formation relative to the wild-type
MCF-7 cells (Fig. 3
). Finally, we tested topo I recruitment in
transgenic mouse cell lines from animals that were heterozygous for p53
(one allele deleted) compared with homozygous null mutants and obtained
consistent results (data not shown). From these collective results, we
conclude that deployment of topo I onto the genome after UV exposure is
related to the status of p53.
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). Three different clones, all expressing p53 (based upon Western
blotting) were examined for topo I recruitment after DNA damage. These
cell lines gave rise to high levels of topo I-DNA complex formation
after UV irradiation (Fig. 4B
), compared with the parental
SK-BR-3 cells (Fig. 4A
). These data clearly demonstrated a
direct link between p53 and the topo I/DNA damage response because the
p53 mutant (parental) line and restored clone should be otherwise
isogenic. In addition, CPT-induced topo I-DNA covalent complexes were
enhanced in the SK-BR-3/hp53 cells. This result suggests that p53 may
be directly stimulating topo I activity in vivo, or possibly
recruiting topo I to sites of DNA damage, which is consistent to
previous reports demonstrating that p53 increases the catalytic
activity of topo I in vitro (11
, 12)
.
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). Controls show that topo I/DNA complexes readily form after CPT
treatment as expected; thus, topo I is clearly active in the null
cells.
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). In contrast, cells that were serum arrested in
G1 clearly demonstrated a strong topo I/DNA
damage response (Fig. 6B
), and upon release from arrest, the
damage response was again lost (Fig. 6C
). Cells containing
this particular p53 mutation actively recruit topo I after UV damage
when arrested in G1. Furthermore, these data
suggest that the topo I/DNA damage response involves at least two
distinct aspects of p53: (a) a cell cycle checkpoint that
facilitates topo I recruitment; and (b) a direct stimulation
of topo I activity by p53 (see below).
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. In p53 wild-type cells, UV treatment reproducibly reduced p170
complexes, whereas p180 complexes were unaffected. Thus, although topo
II is not directly stimulated by DNA damage (like topo I),
VP16-mediated activity was clearly altered in the case of p170.
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). If p53 binds topo I, which itself is covalently trapped on the
genome, then we should detect p53 associated with DNA in the ICT-CsCl
gradient (Fig. 7A
). The presence of topo I and p53 was
examined by Western blotting of the DNA peak fractions (pooled). The
p53 signal, detected in the absence of DMS, reflects the background
signal for p53 (because the signal was not increased with increasing
DNA concentration). In contrast, exposure to DMS resulted in a
significant increase in p53 signal when 2 µg of genomic DNA were
used, and a commensurate increase in p53 signal was observed using 6
µg of DNA (Fig. 7B
). Note, however, that these data do not
unambiguously prove that p53 is physically dragged into the DNA peak by
topo I because p53 could be trapped by linkage to another (as yet
unknown) DNA binding protein. Although this possibility seems remote
given the known association between p53 and topo I in vitro
(11
, 12)
, an additional control was carried out to examine
this possibility. DNA peak fractions off the CsCl gradient were pooled
and digested extensively with DNase I to release bound proteins. We
then performed an immunoprecipitation with an anti-p53 antibody,
recovered the precipitate, and probed with topo I antibody. The data
show clearly a complex formation between topo I and p53 (Fig. 7C
). To the best of our knowledge, this is the first
demonstration that endogenous p53 and topo I are together at the site
of the topo-DNA covalent (cleavable) complex in vivo.
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); however, treatment
with the protein/protein cross-linker stimulated topo I signal in the
DNA peak anywhere from 2- to 5-fold (in different experiments),
suggesting that topo I may (in addition to p53) be interacting with
itself or in some kind of self protein/protein clustering mode as a
complex. |
DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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(a) The kinetics of topo I covalent complex formation after UV irradiation of intact cells closely parallels the nuclear accumulation of p53. In response to DNA damage, cells normally undergo a p53-dependent cell cycle arrest, DNA repair, or apoptosis. Stimulation of topo I activity by p53 (11 , 12) suggests a functional relationship between these two proteins involving in DNA damage response.
(b) MCF-7 cells, expressing wild-type p53, are proficient in the topo I/DNA damage response. In contrast, the SK-BR-3 cells expressing mutant p53 show very little topo I response. These data suggest that p53 may stimulate topo I-DNA interactions, leading to arrested covalent complexes on the genome. It is important to note that cells expressing mutant p53 sustain just as much DNA damage (UV adducts) as do wild-type p53 expressing cells per given UV dose; however, p53 wild-type cells are much more proficient with regard to topo I deployment, as measured by ICT after damage. Lanza et al. (41) and work from this laboratory (29) have confirmed that topo I cleavage/religation equilibrium is sensitive to helical distortions associated with UV lesion formation. Because UV-damaged DNA can stabilize topo I cleavable complexes and because p53 activates topo I activity on DNA, topo I might assist in promoting the cell down a path of apoptosis by inflicting genome damage (see points that follow).
(c) Topo I/DNA damage can be effectively restored in SK-BR-3 (mutant) cells when providing a wild-type p53 gene. Because a single gene replacement is sufficient to restore topo I-DNA complex formation after DNA damage, we conclude that p53 is a key component in the response. All of the p53 wild-type revertants we tested were proficient in the topo I/DNA damage response, thereby arguing against clonal variation independent of p53.
(d) The topo I/DNA damage response is conditionally dependent upon the p53 status. In one particular p53 mutation (point mutation in the DNA binding domain), cells that are exponentially growing do not recruit topo I after DNA damage; however, by blocking cell cycle traverse in G1, topo I-DNA complex formation after UV treatment could be restored. This is consistent with the previous report indicating that post-UV survival of p53 mutant cells is enhanced by imposing a cell cycle checkpoint (7) . In addition, it has been reported that this particular p53 mutant can still physically interact with topo I. Interpretation of these collective results is difficult because of the complexity of the pathways, and additional data will be required to clarify the situation. Basically, there are two models. One model suggests that p53 may promote cell cycle arrest to facilitate DNA repair, as proposed by Linke et al. (7) . Topo I being a repair factor and also responsive to helical distortion or abasic sites (28) could explain why cleavable complexes are elevated during the periods of excision repair. Another model suggests that p53-driven checkpoints stably withdraw DNA-damaged cells from the cycle to ensure that DNA replication does not proceed into or through damaged template. In this case, topo I, perhaps under the stimulatory influence of p53 (11 , 12) , might contribute to the DNA damage by forming covalent complexes that we detect in our ICT bioassay. In this way, topo I and p53 cooperate to inflict irreparable damage and permanent withdrawal of the cell from the replicative pool of cells.
(e) In support of the second model described above, our in vivo cross-linking data show a physical association between p53 and topo I. Although others have reported p53/topo I binding (11 , 12) , this is the first demonstration that p53 and topo I form molecular complexes at the actual site of DNA cleavage/religation in chromatin. Our cross-linking experiments will only detect p53 bound to topo I that has been covalently coupled (trapped) on the genome through its catalytic cycle of action on DNA. This type of experiment reveals that topo I and p53 are either in a complex or close physical contact immediately prior to (or during) the arrest of the covalent "cleavable" complex. We speculate that topo I and p53 are part of a multiprotein complex and that p53 directly stimulates topo I cleavages (12) . Our DMS cross-linking data also suggest that topo I itself is in a self-cluster modality because the amount of topo I detected in the DNA peak can be increased by protein-protein cross-linking. Because topo I is a monomeric protein, it is surprising that it can be detected in a cluster of activity unless its function is to pepper the genome with single strand breaks. It is also possible that topo I interacts with other p53 interactive proteins in chromatin. For example, it has been proposed that p53 plays a direct role in DNA repair by stimulating other DNA binding proteins; p53 binds GADD45, which forms complexes with proliferating cell nuclear antigen, to directly stimulate the DNA synthesis associated with the nucleotide excision repair process (9 , 31) . The reduction of nucleotide excision repair ability in gadd45-/- mouse embryo fibroblasts fits nicely with the idea of a role for Gadd45 in cellular DNA repair (35) . Recently, it was also shown that GADD45 can facilitate topo I in the presence of core histones in vitro (35) . These data suggest that p53/GADD45 might enhance the ability of topo I to recognize or be recruited to altered chromatin structures formed as a result of DNA damage.
(f) The topo I/DNA damage response does not require new
protein synthesis (Fig. 2
), indicating that preexisting topo I is
affected. Elevated topo I-DNA complexes may be explained by the
formation of a higher affinity DNA substrate (i.e., helical
distortions; Refs. 29
and 41
), alterations
that promote topo I access to underlying DNA in chromatin
(e.g., chromatin remodeling; Refs. 35
) or
direct stimulation of topo I by p53 (11
, 12)
. It is not
unreasonable to propose that all these events cooperate to give the
topo I/DNA damage response.
(g) Topo II isoforms are clearly not stimulated to form covalent complexes after DNA damage such as topo I (29) . To examine topo II with the ICT bioassay, it was necessary to treat the cells with a topo II poison to trap complexes. UV irradiation clearly had a strong impact on p170 because covalent complexes were reduced on average 4-fold. There was no detectable effect on p180. The most likely explanation is that DNA damage imposed a cell cycle checkpoint that affected the p170 isoform, which is strongly cell cycle regulated, whereas p180, which is not a periodic enzyme, was not altered.
In summary, there are two models that explain p53 and cell cycle-dependent topo I activity after DNA damage. The first model is that topo I may be an active participant in excision repair. We showed previously that the topo I/DNA damage response is aborted in the repair-deficient XPD cells (29) . Nucleotide excision repair is a complex process that involves damage recognition, incision of the damaged strand, excision of the lesion containing oligonucleotides, synthesis of new DNA, and ligation. Topo I could be involved at any one (or all) of these steps; however, it is also likely that some chromatin activation or restaging is necessary prior to topo I activity at repair patches, because topo I cannot compete with bulk chromatin for DNA access. In this regard, Gadd45, a p53-responsive factor, might drive local chromatin modifications to facilitate topo I accessibility (35) . A second model is that topo I contributes to the general demise of the cell by contributing to genomic damage and subsequent p53-dependent elimination through apoptosis. In this model, topo I assists in forcing cell cycle checkpoints by inflicting DNA cleavage complexes and elimination of cells destined to be precancerous. The clustering of topo I on the genome after DNA damage and the p53 stimulation of topo I cleavage activity support this model. These two models are not mutually exclusive, and it is possible that DNA damaged cells exist in a balance between repair (or resurrection) and apoptosis. The outcome of the process is stochastic and depends on a number of unknown factors in the p53 pathway in addition to the cell cycle phase and degree of damage. Additional experiments are ongoing to elucidate the models.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 This study was supported by Grant RO1-AG16692
from the NIH, Grant GMC-89165 (to L. S. C.) from the American Cancer
Society, and Grant CA16058 from the Ohio State University Comprehensive
Cancer Center. 
2 To whom requests for reprints should be
addressed, at Department of Molecular Genetics, the Ohio State
University, 484 West 12th Avenue, Columbus, OH 43210. Phone:
(614) 292-1914; Fax: (614) 292-4702; E-mail: muller.2{at}osu.edu
3 The abbreviations used are: topo, topoisomerase;
CPT, camptothecin; VP16, etoposide; DMS, dimethylsuberimidate.
Received 10/26/99. Accepted 6/20/00.
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REFERENCES |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-irradiation. Cancer Res., 57: 1171-1179, 1997.This article has been cited by other articles:
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  A. Goswami, S. Qiu, T. S. Dexheimer, P. Ranganathan, R. Burikhanov, Y. Pommier, and V. M. Rangnekar Par-4 Binds to Topoisomerase 1 and Attenuates Its DNA Relaxation Activity Cancer Res., August 1, 2008; 68(15): 6190 - 6198. [Abstract] [Full Text] [PDF]
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C. Bowen, A. Stuart, J.-H. Ju, J. Tuan, J. Blonder, T. P. Conrads, T. D. Veenstra, and E. P. Gelmann NKX3.1 Homeodomain Protein Binds to Topoisomerase I and Enhances Its Activity Cancer Res., January 15, 2007; 67(2): 455 - 464. [Abstract] [Full Text] [PDF]
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