From the Department of Biochemistry and Molecular
Pharmacology, Thomas Jefferson University, Philadelphia,
Pennsylvania 19107 and ¶ Consiglio Nazionale delle Ricerche,
Istituto di Biologia Cellulare, 00137 Rome, Italy
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ABSTRACT |
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The yeast Saccharomyces cerevisiae has been useful in establishing the phenotypic effects of specific mutations on the enzymatic activity and camptothecin sensitivity of yeast and human DNA topoisomerase I. To determine whether these phenotypes were faithfully reiterated in higher eukaryotic cells, wild-type and mutant yeast Top1 proteins were epitope-tagged at the amino terminus and transiently overexpressed in mammalian COS cells. Camptothecin preferentially induced apoptosis in cells expressing wild-type eScTop1p yet did not appreciably increase the cytotoxic response of cells expressing a catalytically inactive (eSctop1Y727F) or a catalytically active, camptothecin-resistant eSctop1vac mutant. Using an epitope-specific antibody, immobilized precipitates of eScTop1p were active in DNA relaxation assays, whereas immunoprecipitates of eScTop1Y727Fp were not. Thus, the enzyme retained catalytic activity while tethered to a support. Interestingly, the mutant eSctop1T722A, which mimics camptothecin-induced cytotoxicity in yeast through stabilization of the covalent enzyme-DNA intermediate, induced apoptosis in COS cells in the absence of camptothecin. This correlated with increased DNA cleavage in immunoprecipitates of eScTop1T722Ap, in the absence of the drug. The observation that the phenotypic consequences of expressing wild-type and mutant yeast enzymes were reiterated in mammalian cells suggests that the mechanisms underlying cellular responses to DNA topoisomerase I-mediated DNA damage are conserved between yeast and mammalian cells.
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INTRODUCTION |
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Eukaryotic DNA topoisomerase I catalyzes the relaxation of supercoiled DNA through the transient breakage and religation of a single DNA strand in a DNA duplex (reviewed in Refs. 1-3). This enzyme plays a role in a number of essential cellular processes, such as replication, recombination, and transcription (1, 3-5). Furthermore, the naturally occurring antitumor drug camptothecin specifically targets this enzyme by stabilizing the covalent enzyme-DNA intermediate (Refs. 6 and 7; reviewed in Ref. 8). During DNA replication (S phase), these stabilized enzyme-DNA adducts are converted into lethal double-stranded DNA breaks due to their interaction with the DNA replication fork (9-12).
Although this enzyme participates in numerous cellular processes,
strains of the yeast Saccharomyces cerevisiae deleted for the gene encoding DNA topoisomerase I (top1) are viable
because other gene products, such as DNA topoisomerase II, can
compensate for the loss of TOP1 (11, 13). These
top1
strains are completely resistant to the cytotoxic
action of camptothecin (14-16). However, expression of either S. cerevisiae or human DNA topoisomerase I restores the sensitivity
of these cells to camptothecin-induced lethality (4, 14-16). These
results demonstrate the specificity of camptothecin for eukaryotic DNA
topoisomerase I and the utility of using yeast as a model system for
the analysis of drug-enzyme interactions. In fact, mutations in yeast
and human TOP1 that render the enzyme resistant to
camptothecin (17, 18) or render the enzyme cytotoxic even in the
absence of camptothecin (19, 20)1,2
have been defined using this yeast system.
These results indicate a significant conservation of function between the yeast and human enzymes, consistent with extensive similarities in TOP1 sequences. Nevertheless, differences between these proteins do exist. For example, expression of a camptothecin-resistant yeast or human DNA topoisomerase I mutant (top1vac) has different effects on the viability of yeast strains defective in the repair of double strand DNA breaks (17). In vitro, these mutant enzymes exhibit different sensitivities to other DNA topoisomerase I poisons, including saintopin, a DNA intercalator that targets both DNA topoisomerase I and II, and the minor groove binding ligand netropsin (21). The human enzyme plays a direct role in transcriptional activation in vitro (22-24) and suppresses the basal level of transcription (22). Although the catalytic activity of human DNA topoisomerase I is dispensable for its role in suppressing transcription, the yeast enzyme could not replace the human enzyme in these assays (22). It is not yet known whether these observations reflect intrinsic differences in enzyme structure or in specific functional domains that mediate enzyme interactions with other cellular factors. However, because these enzymes constitute the cellular targets of clinically important chemotherapeutic agents, it is essential that the mechanisms of DNA topoisomerase I-induced DNA damage be better understood.
Here we investigate the potential differences between yeast and human DNA topoisomerase I with regards to camptothecin-induced cytotoxicity. A family of S. cerevisiae mutants were transiently expressed in mammalian COS cells and examined for their effects on drug-induced apoptosis. These assays included wild-type yeast TOP1 (eScTOP1), the camptothecin-resistant mutant eSctop1vac (17, 21), a catalytically inactive mutant (eSctop1Y727F) (25), and the lethal mutant eSctop1T722A, which mimics that action of camptothecin in stabilizing the covalent enzyme DNA intermediate (20). Given the difficulties inherent in the selection of cytotoxic phenotypes in mammalian cells, the effects of such lethal mutations on mammalian cell viability have not previously been described. In the studies presented here, expression of yeast wild-type protein enhanced COS cell sensitivity to camptothecin, consistent with earlier observations that overexpression of the human enzyme in mammalian cells enhances their sensitivity to the drug (26). The phenotypic consequences of overexpressing the other classes of yeast top1 mutants were also faithfully reiterated in these mammalian cells; no appreciable increase in camptothecin-induced apoptosis was observed in cells expressing eSctop1vac or the inactive mutant eSctop1Y727F, whereas eSctop1T722A expression induced an apoptotic response in the absence of the drug. Moreover, when these yeast enzymes were immunoprecipitated, the activities of the bead-bound enzymes correlated with the observed patterns of drug sensitivity and cell lethality. These results highlight the conservation of enzyme function both in inducing DNA damage and in the cellular responses to this DNA topoisomerase I-mediated damage.
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MATERIALS AND METHODS |
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Plasmids, Cell Culture, and Drug Treatment-- An eight-amino acid residue epitope tag, recognized by monoclonal antibody M2 (Kodak/IBI), was engineered into the amino terminus of yeast DNA topoisomerase I. To avoid confusion with the endogenous mammalian enzyme, the yeast gene and its protein products are prefixed with an eSc to indicate the epitope tag (e.g. eScTOP1 and eScTop1p). Complementary oligos encoding the sequence MDYKDDDDKAI were cloned into an NcoI site, previously engineered into the sequences encoding the initiating methionine residue in the eScTOP1 plasmid YCpGAL1-TOP1 (17), to yield YCpGAL1-eScTOP1. The epitope tag (underlined in the sequence above) was immediately amino-terminal to the original methionine in the fusion protein eScTop1p. The eSctop1Y727F, eSctop1 vac, and eSctop1T722A constructs were prepared by swapping a 989-base pair BamHI-Bsu 36I DNA fragment from YCpGAL1-eTOP1 into the backbones of plasmids YCpGAL1-top1Y727F, YCpGAL1-top1 vac (17, 21), and YCpGAL1-top1T722A (20), respectively. The resultant eSctop1 constructs were all excised by digestion with MluI and PstI, and the blunted-ended DNA fragments were cloned into the blunt-ended EcoRI site of the pMT2 COS cell expression vector (27).
COS cells (African green monkey kidney cells transformed with SV40 T antigen) were maintained in Dulbecco's modified Eagle's medium supplemented with 10% bovine calf serum. All transfections were performed on subconfluent monolayer cultures. Plasmids (30 µg) were transfected into COS cells by the calcium phosphate procedure. The cells were glycerol shocked 5-6 h after DNA addition. Camptothecin (Sigma) was resuspended in Me2SO at a concentration of 10 mM, and aliquots were stored atImmunofluorescence, Confocal Microscopy, and Immunoprecipitation-- Indirect immunofluorescence was performed as described by Logan et al. (28). Briefly, COS cells were plated on 10-cm tissue culture dishes containing glass coverslips at approximately 500,000 cells/plate. The cells were cultured for 24 h and then were transfected with the various eScTOP1 expression plasmids (described above). After the glycerol shock, the cells were treated with various concentrations of camptothecin for an additional 24 h. The cells were then washed twice in PBS and fixed with 4% paraformaldehyde in PBS for 20 min followed by two washes in PBS. To permeabilize the cells, the coverslips were then treated with PBS containing 0.2% Triton X-100 for 15 min followed by three 5-min washes in PBS containing 0.2% gelatin (as described in Ref. 29). The M2 monoclonal antibody (Kodak/IBI) was diluted 1:200 in PBS containing 0.2% gelatin. Coverslips containing the fixed and permeabilized cells were placed cell side down on 50 µl of diluted antibody and incubated for 1.5 h at 37 °C. The coverslips were then washed three times in PBS plus 0.2% gelatin and inverted on 50 µl of fluorescein-conjugated goat anti-mouse IgG (Vector Laboratories) diluted to 30 µg/ml in PBS plus 0.2% gelatin. After 30 min at 37 °C, the coverslips were successively washed in PBS plus 0.2% gelatin (10 min), PBS plus 0.2% gelatin plus 0.05% Tween 20 (10 min), and PBS (10 min). The DNA within the nuclei of the cells was then stained with DAPI.3 The coverslips were rinsed in deionized water, dried, mounted onto glass slides, and analyzed by fluorescence microscopy.
Confocal microscopy was performed on the fluorescein-stained cells, using a Leica TCS 4D confocal microscope with a 100× oil objective lens. The cell thickness was 2.24 µm, and the optical sections taken were 0.24 µm. For the immunoprecipitations, 15 µg of nuclear extract was incubated at 4 °C with 3 µg of M2 monoclonal antibody (Kodak/IBI) for one hour. Two micrograms of Staphylococcus protein A acrylamide beads were added to the extracts for an additional 1.5 h at 4 °C. The beads were then pelleted, washed five times in NTEN (100 mM NaCl, 20 mM Tris pH 8, 1 mM EDTA, 0.5% Nonidet P-40), and resuspended in 50 µl of DNA relaxation assay buffer (see below). The immunoprecipitates were assayed in a plasmid DNA relaxation activity as described below.Generation of Extracts-- Nuclear extracts for Western blots and activity assays were generated as described by Moberg et al. (30), by lysing the cells on ice in 0.1% Nonidet P-40, 10 mM Tris (pH 7.9), 10 mM MgCl2, 15 mM NaCl, and the protease inhibitors phenylmethylsulfonyl fluoride (0.5 mM), pepstatin (2 µg/ml), and leupeptin (1 µg/ml). The nuclei were pelleted by centrifugation at 800 × g for 10 min, resuspended in extraction buffer consisting of 0.5 M NaCl, 20 mM Hepes (pH 7.9), 20% glycerol, phenylmethylsulfonyl fluoride (0.5 mM), pepstatin (2 µg/ml), and leupeptin (1 µg/ml) for 10 min on ice and then centrifuged at 14,000 × g for 8 min to pellet the residual nuclear material. The supernatant fraction was termed nuclear extract.
Western Blot Hybridizations-- Proteins in the nuclear extracts were electrophoretically resolved by SDS-polyacrylamide gel electrophoresis and transferred onto nitrocellulose filters. The blots were then washed in TBST buffer, blocked with 2.5% bovine serum albumin in TBST for 30 min at room temperature and incubated with the primary antibodies for 30-60 min at room temperature in TBST. The blots were incubated with either a rabbit polyclonal antibody specific for yeast DNA topoisomerase I (17, 21) or with the M2 monoclonal. The blots were then incubated with a 1:7500 dilution of secondary antibody (goat anti-rabbit or goat anti-mouse, Vector Laboratories) conjugated to alkaline phosphatase for 30 min at room temperature in TBST. The blot was stained using the Protoblot system from Promega.
DNA Topoisomerase I Activity Assays-- DNA topoisomerase I activity was assayed by the relaxation of negatively supercoiled plasmid DNA as described previously (17). Briefly, nuclear extracts were first equalized for any differences in protein concentration and then serially 10-fold diluted in relaxation assay buffer containing 20 mM Tris (pH 7.5), 10 mM Na2EDTA, 150 mM KCl, and 50 µg/ml gelatin. 2-µl volumes were then incubated in 20-µl reactions containing relaxation buffer and 0.3 µg of pHC624 DNA (2015 base pair) (17) for 1 h at 30 °C (the optimal growth temperature for yeast). The reactions were terminated by the addition of 1% SDS. The extent of plasmid DNA relaxation was assessed following electrophoresis of the reaction products in a 1% agarose gel in 0.1 M Tris-borate buffer at 5 V/cm for 4 h and subsequent visualization by staining with ethidium bromide.
Immunoprecipitates were similarly assayed by incubating the protein-bound beads in a final 100-µl reaction mixture containing relaxation buffer, protease inhibitors (as above), and 0.5 µg of plasmid DNA. Following incubation at 30 °C for 30 min, the beads were pelleted, and the plasmid DNA topoisomers (in the supernatant) were phenol extracted, ethanol precipitated, and resolved in 1% agarose gels as detailed above.Cleavage Assays-- A single 944-base pair DNA fragment representing a high affinity site was 3'-end labeled with 32P-ATP. Approximately 10 ng (5000 cpm) of this labeled fragment was incubated with washed immunoprecipitates, as described above, from cells transfected with either eScTOP1, eSctop1T722A, or the control vector (pMT2). The 50-µl mixtures included 50 mM Tris (pH 7.5), 50 mM KCl, 10 mM MgCl2, and 4% Me2SO. Where indicated, camptothecin was added to a final concentration of 100 µM. After 30 min at 30 °C, the reactions were terminated by the addition of 1% SDS, heating to 75 °C for 10 min, and treatment with 0.4 mg/ml proteinase K. The cleaved DNA fragments were resolved in a 7 M urea, 8% polyacrylamide gel and visualized by autoradiography (as described in Ref. 21).
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RESULTS |
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Expression of Epitope-tagged S. cerevisiae DNA Topoisomerase I (eScTop1p) in COS Cells-- To investigate the function of yeast DNA topoisomerase I in mammalian cells, an epitope tag was introduced at the amino terminus of the yeast enzyme. The epitope is represented by four amino acids (DYKD) that are specifically recognized by the M2 monoclonal antibody (Kodak/IBI). This tag made it possible to efficiently detect the ectopically expressed yeast enzyme by immunoblotting, immunofluorescence, and immunoprecipitation of the yeast protein from COS cell nuclear extracts. Along with the wild-type ScTop1 protein, several mutant yeast DNA topoisomerase I genes were also tagged (see Fig. 1A). One was the catalytically inactive mutant Sctop1Y727F, in which the active site tyrosine was changed to phenylalanine (25). In a second yeast mutant enzyme, Top1vacp, the two amino acid residues immediately amino-terminal to the active site tyrosine, Ile725 and Asn726, were mutated to the Arg and Ala residues found at the corresponding position in the camptothecin-resistant vaccinia virus DNA topoisomerase I (17, 25). These substitutions render the catalytically active yeast Top1vac enzyme resistant to camptothecin-induced DNA cleavage (4, 17, 21). A third yeast mutant, top1T722A, is lethal when overexpressed in yeast cells, even in the absence of camptothecin, as discussed below (20).
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Expression of Catalytically Active eScTop1p in COS Cells Leads to Increased Sensitivity to Camptothecin-- Overexpression of human or yeast DNA topoisomerase I in S. cerevisiae leads to increased cell lethality, following treatment with camptothecin (14-17). In addition, it has been shown that overexpression of human DNA topoisomerase I in baby hamster kidney cells leads to increased sensitivity to camptothecin (26). To determine the extent of functional similarity between the yeast and human enzymes, we examined the camptothecin sensitivity of COS cells overexpressing eScTop1p. The approximately 100-fold increase in DNA topoisomerase I activity, resulting from the overexpression of the epitope-tagged yeast enzyme, should specifically enhance the camptothecin sensitivity of those cells relative to the untransfected COS cells in the same population. Because the ectopically expressed yeast enzyme is epitope-tagged, it allowed us to identify the number of cells expressing eScTOP1 within a given population by immunofluorescence. Treatment of the transfected cells with increasing doses of camptothecin should drive them to apoptosis and be seen as a concentration dependent increase in the percentage of apoptotic immunofluorescent positive cells.
To determine whether yeast DNA topoisomerase I enhanced the sensitivity of COS cells to the cytotoxic action of camptothecin, the cells were plated out onto glass coverslips and then transfected with the eScTOP1-, eSctop1vac-, or eSctop1Y727F-expressing plasmids. Immediately after the glycerol shock, the cells were treated with increasing doses of camptothecin or with a Me2SO control. Twenty-four hours after camptothecin treatment, the coverslips were processed for fluorescent microscopy, using M2 as a primary antibody, followed by DAPI staining. The altered nuclear morphology of an apoptotic cell, as assessed by DAPI staining, has been well defined (34, 35). Examples of camptothecin-induced apoptotic cells that are both fluorescent positive due to expression of eScTOP1 and fluorescent negative are shown in Fig. 7. The well characterized apoptotic features of nuclear blebbing and DNA fragmentation and condensation are evident (34, 35). It is interesting to note that eScTop1p becomes cytoplasmic following camptothecin treatment, which is consistent with the recent report that the enzyme changes its subcellular distribution after drug treatment (32).
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Overexpression of the Lethal Mutant eSctop1T722A in COS Cells Is Cytotoxic in the Absence of Camptothecin-- Substitution of alanine for threonine 722 in S. cerevisiae DNA topoisomerase I produces a lethal phenotype when the mutant enzyme is overexpressed in yeast, even in the absence of camptothecin (20). To determine whether the mechanism of top1 mutant-induced DNA damage is also conserved in higher eukaryotes, eSctop1T722A was cloned into the pMT2 vector and transiently expressed in COS cells. eScTop1T722Ap was found to be expressed to the same levels as eScTop1p by Western blot analysis (data not shown). To determine the potential lethality of this mutant in COS cells, cells were plated onto coverslips and transfected with pMT2 constructs expressing eScTOP1 and eSctop1T722A. At 1 and 4 days after transfection, the cells were processed for immunofluorescent microscopy. As shown in Fig. 9, the percentage of apoptotic cells is significantly increased in the cells transfected with eSctop1T722A compared with cells transfected with eScTOP1. Transfection of the inactive mutant eSctop1Y727F resulted in a similar number of apoptotic cells as eScTOP1 (data not shown). These data indicate that expression of the lethal mutant, eSctop1T722A, induces an apoptotic response in higher eukaryotes, similar to its cytotoxic effect when overexpressed in yeast.
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DISCUSSION |
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To establish the conservation of eukaryotic DNA topoisomerase I function, we have examined the effects of transiently overexpressing an epitope-tagged S. cerevisiae DNA topoisomerase I enzyme (eScTop1p) on the viability of a mammalian cell line. Nuclear extracts of the transfected COS cells contained the ectopically expressed protein as assessed by immunoblotting with either a yeast DNA topoisomerase I antibody or an antibody directed against the epitope tag. The enzyme was also found to be appropriately targeted to the nucleus, as assessed by indirect immunofluorescence.
An important observation was that the ectopically expressed eScTop1 protein enhanced the camptothecin sensitivity of the transfected COS cells because treatment with the drug resulted in preferential killing via apoptosis of those cells expressing the yeast enzyme. Thus, the yeast enzyme appears functional in mammalian cells. Because DNA topoisomerase I has been firmly established as the sole cellular target of camptothecin, overexpression of eScTOP1 in COS cells in the presence of camptothecin likely increases the amount of double-stranded DNA breaks, resulting in apoptotic cell death. Therefore, the conservation of structure and function between the yeast and mammalian enzymes is sufficient to produce lethal DNA damage in mammalian cells in response to the yeast enzyme and the drug. These data highlight the reciprocity in action between the yeast and human enzymes, suggesting that regardless of the source of enzyme or cell type, overexpression of eukaryotic DNA topoisomerase I in a eukaryotic cell will lead to death in the presence of camptothecin.
Along these lines, it is apparent that the phenotypes associated with
overexpression of Sctop1 mutants in yeast were also faithfully reiterated in COS cells. The ScTop1Y727F mutant protein is
catalytically inactive (25), and top1 yeast strains
expressing the eSctop1Y727F mutant are completely resistant
to the effects of camptothecin (4, 17). Although eScTop1Y727Fp was
efficiently targeted to the nucleus in COS cells, as measured by
indirect immunofluorescence, overexpression of this mutant protein
produced no observable changes in DNA relaxation activity in nuclear
extracts or in the camptothecin sensitivity of transfected COS cells.
As has been reported in yeast (20), overexpression of this protein was
slightly cytotoxic in the absence of camptothecin. Whether or not this
effect results from interference with endogenous TOP1 functions has yet to be determined. Nevertheless, these results support
the notion that the increased cytotoxic effects of camptothecin on
eScTOP1-expressing cells is a direct result of the increased DNA topoisomerase I activity in these cells.
The eSctop1vac mutant is a double mutation of
Ile725 to Arg and Asn726 to Ala (25). When
eScTop1vacp is overexpressed in a S. cerevisiae top1
strain, the cells are completely resistant to the lethal effects of
camptothecin (17, 21). Biochemical studies indicate that this
catalytically active mutant enzyme is ~20-fold more resistant to
camptothecin-induced DNA cleavage than the wild-type enzyme (17). This
camptothecin-resistant phenotype is also evident in mammalian cells
because the cytotoxic action of camptothecin on COS cells
overexpressing eSctop1vac is diminished in comparison to COS
cells overexpressing the wild-type eScTop1 enzyme.
One of the more striking DNA topoisomerase I mutants involves a substitution of alanine for threonine at position 722 (20). This substitution (eSctop1T722A) converts the enzyme into a cellular poison when it is overexpressed in yeast, due to the fact that the covalent enzyme-DNA intermediate is stabilized (20). Interestingly, a similar effect on cell viability is evident in COS cells ectopically expressing eSctop1T722A, in contrast to cells expressing eScTOP1. The cells expressing eSctop1T722A die via an apoptotic mechanism, likely the result of the DNA damaging capability of eScTop1T722Ap, as assessed by a DNA cleavage assay (Fig. 10). Further, as in yeast, the cell lethality induced by eSctop1T722A is camptothecin independent. Taken together, these results highlight the conservation in function between yeast and human DNA topoisomerase I and support the use of model systems such as yeast to explore the cytotoxic mode of action of DNA topoisomerase I poisons.
Another novel finding presented in this work concerns the activity of eScTop1p in immobilized immunoprecipitates. Extensively washed Staph-A immobilized immunoprecipitates of the ectopically expressed eScTop1 protein had increased DNA relaxation activity and increased DNA cleavage activity in the presence of camptothecin. This suggests that the enzyme is functional even though its amino terminus is tethered to a support and is immobilized. Control experiments demonstrated that the bulk of the enzyme remains tethered to the Staph-A beads (data not shown). This approach is currently being exploited to assess the effect of specific amino acid substitutions on DNA binding by the enzyme.
Finally, examination of subcellular location of ectopically produced eScTop1p indicated that it can be targeted to a perinuclear region, as demonstrated by confocal microscopy. This is interesting in light of the evidence that the nuclear membrane participates in DNA replication (36) possibly by anchoring chromosomes. The nuclear membrane would therefore be a potential target for DNA topoisomerase I-mediated relaxation of supercoiled DNA generated during replication. A similar perinuclear distribution of human DNA topoisomerase I was reported in CEM cells following leucine starvation (37). Although the enzyme demonstrates a predominantly nucleolar distribution in proliferating cells (37, 38), it is also seen localized to other sites within the nucleus, such as the nuclear membrane (32, 33). Because at a low level of expression, eScTop1p can be seen localized to the nucleoli (data not shown), it is possible that at a higher level of expression the protein occupies available binding sites near the nuclear membrane. The nature of these sites remains to be determined.
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FOOTNOTES |
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* This work was supported in part by National Institutes of Health grants CA67032 (to D. J. H.) and CA70406 (to M. A. B.).
§ Supported by the Foerderer Foundation.
Supported by the Progetto Finalizzato Consiglio Nazionale
delle Ricerche ACRO and the Associazione Italiana Ricerca sul Cancro.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
** To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Pharmacology, Thomas Jefferson University, 233 S. 10th St., Philadelphia, PA 19107. Tel.: 215-503-2035; Fax: 215-923-9162; E-mail: hall{at}hendrix.jci.tju.edu.
1 J. Fertala and M.-A. Bjornsti, unpublished results.
2 P. Fiorani, J. Amatruda, M.-A. Bjornsti, and P. Benedetti, manuscript in preparation.
3 The abbreviations used are: DAPI, 4',6-diamidino-2-phenylindole; TBST, 10 mM Tris, pH 8, 150 mM NaCl, and 0.05% Tween 20.
4 C. Hann and M.-A. Bjornsti, unpublished observations.
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REFERENCES |
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