Introduction
Progression through the mammalian cell cycle requires that gene expression is coordinated with the activity of cell cycle control proteins. A critical period is the transition from the G1 into the S phase, as cells become committed to the division cycle. Binding of free E2F/DP heterodimers to E2F sites generally activates transcription of proteins required for G1 → S transition and DNA synthesis [
1,
2,
3,
4,
5], whereas complex formation with pRb or other pocket proteins including p107 and pRb-2/p130 silences transcriptional activities of downstream target genes [
6,
7,
8,
9,
10]. The resulting retinoblastoma protein (Rb)–E2F interaction not only blocks transcriptional activation by E2F, but also forms an active transcriptional repressor complex at the promoter of cell cycle genes that can block transcription by recruiting histone deacetylase (HDAC) and remodeling chromatin [
11,
12,
13,
14].
Several HDAC inhibitors mediate cell growth arrest and/or differentiation [
15,
16,
17]. We chose to examine the effect of HPCs, which have been reported to induce terminal differentiation and/or apoptosis [
18,
19,
20] in many transformed cells. Although treatment with HMBA induces remission in patients with myelodysplastic syndrome and acute myelogenoud leukemia, it is not currently used therapeutically because of the high dosage required (millimolar blood levels) and the accompanying toxic side effects (thrombocytopenia) [
21].
In this study, we report a novel mechanism of cell growth inhibition by the second generation of HPCs, named SAHA, which is 2000-fold more potent than HMBA and bears at least one hydroxamide in place of the amides in HMBA [
17]. SAHA was reported to be a histone deacetylase inhibitor and caused accumulation of hyperacetylated histone H4 in murine erythroleukemia [
17]. Very little is known about the anticancer mechanism of SAHA in epithelial cells; however, a recent study demonstrated that SAHA diet, at 900 parts per million (ppm), fed to rats reduced methylenitrosouren-induced mammary tumor incidence by 40%, total tumors by 66% and tumor volume by 78% [
22]. In this study, we tested whether SAHA has similar potency to inhibit cell growth in two mouse mammary epithelial cell lines, TM10 (p53 wt) and TM2H (p53 null). We identified a novel mechanism for SAHA in cell growth arrest through inhibition in DNA synthesis, concomitant with significant increases in the nuclear localization of pRb-2/p130 associated with E2F-4, decreases in key molecules in DNA synthesis (E2F-1, PCNA and p21), and increases in histone H3 and H4 protein and acetylation levels. This study discusses the difference in recovery from cell growth inhibition in two mammary epithelial cell lines, TM10 and TM2H, after SAHA removal from cultures.
Materials and methods
Development of cell lines and cell culture
The TM10 and TM2H cell lines chosen for this study were isolated from two different mouse mammary hyperplastic outgrowths: TM10 and TM2H, respectively, as described earlier [
23]. The parental TM10 outgrowth is a moderately tumorigenic outgrowth line
in vivo (time for 50% of the transplants to produce tumors, 11 months) that is karyotypically diploid and maintains wild type p53 expression. TM2H, in contrast, is a highly tumorigenic outgrowth line
in vivo (time for 50% of the transplants to produce tumors, ≤ 4 months), karyotypically aneuploid (DNA index = 1.69) and contains a p53 mutation resulting in a null phenotype [
24]. Exponentially growing TM10 and TM2H cell lines in DMEM/F12 media buffered with 10 mM HEPES at pH 7.6 with 2% adult bovine serum, 10 μg/ml insulin, 5 ng/ml epidermal growth factor (EGF) and 5 μg/ml gentamycin at 60-70% confluence were treated with SAHA (courtesy of Dr Paul Marks and Dr Victoria Richon, Memorial Sloan Kettering Cancer Center, New York, NY, USA). Cells were examined at the time points indicated for cell growth and cell cycle activities.
Analysis of cell growth
Cell growth rates of TM10 and TM2H lines were determined using a [
3H]-thymidine uptake assay, as described earlier [
25]. In initial studies, both cell lines were cultured in the absence or presence of 0.1, 2.5, 5.0, and 10 μM SAHA for 6 days. Both cell lines were also cultured for 2 days in the presence of SAHA at the concentrations already mentioned, followed by removing SAHA from the media for a subsequent 4 days. Based on the results of these studies, subsequent experiments examined asynchronously growing TM10 and TM2H cell lines in the presence or absence of 2.5 μM SAHA for 24 h.
FACS analysis
Exponentially growing cells at 60-70% confluence at control or treated with 2.5 μM SAHA for 24 h were pulse-labeled for 1 h with 10 μM BrdU (Sigma, St Louis, MO, USA). The cell cultures were rinsed once with phosphate buffered saline (PBS), trypsinized for 3 min and rinsed three times with PBS. Cells were resuspended in 200 μl PBS and fixed in 5 ml cold 70% ethanol overnight. Fixed cells were counted and, generally, 4 × 106 cells were transferred to 15 ml polypropylene tubes, centrifuged at 3000 rpm for 5 min and the supernatant removed. Cells were stained for the newly incorporated BrdU for DNA synthesis using BrdU monoclonal antibodies conjugated to fluorescein isothiocyanate (FITC) and stained with propidium iodide for DNA content following the protocol described in Becton and Dickinson's (San Diego, CA, USA) instructions for flow cytometric analysis.
Nuclear and cytoplasmic extracts
To obtain nuclei, (4–6) × 107 cells of each cell line grown in the presence and absence of 2.5 μM SAHA were washed twice in PBS, followed by suspension in 0.3 ml nuclear buffer consisting of 2 mM MgCl2, 5 mM K2HPO4, 0.1 mM EDTA, 1 mM PMSF, 20 μg/ml aprotinin, 20 μg/ml leupeptin, 0.1 mM Na3VO4 and 5 mM β-glycerophosphate. An additional 0.3 ml nuclear buffer containing 0.7% Triton X-100 was then added, after standing on ice for 8-10 min. The suspensions were examined for cell lysis microscopically, centrifuged at 800 rpm for 10 min at 4°C, and the supernatant designated the cytoplasmic fraction. The pellets were washed once with nuclear buffer, and the nuclear extracts were prepared by resuspending the pellets in 0.3 ml buffer containing 20 mM HEPES (pH 7.8), 25% glycerol, 0.42 M NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 0.5 mM PMSF, 0.5 mM DTTl, 0.1 mM Na3VO4, 50 mM NaF, 20 μg/ml leupeptin, and 5 mM β-glycerophosphate. Sonication was carried out on ice using an ultrasonicator processor (PGC Scientific, Caithersburg, MA, USA), and the mixtures were examined microscopically for complete break of the nuclei. The supernatants were designated as nuclear extracts after centrifugation of the mixtures, and the total protein was determined in the nuclear and cytoplasmic fractions.
Western blot and immunoprecipitation analysis
Histones were isolated and lyophilized from nuclear extracts in SAHA treated and untreated TM10 and TM2H cell lines following a protocol described earlier [
26]. Histone samples were assessed for purification quality on 15% SDS acrylamide gel including calf thymus histones as controls before western blot analysis was carried out. Histone samples were resolved by electrophoresis using 15% acid–urea gel containing 36%w/v urea, 5%v/v acetic acid, 600 μl TEMED and 0.7 ml of 10% ammonium persulfate prepared as described elsewhere [
27]. Gels were either stained by Coomassie Brilliant Blue or equilibrated to be transferred into transfer buffer (0.7% acetic acid). The gel sandwich was set up as usual except for the placement of the blotting membrane (because proteins in this type of gel will migrate toward the negative electrode), and the procedure was continued as described earlier [
27]. Each histone was resolved into multiple bands and were visualized in Coomassie blue gel. The acetylated histone isoforms were detected by immunoblot against acetylated histone H3 isoforms using anti-H3 antibodies raised and characterized by Dr Sharon Roth at MD Anderson Cancer Center (personal communication) or against acetylated histone H4 isoforms using anti-H4 polyclonal antibody (Upstate Biotechnology Inc, Lake Placid, NY, USA).
Western blot analysis for all the proteins examined in this study was carried out on equal amounts of cellular fraction protein extracts (100 μg/sample) following a protocol described earlier [
25]. Staining the gel with Coomassie Brilliant Blue for each experiment assessed equal loading control. TM10 and TM2H cells (0.65 × 10
3 cells/cm
2) seeded in 75 T flasks grew for 2 days, and were treated with 2.5 μM SAHA for 24 h. Cellular fraction protein extracts were prepared after SAHA treatment [
25]. Primary antibodies used at 1 or 2 μg/ml were p21/Cip (Pharmingen Inc, San Diego, CA, USA), pRb (IF8), p107 (SD9), p130 (C-20), E2F-1 (C-20), E2F-4(C-20) and PCNA (C-20) (Santa Cruz Biotechnology Inc, Santa Cruz, CA, USA). All antibodies were examined for specificity prior to use. The secondary antibodies conjugated to horseradish peroxidase (1 : 5000-1 : 15,000 dilution) were used followed by enhanced chemiluminescence detection reaction as described by the manufacturer (Amersham Pharmacia Biotechnology, Amersham, Bucks, UK).
The immunoprecipitation assay followed by western blot analysis was as described previously [
25]. Briefly, equal nuclear cell extract (200 μg/sample) was mixed with 3 μg anti-E2F-4 antibodies at 4°C followed by the addition of 50 μl protein A-sepharose beads (Amersham Biotechnology). The immune complex was centrifuged, and the proteins in the immune complex were resolved by 10% SDS-PAGE followed by western blot analysis using anti-pRb-2/p130 antibodies as already described for western blot analysis.
Cyclin-dependent kinase assay
The TM10 and TM2H cell cultures at control and treated with 2.5 μM SAHA for 24 h were examined for cyclin D1, E, and A associated kinase activities as described previously [
28]. Briefly, cellular protein extracts (25 μg) were precleared with 10 μl of 10% preimmune normal rabbit serum followed by immunoprecipitation with either 3 μg anti-cyclin D1, E polyclonal antibodies (Upstate Biotechnology), 2 μg anti-cyclin A polyclonal antibody antibodies (C-19)-G (Pharmingen), or normal rabbit preimmune serum as a negative control. All antibodies were examined for specificity [
28]. Immunoprecipitate complexes were examined for kinase assay following a procedure described previously [
28]. The substrates utilized in the kinase assays were either Histone-H1 (Sigma) or RB protein (Santa Cruz Biotechnology). The phosphorylated H1 and pRb bands were scanned and quantitated densitometrically using a Phosphoimager (Molecular Dynamics, Sunnyvle, CA, USA).
Immunofluorescent staining
Exponentially growing cells on slides were fixed in 4% paraformaldehyde in PEM buffer (80 mM Pipes [pH 7.0], 5 mM EGTA and 2 mM MgCl2) for 30 min, permeabilized in 0.5% Triton X-100 in PEM buffer at room temperature for 15 min and rinsed three times with TBS + 0.1% Tween 20, followed by incubation in 0.5% nonfat dry milk in TBS + 0.1% Tween 20 for 2 h at room temperature. For BrdU and DAPI double immunostaining, cells were incubated for 30 min in media supplemented with 5 μM BrdU. After 30 min, cells were incubated in 2N HCl for 5 min at room temperature prior to incubation with the primary antibody (this step was omitted for pRb-2/p130 immunostaining). Following washing three times, cells were incubated in a (1 : 50) dilution of mouse anti-BrdU monoclonal antibody (Boehringer Mannheim, Indianapolis, IN, USA) for 2 h at 37°C. After washing three times, cells were then incubated in FITC-conjugated anti-mouse secondary antibody (1 : 400) dilution for 1 h at 37°C, followed by washing three times with anti-fade equilibrating buffer and mounting in anti-fade mounting medium (Molecular Probe, Eugene, OR, USA). The same sequential steps were followed for pRb-2/p130 immunostaining using anti-pRb-2/p130 monoclonal antibodies (Santa Cruz Biotechnology).
Apoptosis
Apoptotic activities in TM10 and TM2H cells in the absence and presence of 2.5 μM SAHA were examined by two procedures: the TACS 2TdT In Situ Apoptosis Detection assay following the manufacturer's instructions (Trevingen, Gaithersburg, MD, USA), and the DNA Fragmentation Assay described elsewhere [
29].
Discussion
In this study, we present novel data on the mechanism of SAHA in cell growth arrest on two mouse mammary epithelial cell lines, TM10 (p53 wt) and TM2H (p53 null). SAHA was able to increase histone H3 and H4 protein and acetylation levels, and caused a profound decrease in the protein levels of key molecules, PCNA and E2F-1, essential for DNA synthesis. Furthermore, SAHA significantly enhanced the interaction of pRb2/p130 to E2F-4 and the nuclear localization of the pRb2/p130–E2F-4 complex. SAHA also resulted in the inhibition of G1/S kinase activities and, consequently, hypophosphorylation of the three pRb pocket proteins, which led to G1 cell growth arrest and dramatic decreases in DNA synthesis in both cell lines. TM10 cells continued to be inhibited for 4 days upon removing SAHA after 2 days of treatment, whereas TM2H cells were able to recover their proliferation potentials. A summary of the differences in the molecular status and cell cycle profile between TM10 and TM2H cell lines before SAHA treatment are summarized in Table
1. These differences in p21 protein and BrdU index were predicted based on p53 status of these two cell lines and are in parallel with other reports on mammary tumors, where the absence of p53 results primarily in greater proliferation response in the affected cell [
42]. No differences in histone H3 and H4 acetylation levels were, however, observed in relation to p53 status between the two cell lines. It is not known at this point whether specific mutations of p53 alter the degree of histone acetylation in cells. Histone acetylation and p53 mutation appear not to be correlated in this study; nevertheless, it is necessary and more sensitive to examine histone acetylase and deacetylase activities in correlation to p53 status.
Table 1
The differences between TM10 and TM2H cell lines in the cell cycle profile and protein status
| (p53 wt) | (p53 null) |
Nuclear proteins | | |
E2F-1 | 1 | 4.8 |
PCNA | 1 | 4.0 |
Rb2/p130 | 1 (predominantly | 1 (predominantly |
| hypophosphorylation) | hypophosphorylation) |
p21/Cip1 | Strongly detectable | Weakly detectable |
Cyclin-dependent kinase activity | | |
D1 | 1 | 2.2 |
E | 1 | 1.7 |
A | 1 | 3 |
BrdU index | 16% | 34% |
Cell cycle profile | | |
G0/G1 | 57% | 39% |
S | 18% | 28% |
The mechanism of SAHA in blocking DNA synthesis, as demonstrated by flow cytometric analysis, appeared similar in both TM10 and TM2H cell lines, and implicated several events. First, SAHA increased histones H3 and H4 protein levels after 24 h of treatment, which could result in cell cycle arrest. It has been reported that upregulation of KIAA0128 gene expression, which has been implicated in activation of histone mRNA synthesis, was related to cell cycle arrest in MCF-7 cells after treatment with SAHA [
43]. As SAHA treatment increased histone (H3 and H4) protein and acetylation levels in both cell lines, this may have altered the association of histones with DNA, thereby altering nucleosomal conformation and stability [
27,
44]. Local perturbations of chromatin structure can specifically affect the accessibility and/or function of transcriptional regulatory proteins that bind DNA sequences in the region where histone acetylation or deacetylation took place [
44]. HDAC inhibitors, such as trichostatin A and trapoxin, modulate gene expression in either a positive, negative or neutral fashion [
45]. Ample studies have demonstrated the implication of histone hyperacetylation in gene transcription but also in silencing gene expression of others [
27,
44,
45].
It is well known that E2F-1 regulates transcription of genes predominantly expressed during the G1 → S transition such as cyclins [
1,
5], cdks [
1],
E2F-1 gene [
46], the
RB1 tumor suppressor gene [
8,
47], and genes for DNA replication and repair enzymes and factors [
4]. It appears that SAHA has a profound inhibitory impact on the protein levels of key molecules, E2F-1, PCNA and p21, essential for DNA synthesis. Based on previous reports, disintegration of the cyclin/cdk complexes important for DNA synthesis is correlated to E2F-1 expression level [
1,
5]. It is thus conceivable to interpret that the profound inhibition in E2F-1 and PCNA protein levels after 24 h of exposure to 2.5 μM SAHA may result in disintegration and deactivation of D1, E and A cdk2 complexes, which consequently leads to hypophosphorylation of the three Rb pocket proteins. It is plausible to suggest that the inhibition in E2F-1 protein levels by SAHA was either at the transcription level or induction of the ubiquitin-protein ligase responsible for E2F-1 degradation, but not E2F-4, and this resulted in blocked DNA synthesis. Further work is necessary to prove whether the effect of SAHA is at the RNA transcription level or on stability of E2F-1 protein.
The inhibition in nuclear p21 in SAHA treated TM2H as well as TM10 cells underscores that SAHA-arrested cell growth is through a p53-independent pathway. A recent report indicated that the transcription of p21
Cip1 and accumulation of acetylated histones associated with the promoter and coding regions of that gene were induced after 2 h in 7.5 μM SAHA and fall by 24 h in T24 bladder carcinoma cells [
48]. Although we have utilized 2.5 μM SAHA, our results are in agreement with their data on the fall in p21 level after 24 h of treatment.
A more intriguing and novel mechanism of SAHA-mediated cell growth arrest was the enhanced interaction and nuclear localization of Rb2/p130–E2F-4 complexes in both cell lines after 24 h of treatment. It is well documented that E2F-1 possesses an intrinsic nuclear localization signal whereas E2F-4 is devoid of such signal [
49,
50], and that the mechanism of E2F-4 nuclear localization has been documented to be through its interaction with Rb2/p130 pocket protein, which impedes cell cycle progression [
39,
40]. Furthermore, recent studies demonstrated that Rb2/p130 in complexes with E2F-4 actively represses E2F-1 transcription in cell differentiation and growth arrest, and that this complex was considered the main E2F-1 regulator during the early G1 phase [
39,
51,
52]. Other reports suggest that Rb recruitment of HDAC1 activity repressed E2F-1 [
12,
13]. Although SAHA increases acetylation of histones H3 and H4, it is not known whether SAHA is able to inhibit all HDAC activities of all types of histones, including HDAC1, or whether the profound increase in Rb2/p130–E2F-4 nuclear complex after SAHA treatment may have an alternative pathway other than recruitment of HDAC1 activity.
The difference between TM10 (p53 wt) and TM2H (p53 null) cell cultures in response to removing 2.5 μM SAHA following 2 or 3 days of treatment was significant. We suggest that it might reflect their difference in p53 status. The TM10 cells did not exhibit signs of cell proliferation or 'recovery' during the following 3-4 days. TM2H cells, in contrast, recovered by 88% after 2 days of treatment. Longer treatment may be necessary to inhibit TM2H (p53 null) mammary epithelial cells as preliminary results indicate TM2H cells did not recover after 3 days of SAHA treatment (data not shown). We suggest that the difference in growth recovery between TM10 (p53 wt) and TM2H (p53 null) cell lines after 2 days in 2.5 μM SAHA may be attributed to two factors, both related to their p53 status. Firstly, the p53 in TM10 (p53 wt) cells might have been acetylated upon treatment with 2.5 μM SAHA for 2 or 3 days. Recent studies demonstrated acetylation of p53 in the C-terminal domain increased the DNA-binding capacity of the protein [
53,
54,
55]. This event is obviously not present in TM2H (p53 null). Secondly, although TM10 cells (p53 wt) have lost 64% of their nuclear p21 during SAHA treatment, the remaining 36% of the nuclear p21 plus the continued synthesis of p21 by p53 activity [
55,
56,
57] during the recovery period would maintain TM10 cells in the inhibited state for several days without SAHA. TM2H cells (p53 null), in contrast, lack both negative regulatory potentials of acetylated p53 and the availability of nuclear p21.
We conclude that the mechanisms of SAHA inhibition of DNA synthesis and cell growth arrest at G1 were similar in both TM10 (p53 wt) and TM2H (p53 null) mouse mammary epithelial cell lines. A proposed mechanism of SAHA stresses the involvement of pRb2/p130–E2F-4 interaction and nuclear localization, which ultimately results in cell growth arrest and repression in nuclear E2F-1 and PCNA protein levels, and the subsequent inhibition of DNA synthesis in both cell lines. However, p53 status was critical in maintaining growth arrest in TM10 cells 4 days after removing SAHA treatment, whereas TM2H cells (p53 null) recovered growth arrest under the same conditions. We therefore suggest that the dose and time regimen for histone deacetylase inhibitors, such as SAHA, may have to consider the p53 status of breast cancers.