Cell cycle-related transformation of the E2F4-p130 repressor complex

BBRC Biochemical and Biophysical Research Communications 336 (2005) 762–769 www.elsevier.com/locate/ybbrc

Cell cycle-related transformation of the E2F4-p130 repressor complex q Boris Popov a,b,*, Long-Sheng Chang b, Vladimir Serikov a,c a

Institute of Cytology, Russian Academy of Sciences, 4, Tikhoretsky Ave., St.Petersburg 194064, Russia Department of Pediatrics, ChildrenÕs Hospital, The Ohio State University, Columbus, OH 43205-2696, USA ChildrenÕs Hospital Oakland Research Institute, 5700 Martin Luther King Jr. Way, Oakland, CA 94609-1673, USA b

c

Received 18 August 2005 Available online 30 August 2005

Abstract During G0 phase the p130, member of the pRb tumor suppressor protein family, forms a repressor complex with E2F4 which is inactivated in G1/S by hyperphosphorylation of the p130. The role of p130 after G1/S remains poorly investigated. We found that in nuclear extracts of T98G cells, the p130-E2F4-DNA (pp-E2F4) complex does not dissociate at G1/S transition, but instead reverts to the p130E2F4-cyclin E/A-cdk2 (cyc/cdk-pp-E2F4) complex, which is detected in S and G2/M phases of the cell cycle. Hyperphosphorylation of the p130 at G1/S transition is associated with a decrease of its total amount; however, this protein is still detected during the rest of the cell cycle, and it is increasingly hyperphosphorylated in the cytosol, but continuously dephosphorylated in the nucleus. Both nuclear and cytosol cell fractions in T98G cells contain a hyperphosphorylated form of p130 in complex with E2F4 at S and G2/M cell cycle phases. In contrast to T98G cells, transformation of the p130 containing cyc/cdk-pp-E2F4 complex into the p130-pp-E2F4 repressor does not occur in HeLa cells under growth restriction conditions.
2005 Elsevier Inc. All rights reserved. Keywords: E2F4; p130; Transcription regulation; Cell cycle

Basic function of members of pocket protein family— pRb, p107, and p130—is to regulate cell cycle progression. Pocket proteins coordinate oscillations of cyclin-CDK activity with transcription of the cell cycle-related genes. The main mechanism of this regulation is interaction of pocket proteins with the E2F transcription factors [1]. Though the binding site of E2F family members for pocket proteins is highly conserved, there is a distinct selectivity in binding: E2F1-4 bind pRb, while E2F4-5 bind p107 and p130 [2,3]. It has been shown recently that p130 and p107 can also bind E2F1 and E2F3 under conditions of deficien-

q Abbreviations: pRb, retinoblastoma protein; CDK, cyclin-dependent kinase; EMSA, electrophoresis mobility gel shift assay; FBS, fetal bovine serum; PBS, phosphate-buffered saline; BSA, bovine serum albumin; IP, immunoprecipitation; pp-E2F, pocket protein-E2F-DNA complex; cyc/ cdk-pp-E2F, cyclinE/A-cdk2-pocket protein-E2F-DNA complex; SDS– PAGE, SDS–polyacrylamide gel electrophoresis. * Corresponding author. Fax: +7 812 247 0341. E-mail address: [email protected] (B. Popov).

0006-291X/$ - see front matter
2005 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2005.08.163

cy of the E2F4 [4,5]. E2F4-5 play the most important role in induction of growth arrest and cell differentiation, while E2F1-3 mediate regulation of G1/S transition and further cell cycle progression [6–8]. Under conditions of growth arrest, cells accumulate p130-E2F4 as the major repressor complex, which is inactivated in S and G2/M [9–13]. Mechanisms of active transcriptional repression by pocket proteins include deacetylation, methylation, and phosphorylation of histone tails and modification of nucleosome– DNA interaction [14–16]. During cell cycle progression, growth factors induce cyclin/cdk activities, which phosphorylate multiple sites on pocket proteins inhibiting their ability to associate with E2Fs. All pRb family members are phosphorylated in a cell cycle-dependent manner. Hyperphosphorylation of p130 at G1/S leads to ubiquitin-mediated degradation of one form of the hyperphosphorylated protein [18–20]. There is evidence that p130 exists in S phase [21–23], which suggests that p130 may play an important role in regulation of G1, S, and G2/M phases of cell cycle [24]. However, the

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exact role of p130 in these phases remains uninvestigated. Also, it is not known, whether in different cell types, oscillations of p130 level through the cell cycle follow similar patterns. Though p130 is not detectable after G1/S transition in total cell lysates of some non-transformed cells, it may be detected at S and G2/M phase in transformed T98G cells, sensitive to serum growth factors [18,20,25]. We hypothesized that some amount of distinctly phosphorylated p130 might exist at all stages of cell cycle, including S and G2/M phases, and regulate cell cycle progression through association with E2F4. We further hypothesized that this regulation by p130 might be diverse in different transformed cell lines, for example, in T98G and HeLa cells. Unlike T98G cell line, HeLa cells are unable to enter G0 phase upon serum removal due to functional inactivation of pocket proteins [16]. The goal of our investigation was to determine the presence and kinetics of p130 association with DNA through the cell cycle. Our first aim was to determine oscillations of the p130-E2F4 (pp-E2F4) repressor complex in nuclear extracts of the T98G cells by electrophoresis mobility gel shift assay (EMSA). Our second aim was to determine whether p130-pp-E2F4-associated activity in quiescent T98G cells was related to E2F4-cyclinA/E-cdk2 (cyc/cdk-pp-E2F4) complex after exit from quiescent state. Our third aim was to compare the p130-pp-E2F4 activity in T98G and HeLa. Finally, the fourth aim was to determine oscillations of hypo- and hyperphosphorylat-

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ed forms of the p130, associated with E2F4 in the nucleus, as compared to cytosol by Western blotting and immunoprecipitation. Results Cell cycle-associated oscillations of the pp-E2F activity To answer whether the p130 was functionally important during S and G2/M phases, we investigated cell cycle-related E2F binding activity in the nuclear extracts of synchronized, cycling T98G cells by EMSA. As DNA probe, we used the sequence which included tandem repeat of the E2F binding sites from the human p107 gene promoter [11]. These tests were combined with Western blotting and immunoprecipitation analysis to evaluate both the total amounts of the p130 and E2F4, and the amounts of associated forms of these proteins in the nuclear and cytosol compartments. FACS analysis demonstrated that 87% of the T98G cells, kept for 72 h under serum starvation conditions, were in the G0/G1 phase (Fig. 1A). The cell distribution between different cell cycle stages did not change substantially until 12 h after serum re-stimulation, when cells entered G1/S transition. At 12–24 h, cells were in S phase, while at 30 h, some cells re-entered the G1 phase (Fig. 1A). EMSA showed that in the growth-arrested T98G cells, one major pocket protein-E2F (pp-E2F) complex dominat-

Fig. 1. E2F binding activity oscillates in synchronized T98G cells. (A) Flow cytometry analysis of cell cycle progression of synchronized T98G cells. T98G human glioblastoma cells were growth arrested by 72 h cultivation in DME with 0.1% FBS, restimulated with 10% FBS, and used in synchronization experiments in 6 h intervals after restimulation. (B) Evaluation of the E2F-binding activity in nuclear extracts of synchronized T98G cells by EMSA. The EMSA was performed with the nuclear extracts of T98G cells and the oligonucleotide containing tandem repeat E2F site of the human p107 gene promoter at the same time points as the FACS without (
) or with (+) excess amounts (50-fold) of the unlabeled competitor—C or 0.8% deoxycholate— D; pp-E2F, pocket protein-E2F complexes; *, nonspecific complex. (C) Loading control of the protein samples from the same nuclear extract that were used for EMSA in (B) by immunoblotting. The nuclear extracts containing 20 lg of total protein were loaded on one lane of 8% polyacrylamide gel, resolved by SDS–PAGE, blotted on PVDF membrane, and visualized using rabbit antibody against SP-1 protein.

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ed with minimal amounts of ‘‘free’’ E2F (Fig. 1B). After 6 h following the release from serum starvation, two ppE2F complexes with lower and higher electrophoretic mobility (compared to the major pp-E2F complex in quiescent cells) started to form. The intensity of formation of pp-E2F complexes corresponded to an increase in ‘‘free’’ E2F activity and appearance of two faster migrating additional bands of ‘‘free’’ E2F at 12 h (Fig. 1B). Interestingly, the specific EMSA pattern of all E2F-bound complexes with high activity was finally formed at 12 h, while cells still remained in G1 (Figs. 1A and B). After 12 h, this pattern did not change, although intensity of all complexes (especially the pp-E2F complexes) eventually decreased (Fig. 1B). This pattern suggests that E2F-dependent cell cycle shifts might be predetermined by formation of distinct E2F-containing complexes in G0/G1. The p130-pp-E2F4 complexes in quiescent and cycling cells We further used DOC, which disassembled the ppE2F complexes (but not ‘‘free’’ E2F activity composed by E2F-DP dimers). This analysis demonstrated that at least two pp-E2F complexes and one ‘‘free’’ E2F complex existed in quiescent T98G cells. The second minor and faster migrating pp-E2F complex was clearly seen in quiescent cells after deoxycholate treatment (Fig. 2A). Probing by antibodies to different E2F and DP species revealed that the major pp-E2F complex included p130 and the minor included pRb. Both pocket proteins were complexed with E2F4 and DP1. Antibodies to these proteins abolished ‘‘free’’ E2F4 complex and

induced supershifts of the p130/pRb-containing pp-E2F complexes. ‘‘Free’’ E2F activity in quiescent cells included only E2F4, antibody to which completely abolished this complex (Fig. 2A). The slowest migrating band of pp-E2F activity consisted of two different complexes: the front part of this band was p107-E2F4-DP1-cdk2-(cyclinE/A) (p107-cyc/cdk-ppE2F4), while the rear part was p130-cyc/cdk-pp-E2F4 (Fig. 2B). The cyclin E and cyclin A, associated with the E2F4-pocket proteins, were not detected by conventional EMSA, but were readily detectable by the IPDOC assay (data not shown). p107, p130, and cdk2 were all present in the cyc/cdk-pp-E2F4 complexes at each time point 12 h after serum restimulation, up to the end of the first cell cycle (Fig. 3A). This indicates that some of p130 molecules, not destroyed during G1/S transition, may form complexes with E2F4 and cdk2 during the following period of the cell cycle progression. During exit from quiescence, the p130-pp-E2F4 repressor complex was replaced by two newly formed cyc/cdk-pp-E2F4 complexes with slower electrophoretic mobility, which contained p107 and p130. In cycling T98G cells, we did not observe the p130-E2F4 activity, not bound to cdk2. ‘‘Free’’ E2F4 increased after G1/S transition (Fig. 2B). T98G and HeLa cells In contrast to T98G cells, HeLa cells demonstrated very weak cell cycle response to serum deprivation. The amounts of HeLa cells traversing S phase at 0 and 12 h

Fig. 2. Antibody analysis of the E2F-binding activity by EMSA in quiescent and cycling T98G cells. (A) p130-E2F4-DP1 complex forms major E2Fbinding activity in quiescent T98G cells. Nuclear extracts of growth arrested T98G cells were used for the EMSA. The samples were treated with competitor—C, deoxycholate—D, or different antibodies as indicated. The protein–DNA bands included different proteins, designated with (+), the DNA–protein complex supershifts induced by specific antibodies designated with (*), and the complex abolishment designated with (
). (B) p130-DNA complexes in cycling T98G cells associate with cyclin-CDK2 activity. Nuclear extracts of the synchronized at 18 h point T98G cells were used for EMSA. The abbreviations are the same as in (A).

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Fig. 3. The p130-containing protein complexes bind E2F sites at G1/S, S, and G2/M phases. (A) Antibody analysis of the 130-E2F4 and p107-E2F4 activity bound to the p107 human gene promoter at G1/S, S, and G2/M cell cycle phases. EMSA was performed on nuclear extracts of synchronized T98G cells. The abbreviations are the same as in Fig. 2A. (B) Serum deprivation conditions do not induce formation of the p130-E2F4-DP1 complex in HeLa cells. The abbreviations are the same as in Fig. 2A. (C) Comparison of cell cycle response to serum deprivation in HeLa and T98G cell lines.

after serum restimulation (the latter corresponds to the S phase peak in this cell line) were not significantly different, in contrast to T98G cells (Fig. 3C). Insensitivity to serum deprivation correlated well with the absence of reformation of the cyc/cdk-pp-E2F complexes into the p130-E2F4 suppressor under growth arrest conditions (Fig. 3B). Cell cycle-dependent p130 oscillation in different cell compartments In quiescent T98G cells (0 and 6 h), the p130 consisted of several similar phosphorylated forms in nuclear and cytosol fractions. The amounts and variety of phosphorylated p130 forms increased at 6 h and even more at 12 h. Interestingly, newly hypophosphorylated p130 were seen in nuclear extract, while newly hyperphosphorylated forms appeared in cytosol fraction (Fig. 4A). This increase in diversity of the p130 phosphorylation coincided with the strong decline in the total protein level at 12 h. During the next stages of cell cycle progression (18– 24 h), pp130 levels continued to decrease in the nuclear extracts. In the cytosol, the levels of p130 phosphorylation increased, while the total protein levels did not change (Fig. 4A). At 30 h, the phosphorylation pattern of the p130 in both nucleus and cytosol was similar to that in asynchronously growing cells. E2F4 was abundant in both the nucleus and cytosol fractions in quiescent cells evaluated in the same extracts as the p130, also protein was highly phosphorylated. The E2F4 phosphorylation

pattern included several additional hyperphosphorylated bands at 0 and 6 h, which disappeared during the cell cycle progression (Fig. 4A). Amounts of E2F4 also decreased in parallel with disappearance of the hyperphosphorylated forms in both nuclear and cytosol compartments. In quiescent cells, anti-E2F4 antibody co-precipitated two distinctly phosphorylated p130 forms in both nuclear and cytosol fractions. In cycling cells, the nuclear fraction also contained two different forms of the p130 co-immunoprecipitated with E2F4, while only hyperphosphorylated p130 was co-immunoprecipitated in the parallel cytosol fractions (Fig. 4B). Amounts of the co-immunoprecipitated p130 were maximal at 6–12 h and decreased at 30 h, both in nucleus and cytosol. HeLa cells, in contrast to the T98G cells, did not show reversion of the cyc/cdk-pp-E2F complexes into the p130 containing pp-E2F4 repressor complex under serum deprivation (Fig. 3B). Pocket proteins-E2Fs-DPs-DNA associations in T98G cells are summarized in Fig. 5. Discussion Here we demonstrated that, although the total amount of the p130 gatekeeper decreased after G1/S transition, some distinctly phosphorylated forms of this protein bound to DNA existed during all stages of cell cycle. In the resting cells, p130 was associated with E2F4 and formed the pocket protein-E2F4 (pp-E2F4) repressor

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Fig. 4. Both hypo- and hyperphosphorylated p130 are able to associate with E2F4 in nuclear extracts of cycling T98G cells at different stages of cell cycle progression. (A) Western blotting analysis of the p130 and E2F4 oscillations in total extract, nuclear, and cytosol compartments of synchronized T98G cells at different time points after serum restimulation. The extracts containing 30 lg of total protein prepared from total cell lysates, or from separated nuclear and cytosol lysates, were resolved on 8% SDS–PAGE for the following Western blotting. P-pp designate differently phosphorylated protein forms. As, asynchronously growing cells. (B) Antibody to E2F4 co-immunoprecipitated both hyperphosphorylated- and hypophosphorylated p130 from the nuclear extracts of synchronously growing T98G cells. Two hundred and fifty micrograms of the T98G nuclear and cytosol extracts was treated with antibody to E2F4 and the precipitated proteins were visualized after SDS–PAGE and Western blotting by antibody against p130. P-pp designates differently phosphorylated p130 molecules.

Fig. 5. Nuclear E2F-associated complexes bound to the E2F sites of the human p107 gene promoter in cycling T98G cells. Scheme represents appearance of different complexes during cell cycle according to own experimental data.

complex. In the nuclear extracts of T98G cells, this complex did not dissociate after exit from quiescent state, but reverted to the p130 containing cyc/cdk-pp-E2F4 complex, which was detected at G1, S, and G2/M phases. The results of immunoblotting and immunoprecipitation experiments were consistent with EMSA. These results suggested that in nuclear compartment of cycling T98G cells, the p130

existed in hypo- and hyperphosphorylated forms, both of which might physically associate with E2F4 and be recruited to the cyc/cdk-pp-E2F4 complex persisting through G1, S, and G2/M phases. This notion is in agreement with observations that p130 was not detected by immunoblotting after G1/S transition in some cell lines. In rat embryonic fibroblasts (REF52)

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and immortalized human fibroblasts (hTERT) sensitive to the growth arrest under serum deprivation, the p130 was hyperphosphorylated and ubiquitylated at G1/S transition which resulted in its disappearance during S and G2/M phases [19,26,27]. In transformed cells, still sensitive to the growth arrest under serum deprivation like T98G cells, the p130 did not disappear after G1/S transition [25]. Levels of p130 may vary when evaluated by the methods which utilize total cell extracts for analyses, compared to those utilizing extracts from the separated nuclear and cytosol cell compartments. In chromatin immunoprecipitation assay of nuclear compartment, p130 was found as recruited to promoters of many cell cycle-related genes in cycling fibroblast cell lines [24]. Immunoblotting did not reveal p130 in total extracts of primary diploid cells or even in some transformed cycling cells [18–20]. Some studies suggested that p130 was present in nuclei of the S- and G2/M-phased T98G cells, but the phosphorylation status was not reported [28]. EMSA results suggest that the p130 containing pp-E2F4 suppressor complex in quiescent cells was modified during G0-G1 transition by binding to cdk2-cyclinA/E and formation of the cyc/cdk-pp-E2F4 complex. In contrast to primary T-lymphocytes, which showed disappearance of the p130 pp-E2F4 after G1/S transition [8], the T98G cells demonstrated reformation of this activity into the cyc/ cdk-pp-E2F4 complex (Fig. 3A). Presumably, the cdk2 bound to this complex may phosphorylate p130. Phosphorylation of the p130 may also result in dissociation of the complex and appearance of both the cycE/cdk2 and ‘‘free’’ E2F in S and G2/M phases (Fig. 1B). High level of the ‘‘free’’ E2F4 (which has strong affinity to p107) promotes formation of a new cyc/cdk-pp-E2F4 complex with p107. Level of p107 is elevated during G1/S transition due to transcription activation of this gene [11]. Entire p107 and p130 activities associated with DNA in cycling cells were bound to cyc/cdk2. In contrast to abundant pp-E2F complexes containing pRb, the cycling cells did not reveal any p130/p107 pp-E2F4 activity, not bound to cdk2 (Figs. 2B and 3A). Absence of such complexes may indicate either full inactivation of the p107/p130 suppressor activity or full saturation of their binding capacity by the cycE/cdk2. Presumably, p107/p130 and cyc/cdk in the context of the cyc/ cdk-pp-E2F4 complexes may regulate activity of each other. This is consistent with previously published findings that the p130/p107-E2F4-DP1-cdk2 complexes in cycling ML1 cells exhibit low level of associated kinase activity, which may be increased by dissociation of the pocket proteins [29]. In our experiments, all E2F activity in quiescent and cycling T98G cells was associated with DP1, while DP2 was never detected as partner of any E2Fs (Figs. 2A, B and 3A). Our experiments using nuclear cell extracts for EMSA showed that in growth arrested T98G cells, the p130 and pRb form accordingly major and minor suppressor complexes including E2F4 (pp-E2F4). The quiescent cells have minimal amounts of ‘‘free’’ E2F4 and no other ‘‘free’’ E2F

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activity (Fig. 2A). At G1/S transition the pRb composing the pp-E2F4 complex changed its partner from E2F4 to E2F1 and E2F3. Apparently, the reformation of the pRb pp-E2F4 complex was associated with G1/S expression of E2F1 and E2F3, which had higher affinity to pRb. PRbE2F1 and pRb-E2F3 complexes in cycling cells are important for transcription regulation of cyclin E. Promoter of this gene is regulated partly by a bipartite E2F-Sp1 site, occupied on one part by the E2F-1,3 bound to pRb [14,30]. Pocket proteins-E2F network is involved in control of DNA replication, reparation, and mitosis [16,17,31,32]. Our results suggest that ability of dividing cell to regulate reversion of the p130 containing pp-E2F4 suppressor into the cyc/cdk-pp-E2F4 complex and back allows cell to control G0 exit and entry. However, the function of p130 is not limited to the G0/G1 phases, it is employed by cycling cells as a ‘‘full time operator’’ acting also during S and G2/M phases. The p130 functions may be performed in association with cycE/A-cdk2. The cycE/cdk2 kinase activity plays direct role in activation of transcription [33] and is central in initiation of DNA replication, blocking of rereplication and resetting of replication origins after mitosis [34]. Although p130 and p107 may decrease the associated cyc/cdk activity in the cyc/cdk-pp-E2F complexes, we speculate that these proteins may also function as tools for delivery of the cycE/cdk2 activity to DNA. Only nuclear, but not cytosol, fraction of cycling T98G cells contained differently phosphorylated forms of the p130 co-immunoprecipitated with E2F4. Phosphorylation of p130 at G1 results in appearance of its hyperphosphorylated form which does not associate with E2F4 [5,25–27]. We suggest that regulation of the p130 might be distinct in nucleus and cytosol, as only nuclear p130 fraction is utilized by transcription machinery. Oscillations of the p130 in the nucleus and cytosol may be different. Phosphorylation of the p130 may result in dissociation of this complex and liberation of the associated cycE/cdk2, required for initiation of DNA replication. Concomitant with initiation of replication, the level of cycE in nucleus increases about 200-fold [35]. The cycE/cdk2 liberated due to dissociation of the cyc/ cdk-pp-E2F4 complexes might be accumulated in a nuclear compartment. P130 included into the cyc/cdk-pp-E2F4 may also contribute to reorganization of chromatin in cycling cells. P130 co-localized with replication proteins PCNA and CAF-1 in small number of primary replication foci at the beginning of S phase, suggesting that pocket proteins might link special organization of chromatin with DNA replication [36,37]. To summarize, our results demonstrated that in the nuclear extracts of quiescent T98G cells, the p130 containing pp-E2F4 suppressor complex did not dissociate after exit from the G0 state but reverted to cyc/cdk-pp-E2F4 complex persisting during G1, S, and G2/M phases. The gatekeeper function of the p130 may be reduced in transformed cell lines like HeLa cells which did not show reversion of the p130 containing cyc/cdk-pp-E2F4 into pp-E2F4

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tumor suppressor under serum deprivation. PRb forming minor pp-E2F4 suppressor in quiescent cells shifted from E2F4 to E2F1 and E2F3 in cycling cells. Both nuclear and cytosol cell fractions in T98G cells contain a hyperphosphorylated form of the p130, co-immunoprecipitated with the E2F4. After G1/S transition, the p130 is increasingly hyperphosphorylated in cytosol but continuously dephosphorylated in nucleus of T98G cells. Materials and methods Cell culture, synchronization, and flow cytometry analysis. The T98G human glioblastoma cells and HeLa human epithelial cells were obtained from ATCC (American Type Culture Collection) and cultured in DulbeccoÕs modified EagleÕs medium (DMEM) with 10% of fetal bovine serum (FBS). For growth arrest, subconfluent cells were grown for 72 h in DMEM with 0.1% FBS, restimulated with 10% FBS, and used in synchronization experiments in 6-h intervals after restimulation. For flow cytometry analysis, the synchronized cells were washed twice with phosphate-buffered saline (PBS) and trypsinized. Then, the cells from one 100 mm plate were washed twice in PBS, containing 1% of bovine serum albumin (BSA), resuspended in 0.3 ml of the PBS and fixed in 70% ethanol by adding dropwise its 5 ml aliquot to the cells while vortexing, followed by incubating the cells at
20 C. For nuclear staining, cells were collected by centrifugation, resuspended in appropriate volume of PBS, containing 100 lg/ml RNAse A, and 40 lg/ml of propidium iodide, to make the 5 · 105 cell/ml concentration, and then incubated at 37 C for 30 min. Flow cytometry analysis was performed using Beckton–Dickinson FACScan. The DNA content and cell cycle profiles were determined using CellFIT Cell Cycle analysis software. Subcellular fractionation. Preparation of nuclear and cytosol fractions was performed as described earlier [38], with small modifications. The cells were washed twice with ice-cold PBS, scraped by rubber policeman, and collected by spinning for 5 min at 1500 rpm. Cell pellets were swelled for 20 min in two packed volumes of hypotonic buffer (10 mM Hepes (pH 7.5), 10 mM KCl, 3 mM MgCl2, 1 mM EDTA (pH 8.0), 10 mM b-glycerolphosphate, 10 mM NaF, 0.1 mM Na3VO4, 1 mM PMSF, 1 mM DTT, and 1 mg/ml aprotinin and leupeptin. Then the cells were subjected to 10 slow strokes with a Dounce homogenizer. Nuclei were separated by a 500g spin for 5 min and washed twice in the hypotonic buffer containing 0.1% of NP-40. Nuclear and cytosol fractions were then treated with the lysis buffers for Western blotting or immunoprecipitation, followed by the protein assay analysis. SDS–PAGE, immunoblotting, and immunoprecipitation. SDS–gel electrophoresis (SDS–PAGE) was performed in 8% polyacrylamide gel. Cells were removed from 100 mm Petri dishes with a rubber policeman, washed twice in PBS, and lysed in Western blotting buffer (25 mM Tris (pH 7.4), 250 mM NaCl, 0.25% NP-40, 10 mM b-glycerolphosphate, 10 mM NaF, 0.1 mM sodium vanadate, 1 mM PMSF, 1 mg/ml aprotinin and leupeptin, and 1 mM DTT), placed on ice bath, and shaken vigorously every 5 min for a total of 30 min. The cell debris was spun down at 4 C for 15 min, total protein concentration was determined for each sample, and lysates were normalized to equivalent total protein levels, as determined by the Bradford assay. After the SDS–PAGE, the gel was blotted to a sheet of Hybond-C nitrocellulose paper (Amersham). After 2 h of blotting, proteins were visualized using standard techniques. Immunoprecipitation. Cells were washed twice and lysed for 30 min on ice. One microgram of antibody was added and mixture was rocked for 2 h at 4 C. Then 15 ll of the protein A/G–Sepharose was added to the lysates with primary antibodies for 1 h. The pellets were spun down, washed five times in the IP buffer and two times in the same buffer without NP-40. Immunoprecipitation-DOC. Two hundred and fifty micrograms of the cell extracts was incubated on a rocking platform for 60 min at 4 C with 200 ll of hybridoma supernatant coupled with protein A–Sepharose in 1· IP-DOC buffer—20 mM Hepes (pH 7.5), 40 mM KCl, 1 mM MgCl2,

0.1 mM EDTA, containing 3 mg of BSA per milliliter, and all protein and phosphatase inhibitors as described above; washed three times in 1· IPDOC buffer. The associated proteins were released by the addition of 10 ll of 0.8% sodium DOC in 1· IP-DOC buffer and NP-40 at concentration of 1.5%, and the supernatants were assayed in EMSA. Electrophoresis mobility gel shift assay. For electrophoresis mobility gel shift assay (EMSA) T98G cells and HeLa cells were grown on 100 mm plastic dishes and synchronized as described above. To prepare nuclear extracts cells were lysed in buffer containing 10 mM Hepes (pH 7.9), 0.1 mM EGTA (pH 8.0), 0.1 mM EDTA (pH 8.0), 10 mM KCl, and 0.5 mM PMSF. One millimolar NP-40 was added to the final concentration of 0.6%. The lysates were shaken on ice for 5 min and centrifuged at 14000 rpm for 1 min. The pellets were resuspended on ice in the following buffer—20 mM Hepes (pH 7.9), 1 mM EDTA (pH 8.0), 1 mM EGTA (pH 8.0), 0.4 M NaCl, 1 mM DTT, 1 mM PMSF, and 1 mg/ml aprotinin, leupeptin, and pepstatin for 30 min and spun down at 14,000 rpm for 20 min at +4 C. The protein concentration in the nuclear extracts was determined by the Bradford reagent and extracts containing 20 lg of total protein were incubated in the volume of 10 ll in the loading buffer—1 mg/ml of salmon sperm DNA in 10 mM Hepes (pH 7.9), 15% glycerol, 1 mM EDTA, 8 mM MgCl, and 1 mM DTT, for 15 min on ice. The oligonucleotides with 5 0 C residues overhanged for both E2F sites of the human p107 gene promoter were synthesized, annealed and labeled using Klenow fragment of DNA polymerase and [a-32P]dGTP. The p107 DNA probes with the sequence 5 0 -CAGATTTTCGCGCGCTTTGGCG CAGGT-3 0 and 5 0 -CCACCTGCGCCAAAGCGCGCGAAAATCT-3 0 contained both E2F tandem repeat sites shown in bold (nucleotide position
20+7). Competition and deoxycholate (DOC) treatment were carried out by addition of 100 gg of unlabeled double stranded wild type oligonucleotides or 0.8% DOC prior to addition of cell extract. When indicated, a specific antibody was added for 30 min at room temperature after addition of labeled oligonucleotides. E2F-binding complexes were resolved by electrophoresis in 6% (29:1) acrylamide–bisacrylamide. We used the following antibodies—Rb(IF8)x-sc-102; p107(SD-9)-sc250; p130(C-20)sc-317; E2F1(KH-95)-sc-251x; E2F2(C-20)x-sc-633x; E2F3 (N-20)x-sc-879x; E2F4(C-20)x-sc-866x; E2F5(E19)x-sc-999x; DP1(K-20)x-sc610x; DP2(G12)-sc-849; cyclinE(HE111)x-sc-248; cyclinA (C19)-sc-596G; cdk2(M2)-sc-163; Sp1(PEP2)-sc-59 all purchased from Santa Cruz Biotechnology, and monoclonal anti b-actin, clone AC-15— purchased from Sigma (St. Louis, MO).

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