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Department of Pharmacology/Toxicology and Medicine, Medical College of Virginia, Virginia Commonwealth University, Richmond, Virginia 23298
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Summary |
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Summary
Introduction
Procedures
Results
Discussion
References
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Ionizing radiation and the topoisomerase II inhibitor, teniposide (VM-26) both increase levels of the cyclin dependent kinase inhibitor, p21waf1/cip1 and promote dephosphorylation of the retinoblastoma tumor suppressor protein, Rb, in MCF-7 breast tumor cells, perturbations associated with suppression of the activity of the transcription factor, E2F. However, studies using an E2F binding site-luciferase reporter plasmid transfected into MCF-7 cells failed to demonstrate a reduction in E2F activity in response to VM-26 or to ionizing radiation. In contrast, E2F activity (both basal and E1A stimulated) could be suppressed by transfection with a plasmid expressing Rb, indicating that the capacity of E2F to bind to Rb and to be inactivated by Rb is functionally intact in MCF-7 cells. These findings in MCF-7 breast tumor cells suggest that E2F activity may not be directly susceptible to modulation by endogenous p21waf1/cip1 and Rb.
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Introduction |
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Summary
Introduction
Procedures
Results
Discussion
References
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Cellular
progression through the restriction point
(the G1 to S transition) is stringently
controlled through the successive activation of cyclin-dependent
kinases (1) that modulate the activity of the Rb family of tumor
suppressor proteins, including Rb, p107, p130, and p300 (1-3). In
turn, binding of the Rb proteins to a family of E2F transcription
factors (4) influences the activity of E2F (2, 5) and the transition to
S phase (6). Initiation of the S phase is thought to be a consequence
of E2F transactivation of genes such as dihydrofolate reductase, DNA polymerase 
, thymidylate synthase, thymidine kinase,
c-myc, and c-myb (7). Recent studies have
determined that, in addition to the Rb family of proteins, E2F activity
may be influenced directly by p53 (8), by the mdm2 protein that is
downstream of p53 (9), by the cyclin-dependent kinase inhibitory
protein p21waf1/cip1 (10), and through
alterations in E2F phosphorylation by the cyclin E/cdk2 and cyclin
A/cdk2 complexes (11).
In addition to promoting the G1 to S transition, there is indirect evidence suggesting that E2F could be involved in arresting cell growth in response to DNA damage. Alterations in elements putatively upstream of E2F in response to DNA damage include an increase in the levels of the p53 tumor suppressor (12-14) and the p21waf1/cip1 proteins (15, 16), as well as dephosphorylation of Rb (14, 17). However, other findings argue (again indirectly) against a role for E2F in growth arrest. Although there is evidence for a growth-suppressive action of Rb (18), presumably through inactivation of E2F, transfection of Rb-negative cells with vectors constitutively expressing Rb does not uniformly result in abrogation of cell growth (19, 20). Furthermore, although DNA damage generally results in the up-regulation of p53 and p21waf1/cip1 and the induction of Rb dephosphorylation, events that should lead to G1 arrest through suppression of E2F activity, tumor cells in which DNA is damaged generally arrest in G2 (21-23). However, to our knowledge, no studies have been reported assessing the response to DNA damage at the level of E2F activity in the intact cell.
The studies in this report describe the effects of ionizing radiation and teniposide on E2F activity in MCF-7 breast tumor cells. Ionizing radiation has been reported to induce p53, to increase p21waf1/cip1 levels (14, 15), and to promote dephosphorylation of Rb in MCF-7 cells (15). We have substantiated the influence of ionizing radiation on p21waf1/cip1 levels and Rb dephosphorylation and demonstrated similar effects by the topoisomerase II inhibitor, VM-26. To further extend these findings, we evaluated the influence of VM-26 and ionizing radiation on the activity of E2F by assessing the expression of an E2F binding site-luciferase reporter plasmid transfected into MCF-7 cells. The studies described below indicate that an increase in endogenous p21waf1/cip1 protein levels and/or the dephosphorylation of endogenous Rb in MCF-7 cells may not be sufficient to suppress E2F activity. Consequently, it remains to be determined what role is played by E2F in the signal transduction pathway that responds to DNA damage through p53, p21waf1/cip1, and Rb.
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Experimental Procedures |
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Summary
Introduction
Procedures
Results
Discussion
References
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Materials.
Dulbecco's modified Eagle's medium (56-439) was
obtained from Hazelton Research Products (Denver, PA);
L-glutamine, penicillin/streptomycin (10,000 units
penicillin/ml and 10 mg/ml streptomycin), and fetal bovine serum were
obtained from Whittaker Bioproducts (Walkersville, MD); defined bovine
calf serum was obtained from Hyclone Laboratories (Logan, UT).
Trypsin-EDTA (10×; 0.5% trypsin, 5.3 mM EDTA) was obtained from GIBCO/BRL (Grand Island, NY) and maintained as a frozen
stock. VM-26 (teniposide) was generously provided by Bristol-Myers. (Wallingford, CT). VM-26 was dissolved in dimethylsulfoxide (Aldrich Biochemicals, Milwaukee, WI) and maintained as a frozen stock solution
for a maximum period of 2-3 weeks. Drug was diluted in incubation
medium on the day of the experiment.
[-32P]dCTP (3000 Ci/mmol) was obtained from
DuPont NEN Research Products (Boston, MA). The restriction
endonucleases were obtained from New England Biolabs, Beverly, MA, and
the nick-translation kit was obtained from GIBCO/BRL. Anti-Cip1 (p21)
was obtained from Transduction Laboratories (Lexington, KY). Anti-Rb
was obtained from Pharmingen (San Diego, CA).
Probes and plasmids.
A plasmid containing functional E2F
consensus binding sites linked to a luciferase reporter (PGL2-E2F/Luc)
and a plasmid containing mutated E2F binding sites linked to a
luciferase reporter (PGL2-E2Fmut/Luc) were generously provided to our
laboratory by Dr. Srikumar Chellappan (Columbia University College of
Physicians and Surgeons, New York, NY). The wild-type pGL2 plasmid
contains four E2F consensus sites (---TTTCGCGC---), whereas the mutant
plasmid contains four E2F sites mutated at 3 base pairs
(---TTTCTATC---) blocking the binding of the E2F protein. The normal
and the mutated E2F consensus sequences were linked to a
(---GATCTGTCCGAGTTATAA---) sequence containing a TATAA box, which was
then attached to a luciferase reporter gene. The pCEP4 vector
containing the 
-galactosidase gene under the control of the CMV
promoter was obtained from Dr. Eric Westin's laboratory at the Medical
College of Virginia (Richmond, VA). The pSVE-Rb plasmid was generously
provided by Dr. Lila Adnane at the Molecular Genetics Department,
University of Pittsburgh. The pSVE-Rb plasmid contains the 4.7-kb human
retinoblastoma gene placed in the BamHI site of the pSVE
vector. This plasmid overexpresses Rb through the SV-40 promoter that
is upstream from the BamHI site. The pGE1A plasmid, which
expresses E1A through the SV-40 promoter, was a gift from Dr. Joseph
Nevins (Duke University Medical Center, Durham, NC).
Cell line. The MCF-7 breast tumor cell line was kindly provided by the laboratory of Dr. Kenneth Cowan at the National Cancer Institute (Bethesda, MD). Cells were maintained as monolayers in Dulbecco's modified Eagle's medium supplemented with glutamine (0.292 mg/ml), penicillin/streptomycin (0.5 ml/100 ml of medium), 5% fetal bovine serum, and 5% defined bovine serum. All cells were cultured at 37° in an atmosphere of 5% CO2 and 100% humidity. For the studies described in this report, cells were subcultured at densities in which the cells were maintained in logarithmic growth during the assay procedure.
Cell cycle analysis. MCF-7 breast tumor cells were grown in 75-cm2 flasks to approximately 30-40% confluence. Cells were irradiated with the indicated dose of ionizing radiation or incubated with VM-26 for 3 hr at 37°. Cells were washed twice with fresh medium and permitted to grow in fresh medium. Samples were isolated at 0-, 24-, 48-, and 72-hr intervals postexposure. At appropriate times, the cells were washed once with 1 × PBS and detached from the flasks by trypsinization. Cells were centrifuged for 5 min at 1500 rpm and resuspended in ice cold 1 × PBS. The cell number was determined, and 5 × 106 cells/ml were resuspended in 500 µl of 1 × PBS and 500 µl of ethanol from a 100% stock. Cells were either incubated for 15 min on ice or stored at 2-8°C until the day of use. The samples that had been fixed in ethanol were centrifuged for 15 min at 1200 rpm, and the supernatant was discarded. The pellet was resuspended in 1 ml of filtered 1 × PBS (0.2-µm filter) and transferred to a specimen collection vial. Using a 3-ml syringe and a 26-gauge needle, the samples were passed through the syringe six times. One milliliter of solution A (3.4 mM trisodium citrate dihydrate, 1% (v/v) Nonidet P-40, 1.5 mM spermine tetrahydrochloride, and 500 µM Tris, pH 7.6) was added to the cells, and the cell suspension was incubated for 10 min at room temperature with gentle agitation. One milliliter of solution B (0.5 mg/ml trypsin inhibitor, 0.1 mg/ml ribonuclease A, 3.3 mM spermine tetrahydrochloride, pH 7.6) was added, and the cell suspension was incubated for an additional 10 min at room temperature with gentle agitation. Finally, solution C (748 µM propidium iodide, 3.33. mM spermine tetrahydrochloride, pH 7.6) was added to the samples and incubated for 10 min at room temperature with gentle agitation. The samples were then centrifuged for 5 min at 1200 rpm. The supernatant was removed, and the pellets were resuspended in 1 ml of filtered PBS and transferred to 12 × 75-mm tubes. The samples were maintained on ice or at 2-8°C and in the dark for up to 4 days until analysis by flow cytometry. For flow cytometric analysis, an argon laser emitting at 488 or 514 nm was used to determine DNA content, and the Verity Mod Fit LT Software was used to analyze the results obtained from the Becton Dickinson FACScan, model FC.
Western blot analyses. Cells were grown in T-75 flasks to approximately 60% confluence before irradiation or treatment with VM-26. Cells were then washed with ice-cold PBS, and crude cellular protein was extracted using hot lysis buffer (50 mM Tris·HCl, pH 6.8, containing 0.1% sodium dodecyl sulfate) at 0, 3, 6, 9, and 24 hr. Samples were denatured by boiling for 10 min followed by centrifugation. Total protein was quantified using the Pierce Coomassie Protein Assay Reagent (Pierce, Rockford, IL). Proteins (25 µg per lane) were electrophoresed on sodium dodecyl sulfate polyacrylamide gels (6% for Rb and 15% for p21) and transferred to nitrocellulose membranes. The blots were blocked with TBS containing 5% nonfat dry milk and 0.1% Tween 20 for at least 4 hr at room temperature. The blocking buffer was then decanted, and the blots were probed with the respective primary antibody diluted in TBS-Tween (containing 2% nonfat dry milk) for 1 hr at room temperature. p21 and Rb proteins were detected using 0.5 µg/ml of the respective antibodies. After washing in TBS-Tween, the blots were incubated with horseradish peroxidase-conjugated goat anti-mouse antibody (0.2 µg/ml; Kirkegaard & Perry Laboratories, Gaithersburg, MD), then washed again in TBS-Tween. Antibody binding was detected using a chemiluminescence kit (Pierce). The blots were exposed to X-ray film and quantified using NIH Image software.
Luciferase reporter assay for E2f activity.
Approximately
1.5 × 105 cells/ml were plated in each
cylinder of a 6-well plate containing 2 ml of growth medium, and the
cells were allowed to proliferate overnight. The following day, the various plasmids were diluted in 100 µl of Opti-MEM (GIBCO BRL); to
keep the total concentration of DNA transfected in each condition constant, Bluescript (KSP+) plasmid containing
nonspecific sequences was added as needed. The LipofectAMINE
transfection procedure by GIBCO BRL was performed as described by the
manufacturer. After either irradiation or drug treatment (VM-26 or the
vehicle, DMSO), the cells were washed twice with 2 ml of
Ca+2/Mg+2-free PBS, and
then lysed using 235 µl/well reporter lysis buffer (Promega, Madison,
WI) containing 125 mM Tris, pH 7.8, with
H3PO4, 10 mM EDTA, 10 mM
dithiothreitol, 50% glycerol, and 5% Triton X-100 (diluted 1:4) for
15 min at room temperature. The cell lysate was scraped using a rubber
policeman, collected in 1.5-ml microcentrifuge tubes, and centrifuged
at 10,000 rpm for 2 min at 4°. The supernatant was transferred to a
1.5-ml Eppendorf tube and stored at 
70° until the extract was used
for the determination of luciferase and -galactosidase activities.
-galactosidase from a
cytomegalovirus promoter. -Galactosidase activity was determined as
described in Sambrook et al. (24). Briefly, 5-20 µl of
cell extract were brought up to a final volume of 30 µl by the
addition of lysis buffer (Promega). A substrate mixture (270 µl)
containing
O-nitrophenyl--D-galactopyranoside, 0.1 M MgCl2, 4.5 M
-mercaptoethanol, and 0.1 M sodium phosphate (pH 7.5)
was added to 30 µl of the cell extract. The solution was then
incubated for 30 min at 37°. The reaction was stopped by the addition
of 500 µl of 1 M
Na2CO3. The product of
-galactosidase activity, O-nitrophenol, was measured
spectrophotometrically at 420 nm.
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Results |
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Summary
Introduction
Procedures
Results
Discussion
References
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Effects of VM-26 and ionizing radiation on cell cycle progression. A primary goal of the present work was to determine whether suppression of E2F activity is a component of the pathway leading to growth arrest in MCF-7 breast tumor cells. This first series of experiments was designed to assess the nature of growth arrest in MCF-7 breast tumor cells exposed to either ionizing radiation or to VM-26.
Drugs that inhibit topoisomerase II, such as VM-26, have generally been reported to produce arrest in the G2 phase of the cell cycle (22-23). Asynchronously growing MCF-7 breast tumor cells exposed to 10 µM VM-26 for 3 hr were analyzed for cell cycle distribution at 24-hr intervals subsequent to the drug exposure. Table 1 indicates that within 24 hr the percentage of cells in the G2M phase of the cell cycle more than doubled, whereas the proportion of cells in the G0-G1 phase was reduced. By 72 hr, 35.7 ± 8.7% of the cell population was in the G2M phase compared with 13.2 ± 1.3% of cells in G2M before treatment. There was a modest but significant decline in the proportion of cells in the G0-G1 phases of the cell cycle, whereas the S phase fraction remained essentially unchanged.2
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Influence of VM-26 on the levels of p21waf1/cip1 and on the phosphorylation state of Rb. The induction of p21waf1/cip1 in response to DNA damage has been demonstrated to occur in cells exposed to ionizing radiation as well as to other modalities that induce DNA damage (12-14, 16). The influence of 10 µM VM-26 on the levels of the p21waf1/cip1 protein was assessed over a time frame of 24 hr by Western analysis. Fig. 1A indicates that there was an approximately 2-fold increase in p21waf1/cip1 levels by 3 hr, and a 3-fold elevation within 6 hr that was sustained over 24 hr. This represents a significant, though far from imposing, increase in p21waf1/cip1 levels.
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Effects of ionizing radiation on the levels of p21waf1/cip1 and on the phosphorylation of Rb. Exposure of MCF-7 cells to 10 Gy of ionizing radiation resulted in a time-dependent increase in p21waf1/cip1 levels (Fig. 2A) that was similar to that induced by VM-26. As was the case with VM-26, the increase in p21 was roughly 2-fold after 3 hr; a 3-fold increase, which was evident by 6 hr, was maintained over a period of 24 hr. These observations are similar to the findings of other investigators using a dose of 5 Gy in MCF-7 cells (14, 15).
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Effects of VM-26 and ionizing radiation on E2F activity. The drug and radiation induced increases in p21waf1/cip1 levels and the dephosphorylation of Rb should result in a concomitant inactivation of the transcription factor E2F (10, 29). However, this possibility has, to our knowledge, not been assessed directly in cells responding to DNA damage. To assess the effects of ionizing radiation and teniposide on E2F activity, an E2F-luciferase reporter plasmid (PGL2-E2F/luc; see Experimental Procedures) that permits assessment of the activity of the multiple forms of E2F in the cell (4) was transfected into MCF-7 cells. Fig. 3A presents values for E2F activity in MCF-7 cells exposed to VM-26 for various time periods as well as E2F activity in control cells exposed to volume equivalents of DMSO (the vehicle for VM-26). Even continuous exposure of MCF-7 cells to VM-26 for up to 12 hr failed to reduce the transactivation activity of E2F.3 In fact, at 6 and 9 hr, there was a small but significant increase in E2F activity (when compared with cells exposed to DMSO). Fig. 3 indicates that a reporter plasmid containing mutations in the E2F binding site (pGL2-E2F-mut/luc; see Experimental Procedures) that was utilized as a negative control at 12 hr had relatively low expression of luciferase as compared with the wild-type plasmid containing intact E2F binding sites.
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Determination of the functional interaction between Rb and E2F in the MCF-7 cell line
The absence of suppression of E2F activity by either VM-26 or ionizing radiation in cells with elevated levels of the p21waf1/cip1 protein and dephosphorylation of Rb might be ascribed to problems with the reporter assay or to defects in the association between Rb and E2F. These issues were addressed by transfecting cells with vectors constitutively expressing exogenous Rb or the adenoviral protein, E1A, either alone or in combination. Transfection with Rb was designed to demonstrate that endogenous E2F activity could be inhibited by its binding to the Rb tumor suppressor protein. Transfection with E1A, which dissociates E2F complexed with the Rb family of proteins (32, 33) was designed to further validate the utility of the reporter assay in detecting alterations in E2F activity. As expected in cells with functional E2F, the Rb expressing vector produced a marked reduction in E2F activity (Fig. 4A). The average reduction in E2F activity was 67.7 ± 16.5%. Conversely, as shown in Fig. 4B, transfection with E1A stimulated E2F activity, indicating the dissociation of endogenous complexes involving E2F and the Rb family of proteins (i.e., p130, p107, and Rb) in MCF-7 cells. Transfection with Rb was able to reverse the stimulation of E2F activity induced by E1A, a finding that is consistent with the capacity of E2F to be bound to and inhibited by Rb. In both sets of experiments, the activity of the pGL2-E2F-mut/luc plasmid was included as a negative control.
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Discussion |
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Summary
Introduction
Procedures
Results
Discussion
References
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It is generally accepted that DNA damage, such as that induced by ionizing radiation, increases levels of the p53 tumor suppressor protein (12-15), up-regulates the cyclin-dependent kinase inhibitory protein, p21waf1/cip1 (13-16) and promotes dephosphorylation of Rb (14, 15, 17). However, although these perturbations are thought to suppress the activity of the transcription factor E2F (2, 5, 10, 28, 29) the influence of DNA damage on E2F activity has not previously been assessed in the intact cell. Consequently, a primary focus of the studies described in this report was to determine whether exposure of MCF-7 breast tumor cells to ionizing radiation or to VM-26 would alter the transactivational activity of E2F in concert with their effects on p21waf1/cip1 levels and on the phosphorylation state of Rb.
Gudas et al. (14) and Wosikowski et al. (15) have reported that ionizing radiation increases levels of p53 and of p21waf1/cip1 and promotes the dephosphorylation of Rb in the MCF-7 breast tumor cell line. We have substantiated the effects of ionizing radiation on p21waf1/cip1 levels and Rb dephosphorylation and further demonstrated that the topoisomerase II inhibitor VM-26 produces similar (and in the case of Rb, more pronounced) alterations in these growth-regulatory proteins in the MCF-7 breast tumor cell line. However, despite the 2-3-fold increase in p21waf1/cip1 levels and the dephosphorylation of Rb, neither VM-26 nor ionizing radiation suppressed E2F activity. Thus, it appears that in MCF-7 cells, the damage responses involving up-regulation of p21waf1/cip1 and dephosphorylation of Rb occur as expected, but that these effects, either alone or in combination, are not sufficient to suppress E2F activity. The lack of effect on E2F activity might explain, in part, the absence of G1 arrest in MCF-7 cells exposed to VM-26; however, this observation is not consistent with the combined G1 and G2 arrest induced by ionizing radiation.
Dbaibo et al. (34), using gel-shift analysis, demonstrated that treatment of the MOLT-4 human lymphoblastic leukemia cell line with sphingosine, a breakdown product of membrane sphingolipids, promoted the loss of association between E2F and its specific DNA sequence elements; this effect was associated with sphingosine-induced dephosphorylation of Rb (35). These observations suggest that the relationship between Rb and E2F may be intact in other tumor cell lines under different experimental conditions, although transcriptional repression by the Rb-E2F complex is generally thought to require occupation by E2F of its binding site on the promoter.
Our studies raise the question of why p21waf1/cip1 and the dephosphorylated form of Rb fail to suppress E2F activity in MCF-7 cells. We considered the possibility that the existence of a mutation in the E2F family of proteins in MCF-7 cells might prevent their association with Rb. This possibility appears to have been ruled out by the studies in which E2F activity was suppressed by transfection with a plasmid expressing Rb; these studies indicate that the capacity of endogenous forms of E2F to bind to Rb is intact in MCF-7 cells. Although this approach does not identify the forms of E2F bound to Rb, these should include E2F-1, E2F-2, E2F-3 (36), as well as E2F-4 (37).
One explanation that might account for the failure of VM-26 and ionizing radiation to inactivate E2F activity in the MCF-7 breast tumor cell is that endogenous Rb levels are too low to suppress E2F. An and Dou (38) have reported proteolytic cleavage of Rb in HL-60 and U937 leukemic cells exposed to VP-16, whereas we observe what appears to be an overall reduction in Rb protein levels in irradiated MCF-7 cells after 24 hr. Although the MCF-7 cells contain a wild-type Rb protein, we must consider the possibility of defects in the capacity of the endogenous Rb protein to either bind to E2F or, once bound, to inactivate E2F. Consequently, it is possible that in MCF-7 cells exposed to VM-26 or ionizing radiation, where Rb (and possibly p107 and/or p130) are dephosphorylated, inactivation of E2F does not occur because Rb is functionally inactive. This might occur if the dephosphorylation is incomplete or occurs at sites that fail to confer activity on the Rb protein.
The current studies are the first to suggest that dephosphorylation of Rb as well as a 3-fold increase in p21waf1/cip1 levels and an increase in p53 in concert with dephosphorylation of Rb (14, 15) are not sufficient to predict a concomitant suppression of E2F activity in the MCF-7 breast tumor cell. Although we cannot yet predict the relevance of the current findings to other experimental tumor cell lines or to the clinical situation, these findings suggest that the putative signaling pathway linking p53, p21waf1/cip1, and Rb to E2F could be dysfunctional or dysregulated at the level of the Rb-E2F complex, at least in the MCF-7 cell line. Although mutations in p53 represent one well established mechanism for loss of restriction point control (39, 40), the failure of signals downstream of p53 (e.g., increased p21waf1/cip1 and Rb dephosphorylation) to abrogate E2F function (7) could contribute to loss of restriction point control in cells with functional p53.
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Acknowledgments |
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We are grateful to Dr. Srikumar Chellappan for proving the E2F
reporter plasmids, Dr. Eric Westin for the -galactosidase reporter
plasmid, Dr. Lila Adnane for the plasmid expressing Rb, and Dr. Joseph
Nevins for the plasmid expressing E1A. We thank Dr. Karen Magnet for
helpful suggestions relating to the writing of this manuscript.
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Footnotes |
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Received March 14, 1997; Accepted May 19, 1997
1 Current affiliation: National Cancer Institute, National Institutes of Health, Bethesda, MD 20892.
2 In separate studies, in which cell number was assessed by trypan blue exclusion over a period of 3 days, we independently verified that both VM-26 and ionizing radiation interfere with the growth of MCF-7 breast tumor cells.
3 In a separate study using four sample replicates, we found no decrease in E2F activity by VM-26 even after 24 hr.
This research was supported in part by Grant NRC0493080 from the Nuclear Regulatory Commission, Grant CA55815 from the National Cancer Institute, and United States Army Medical Research and Material Command Award DAMD 17-96-1-6167.
Send reprint requests to: Dr. David A. Gewirtz, Department of Medicine, Medical College of Virginia, P.O. Box 980230, Richmond, VA 23298. E-mail: gewirtz{at}gems.vcu.edu
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Abbreviations |
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PBS, phosphate-buffered saline; TBS, Tris-buffered saline.
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References |
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Summary
Introduction
Procedures
Results
Discussion
References
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