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DNAX Research Inc., Palo Alto, California 94304-1104
Received 28 July 2003/ Returned for modification 2 September 2003/ Accepted 15 September 2003
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ABSTRACT |
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INTRODUCTION |
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In mammalian cells BRG1 is known to play a role in the regulation of cellular proliferation (8, 49). This activity of BRG1 has been shown to be dependent on its association with the retinoblastoma tumor suppressor gene product (pRB) and, consequently, via the repression of E2F target gene expression (29). Specifically, it was demonstrated that BRG1/pRB complexes repress certain E2F target genes such as cyclin E, cyclin A, and CDC2 (48, 61). The temporally ordered association of BRG1 with histone deacetylase and pRB complexes ensures sequential activation of cyclin E and then cyclin A and maintains the order of G1 and S phases of the cell cycle (61).
Studies have now revealed that BRG1 is frequently deleted or mutated in a variety of tumor cell lines (59), implicating BRG1 as a potential tumor suppressor gene in its own right. The tumor suppressor function of BRG1 is supported by the generation of mice harboring a BRG1 null mutation. Heterozygotes for BRG1 are susceptible to neoplasia and display large subcutaneous tumors, suggesting that a partial reduction in cellular BRG1 level leads to critical loss of proliferation control (5). More recent work has shown loss of expression of BRG1/BRM expression in a subset of primary lung tumors associated with poor prognosis (39). Several studies have also revealed that the INI1/SNF5 conserved component of the SWI/SNF complex is also mutated in human tumors, providing further evidence of a role for the SWI/SNF complex in growth regulation and tumor suppression (3, 21, 57).
It has been demonstrated that BRG1 regulates the expression of other non-E2F-regulated genes that are important in cancer. Murphy et al. demonstrated that BRG1 repressed transcription of the oncogene c-FOS in an E2F-independent manner (32), and recent work from Strobeck et al. suggests that BRG1 regulates CD44 expression (47). Misregulation of CD44, encoding a transmembrane glycoprotein involved in cell-cell interactions and cell-matrix adhesion, has been implicated in tumor growth and metastasis. The recent identification of distinct classes of transcription factors that associate with BRG1 and BRM may provide clues as to which of and how these factors are recruited to specific promoters (19). Although the total number of genes regulated by mammalian Swi/Snf is unknown, the yeast Swi/Snf complex is estimated to control expression of 5 to 6% of genes (18, 50).
The majority of studies of BRG1 function in mammalian cells have relied on two cell lines, C33A and SW13, that lack BRG1 expression for unknown reasons. Our recent work has identified a collection of cell lines with defined mutations in the BRG1 gene, providing a broader spectrum of cell lines to dissect out BRG1's role in gene regulation (59). Using the breast tumor cell line ALAB, which contains a mutation (C
T) at nucleotide 1630 in exon 10 of BRG, resulting in a STOP codon and consequently no detectable protein expression, we have studied global gene induction and repression using microarray and real-time PCR analysis by reintroduction of BRG1. By comparison with earlier studies, we show here that BRG1 induces a number of novel genes in this cell type that are involved in cell adhesion, motility, and proliferation. Gene expression profiles in different cellular contexts will help provide a more comprehensive understanding of the mechanism of action of BRG1 in the control of tumorigenesis and metastasis.
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MATERIALS AND METHODS |
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Cell culture and adenovirus infection. ALAB, SW13, and SAOS2 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% serum, nonessential amino acids, and penicillin-streptomycin. 293A cells (QBIOGENE) were cultured in DMEM supplemented with 5% serum, nonessential amino acids, and penicillin-streptomycin. In experiments to optimize infection conditions, BRG1 was visualized by immunofluorescence with a monoclonal antibody (MAb) to BRG1 (5D11) (59). For experiments presented here, cells were infected with 1,000 particles per cell (multiplicity of infection = 10) using cationic lipids for enhanced infection (10). The cells were incubated with adenovirus at 37°C for 3 h. Following the incubation, the virus cocktail was replaced with fresh medium and cells were harvested at the indicated hour postinfection (p.i.). Time zero corresponds to virus addition to cells.
Proliferation assay. Cells were plated in Cytostar-T scintillating microtiter plates (Amersham), incubated for 24 h at 37°C, and then incubated with the indicated viruses for 3 h at 37°C. The virus cocktails were removed at 3 h p.i., and medium was replaced on all samples with medium containing [14C]thymidine. Counts per minute were measured using a Packard TopCount plate reader at 0, 24, 48, and 72 h after addition of medium containing [14C]thymidine. Staurosporine (Calbiochem) was used at 500 nM.
Cell cycle analysis. Cells were plated in 10-cm dishes, incubated for 24 h at 37°C, and then infected with the adenoviruses as described above. AdVector was used as control virus because of AdGFP interference with the fluorescence of the DNA-binding dye, 7-amino-actinomycin D (7-AAD; BD Biosciences). At the indicated times, cells were pulse-labeled with 10 µM bromodeoxyuridine (BrdU) for 30 min, harvested, fixed in 70% ethanol, and stained with anti-BrdU fluorescein isothiocyanate (Becton Dickinson) for flow cytometric analysis following the manufacturer's recommendations.
mRNA analysis. Total RNA was extracted using an RNeasy mini kit (Qiagen) with an on-column DNase treatment (Qiagen). Five micrograms of RNA in a 100-µl reaction volume was used for first-strand cDNA synthesis. The reaction mixture included 2.5 µg of oligo(dT)12-18 (Gibco-BRL), 250 ng of random hexamers (Promega), 10 mM dithiothreitol (Gibco-BRL), 1x First-Strand buffer (Gibco-BRL), 500 µM deoxynucleoside triphosphate mix (Pharmacia), 80 U of RNasin (Roche), and 500 U of Superscript II reverse transcriptase (Gibco-BRL) and was incubated for 50 min at 42°C. The cDNA was diluted and used as template in a quantitative real-time PCR (Taqman) using a GeneAmp 5700 sequence detection system (Perkin-Elmer). Reactions were done in a final volume of 20 µl in SYBR Green PCR mix (Perkin-Elmer) and included 10 ng of cDNA and primers, each at a concentration of 400 nM. Primer sequences are listed in the Appendix. The mRNA expression level for all samples was normalized to the housekeeping gene S9. Data from triplicate samples of a representative experiment are shown.
Immunoblot analysis and immunoprecipitations. Cell lysates were prepared as described previously (45), subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and blotted to Immobilon (Millipore) or nitrocellulose (Schleicher & Schuell). Protein concentration in each clarified lysate was determined by the dye-binding method using bovine serum albumin as the standard. For CDK2 immunoprecipitations, 1 mg of protein was diluted to a final volume of 400 µl in lysis buffer and mixed with 2 µl of normal rabbit serum and protein A-agarose beads to preclear the lysate. After incubation at 4°C on a rotating shaker for 45 min, the precleared supernatant was collected and mixed with either 2 µg of protein A-purified anti-CDK2 antibody or 2 µg of protein A-purified normal rabbit antibody and incubated as before for 45 min. Protein A-agarose beads were added to the mixture, and incubation was continued for another 45 min. Beads were collected by brief centrifugation, washed three times with 1 ml of lysis buffer, and recollected. Bead-bound immune complexes were solubilized in SDS-PAGE sample buffer, immediately boiled for 5 min, resolved by SDS-PAGE, and analyzed by immunoblotting. Immunoblotting (50 to 100 µg of protein for each sample) was performed as described previously (59). Antibodies to BRG1, cyclin E (HE12), CDK2, and p15 have been described elsewhere (35, 45, 59). Other antibodies were purchased from the following sources: anti-RB polyclonal antibody (C-15; Santa Cruz); anti-p21 Ab-11 MAb (clone CP74; Neomarkers); anti-Waf1 Ab-1 MAb (clone EA10; Oncogene Research Products); anti-p21 MAb (clone 70; Transduction Labs); anti-pSTAIR (P-7962) MAb (Sigma); anti-p53 MAb (Ab-6 clone DO-1; Oncogene Research Products); anti-rabbit HRP (Amersham); anti-mouse HRP (Amersham).
Immobilized template assay. Template assays were performed by the method of Holloway et al. (17). Fragments of the human p21 promoter (-2328 to -2224 and -306 to -5) or the p21 open reading frame (+314 to + 631) were amplified by PCR with either pWWP-Luc (9) or CMV-p21 as template and biotinylated primers (-2328, -5, +314). PCR products were resolved by gel electrophoresis and purified with a Qiagen QIAquick gel extraction kit. For each reaction, 150 ng of template was bound to Dynabeads M280 streptavidin (Dynal) and blocked as described previously (38). Nuclear extracts were prepared (100 µg per reaction mixture) and supplemented with sheared salmon sperm DNA and poly(dI-dC) and allowed to bind the templates as described elsewhere (17). Precipitated proteins were eluted from the beads with SDS-PAGE sample buffer and subjected to SDS-PAGE and immunoblotting with anti-BRG1 or anti-p53 antibodies.
cDNA microarray analysis. ALAB cells were seeded in 500-cm2 plates and incubated for 24 h at 37°C. Cells were infected with either AdBRG1 or AdGFP by the cationic lipid method at a multiplicity of infection of 10 (see above). For one experiment cells were harvested at 18 h p.i., and for the other experiment cells were harvested at 24 h p.i. Cells were washed in DMEM without additives, and cell pellets were frozen at -80°C. RNA preparations, probe synthesis, and microarray hybridizations were performed as described previously (62). The probe pairs were used for hybridization to six microarrays that contained more than 40,000 human cDNA elements (Incyte Genomics).
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RESULTS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionAppendixReferences |
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T) at nucleotide 1630 in exon 10 of BRG1, resulting in a STOP codon and consequently no detectable protein expression (59), was selected for our studies. SW13 cells were used as a means of correlating our data with those published by other investigators. Using the BRG1 adenovirus, we were able to achieve BRG1 expression in all cells of an infected population as determined by anti-BRG1 immunofluorescence (data not shown). In a time-course immunoblot analysis of BRG1 expression, BRG1 could be detected as early as 6 h p.i., and expression increased dramatically at 11 and 24 h p.i. and was maintained for at least 72 h p.i. (Fig. 1A).
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Differential gene induction profile in ALAB cells. To validate our system for the study of gene regulation by BRG1, we tested the expression of CD44, mCSF-1, and osteonectin, three genes which have previously been shown to be regulated by BRG1 in SW13 cells (25, 47). ALAB and SW13 cells were infected with either AdGFP or AdBRG1, and gene expression was examined by real-time PCR. As shown in Fig. 2A, we observed a 42-fold induction over AdGFP of CD44 by AdBRG1 in SW13 cells and a similar albeit smaller induction (8- to 9-fold) in ALAB cells at 18 h p.i. Osteonectin was robustly induced in both SW13 cells and ALAB cells, exceeding 600-fold induction at the 24-h time point in both cell types (Fig. 2B). In contrast, the regulation of mCSF-1 expression was not conserved between cell lines. In SW13 cells, mCSF-1 was induced sixfold over AdGFP at 18 h p.i., in agreement with the published microarray data (25); however, no induction was seen in ALAB cells (Fig. 2C). BRG1 was expressed at similar levels in both cell lines (data not shown).
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R) as shown in Fig. 3B. Immunoblot analysis with an antibody specific for hBRM (40) verified the induction of hBRM in ALAB cells at the protein level (data not shown). Confirming published data, hBRM mRNA levels were unchanged by BRG1 reintroduction into SW13 cells, demonstrating a cell type selectivity to this response.
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Consistent with an antiproliferative phenotype, we also detected up-regulation in expression of p21. p21Cip1,Waf1 was induced in both hybridizations: 2.9-fold in the 18-h sample and 5.1-fold in the 24-h sample. p21 is one of several cyclin-dependent kinase inhibitors (CKIs), which are key negative regulators of the cell cycle by inhibition of cyclin-dependent kinases (CDKs) (14, 60). We were able to confirm the effect of AdBRG1 on expression of p21 in ALAB cells by real-time PCR, with p21 mRNA up-regulated 3-fold by 6 h p.i. and nearly 50-fold at 24 h p.i. (Fig. 4A). We also examined the expression of the related CKI p27 (37) but saw no change in mRNA levels by BRG1 (Fig. 4A). Interestingly, the ATPase mutant BRG(KR) was able to induce p21, but to a lower level than the wild type (Fig. 4C). This suggests that BRG1 may have a function, in addition to chromatin remodeling, in the activation of p21. To complete our analysis of cell cycle inhibitors, we also examined the effect of AdBRG1 on the expression of the INK family of CKIs (13) (Fig. 4B). AdBRG1 induced a fivefold up-regulation of p15INK4b mRNA as early as 11 h p.i. Induction of p15 increased to 20-fold at 20 h p.i. In contrast, mRNA expression of the three other INK family members, p16, p18, and p19, remained unchanged. Thus, BRG1 up-regulates mRNA levels of both p15 and p21, with kinetics that precede the eventual cell cycle arrest induced by BRG1 reintroduction.
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Mechanism of p21 induction by BRG1.
p21 is a known p53-responsive gene (9). Three other p53-regulated genes, GADD45, S100A2, and DRAL, were also induced by BRG1 (Table 1) (20, 43, 53). GADD45 induction (40-fold at 24 h p.i.) was confirmed by real-time PCR (data not shown), supporting a potential p53 link. As an alternative connection, both p21 and p15INK4b have also been identified as transforming growth factor ß (TGF-ß)-induced genes (13, 42). It was therefore of note that several other genes induced by BRG1 in the microarray experiments have been reported to be TGF-ß responsive (Table 1): insulin-like growth factor binding protein-4 (2.5- to 3.4-fold), integrin alpha-5 (2.6- to 4.8-fold), integrin alpha-3 (2.1- to 3.2-fold), matrix metalloproteinase-1 (2.3- to 3.4-fold), c-JUN (2.2- to 2.6-fold), and tropomyosin 
(3.3- to 4.5-fold) (2, 4, 6, 7, 12, 27, 56).
To further investigate the mechanism of p21 induction by BRG1, we first examined the status of p53 in the ALAB cell line. The p53 protein was undetectable by immunoblot analysis, even after treatment of cells with ionizing radiation or camptothecin, agents known to induce stabilization of p53, suggesting that these cells are functionally null for p53 (Fig. 5A). As further confirmation of the absence of functional p53, p21 was not induced either (Fig. 5A). Normal fibroblasts containing functional p53 (WS1) and a cell line containing mutant p53 (DU145) were used as controls. Reintroduction of p53 was able to induce expression of p21 in ALAB cells (Fig. 5B), suggesting that the p53 element in the p21 promoter in ALAB cells is functional. We also tested whether expression of p53 itself was regulated by BRG1, and we saw no induction of p53 mRNA (Fig. 5C). Taken together, the results in Fig. 5 demonstrate that it is unlikely that BRG1 exerts its effect on p21 via p53 in this system.
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pRB hyperphosphorylation is inhibited in AdBRG1-infected cells. The anticipated result of p21 and p15 induction would be the inhibition of CDK2, resulting in cell cycle arrest prior to S phase. To determine if CDK activity was affected by BRG1 expression, we examined the phosphorylation status of pRB, which is a substrate of cyclin D-CDK4/6 and cyclin E-CDK2 in vivo and is hyperphosphorylated at the G1-to-S transition. Immunoblot analysis (Fig. 7C) using a polyclonal pRB antibody that recognizes all forms of pRB demonstrated that in cells expressing BRG1, very little hyperphosphorylated pRB was detected by 20 h p.i., and almost none was detected at 24 h p.i. while hyperphosphorylated pRB was still present in cells expressing AdGFP. The reduction in hyperphosphorylated pRB follows the appearance of p21 in AdBRG1-infected cell lysates and the association of p21 with CDK2, which is a consistent timeframe of molecular events. The hypophosphorylated, growth-suppressive form of pRB persisted in the AdBRG1-infected samples. These data suggest that CDK activity is inhibited in cells expressing BRG1.
BRG1 effect on E2F target genes. Our data strongly support a major role for p21 in the growth arrest seen upon BRG1 reintroduction into ALAB cells. Existing models of BRG1 function also predict a role for E2F gene regulation in this activity (48, 61). We did not see E2F target genes substantially modulated by microarray, so we took a number of classic E2F-regulated genes (30) and analyzed them more thoroughly by real-time PCR in ALAB cells infected with either AdBRG1 or AdGFP. Data from the 24-h time point are presented in Fig. 8A. Consistent with the microarray data, the expression of most E2F target genes examined was reduced only modestly by BRG1. We observed the same modest repression of some E2F target genes in SW13 cells (Fig. 8B).
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DISCUSSION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionAppendixReferences |
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We demonstrated that the CKIs p21 and p15 were induced upon reintroduction of BRG1 into BRG1-/- ALAB cells. Both p21 and p15 protein levels increased dramatically, and p21 protein was recruited to CDK2 complexes. Concomitantly, a marked reduction in hyperphosphorylated pRB was observed, consistent with CDK inhibition. Given the kinetics of CKI induction and subsequent cell cycle arrest, it appears that growth arrest by BRG1 in ALAB cells is likely to occur through direct induction of CKIs and inhibition of CDK activity. We also saw significant induction of the plasminogen activator inhibitor-1 (PAI-1) by 24 h (data not shown). PAI-1 has been shown to play a role in arrest of cellular proliferation during senescence (31). Subsequent down-regulation of E2F-mediated transcriptional activation and other genes may contribute to the maintenance of the arrest phenotype. Our results do not rule out an involvement from direct E2F-mediated events, such as lowering of cyclin E levels, but they contrast significantly from what is observed for pRB. Interestingly, arrest induced by pRB in SAOS2 cells was quite different, with a more dramatic down-regulation of E2F targets and no induction of CKIs (Fig. 8C and data not shown). This supports the notion that BRG1 and pRB functions may overlap but are distinct.
The observation that hBRM is induced upon BRG1 expression is interesting, considering the phenotype of mice harboring homozygous null mutations in either BRG1 or hBRM (5, 41, 51). hBRM-/- mice are viable, whereas BRG1-/- mice are not, suggesting that BRG1 has an important and essential role during development whereas hBRM is dispensable. The difference between the knockout mice can be explained by a model wherein BRG1 regulates the expression of hBRM. This model is further supported by a study of mRNA and protein levels of BRG1 and hBRM in mice. mBRG1 mRNA and protein are present throughout preimplantation development, whereas mbrm mRNA and protein are detectable only when differentiation occurs at the blastocyst stage (23). Interestingly, in other studies we and others have found that in several BRG1 mutant cell lines and in primary tumors, hBRM levels were generally much lower, or undetectable, suggesting that hBRM expression may be lost when BRG1 is mutated (39, 40, 59). Our conclusions are in disagreement with a report by Reisman et al., who failed to see hBRM regulation by BRG1 in SW13 and H522 cells (40). Our data in ALAB cells are to the contrary and illustrate that there is a cell type difference in BRM induction and that BRG1 can indeed regulate BRM expression under certain circumstances. It is of interest that the defect in BRG1 in SW13 and H522 cells that leads to loss of BRG1 expression remains undefined and may be a reason for this difference in response. The fact that we see both BRG1 and hBRM induction in our system may provide some explanation into the gene expression profile differences that we saw, compared to those in the earlier studies with SW13 cells. While earlier studies showed that BRM and BRG1 are essentially interchangeable, at least in restoring cell cycle arrest (40), there is growing evidence to suggest that BRG1 and BRM may have transcriptional specificity by virtue of unique protein-protein interactions with distinct classes of transcription factors (19).
We tested the effect of the ATPase mutant (KR) BRG on gene expression and observed that for the majority of genes, hBRM and CD44 for example, the ATPase mutant did not affect gene expression, suggesting that ATPase activity (and by inference, chromatin remodeling) is necessary for the activation by BRG1. In contrast, p21 was modestly induced by the ATPase mutant (Fig. 4C). This suggests that chromatin remodeling may not be the only contribution that BRG1 makes in the activation of p21. This is in agreement with earlier observations that the ATPase mutant causes growth arrest in BRG1 mutant cells, though not as well as wild-type BRG1 (8, 45).
Several of the genes induced by BRG1 in the microarray and real-time PCR experiments encode proteins with functions in cell adhesion and differentiation that may play a role in tumor suppression. These were of interest considering the dramatic effect that BRG1 reintroduction has on cellular morphology. Typically, cells become flattened and exhibit altered cytoskeletal organization (1, 45). CD44 encodes a hyaluronic acid cell surface receptor with important implications for growth and metastasis of tumors and is a known BRG1-regulated gene (47). CD44 associates with moesin (54), an ERM (ezrin, radixin, moesin) protein family member which links the plasma membrane with the actin cytoskeleton. Reduction of ERM protein expression by antisense oligonucleotides in cultured cells destroys cell adhesion sites (52). Thus, maintaining adequate levels of ERMs may be critical for cell adhesion. The expression of both CD44 and moesin was induced by BRG1 in ALAB cells (Fig. 2A and data not shown). These observations, taken together, raise important questions regarding a role for BRG1 in maintaining cell integrity and preventing cell spreading and metastasis. In a study using subtractive hybridization to survey genes which may be repressed during the development of neuroblastoma, Shtivelman and Bishop identified CD44 as well as AHNAK/PM227 (46). We found that AHNAK was also induced by BRG1 in the microarray experiments (Table 1). S100A2, a putative tumor suppressor that is down-regulated in tumor cells and appears to inhibit cell motility (33), was induced by BRG1 (Table 1). In addition, the breast epithelial antigen BA46, integrin alpha-5 (fibronectin receptor subunit), and integrin alpha-3 were also induced, indicating that BRG1 overexpression affected genes involved in cell adhesion, also.
It is of interest that several cell surface receptors were down-regulated by BRG1, including the chemokine receptors CCR1, CCR2, and CCR6. Chemokines and their receptors are involved in directed migration of immune cells by chemoattraction. Two other G protein-coupled receptors, GPR56 and GPR43, were also repressed by BRG1 expression.
We have not yet elucidated the precise mechanism by which BRG1 promotes induction of p21 and p15. BRG1 has been implicated in the induction of p21 by p53 in another report (22). Those authors demonstrated a physical association of BRG1 with the p21 promoter in a p53-dependent manner in SAOS2 cells and observed stimulation of p53 transactivation by BRG1 overexpression. While we have clearly shown that BRG1 can bind to elements within the p21 promoter in the absence of p53, it is plausible that in normal cells there is cooperativity in the activities of BRG1 and p53 on p21 induction, given that we observed BRG1 binding to the p21 promoter in the vicinity of the p53 element.
Both p15INK4b and p21 have also been described as TGF-ß-responsive genes. In addition, a number of other TGF-ß-regulated genes were detected in the microarray analysis, including insulin-like growth factor binding protein-4, integrin alpha-5, integrin alpha-3, matrix metalloproteinase-1, c-JUN, c-myc, and tropomyosin . If the effect of BRG1-mediated gene induction in BRG1-/- cells is related to restoration of a TGF-ß response, it may occur at any of several levels, including direct association with the promoter. We did not observe BRG1 on the proximal region of the p21 promoter that contains a TGF-ß-responsive element. Transcriptional induction of p15 and p21 by TGF-ß is in part Smad dependent (11, 24, 28, 34). Interactions between Smad and BRG1 have not been reported, but it would be interesting to test that hypothesis.
In conclusion, our studies elucidate a complex gene regulation profile for BRG1. Some of the effects on cell cycle regulation can be accounted for by induction of CKIs, which would be potentiated by indirectly affecting E2F transactivation of S-phase genes after the inhibition of CDK2. An array of other BRG1-regulated genes involved in cell adhesion, migration, and possible TGF-ß responses suggest overlapping but nonredundant pathways of tumor suppression for BRG1 and pRB.
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APPENDIX |
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ACKNOWLEDGMENTS |
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DNAX Research Inc. is supported by Schering Plough Corporation.
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FOOTNOTES |
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REFERENCES |
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), treated with staurosporine (
), or infected with adenoviruses expressing BRG1 (•) or GFP (
). Three hours after virus addition, medium was replaced on all samples with medium containing [14C]thymidine, and incorporated 14C was measured at the indicated times (after [14C]thymidine addition) to assay proliferation. (C) Asynchronous ALAB cells infected with the indicated adenoviruses for the indicated times were pulse-labeled with BrdU for 30 min prior to harvest for anti-BrdU flow cytometry cell cycle analysis. The x axis represents DNA content as assayed by 7-AAD (BD Biosciences). The y axis represents DNA synthesis as assayed by BrdU content.
) or camptothecin (CPT), were subjected to SDS-PAGE and analyzed by immunoblotting with anti-p53 or anti-p21 MAbs. (B) Lysates from asynchronous ALAB cells infected for 24 h with either AdGFP, Adp21, AdBRG1, Adbrg1K
