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J. Biol. Chem., Vol. 280, Issue 1, 361-374, January 7, 2005
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From the
Department of Microbiology-Immunology, Feinberg School of Medicine, Northwestern University, Chicago, Illinois 60611 and the **Department of Biochemistry and Molecular Genetics, University of Illinois, Chicago, Illinois 60607
Received for publication, July 29, 2004 , and in revised form, October 29, 2004.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Previous adenovirus (Ad) E1A studies have indicated that p300 may play a role in maintaining cells in the G1 phase of the cell cycle. For example, E1A can stimulate DNA synthesis in quiescent baby rat kidney cells, which is dependent on its binding to p300 (13–15). E1A can also re-stimulate DNA replication in terminally differentiated cardiac myocytes through its p300/CBP-binding domain (16). There is evidence that p300 is involved in terminal differentiation in several cell types, including muscles, neurons, and enteroendocrine cells (reviewed in Ref. 1). During terminal differentiation, p300 transactivates p21 by cooperating with Sp1, Sp3, or tissue-specific transcription factors, suggesting that p300 may play a role in keeping cells in G0/G1 (1, 17, 18). However, fibroblasts from p300 knockout mice are unable to replicate and appear to undergo cell cycle arrest, indicating that some p300 function may be necessary for cell cycle progression (5). Furthermore, CBP has been shown to be associated with Cdk2 in vivo and may be a substrate for cyclin E/Cdk2 (19). p300 provides a coactivator function for both growth stimulatory transcription factors such as E2F (3) and growth inhibitory proteins such as p53 (2, 11, 12). p300 is underphosphorylated during G0/G1 and hyperphosphorylated during S and G2/M phases (20). Therefore, the overall roles played by p300/CBP at different stages of the cell cycle remain to be determined.
Although, in most studies, p300 was able to substitute for CBP functions, not all p300 functions can be substituted by CBP (reviewed in Ref. 1). For example, in F9 cells, both differentiation and cell cycle arrest induced by retinoic acid are dependent on normal levels of p300, but not of CBP (21). Mice lacking one copy of the CBP gene display some of the characteristics found in Rubinstein-Taybi syndrome patients, including the increased development of hematological neoplasm (6). In striking contrast, p300 heterozygous mice do not display this phenotype (5). These proteins also appear to play distinct roles in maintaining normal hematopoiesis (22). Mouse p300-/- fibroblasts are also defective in retinoic acid-induced transcription despite the presence of normal level of CBP in these cells. In contrast, these cells maintain normal levels of CREB-activated transcription (5). In transient assays, transcription of the cyclin D1 gene can be induced by p300, but not by CBP (23). Other studies related to functional differences between p300 and CBP include the involvement of p300 (but not CBP) acetyltransferase activity during myogenesis (24) and complexes containing p300 (but not CBP) and the nuclear proto-oncoprotein SYT in the proper activation of 
1 integrin and control of cell adhesion (25).
Recently, we showed that quiescent human cells depleted of p300 enter S phase without serum and that this premature DNA synthesis is due to up-regulation of c-myc (26). Similarly, we also showed that conditional overexpression of p300 represses c-myc and S phase entry (27). In view of the above-noted functional differences between p300 and CBP, we investigated the effects of depletion of CBP on c-myc regulation and G1-S transition. We now show that reduced synthesis of CBP in quiescent cells also results in the induction of c-myc and S phase entry. However, p300- or CBP-depleted cells with or without serum stimulation did not undergo normal cell cycle progression. Such cells expressed c-myc to much higher levels compared with control cells, and a significant portion of them died of apoptosis after serum stimulation, which did not appear to be related to p53 levels. Furthermore, we also show that in growth-arrested p300/CBP-depleted cells, the Cdk inhibitor p21 is dramatically down-regulated, and p107 and p130 (but not pRb) are hyperphosphorylated at low levels, suggesting that in addition to the induction of c-myc, the alterations in these negative regulators of the cell cycle may also contribute to the induction of S phase. Finally, we show that the inappropriate up-regulation of c-myc in CBP-depleted cells is primarily responsible for the induction of S phase and apoptosis, as inhibition of c-myc expression reverses both these effects. Thus, our results suggest that despite their structural and functional similarities, normal levels of both p300 and CBP are required for keeping c-myc in a repressed state and maintaining cells in the G1 phase of the cell cycle. These two proteins may have a universal role as checkpoint proteins, preventing the untimely onset of DNA synthesis in senescent or differentiated cells. Our results also indicate that normal levels of both these proteins are essential for coordinated cell cycle progression.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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-gal expresses -galactosidase from the cytomegalovirus promoter (26). AdM4 is an Ad vector that contains a promoter-luciferase reporter cassette with four copies of the c-Myc-binding site (E-box element) cloned upstream of the herpes simplex virus thymidine kinase minimal promoter and linked to the luciferase reporter gene (27). AS-Myc is an Ad vector that expresses antisense c-myc sequences (26). Cell Cycle Analysis—Cells were seeded overnight at a density of 1 x 106/100-mm dish and serum-starved. Thirty-six hours after serum starvation, they were infected with appropriate Ad vectors at a multiplicity of infection of 200 and maintained under serum-starved conditions for the duration of the experiments shown in the figure legends. For the serum stimulation experiments, cells were stimulated with serum 18 h after vector infection and harvested at the indicated time points. For experiments in which cell cycle analysis was carried out under continued serum starvation, cells were maintained in serum-free medium for the duration of the experiments and then harvested and processed for FACS analysis as described (29).
RNA Analysis—MCF10A cells were seeded, starved, and infected with Ad vectors as described above; starvation was continued for 18 h after virus infection; and the cells were stimulated with growth medium containing 5% horse serum. Total RNA was isolated from cells using an RNA isolation kit (Promega catalog no. Z3100). Poly(A)-containing RNA was isolated from total RNA using a poly(A)+ RNA isolation kit (QIAGEN catalog no. 72012). Equal quantities of RNA were analyzed by Northern blotting using a human c-myc probe corresponding to 1 kb of N-terminal coding sequence.
Apoptosis Assays—Apoptosis assays for p300- or CBP-depleted serum-starved cells were carried out using the annexin V binding assay to assess the loss of phospholipid asymmetry of the plasma membrane (32) and by staining the mitochondria with chloromethyl-X-rosamine to assess the changes in the mitochondrial membrane potential (33). Cells were serum-starved, infected with Ad vectors, and serum-stimulated as described above for the cell cycle experiment. At the indicated times, the adherent cells were harvested after trypsinization and pooled with the previously collected floating cells, which were washed once with calcium/magnesium-free phosphate-buffered saline. Annexin V binding assay was then carried out using a commercially available kit (Immunotech catalog no. PN IM3546). Briefly, the phosphate-buffered saline-washed cell pellet was resuspended in 100 µl of 1x binding buffer at a concentration of 5 x 106/ml. One microliter of FITC-conjugated annexin V and 5 µl of propidium iodide were then added from the stock solutions provided in the kit. The cell suspension was mixed gently and incubated in the dark for 10 min on ice. It was then diluted to 5-fold with 1x binding buffer and analyzed by flow cytometry within 1 h using a Beckman Coulter Epics XL-MCL counter. Cells were analyzed at an excitation emission wavelength of 488 nm and at detection emission wavelengths of 620 nm for propidium iodide-positive cells and 525 nm for FITC-conjugated annexin V-positive cells.
Mitochondrial membrane potential was assessed by staining the mitochondria with chloromethyl-X-rosamine using a commercially available kit (Molecular Probes catalog no. M-7512). Briefly, cells were infected and harvested at the indicated times as described above for the annexin V binding assays, resuspended in 3 ml of culture medium containing 100 nM MitoTracker Red CMX-Ros (chloromethyl-X-rosamine), and incubated for 20 min at 37 °C in a 5% CO2 incubator. MitoTracker Red CMX-Ros is a modified chloromethyl-X-rosamine (as described in the Molecular Probes product information sheet). This modified chloromethyl-X-rosamine labels mitochondria with a longer wavelength fluorescence emission that can be separated from FITC-conjugated annexin V fluorescence. The cell suspension containing chloromethyl-X-rosamine was spun down, washed once with calcium/magnesium-free phosphate-buffered saline, and resuspended in 100 µl of annexin V binding buffer. One microliter of FITC-conjugated annexin V was then added, and the cell suspension was incubated on ice in the dark for 10 min. At the end of the 10-min incubation, the cell suspension was diluted to 5-fold with 1x annexin V binding buffer and analyzed by flow cytometry at an excitation emission wavelength of 488 nm and at detection emission wavelengths of 620 nm for chloromethyl-X-rosamine-positive cells and 525 nm for FITC-conjugated annexin V-positive cells (33).
Myc Activity Assays—Serum-starved MCF10A cells were infected with Ad vectors as described above for cell cycle analysis and superinfected 1 h later with AdM4 at 10 plaque-forming units/cell. Infection was continued for another 18 h, and cells were either stimulated with serum or maintained under starvation conditions as appropriate. At the indicated times, cells were harvested in lysis buffer, and the luciferase activity was measured using a luciferase assay kit (Promega catalog no. E1501) as described (27). A parallel infection was carried out to assay the distribution of the vector-infected cells in G1, S, and G2/M phases to ensure that the p300- or CBP-depleted cells exited G1 as expected.
Western Blot Analysis—To determine the levels of p300 and CBP, quiescent MCF10A cells were infected with antisense vectors, lysed, and immunoblotted using anti-p300 or anti-CBP antibodies as described (26). c-Myc, p53, p21, and the three Rb family proteins were analyzed by Western immunoblotting using appropriate antibodies (see figure legends for antibodies and other details).
Cdk Assays—To determine cyclin D kinase activity, antisense-expressing cells were lysed at the indicated times, immunoprecipitated with anti-cyclin D antibody, and assayed for kinase activity using a recombinant truncated Rb protein as a substrate (Upstate Biotechnology catalog no. 12-439) (34). Cyclin E kinase activity was assayed using histone H1 as a substrate (27).
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RESULTS |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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-galactosidase from the cytomegalovirus promoter (Ad-gal) was used as a control in these and all other experiments in this work. Fig. 1A shows that AS-CBP infection resulted in a significant reduction in CBP levels without affecting p300 levels. Consistent with our previous report, infection of cells with AS-p300 resulted in a considerable reduction in p300 levels, but not CBP levels. This result suggests that our antisense vectors are effective in reducing the synthesis of respective gene products with a high degree of specificity despite the fact that these two proteins are highly homologous (35–37).
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-gal, and serum starvation was continued (see Fig. 1B for the time course of infection and harvesting). After a total of 52 h of serum starvation, cells were harvested at 4-h time intervals up to 36 h post-infection. Fig. 1C shows that both CBP- and p300-depleted cells exited G1 at similar rates, although AS-CBP was slightly more effective in this regard. For example, at 36 h after infection, 
35% of the AS-CBP-infected cells were in S phase fractions, whereas at this time point, 28% of the AS-p300-infected cells were found in S phase. In contrast, the number of Ad-gal-infected cells in S phase at this time point was <5%, ruling out the effects of genes expressed by the vector backbone contributing to the induction of S phase. Both AS-CBP- and AS-p300-infected cells steadily accumulated in S phase, but did not traverse into G2/M, suggesting that these cells lack the ability to complete the cell cycle. This aspect is addressed below. Thus, our results show that both p300 and CBP provide an activity that allows the cells to remain in G0/G1 phase. Surprisingly, we did not observe a significant increase in the number of S phase-specific cells when cells expressed both types of antisense sequences simultaneously as opposed to cells expressing only antisense p300 or CBP sequences (data not shown). At present, we do not know the reasons for the lack of an additive or synergistic effect.
c-myc Is Up-regulated at Similar Levels in Antisense CBP- and p300-expressing Cells—Our previous study showed that premature DNA synthesis that occurs in cells expressing antisense p300-specific sequences is due to up-regulation of c-myc (26). To determine whether the S phase entry of cells infected with AS-CBP was also due to up-regulation of c-myc, we assayed the levels of c-Myc protein in serum-starved cells expressing antisense CBP (see Fig. 2A for the time course of infection and harvesting). As a comparison, the levels of Myc in cells expressing antisense p300 were also determined in parallel. Myc levels increased significantly in both antisense CBP and p300-expressing cells compared with control cells infected with Ad-gal (Fig. 2, B and C, respectively). To determine the endogenous Myc transcriptional activation activity, we used an Ad vector (AdM4) that expresses a promoter-luciferase reporter cassette containing four copies of the c-Myc protein-binding site (E-box element) (27). We showed previously that an increase in luciferase activity in AdM4-infected cells directly correlates with an increase in c-Myc protein levels (27). To assay the induction of c-myc in p300-depleted quiescent cells, serum-starved cells were first infected with AS-CBP, AS-p300, or Ad-gal and again infected 1 h later with AdM4. Seventeen hours following AdM4 infection and every 2 h thereafter, they were harvested, and the luciferase activities of the infected cells were determined. As shown in Fig. 2 (D and E), the reporter activity in the antisense-expressing cells began to rise at 19 h post-infection and continued to increase up to 25 h (later time points were not tested). The luciferase activity of Ad-gal-infected cells increased only at moderate levels, indicating that the 3–4-fold increase in luciferase activity observed in antisense-infected cells was due to an increase in Myc protein levels. Thus, we conclude that depletion of either p300 or CBP leads to up-regulation of c-myc. Furthermore, the above results indicate that both proteins are essential for the negative regulation of c-myc and the prevention of cells from exiting G1. Using antisense c-Myc, we have shown previously that the induction of DNA synthesis in p300-depleted cells is due to c-myc up-regulation (26). Using this strategy, we show below that S phase induction in CBP-depleted cells is also due to c-myc up-regulation. Consistent with the lack of additive effect of the two antisense viruses on DNA synthesis discussed above, we did not observe an increase in Myc activity when cells were infected simultaneously with two antisense vectors in our several independent experiments (data not shown).
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-gal lanes, and a new band migrating slower than the two phosphorylated forms was evident in serum-stimulated cells, which served as a positive control (Fig. 3D, phosphorylated form shown by the arrow). A faint slower migrating band was evident in AS-CBP-infected cells at 24 h after infection, suggesting a low level phosphorylation of p130 (Fig. 3D; the 28-h sample could not be completed in this experiment). Similarly, we observed a faint band migrating slower than the p107 band in AS-CBP-infected cells at the 24- and 28-h time points, indicating that p107 was also phosphorylated at low levels. Note that in serum-stimulated cells serving as a positive control, the band corresponding to phosphorylated p107 was merged with the p107 band (Fig. 3E). Because pRb was not hyperphosphorylated in antisense-expressing cells, we determined whether the cyclin D/Cdk activity was elevated in these cells. As shown in Fig. 3F, there was no change in the levels of cyclin D/Cdk activity at 20 and 24 h post-infection. In contrast, AS-CBP-infected cells showed elevated levels of cyclin E/Cdk activity beginning 20 h after virus infection (Fig. 3G). We believe that cyclin A/Cdk activity is also elevated in these cells, as we showed previously that both cyclin E and cyclin A kinase activities are induced in antisense p300-expressing cells (26). The lack of induction of cyclin D/Cdk activity in these cells could be attributed to the lack of growth factors in the medium because induction of cyclin D is dependent on growth factors (reviewed in Ref. 44).
Antisense c-Myc Reverses the Induction of S Phase and the Phosphorylation of p107 and p130 —To determine whether the induction of S phase and phosphorylation of the Rb family proteins in CBP-depleted cells was the result of induction of c-myc, we inhibited c-myc expression by expressing antisense c-Myc sequences as described previously (26), followed by monitoring the capacity of the CBP-depleted cells to exit G1. Serum-starved cells were co-infected with AS-CBP and an Ad vector that expresses antisense c-myc sequences (AS-Myc) (26). To maintain a constant multiplicity of infection, the Ad-gal vector was included in the protocol where appropriate. To confirm the effectiveness of AS-Myc in blocking the synthesis of c-Myc, c-myc transcriptional activity was monitored using the reporter assay described above. As shown in Fig. 4B (Fig. 4A shows the time course for infection and harvesting of cells), at 16 h post-infection, the luciferase activity in AS-CBP-infected cells was comparable with that detected in different control samples. In contrast, at 25 h post-infection, the AS-CBP-infected cells expressed 3-fold more luciferase activity compared with cells infected with Ad-gal or with Ad-gal and AS-Myc. Importantly, in cells co-infected with AS-Myc and AS-CBP, Myc activity was reduced to 50% of that observed in AS-CBP-infected cells, indicating that AS-Myc was effective in blocking c-myc expression.
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33% of the AS-CBP-infected cells accumulated in S phase, whereas <5% of the Ad-gal- and Ad-gal/AS-Myc-infected cells accumulated in S phase. Importantly, the number of S phase-specific cells co-infected with AS-CBP and AS-Myc was reduced to <10%, indicating that AS-Myc efficiently reversed the induction of S phase in CBP-depleted cells. Thus, we conclude that the induction of S phase in CBP-depleted cells is the result of induction of c-myc.
To determine whether AS-Myc would also reverse the phosphorylation of p107 and p130, cell extracts were prepared from cells infected with AS-CBP and with AS-CBP and AS-Myc for 28 h along with appropriate controls as shown in Fig. 4C. The phosphorylation status of p107 and p130 was then determined by Western immunoblotting as described in the legend to Fig. 3. Fig. 4D shows that p107 was not phosphorylated in cells infected with Ad-gal or with Ad-gal and AS-Myc. p107 was phosphorylated at moderate levels in AS-CBP-infected cells, which was reversed by AS-Myc (Fig. 4D, AS-CBP + AS-Myc lane). Similarly, low level phosphorylation of p130 in CBP-depleted cells observed at 28 h post-infection was reversed by AS-Myc. In summary, we conclude that the induction of S phase and the phosphorylation of p107 and p130 in CBP-depleted cells are the result of induction of c-myc.
Serum-starved p300- and CBP-depleted Cells Fail to Progress beyond S Phase after Serum Stimulation—Our FACS analysis of the serum-starved antisense-expressing cells suggested to us that these cells accumulated in S phase steadily, but did not traverse beyond S phase (data not shown). As growth factor-stimulated activation of cyclin D is essential for the G1-S transition of serum-starved cells, we considered it possible that serum stimulation of the cells accumulating in S phase would allow them to progress into G2/M and complete the cell cycle. Accordingly, cells were serum-starved for 36 h and infected with antisense vectors or Ad-gal, and starvation was continued for another 18 h as described above (Fig. 5A). Cells were then stimulated with serum, and the distribution of cells in G1, S, and G2/M phase fractions was determined by flow cytometry. Fig. 5B shows that control cells exited G1 steadily beginning at 14 h after serum stimulation, continued to accumulate in S phase up to 26 h, and then declined. Quantification of these cells in the G2/M phase fraction indicated that at 26 h after serum stimulation, the control cells began to accumulate in the G2/M phase fraction, and this accumulation continued up to 29 h (Fig. 5C). After 29 h, the number of cells in the G2/M phase fraction began to decline, with a corresponding increase in the G1 phase fraction (data not shown). This pattern of accumulation was comparable with that observed for mock-infected cells, indicating that the Ad-gal-infected cells cycled normally. However, the distribution of antisense-expressing cells during these time points was dramatically different. For example, significantly more antisense-expressing cells appeared in S phase at 14 h after serum stimulation compared with control cells and continued to accumulate, reaching 50% by 35 h (Fig. 5B). Surprisingly, unlike control cells, even after prolonged serum stimulation, neither antisense p300- nor CBP-expressing cells accumulated in the G2/M fraction (Fig. 5C), indicating that these cells were unable to traverse beyond S phase. These results suggested to us that a substantial number of the antisense-expressing cells did not survive beyond S phase and might be undergoing apoptosis.
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-gal, AS-p300, or AS-CBP at a multiplicity of infection of 200 and maintained under serum starvation conditions for a total of 52 h. Cells were then stimulated with growth medium containing 5% serum as described above for the cell cycle analysis. At the indicated times shown in the figure legends, cells were incubated with FITC-conjugated annexin V and propidium iodide as described under "Experimental Procedures" and analyzed by flow cytometry (Fig. 6, A-D). The signals that are registered in the upper right quadrangle (propidium iodide-positive and annexin V-positive; late stages of apoptosis), which are indicative of necrotic and apoptotic cells, and those in the lower right quadrangle (propidium iodide-negative and annexin V-positive; early stages of apoptosis), which are indicative of apoptotic cells, together constitute the percentage of apoptotic cells in a total population (Fig. 6D). These data indicate that the percentage of Ad-gal-infected cells undergoing apoptosis before serum stimulation (0-h time point) (Fig. 6, A and D) was 6–8%, which is comparable with the percentage of AS-p300- and AS-CBP-infected cells undergoing apoptosis before serum stimulation (Fig. 6, B-D; scatter diagrams for the 9- and 12-h samples are not shown). The percentage of cells undergoing apoptosis upon Ad-gal infection remained 10% at the 9-, 12-, and 16-h time points in the presence or absence of serum (Fig. 6, A and D). The percentage of AS-p300- and AS-CBP-infected cells undergoing apoptosis in the absence of serum stimulation also remained <10%. In contrast, at 16 h after serum stimulation, the percentage of apoptotic cells expressing antisense sequences increased up to 18–20% (Fig. 6, B–D).
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10% of the control cells underwent apoptosis during the time points tested, whereas the percentage of antisense-expressing cells undergoing apoptosis increased to 20% at 16 h after serum stimulation. These results are in agreement with the annexin V binding assay results. Thus, we conclude that at least a considerable number of serum-starved antisense-expressing cells undergo apoptosis within 12–16 h after serum stimulation, and this may contribute at least in part to their inability to travel beyond S phase. Both annexin V binding assays and trimethyl-X-rosamine staining assays were repeated twice, and the data were very reproducible. c-Myc Is Further Induced in Quiescent p300- or CBP-depleted Cells after Serum Stimulation—Studies have shown that high levels of c-Myc can cause apoptosis in several cell types (45, 46). Our previously published data (26) and those presented above clearly show that depletion of p300 or CBP in serum-starved cells results in the induction of c-myc. Thus, we considered it possible that c-myc that is already induced in the serum-starved antisense-expressing cells is further induced upon serum stimulation. The presence of high levels of c-Myc in cells deprived of growth factors and a further increase in its levels after serum stimulation would lead to non-physiological levels of Myc in the cell, which may contribute to the apoptotic effects. We show below that c-myc induced as a result of p300/CBP depletion in antisense-expressing cells was further induced by serum stimulation.
First, increases in c-Myc RNA levels in antisense-expressing cells were determined by Northern blot hybridizations. Cells were serum-starved for 36 h and infected with AS-CBP, AS-p300, or Ad-gal, and serum starvation was continued. After a total of 54 h of starvation (18 h after antisense virus infection), one set of plates was stimulated with serum, and the other set was maintained under starvation conditions (schematically shown in Fig. 7A). Two and 4 h after stimulation, serum-stimulated and unstimulated cells were harvested, and poly(A)+ RNA was isolated and subjected to Northern blot hybridizations using a human c-myc probe. Fig. 7B shows that Myc RNA levels began to rise by 18 h post-infection in AS-CBP- and AS-p300-infected cells in the absence of serum (0-h time point) (Fig. 7B). At this time point, Myc RNA was barely detectable in control cells. Myc RNA levels began to rise by 2 h after serum stimulation in cells infected with Ad-gal. Quantitation of RNA bands of the autoradiogram using a densitometer indicated that the RNA levels in unstimulated AS-p300- and AS-CBP-infected cells increased by 2–3- and 4–5-fold, respectively, compared with the serum-stimulated Ad-gal-infected cells. After serum stimulation, the antisense-expressing cells showed a further increase of 2–3-fold in Myc RNA levels compared with those without serum stimulation (Fig. 7B, compare the 0, 2, and 4 lanes in the Unstimulated panels with those in the Stimulated panels). These RNA levels were much higher than those observed in Ad-gal-infected cells at these time points and were also higher than those found in antisense-expressing cells without serum stimulation. Thus, we conclude that c-myc induced in serum-starved p300/CBP-depleted cells is further induced upon serum stimulation.
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To determine whether the induction pattern of c-myc correlates with its transcriptional activation activity, an experiment identical to that described above was performed, but including AdM4 infection following AS-CBP infection as shown in Fig. 2A. Luciferase activities in the stimulated and unstimulated cells at 0 and 4 h (time points in relation to serum stimulation) were assayed. As shown in Fig. 8B, at 4 h after serum stimulation, the luciferase activity in control samples (Ad-gal infection) increased by 2.5-fold compared with that in unstimulated control cells (compare bars 1 and 3 at the 4-h time point). At this time point, the luciferase activity in the unstimulated antisense-expressing cells had already increased by 2.5-fold compared with that in the unstimulated control cells (compare bars 1 and 2 at the 4-h time point). Upon serum stimulation, the Myc activity of the AS-CBP-infected cells further increased by another 2-fold (compare bars 2 and 4 at the 4-h time point). This would translate into an overall increase of 2.5-fold in Myc activity in the serum-stimulated antisense CBP-expressing cells compared with the unstimulated antisense-expressing cells and an 8-fold increase compared with the unstimulated control cells. This pattern of increase in Myc activity was also observed 10 h after serum stimulation (data not shown). This experiment was repeated using the AS-p300 vector. The results shown in Fig. 8C indicate that cells infected with AS-p300 also showed a similar pattern of increase in Myc activity, with the highest level of Myc activity in serum-stimulated AS-p300-infected cells. These results were reproducible in at least three independent experiments. In summary, these results show that the c-myc induction pattern was similar in both antisense AS-CBP- and AS-p300-expressing cells and that both cells contained higher levels of Myc after serum stimulation compared with serum-stimulated control cells undergoing normal G1-S transition.
Antisense c-Myc Reverses Apoptosis of CBP-depleted Cells—If the superinduction of c-myc observed in serum-stimulated p300/CBP-depleted cells contributes to apoptosis, then inhibition of c-myc expression should reverse this effect. To test this prediction, serum-starved cells were infected with AS-CBP and with AS-CBP and AS-Myc along with appropriate control infections as shown in Fig. 9B (Fig. 9A shows the time course of infection and harvesting). Cells were harvested at 8 and 16 h after serum stimulation, and the percentage of apoptotic cells in the total population was determined by annexin V binding assays as described under "Experimental Procedures." As shown in Fig. 9B, at 8 h after serum stimulation, 7–8% of the cells in the control samples (Ad-gal and Ad-gal + AS-Myc) showed apoptosis. In contrast, the percentage of apoptosing cells in the CBP-depleted population increased to 15%. At 16 h post-infection, 10% of the cells in control samples showed apoptosis, whereas the percentage of apoptotic cells in CBP-depleted population increased to 25%. Importantly, at both time points, the percentage of apoptotic cells coexpressing antisense CBP- and c-Myc-specific sequences decreased to control levels, indicating that inhibition of c-Myc expression prevented the apoptosis of the CBP-depleted cells. These results were reproducible in two independent experiments. Thus, we conclude that high levels of Myc are primarily responsible for the apoptosis of CBP-depleted cells.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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To gain insight into the mechanism of induction of S phase in antisense-expressing cells, we determined the levels of p21, a general inhibitor of cyclin/Cdk activities (50), and the phosphorylation of three pocket proteins, pRb, p130, and p107, which sequester E2F (51). We showed that p21 levels are reduced in antisense-expressing cells before a rise in c-Myc levels. The reduction of p21 levels might have contributed to the S phase induction, as cyclin-Cdk complexes are not under the influence of p21. Besides, p21 also has been shown to interact with proliferating cell nuclear antigen and to inhibit DNA replication (52). Therefore, decreased levels of p21 in antisense-expressing cells would provide a favorable environment for DNA synthesis. Down-regulation of the p21 genes most likely occurs at the transcriptional level, as we showed previously that p21 RNA levels are reduced in antisense p300-infected cells (26). Because p53 levels did not significantly change in serum-starved antisense-expressing cells (Fig. 10), coactivation of other transcription factors related to p21 gene activation might have been impaired (17). Furthermore, at later time points after antisense virus infection, high levels of Myc that accumulate in the cell also might contribute to repression of the p21 gene (53).
We have shown that in antisense-expressing cells, cyclin D/Cdk activity did not change, whereas cyclin E and A/Cdk activities were elevated to considerable levels. Furthermore, pRb was not phosphorylated, and p107 and p130 were phosphorylated in antisense-expressing cells at low levels. Because pRb is a major player in sequestering E2F, this suggests that only limited amounts of E2F might have been released from the repressor complexes, presumably by the phosphorylation of p107 and p130. Thus, E2F might play a limited role in the induction of S phase. For reasons that we cannot explain at present, even though there was a considerable induction of cyclin E and cyclin A kinases in antisense p300/CBP-expressing cells (Fig. 3G and data not shown) (26), there were negligible levels of pRb phosphorylation.
It is likely that induction of c-myc in p300/CBP-depleted cells is an initial event that leads to the phosphorylation of p107 and p130 and the induction of S phase. This is based on our observation that blocking the induction of c-myc in antisense CBP-expressing cells reversed S phase induction as well as phosphorylation of p107 and p130 (Fig. 4, C and D). These results are in agreement with our previous report in which we showed that inhibiting the induction of c-myc can reverse the induction of S phase in p300-depleted cells (26). Similarly, inhibition of G1 exit in p300-overexpressing cells as a result of repression of c-myc can be reversed by overexpression of c-myc (27). A number of recent reports suggest the existence of two pathways that promote mitogen-stimulated G1-S transition. One mechanism involves induction of c-myc, which then transcriptionally targets several cell cycle-related genes, including cyclin E, cdc25A, and the genes that are responsible for DNA synthesis (reviewed in Refs. 54–57). In the second well understood mechanism, mitogenic signals inactivate Rb and release E2F, which then activate cyclin E, cdc25A, and other E2F target genes involved in DNA synthesis. Both these pathways operate independently and mutually cooperate in inducing DNA synthesis and coordinated cell cycle progression. The contribution of both these pathways is essential for efficient S phase induction (58–61). Thus, we believe that in antisense-expressing cells, DNA synthesis is primarily due to the Myc pathway, and insufficient contribution by the Rb/E2F pathway likely results in inefficient S phase induction.
In this study, we have also shown that the antisense-expressing cells that accumulated in S phase in the absence of serum stimulation did not transit into G2/M phase. Serum stimulation of these cells also did not promote their transition from S to G2/M phase. In contrast, cell cycle progression was normal in cells infected with the Ad-gal control. For many reasons, the antisense-expressing cells may have failed to transit from S to G2/M phase. For example, both p300 and CBP provide coactivator functions for a number of transcription factors, many of which are directly involved in the activation of genes that control DNA synthesis. These include activator protein-1 (cyclin D1) (23), E2F (cyclins E and A) (62), and activating transcription factor (cyclin A) (63). These coactivators also acetylate nucleosomes of transcriptionally active promoters and stimulate transcription. The histone acetyltransferase activity of these proteins is also known to acetylate E2F and thereby increase its DNA binding and transcriptional activation activities (3). Other studies have suggested that the histone acetyltransferase activity of CBP increases during the G1-S transition (64). Therefore, lack of p300 or CBP, combined with lack of a contribution by the Rb pathway discussed above, would result in incomplete DNA synthesis, and such cells would not transit to G2/M phase.
We have shown that a significant number of serum-starved cells expressing antisense p300/CBP sequences underwent apoptosis upon serum stimulation. At 16 h after serum stimulation, the number of apoptotic cells increased by 2-fold for antisense p300- or CBP-expressing cells compared with Ad-gal-expressing cells (Fig. 6). We provided genetic evidence that apoptosis of the CBP-depleted cells could be reversed if expression of c-myc is blocked by antisense c-Myc sequences (Fig. 9). This shows that c-Myc plays an important role in the induction of apoptosis in CBP-depleted cells. A number of studies have shown that high levels of c-Myc can cause apoptosis (reviewed in Refs. 57, 65, and 66). In serum-starved antisense p300- and CBP-expressing cells, c-myc was induced at considerable levels. This induced c-myc was further induced upon serum stimulation (Fig. 7 and 8). For example, at 4 h after serum stimulation, the AS-p300- and AS-CBP-infected cells contained 3-fold higher levels of c-Myc activity compared with serum-stimulated control cells undergoing normal cell cycle progression (Fig. 8, B and C). It is conceivable that the inappropriate induction of c-myc and the unscheduled DNA synthesis that occur in serum-starved cells prime these cells for apoptosis. Further induction of c-myc by serum and perhaps other yet to be identified factors contribute to apoptosis. This interpretation is consistent with the view that induction of apoptosis by E1A, Myc, or E2F is the result of a conflict between the growth-promoting action of the oncoprotein and simultaneous growth inhibitory signals such as low serum (67, 68). We believe that p53 does not play a role in this apoptosis, as the levels of p53 in antisense-expressing cells dropped beginning at 4–6 h after serum stimulation (Fig. 10B). This is consistent with published studies suggesting that c-myc can induce apoptosis in certain cell types by both p53-dependent and p53-independent mechanisms (69–71). Other studies have shown that during myc-induced apoptosis, Myc targets the pro-apoptotic gene bax, which mediates apoptosis (72, 73). Western blot experiments did not detect a significant increase in the levels of Bax protein in antisense-expressing cells after serum stimulation (data not shown). Thus, at the present time, we do not know the Myc target genes in antisense-expressing cells that may be responsible for apoptosis.
In summary, normal levels of both p300 and CBP are essential to keep c-myc in a repressed state in G1 and to prevent inappropriate DNA synthesis. Similarly, normal levels of both these proteins are also needed in S phase. Previously knockout mice studies showed that embryos lacking p300 are significantly smaller than control embryos (5). In vivo bromodeoxyuridine labeling studies showed a striking reduction of the number of cells in S phase in the embryos of these mice (5). Fibroblasts derived from p300-/- mice also grow slowly and cease to divide after a few generations (5). These data indicate a defect in cell cycle progression in these mice and are in agreement with the data presented here.
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Both authors contributed equally to this work.
¶ Present address: Dana-Farber Cancer Inst., Boston, MA 02115.
|| Present address: Dept. of Pharmacology, Yale University School of Medicine, New Haven, CT 06520-8066.
Present address: Dept. of Pediatrics, Feinberg School of Medicine, Northwestern University, Chicago, IL 60611.
To whom correspondence should be addressed: Lurie Cancer Center, Olson 8452, Feinberg School of Medicine, Northwestern University, 303 E. Chicago Ave., Chicago, IL 60611. Tel.: 312-503-5224; Fax: 312-908-1392; Email: b-thimmapaya{at}northwestern.edu.
1 The abbreviations used are: CBP, cAMP-responsive element-binding protein-binding protein; Ad, adenovirus; Cdk, cyclin-dependent kinase; CREB, cAMP-responsive element-binding protein; FACS, fluorescence-activated cell sorting; FITC, fluorescein isothiocyanate.
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