Hdacs are required for growth and morphogenesis in zebrafish exocrine pancreas
Our recent study indicates a crucial role of Hdac1 in exocrine pancreatic epithelial proliferation (Zhou et al., 2011
). Here, we determined the role of Hdacs in the developing exocrine pancreas by treating WT zebrafish larvae with TSA between 48 and 72 hours post-fertilization (h.p.f.) when the pancreatic epithelia maximally proliferate during this period (Yee et al., 2007
). First, TSA at various concentrations was added at 48 h.p.f., and acetylation of histones H3 and H4 was analyzed at 72 h.p.f. At a concentration of 165 nM, TSA induced maximal level of acetylated histone H3 and near-maximal level of acetylated histone H4 (). The effect of TSA on exocrine pancreas was then determined by incubating WT zebrafish larvae with 165 nM TSA for 24 hours. The TSA-treated larvae appeared grossly normal. They developed exocrine pancreas of reduced size, and acinar morphogenesis was disrupted (). While TSA significantly reduced the number of pancreatic epithelia (4′6-diamidino-2-phenylindole or DAPI containing nuclei) by 34%, the proliferative rate as determined by the proportion of epithelia in S-phase (5-bromo-2′-deoxyuridine or BrdU containing nuclei) was not significantly decreased (). The effect of TSA on exocrine pancreas was associated with increased levels of acetylated histones H3 and H4 (). Therefore, Hdacs are required for normal growth and morphogenesis of exocrine pancreas through regulating the acetylation status of histones in zebrafish.
TSA at 165 nM induces maximal acetylation of histone H3 and near-maximal acetylation of histone H4.
TSA impairs growth and disrupts morphogenesis of exocrine pancreas in zebrafish larvae with hyperacetylation of nucleosomal histones.
Combination of inhibitors of Hdacs and Polr3 arrests expansion of exocrine pancreas during morphogenesis
Considering the growth requirement of Hdacs (, ) and our previous findings of Polr3 in the proliferation of pancreatic epithelia (Yee et al., 2007
), we hypothesize that Hdacs and Polr3 cooperatively regulate the growth of exocrine pancreas. To test this hypothesis, we examined the effects of a combination of TSA and the Polr3 inhibitor ML-60218 on the growth of exocrine pancreas during morphogenesis. First, we determined the optimal concentrations of TSA or ML-60218 by incubating WT zebrafish larvae at various concentrations of each small molecule and observing for signs of toxicity and lethality. The zebrafish larvae incubated in the medium containing TSA at a maximal concentration of 330 nM appeared grossly normal. The larvae treated with ML-60218 at a concentration of 110 µM or below appeared normal without precipitation of ML-60218 (supplementary material Table S1). Thus, TSA at 165 nM and ML-60218 at 110 µM were used for the subsequent experiments.
Addition of TSA and ML-60218 at 48 h.p.f. to WT zebrafish larvae for 24 hours completely arrested expansion of the exocrine pancreas, such that the size of the exocrine pancreas remained the same as that in untreated WT larvae at 48 h.p.f. (; Yee et al., 2005
). Neither TSA nor ML-60218 at the concentrations being used alone caused any apparent reduction of organ size. No apparent effect was observed by treatment with either TSA + dimethyl sulfoxide (DMSO, the solvent used to dissolve TSA and ML-60218) or DMSO alone (N. S. Yee, unpublished). The same effects of these small molecules were observed in WT zebrafish larvae of various strains including AB, TLF, and WIK (N. S. Yee, unpublished).
TSA and ML-60218 synergistically inhibit expansion of exocrine pancreas in zebrafish by impeding cell cycle progression, with enhanced induction of histone acetylation and cyclin-dependent kinase inhibitors.
To determine the cellular basis of the growth-suppressive effect of the combination of inhibitors on exocrine pancreas, the rate of epithelial proliferation was assessed. The combination of TSA and ML-60218 caused a significant reduction in the proportion of S-phase (BrdU+) nuclei by 23% of that in control (). Neither TSA nor ML-60218 significantly reduced the proportion of BrdU+ nuclei. Moreover, TSA + ML-60218 supra-additively reduced the number of DAPI positive nuclei by 48% (P<0.01). Using morphometric analysis, the combination of TSA + ML-60218 significantly reduced cell growth by 22%, whereas neither TSA nor ML-60218 alone caused any significant impairment of cell growth (). No increase in apoptotic cell death was observed in the exocrine pancreas of the larvae treated with TSA + ML-60218 using Apoptag® (N. S. Yee, unpublished). These results indicate that the combination of TSA and ML-60218 produces enhanced suppression of exocrine pancreatic growth by inhibiting epithelial proliferation with impaired progression from G1 to S phase of the cell cycle and reduced cell growth.
To explore the mechanism by which the combination of TSA and ML-60218 perturbed cellular proliferation, the acetylation status of histones H3 and H4 and expression of p21cdkn1a and p27cdkn1b as molecular markers of progression of cell cycle from G1 to S phase were assessed by immunoblotting and real-time polymerase chain reaction (PCR), respectively. TSA caused a slight accumulation of acetylated histones H3 and H4, and combination with ML-60218 enhanced this effect by 2-fold and 70%, respectively (). Consistent with the effects on acetylated histones, the combination of TSA and ML-60218 significantly up-regulated the levels of both p21cdkn1a and p27cdkn1b mRNA by about 1.3-fold over control, and either TSA or ML-60218 alone caused relatively little changes (). No significant alteration of total tRNA and 5s rRNA levels was detected among the experimental groups and controls as revealed by electrophoretic separation of total RNA extracted from whole larvae in each group (N. S. Yee, unpublished). Taken together, the combination of TSA and ML-60218 causes growth arrest of exocrine pancreas by suppressing pancreatic epithelial proliferation that is associated with enhanced accumulation of acetylated histones and up-regulated expression of p21cdkn1a and p27cdkn1b.
Combination of SAHA and ML-60218 produces enhanced suppression of proliferation in human pancreatic adenocarcinoma by impairing cell cycle progression and inducing apoptosis
To translate the findings of the zebrafish studies, we examined the effects of the clinically used HDAC inhibitor SAHA in combination with ML-60218 in the human pancreatic adenocarcinoma cell lines PANC-1 and BxPC-3. These cells have been shown to carry mutations in K-RAS
(Berrozpe et al., 1994
; Moore et al., 2001
), and they are resistant to the cytotoxic effects of the chemotherapeutic agent gemcitabine at clinically used concentrations (1 µM and 10 µM) and even up to 50 µM (supplementary material Table S2A). SAHA is a synthetic inhibitor of classes I and II HDACs. It was used at 5 µM in this study based on the dose–response in cellular proliferation of PANC-1 and BxPC-3 (Chun et al., 2009
). ML-60218 was used at 100 µM, which is approximately the same concentration used in the experiments with zebrafish (). In a soft agar assay that mimics in vivo conditions of tumor growth, the combination of SAHA and ML-60218 produced enhanced reduction of anchorage-independent colony formation (). Neither SAHA nor ML-60218 alone significantly inhibited PANC-1 colony formation; but when combined, they reduced PANC-1 colony formation by 47%. In BxPC-3, SAHA reduced colony formation by 35%, ML-60218 had no effect, and SAHA + ML-60218 reduced colony formation by 70%, which was significantly different compared to SAHA alone. DMSO did not significantly affect colony formation either alone or in combination with SAHA (N. S. Yee, unpublished).
SAHA and ML-60218 inhibit anchorage-independent colony formation and induce cellular morphology consistent with cellular senescence and cell death.
To gain insights into the effects on colony formation, cellular morphology was analyzed by phase contrast microscopy (). In both PANC-1 and BxPC-3, the combination of SAHA and ML-60218 produced morphologic changes including cell enlargement and flattening, irregular shapes with cytoplasmic projections, accumulation of cytoplasmic vacuoles, and nuclear fragmentation. These morphologic changes suggest that the enhanced suppression of colony formation might involve induction of cytodifferentiation, apoptotic cell death, and senescence-like cytostasis.
To investigate the cellular mechanisms underlying the suppression of colony formation, the effects of SAHA and ML-60218 on cellular proliferation were analyzed using the MTS assay. The combination of SAHA and ML-60218 produced enhanced inhibition of cellular proliferation (). In PANC-1, neither SAHA nor ML-60218 had any effect, but the combination significantly reduced proliferation by 23%. In BxPC-3, SAHA reduced proliferation by 49%, ML-60218 had no effect, and the combination of SAHA and ML-60218 reduced proliferation by 53%. To determine the cytotoxicity of SAHA and ML60218 in normal pancreatic epithelia, the immortalized human pancreatic ductal epithelia H6c7 were treated with the inhibitors and analyzed for proliferation. Similar to their effects on PANC-1 and BxPC-3, the combination of SAHA and ML-60218 produced enhanced cytotoxicity in H6c7 cells (supplementary material Table S2B). However, the proliferation-suppressing effect of the combination of SAHA and ML-60218 in H6c7 cells (reduced by 54%) (supplementary material Table S2B) is less than that of gemcitabine at 1 µM and 10 µM (reduced by 64% and 82%, respectively) (supplementary material Table S2A).
Combination of SAHA and ML-60218 produces augmented suppression of cellular proliferation with impaired cell cycle progression, enhanced apoptotic cell death; SAHA either alone or in combination with ML-60218 induces nuclear localization of survivin.
Next, the effect on cell cycle progression was analyzed by flow cytometric determination of DNA content (). In PANC-1 and BxPC-3, SAHA increased the proportion of cells in G0/G1 (28%), and decreased the proportion of cells in the S phase (28% and 24%, respectively). ML-60218 alone produced no or little effect. The combination of SAHA and ML-60218 caused a further accumulation of cells in G0/G1 (additional 3% and 5% in PANC-1 and BxPC-3, respectively) as compared to SAHA alone. The effect on cell death was then determined by flow cytometric detection of Annexin-V binding. Compared to control PANC-1, SAHA caused 13% more cells to undergo apoptosis, ML-60218 had no effect, and SAHA + ML-60218 induced apoptosis in an additional 21% of cells (). In H6c7 cells, the combination of SAHA and ML-60218 produced a smaller proportion of apoptotic cells than gemcitabine (3-fold and 4-fold, respectively) relative to control (supplementary material Table S2C). Moreover, the combination of SAHA and ML-60218 produced fewer dead cells than gemcitabine (no increase and 4-fold, respectively) as compared with no treatment (supplementary material Table S2C).
To further explore the mechanisms by which SAHA and ML-60218 induced apoptosis, expression of the anti-apoptotic protein survivin was evaluated by confocal microscopy. In the untreated or DMSO-treated BxPC-3 cells, survivin was present diffusely throughout the cells. Either SAHA or the combination of SAHA and ML-60218 caused survivin to be expressed almost exclusively in the nuclei (), and this effect presumably abolishes the anti-apoptotic action of survivin by accelerating its degradation in the nucleus (Connell et al., 2008a
These results show that SAHA induces cytotoxicity in pancreatic adenocarcinoma by causing cell cycle arrest and apoptotic cell death, and these effects are enhanced by ML-60218. The combination of SAHA and ML-60218 also produces enhanced cytotoxicity in the pancreatic ductal epithelia, but the extent of of cytotoxicity is less than that produced by gemcitabine.
SAHA and ML-60218 cooperatively up-regulate expression of BAX and p21CDKN1A protein
In an attempt to gain insights into the mechanism underlying the enhanced actions of the combination of SAHA and ML-60218, we analyzed histone acetylation by immunoblotting (). SAHA alone increased the levels of acetylated histones H3 and H4 in PANC-1 by 40% and 110%, respectively. In BxPC-3, SAHA increased acetylated histones H3 and H4 by 20% and 4.3-fold, respectively. The combination of SAHA and ML-60218 produced similar effects as SAHA alone in both cell lines. The increased level of acetylated histones is consistent with the established action of SAHA and expected to modulate gene expression.
Effect of SAHA and ML-60218 on histone acetylation, BAX, and p21CDKN1A expression.
To further characterize the growth-suppressive actions of SAHA and ML-60218, the level of the pro-apoptotic regulator BAX was analyzed by immunoblotting (). Consistent with the results of the flow cytometric detection of apoptosis, the combination of SAHA and ML-60218 produced enhanced expression of BAX by 2.1-fold and 6.2-fold over control in PANC-1 and BxPC-3, respectively. Either SAHA or ML-60218 alone produced relatively little or no increase in the level of BAX. Expression of the cyclin-dependent kinase inhibitor p21CDKN1A was then examined. In PANC-1 and BxPC-3, SAHA caused elevation of the p21CDKN1A protein levels by 60% and 80%, respectively, and these were further increased by 1.6-fold and 5-fold over control, respectively, when SAHA was combined with ML-60218.
These results indicate that the combination of SAHA and ML-60218 suppresses cellular proliferation by impairing cell cycle progression from G1 to S phase with up-regulation of p21CDKN1A and by inducing apoptosis through the BAX-mediated mitochondrial pathway with nuclear localization of survivin.
ML-60218 reverses SAHA-stimulated tRNA expression
We attempted to understand the mechanism that mediates the enhanced cytotoxicty of the combination of SAHA and ML-60218 by examining their effects on tRNA levels. It has been shown that histone acetylation influences chromatin structure and organization that play a role in protein–DNA interactions and regulate expression of POLR3-transcribed genes (Lee et al., 1993
; Boyd et al., 2000
; Noma et al., 2006
). The activity of POLR3 transcription complex is up-regulated in most of the examined pancreatic adenocarcinoma cell lines (), and over-expression of tRNA has been implicated in malignant transformation (Marshall and White, 2008
). We hypothesize that inhibition of HDACs by SAHA leads to up-regulated expression of POLR3-mediated transcripts including tRNAs, which in turn may antagonize its anti-tumor activity. To test this hypothesis, we quantified tRNAs in the pancreatic cancer cells treated with SAHA alone or in combination with ML-60218 using real-time PCR. SAHA significantly increased the levels of tRNAMet
in both PANC-1 and BxPC-3 by 71% and 85%, respectively, and increased tRNASer
in PANC-1 and BxPC-3 by 2.2-fold and 1.9-fold, respectively (). In control cells, ML-60218 alone had slight effect on the levels of tRNAMet
. When added to SAHA-treated cells, ML-60218 significantly reduced SAHA-stimulated expression of tRNAMet
by 55% and 64%, respectively in PANC-1, and by 43% and 77%, respectively, in BxPC-3.
Relative levels of tRNAMet and tRNASer in human pancreatic adenocarcinoma cell lines.
ML-60218-mediated suppression of SAHA-induced up-regulation of tRNA as a potential mechanism that contributes to the enhanced anti-cancer actions in pancreatic adenocarcinoma.
Therefore, SAHA inhibits HDACs and increases acetylation of histones H3 and H4, resulting in up-regulated expression of the pro-apoptotic regulator (BAX), the CDK inhibitor (p21CDKN1A), and tRNAs (tRNAMet and tRNASer). At a concentration that it does not significantly affect tRNA level, ML-60218 reverses SAHA-induced up-regulation of tRNAs, and this may contribute to the enhanced cell cycle arrest and apoptosis in pancreatic adenocarcinoma by the combination of SAHA and ML-60218, as illustrated in .